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Hydrogen alleviates osteoarthritis via JNK signaling pathway

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Hydrogen alleviates osteoarthritis via JNK signaling pathway

Introduction

As an irreversible condition of arthritic degeneration that can result in seriously unsteady joints, osteoarthritis (OA) is pathologically characterized by degenerative articular cartilage, progressive subchondral sclerosis, genesis of osteophytes, as well as inflammatory synovium.1 Being the most widespread type of chronic arthritic condition, its impact on everyday activities is especially prominent in elderly populations.2,3 Plentiful studies have identified a range of influencing factors of OA development, including inflammation, aging, obesity, trauma, deformed joints, osteoporosis, among others.4 In spite of these, the pathogenesis of OA remains hardly understood. Oxidative stress and inflammation have been proven as crucial risk factors for the OA progression.5 Besides, a large number of OA factors are capable of causing oxidant-antioxidant level imbalance, which facilitates chondrocyte stimulation to result in inflammatory cytokine generation.6–8 The current clinical therapies for OA mainly aim at improving joint functions and relieving pain. However, this treatment regime does not alleviate the progression of OA. Therefore, it is essential to explore new strategies for OA treatment.

Hydrogen (H2) is a colorless and odorless gas. It exhibits diverse biological activities, including anti-inflammatory and anti-oxidative properties.9–13 In 1975, Dole et al found that 97.5% H2 could be used as therapy for cancer.14 Ohta S found that a 2% concentration of H2 could ameliorate mitochondrial diseases due to its rapid diffusion into the tissues and cells.15 Ohsawa et al observed that the inhalation of 1–4% H2 could significantly alleviate cerebral ischemia-reperfusion injury by selectively reducing cytotoxic oxygen radicals.16

Previous studies revealed that the JNK signaling pathway can regulate inflammatory and apoptosis. When initiated by certain stimuli such as TBHP (tert-butyl hydroperoxide), the activation of JNK causes the phosphorylation of c-Jun. The c-Jun then decreases proteoglycan synthesis and enhances the production of matrix metalloproteinase-13 (MMP-13).17 Gao et al found that LncRNA MALAT-1 can suppress apoptosis and cartilage matrix degradation via the JNK signaling pathway.18 Jiang et al revealed that Nesfatin-1 inhibited the IL-1β-induced activation of NF-κB, the mitogen-activated protein kinase (MAPK), and the Bax/Bcl-2 signal pathway in chondrocytes.19 The activation of the JNK pathway stimulates the production of apoptosis mediators like Bax, Cytochrome c, cleaved caspase-3, etc.20,21 Therefore, inhibiting the activation of the JNK pathway might be a promising therapeutic strategy for the treatment of OA. A recent study indicated that H2 could suppress oxidative damage by decreasing MMP-13 and increasing collagenase type II (Col II) expression.22 However, it is unclear whether H2 mediates its protective effect in OA by utilizing the JNK pathway.

In this study, we found that H2 exerted a chondroprotective effect in OA and explored the potential mechanism under in vivo as well as in vitro conditions. Here, chondrocytes were exposed to tert-butyl hydroperoxide (TBHP) for the in vitro induction of oxidative stress. We found that H2 may suppress TBHP-induced apoptosis and inflammation in chondrocytes through the JNK pathway. Moreover, the activation of JNK by Anisomycin eliminated the anti-apoptotic and anti-inflammatory effects of H2. Thus, H2 suppressed apoptosis and inflammation and attenuated OA through the JNK signaling pathway. Our study highlighted the therapeutic potential of H2 in OA and explored the mechanism of anti-apoptotic and anti-inflammatory effects of H2 in the chondrocytes.

Materials and Methods

Ethics Statement and Experimental Animals

All Experimental procedures involving animal care strictly followed the guidelines for the Animal Care and Use outlined by the Committee of Wenzhou Medical University and approved by the Animal Care and Care Committee of Wenzhou Medical University. Tissue collection and experiments involving human OA were approved by the Second Affiliated Hospital of Ethics Committee of Wenzhou Medical University (ethic cord: LCKY-2019–67) and Yuying unknown Children’s Hospital and followed the guiding principles of the Helsinki Declaration. Informed consent was obtained from the human participants of this study.

Reagents

Anisomycin, Collagenase-II, Safranin-O/Fast Green, and dimethylsulfoxide (DMSO) were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). Primary antibodies of Collagenase II, Aggrecan, ADAMTS-5, and MMP-13 were gained from Abcam (Cambridge, MA, USA). Primary antibodies directed against P-JNK and JNK were procured from Cell Signaling Technology (MA, USA). Primary antibodies against Bcl-2 and GAPDH were procured from ProteinTech Group (Wuhan, China). Primary antibodies against cleaved caspase-3 and Bax were procured from Affinity Biosciences (Cincinnati, OH, USA). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM)/F12 were obtained from Gibco (Grand Island, NY, USA). Tert-Butyl hydroperoxide solution (TBHP) was procured from Sigma-Aldrich (St Louis, MO, USA). The secondary antibodies of Goat Anti-Mouse IgG, Goat Anti-Rabbit IgG, Alexa Fluor®488 and labeled Alexa Fluor®594 were obtained from bioWORLD (OH, USA). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). The BCA protein assay kit was procured from Beyotime Biotechnology (Shanghai, China). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan).The In Situ Cell Death Detection Kit was purchased from Roche (San Francisco, CA, USA). Caspase-3 colorimetric assay kit was obtained from Keygen Biotech Co., Ltd. (Nanjing, China).

Development of Mice OA Models

For the experiment, forty-five 10-week-old C57BL/6 female mice were obtained from the Animal Center of Chinese Academy of Sciences Shanghai. All mice procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Wenzhou Medical University and approved by the Animal Ethics Committee of Wenzhou Medical University (ethics code: wydw2019–0808). After injection with 2% pentobarbital sodium (40 mg kg−1) intraperitoneally for anesthesia, we established OA model by the method described in the previous study.23 The tibial ligament of the medial meniscus was cut with microsurgical scissors. An operation of arthrotomy without the transaction of medial meniscus ligament was also performed in the left knee joint of the mice in the sham operation group. After the operation, the mice were randomly divided into three groups (n = 15): sham group, an OA group (DMM), and an OA group treated with H2 (DMM + 75% H2). The gas was inhaled for 1 h per day.

X-Ray Imaging Assay

The mice were subjected to radiographic assessment at 8 weeks after surgery. Kubtec Model XPERT.8 X-ray machine (KUB Technologies Inc.) was utilized to determine the formation of osteophyte, joint clearance, and calcification of the cartilage surface. The settings of the machine were as follows: 160 µA and 50 kV.

Histopathologic Analysis

Safranin-O/Fast Green was used to measure the articular cartilage destruction. A light microscope was utilized to assess the morphological changes in the mice chondrocytes and the surrounding tissues. The destruction of articular cartilage was evaluated by the Osteoarthritis Research Society International (OARSI) scoring system for the medial tibial condyle and medial femoral plateau.

TUNEL Staining

After fixing the chondrocytes or cartilage sections, TUNEL staining was performed in a dark, humidified room using an In Situ Cell Death Detection kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Next, DAPI (4′,6-diamidino-2-phenylindole) was used to stain the cell nuclei. Positive staining of DNA strand breaks in the apoptotic cells was detected under a fluorescence microscope.

Immunohistochemical Assay

As a first step, the knee joints were subjected to 4% paraformaldehyde (PFA) fixation, decalcification, paraffin-embedment, as well as cutting into 7 µm sections for deparaffinization and rehydration processes. Then, 30 min treatment of histological sections was carried out at 37 °C using hydrogen peroxide (3% v/v) and trypsin-EDTA solution (0.25%). After a further 60 min incubation of these sections at 37 °C in BSA (10%), they were treated with the primary antibodies at 4 °C against the cleaved caspase-3 and P-JNK for a 24 h duration. Afterwards, 1 h section incubation was performed on the 2nd d, at 4 °C using an HRP-conjugated second antibody. Aided by the Image-Pro Plus version 6.0 (Media Cybernetics, MD, USA), image analysis was accomplished, while in the quantitative analysis, five sections from every group were used.

