PCB153 suppressed autophagy via PI3K/Akt/mTOR and RICTOR/Akt/mTOR signaling by the upregulation of microRNA-155 in rat primary chondrocytes.

Polychlorinated biphenyls (PCBs) are a typical type of persistent organic pollutant. PCB exposure is associated to the occurrence and development of osteoarthritis (OA); however, the involved mechanisms have yet to be elucidated. Here, we investigated the pro-osteoarthritic effect of 2, 2', 4, 4', 5, 5'-hexachlorobiphenyl (PCB153), and the involvement of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) and the RICTOR/Akt/mTOR signaling pathways. PCB153 of 20 and 30 µM increased the expression of MMP13 and decreased the expression of type II collagen, in a concentration-dependent manner. PCB153 treatment reduced the expression of Beclin 1 and LC3B, but increased the expression of p62 by upregulating miR-155 levels. PCB153 treatment activated the PI3K/Akt/mTOR signaling pathway by upregulating miR-155 levels. RICTOR was involved in activating the Akt/mTOR signaling pathway, and was also regulated by miR-155. In conclusion, PCB153 could promote the degradation of the extracellular matrix of chondrocytes by upregulating miR-155 via a mechanism related to the activation of the PI3K/Akt/mTOR and RICTOR/Akt/mTOR signaling pathway, which suppressed autophagy and facilitated the development of OA. MiR-155 may represent potential therapeutic targets to alleviate the development of OA.

Protective effect of resveratrol on obesity-related osteoarthritis via alleviating JAK2/STAT3 signaling pathway is independent of SOCS3.

Resveratrol (RES) has a protective effect on osteoarthritis (OA), nevertheless, the underlying mechanisms of RES towards obesity-related OA are still unclear. This study is aimed to determine whether leptin resistant mechanism presents in articular cartilage of obesity-related OA and whether the protective effect of RES is involved in the regulation of leptin signal by affecting suppressor of cytokine signaling 3 (SOCS3). Male C57BL/6 J mice were fed with standard chow diet, high fat diet (HFD) or high fat diet with RES (45 mg/kg.bw) for 22 weeks. Knee joints of mice were evaluated by histological and immunohistochemistry analysis. Serum level of leptin was measured by ELISA. The leptin, leptin receptor (OB-Rb), SOCS3 mRNA expression and JAK2, STAT3, OB-Rb and SOCS3 protein expression in cartilage were determined by real-time RT-PCR and western blot. In addition, SW1353 cells were pretreated with or without AG490, and stimulated with leptin in the presence or absence of RES to detect JAK2, STAT3, matrix metalloproteinase-13 (MMP-13) and SOCS3 expression. We found that HFD could induce obesity-related OA and RES prevented its progression. Serum leptin and mRNA expression in cartilage was up-regulated by HFD, while RES ameliorated the elevation. Besides, RES significantly inhibited the JAK2/STAT3 signaling pathway in cartilage, as well as SOCS3. In in vitro study, RES exhibited the same effect in SW1353 cells which stimulated with leptin. In conclusion, no significant leptin resistance existed in cartilage of obesity-related OA and the inhibitory effect of RES on obesity-related OA via alleviating JAK2/STAT3 signaling pathway is independent of SOCS3.

Resveratrol inhibits the development of obesity-related osteoarthritis via the TLR4 and PI3K/Akt signaling pathways.

Aim of the study: Obesity leads to mild, chronic inflammation which is a primary risk factor for osteoarthritis (OA). Resveratrol exerts a protective effect on OA through its anti-inflammatory properties, but the precise mechanism remains unknown. The present study aimed to investigate the mechanism by which resveratrol alleviates obesity-related OA, and whether it is linked to the TLR4 and PI3K/Akt signaling pathways. Materials and methods: C57BL/6J male mice were fed a high-fat diet (HFD) with or without resveratrol treatment and knee joints were collected for analysis. In addition, IL-1ß-induced SW1353 cells were used to study in vitro the reciprocal effects of TLR4 and PI3K/Akt pathways. Results: Resveratrol inhibited the development of OA in mice fed a HFD. TLR4 and PI3K/Akt signaling pathways were both activated in the articular cartilage; resveratrol treatment down-regulated TLR4 but up-regulated PI3K/Akt signaling. Further in vitro results showed that the effect of resveratrol alone on activation of PI3K/Akt was attenuated but not abolished by the TLR4 inhibitor CLI-095, and resveratrol failed to reduce TLR4 protein expression in IL-1ß stimulated cells pretreated with the PI3K inhibitor LY294002. Conclusions: Resveratrol may exert an anti-osteoarthritic effect by inhibiting TLR4 via the activation of PI3K/Akt signaling pathways. Resveratrol has potential as a drug for OA prevention.

