Effects of different sizes of ambient particulate matter and household fuel use on physical function: National cohort study in China.

BACKGROUND: Impact of outdoor and household air pollution on physical function remains unelucidated. This study examined the influence of various ambient particulate sizes (PM1, PM2.5, and PM10) and household fuel usage on physical function. METHODS: Data from the China Health and Retirement Longitudinal Study (CHARLS) spanning 2011 and 2015 were utilized. The physical functional score was computed by summing scores from four tests: grip strength, gait speed, chair stand test, and balance. Multivariate linear and linear mixed-effects models were used to explore the separate and combined effects of PM1, PM2.5, PM10 and household fuel use on physical function in the cross-sectional and longitudinal analyses, respectively, and to further observe the effects of fuel cleanup on physical function in the context of air pollution exposure. RESULTS: Both cross-sectional and longitudinal analyses revealed negative correlations between PM1 (ß = -0.044; 95% CI: -0.084, -0.004), PM2.5 (ß = -0.024; 95% CI: -0.046, -0.001), PM10 (ß = -0.041; 95% CI: -0.054, -0.029), and physical function, with a more pronounced impact observed for fine particulate matter (PM1). Cleaner fuel use was associated with enhanced physical function compared to solid fuels (ß = 0.143; 95% CI: 0.070, 0.216). The presence of air pollutants and use of solid fuels had a negative impact on physical function, while cleaner fuel usage mitigated the adverse effects of air pollutants, particularly in areas with high exposure. CONCLUSION: This study underscores the singular and combined detrimental effects of air pollutants and solid fuel usage on physical function. Addressing fine particulate matter, specifically PM1, and prioritizing efforts to improve household fuel cleanliness in regions with elevated air pollution levels are crucial for preventing physical disability.

Ultra-processed food consumption, genetic susceptibility, and the risk of hip/knee osteoarthritis.

BACKGROUND: The associations between ultra-processed food (UPF) consumption, genetic susceptibility, and the risk of osteoarthritis (OA) remain unknown. This study was to examine the effect of UPF consumption, genetic susceptibility, and their interactions on hip/knee OA. METHODS: Cohort analyses included 163,987 participants from the UK Biobank. Participants' UPF consumption was derived from their 24-h dietary recall using a questionnaire. Genetic risk scores (GRSs) of 70 and 83 single nucleotide polymorphisms (SNPs) for hip and knee OA were constructed. FINDINGS: After 1,461,447 person-years of follow-up, 11,540 patients developed OA. After adjustments, compared to participants in the low quartile of UPF consumption, those in the high quartile had a 10 % (hazard ratio [HR], 1.10; 95% confidence interval [CI], 1.03-1.18) increased risk of knee OA. No significant association was found between UPF consumption and hip OA. Replacing 20% of UPF diet weight with an equivalent proportion of unprocessed or minimally processed food caused a 6% (HR, 0.94; 95% CI, 0.89-0.98) decreased risk of knee OA, respectively. A significant interaction was found between UPF consumption, genetic predisposition, and the risk of knee OA (P = 0.01). Participants with lower OA-GRS scores experienced higher knee OA risks due to UPF consumption. INTERPRETATION: UPF consumption was associated with a higher risk of knee OA but not hip OA, particularly in those with lower genetic susceptibility. These results highlight the importance of reducing UPF consumption to prevent knee OA.

Association of loneliness and social isolation with mental disorders among medical residents during the COVID-19 pandemic: A multi-center cross-sectional study.

BACKGROUND: Loneliness and social isolation usually increase the risk of mental disorders. However, this association among Chinese medical residents during the COVID-19 pandemic remains unclear. METHODS: This study was conducted in September 2022; 1,338 medical residents from three hospitals in Northeastern China were included in the final analysis. The data were collected via online self-administered questionnaires. Adjusted odds ratios and 95% confidence intervals were determined for adjusting for potential confounders by binary logistic regression. RESULTS: Among the 1,338 participants, 12.93% (173), 9.94% (133), and 9.72% (130) had experienced major depression, major anxiety, and suicidal ideation, respectively. Further, 24.40% (327) and 44.50% (596) of the total participants had experienced loneliness and social isolation. Loneliness increased the risk of major depression, major anxiety, and suicidal ideation (all p<0.001); Compared with the lowest quartile, the odds ratios of the highest quartile were 4.81, 4.63, and 5.34. The same result was obtained in relation to social isolation (all p<0.001). CONCLUSIONS: The findings of this study revealed a considerable prevalence of loneliness, social isolation, and mental disorders among Chinese medical residents during the COVID-19 pandemic. Both loneliness and social isolation increased the risk of major depression, major anxiety, and suicidal ideation.