Primary Human Chondrocyte Isolation and Culture

Initially, from 10 patients with OA (half males and half females, age of 54 ± 8 years), who had received complete knee arthroplasty at the Second Hospital Affiliated to Wenzhou Medical University, we collected the human cartilage tissues, which were cut into pieces (1×1×1 mm3) and thrice washed using PBS. Then, 4 h incubation of the cut pieces was performed at 37 °C using collagenase II (2 mg/mL). After 6 min centrifugation at 800 rpm, suspension of the digested cartilage tissues was carried out, which was followed by plating into flasks for tissue cultures. At 37 °C, chondrocyte incubation was achieved under a 5% CO2 atmosphere using DMEM/F12 medium involving 10% FBS. Medium replacement was implemented every alternate day. We utilized trypsin-EDTA (0.25%) to subculture the human cells till reaching 80 to 90% confluences.

The Application of H2

H2 was stored in gas cylinders before the experiment. Then, hydrogen, oxygen, and nitrogen were mixed, and the H2 gas concentration (6.25, 12.5, 25, 50, and 75% (vol/vol)) was adjusted through a three-way connection and measured with TRACE GC Ultra gas chromatography (Thermo Fisher, MA, USA). After a volume of H2 was removed in each group, the remaining volume was mixed according to the ratio of nitrogen and oxygen in the air, that is, 0.78:0.21. Chondrocytes were pretreated with 25 µM TBHP for 24 h, and H2 was introduced into the cultured cells for 4 h.

CCK-8 Assay

The cytotoxicity of H2 on chondrocytes was detected with CCK-8 kits by following the manufacturer’s instruction. Firstly, cells were plated in 96-well plates at a density of 50,000 cell per cm2 for 24 h and incubated with various concentrations of H2 (0, 12.5, 25, 50, and 75%) for 4 h. Later, the chondrocytes were rinsed thrice in PBS. Finally, 10 mol/L CCK-8 solution was added to each well for 2 h, and the optical density was observed at 450 nm with a spectrophotometer (Thermo Fisher Scientific).

qRT-PCR

After stimulation with TBHP (25 µM) and treatment with H2 (75%), total RNA was extracted with the aid of TRIzol (Invitrogen) from the human chondrocytes. Regarding the RT-qPCR procedure, the CFX96 PCR System (Bio-Rad Laboratories, California, USA) was utilized under the following PCR cycling conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The reaction mixture comprised, in a 10 µL total volume, 5 µL of 2 X SYBR Master Mix, 4.5 µL of diluted cDNA, as well as each 0.25 µL of primer. Collection and normalization of cycle threshold (Ct) values were carried out towards the expression levels of GAPDH. To achieve computation of relative mRNA levels for every target gene, the 2−ΔΔCt method was employed. The primer creation for Bax, Bcl-2 and GAPDH was accomplished via the NCBI Primer-Blast program. In Table 1, the sequences of forward and reverse primers are detailed.

Table 1 Primers Used in the Studies

Western Blotting

The expression level of the proteins was measured by Western blotting. The proteins were separated with the RIPA lysis buffer, sonicated on ice for 10 min, and then centrifuged at 12,000 rpm for 15 min at 4 °C. The BCA protein detection kit was used to estimate the protein concentration. After protein isolation by gel electrophoresis with sodium dodecyl sulfate-polyacrylamide (SDS-PAGE), the isolated 40 mg of proteins were shifted to the polyvinylidene difluoride (PVDF) membranes (Millipore), and subjected to 3 h blockage using non-fat milk (5%). Incubation of the resulting membranes was performed against such primary antibodies as JNK (1:2000), P-JNK (1:2000), Bax (1:2000), Bcl-2 (1:2000), GAPDH (1:2000), cleaved caspase-3 (1:2000), MMP-13 (1:2000), ADAMTS-5 (1:2000), Collagenase II (1:2000), as well as Aggrecan (1:2000). Next, an additional 2.5 h incubation was carried out at room temperatures against secondary antibodies. For blots visualization, the Image Lab Touch 3.0 (Bio-Rad, Hercules, CA, USA) was utilized, followed by washing thrice using TBST.

Analysis of Immunofluorescence

The cells were rinsed with PBS and fixed with 4% paraformaldehyde for 15 min. Next, the chondrocytes were incubated with 0.1% Triton X-100 at room temperature. Further, 10% goat serum solution was used to block in 37 °C water bath for 30 minutes and incubated with primary antibodies against Collagenase II (1:300) for the entire night at 4 °C. The next day, the cells were exposed to Alexa Fluor® 488-labeled conjugated secondary antibodies (1:400) for 1.5 h. Finally, the cells were exposed to DAPI (Beyotime) for 1 min. Ultimately, the cell samples were detected on the Olympus fluorescence microscope (Tokyo, Japan). The fluorescence intensity was observed by using the Image J software.

Statistical Analysis

The experiments were performed at least three times. The data obtained were expressed as the mean ±SEM. The data were analyzed via GraphPad Prism (United States). Inter-group comparisons were performed using a one-way ANOVA followed by the Tukey’s test. Probability values of P < 0.05 were considered statistically significant.

Results

H2 Inhibits the Cartilage Deterioration in a Murine DMM Model

A murine OA model was created through surgical operation, which was achieved by making the medial meniscus unstable In order to figure out whether H2 exerted protective functions on the progression of OA in vivo, we examined the cartilage histology in OA mice based on Safranin–O (S–O) staining in conjunction with X-radioscopy. As a result of the surgery, the density of cartilage surface was elevated and the joint space was narrowed. In the H2 group, on the contrary, improvements in the above impairments were noticed (Figure 1A). Based on the S–O staining outcomes presented in Figure 1B, the H2 therapy reduced the erosion of superficial cartilage and the substantial loss of proteoglycan. Plus, both the synovitis and OARSI scores agreed with the S–O staining outcomes. As shown in Figure 1C and D, the two types of scores were lower in the group treated with H2 than the untreated OA group.

Figure 1 H2 inhibits the progression of OA in the mouse DMM model. The digital X-ray images of mouse knee joints. (A) The white arrow indicates the narrowing of the joint space; the black arrow implies the calcification of the cartilage surface. Typical Safranin O staining of the cartilage and subchondral cortical bone (n = 15, scale bar: 200 µm and 50 µm) (B). Synovitis scores. H2 reduced synovitis scores compared to DMM. (n = 15) (C). Diagrams indicate the cartilage OARIS scores (n = 15) (D). All data are presented as mean ±SEM. ###P < 0.001 vs the sham group; ***P < 0.001 vs the DMM group; n = 15.

H2 Attenuates the Expression of Cytokines and Apoptosis-Related Proteins Following DMM in Mice

The apoptosis of chondrocytes was measured by TUNEL staining and cleaved caspase-3 immunohistochemical staining. As shown in Figure 2A and B, a higher proportion of cellular apoptosis was observed in the DMM group than in the sham group. However, the H2 group significantly reversed this pathological phenomenon. Besides, immunohistochemical staining of cleaved caspase-3 was performed to detect the in vivo protective effect of H2. As shown in Figure 2C and E, H2 decreased the expression of cleaved caspase-3 in mouse articular cartilage. Furthermore, immunohistochemical staining of P-JNK demonstrated increased levels of P-JNK in the DMM group than in the sham group. Again, the H2 treatment significantly reduced the expression of P-JNK (Figure 2C and D). These data indicated that H2 was a potential agent of treatment of OA under in vivo conditions.

Figure 2 H2 suppresses the apoptosis of cartilage in OA mice. TUNEL staining assay in the mouse cartilage (n = 15) (A and B). Immunohistochemistry of cleaved caspase-3 and P-JNK identified the effect of H2 on the degradation of cartilage matrix in OA mice (n = 15) (C). Quantification of cleaved caspase-3 and P-JNK-positive cells in the cartilage samples (D and E). All data are presented as mean ±SEM. ###P < 0.001 vs the sham group; ***P < 0.001 vs the DMM group; n = 15.