Oral Administration of Resveratrol Alleviates Osteoarthritis Pathology in C57BL/6J Mice Model Induced by a High-Fat Diet.

Obesity has been associated with osteoarthritis (OA) due to increased mass and metabolic factors which are independent of the biomechanical contribution to joint load. Resveratrol, a natural polyphenolic compound, exerts protective effects on OA through its anti-inflammatory property. However, the mechanism of resveratrol on obesity-related OA is unclear. To investigate the effect and possible mechanism of oral resveratrol on obesity-related OA, we fed C57BL/6J mice with a high-fat diet (HFD) for 16 weeks to establish obesity-related OA model; then two doses (22.5 mg/kg and 45 mg/kg) of resveratrol were given by gavage for additional 12 weeks. Mice with HFD significantly increased body weights compared to the control mice, while resveratrol treatment did not cause obvious weight loss. Histological assessments showed that resveratrol at 45 mg/kg significantly improved OA symptoms. Levels of serum IL-1ß and leptin were decreased by resveratrol treatment and positively correlated with Mankin scores. Moreover, resveratrol significantly inhibited the expression of TLR4 and TRAF6 in cartilage. These results suggest that HFD induced obesity can lead to the occurrence of OA, and resveratrol may alleviate OA pathology by decreasing the levels of systematic inflammation and/or inhibiting TLR4 signaling pathway in cartilage. Thus, resveratrol might be a promising therapeutic treatment for obesity-related OA.

Protective effect of resveratrol against IL-1ß-induced inflammatory response on human osteoarthritic chondrocytes partly via the TLR4/MyD88/NF-κB signaling pathway: an "in vitro study".

Resveratrol is a natural polyphenolic compound that prevents inflammation in chondrocytes and animal models of osteoarthritis (OA) via yet to be defined mechanisms. The purpose of this study was to determine whether the protective effect of resveratrol on IL-1ß-induced human articular chondrocytes was associated with the TLR4/MyD88/NF-кB signaling pathway by incubating human articular chondrocytes (harvested from osteoarthritis patients) with IL-1ß before treatment with resveratrol. Cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and TNFα levels in culture supernatants were measured by ELISA(Enzymelinked immunosorbent assay). The levels of TLR4 and its downstream signaling targets (MyD88 and TRAF6) and IL-1ß were assessed by measuring the levels of mRNA and protein expression by real-time RT-PCR and western blot analysis, respectively, in addition to assessing NF-кB activation. In addition, TLR4 siRNA was used to block TLR4 expression in chondrocytes further demonstrating that resveratrol prevented IL-1ß-mediated inflammation by TLR4 inhibition. We found that resveratrol prevented IL-1ß-induced reduction in cell viability. Stimulation of chondrocytes with IL-1ß caused a significant up-regulation of TLR4 and its downstream targets MyD88 and TRAF6 resulting in NF-кB activation associated with the synthesis of IL-1ß and TNFα. These IL-1ß-induced inflammatory responses were all effectively reversed by resveratrol. Furthermore, activation of NF-кB in chondrocytes treated with TLR4 siRNA was significantly attenuated, but not abolished, and exposure to resveratrol further reduced NF-кB translocation. These data suggested that resveratrol prevented IL-1ß-induced inflammation in human articular chondrocytes at least in part by inhibiting the TLR4/MyD88/NF-кB signaling pathway suggesting that resveratrol has the potential to be used as a nutritional supplement to counteract OA symptoms.

n-3 polyunsaturated fatty acids alleviate the progression of obesity-related osteoarthritis and protect cartilage through inhibiting the HMGB1-RAGE/TLR4 signaling pathway.