Natural anti-inflammatory products for osteoarthritis: From molecular mechanism to drug delivery systems and clinical trials.

Osteoarthritis (OA) is a degenerative joint disease that affects millions globally. The present nonsteroidal anti-inflammatory drug treatments have different side effects, leading researchers to focus on natural anti-inflammatory products (NAIPs). To review the effectiveness and mechanisms of NAIPs in the cellular microenvironment, examining their impact on OA cell phenotype and organelles levels. Additionally, we summarize relevant research on drug delivery systems and clinical randomized controlled trials (RCTs), to promote clinical studies and explore natural product delivery options. English-language articles were searched on PubMed using the search terms "natural products," "OA," and so forth. We categorized search results based on PubChem and excluded "natural products" which are mix of ingredients or compounds without the structure message. Then further review was separately conducted for molecular mechanisms, drug delivery systems, and RCTs later. At present, it cannot be considered that NAIPs can thoroughly prevent or cure OA. Further high-quality studies on the anti-inflammatory mechanism and drug delivery systems of NAIPs are needed, to determine the appropriate drug types and regimens for clinical application, and to explore the combined effects of different NAIPs to prevent and treat OA.

PGC-1α in osteoarthritic chondrocytes: From mechanism to target of action.

Osteoarthritis (OA) is one of the most common degenerative joint diseases, often involving the entire joint. The degeneration of articular cartilage is an important feature of OA, and there is growing evidence that the mitochondrial biogenesis master regulator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) exert a chondroprotective effect. PGC-1α delays the development and progression of OA by affecting mitochondrial biogenesis, oxidative stress, mitophagy and mitochondrial DNA (mtDNA) replication in chondrocytes. In addition, PGC-1α can regulate the metabolic abnormalities of OA chondrocytes and inhibit chondrocyte apoptosis. In this paper, we review the regulatory mechanisms of PGC-1α and its effects on OA chondrocytes, and introduce potential drugs and novel nanohybrid for the treatment of OA which act by affecting the activity of PGC-1α. This information will help to further elucidate the pathogenesis of OA and provide new ideas for the development of therapeutic strategies for OA.

Co-interventions with Clostridium butyricum and soluble dietary fiber targeting the gut microbiota improve MAFLD via the Acly/Nrf2/NF-κB signaling pathway.

Purpose: The pathogenesis of metabolic associated fatty liver disease (MAFLD) is complex. Lipid metabolic disorder, chronic inflammation, and oxidative stress are the core events for MAFLD. Dietary intervention is an important treatment strategy for preventing the onset and progression of MAFLD. Clostridium butyricum (CB) and soluble dietary fiber (SDF) are often considered beneficial for health. We explored how two microbiota-targeted interventions (SDF and CB) influence the hepatic immune system, oxidative stress, and lipid metabolism in MAFLD mice. Methods: To explore the role of SDF and CB in MAFLD, we generated MAFLD mouse models by feeding C57BL/6 mice with a high-fat diet (HFD). After 8 weeks of intervention, we measured immune cell function, lipid metabolism, and oxidative stress levels in the livers of mice. Results: Single intervention with SDF or CB was not effective in improving MAFLD; however, co-interventions with SDF and CB increased microbiota diversity and decreased inflammation, oxidative stress, and lipid synthesis. Moreover, we determined that co-intervention with SDF and CB mediated fatty acid oxidation by activating the Acly/Nrf2/NF-κB signaling pathway. Most importantly, co-intervention exerted anti-inflammatory effects by inhibiting the differentiation of macrophages into pro-inflammatory M1 macrophages. Conclusion: This study show that co-intervention with SDF and CB can improve MAFLD, and co-intervention with  SDF and CB are suggested to be potential gut microbiota modulators and therapeutic substances for MAFLD.

Curcumin exerts chondroprotective effects against osteoarthritis by promoting AMPK/PINK1/Parkin-mediated mitophagy.