H2 Protects Chondrocytes from TBHP Treatment

We used toluidine blue, immunofluorescence, and Safranin O staining to characterize the isolated human chondrocytes. (Figure 3A). The chondrocytes were stained red by Safranin O staining, while their cytoplasm was stained purple by toluidine blue. The collagen II in the cytoplasm of human chondrocytes was stained green by immunofluorescence without any positive staining in the nucleus (Figure 3B and C). Both stains demonstrated that the cells extracted from the articular cartilage were chondrocytes. The cytotoxic effects of H2 on human chondrocytes were detected by the CCK-8 assay. The cells were treated with a concentration gradient (0,12.5, 25, 50, and 75%) of H2 for 4 h. The CCK-8 analysis revealed that H2 did not show any obvious cytotoxicity toward human chondrocytes (Figure 3D). Furthermore, the chondrocytes were treated with different concentrations of H2 (0,12.5, 25, 50, and 75%) for 4 h after stimulation with TBHP for 24 h. As shown in Figure 3E, the viability of the chondrocytes was reduced in the TBHP group whereas H2 significantly increased the viability of the cells in a dose-dependent manner. Thus, we adopted 75% H2 as the concentration to conduct subsequent experiments.

Figure 3 Effect of H2 on human chondrocyte viability. Chondrocytes were stained by toluidine blue, and proteoglycans in chondrocytes were stained purple (n = 3) (A). Safranin O staining of human primary chondrocytes (n = 3) (B). Collagen II immunofluorescence staining of human primary chondrocytes (n = 3) (C). The cytotoxicity and the cell viability of H2 on the TBHP-induced chondrocytes by using the CCK-8 assay (n = 3, 24 h) (D and E). All data are presented as mean ±SEM. *P < 0.05, **P < 0.01, ***P < 0.001, vs the control group, n = 3.

H2 Exerts Anti‑Apoptotic Effects in TBHP-Induced Human Chondrocytes

To identify the effects of TBHP on the human chondrocytes, different concentrations (0, 12.5, 25, and 50 µM) were used for stimulation. As pictured in Figure 4A and B, the apoptosis-related protein expression of cleaved-caspase 3 upregulated with increasing concentrations of TBHP. Moreover, TBHP induced the apoptosis of chondrocytes (Figure 4C and D). Therefore, the TUNEL assay was used to detect the anti-apoptotic effect of H2 on human chondrocytes. As shown in Figure 4E and F, a greater incidence of apoptosis was recorded in the chondrocytes in the TBHP (25 µM) group compared with the control group; the H2 treatment significantly reversed this trend. The activation of caspase-3 was evaluated by the caspase colorimetric assay kit. As shown in Figure 4G, the activation of caspase-3 was significantly upregulated after stimulation with TBHP. However, H2 treatment downregulated this activation of caspase-3.

Figure 4 Effects of H2 on TBHP-induced apoptosis in human chondrocytes. The protein expression of cleaved caspase-3 in chondrocytes treated with/without TBHP was detected by Western blotting and TUNEL assay (A–D). H2 exerts the anti-apoptosis effect in TBHP-induced chondrocytes as seen from the TUNEL assay (scale bar: 50 µm) and the quantification of apoptotic positive cells (E and F). The activity of caspase-3 was determined using the caspase colorimetric assay kit (n = 3) (G). All data are presented as mean ±SEM. ###P < 0.001 vs the control group; ***P < 0.001 vs the TBHP group; n = 3.

H2 Decreases the Expression of Cytokines and Apoptosis‑Related Proteins in TBHP-Induced Human Chondrocytes

To determine the effect of H2 on apoptosis in TBHP-induced human chondrocytes, the expression of cleaved caspase-3, Bcl-2, and Bax was evaluated by Western blot. As pictured in Figure 5A–E, TBHP increased the expression of Bax, cytochrome c, and cleaved caspase-3 and downregulated the expression of Bcl-2. On the contrary, H2 suppressed the expression of Bax, cytochrome c, and cleaved caspase-3 and improved the Bcl-2 expression. In addition, the results of qRT-PCR showed that H2 suppressed the generation of Bax, which had increased after TBHP stimulation (Figure 5F and G). Moreover, the immunofluorescence of cleaved caspase-3 also indicated that H2 suppressed the expression of cleaved caspase-3 in TBHP-induced chondrocytes (Figure 5H). Therefore, these data demonstrated that H2 was capable of preventing the TBHP-induced apoptosis of chondrocytes.

Figure 5 Effects of H2 on the TBHP-induced expression of cytokines and apoptosis-related proteins in human chondrocytes. The levels of cleaved caspase-3, Bcl-2, cytochrome c, and Bax were evaluated by Western blotting (A–E). The mRNA expression levels of Bcl-2 and Bax were assessed by qRT-PCR (F and G). Immunofluorescence of cleaved caspase-3 was observed with a fluorescence microscope (OLYMPUS)(Scale bar: 50 µm) and assayed by Image J (H). All data are presented as mean ±SEM. ##P < 0.01, ###P < 0.001, vs the control group; **P < 0.01, ***P < 0.001, vs the TBHP group; n = 3.

H2 Exerts Anti‑Inflammatory Effects in TBHP-Induced Human Chondrocytes

To investigate the role of H2 in inhibiting the degradation of extracellular matrix (ECM) in TBHP-induced human chondrocytes, Western blotting was utilized. We, respectively, assessed the protein levels of Collagen II, Aggrecan, ADAMTS-5, and MMP-13 in the chondrocytes. As pictured in Figure 6A–E, chondrocytes exerted an apparent downregulation of the expression of collagen II and Aggrecan following TBHP-induction. In contrast, an upregulation of the expression of ADAMTS-5 and MMP-13 was observed. Moreover, H2 treatment could reverse this trend. Further, the immunofluorescence outcomes demonstrated that H2 attenuated the degradation of collagen II (Figure 6F and G). Therefore, these results indicated that H2 can inhibit inflammation induced by TBHP in human chondrocytes.

Figure 6 Effects of H2 on influencing ECM synthesis on TBHP-induced chondrocytes. The levels of aggrecan, ADAMTS5, Collagen II, and MMP13 were evaluated by Western blotting (A–E). A representative image of immunofluorescence staining of collagen II in the chondrocytes was detected by a fluorescence microscope (OLYMPUS) (Scale bar: 50 µm) and assayed by the Image J software (F and G). All data are presented as mean ±SEM. ##P < 0.01, ###P < 0.001, vs the control group; *P < 0.05, **P < 0.01, ***P < 0.001, vs the TBHP group; n = 3.

H2 Exerts Anti-Apoptotic and Anti‑Inflammatory Effects in the TBHP-Induced Human Chondrocytes by Suppressing the JNK Pathway

Western blot was used to investigate the effect of H2 on the JNK signaling pathway. The expression of P-JNK significantly increased in the chondrocytes after stimulation with TBHP. Conversely, H2 notably suppressed the TBHP-induced activation of JNK in the human chondrocytes (Figure 7A–C).

Figure 7 H2 inhibits the TBHP–induced JNK signaling pathway in the chondrocytes. The levels of JNK, P-JNK, Collagen II, MMP-13, cytochrome c, and cleaved caspase-3 in the human chondrocytes were assayed by Western blotting (A–J). The results of the TUNEL assay of the above-treated chondrocytes (scale bar: 50 µm) (K) and the quantification of apoptotic positive cells (L). All data are presented as mean ±SEM. ##P < 0.01, ###P < 0.001 vs the control group; *P < 0.05, **P < 0.01, and ***P < 0.001, vs the TBHP group; &P < 0.05, &&P < 0.01, and &&&P < 0.001 vs the TBHP + H2 group; n = 3.

