| Objective : Osteoarthritis(OA)is one of the most common joint diseases and characterized by articular cartilage degeneration and secondary bone hyperplasia,eventually leading to joint dysfunction and disability.In recent years,the sharp increase of obesity in the global scope has become one of the most important reasons for the increase of OA,while the chronic low-grade inflammatory state caused by obesity is closely related to the occurrence and development of OA.Toll-like receptor 4(TLR4)is a member of innate immune receptors family,TLR4-mediated signaling pathways play crucial roles in the development of obesity-related OA.Inhibiting TLR4 signaling pathways can play the anti-obesity-related OA effect.Resveratrol(RES),a natural polyphenolic compound,perform anti-apoptotic,anti-inflammatory and anti-oxidative effects.Previous studies have shown that the anti-obesity-related OA effect of resveratrol is related to the inhibition of the TLR4/NF-kB signaling pathway.However,the exact mechanism of its regulation is not very clear.Endogenous Forkhead box O1(FoxO1)is necessary to maintain normal TLR4 level.In macrophages,FoxO1 up-regulates the expression of TLR4 and potentiates TLR4 signaling by binding to multiple enhancer-like elements,while the activation of the TLR4 induces protein kinase B(Akt)phosphorylation which inactivates FoxO1,establishing a self-limiting mechanism of inflammation,suggesting that these mechanisms may also exist in OA chondrocytes in obesity,and that the obesity-related OA inflammatory state may be improved by enhancing this negative feedback effect.Therefore,this study explored the role of TLR4/Akt/FoxO1 axis in obesity-related OA by “in vivo” and “in vitro” experiments,and whether resveratrol down-regulates the expression of TLR4 by enhancing the self-limitation of TLR4/Akt/FoxO1 and subsequent inactivation of NF-k B,eventually improving inflammation state of obesity-related OA chondrocytes,which will provid a new target for the nutritional prevention of obesity-related OA.Methods:1.Animal grouping and treatment: 60 male C57BL/6 mice(18-22 g,7 weeks old)according to their weight were randomly divided into control group(CON),high-fat diet group(HFD),and resveratrol treatment group(HFD+RES).Mice in the control group were given common diet.Mice in High-fat diet group(HFD)and resveratroltreatment group(HFD+RES)were fed high-fat diet.Mice in the CON group and HFD group were gavaged with 0.5% carboxymethyl cellulose sodium(CMC),while mice in the HFD+RES group were administered 0.5% CMC and resveratrol(45mg/kg/d)suspension by gavage.All animals were fed ad libitum for 22 weeks.At the end of the experiment,all mice were killed.The knee joints were taken for subsequent examination.2.Pathological changes and evaluation of the knee joint:(1)Hematoxylin-Eosin(HE),Safranin O and Safranin O/Fast Green staining were performed.(2)Mankin scores were used for assessment of cartilage damage.(3)The expression of collagen ? in articular cartilage: Collagen ? expression in the knee joint cartilage was detected by immunohistochemical analysis.(4)MMP13 expression of gene and protein in the knee joint cartilage: Real-time RCR and Western blot were used for the detection.3.Expression of TLR4/Akt/FoxO1 axis-related genes and proteins in articular cartilage of mice:(1)TLR4 mRNA expression of articular cartilage was detected by Real-time RCR.(2)The protein expression levels of TLR4,MyD88,TRIF,IL-1b,p-NF-?B p65,PI3 K,p-PI3 K,Akt,p-Akt,FoxO1 and p-FoxO1 in the knee cartilage were examined by the Western blot.(3)Immunofluorescence staining of articular cartilage: The expression of p-Akt in articular cartilage and the nuclear localization of FoxO1 were detected by immunofluorescence method.4.Cell grouping and treatment:(1)SW1353 cell line was used and divided into(1)CON group(2‰ DMSO),IL-1b group(10 ng/ml)and IL-1b +RES group(IL-1 b :10 ng/ml,RES: 50 ?mol/L).Expression levels of TLR4,MyD88,TRIF,p-NF-?B p65,PI3 K,PI3K,Akt,p-Akt,FoxO1 and p-FoxO1 were detected by Western blot.