Investigate Mechanism Of Rongjin Niantong Fang Treatment Knee Osteoarthritis By LncRNA GAS5/miR-21 Regulating Extracellular Matrix Metabolism

ObjectiveTo explore the mechanism of Rongjin Niantong Fang(RJNTF)treatment knee osteoarthritis by LncRNA GAS5,miR-21 and its target gene TIMP-3 regulating the cartilage matrix metabolic factors MMP-3,MMP-9,MMP-13,ADAMTS-5,Collagen Ⅱ and Aggrecan.Methods1.Knee osteoarthritis model of 66 SPF-grade SD male rats aged 8 weeks was established by modifying Hulth method and identified by 7.0T MRI.Experimental rats were divided into blank group,model group,treatment group and the positive control group by random number table grouping method.The blank group and the model group were intragastrically administered with saline at 1m L/(100g·d),the treatment group was intragastrically administered with water extract of RJNTF at 0.45g/(100g·d),and control group was intragastrically administered with glucosamine hydrochloride capsules(GHC)at0.015g/(100g·d)for 12 weeks.Imageology changes of knee joint,morphological changes of cartilage were observed by 7.0T MRI,HE staining and Safranin-O staining.The expression of LncRNA GAS5,miR-21 in cartilage was detected by Real-time PCR.The expression of TIMP-3,MMP-3,MMP-9,MMP-13,ADAMTS-5,Collagen Ⅱ,Aggrecan m RNA and protein in cartilage was detected by Real-time PCR and Western blot.2.Chondrocytes were isolated and extracted by mechanical-type II collagenase digestion from the cartilage of 42 SPF-grade SD male rats aged 4 weeks.The phenotype of chondrocytes were identified by type II collagen immunohistochemical staining and toluidine blue staining,and culture system of rat chondrocytes was established in vitro.The second-generation chondrocytes were induced by IL-1β to establish the model of degenerative chondrocytes,which were identified by type II collagen immunohistochemical staining and selected for follow-up experiments.Degenerative chondrocytes were randomly divided into model group(IL-1β + blank serum),treatment group(IL-1β + RJNTF serum),and control group(IL-1β + GHC serum).After the intervention,the expression of LncRNA GAS5,miR-21 in chondrocytes was detected by Real-time PCR.The expression of TIMP-3,MMP-3,MMP-9,MMP-13,ADAMTS-5,Collagen Ⅱ,Aggrecan m RNA and protein in chondrocytes was detected by Real-time PCR and Western blot.3.The chondrocytes were infected with overexpression of LncRNA GAS5,miR-21 inhibitor and miR-21 mimics lentivirus.The infected chondrocytes were screened by puromycin and divided into blank group(blank serum)and treatment group(RJNTF serum).After intervention,the expression of LncRNA GAS5,miR-21 and the expression of TIMP-3,MMP-9,MMP-13,ADAMTS-5,Collagen Ⅱ,Aggrecan m RNA in infected chondrocytes was detected by Real-time PCR.Results1.In vivo,the knee joint MRI after 6 weeks showed that early and middle term KOA imaging changes containing the joint effusion and the swollen joint surrounding soft tissue.The knee joint MRI after 12 weeks showed that late term KOA imaging changes containing worn and sunken articular surface,obvious effusion,osteophyte formation and muscle atrophy in model group;the imaging changes of treatment group and control group were lighter than model group.The articular surface of the model group is covered with connective tissue hyperplasia and is indistinguishable;the part articular surfaces of the treatment group and the control group are still distinguishable on general view of the articular surface of the tibial plateau.According to the observation of cartilage microstructure,the four layers of cartilage were disordered and arranged irregularly,the tide line and adhesion line were irregular or disappeared,the chondrocytes proliferated and arranged disorderly,the subchondral bone proliferated,the collagen and proteoglycan were lost in large quantities in the model