However, treatment with Anisomycin (a JNK activator) inhibited the dephosphorylation of JNK. Moreover, H2 mediated a reduction in the expression of MMP-13, cytochrome c, and cleaved caspase-3, and this reduction was abolished by the addition of Anisomycin in the H2+Anisomycin group (Figure 7D–J). Moreover, the TUNEL assay showed that H2 decreased the incidence of apoptosis in TBHP-induced chondrocytes compared to the TBHP group, whereas the Anisomycin treatment could significantly reverse this trend (Figure 7K and L). These results revealed that H2 suppressed the apoptosis induced by TBHP in human chondrocytes via the JNK signaling pathway.

Discussion

Osteoarthritis (OA) is a chronic joint degradation disease that is characterized by long-term pain and joint limitation.2 The current options for the treatment of OA are an early application of non-steroidal anti-inflammatory drugs (NSAIDs), but they only relieve the clinical symptoms.24,25 Moreover, they also result in a series of side effects such as heart attack and stroke.26 Artificial joint replacement remains the only choice for the treatment of the final stages of OA.27 Therefore, an agent that prevents the progress of OA and is accompanied by fewer side effects is a potential therapeutic strategy for OA. Recently, increasing attention has been focused on anti-apoptosis compounds that may be capable of treating OA in the absence of harmful side effects.28 Biochemical and biomechanical events induce apoptosis and imbalance between the catabolism and anabolism of the ECM in the chondrocytes.29 Several pharmacological treatments for the genetic regulation of apoptosis have shown therapeutic effects against OA development in cellular and animal models.30 Therefore, studying the underlying mechanism of apoptosis in the chondrocytes may result in a potential therapy for OA.

Oxidative stress is the main process in chondrocyte apoptosis. When chondrocytes are stimulated with TBHP, they generate ROS like superoxide anion (O2−), H2O2, and hydroxyl radical (OH); this leads to mitochondrial damage and eventually apoptosis.31 An increasing number of studies have found that excessive ROS leads to the degradation of cartilage and apoptosis of chondrocytes.32,33 Therefore, decreasing the production of ROS may be an effective strategy for OA treatment. Interestingly, hydrogen is an anti-oxidative compound.

H2 is the lightest element and characteristic of the simplest and abundant elements in nature.16 Many studies have reported the anti-inflammatory, anti-apoptotic, and anti-oxidative effects of H2 in several diseases.10,34 They demonstrated that inhalation of 4% H2 was a safe and efficient treatment for human diseases. Hydrogen has aroused the attention of researchers due to its simple preparation, high safety, strong permeability, and ease of carrying it.16 Peng Guan et al found that H2 improved chronic intermittent hypoxia (CIH)-induced kidney injury via the JNK signaling pathway.35 Zhao et al revealed that H2 alleviated ER stress and apoptosis of cardiac myocytes by blocking the c-Jun N-terminal kinase(JNK)-MAPK.36 Wu et al uncovered that H2 attenuated the hypoxic-ischemic brain damage by suppressing apoptosis and inflammation in neonatal rats.37 Chen et al found that Inhalation of hydrogen could ameliorate apoptosis and oxidative stress of spinal cord neurons in spinal cord injury in mice.38 The JNK pathway is a classical pro-apoptosis pathway and has been demonstrated to play a pivotal role in the regulation of apoptosis of chondrocytes with OA development. Previous studies have demonstrated that some drugs such as Urolithin A, Kinsenoside, and Luteolin protected the chondrocytes by inhibiting the activation of the JNK signaling pathway.39–41 Hence, targeted downregulation of phosphorylation of JNK may be deemed to cure OA effectively.

Therefore, in the present study, we explored the anti-apoptotic capability of H2 on human chondrocytes with the JNK signaling pathway. In the TBHP-treated chondrocytes of humans, remarkable inhibition of JNK phosphorylation by H2 was demonstrated. Due to the inhibited activity of JNK pathway, the production of such apoptosis factors as Bax and cleaved caspase-3 was downregulated, which in turn led to improvements in the OA progression. Collectively, our data somewhat suggested that the H2 possessed an anti-apoptotic activity during OA development, which was demonstrated on THBP-treated chondrocytes, and that the JNK pathway might be involved in its mechanism of action. The potential value of H2 was thus apparent. Moreover, shrinking space of joint, degradative ECM, seriously eroded cartilage and vastly deficient proteoglycan were noticed in the DMM groups in contrast to the sham group. Upon the H2 therapy, the aforementioned symptoms all got improved, and the murine DMM model also exhibited lower grading score of OARSI. According to the TUNEL stain of cartilage and IHC stain of cleaved caspase-3, the apoptosis of cartilage was mitigated by H2. Furthermore, the OA progression got improved by H2, which involved the signaling pathway of JNKs. Collectively, these outcomes and the preparatory findings suggest the ameliorating activity of H2 against the OA development, which was achieved by suppressing the JNK pathway activation (Figure 8).

Figure 8 Schematic reveals the involvement of H2 via the JNK pathway and the potential protective effects in the progression of osteoarthritis.

Interestingly, hydrogen has been studied for its therapeutic effects on various diseases at different concentrations. In this study, high H2 concentration (6.23–75%) was used to study the protective effect of H2 on osteoarthritis. However, the role of H2 in osteoarthritis still needs to be investigated as this study has some limitations. For example, we do not know whether a high concentration of H2 has adverse effects on other systems of the body. Moreover, the effects of long-term low concentration of hydrogen on osteoarthritis have not been studied; these need to be further studied in the future.

In conclusion, we uncovered that H2 alleviates the apoptotic reaction and degradative ECM by deactivating the signaling pathway of JNKs in chondrocytes of humans. In the meantime, a vital function of H2 therapy is also found against the OA development in the murine model of surgically-induced DMM. Our data collectively imply an anti-OA protective activity exerted by H2.

Funding

This study is supported by Zhejiang Provincial Natural Science Foundation of China (LY16H060010).

Disclosure

The authors have declared that no competing interests exists.

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ampk-progeria-hiv · 7 years

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Antibiotics produced by Bacteria activate Human Oocytes, creating Healthy Babies: AMPK links the Creation of Human Life with HIV, Progeria, & Cancer

CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/)], via Wikimedia Commons;By De Wood, Pooley, USDA, ARS, EMU. [Public domain], via Wikimedia Commons

A recent study published online in the journal Fertility and Sterility in September of 2017 systematically reviewed for the first time evidence for the effect of two compounds, ionomycin and A23187 (also known as calcimycin), on fertilization rates and pregnancy outcomes in infertile couples undergoing an in vitro fertilization procedure known as intracytoplasmic sperm injection (ICSI) [1]. ICSI involves the direct deposition of sperm into the oocyte cytoplasm, which typically leads to high rates of fertilization. However, fertilization failure despite repeated ICSI is likely caused by a failure of the oocyte to activate [1]. Physiological oocyte activation is accomplished by the delivery of a sperm-borne oocyte activating factor called phospholipase C zeta (PLCζ). PLCζ activates human oocytes by inducing an intracellular signaling cascade that ultimately results in increased calcium (Ca2+) oscillations in the oocyte, which drives oocyte activation to completion [1]. As oocyte activation is an indispensable prerequisite for the creation of all human life, every human being alive today and any human being that has ever lived began their existence as an activated oocyte [2]. Ionomycin and A23187 increase the levels of intracellular Ca2+ and are thus commonly known as Ca2+ ionophores [1]. The authors of the Fertility and Sterility study showed that over a total of 1,521 ICSI cycles, calcium ionophores including ionomycin and A23187 led to a statistically significant improvement in fertilization, cleavage, blastulation, implantation rates, overall pregnancy, and live-birth rates [1]. Ionomycin and A23187 have also been shown in several independent studies to effectively induce human oocyte activation, leading to the birth of normal, healthy children [3,4].

