Study On The Role And Mechanism Of Leptin Signaling In The Pathogenesis Of Obesity-related OA By Regulating TLR4/TLR2

Objective: Obesity is considered to be a risk factor for osteoarthritis(OA),and OA caused by obesity has attracted more and more attention.Obesity-related OA is a kind of metabolic OA.In addition to the increased joint load,the high prevalence of OA in obese people is also significantly related to excessive adipokines in the body,among which leptin is one of the most relevant factors secreted by adipose tissue.However,is leptin a necessary condition in the pathogenesis of obesity-related OA? Or is it just a contributing factor to the progress of OA? What mechanism does leptin promote/promote OA? The solution of these problems is of vital importance to reveal the mechanism of obesity-related OA.Toll-like receptors(TLR)are an important class of protein molecules involved in non-specific immunity(innate immunity).Among them,TLR2 and TLR4 are closely related to maintaining the homeostasis of articular cartilage cells.TLR2 activation leads to the activation of downstream transcription factors,thereby regulating the expression of survival genes or pro-inflammatory cytokines and chemokines.TLR4 is the Toll-like receptor that causes obesity-related inflammation in the innate immune receptor family,and is considered to be the Toll-like receptor that causes obesity-related inflammation.We speculate that leptin may play a key role in promoting/promoting obesity-related OA by regulating the TLR4/TLR2 signaling pathway.Suppressor of cytokine signaling(SOCS)3 is a negative feedback inhibitor of leptin signaling.During the occurrence and development of obesity-related OA,SOCS3 may be a key factor for leptin signaling to regulate the TLR4/TLR2 signaling pathway..Therefore,this study used high-fat diet to feed SD rats to induce obesity-related OA and conduct in vivo and in vitro experiments such as primary chondrocyte culture in rats to explore whether leptin plays a role as an initiating factor in the occurrence of obesity-related OA.The role and possible mechanism of action provide new ideas for finding intervention targets and nutritional prevention and treatment measures for obesity-related OA.Methods: 1.Grouping and handling of experimental animals: We got 6 weeks old male SD rats(n=72).After one week of adaptive feeding,rats were randomly divided into the CON group(n = 36,fed a control diet with 10% of the calories from fat)or the HFD group(n = 36,fed a high fat diet with 60% of the calories from fat).At the end of W5,W15 and W27,12 rats were randomly taken out of the two groups,fasted overnight.The blood was taken from the abdominal aorta after ether anesthesia,and sacrificed with the spinal dislocation.The taken-out serum was aliquoted and stored in the refrigerator at-80?,and the serum leptin level was tested by ELISA.Five rats in each group were taken from the right lower limb knee joint to extract synovial fluid,and the left lower limb knee joint was stained with safranine O,HE stain,safranine O-fast green stain and MMP-13 immunohistochemical staining,and the joint tissues were stained.Physical examination and double-blind method for Mankin score.The joints of 7 rats in each group were stripped clean,cartilage RNA was extracted from one joint,and the m RNA levels of leptin,OB-Rb,CD14,TLR2 and TLR4 were determined by q RT-PCR method;the cartilage protein was extracted from the other joint joints,and western blotting method was used to determine the protein expression levels of JAK2(p-JAK2),STAT3(p-STAT3),CD14,TLR2,TLR4,SOCS3 and MMP-13.2.The effect of AG490 inhibitor on the expression of chondrocyte related proteins: Rat primary chondrocytes were used to treat the cells with leptin alone and the combined action of leptin and IL-1?respectively.The specific groups are as follows:(1)Treatment with leptin alone : CON group(0.1% DMSO),leptin group(200 ng/m L leptin+0.1% DMSO),AG490 group(10?M AG490+0.1% DMSO)and leptin+AG490 group(200 ng/m L leptin+10 ?M AG490+ 0.1% DMSO);(2)Leptin combined with IL-1? treatment: CON group,IL-1?alone group(10ng/ml),leptin+IL-1? group(200 ng/m L leptin+10ng/ml IL-1?),AG490 alone Group(10 ?M),IL-1?+AG490 group(10ng/ml IL-1?+10 ?M AG490)and leptin+IL-1?+AG490 group(200 ng/m L leptin+10ng/ml IL-1?+10 ?M AG490).After pretreatment with 10?M AG490 for 2 hours,cells in the intervention group were added with leptin at a final concentration of 200ng/ml and/or IL-1? at 10ng/ml,and cultured for2 hours or 24 hours.Western blotting was used to detect the expression of related proteins.3.Chondrocyte protein expression after silencing SOCS3: Use lentivirus carrying sh SOCS3 gene titer of 30 MOI and negative control lentivirus to infect chondrocytes,and add 200ng/ml leptin and/or 10ng/ml IL-1? at the same time.After culturing for 2 hours or 24 hours,western blotting was used to detect the expression of related proteins.Results:1.From the W3,the body weight of the HFD group was significantly higher than that of the CON group,and the body weight difference between the HFD group and the CON group increased over time(P<0.05).The body fat ratio of rats in the HFD group at W5,W15 and W27 were significantly higher than that of the CON group(P<0.0012).2.The serum leptin level of rats in the HFD group was significantly higher than that in the CON group at W5,W15 and W27(P<0.001).In addition,the