(2)CON group(2‰ DMSO),IL-1b group(10 ng/ml)or RES group(RES:50 ?mol/L),the cells were treated with IL-1?(10 ng/ml)or RES(50 ?mol/L)for different time.Then,the expression levels of PI3 K,p-PI3 K,Akt,p-Akt,FoxO1 and p-FoxO1 were examined by Western blot,which were used to determine the follow-up cell treatment time.5.TLR4 gene silencing,TLR4 inhibitor CLI-095,PI3 K inhibitor LY294002 and FoxO1 gene silencing were used to treat cells,respectively,and the related proteins expression levels were detected:(1)TLR4 or FoxO1 gene silencing: The transfection efficiency was examined by the fluorescence method,and the silencing efficiency was detected by Western blot.(2)TLR4 gene silencing: The expression levels of TLR4,MyD88,TRIF,p-NF-?B p65,PI3 K,p-PI3 K,Akt,p-Akt,FoxO1 and p-FoxO1 were detected by Western blot.(3)TLR4 inhibitor,PI3 K inhibitor and FoxO1 gene silencing: Western blot was used to detect the expression levels of TLR4,PI3 K,p-PI3 K,Akt,p-Akt,FoxO1 and p-FoxO1.6.The level of IL-6 in the culture supernatants of cell:The level of IL-6 was detected by ELISA.7.SPSS 13.0 software was used for statistical analysis.The data obtained in the experiment were expressed as mean ± standard deviation((?)±s),and the differences between groups were analyzed by one-way ANOVA followed by Fisher's least significant difference(LSD)test for multiple comparisons.For all experiments,P < 0.05 was considered statistically significant.Results: 1.During the whole experiment,all groups of mice gained weight,and the body weight and body fat percentage in HFD and HFD+RES groups at week 22 were significantly higher than those in the CON group(P < 0.01),while there was no significant difference between HFD and HFD+RES groups.2.The pathological examination of mice knee joint cartilage in CON group showed that the cartilage surface was flat,arrangement of cartilage cells was orderly,and tidal line was clear and complete.The pathological examination in HFD group showed that the cartilage surface was slightly damaged,the number of chondrocytes locally was reduced,the color of the tide line became lighter,and occasionally the tide line was lost.In contrast,resveratrol treatment inhibited these histological changes of the HFD group.Mankin score in HFD group was significantly higher than that of CON group(P < 0.01).Compared with the HFD group,the Mankin score in HFD+RES group was decreased significantly(P < 0.01).3.Collagen ? expression in articular cartilage showed that: Compared with the CON group,Collagen ? expression in HFD group was decreased significantly.Compared with the HFD group,Collagen ? expression was increased in HFD+RES group;Average optical density analysis showed that: Compared with the CON group,the average optical density values were decreased significantly in HFD group and HFD+RES group(P <0.01);Compared with the HFD group,the average optical density value was increased significantly in HFD+RES group(P < 0.01).4.Real-time RCR and Western blot analysis showed a significant increase of MMP13 mRNA and protein in the HFD group and HFD+RES group as compared with the CON group(P < 0.01,P < 0.05),while a reduction of MMP13 mRNA and protein expression was observed in resveratrol-treated group as compared with the HFD group(P < 0.01).Compared with the CON group,theexpression levels of TLR4 mRNA and protein,MyD88,TRIF,p-NF-?B p65 and IL-1bprotein were significantly up-regulated in articular cartilage of HFD group and HFD+RES group(P <0.01,P <0.05),whereas the expression levels of TLR4 mRNA and these proteins in the resveratrol-treated group were down-regulated compared with the HFD group(P < 0.05,P < 0.01).5.Compared with the CON group,the phosphorylation levels of PI3 K,Akt and FoxO1 were significantly higher in HFD group and HFD+RES group(P < 0.05,P < 0.01),and even higher in HFD+RES