Strikingly, as described further below, both ionomycin and A23187 are antibiotics that are naturally produced by certain species within the bacterial genus Streptomyces [5,6]. Other structurally distinct compounds and methods have also been shown to induce human oocyte activation, including ethanol, puromycin (an antibiotic and protein synthesis inhibitor produced by Streptomyces alboniger), as well as mechanical manipulation and electrical stimulation, both of with have been reported to result in the creation of normal children [7-11]. Interestingly, as mouse oocytes are considered models for human oocytes, ionomycin, A23187, anisomycin (an antibiotic and protein synthesis inhibitor produced by Streptomyces griseolus), mycophenolic acid (an immunosuppressant produced by the fungus Penicillium brevicompactum), cycloheximide (a protein synthesis inhibitor produced by Streptomyces griseus), carvacrol (a secondary plant metabolite produced by Origanum vulgare{oregano}), and phorbol 12-myristate 13-acetate (PMA, a secondary plant metabolite produced by Croton tiglium) each induce activation of mouse oocytes [12-22]. Ionomycin, A23187, PMA, and reactive oxygen species (ROS) also induce the acrosome reaction in human sperm, a process characterized by the release of hydrolytic enzymes from the head of sperm which is necessary for oocyte penetration and thus indispensable for the creation of all human life outside of a clinical setting (ICSI bypasses the need for oocyte penetration) [23,24]. Additionally, although an over-production of ROS, similar to Ca2+, may lead to deleterious effects including cell death/apoptosis, low levels of ROS have been shown to act as signaling molecules and ROS is significantly increased on or immediately following mouse oocyte activation [25,26].

Furthermore, the master metabolic regulator AMPK is critical for oocyte meiotic resumption and maturation (a process that precedes and is essential for oocyte activation), is found located across the entire acrosome in the head of human sperm, and is activated by increases in ROS and Ca2+ [27-29]. Ionomycin, A23187, ethanol, puromycin, mechanical force, electrical stimulation, anisomycin, mycophenolic acid, carvacrol, and PMA also induce AMPK activation, indicating that a common mechanism of action links chemically distinct compounds with the creation of human life [30-39]. This common mechanism of action likely centers on the induction of cellular stress, mediated by indirect increases in intracellular Ca2+, ROS, and/or the AMP(ADP)/ATP ratio, etc. as I originally proposed in 2016 [40]. Because the bacterial-derived antibiotics ionomycin and A23187 induce both the acrosome reaction in human sperm and human oocyte activation, producing normal, healthy children, it can be said that “non-human organisms have the power to create human life or the power to end life.” As explained below, the beneficial effects of cellular stress induction (i.e. a “shock”) crosses species boundaries and may indeed play a role in facilitating natural selection, a process that underlies and drives evolution.

A number of bacterial species residing within the genus Streptomyces have proven to be extremely important and medicinally valuable as approximately 70% of clinically useful antibiotics are derived from Streptomyces [41]. The antibiotics ionomycin and A23187 are naturally produced by Streptomyces conglobatus and Streptomyces chartreusensis, respectively [5,6]. Other important examples include the antibiotic tetracycline (produced by Streptomyces aureofaciens), the immunosuppressant rapamycin (produced by Streptomyces hygroscopicus), and the anti-helminthic avermectins (produced by Streptomyces avermitilis) [42]. Many soil and aquatic-dwelling species of Streptomyces can be found in harsh environments and are characterized by a unique life cycle, including spore germination followed by vegetative mycelium production, aerial hyphae formation, sporulation (i.e. spore formation), and antibiotic production [43,44]. Curiously, just as cellular stress induction leads to the creation of human life and other beneficial effects in human cells (see below), stress induction also promotes the induction of aerial hyphae formation, sporulation, and antibiotic production in many Streptomyces species (spp.). Indeed, a decrease in the levels of ATP and bacterial growth is associated with sporulation, aerial hyphae formation, and antibiotic production [42,45]. A reduction in glucose/nutritional deprivation, the preferred sugar/carbon source for many Streptomyces spp., also significantly increases antibiotic production [46]. An increase in intracellular ROS and Ca2+ is associated with spore germination, aerial hyphae formation, and antibiotic production [47-49]. Other cellular stressors, including heat shock and ethanol, also significantly increase antibiotic production, provocatively indicating that the effects of cellular stress crosses species boundaries, enhancing bacterial survival and facilitating the creation of human life [50,51].

The beneficial effects of low-level cellular stress induction also extends to plants, as many plants produce secondary metabolites partly for the purpose of self-defense, analogous to antibiotics. Similar to the harsh, stressful environments often inhabited by Streptomyces spp., the Great Basin Bristlecone Pine (Pinus Longaeva), considered the oldest living non-clonal organism on the planet ( >5000 years old), thrives in an exceptionally harsh environment, characterized by increased elevations and exposure to UV radiation, nutritionally-deprived soils, harsh temperatures, and mechanical stress due to wind variances, leading early researchers to conclude that it’s longevity is intimately associated with adversity [52-54]. Conversely, Pinus Longaeva species that are located in less stressful environments (i.e. lower elevations) are strongly associated with younger age classes (<875 years) [55]. Similarly, the Creosote bush (Larrea tridentate), considered one of the oldest living clonal organisms on the planet (>11,000 years old), also thrives in harsh environments including the Mohave Desert [56]. AMPK, which increases lifespan and healthspan in several model organisms, is the primary sensor of cellular stress in eukaryotic organisms (e.g. plants and humans) and the plant AMPK orthologue SnRK1 as well as Ca2+ and ROS are critical for seed germination, fertilization, root gravitropism, and secondary metabolite production [57-64]. The secondary plant metabolites PMA (which activates mouse oocytes and promotes the acrosome reaction in human sperm) and artemisinin (an anti-malarial drug) both activate AMPK and the antibiotic A23187 also increases production of the secondary metabolite resveratrol in grape cell cultures, again indicating that exposure to low-level stressors may promote extension of lifespan and initiate the creation of human life [17,23,39,65,66].

Organismal exposure to beneficial levels of stress may also play a critical role in evolution. As first noted by Charles Darwin, evolution is driven by natural selection, a process characterized by environmentally-induced phenotypic changes that may lead to inheritable survival and reproductive advantages [67]. From “On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life”, Darwin explained that “if there be, owing to the high geometrical powers of increase of each species, at some age, season, or year, a severe struggle for life, and this certainly cannot be disputed;……But if variations useful to any organic being do occur, assuredly individuals thus characterised will have the best chance of being preserved in the struggle for life;” [67]. This “struggle for life” Darwin spoke of is embodied by selective pressures which may be abiotic (i.e. light, wind, temperature, etc.) or biotic (predation, disease, competition, etc.) [68,69]. As alluded to above, such selective pressures are indeed sources of cellular stress, sensed by both prokaryotes and eukaryotes, that induce beneficial responses (at appropriate levels), leading to the acquisition of phenotypes conducive for continued survival. Both biotic (e.g. infection) and abiotic (e.g. heat) stressors/selective pressures activate AMPK (which is evolutionarily conserved among eukaryotes) in human cells [70,71]. A phenomenon often cited as an example of natural selection on a readily observable timescale is the development of bacterial resistance to antibiotics, resulting in problematic mutant strains that may be life-threatening for some individuals (i.e. the elderly and immunocompromised) [72]. Intriguingly, lethal levels of bactericidal antibiotics have been shown to kill microorganisms via the induction of ROS, sub-lethal levels of bactericidal antibiotics however increase mutagenesis and bacterial resistance via induction of lower levels of ROS, and heat as well as nutritional stress increase bacterial resistance to antibiotics, providing compelling evidence that continuous exposure to low levels of stress likely plays a significant role in natural selection and evolution [73-75].

Moreover, gravity itself likely functions as a cellular stressor/selective pressure that has influenced the development of organisms on Earth since the emergence of the very first lifeform. Gravity exerts its effects on living organisms via the application of force, which is experienced by human cells in the form of mechanical loading or stress [76]. The application of force or a mechanical load has recently been shown to activate AMPK and simulated microgravity (i.e. hind limb unloading in mice) significantly decreases AMPK activation [77,78]. Spaceflight also inhibits the activation of T cells (immune cells essential for adaptive immunity), whereas the application of force and AMPK activation promote T cell activation [79-81]. Interestingly, spaceflight has recently been shown to decrease the levels of the master antioxidant transcription factor Nrf2 and the heat shock-inducible protein HSP90a but increase the levels of the growth-promoting kinase mTOR in mice [82]. AMPK however inhibits mTOR but increases the phosphorylation, nuclear retention, and transcriptional activity of Nrf2 [57,83,84]. Also, HSP90 interacts with and maintains AMPK activity and HSP90 is necessary for progesterone-induced human sperm acrosome reaction [85,86]. Interestingly, rapamycin, an immunosuppressant produced by Streptomyces hygroscopicus, extends lifespan in genetically heterogeneous mice, activates AMPK in vivo in normal aged mice, and increases human sperm motility [42,87,88]. Simulated microgravity via the use of NASA-designed rotating wall vessels (RWVs) however drastically reduces rapamycin production (~90%) whereas the antibiotic gentamycin increases rapamycin production by Streptomyces hygroscopicus, providing further evidence that cellular stress, in the form of mechanical loading induced by gravity, is essential for development, function, and survival of Earth-bound organisms [89,90].