Osteoarthritis (OA) is a common joint degenerative disease. There is currently no cure for OA. Dietary fatty acids have potential value in the prevention and treatment of OA. n-3 polyunsaturated fatty acids (PUFAs) have anti-inflammatory effects, but their anti-OA mechanism remains unclear. High-mobility group box 1 (HMGB1) promotes inflammation and participates the pathogenesis of OA. The purpose of this study was to investigate the protective effect of n-3 PUFAs on cartilage and whether n-3 PUFAs could exert an anti-OA effect through inhibiting HMGB1-RAGE/TLR4 signaling pathway. We established an obesity-related post-traumatic OA mice model and an in vitro study was conducted to explore the regulatory mechanism of n-3 PUFAs on HMGB1 and its signal pathway against OA. We found that diet rich in n-3 PUFAs alleviated OA-like lesions of articular cartilage with the decrease of HMGB1-RAGE/TLR4 signaling protein in mice. In SW1353 cells, DHA significantly reduced the expression of HMGB1-RAGE/TLR4 signaling protein which was up-regulated by IL-1ß stimulation. HMGB1 overexpression reversed the inhibitory effect of DHA on HMGB1-RAGE/TLR4 signaling pathway. The activation of SIRT1 may participate the inhibitory effect of DHA on HMGB1-RAGE/TLR4 signaling pathway. In conclusion, n-3 PUFAs could attenuate the progression of obesity-related OA and exert protective effect on cartilage by inhibiting HMGB1-RAGE/TLR4 signaling pathway, which may be associated with the activation of SIRT1. Dietary n-3 PUFAs supplements can be considered as a potential therapeutic substance for OA.

Application of 3-dimensional printing technology in the treatment of postoperative nonunion of femoral neck fracture: A case report.

Nonunion of the femoral neck represents the main complication following fractures at this location. Few studies have documented the application of 3-dimensional printing technology for the surgical treatment of postoperative nonunion of femoral neck fractures. The present manuscript describes a case of a particular 3-dimensional printing technology method for designing a custom-made guide plate for revision surgery. A 46-year-old man suffered a nonunion of the femoral neck following an internal fracture fixation. Using 3-dimensional printing technology, we preoperatively printed a femur model and a custom-made guide plate. Using the model, we performed a simulated operation before the surgery, and the guide plate was used for accurate osteotomy during the operation. With this method, we achieved the desired results: the fracture healed, the operation time was shortened, and there was no femoral head necrosis. Our case suggests that 3-dimensional printing technology provides an effective aid in the treatment of patients with nonunion following a femoral neck fracture and recommends 3-dimensional printing technology for such cases.

Dietary Fatty Acid Regulation of the NLRP3 Inflammasome via the TLR4/NF-κB Signaling Pathway Affects Chondrocyte Pyroptosis.

Dietary fatty acid (FA) content and type have different effects on obesity-associated osteoarthritis (OA), but the mechanisms underlying these differences are not fully understood. Inflammation activated by toll-like receptor 4 (TLR4)/nuclear factor- (NF-) κB signaling and pyroptosis induced by the NLRP3/caspase-1/gasdermin D (GSDMD) signaling pathway play important roles in OA development. Our aim in this study was to observe the effects of dietary FAs on the articular cartilage of obese post-traumatic OA model mice and on chondrocytes stimulated by lipopolysaccharide (LPS) and to determine whether the underlying mechanisms involve TLR4/NF-κB and NLRP3/caspase-1/GSDMD signaling pathways. Mice were fed high-fat diets rich in various FAs and underwent surgical destabilization of the medial meniscus to establish the obesity-related post-traumatic OA model. LPS-induced SW1353 chondrosarcoma cells were used to mimic OA status in vitro, and TLR4 inhibitors or TLR4 overexpressing lentivirus was administered. Analysis using weight-matched mice and multiple regression models revealed that OA was associated with dietary FA content and serum inflammatory factor levels, but not body weight. Diets rich in n-3 polyunsaturated fatty acids (PUFAs) attenuated OA and inhibited the TLR4/NF-κB and NLRP3/caspase-1/GSDMD signaling pathways, whereas diets rich in saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), or n-6 PUFAs increased OA severity and activated these pathways. In vitro results for SFAs, n-6 PUFAs, and n-3 PUFAs were consistent with the animal experiments. However, those for MUFAs were not. FA effects on the NLRP3/caspase-1/GSDMD pathway were associated with the inhibition or activation of the TLR4 signaling pathway. In conclusion, diets rich in SFAs or n-6 PUFAs can exacerbate obesity-associated OA, whereas those rich in n-3 PUFAs have protective effects against this disease, due to their respective pro-/anti-inflammatory and pyroptotic effects. Further research on dietary FA supplements as a potential therapeutic approach for OA is needed.

