High Fat Diet Promotes The Devolopment Of Osteoarthritis Through PERK-Mediated Endoplasmic Reticulum Stress Pathway

IntroductionOsteoarthritis(OA)is a chronic joint disease which eventually leads to loss of joint function.In recent years,the incidence of OA has been rising,and its high rate of disability has become a major public health problem at home and abroad,but there is no effective means to slow or stop its progression.Therefore,explore and clarify the pathogenesis and risk factors of OA is an urgent problem for fundamental and clinical research.The latest view is that OA is a metabolic disease,which is related to "metabolic disorder","inflammatory aging" and other metabolic factors.Clinical studies have confirmed that hypertriglyceridemia is closely related to the occurrence and development of OA.The increase of free fatty acid(FFAs)caused by hypertriglyceridemia can cause lipotoxicity to tissues and cells of the body.Lipotoxicity is related to the pathogenesis of many metabolic diseases and is also an important factor of OA,but its exact mechanism is still unclear.Apoptosis induced by endoplasmic reticulum stress(ERS)activation is related to the pathogenesis of many diseases.ERS mainly involves three pathways:IRE1 pathway,ATF6 pathway and PERK pathway.Basic research has confirmed that ERS is related to the pathogenesis of OA,but whether the lipotoxicity caused by high-fat diet causes chondrocyte apoptosis through ERS and its exact mechanism in the occurrence and development of MetSOA is still unclear.This study explored the effect of high-fat diet on OA,and the role and related mechanism of ERS in hyperlipidemia related OA through the combination of in vivo(establishment of hypertriglyceridemia OA model)and in vitro(palmitic acid treatment of articular chondrocytes),providing reference basis for clinical research of Mets-OA.Our hypothesis is that lipotoxicity promotes the occurrence and development of hypertriglyceridemia related OA by activating ERS and inducing chondrocyte apoptosis.MethodsModeling method:After being approved by the Animal Ethics Committee of Shandong Provincial Hospital,male SD rats aged 6 to 7 weeks were randomly divided into control group(normal diet),15%HFD group(customized diet)and 25%HFD group(customized diet).The diet of rats in the latter two groups contained different concentrations of lard,15%and 25%respectively,which could induce hypertriglyceridemia.We also established osteoarthritis models on three groups of rats through medial meniscus(DMM)surgery.In vitro,we treated rat primary articular chondrocytes with different concentrations of palmitic acid,which can effectively produce lipotoxicity on chondrocytes,thus causing different degrees of apoptosis.We used specific inhibitors to inhibit three ERS-related pathways,including IRE1 pathway,ATF6 pathway and PERK pathway,in vitro and in vivo to detect the speci fic mechanism.Knee joint degeneration/inflammatory recovery test:H&E staining and Safranine O(SO)staining.Expression of mRNA and protein expression levels of inflammatory factors:Western blotting was used to detect the expression of inflammatory factors.qRT-PCR was used to detect the expression of mRNA of inflammatory factors.Expression of protein expression levels of ERS marker:Western blotApoptosis test:Western blot,TUNEL staining and flow cytometryResultsHigh fat diet aggravates the damage of OA to articular cartilage:Compared with CON group,the OARSI score of knee joint in 15%HFD group was significantly higher(p<0.05);Compared with CON group and 15%HFD group,the OARSI score of knee joint in 25%HFD group was significantly higher(p<0.05).High fat diet promotes OA inflammatory response:Compared with the CON group,the protein and mRNA expression of OA-associated matrix metalloproteinases(MMP-3 and MMP13)in the 15%HFD group were significantly up-regulated(p<0.05),and the protein and mRNA expression of inflammatory suppressor protein(COL2A1)was significantly down-regulated(p<0.05).Compared with CON group and 15%HFD group,the expression of protein and mRNA of OA-related matrix metalloproteinases(MMP-3 and MMP-13)in 25%HFD group was significantly up-regulated(p<0.05),and the expression of protein and mRNA of inflammatory inhibitory protein(COL2A1)was significantly down-regulated(p<0.05).Lipotoxicity induced by high fat diet promotes apoptosis in rat cartilage tissue:In hypertriglyceride-related OA rats,compared with the CON group,the expression of proapoptotic factors(GADD153,p-JNK,Bax)in the 15%HFD group was up-regulated(p<0.05),and the expression of anti-apoptotic factor Bcl-2 was down-regulated(p<0.05);Compared with CON group and 15%HFD group,the expression of proapoptotic factors(GADD153,p-JNK,Bax)in 25%HFD group was significantly up-regulated(p<0.05),and the expression of anti-apoptotic factor Bcl-2 was significantly down-regulated(p<0.05).Lipotoxicity caused by high fat diet promotes the occurrence of ERS in rat cartilage tissue cells:In hypertriglyceride-related OA rats,compared with the CON group,the expression of ERS markers(BIP,p-IRE1,XBP1s,ATF6,p-PERK,p-elf2α,ATF4)in the 15%HFD group was up-regulated(p<0.05);Compared with CON group and 15%HFD group,the expression of ERS markers(BIP,p-IRE1,XBPIs,ATF6,p-PERK,p-elf2α,ATF4)in 25%HFD group was significantly up-regulated(p<0.05).Inhibition of the PERK pathway can maximize the damaging effect of a high-fat diet on cartilage tissue cells:After administration of ERS pathway inhibitor,Inhibition of PERK pathway showed the most significant down-regulation of chondrocyte apoptosis factors in 25%HFD DMM rats(P<0.05).Inhibition of PERK pathway can also reduce the inflammatory of articular cartilage in 25%HFD rats(P<0.05).The results of flow cytometry and TUNEL staining showed that inhibition of PERK pathway significantly reduced the apoptosis of chondrocytes induced by palmitic acid(P<0.05).Conclusions1.The severity of osteoarthritis in HFD rats increased with the accumulation of FFAs.2.Chondrocyte apoptosis induced by lipid toxicity activating ERS stress pathway plays an important role in hypertriglyceride-related OA.3.Inhibition of PERK pathway can protect chondrocyte apoptosis induced by lipotoxicity to the greatest extent,thus reducing the high triglyceride-related OA.