The induction of cellular stress also links seemingly dissimilar physiological and pathological states with the activation of AMPK. As discussed above, both ionomycin and ROS activate AMPK and promote oocyte meiotic resumption, a process that is AMPK-dependent and is essential for efficient oocyte activation [27,30,91]. ROS is also critical for ovulation, PMA and ionomycin both activate mouse oocytes, and ionomycin is extensively used during ICSI procedures, creating normal healthy children, suggesting that cellular stress-induced AMPK activation is also essential for oocyte activation [3,4,12,17,92]. The activation of oocytes and T cells share strikingly similar intracellular signaling mechanisms (e.g. PLC-PIP2-DAG-PKC-IP3-Ca2+) and ionomycin combined with PMA are extremely effective in activating T cells and are often used as positive controls in HIV-1 latency reversal studies [93-95]. Reactivating latent/dormant HIV-1 in CD4+ T cells, potentially facilitating immune system detection and virus destruction, is currently being pursued as a method for the potential eradication of HIV-1 (called the “shock and kill” approach) [96]. Similar to oocyte activation, both Ca2+ and ROS are critical for T cell activation (and hence latent HIV-1 reactivation) and other cellular stress-inducing compounds, including NDGA derived from the Creosote bush, butyrate derived from bacteria, as well as ROS and HSP90 have been shown to reactivate latent HIV-1 [26,93,94,97-101]. Interestingly, AMPK inhibition leads to cell death on T cell activation, knockdown of AMPK significantly decreases HIV-1 replication, and metformin (a well-studied AMPK activator derived from the French Lilac plant) increases butyrate production in human diabetic patients [81,102,103]. Perhaps most convincingly, early preliminary data showed that metformin significantly reduced several markers preferentially associated with cells latently infected with HIV-1 (e.g. PD-1, TIGIT, TIM-3) and also destabilized the latent HIV-1 reservoir in chronically-infected HIV patients, indicating that cellular-stress induced AMPK activation likely links the creation of human life with the potential eradication of HIV-1, as I originally proposed in 2016 [40,104,105].

AMPK activation may also link the disparate disease states of HIV-1 latency and Hutchinson-Gilford progeria syndrome (HGPS). HGPS is a genetic disorder caused by aberrant alternative splicing of the LMNA gene, generating a toxic protein called progerin that induces an accelerated aging phenotype and premature death at approximately 14 years of age [106]. Excessive activity of the gene splicing factor SRSF1 has been shown to prevent reactivation of latent HIV-1 and contribute to aberrant splicing of the LMNA gene in HGPS [107-109]. Metformin however has recently been shown to ameliorate the accelerated aging phenotype in cells derived from children with HGPS by reducing the levels of both SRSF1 and progerin and activating AMPK, as I first proposed in 2014 [110-112]. Interestingly, both Ca2+ and ROS induce autophagy (a process of disposing of damaged/toxic proteins and organelles) and rapamycin, which activates AMPK in vivo and increases intracellular Ca2+ levels, improves accelerated aging in progeria cells by inducing autophagic degradation of progerin [87,113-116]. Temsirolimus, an analog of rapamycin, also alleviated accelerated aging defects in progeria cells but also increased the levels of ROS and superoxide within the first hour of treatment [117]. Such evidence strongly suggests that cellular stress-induced AMPK activation links the reversal of HIV-1 latency and alleviation of accelerated cellular aging defects in HGPS.

Cellular stress-induced AMPK activation also links the potential elimination of cancer stem cells (CSCs) with HIV-1 latency reversal and viral eradication. CSCs, which are largely resistant to chemoradiation therapy, are a subpopulation of cancer cells that exhibit characteristics similar to embryonic stem cells (ESCs), including self-renewal, multi-lineage differentiation, & the ability to initiate tumorigenesis [118,119]. Mechanisms that sustain quiescence & promote self-renewal in adult stem cells (ASCs) & CSCs likely also function to maintain latency of HIV-1 in CD4+ memory T cells. Indeed, HIV-1 has been found to establish long-lasting latency in a recently discovered subset of CD4+ T cells that exhibit stem cell-like properties known as T memory stem (TSCM) cells and increases in Ca2+, ROS, and AMPK activation have been shown to promote T cell activation and ESC, ASC, and CSC differentiation [119,120]. Additionally, A23187 and PMA have been shown to promote CSC differentiation (causing CSCs to become more susceptible to chemoradiation) and metformin induces CSC differentiation and/or apoptosis in an AMPK-dependent manner in the deadliest of cancers, including glioblastoma and pancreatic cancer, providing support for my publication in 2017 in which I first proposed that CSC differentiation and/or apoptosis and HIV-1 latency reversal/viral eradication may be linked by cellular stress-induced AMPK activation [119,121-124].

In conclusion, the ability of non-human organisms including certain Streptomyces spp. to initiate the creation of human life is predicated on the induction of cellular stress, mediated by increases in intracellular ROS, Ca2+, AMP(ADP)/ATP ratio increase, etc. The beneficial effects of transient cellular stress induction, which may be likened to selective pressures, crosses species boundaries and may indeed play a role in facilitating natural selection, a process that underlies and drives evolution, as evidenced by stress-induced increases in antibiotic production by Streptomyces spp. and stress-induced mutagenesis and antibiotic resistance in various bacterial strains. Because AMPK, a primary sensor of cellular stress in eukaryotic cells that increases lifespan and healthspan, plays a critical role in oocyte meiotic resumption/maturation, T cell activation, and stem cell differentiation, the creation of human life, the potential eradication of HIV-1, amelioration of accelerated aging in HGPS cells, and CSC differentiation/apoptosis are likely linked by a “Shock to Live”, or a “Shock to Kill”.

https://www.linkedin.com/pulse/antibiotics-produced-bacteria-activate-human-oocytes-creating-finley/

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Bays JL, Campbell HK, Heidema C, Sebbagh M, DeMali KA. Linking E-cadherin mechanotransduction to cell metabolism through force-mediated activation of AMPK. Nat Cell Biol. 2017 Jun;19(6):724-731.

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Li YC, Chen BM, Wu PC, et al. Cutting Edge: mechanical forces acting on T cells immobilized via the TCR complex can trigger TCR signaling. J Immunol 2010;184(11):5959–63.

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Blaber EA, Pecaut MJ, Jonscher KR. Spaceflight Activates Autophagy Programs and the Proteasome in Mouse Liver. Int J Mol Sci. 2017 Sep 27;18(10). pii: E2062.

Mo C, Wang L, Zhang J, et al. The crosstalk between Nrf2 and AMPK signal pathways is important for the anti-inflammatory effect of berberine in LPS-stimulated macrophages and endotoxin-shocked mice. Antioxid Redox Signal. 2014 Feb 1;20(4):574-88.

Joo MS, Kim WD, Lee KY, Kim JH, Koo JH, Kim SG. AMPK facilitates nuclear accumulation of Nrf2 by phosphorylating at serine 550. Mol Cell Biol. 2016 Jun 29;36(14):1931-42.

Zhang L, Yi Y, Guo Q, et al. Hsp90 interacts with AMPK and mediates acetyl-CoA carboxylase phosphorylation. Cell Signal. 2012 Apr;24(4):859-65.