Osteoarthritis (OA), a chronic degenerative disease with heterogeneous properties, is difficult to cure due to its complex pathogenesis. Curcumin possesses excellent anti-inflammatory and antioxidant properties and may have potential therapeutic value in OA. In this study, we investigated the action targets of curcumin and identified potential anti-OA targets for curcumin. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analyses were performed to evaluate these targets. Furthermore, we established a sodium monoiodoacetate-induced rat knee OA model and IL-1ß induced OA chondrocyte model to verify the effect and mechanism of curcumin against OA. The GO and KEGG analyses screened seven hub genes involved in metabolic processes and the AMPK signaling pathway. Curcumin can significantly attenuate OA characteristics according to Osteoarthritis Research Society International (OARSI) and Mankin scores in OA rats. Additionally, curcumin is notably employed as an activator of mitophagy in maintaining mitochondrial homeostasis (ROS, Ca2+, ATP production, and mitochondrial membrane potential). The expression levels of mitophagy-related proteins were increased not only in articular cartilage but also in chondrocytes with curcumin intervention. Combining validation experiments and network pharmacology, we identified the importance of mitophagy in the curcumin treatment of OA. The chondroprotective effects of curcumin against OA are mediated by the AMPK/PINK1/Parkin pathway, and curcumin may serve as a potential novel drug for OA management.

Mechanical Stimulation Protects Against Chondrocyte Pyroptosis Through Irisin-Induced Suppression of PI3K/Akt/NF-κB Signal Pathway in Osteoarthritis.

Irisin, a myokine secreted by muscle during physical exercise, is known to have biological activities in different cell types. Chondrocyte inflammation and pyroptosis have been shown to play important roles in osteoarthritis (OA). In this study, we investigated the effects of exercise-induced irisin during different intensities of treadmill exercise in a rat OA model and the anti-inflammatory and antipyroptosis mechanism of irisin in OA chondrocytes. Forty-eight SD rats (n = 8) were randomly assigned to control (CG), OA (OAG), OA groups under different intensities of treadmill exercise (OAL, OAM, and OAH), OAM + irisin neutralizing antibodies group (OAM + irisin (NA)). The levels of irisin and the severity of OA between groups were detected using ELISA, histology, immunohistochemistry, X-ray and computed tomography and magnetic resonance imaging. The anti-inflammatory and antipyroptosis mechanisms of irisin were investigated in vitro in OA chondrocytes preincubated with recombinant irisin (0, 5, or 10 ng/ml) for 1 h before treatment with interleukin-1ß (IL-1ß) for 24 h mRNA and protein expression levels were determined using quantitative reverse transcription polymerase chain reaction, and western blot analyses. Morphological changes and cell death associated with pyroptosis were examined using transmission electron microscopy, flow cytometry and immunofluorescence. Moderate-intensity treadmill exercise increased the levels of irisin, exhibiting the best therapeutic effects on OA which could be suppressed by irisin neutralizing antibodies. Irisin not only recovered the expression of collagen II and attenuated that of MMP-13 and ADAMTS-5 in IL-1ß-induced OA chondrocytes by inhibiting the PI3K/Akt/NF-κB signaling pathway, but also inhibited the activity of nod-like receptor protein-3 (NLRP3)/caspase-1, thus ameliorating pyroptosis in chondrocytes. In conclusion, moderate mechanical stimulation protects against chondrocyte pyroptosis through irisin-induced suppression of PI3K/Akt/NF-κB signal pathway in osteoarthritis.

Bidirectional Longitudinal Study of Frailty and Depressive Symptoms Among Older Chinese Adults.

OBJECTIVE: Frailty and depression, as two common conditions among older adults in China, have been shown to be closely related to each other. The aim of this study was to investigate the bidirectional effects between frailty and depressive symptoms in Chinese population. METHODS: The bidirectional effect of frailty with depressive symptoms was analyzed among 5,303 adults ≥ 60 years of age from the China Health and Retirement Longitudinal Study (CHARLS). Phenotype and a frailty index were used to measure frailty. Depressive symptoms were evaluated using the Epidemiological Studies Depression Scale (CES-D). Logistic regression and Cox proportional hazard regression models were used to determine the bidirectional effects of frailty and depressive symptoms in cross-sectional and cohort studies, respectively. Subgroup and sensitivity analyses were further used to further verify the associations. RESULTS: In the cross-sectional study, the multivariate-adjusted ORs (95% CIs) for depressive symptoms among pre-frail and frail adults, as defined by the frailty index and phenotype, were 3.05 (2.68-3.49), and 9.78 (8.02-12.03), respectively. Depressed participants showed higher risks of pre-frailty and frailty [frailty index, 3.07 (2.69-3.50); and phenotypic frailty, 9.95 (8.15-12.24)]. During follow-up, the multivariate-adjusted HRs (95% CIs) for depressive symptoms among pre-frail and frail participants, as defined by the frailty index and phenotype, were 1.38 (1.22-1.57), and 1.30 (1.14-1.48), respectively. No significant relationship existed between baseline depressive symptoms and the incidence of frailty. Moreover, the results from subgroup and sensitivity analyses were consistent with the main results. CONCLUSION: Although a cross-sectional bidirectional association between depressive symptom and frailty has been observed in older (≥60 years old) Chinese adults, frailty may be an independent predictor for subsequent depression. Moreover, no effect of depressive symptoms on subsequent frailty was detected. Additional bidirectional studies are warranted in China.