group;the four layers of cartilage were recognizable,the cartilage cells arranged regularly,and the subchondral bone structure was relatively complete,part of collagen and proteoglycan were lost in the treatment group and the control group.Compared with the blank group,the expression of LncRNA GAS5 were increased(P<0.05),miR-21 were decreased(P<0.05),MMP-3,MMP-9,MMP-13 and ADAMTS-5 m RNA and protein were increased(P<0.01 or P<0.05),TIMP-3,Collagen Ⅱ,Aggrecan m RNA and protein were decreased(P<0.01 or P<0.05)in cartilage of model group.Compared with the model group,the expression trends of these genes and proteins were changed in reverse in cartilage of treatment group and the control group.2.In vitro,immunocytochemical staining of Collagen Ⅱ and toluidine blue staining showed that the cytoplasm of the extracted cells was brownish yellow and blue-purple.CCK-8 results showed that the effective concentration of IL-1β was 10ng/ml and the time was24 h.The effective concentration of RJNTF serum was 10%,the effective concentration of GHC serum was 15% and the time was 48 h.Compared with the blank group,the expression of LncRNA GAS5 were decreased(P<0.05),miR-21 were increased(P<0.05),MMP-3,MMP-9,MMP-13 and ADAMTS-5 m RNA and protein were decreased(P<0.01 or P<0.05),TIMP-3,Collagen Ⅱ,Aggrecan m RNA and protein were increased(P<0.01 or P<0.05)in chondrocytes of treatment group and the control group.Compared with the model group,the expression trends of these genes and proteins were changed in reverse in chondrocytes of treatment group and the control group.3.In vitro of infection experiment,the effective MOI of chondrocytes infected by LncRNA GAS5,miR-21 inhibitor and mimics lentivirus was 100,and the infection enhancing reagent was Hitransg P.(1)Compared with NC group in chondrocytes overexpressed by LncRNA GAS5 lentivirus,the expression of LncRNA GAS5 was significantly increased(P<0.01).Compared with blank group,the expression of LncRNA GAS5 decreased(P<0.05);miR-21 increased(P<0.05);TIMP-3,collagen Ⅱ and aggrecan m RNA increased(P<0.05);MMP-9,MMP-13 and ADAMTS-5 m RNA decreased(P<0.05).(2)Compared with NC group in chondrocytes inhibited by miR-21 lentivirus,the expression of miR-21 decreased significantly(P<0.01).Compared with blank group,the expression of miR-21 increased(P>0.05);TIMP-3,collagen Ⅱ and aggrecan m RNA increased(P>0.05);LncRNA GAS5,MMP-13 and ADAMTS-5 decreased(P>0.05);the expression of MMP-9 m RNA decreased(P<0.05).(3)Compared with NC group in chondrocytes overexpressed by miR-21 lentivirus,the expression of miR-21 increased significantly(P<0.01).Compared with blank group,the expression of Aggrecan m RNA increased(P<0.05),MMP-9 and ADAMTS-5 m RNA decreased(P<0.05);miR-21,TIMP-3,Collagen Ⅱ increased(P>0.05),LncRNA GAS5 and MMP-13 decreased(P>0.05).Conclusions1.RJNTF can reduce the loss of collagen and proteoglycan in cartilage matrix,delay the degradation of cartilage matrix,and reduce the damage of cartilage tissue morphology and structure by down-regulating the expression of LncRNA GAS5,MMP-3,MMP-9,MMP-13 and ADAMTS-5,up-regulating the expression of miR-21,TIMP-3,Collagen Ⅱ and Aggrecan in cartilage of KOA rats.2.RJNTF serum can delay the degradation of extracellular matrix of degenerated chondrocytes by down-regulating the expression of LncRNA GAS5,miR-21 and TIMP-3,inhibiting the expression of MMP-3,MMP-9,MMP-13 and ADAMTS-5,and promoting the expression of Collagen Ⅱ and Aggrecan.3.RJNTF serum can inhibit the catabolism of matrix and promote the synthesis of matrix to delay the degradation of chondrocytes by down-regulating the expression of LncRNA GAS5,up-regulating the expression of miR-21 and TIMP-3,inhibiting the expression of MMP-3,MMP-9,MMP-13 and ADAMTS-5,and promoting the expression of Collagen Ⅱand Aggrecan in the chondrocytes infected by LncRNA GAS5 and miR-21 lentivirus.