Curcumin-Alleviated Osteoarthritic Progression in Rats Fed a High-Fat Diet by Inhibiting Apoptosis and Activating Autophagy via Modulation of MicroRNA-34a.

PURPOSE: The mechanism underlying curcumin's protective effect on osteoarthritis (OA) has not been clarified. This study aimed to determine whether curcumin exerts a chondroprotective effect by inhibiting apoptosis via upregulation of E2F1/PITX1 and activation of autophagy via the Akt/mTOR pathway by targeting microRNA-34a (miR-34a). METHODS: Male Sprague-Dawley rats were fed a normal diet (ND) or high-fat diet (HFD) for 28 weeks. Five rats from each diet group were selected randomly for histological analysis of OA characteristics. Rats fed a HFD were given a single intra-stifle joint injection of the miR-34a mimic agomir-34a or negative control agomir (NC), followed by weekly low-dose (200 µg/kg body weight) or high-dose (400 µg/kg body weight) curcumin intra-joint injections from weeks 29 to 32. The rats' stifle joints were submitted to histological analysis and to an apoptotic assay. Expression of miR-34a was detected using a real-time RT-PCR. E2F1 and PITX1 protein levels were determined by Western blot analysis, and the expressions of Beclin1, LC3B, p62, phosphorylated (p)-Akt, and p-mTOR were measured using immunofluorescence analysis. RESULTS: We found that rats fed a HFD had OA-like lesions in their articular cartilage and had increased apoptosis of chondrocytes and decreased autophagy compared to rats fed a ND. Curcumin treatment alleviated OA changes, inhibited apoptosis, and upregulated autophagy. Agomir-34a treatment reduced E2F1, PITX1, Beclin1, and LC3B expression and increased p62, p-Akt, and p-mTOR expression in HFD-fed rats given low- or high-dose curcumin. Greater numbers of apoptotic cells, lesser expression of p62, p-Akt, and p-mTOR, and greater expression of E2F1, PITX1, and LC3B were observed in the agomir-34a and high-dose curcumin-treated group than in agomir-34a and low-dose curcumin-treated group. CONCLUSION: Curcumin's chondroprotective effect was mediated by its suppression of miR-34a, apparently by reducing apoptosis, via upregulation of E2F1/PITX1, and by augmenting autophagy, likely via the Akt/mTOR pathway.

Selection of internal fixation method for femoral intertrochanteric fractures using a finite element method.

BACKGROUND: Failure to fix unstable intertrochanteric fractures impairs return to daily activities. AIM: To simulate five different internal fixation methods for unstable proximal femoral fractures. METHODS: A three-dimensional model of the femur was established from sectional computed tomography images, and an internal fixation model was established. Finite element analysis of the femur model was established, and three intertrochanteric fracture models, medial defect, lateral defect, and medial-lateral defects, were simulated. Displacement and stress distribution after fixation with a proximal femoral anti-rotation intramedullary nail (PFNA), integrated dual-screw fixation (ITN), PFNA + wire, PFNA + plate, and PFNA + wire + plate were compared during daily activities. RESULTS: The maximum displacement and stress of PFNA and ITN were 3.51 mm/473 MPa and 2.80 mm/588 MPa for medial defects; 2.55 mm/288 MPa and 2.10 mm/307 MPa for lateral defects; and 3.84 mm/653 MPa and 3.44 mm/641 MPa for medial-lateral defects, respectively. For medial-lateral defects, reconstructing the medial side alone changed the maximum displacement and stress to 2.79 mm/515 MPa; reconstructing the lateral side changed them to 3.72 mm/608 MPa, when both sides were reconstructed, they changed to 2.42 mm/309 MPa. CONCLUSION: For medial defects, intramedullary fixation would allow early low-intensity rehabilitation exercise, and ITN rather than PFNA reduces the risk of varus and cut-out; for lateral wall defects or weakness, intramedullary fixation allows higher-intensity rehabilitation exercise, and ITN reduces the risk of varus. For both medial and lateral defects, intramedullary fixation alone will not allow early functional exercise, but locating lateral or medial reconstruction will. For defects in both the inner and outer sides, if reconstruction cannot be completed, ITN is more stable.