Sagare-Patil V, Bhilawadikar R, Galvankar M, Zaveri K, Hinduja I, Modi D. Progesterone requires heat shock protein 90 (HSP90) in human sperm to regulate motility and acrosome reaction. J Assist Reprod Genet. 2017 Apr;34(4):495-503.

Lesniewski LA, Seals DR4, Walker AE, et al. Dietary rapamycin supplementation reverses age-related vascular dysfunction and oxidative stress, while modulating nutrient-sensing, cell cycle, and senescence pathways. Aging Cell. 2017 Feb;16(1):17-26.

Aparicio IM, Espino J, Bejarano I, et al. Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility. Sci Rep. 2016 Sep 16;6:33647.

Fang A, Pierson DL, Mishra SK, Demain AL. Growth of Steptomyces hygroscopicus in rotating-wall bioreactor under simulated microgravity inhibits rapamycin production. Appl Microbiol Biotechnol. 2000 Jul;54(1):33-6.

Cheng YR, Huang J, Qiang H, Lin WL, Demain AL. Mutagenesis of the rapamycin producer Streptomyces hygroscopicus FC904. J Antibiot (Tokyo). 2001 Nov;54(11):967-72.

Heindryckx B, Lierman S, Combelles CM, Cuvelier CA, Gerris J, De Sutter P. Aberrant spindle structures responsible for recurrent human metaphase I oocyte arrest with attempts to induce meiosis artificially. Hum Reprod. 2011 Apr;26(4):791-800.

Shkolnik K, Tadmor A, Ben-Dor S, Nevo N, Galiani D, Dekel N. Reactive oxygen species are indispensable in ovulation. Proc Natl Acad Sci U S A. 2011 Jan 25;108(4):1462-7.

Amdani SN, Jones C, Coward K. Phospholipase C zeta (PLC ζ): oocyte activation and clinical links to male factor infertility. Adv Biol Regul 2013;53(3):292–308.

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Spina CA, Anderson J, Archin NM, et al. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog 2013;9(12):e1003834.

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Sena LA, Li S, Jairaman A, et al. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling. Immunity. 2013 Feb 21;38(2):225-36.

Barquero AA, Dávola ME, Riva DA, Mersich SE, Alché LE. Naturally occurring compounds elicit HIV-1 replication in chronically infected promonocytic cells. Biomed Res Int. 2014;2014:989101.

Imai K, Ochiai K, Okamoto T. Reactivation of latent HIV-1 infection by the periodontopathic bacterium Porphyromonas gingivalis involves histone modification. J Immunol. 2009 Mar 15;182(6):3688-95.

Piette J, Legrand-Poels S. HIV-1 reactivation after an oxidative stress mediated by different reactive oxygen species. Chem Biol Interact. 1994 Jun;91(2-3):79-89.

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Zhou H, Xu M, Huang Q, et al. Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe. 2008 Nov 13;4(5):495-504.

Wu H, Esteve E, Tremaroli V, et al. Metformin alters the gut microbiome of individuals with treatment-naive type 2 diabetes, contributing to the therapeutic effects of the drug. Nat Med. 2017 Jul;23(7):850-858.

G.M. Chew, D.C. Chow, S.A. Souza, et al. Impact of adjunctive metformin therapy on T cell exhaustion and viral persistence in a clinical trial of HIV-infected adults on suppressive ART. Journal of Virus Eradication 2017; 3 (Supplement 1): 6–19.

http://viruseradication.com/supplement-details/Abstracts_of_the_IAS_HIV_Cure_and_Cancer_Forum_2017/

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#ampk #gut bacteria #metformin #oocyte #HIV/AIDS #progeria #cancer stem cell #evolution #charles darwin #sperm #acrosome reaction #fertilization #reproduction

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dehnfrederiksen · 2 years

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Your Qualifications Procedure Ensures Good quality of Education within Biomedical and also Wellbeing Informatics

Bone tissue Marrow Transplantation (Next year) 48, 716-724; doi:15.1038/bmt.This year.169; published click here on-line Twenty nine August This yearTrastuzumab can be a fully humanised monoclonal antibody inclined to a persons epidermal progress issue receptor-2 (HER-2) which has been a component of normal remedy pertaining to superior and resected HER-2-positive busts cancer for nearly several years. HER-2 over-expression, looked as HER-2 proteins over-expression using immunohistochemistry obtained as 3+ and/or erbB-2 boosting discovered simply by phosphorescent inside situ hybridisation, ended up being detected in Twenty-two.1% regarding 3807 patients with superior stomach along with oesophagogastric junction (OGJ) adenocarcinoma screened regarding membership to the cycle III ToGA review. The actual confirmed credit scoring system pertaining to HER-2 positivity throughout stomach types of cancer is different from which suitable for cancers of the breast because of a heightened consistency of partial membranous immunoreactivity and heterogeneity associated with HER-2 appearance throughout stomach cancers. The highest costs of HER-2 over-expression tend to be observed in individuals along with OGJ as opposed to abdominal tumours and also intestinal-type rather than diffuse as well as put together histology. The worldwide multicentre randomised period Three ToGA study evaluated adding trastuzumab to a cisplatin plus fluoropyrimidine (FP) radiation treatment doublet with regard to people with HER-2-positive innovative stomach or perhaps OGJ adenocarcinoma. Your investigators described a medically as well as mathematically considerable gain regarding result fee (50.3% compared to Thirty four.5%, g Is equal to Zero.0017), typical progression-free emergency (Some.7 versus A few.Five weeks, s = 3.0002) as well as median all round success (13.7 versus 11.One weeks, s Equals 0.0046). Trastuzumab in addition FP chemotherapy is currently the standard of look after patients using sophisticated abdominal along with OGJ types of cancer which in turn over-express HER-2. Further analysis to judge trastuzumab sent beyond further advancement, together with option first-line radiation regimens, as well as in your perioperative and adjuvant environment is actually urgently necessary. In addition, analysis directly into mechanisms of weight and methods to get over main or purchased potential to deal with trastuzumab ought to easily be fast, making use of lessons discovered in the last 10 years throughout HER-2-positive breast cancer to optimize the advantage from this adviser. (C) This year Elsevier Limited. Most privileges reserved.The particular Drosophila phototransduction cascade terminates in the opening up with the route temporary receptor possible (TRP) along with TRP-like (TRPL). Unlike TRP, TRPL undergoes light-dependent subcellular trafficking involving rhabdomeric photoreceptor filters with an intracellular storage area inner compartment, leading to long lasting mild edition. Here, all of us determined throughout vivo phosphorylation internet sites of TRPL that affect TRPL steadiness and also localization. Quantitative bulk spectrometry uncovered any light-dependent alteration of the actual TRPL phosphorylation pattern. Mutation regarding ten C-terminal phosphorylation web sites not affected multimerization in the channels neither the particular electrophysiological result regarding flies expressing the mutated channels. Nevertheless, these kind of mutations led to mislocalization that has been enhanced deterioration of TRPL after extended dark-adaptation. Mutation regarding subsets of the eight C-terminal phosphorylation websites also triggered a discount of TRPL written content along with part mislocalization after dark.

