The Study Of The Alleviate Effects And Mechanisms Of β-Hydroxybutyrate On Osteoarthritis

Background:Osteoarthritis(OA)is a common age-related disease(ARD)and a major cause of disability in elderly.Aging is one of the most important risk factors for OA,chondrocyte senescence is the main cause of articular cartilage dysfunction.β-Hydroxybutyrate(BHB)is a natural fatty acid metabolite which participates in a variety of signaling pathways to plays an anti-aging and anti-inflammatory role.Although the critical role of senescent cells and their pro-inflammatory properties in the pathological mechanisms of OA have been recognized,intervention against these adverse effects still faces huge challenges.At present,in the process of prevention and treatment of OA,how to effectively intervene senescent cells still faces great challenges.whether BHB can alleviate the progression of OA by improving chondrocyte senescence has not been reported.Purpose:To explore the effect of BHB on the aging phenotype and function of chondrocytes,and to elucidate the molecular mechanism of its improvement of osteoarthritis progression.Methods:In order to study the effect of BHB on the aging phenotype of chondrocytes,the most suitable concentration of BHB was obtained for the intervention of OA chondrocytes(CCK8 was used to detect the activity of chondrocytes,PCR and WB were used to detect the function of chondrocytes).To determine the effect of BHB on the senescence phenotype and function of chondrocytes,senescence-positive cells were detected by SA-β-gal staining.Senescence-related paracrine markers(IL1,IL6,MMP1,MMP13)and senescence markers(p16,p21)were detected by PCR.Western blot was used to detect the function of chondrocytes(Col Ⅰ,Col Ⅱ,Col Ⅹ,and ACAN),and Ed U was used to detect the proliferation of chondrocytes.To study the effect of BHB on OA in rats,the Hulth(anterior and posterior cruciate ligament and medial collateral ligament were cut and the medial meniscus was removed)OA rat model was constructed,and PBS or BHB was injected into the joint.Safranin fast green staining and immunohistochemical staining(MMP13,Col Ⅱ,p16,p21)and Western blotting(Col Ⅰ,Col Ⅱ,Col Ⅹ,ACAN,MMP13,p16,p21)were used to detect senesence-related indicators and chondrocyte function.El ISA(PINP,CTx)was used to detect the effect of BHB on bone metabolism.To investigate the molecular mechanism by which BHB improves OA,human primary chondrocytes from 3 knee arthroplasty patients were cultured and treated with BHB(8m M)for 3 days.After transcriptome analysis,the most significantly affected gene(PTEN)was analyzed and obtained.In order to determine the expression of PTEN in OA cartilage and senescent chondrocytes,the expression of PTEN was detected by immunohistochemistry,cell immunofluorescence staining,PCR and WB.In order to further clarify the role of PTEN in the improvement of OA by BHB,human chondrocytes were divided into 4 groups and treated with si-PTEN and BHB for 3 days or without intervention.The expression of PTEN in chondrocytes was detected by PCR and WB.The senescence-associated secretory phenotype(SASP)(IL1,IL6,MMP1,and MMP13)senescence markers(p16,p21)were detected by PCR,and Col Ⅱ,Col Ⅹ,and MMP13 were detected by WB.Western blot was used to detect the downstream products of PTEN(Akt,p-Akt).In order to further explore the regulatory mechanism of BHB on PTEN,RNA immunoprecipitation(RIP)assay was used to verify the effect of BHB on the binding of hn RNP A1 to PTEN-mrna.The expression of hn RNP A1 and PTEN was detected by PCR and WB.SASP(IL1,IL6,MMP1,and MMP13)and senescence markers(p16 and p21)were detected by PCR.Cartilage function(Col Ⅱ and Col Ⅹ),Akt and p-Akt were detected by WB.Result:SA-β-gal staining,PCR and WB results showed that BHB at the concentration of 8 m M could promote the growth and anabolism of chondrocytes(p < 0.05),and the inhibition effect on catabolism was the most obvious(p < 0.05).BHB treatment reduced the positive rate of senescent cells staining(p < 0.05)and the expression of senescence-related paracrine markers(IL1,IL6,MMP1,MMP13)and senescence markers(p16,p21)(p < 0.05).Chondrocyte function(Col Ⅰ,Col Ⅱ,Col Ⅹ,ACAN)and proliferation ability were increased after BHB intervention(p < 0.05).Safranin fast green staining of rat knee joints showed that BHB intervention reduced cartilage wear(p < 0.05).The results of immunohistochemical staining and WB showed that BHB intervention reduced the expression of cellular senescence markers(p16 and p21)and catabolic factors(MMP13),Col Ⅰ(WB)and Col Ⅹ(WB)in cartilage(p <0.05),and increased the expression of anabolic factors(immunohistochemistry: Col Ⅱ;WB: ACAN)expression(p < 0.05).Elisa results showed that BHB intervention promoted bone formation(PINP)and inhibited bone resorption(CTx)(p < 0.05).Transcriptome data showed that 77 genes were up-regulated and 13 genes were down-regulated after BHB treatment.Among the functional categories most significantly affected by BHB,PTEN expression was upregulated most significantly,and PTEN protein was the most prominent node in protein interactions.The results of immunohistochemistry and cellular immunofluorescence showed that OA model significantly reduced the expression of PTEN in cartilage(p <0.05).However,BHB intervention significantly increased the expression of PTEN(p < 0.05).The results of PCR and WB showed that BHB intervention significantly promoted the expression of PTEN in human chondrocytes(p < 0.05).The results of β-galactosidase staining showed that the senescent cells increased significantly after PTEN knockdown,and BHB intervention could not alleviate the senescence of chondrocytes after PTEN knockdown(p > 0.05).PCR also showed that SASP(IL1,IL6,MMP1,MMP13)and senescence markers(p16,p21)were significantly increased after PTEN knockdown(p < 0.05).WB results showed that the expression of anabolic factor(Col Ⅱ)was significantly decreased,and the catabolic factor(MMP13)and cartilage hypertrophy(Col Ⅹ)were significantly increased,and the downstream p-Akt signal activation was increased after PTEN knockout(p < 0.05).The results of RIP showed that the binding of BHB to hn RNP A1 promoted the expression of PTEN in chondrocytes(p < 0.05),and BHB intervention did not affect the expression of hn RNP A1 in chondrocytes(p > 0.05),but the expression of PTEN was significantly reduced after knocking out hn RNP A1(p < 0.05).Moreover,the expression of SASP(IL1,IL6,MMP1,MMP13)and senescence markers(p16,p21,p < 0.05)increased significantly after knocking out hn RNP A1(p < 0.05),and BHB intervention could not alleviate the reduction of the above SASP and senescence markers(p > 0.05).WB showed that the expression of anabolic factor(Col Ⅱ)decreased significantly,and the expression of hypertrophy factor(Col Ⅹ)and p-Akt increased significantly after knocking down hn RNP A1,which could not be reversed by BHB intervention.Conclusions:β-hydroxybutyrate can alleviate the progression of OA by improving the senescence phenotype of chondrocytes,and the mechanism may be related to the promotion of PTEN expression by β-hydroxybutyrate through hn RNP A1.