Leptin Induced TLR4 Expression via the JAK2-STAT3 Pathway in Obesity-Related Osteoarthritis.

Obesity is considered as a risk factor of osteoarthritis (OA), but the precise relationship is still poorly understood. Leptin, one of the most relevant factors secreted by adipose tissues, plays an important role in the pathogenesis of OA. Our aim was to investigate the regulation and molecular mechanism of the leptin signaling pathway in obesity-related OA. SD rats were fed with a high-fat diet (HFD) for 5, 15, and 27 weeks. The levels of leptin in serum increased from W5, while in the synovial fluid increased from W15. The histological evaluation showed that the pathological changes of OA occurred at 27 weeks rather than 5 or 15 weeks. We also found that leptin induced CD14/TLR4 activation by the JAK2-STAT3 signaling pathway to promote OA. Moreover, silencing SOCS3 enhanced leptin-induced JAK2-STAT3-CD14/TLR4 activation in rat primary chondrocytes. Our findings indicated that leptin may be one of the initiating factors of obesity-related OA. TLR4 is at least partially regulated by leptin through the JAK2-STAT3-CD14 pathway. Meanwhile, SOCS3 acting as a negative feedback inhibitor of leptin signaling presented a potential therapeutic prospect for obesity-related OA. Our study provided new evidence suggesting the key role of leptin in mediating obesity-related OA process and its underlying mechanisms.

Adipogenic differentiation is not influenced by lentivirus-mediated shRNA targeting the SOCS3 gene in adipose-derived stromal cells.

The adipose-derived stromal cells (ADSCs) are capable of adipogenic differentiation by which they can be used as the seed of adipocytes and as the model to study the mechanism of differentiation. Suppressor of cytokine signaling 3 (SOCS3), a negative regulator of leptin signaling, perhaps involves in cell survival and differentiation. However, to date, rare studies are performed on ADSCs differentiation and SOCS3 regulation. In this study, a lentiviral vector expressing a shRNA targeting SOCS3 gene (Lv-shSOCS3) is developed and tested. To observe the effect of Lv-shSOCS3 on ADSCs differentiation, we infected ADSCs with Lv-shSOCS3 or Lv-shSOCS3-NC (non-silenced control lentivirus) and examined adipocyte differentiation using morphology and the expression of key gene for adipogenic differentiation using real-time PCR. The results show that Lv-shSOCS3 can significantly suppress the expression of SOCS3 mRNA and protein in ADSCs. There are no distinguishable differences on adipogenic differentiation and the expression of peroxisome proliferators-activated receptor gamma (PPARgamma) mRNA for ADSCs between infection with Lv-shSOCS3 and Lv-shSOCS3-NC. We conclude that SOCS3 knock-down does not influence the growth and adipogenic differentiation feature of ADSCs. Our study provides a new molecular basis for understanding the differentiation mechanism of ADSCs and lays the foundation for further study of the biological functions of SOCS3.

Resveratrol Exerts Anti-Osteoarthritic Effect by Inhibiting TLR4/NF-κB Signaling Pathway via the TLR4/Akt/FoxO1 Axis in IL-1ß-Stimulated SW1353 Cells.