group as compared with the HFD group(P < 0.05);Immunofluorescence and fluorescence intensity analysis of joint tissues revealed that the levels of red fluorescence intensity of p-Akt in HFD group and HFD+RES group were higher than that of the CON group(P < 0.01),and additional resveratrol treatment further increased its intensity as compared with the HFD group(P <0.05).Immunofluorescence of FoxO1 in knee joints showed that red fluorescence of FoxO1 in HFD group and HFD+RES group expressed in cytoplasm.6.IL-1b treatment activated TLR4,MyD88,TRIF and p-NF-?B p65 in SW1353 cells(P < 0.01),while resveratrol treatment significantly down-regulated the levels of these proteins(P < 0.05,P < 0.01).IL-1b increased the expresstion levels of p-PI3 K,p-Akt and p-FoxO1 in SW1353 cells(P < 0.01),and resveratrol treatment further up-regulated the expresstion levels of p-PI3 K,p-Akt and p-FoxO1(P < 0.01).IL-6 level in the culture supernatants was obviously up-regulated in IL-1b-induced SW1353 cells(P < 0.01),while a marked reduction of IL-6 level was observed in the addition of resveratrol(P < 0.01).7.The treatment of IL-1 b(10 ng/ml)or resveratrol(50?mol/L)at different time had no significant effect on the levels of total protein of PI3 K,Akt and FoxO1 in SW1353 cells,while the expression levels of p-PI3 K,p-Akt and p-FoxO1 were increased,and the peak levels appeared in 30 min(P < 0.01).8.TLR4-knockdown significantly down-regulated the expression levels of MyD88,TRIF and NF-kB in IL-1b-induced cells or IL-1? and resveratrol treated cells(P < 0.05,P < 0.01).9.The expression levels of p-PI3 K,p-Akt and p-FoxO1 protein were significantly decreased by CLI-095 treatment in IL-1 b-induced cells or IL-1? and resveratrol treated cells(P < 0.01).CLI-095down-regulated the level of IL-6 in the culture supernatants of IL-1b-induced cells(P <0.01),and resveratrol treatment further reduced the level of IL-6(P < 0.01).10.TLR4-specific siRNA significantly attenuated PI3 K,Akt and FoxO1 phosphorylation inSW1353 cells treated with IL-1 b or cells cultured in the presence of IL-1? and resveratrol(P < 0.01).Morever,TLR4-knockdown decreased IL-6 production in the IL-1b-induced cells(P < 0.01),and additional resveratrol further reduced IL-6 level(P <0.01).11.After the PI3K/Akt pathway was inhibited,the expression levels of p-PI3 K,p-Akt and p-FoxO1 were attenuated in IL-1?-induced cells or IL-1? and resveratrol treated cells(P < 0.01),while TLR4 protein expression was significantly increased(P <0.01).In addition,compared cells exposed to IL-1? or IL-1? and resveratrol,LY294002 treatment led to a marked increase of IL-6 level(P < 0.01).12.FoxO1-knockdown significantly attenuated p-PI3 K,p-Akt and TLR4 protein expression in IL-1?-treated cells or in IL-1?-induced cells in the presence of resveratrol(P < 0.01).Consistently,IL-6 level in the culture supernatants of IL-1b-induced SW1353 cell was also reduced by the FoxO1-specific siRNA(P < 0.01),and the addition of resveratrol further decreased IL-6 level(P < 0.01).Conclusion: 1.High-fat diet feeding C57BL/6 mice could cause obesity-related OA,and oral administration of resveratrol could inhibit the occurrence of obesity-related OA in mice induced by high-fat diet.2.The anti-obesity-related OA effect of resveratrol may be related to the inhibition of TLR4/NF-?B signaling pathways including the MyD88-dependent and-independent signaling pathway,activation of the PI3K/Akt signaling pathway and inactivation of FoxO1.3.TLR4/Akt/FoxO1 inflammatory self-limiting mechanism may exsit in obese-related OA chondrocytes.Resveratrol may exert anti-OA effect by enhancing this self-limiting effect: Resveratrol inactivates FoxO1 by activating the PI3K/Akt pathway and subsequent inactivation of FoxO1,and then downregulates TLR4 expression following inactivating TLR4/NF-?B signaling pathway,thereby exerts anti-OA effect. |
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