The effect of water source on cognitive functioning in Chinese adults: A cross-sectional and follow-up study.

The effect of water source on cognitive functioning is poorly understood. The present study explored the associations between water source and cognition in 9921 participants from the China Health and Retirement Longitudinal Study (CHARLS). Cognitive functioning was measured from three aspects: orientation and attention, episodic memory, and visuo-construction. Water sources included tap and non-tap water. Generalized linear models and multiple logistic regression models were conducted to investigate the associations of cognitive scores with water source among the whole population and different subgroups. Results from cross-sectional analysis reported that participants without access to tap water showed a lower cognitive score (ß = - 0.57; 95% CI: - 0.74, - 0.39) than those with tap water as a water source; and this phenomenon was pronounced for both sexes and across all residences. During 4-years' follow-up, a greater decline of cognitive score was associated with no tap water use in the lowest quartile of baseline cognitive scores (ß = - 0.67; 95% CI: - 1.26, - 0.08). Additionally, the utilization rate of tap water was lower in rural areas compared to urban areas. The lowest utilization rates were observed in urban areas of the Anhui province (0.38 in 2011 and 0.55 in 2015) and in rural areas of Inner Mongolia (0.09 in 2011 and 0.20 in 2015). These findings suggest that having no tap water may be a risk factor for cognition impairment, particularly for those with a low basic cognition score. Additionally, our results support the need to expand tap water use in China.

Cemented hemiarthroplasty versus proximal femoral nail antirotation in the management of intertrochanteric femoral fractures in the elderly: a case control study.

BACKGROUND: The treatment for intertrochanteric femoral fractures (IFF) among the elderly has been a controversial topic. Hemiarthroplasty (HA) and proximal femoral nail antirotation (PFNA) have their own advantages in the management of IFF. Hence, this study aims to compare and analyze differences in the effectiveness of both procedures on IFF among the elderly. METHODS: Overall, 99 patients (81.09 ± 8.29 years; 68 women) underwent HA or PFNA from January 2016 to May 2020. IFF were classified according to the Arbeitsgemeins für Osteosynthesefragen (AO) classification. The difference in underlying diseases, the American Society of Anesthesiologists (ASA) grade, Singh index, Harris scores, surgical time, intraoperative bleeding, postoperative blood test results, postoperative number of days to partially bearing weight, and survival outcomes were analyzed. Postoperative follow-ups were performed every 3 months. RESULTS: There was no significant difference in the AO classification, underlying diseases, ASA grade, Singh index, surgical time, and survival outcomes of the HA (45 patients) group and PFNA group (54 patients). The HA group was associated with earlier partial weight-bearing (HA: 4 [2 ~ 4.5] days, PFNA: 10 [8~14] days). It also had a higher total Harris score than the PFNA group at the 6-month follow-up visit (HA: 86.8 [81.90 ~ 90.23], PFNA: 83.48 [75.13 ~ 88.23]). Harris scores decreased more in patients aged ≥90 years in the PFNA group than in the HA group. The postoperative stress recovery rate in the HA group was faster based on postoperative blood test results. CONCLUSIONS: PFNA and HA have good therapeutic effects in the treatment of IFF. The advantages of HA were reflected in short-term weight bearing, faster recovery from stress, and better joint function in the long term. This advantage is more obvious in the patient population aged over 90 years. Therefore, we suggest that surgeons should consider the benefit of HA in the treatment of IFF among the elderly. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR2000035814. Registered 17 August 2020, https://www.chictr.org.cn/showproj.aspx?proj=57083.

IRE1-mTOR-PERK Axis Coordinates Autophagy and ER Stress-Apoptosis Induced by P2X7-Mediated Ca2+ Influx in Osteoarthritis.