PURPOSE: Osteoarthritis (OA) is associated with chronic low-grade inflammation. Resveratrol exerts protective effects on OA through its anti-inflammatory property; however, the mechanism of resveratrol on anti-inflammatory signaling pathways has not been fully elucidated yet. The aim of the present study was to investigate whether resveratrol-mediated PI3K/Akt expression is linked to TLR4/NF-κB pathway and the role of TLR4/Akt/FoxO1 axis in the anti-osteoarthritic effect of resveratrol. METHODS: SW1353 cells stimulated by IL-1ß (10 ng/mL) were cultured in the presence or absence of resveratrol (50 µM) and then treated with TLR4 siRNA, PI3K inhibitor LY294002 or FoxO1 siRNA, respectively. The associated proteins of TLR4 signaling pathways and TLR4/Akt/FoxO1 axis were evaluated by Western blot. The level of IL-6 in the supernatant was detected by ELISA. RESULTS: IL-1ß treatment increased the expression of TLR4/NF-κB and phosphorylation of PI3K/Akt and FoxO1, while additional resveratrol further upregulated the expression of PI3K/Akt and FoxO1 phosphorylation but downregulated TLR4 signals in SW1353 cells. Further analyses by the inhibition of TLR4, PI3K/Akt and FoxO1 signaling pathways, respectively, showed that the activation of TLR4 can induce PI3K/Akt phosphorylation, which increases the phosphorylation of FoxO1 and inactivates it. Next, inactivated-FoxO1 can reduce the expression of TLR4, which forms a self-limiting mechanism of inflammation. Resveratrol treatment can upregulate PI3K/Akt phosphorylation and inactivate FoxO1, thereby reducing TLR4 and inflammation. CONCLUSION: This study reveals that TLR4/Akt/FoxO1 inflammatory self-limiting mechanism may exist in IL-1ß-stimulated SW1353 cells. This study reveals a novel cross-talk mechanism which is between integrated PI3K/Akt/FoxO1 signaling network and TLR4-driven innate responses in IL-1ß-stimulated SW1353 cells. Resveratrol may exert anti-OA effect by enhancing the self-limiting mechanism of inflammation through TLR4/Akt/FoxO1 axis.

[Effect of rat recombinant leptin on expression of SOCS-3 in mature adipocytes].

OBJECTIVE: To study the effect of rat recombinant leptin on expressions of suppressor of cytokine signaling 3 (SOCS-3) mRNA and protein in mature adipocytes . METHODS: Mature adipocytes were separated from epididymal adipose tissues of male SD rats, and the expressions of long-form leptin receptor mRNA were detected. The separated adipocytes were then treated with rat recombinant leptin at the doses of 0.5-500 nmol/L for 0.5 to 6h. The expressions of SOCS-3 mRNA were measured with RT-PCR and proteins were measured with immuniprecipitation combined with Western blot analysis. RESULTS: Low-level expressions of long-form leptin receptor mRNA could be measured in mature adipocytes. The adipocytes expressed small amounts of SOCS-3 mRNA and protein witbout leptin treatment, and no significant changes were observed at 0.5 Snmol/L of leptin. However, tbe expressions of SOCS-3 mRNA and protein increased significantly at the doses of 5-500 nmol/L of leptin in the consistent with treatment time. CONCLUSION: Leptin could have direct effects on adipocytes. High leptin concentration could induce the expressions of SOCS-3 mRNA and protein in mature adipocytes.

[Study on SOCS-3, PPARgamma and ACO mRNA expression of adipose tissues in obese rats with leptin resistance].

OBJECTIVE: To study the difference of socs-3, PPARgamma and ACO mRNA expression in obese rats with leptin resistance induced by high-fat diet. METHODS: 6 Wistar male rats were fed with basic diet as control, and 24 rats were fed with high-fat diet as high-fat diet group. At the end of the 8th week, 8 obese rats were selected by body weight from high-fat diet group as obese group. Serum leptin, socs-3, PPARgamma and ACO mRNA were detected. RESULTS: In compare with control group, body weight, serum leptin, socs-3 and PPARgamma mRNA expressions of epididymal adipose tissues were significantly increased in obese group, while ACO mRNA were markedly decreased. CONCLUSION: Obese rats induced by high-fat diet showed the suppression of leptin signaling transduction pathway, decreases in fatty acid oxidation and increases in fat synthesis.

