Study On Improving Cartilage Matrix Matrix Metabolism Of Knee Osteoarthritis By Chonggu Granules Based On MiR-148a-3p/wnt/β-catenin Pathway

1 Objective The clinical effect of Chonggu granules on Knee osteoarthritis(KOA)patients with TCM dialectical liver and kidney deficiency was observed in this study,and the regulation of extracellular matrix metabolism of chondrocytes based on the miR-148a-3p/Wnt/β-catenin pathway was investigated experimentally.To take part in the pathogenesis of KOA and investigate the potential mechanism of Chonggu granules.2 Methods2.1 Study 1 —— Theoretical AnalysisBy reviewing a large number of literature and searching databases,the relationship between miR-148a-3p,wnt1/β-catenin pathway,cartilage matrix metabolism and KOA was explored according to the research progress at home and abroad,so as to provide a theoretical basis for the intervention study of the clinical efficacy of heavy bone granules on KOA and the mechanism of cartilage matrix metabolism.2.2 Study 2 ——Intervention of Chonggu Granules on clinical efficacy of KOA patientsSixty KOA patients from our department were chosen,and twenty healthy adults from the physical examination facility were chosen as the control group.By using a random number table classification approach,60 patients with KOA were separated into two groups: treatment and control.Each group had 30 patients.The control group received glucosamine hydrochloride pills,whereas the treatment group received Chonggu granules.The relative expression of miR-148a-3p,wnt1,β-catenin,and GSK3 mRNA,IL-6,TNF-a,and IL-1β levels,ESR,and hs-CRP were measured and compared between the KOA and healthy groups.The relative expression levels of miR-148a-3p,wnt1,β-catenin,and GSK3 mRNA,IL-6,TNF-a,and IL-1β levels,ESR,hs-CRP values,and TCM syndrome scores were measured and compared within and between the treatment group and the control group before and after treatment.Efficacy evaluation scale scores(WOMAC score,VAS score,Lequesne index score),and SF-36 quality of life assessment scale score changes.2.3 Study 3——Based on miR-148a-3p/wnt/β-catenin pathway,the mechanism of Chonggu granules affecting cartilage matrix metabolism in KOA rats was studied36 SPF grade male rats were randomly divided into 6 normal group A,and the remaining 30 KOA rats were modeled by papain method,and then randomly divided into model control group B after successful modeling.Chonggu Granules small dose group C;Chonggu Granules medium dose group D;Group E with high dose of Chonggu Granules;Glucosamine hydrochloride F group,6 in each group,were given drug therapy after adaptive feeding for 1 week.Normal and model control groups were treated with distilled water,while the remaining groups were treated with small and medium doses of Chonggu granules and glucosamine hydrochloride once a day for 4 weeks.The ECM and chondrocytes of 6 groups were observed after 4 weeks of anesthesia.ELISA determined the expression of IL-6,TNF-a and IL-1β in peripheral blood.Expression of MMP-3,MMP-3 and Col2 al in rat cartilage was determined by IF.WB detected protein expression of wnt1,GSK3β,β-catenin,and Aggrecan in rat cartilage.Relative mRNA expression of miR-148a-3p,wnt1,β-catenin and GSK3β in rat cartilage was detected by RT-PCR.Luciferase reported that gene analysis confirmed the presence of miR-148a-3p target genes.3 Results3.1 Results of Study 1(1)The incidence and progression of KOA is linked to an imbalance in cartilage matrix metabolism.(2)The wnt/β-catenin signaling pathway and miR-148a-3p regulate extracellular matrix metabolism in KOA cartilage.The wnt/β-catenin signaling pathway and miR-148a-3p have a targeting interaction.(3)A prior study indicated that Chonggu granules might increase the metabolism of cartilage extracellular matrix,which provided a theoretical foundation for our work.3.2 Results of Study Ⅱ(1)When compared to the healthy group,the levels of miR-148a-3p and GSK3β in the KOA group were considerably lower(P<0.01 or P<0.05),while wnt1 and β-catenin were significantly higher(P<0.01).(2)When compared to the control group,the levels of IL-1β,IL-6,TNF-a,hs-CRP,and ESR in the KOA group were substantially higher(P<0.01).(3)Following treatment,mRNA levels of miR-148a-3p and GSK3β were significantly increased in both the treatment and control groups(P<0.01),while mRNA levels of wnt1 and β-catenin decreased significantly(P<0.05).