Objective: Knee osteoarthritis(KOA)is a common chronic bone and joint disease.In clinical orthopaedics,it is closely related to articular cartilage injury,osteophyte formation and inflammation,and the elderly are more likely to suffer from this disease.Modified Yanghe Decoction(JWYHD)is an effective compound prescription for clinical treatment of KOA,but its specific pharmacological mechanism and intervention targets need to be further studied.The compound prescription of traditional Chinese medicine can regulate and stimulate the regeneration of bone marrow mesenchymal stem cells(BMSCs)in different degrees,which provides a new possibility for the repair of cartilage tissue.The purpose of this study is to observe the effect of JWYHD medicated serum on BMSCs from the point of view of senescence,apoptosis,differentiation dysfunction and chondrogenic differentiation of JWYHD cells in vitro,and to explore its mechanism based on p38 MAPK signal pathway.Methods: The JWYHD medicated serum was prepared;The rat BMSCs was isolated,cultured and purified;Double identification of BMSCs by flow cytometry and chemical induction in vitro;Construction of inflammatory microenvironment simulated in vivo KOA model induced by 10ng/m L IL-1β in vitro;Observation indicators:(1)Identification of BMSCs by chemical induction and flow cytometry in vitro;(2)Drug toxicity test to determine the intervention concentration of JWYHD containing serum(5%,10%,15%);(3)The KOA model was induced by 10 ng/m L IL-1β,the activity of BMSCs was detected by CCK8 method,and the best intervention time was determined;(4)JWYHD medicated serum(5%,10%,15%)interfered with KOA model.After BMSCs 24 h,BMSCs activity was detected by CCK 8 method,and cell grouping was determined(A.Blank group B.Model group C.5% medicated serum group D.10% medicated serum group E.15%medicated serum group F.Celecoxib group);(5)ELISA detected TNF-α and IL-6expression in supernatants of BMSCs;(6)SA-β-Gal staining detected the aging of BMSCs in each group;(7)The apoptosis distribution of BMSCs in each group was observed by TUNEL staining,and the apoptosis rate of BMSCs in each group was detected by flow cytometry;(8)Alcian blue staining detected the production of extracellular matrix proteoglycans(glycosaminoglycans)in each group;(9)The Western Blot method detected the relative expression of p38,p-p38,JNK,p-JNK,IL-6,Bcl-2,IL-1β,TNF-α,SOX-9,Bax,Col II.and Aggrecan proteins in BMSCs in each group.Results:(1)Isolated,cultured and purified rat BMSCs,and the flow cytometry results showed that the expression of relevant specific markers on the surface of BMSCs,such as CD29,CD44,CD90,CD105,etc.,was strongly positive,with positive rates of99.12%,99.47%,99.47% and 99.50%,respectively.CD34 and CD45 expression were strongly negative,and the positive rates were 0.01% and 0.36%.And under in vitro chemical induction,it can differentiate in the direction of osteogenesis and fat,and has the potential for multidirectional differentiation.The results show that our isolated and cultured mesenchymal stem cells meet the characteristics of BMSCs.(2)The results of drug toxicity test showed that the 5%,10% and 15% concentrations of drug-containing serum group basically met the energy required for the proliferation of BMSCs,and there was no significant difference in cell viability between the groups compared with the 10%FBS normal group(P>0.05).(3)10 ng/ml IL-1β induced the construction of KOA model,and the results showed that the cell survival rate was 69.486% at 12 h of intervention,which was the best effect.(4)The results of ELISA test showed that compared with the model group,the expression of IL-6 and TNF-α in the JWYHD medicated serum group(5%,10%,15%)and celecoxib group showed a significant downward trend(P<0.01).(5)The results of SA-β-Gal staining showed that compared with the model group,the number of positive senescent cells(blue)in the JWYHD medicated serum group(5%,10%,15%)and celecoxib group were reversed to varying degrees,showing a significant downward trend(P<0.01).(6)The results of TUNEL staining showed that compared with the model group,the TUNEL-positive cells(green fluorescence)in the JWYHD medicated serum group(5%,10%,15%)and celecoxib group had different degrees of significant decrease,and the apoptotic cells in the field of view of each group were significantly reduced,normal cells increased,and the gap became smaller.The results of flow apoptosis showed that compared with the model group,the apoptosis rate of BMSCs in the JWYHD medicated serum group(5%,10%,15%)and celecoxib group was reversed by different amplitudes,showing a significant downward trend(P<0.01).(7)The staining results of Alcian blue showed that the cartilage-specific matrix protein glycosaminoglycans(blue)produced by cells in the 5%,10% and 15% drug-containing serum intervention groups increased sequentially,and the staining and coloring deepened sequentially,which was positively correlated with drug concentration,and the number of cells increased sequentially compared with the model group.(8)The results of Western Blot showed that compared with the model group,the relative expression of p-p38,pJNK,p-p38/p38,Bax,TNF-α,IL-1β,IL-6,p-JNK/JNK proteins in the JWYHDcontaining serum group(5%,10%,15%)was reduced to varying degrees(P<0.05),and the relative expression of Bcl-2/Bax,SOX-9,Col II.and Aggrecan proteins gradually increased(P<0.05),and dose-dependent.Conclusion: JWYHD can affect the expression balance of Bcl-2/Bax protein(ratio)in BMSCs,thereby regulating the occurrence of apoptosis.By inhibiting the phosphorylation of the p38 MAPK signaling pathway,downregulating the expression of inflammatory proteins such as IL-1β,TNF-α and IL-6,and upregulating the expression of related cartilage-specific matrix SOX-9,Col II.,Aggrecan and other proteins,reducing the damage,differentiation dysfunction and apoptosis caused by inflammatory stimulation to BMSCs,and promoting the differentiation of chondrocytes to repair early KOA cartilage damage.This may be one of the mechanisms of action of the multichannel multi-target of JWYHD compound. |