Moderate-intensity exercise can help delay the development of osteoarthritis (OA). Previous studies have shown that the purinergic receptor P2X ligand gated ion channel 7 (P2X7) is involved in OA development and progression. To investigate the effect of exercise on P2X7 activation and downstream signaling in OA, we used the anterior cruciate ligament transection (ACLT)-induced OA rat model and primary chondrocyte culture system. Our in vivo experiments confirmed that treadmill exercise increased P2X7 expression and that this effect was more pronounced at the later time points. Furthermore, P2X7 activation induced endoplasmic reticulum (ER) stress and increased the expression levels of ER stress markers, such as 78 kDa glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE1), and activating transcription factor 6 (ATF6). At the early time points, IRE1 and PERK were activated, and mTOR was inhibited. At the later time points, mTOR was activated, mediating PERK to promote ER stress-apoptosis, whereas IRE1 and autophagy were inhibited. To confirm our observations in vitro, we treated primary chondrocytes with the P2X7 agonist benzoylbenzoyl-ATP (Bz-ATP). Our results confirmed that P2X7-mediated Ca2+ influx activated IRE1-mediated autophagic flux and induced PERK-mediated ER stress-apoptosis. To further investigate the role of P2X7 in OA, we injected mTOR antagonist rapamycin or P2X7 antagonist A740003 into the knee joints of ACLT rats. Our results demonstrated that mTOR inhibition induced autophagy, decreased apoptosis, and reduced cartilage loss. However, injection of mTOR agonist MHY1485 or Bz-ATP had the opposite effect. In summary, our results indicated that during the early stages of moderate-intensity exercise, P2X7 was activated and autophagic flux was increased, delaying OA development. At the later stages, P2X7 became over-activated, and the number of apoptotic cells increased, promoting OA development. We propose that the IRE1-mTOR-PERK signaling axis was involved in the regulation of autophagy inhibition and the induction of apoptosis. Our findings provide novel insights into the positive and preventative effects of exercise on OA, suggesting that the intensity and duration of exercise play a critical role. We also demonstrated that on a molecular level, P2X7 and its downstream pathways could be potential therapeutic targets for OA.

P2X7 Receptor Induces Pyroptotic Inflammation and Cartilage Degradation in Osteoarthritis via NF-κB/NLRP3 Crosstalk.

Osteoarthritis (OA) is an urgent public health problem; however, the underlying causal mechanisms remain unclear, especially in terms of inflammatory mediators in cartilage degradation and chondrocyte imbalance. P2X7 receptor (P2X7R) is a critical inflammation switch, but few studies have examined its function and mechanisms in OA-like pyroptotic inflammation of chondrocytes. In this study, Sprague-Dawley rats were injected in the knee with monosodium iodoacetate (MIA) to induce OA, followed by multiple intra-articular injections with P2X7R antagonist A740003, P2X7R agonist BzATP, NF-κB inhibitor Bay 11-7082, and NLRP3 inhibitor CY-09. Primary rat chondrocytes were harvested and treated similarly. We assessed cell viability, damage, and death via cell viability assay, lactate dehydrogenase (LDH) release, and flow cytometry. Concentrations of adenosine triphosphate (ATP) and interleukin- (IL-) 1ß in cell culture supernatant and joint cavity lavage fluid were analyzed by enzyme-linked immunosorbent assay. Changes in expression levels of P2X7 and inflammation-related indicators were analyzed by immunofluorescence, quantitative reverse-transcription polymerase chain reaction, and western blotting. Cell morphology changes and pyroptosis were observed using transmission electron microscopy. Histology, immunohistochemistry, and microcomputed tomography were used to analyze damage to bone and cartilage tissues and assess the severity of OA. Similar to MIA, BzATP reduced cell viability and collagen II expression in a dose-dependent manner. Conversely, A740003 ameliorated MIA-induced cartilage degradation and OA-like pyroptotic inflammation by rescuing P2X7, MMP13, NF-κB p65, NLRP3, caspase-1 (TUNEL-positive and active), and IL-1ß upregulation. Additionally, A740003 reduced the caspase-1/propidium iodide double-positive rate, LDH concentration, and reactive oxygen species production. These effects also occurred via coincubation with Bay 11-7082 and CY-09. In conclusion, activated P2X7 promoted extracellular matrix degradation and pyroptotic inflammation in OA chondrocytes through NF-κB/NLRP3 crosstalk, thus, aggravating the symptoms of OA. The study findings suggest P2X7 as a potential target for inflammation treatment, providing new avenues for OA research and therapy.

Maresin-1 suppresses IL-1ß-induced MMP-13 secretion by activating the PI3K/AKT pathway and inhibiting the NF-κB pathway in synovioblasts of an osteoarthritis rat model with treadmill exercise.