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ludvigsenbowles · 2 years

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Determination of levetiracetam throughout individual plasma tv's simply by online heart-cutting liquid chromatography: Program for you to healing substance monitoring

With this cardstock, we illustrate the level to which present HIV-specific health-status tools catch the experience of incapacity regarding grown ups living with Aids. Methods: Many of us searched sources through 1980 to be able to '06 with regard to English words, HIV-specific, self-reported surveys comprising a minimum of a couple of items that were examined regarding trustworthiness along with truth. We then conducted a new articles evaluation to guage how well present questionnaires explain disability as determined by the actual Episodic Impairment Platform, the construction which conceptualizes this kind of experience through the outlook during older people experiencing Human immunodeficiency virus. All of us harmonized pieces of the devices together with types of the platform to evaluate the actual extent this agreement your instruments catch significant proportions of handicap in the composition. Results: We evaluated 4274 abstracts, of which 40 equipment met the inclusion requirements as well as were recovered. Of the a number of key size of incapacity, symptoms/impairments were contained in just about all Thirty devices, complications with day-to-day activities inside Of sixteen, issues for you to sociable add-on within Sixteen, as well as anxiety throughout 9. 7 instruments included selleck inhibitor at least One object all Four measurements of disability (breadth) nevertheless, the comprehensiveness with which the dimensions have been manifested (level) diverse among the equipment. Conclusions: Generally speaking, symptoms/impairments and difficulties undertaking day-to-day activities ended up the actual handicap dimensions recognized within best depth even though uncertainness and also problems in order to cultural addition were less nicely represented. Though no devices explained the full breadth along with level involving handicap while designed from the Episodic Incapacity Framework, they provide the groundwork out of which to develop a way of measuring impairment for grownups experiencing Aids.Introduction: The purpose of these studies ended up being compare the chance involving actual chips seen on the apical main surface area and/or in the tunel walls following channel instrumentation along with Three or more single-file systems along with the ProTaper program (Dentsply Maillefer, Ballaigues, Switzerland). Methods: A hundred mandibular incisors were picked. Something like 20 management the teeth had been coronally flared with Gates-Glidden soccer drills for kids (Dentsply Maillefer). Absolutely no more preparation was developed. One other 50 teeth have been installed within resin prevents using simulated gum structures, along with the height has been exposed. They were split into Four experimental groupings (n Is equal to Something like 20); the root pathways ended up 1st coronally flared together with Gates-Glidden soccer drills for kids and then instrumented to the full functioning length together with the ProTaper, OneShape (Micro-Mega, Besancon, Italy), Reciproc (VDW, Munich, Belgium), or Self-Adjusting Document (ReDent-Nova, Ra'anana, Israel). Your apical underlying surface area as well as side to side portions Two, Some, along with Half a dozen mm through the height ended up noticed under a microscopic lense.

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choateholmes · 2 years

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Regenerating frontal EEG asymmetry as well as schizotypal qualities: a test-retest review.

Quetiapine inside low amounts seemed attractive sudden expenses from the adults together with extreme FGIDs who stayed at on treatment method. This particular reply in otherwise refractory patients suggests quetiapine may possibly increase the potency of antidepressant medications within severe FGIDs.Localised lymphadenectomy, with both it's diagnostic and restorative jobs, may serve as a vital component of the management of numerous types of cancer. Prolonged pelvic lymph node dissection (LND) could be the regular regarding care for urothelial carcinoma of the kidney, with all the magnitude associated with lymphadenectomy correlating absolutely using cancer-related outcomes. Provided their particular histologic parallels with bladder cancer malignancy, it can be sensible to be able to hypothesize that a related connection exists for top region urothelial carcinomas (UTUCs). Nonetheless, your data printed up to now failed CHR2797 for you to constantly display a new healing good thing about localised LND pertaining to UTUC. As a result, usage of localised lymphadenectomy continues to be at the attention from the cosmetic surgeon, the actual magnitude involving LND falls short of standardization, and its particular medical energy may be discussed. So that you can far better describe the prevailing data, we present overview of the function regarding localized lymphadenectomy inside patients together with UTUC.The Arabian Sea oxygen bare minimum zone (OMZ) impinges around the american American indian continental perimeter among One hundred fifty and also 2500 m, creating gradients in air accessibility along with sediment geochemistry in the seashore floor. Air supply along with deposit geochemistry are essential factors structuring macrofaunal assemblages inside maritime sediments. Even so, relationships between macrofaunal montage construction as well as sea-floor carbon dioxide as well as nitrogen bicycling are generally inadequately recognized. We all conducted within situ C-13:N-15 tracer findings inside the OMZ core (540 m [O-2] = 0.Thirty five mu mol d(-1)) and minimize OMZ border (800-1100 m, [O-2] Equals A couple of.2-15.Zero mu mol d(-1)) to research exactly how macrofaunal installation structure, suffering from diverse o2 quantities, along with D:D direction influence the fortune involving particulate organic and natural issue. Absolutely no macrofauna had been contained in the actual OMZ core. Inside the OMZ perimeter, fairly high abundance along with biomass led to the greatest macrofaunal ingestion of air particle organic and natural co2 (POC) as well as nitrogen (PON) on the lower fresh air Eight hundred m stations ([O-2] Is equal to Two.2-2.Thirty six mu mol t(-1)). From these kinds of programs your numerically prominent cirratulid polychaetes displayed best POC as well as PON usage. By comparison, on the larger fresh air 1100 mirielle stop ([O-2] Equates to Fifteen.3 mu mol m(-1)) macrofaunal D along with N assimilation ended up being lower, using POC compression covered with a single big one ascidian. Macrofaunal POC as well as PON ingestion ended up influenced by changes in oxygen accessibility, as well as drastically associated to variants macrofaunal assemblage construction in between areas. Even so, macrofaunal giving reactions ended up ultimately characterised by preferential natural and organic nitrogen intake, relative to their own internal D:N costs.

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combswoodward · 2 years

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Countercurrent using associated with Ni, Company, Minnesota, as well as Li via spent lithium-ion power packs.

(Chemical) The year 2010 Elsevier Inc. All rights reserved.Your genus Chili peppers is completely new Entire world throughout origins and represents a fancy of the wide variety of the two crazy as well as domesticated taxa. All kinds of peppers or perhaps fruits involving Chili peppers kinds almost never have been determined inside the paleoethnobotanical report in either Meso- or Latin america. All of us report the following confirmation associated with Chili peppers sp. remains coming from pottery trials excavated in Chiapa delaware Corzo throughout the southern area of Mexico dated via Midst for you to Overdue Preclassic times (400 BCE to be able to More than 200 CE). Remains from Tough luck diverse art sorts were accumulated along with taken out utilizing normal strategies. Presence of Chili peppers was established through ultra-performance liquefied chromatography (UPLC)/MS-MS Analysis. A few art types exhibited substance highs with regard to Capsicum in comparison to the standard (dihydrocapsaicin). Zero highs were observed in the remaining nine biological materials. Results of the chemical extractions offer conclusive proof pertaining to Capsicum employ from Chiapas signifiant Corzo within a 700 calendar year interval (300 BCE-300 CE). Existence of Capsicum in different varieties of culinary-associated ceramics increases questions just how chili spice up could have been utilized during this early time period. Because Pre-Columbian cocoa goods at times were flavored making use of Capsicum, the identical ceramic taste set ended up being screened for evidence chocolate using a theobromine sign: these outcome was negative. As every single vessel that screened optimistic regarding Chili peppers had a cookery employ we propose here the chance that chili residues from your Chiapas p Corzo ceramic examples reflect sometimes stick or drink products with regard to spiritual, event, or even each day cookery use. Additionally, several vessels which analyzed good simply may have been employed to keep red and green peppers. Very best through a great archaeological context was the presence of Chili peppers residue from a spouted container, the pottery sort in the past believed only to provide pertaining to flowing beverages.Goal: Strains in the myocilin gene (MYOC) tend to be related to principal open-angle glaucoma (POAG) in many different numbers. These studies signifies the very first large study regarding MYOC strains within an African American populace. Methods: Many of us recruited 529 Dark topics with POAG and also 270 African American manage topics in this buy BMS-387032 examine. A total vision evaluation as well as body series had been executed in all research subjects. Genomic Genetic has been taken out. The entire coding string associated with MYOC has been amplified as well as sequenced while using the Sanger strategy. Determined MYOC alternatives have been weighed against formerly described MYOC versions. Results: We all discovered a total of 28 MYOC alternatives such as six to eight potential MYOC strains. A pair of strains (Thr209Asn as well as Leu215Gln) are fresh and so are found only in the event with out handles.

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ampk-cancer-stem-cell-hiv · 7 years

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Antibiotics produced by Bacteria activate Human Oocytes, creating Healthy Babies: AMPK links the Creation of Human Life with HIV, Progeria, & Cancer

CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/)], via Wikimedia Commons;By De Wood, Pooley, USDA, ARS, EMU. [Public domain], via Wikimedia Commons

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