Resveratrol inhibits the IL-1ß-induced expression of MMP-13 and IL-6 in human articular chondrocytes via TLR4/MyD88-dependent and -independent signaling cascades.

The natural polyphenolic compound, resveratrol, has been shown to exhibit anti-osteoarthritic activity. Therefore it is hypothesized that resveratrol may serve as a nutritional supplement to counteract osteoarthritis (OA). However, the mechanisms responsible for these anti-osteoarthritic effects have not yet been fully elucidated. The aim of this study was to determine whether the biological effects of resveratrol against interleukin (IL)-1ß­induced inflammation in human articular chondrocytes involved both Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)-dependent and -independent signaling pathways. Human articular chondrocytes derived from patients with OA were stimulated with IL-1ß, and then co-treated with resveratrol. Cell viability was subsequently evaluated by MTS assays, and the concentrations of matrix metalloproteinase (MMP)-13 and the pro-inflammatory factor, IL-6, were detected in culture supernatants using ELISA. The mRNA and protein levels of downstream mediators of TLR4/MyD88-dependent and -independent signaling pathways were also assayed by RT-qPCR and western blot analysis, respectively. Our results revealed that resveratrol prevented the IL-1ß-induced reduction in cell viability. Furthermore, stimulation of the chondrocytes with IL-1ß resulted in a significant upregulation of TLR4 and downstream targets of both TLR4/MyD88-dependent and -independent signaling pathways that are associated with the synthesis of MMP-13 and IL-6. Correspondingly, IL-1ß-induced catabolic and inflammatory responses were effectively reversed by resveratrol. Taken together, these data suggest that resveratrol exerted protective effects against matrix degradation and inflammation in OA-affected chondrocytes by inhibiting both TLR4/MyD88-dependent and -independent signaling pathways. Thus, resveratrol represents a potential treatment for OA and warrants further investigation.

In utero exposure to di-(2-ethylhexyl) phthalate induces metabolic disorder and increases fat accumulation in visceral depots of C57BL/6J mice offspring.

Excessive visceral fat accumulation is associated with metabolic disorders. Di-(2-ethylhexyl) phthalate (DEHP), a candidate environmental obesogen, affects lipid metabolism and adipogenesis. Perinatal exposure to DEHP may be associated with metabolic disorders of dams and offspring. The aim of the present study was to explore the effects of exposure of pregnant dams to DEHP on the metabolism and fat distribution of their offspring, and to determine the mechanisms for these effects. Pregnant C57BL/6J mice were administered DEHP via gavage (0.05 or 500 mg/kg/day) from gestational days 1-19. Pups were sacrificed at nine weeks of age. Serum leptin, insulin, lipid and fasting glucose levels, and the weights of the inguinal (subcutaneous) and gonadal (visceral) fat pads were determined. mRNA expression levels of two developmental genes, T-box 15 (Tbx15) and glypican 4 (Gpc4) were detected in fat tissues. A 100% abortion rate was exhibited in 500 mg/kg DEHP-treated dams, whereas exposure to 0.05 mg/kg DEHP did not affect reproductive outcomes. Pups from the 0.05 mg/kg exposure group were used for subsequent experimentation. Serum leptin, insulin, lipid and fasting glucose concentrations in these pups were significantly higher than those of control pups (P<0.05). Although no significant change in body weight was detected, the visceral fat weights of DEHP-exposed pups were significantly higher than those of control pups (P<0.05). Compared with controls, mRNA expression levels of Tbx15 in subcutaneous fat and Gpc4 in visceral fat were significantly increased among DEHP-exposed pups (P<0.01). The present results suggest that in utero exposure to an environmentally safe dose of DEHP may lead to excessive visceral fat accumulation and metabolic disorders in offspring and that aberrant expression of Tbx15 and Gpc4 may have an important role in these effects.

Oral Resveratrol Prevents Osteoarthritis Progression in C57BL/6J Mice Fed a High-Fat Diet.