Aim: Maresin-1 is a metabolite of docosahexaenoic acid (DHA) that has potential anti-inflammatory effects. To explore whether maresin-1 changes and has a therapeutic effect in osteoarthritis (OA) model rats undergoing treadmill exercise, we examined endogenous maresin-1 in a single-session treadmill experiment and OA model rats were treated with maresin-1, moreover, we examined the effects of maresin-1 on IL-1ß induced rat fibroblast-like synoviocytes (FLSs) and possible mechanisms.Methods: In single-session treadmill experiment, 48 rats were randomly divided into 3 groups and performed three different intensities of exercise (15.2 m/min, 0°; 19.3 m/min, 5°; 26.8 m/min, 10°) for 60 min. Intra-articular lavage fluid (IALF) samples were harvested after 0, 2, and 4 h from each group (n = 4) and maresin-1 levels were evaluated by ELISA. Another 30 rats were treated with monosodium iodoacetate (MIA) to induce osteoarthritis and exogenous maresin-1 (MaR-1) and were divided into three groups (n = 10, OA: MIA, OAM: MIA+MaR1, and CG: control group). The level of injury was evaluated by OARSI and Mankin scores, and the levels of type II collagen and MMP13 were evaluated by immunohistochemistry. FLSs were obtained from the knee joint of SD rats, and the expression of MMP13 and activation of the PI3k/Akt and NF-κB p65 pathways in IL-1ß-induced FLSs were evaluated by western blotting.Results: Maresin-1 levels were increased in IALF at 4 h after exercise, and type II collagen increased in cartilage and MMP13 decreased in the synovium after treatment with maresin-1 in MIA-induced osteoarthritis. The results of vitro experiment showed decreased MMP13, activation of the PI3k/Akt pathway, and suppression of the NF-κB p65 pathway upon treatment with maresin-1 in IL-1ß-induced FLSs.Conclusions: The changes in maresin-1 in IALF, as seen in our single-section treadmill exercise, provides an explanation for the therapeutic effect of appropriate-strength treadmill exercise on osteoarthritis, and our experiments confirmed the therapeutic effect of maresin-1 both in vivo and in vitro.

Protectin DX attenuates IL-1ß-induced inflammation via the AMPK/NF-κB pathway in chondrocytes and ameliorates osteoarthritis progression in a rat model.

Protectin DX (PDX) has been reported to have extensive anti-inflammatory effects. However, it is unknown whether PDX acts as an anti-inflammatory agent in the context of osteoarthritis (OA). This study aimed to evaluate the anti-inflammatory activity of PDX in vitro and in vivo in a model of OA. Primary rat chondrocytes were preincubated with PDX 1 h prior to IL-1ß treatment for 24 h. We found that PDX was nontoxic, and pretreatment with PDX increased cell viability in IL-1ß-induced chondrocytes. Preincubation with PDX also efficiently inhibited the degradation of type II collagen dose-dependently. Additionally, the expression of MMP-3, MMP-13, ADAMTS4, iNOS, COX-2, NO, and PGE2 decreased after IL-1ß stimulation when cells were preincubated with PDX. Moreover, PDX inhibited the increase in phosphorylated NF-κB p65 and IκBα upon IL-1ß stimulation, and the negative effects of IL-1ß on chondrocytes were partially blocked by treatment with pyrrolidine dithiocarbamate (PDTC), a selective NF-κB inhibitor. In addition, we found that PDX increased AMPK phosphorylation in IL-1ß-mediated chondrocytes. The phosphorylation of AMPK could be inhibited by compound C, a classic AMPK inhibitor. Compound C also remarkably reversed the decrease in p65 phosphorylation and MMP-13 expression caused by PDX. Furthermore, nuclear translocation of NF-κB was visible by immunofluorescence after PDX-induced AMPK activation. Additionally, we verified that PDX ameliorated cartilage degradation in monosodium iodoacetate (MIA)-induced OA rats through histological evaluation and ELISA of TNF-α in the serum and intra-articular lavage fluid. In conclusion, we have shown that PDX suppresses inflammation in chondrocytes in vitro and in vivo, likely through the AMPK/NF-κB signaling pathway. Our results suggest that PDX could be a useful novel therapeutic agent for OA treatment.

The Therapeutic Effects of Treadmill Exercise on Osteoarthritis in Rats by Inhibiting the HDAC3/NF-KappaB Pathway in vivo and in vitro.