(4)After treatment,the levels of IL-1β,IL-6,and TNF-a in both the treatment and control groups were considerably reduced(P<0.01 or P<0.05),and the levels of IL-1β,IL-6,and TNF-a in the treatment group were lower than those in the control group(P<0.01 or P<0.05).Following the same therapy,the treatment group’s ESR and hs-CRP reduced considerably more than the control group’s(P<0.05).(5)Both groups’ clinical symptoms were eased to some extent,but the treatment group considerably outperformed the control group in terms of lowering the TCM syndrome score,WOMAC score,Lequesne index score,VAS score,and other scores(P<0.05).After therapy,the scores of each dimension of the SF-36 scale in the two groups were compared,improved in different degrees except RP and MH.Compared with the control group,the treatment group could significantly improve GH,PF,RP,RE,SF,VT,and reduce BP(P<0.01).3.3 Results of Study Ⅲ(1)The pathological results confirmed that the structure of the knee joint cartilage of the rats after papain enzymatic modeling was changed to varying degrees compared with the blank group,and the degradation of cartilage matrix was disordered.The changes of the cartilage structure of the rats in each administration group were lighter than those in the model control group,however,the degree of cartilage changes was the lightest and the degradation of cartilage matrix was significantly improved in the rats treated with medium-dose heavy bone particles.(2)The ELISA results demonstrated that IL-6,IL-1β,and TNF-a levels in the peripheral blood of rats in the model group were considerably higher than those in the control group(P<0.01);however,when compared to the blank group,the levels of these molecules were significantly decreased(P<0.01);and the levels of these molecules in the Chonggu were the lowest(P<0.01).(3)MMP-3 and MMP-13 protein expression levels in the cartilage tissue of rats in the KOA model group were significantly elevated,however Col2 al protein expression levels were dramatically lowered,according to the IF detection data.Comparing each administration group to the model group,MMP-3 and MMP-13 protein expression levels were noticeably lower,whereas Col2 al protein expression levels were noticeably greater.Nevertheless,MMP-3 and MMP-13 protein expression levels were lowest in the Chonggu granule medium dose group when compared to each treatment group,but Col2 al protein expression levels were greatest.(4)Wb examination revealed that the expression levels of Wnt pathway-related proteins were aberrant in the cartilage tissues of rats in the KOA model group,for example,wnt1 and β-catenin were dramatically raised,where as GSK3β and Aggrecan were significantly reduced(P<0.01).The protein contents of wnt1 and β-catenin were lowered,whereas the protein expressions of GSK3β and Aggrecan were elevated to varied degrees in all treatment groups,particularly in the Chonggu granule medium dosage group(P<0.01).(5)RT-PCR results revealed that the mRNA expression levels of miR-148a-3p and GSK3β were decreased in the cartilage tissues of rats in the group B,while wnt1 andβ-catenin were significantly increased.Mi R-148a-3p and GSK3β mRNA expression levels were significantly higher in each administration group compared to the model group,while wnt1 and β-catenin mRNA expression levels were lower.The relative expression levels of miR-148a-3p and GSK3β were increased in CGG medium dose group,while the mRNA expression levels of wnt1 and β-catenin were most significantly decreased(P<0.01).(6)Luciferase gene reports confirmed that Rno-miR-148a-3p significantly down-regulated the expression of luciferase in Rno-Wnt1-wt compared with NC mimic group(P<0.01),indicating that miR-148a-3p could target wnt1.4 conclusion(1)miR-148a-3p/wnt/β-catenin signaling pathway is closely related to the metabolism of extracellular matrix in KOA cartilage.(2)Low expression of miR-148a-3p and GSK3β,high expression of wnt1 andβ-catenin in KOA patients.(3)After intervention of Chonggu Granules in KOA patients,it has significant clinical effects on reducing disease activity and inflammatory indicators,improving TCM symptom scores,and improving patients’ quality of life.(4)Chonggu Granules alleviated the inflammatory response of rat knee cartilage,inhibited the secretion of chondrocyte matrix degrading enzyme in KOA rats,improved the synthesis of type Ⅱ collagen and agglutinoglycan,and improved the extracellular matrix environment of cartilage.(5)Chongu granules may inhibit wnt1 and wnt1/β-catenin pathway by increasing the expression of miR-148a-3p in cartilage tissue,thereby improving the metabolism of chondrocyte extracellular matrix,reducing cartilage inflammatory response,and improving the prognosis of knee osteoarthritis.