The effects of resveratrol on osteoarthritis (OA) pathogenesis have been demonstrated in vitro and in animal models employing intra-articular injections. However, the potential for oral resveratrol supplements to mediate protective effects on OA have not been examined. Therefore, the aim of the present study was to investigate the potential anti-OA effects of oral resveratrol on mice fed a high-fat diet (HFD). C57BL/6J male mice were fed either a standard diet or a HFD, and a subset of the latter also received varying doses of resveratrol. Twelve weeks later, all of the animals were sacrificed and knee joints were evaluated with histological, immunohistochemical, and TUNEL analyses. Mice that received a HFD had significantly greater body weights than the control mice and also exhibited features consistent with knee OA. The mice that received a HFD in combination with low, intermediate, or high doses of resveratrol were only slightly heavier than the control mice at the end of 12 weeks. Quantitative histological assessments indicated that resveratrol treatment partly recovered joint structure in the mice that received a HFD, while high doses of resveratrol prevented the degradation of type II collagen into C-telopeptide of type II collagen (CTX-II) and retained type II collagen expression in cartilage. Furthermore, TUNEL analyses revealed a reduction in chondrocyte apoptosis in the resveratrol-treated mice compared with the HFD mice. Thus, oral resveratrol appears to exert anti-OA effects in a mouse model of HFD-induced OA, thereby highlighting the potential preventive and therapeutic value of administering resveratrol for obesity-associated OA.

Glypican 4 may be involved in the adipose tissue redistribution in high-fat feeding C57BL/6J mice with peroxisome proliferators-activated receptor γ agonist rosiglitazone treatment.

Fat distribution affects the risk of developing obesity-related chronic diseases. Glypican 4 (Gpc4) may be involved in the regulation of obesity and body fat distribution. The aim of the study was to explore whether Gpc4 affects fat accumulation and the possible mechanism. C57BL/6J mice were fed with a high-fat diet for eight weeks and treated with a peroxisome proliferators-activated receptor γ (PPARγ) agonist, rosiglitazone, for another four weeks. The weight of inguinal and epididymal fat pads was determined. The Gpc4 mRNA and protein expression and two probable regulators of the Gpc4 gene, specificity protein 1 (Sp1) and Sp3 mRNA, were also measured. Mice treated with rosiglitazone showed a significant increase in subcutaneous fat weight compared with the untreated mice. The expression of Gpc4 mRNA and protein was significantly higher in visceral than in subcutaneous fat in all the groups. Compared with untreated mice the expression of Gpc4 and Sp3 mRNA in subcutaneous fat and the expression of Sp1 and Sp3 mRNA in visceral fat in mice treated with rosiglitazone increased significantly. The Sp3/Sp1 ratio was consistent with the expression of Gpc4 mRNA and protein in subcutaneous and visceral fat. The present study indicated that Gpc4 may play an important role in fat distribution, and this effect is perhaps regulated by the ratio of Sp3/Sp1 in the subcutaneous and visceral fat tissues.

Inhibition of SOCS-3 in adipocytes of rats with diet-induced obesity increases leptin-mediated fatty acid oxidation.

Rats with diet-induced obesity (DIO) usually experience hyperleptinemia. Thus, leptin produced by adipocytes does not deplete adipocyte fat, which implying a leptin resistance in adipocytes during overnutrition. Here, we induced hyperleptinemia in rats by feeding them a diet consisting of 45% fat. In epididymal adipose tissues, the mRNA and protein levels of a putative leptin resistant factor, suppressor of cytokine signaling 3 (SOCS-3), were increased. The mRNA levels of SOCS-3 in adipocytes differentiated from adipose-derived stromal cells (ADSCs) were higher in DIO rats than in rats on a 10% fat diet. Using SOCS-3 short hairpin RNA lentivirus interference, we found decreased expression of acetyl-CoA carboxylase mRNA (a marker of de novo lipogenesis) and increased expression of acetyl-CoA oxidase mRNA (a marker of fat oxidation) in SOCS-3-knockdown adipocytes after incubation with 50 nM leptin for 6 h. We conclude that the SOCS-3 knockdown may have increased the leptin-mediated in situ fatty acid oxidation in the DIO adipocytes, and therefore, SOCS-3 might be an excellent target for therapeutic intervention for obesity.