Osteoarthritis (OA) is a disease characterized by non-bacterial inflammation. Histone deacetylase 3 (HDAC3) is a crucial positive regulator in the inflammation that leads to the development of non-OA inflammatory disease. However, the precise involvement of HDAC3 in OA is still unknown, and the underlying mechanism of exercise therapy in OA requires more research. We investigated the involvement of HDAC3 in exercise therapy-treated OA. Expression levels of HDAC3, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), matrix metalloproteinase-13 (MMP-13), HDAC3 and nuclear factor-kappaB (NF-kappaB) were measured by western blotting, reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Cartilage damage and OA evaluation were measured by hematoxylin and eosin staining and Toluidine blue O staining according to the Mankin score and OARSI score, respectively. We found that moderate-intensity treadmill exercise could relieve OA. Meanwhile, the expression of HDAC3, MMP-13, ADAMTS-5 and NF-kappaB decreased, and collagen II increased in the OA + moderate-intensity treadmill exercise group (OAM) compared with the OA group (OAG) or OA + high- or low-intensity treadmill exercise groups (OAH or OAL). Furthermore, we found the selective HDAC3 inhibitor RGFP966 could also alleviate inflammation in OA rat model through inhibition of nuclear translocation of NF-kappaB. To further explore the relationship between HDAC3 and NF-kappaB, we investigated the change of NF-kappaB relocation in IL-1ß-treated chondrocytes under the stimulation of RGFP966. We found that RGFP966 could inhibit the expression of inflammatory markers of OA via regulation of HDAC3/NF-kappaB pathway. These investigations revealed that RGFP966 is therefore a promising new drug for treating OA.

The Transient Receptor Potential Channel, Vanilloid 5, Induces Chondrocyte Apoptosis via Ca2+ CaMKII-Dependent MAPK and Akt/ mTOR Pathways in a Rat Osteoarthritis Model.

BACKGROUND/AIMS: Chondrocyte apoptosis is a central pathological feature of cartilage in osteoarthritis (OA). Accumulating evidence suggests that calcium ions (Ca2+) are an important regulator of apoptosis. Previously, we reported that the transient receptor potential channel vanilloid (TRPV5) is upregulated in monoiodoacetic acid (MIA)-induced OA articular cartilage. METHODS: The protein levels of TRPV5, phosphorylated Ca2+/calmodulin-dependent kinase II (p-CaMKII), and total CaMKII were detected in vivo using western blotting techniques. Primary chondrocytes were isolated and cultured in vitro. Then, p-CAMKII was immunolocalized by immunofluorescence in chondrocytes. Fluo-4AM staining was used to assess intracellular Ca2+. Annexin V-fluorescein isothiocyanate / propidium iodide flow cytometric analysis was performed to determine chondrocyte apoptosis. Western blotting techniques were used to measure the expression of apoptosis-related proteins. RESULTS: We found that ruthenium red (aTRPV5inhibitor)or(1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperaze (KN-62) (an inhibitor of Ca2+/calmodulin-dependent kinase II (CaMKII) phosphorylation) can relieve or even reverse OA in vivo. We found that TRPV5 has a specific role in mediating extracellular Ca2+ influx leading to chondrocyte apoptosis in vitro. The apoptotic effect in chondrocytes was inhibited by KN-62. We found that activated p-CaMKII could elicit the phosphorylation of extracellular signal-regulated protein kinase 1/2, c-Jun N-terminal kinase, and p38, three important regulators of the mitogen-activated protein kinase (MAPK) cascade. Moreover, we also showed that activated p-CaMKII could elicit the phosphorylation of protein kinase B (Akt) and two important downstream regulators of mammalian target of rapamycin (mTOR): 4E-binding protein, and S61 kinase. CONCLUSION: Our results demonstrate that upregulated TRPV5 may be an important initiating factor that activates CaMKII phosphorylation via the mediation of Ca2+ influx. In turn, activated p-CaMKII plays a critical role in chondrocyte apoptosis via MAPK and Akt/mTOR pathways.

Transient Receptor Potential Channel, Vanilloid 5, Induces Chondrocyte Apoptosis in a Rat Osteoarthritis Model Through the Mediation of Ca2+ Influx.