leptin level in the HFD group increased significantly over time,and the serum leptin level in the HFD group at W27 was significantly higher than that at W5 and W15(P<0.001).However,there was no difference in leptin levels between the CON groups at different time points(P>0.05);at W5,there was no difference in the leptin levels in the synovial fluid between the two groups(P>0.05).At W15 weeks and W27,the leptin level in the joint fluid of the HFD group was significantly higher than that of the CON group,and the difference was statistically significant(P<0.01,P<0.001).Compared with W5,the level of leptin in the joint fluid of the HFD group at W15 and W27 was significantly increased(P<0.01,P<0.001).However,there was no difference in the level of leptin in the joint fluid of the CON group at different time points(P>0.05).At W5,there was no correlation between serum and joint fluid leptin in HFD group and CON group(P=0.2029,P=0.1222).At W15 and W27,there was a positive correlation between serum and synovial leptin in the HFD group(R2=0.8019,P=0.2029;R2=0.9613,P=0.0033).There was no correlation between serum and synovial leptin in CON group at different time points(P>0.05).At W15 and W27,the level of IL-1? in serum and synovial fluid of the HFD group was significantly higher than that of the CON group(P<0.01,P<0.001).In addition,the concentration of IL-1? in the synovial fluid of the HFD group gradually increased over time.3.Compared with the CON group,the HFD group had no obvious OA characteristics at 5and 15 weeks.At week 27,OA-like lesions were observed in the knee joints of rats in the HFD group.In addition,the Mankin score of HFD rats gradually increased over time.The Mankin score of the HFD group at W27 was significantly higher than the score at week 5(P<0.01),which was consistent with the pathological staining results.In addition,there was no difference in the expression of MMP-13 between the HFD group and the CON group at 5 and 15 weeks(P>0.05).At W27,the expression level of MMP-13 in the HFD group was significantly higher than that in the CON group(P<0.001).At W5,there was no difference in the expression of COMP protein between the HFD group and the CON group(P>0.05).At W15 and W27,the expression of COMP in the HFD group was significantly higher than that in the CON group(P<0.05,P<0.01).4.The expressions of leptin and leptin receptor m RNA in the knee cartilage of rats in the HFD group increased gradually with the intervention time(P<0.05).Compared at the same time point,HFD was at W5,W15 and W27.The expressions of leptin and leptin receptor m RNA in the knee cartilage of rats in the group were higher than those in the CON group(P<0.05,P<0.01).Compared with the CON group,the JAK2-STAT3 signaling pathway in the knee cartilage of the HFD group was not activated at 5 weeks(P>0.05).At W15 and W27,HFD significantly increased the expression of P-JAK2 and P-STAT3(P<0.05,P<0.01).In addition,compared with the CON group,the expression of the negative feedback regulator SOCS3 of the leptin signaling pathway in the HFD group was also significantly increased at W15 and W27(P<0.05,P<0.01).CD14 and TLR2 in the HFD group And TLR4 m RNA and protein expression were significantly higher than the CON group(P<0.05,P<0.01).5.In chondrocytes,compared with the blank CON group,leptin treatment alone can activate the JAK2-STAT3 signaling pathway(P<0.05).However,when AG490 was added,the activation of JAK2-STAT3 signaling pathway by leptin was inhibited(P<0.05,P<0.01).Leptin can up-regulate the expression of CD14,TLR2,TLR4,MMP-13 and SOCS3 in primary rat chondrocytes(P<0.01,P<0.001).However,AG490 can inhibit the up-regulation of these proteins by leptin(P<0.05,P<0.001).Compared with IL-1? alone treatment,leptin combined with IL-1? treatment of rat primary chondrocytes has a more obvious activation effect on JAK2-STAT3 signaling pathway.After adding AG490,it has a significant inhibitory effect on the activation of JAK2-STAT3 signaling pathway in rat primary chondrocytes treated with IL-1? alone and with leptin combined with IL-1?(P<0.01,P<0.001).Silencing SOCS3 enhanced the activation of leptin on the JAK2-STAT3 signaling pathway(P<0.01,P<0.001).Compared with the cells in the CON and NC groups,the interference of SOCS3 with lentivirus can significantly enhance the up-regulation of IL-1? and leptin combined with IL-1? treatment on the JAK2-STAT3 signaling pathway and downstream related proteins in rat primary chondrocytes(P<0.01,P<0.001).Conclusions:1.High-fat diet can induce obesity-related OA.The increase in serum leptin precedes the pathological changes of joint leptin and OA,suggesting that the increase in serum leptin level may be the initiating factor for obesity-related OA induced by high-fat diet.2.In high-fat diet-induced obesity-related OA,leptin can activate the JAK2-STAT3 signaling pathway,and regulate the expression of CD14/TLR4 through the JAK2-STAT3 signaling pathway to participate in the occurrence and development of OA,while the increased SOCS3 expression does not significantly inhibit leptin signaling,suggesting that there is no obvious leptin resistance in articular cartilage.3.The in vitro results were consistent with in vivo experiments,which indicated that leptin at least partially regulated TLR4 through the JAK2-STAT3 pathway,and silencing SOCS3 can enhance the sensitivity of chondrocytes to leptin.