BACKGROUND/AIMS: Chondrocyte apoptosis is the most common pathological feature in cartilage in osteoarthritis (OA). Transient receptor potential channel vanilloid 5 (TRPV5) is important in regulating calcium ion (Ca2+) influx. Accumulating evidences suggest that Ca2+ is a major intracellular second messenger that can trigger cell apoptosis. Therefore, we investigate the potential role of TRPV5 in mediating Ca2+ influx to promote chondrocyte apoptosis in OA. METHODS: The monoiodoacetic acid (MIA)-induced rat OA model was assessed by macroscopic and radiographic analyses. Calmodulin protein immunolocalization was detected by immunohistochemistry. The mRNA and protein level of TRPV5, calmodulin and cleaved caspase-8 in articular cartilage were assessed by real time polymerase chain reaction and western blotting. Primary chondrocytes were isolated and cultured in vitro. TRPV5 small interfering RNA was used to silence TRPV5 in chondrocytes. Then, calmodulin and cleaved caspase-8 were immunolocalized by immunofluorescence in chondrocyte. Fluo-4AM staining was used to assess intracellular Ca2+ to reflect TRPV5 function of mediation Ca2+ influx. Annexin V-fluorescein isothiocyanatepropidium iodide flow cytometric analysis was performed to determine chondrocytes apoptosis. Western blotting techniques were used to measure the apoptosis-related proteins in chondrocyte level. RESULTS: Here, we reported TRPV5 was up-regulated in MIA-induced OA articular cartilage. Ruthenium red (a TRPV5 inhibitor) can relieve progression of joint destruction in vivo which promoted us to demonstrate the effect of TRPV5 in OA. We found that TRPV5 had a specific role in mediating extracellular Ca2+ influx leading to chondrocytes apoptosis in vitro. The apoptotic effect was inhibited even reversed by silencing TRPV5. Furthermore, we found that the increase Ca2+ influx triggered apoptosis by up-regulating the protein of death-associated protein, FAS-associated death domain, cleaved caspase-8, cleaved caspase-3, cleaved caspase-6, and cleaved caspase-7, and the up-regulated proteins were abolished by silencing TRPV5 or 1, 2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (a Ca2+ chelating agent). CONCLUSION: The up-regulated TRPV5 could used be as an initiating factor that induces extrinsic chondrocyte apoptosis via the mediation of Ca2+ influx. These findings suggested TRPV5 could be an intriguing mediator for drug target in OA.

Reactive oxygen species induce injury of the intestinal epithelium during hyperoxia.

Long-term therapeutic hyperoxia may exert serious toxic effects on intestinal epithelial cells in vitro and in vivo. The aim of the present study was to investigate the cause of this intestinal injury under conditions of hyperoxia. Caco-2 cells were treated with different concentrations of hydrogen peroxide (H2O2) and 85% hyperoxia for 24 h. higher rates of injury of Caco-2 cells were observed in the hyperoxia and H2O2 groups compared with the control group. The reactive oxygen species (ROS) level of the hyperoxia group was significantly higher compared with that of the 400 µM H2O2 group. The protein and gene levels of RelA, RelB, hypoxia­inducible factor-1α, tumor necrosis factor-α and apoptosis signal­regulating kinase 1 were significantly higher in the hyperoxia and H2O2 groups compared with those in the control group. In conclusion, during hyperoxia, intestinal epithelial cells were destroyed and the levels of ROS were increased. Therefore, ROS may play an important role in intestinal injury in a hyperoxic environment.

Transient Receptor Potential Vanilloid 5 Mediates Ca2+ Influx and Inhibits Chondrocyte Autophagy in a Rat Osteoarthritis Model.

BACKGROUND: Autophagy, a self-protective mechanism of chondrocytes, has become a promising target to impede the progress of osteoarthritis (OA). Autophagy is regulated by cytosolic Ca2+ activity and may thus be modified by the Ca2+ permeable transient receptor potential channel vanilloid 5 (TRPV5). Therefore, we investigated the potential role of TRPV5 in mediating Ca2+ influx and in inhibiting chondrocyte autophagy in a rat OA model. METHODS: The rat OA model was assessed by macroscopic and histological analyses. light chain 3B (LC3B) immunolocalization was detected by immunohistochemistry. TRPV5, LC3B and calmodulin in OA articular cartilage were assessed by real time polymerase chain reaction (RT-PCR) and western blotting. TRPV5 small interfering RNA (TRPV5 siRNA) were transfected into rat primary chondrocyte then the calmodulin and LC3B was detected by immunofluorescence. The functionality of the TRPV5 was assessed by Ca2+ influx. Western blot was used to measure autophagy-related proteins. RESULTS: We constructed a monosodium iodoacetate (MIA) -induced rat OA model and found that ruthenium red (TRPV5 inhibitor) slowed the progression of joint destruction. We found that the TRPV5 and calmodulin were up-regulated but LC3B was down-regulated in articular cartilage following prolonged progression of OA. Furthermore, the up-regulated TRPV5 channel caused an increase in the Ca2+ influx in chondrocytes. The up-regulation of TRPV5 stimulated Ca2+ influx, which inhibited autophagy by increasing the production of calmodulin, phosphorylation of calmodulin dependent protein kinases II (p-CAMK II), phosphorylation of Beclin1 (p-Beclin1), and protein of B-cell lymphoma-2 (Bcl-2), and attenuating ratio of LC3-II/ LC3-. CONCLUSION: Up-regulated TRPV5 as an initiating factor inhibited chondrocyte autophagy via the mediation of Ca2+ influx.