| ObjectiveUsing bioinformatics and network pharmacology technology to construct the target network of the effect of Rongjin Niantong Fang on osteoarthritis cartilage degeneration.Then,the action mechanism of Rongjin Niantong Fang on osteoarthritis cartilage degeneration was to be described.Based on the aforementioned results,the effective pathways were determined for subsequent animal and cell experiments,and an experimental basis for clinical application of Rongjin Niantong Fang was provided.Methods1.The TCMSP,BATMAN-TCM,and ETCM databases were used to collect the binding targets of Rongjin Niantong Fang traditional chinese medicine compounds.The chip data set of cartilage of osteoarthritis rats was retrieved from the GEO database,and the differential genes of osteoarthritis cartilage degeneration were analyzed by R language.The differential genes were analyzed by DAVID and STRING databases for enrichment analysis and PPI analysis.Venn diagram analysis was used to obtain the same molecules of Rongjin Niantong Fang traditional chinese medicine compound binding targets and osteoarthritis cartilage degeneration differential genes.In the STRING database,PPI analysis and enrichment analysis were performed on the same molecules.Comprehensive bioinformatics results,network pharmacology results and existing literature reports,the follow-up experimental research direction was determined.2.Forty male SPF-grade SD male rats aged 8 weeks were used in this experiment.Knee osteoarthritis model of rat was established by modifying Hulth method.Experimental rats were divided into blank group,model group,treatment group and control group by random number table grouping method.The blank group and the model group were intragastrically administered with saline,the treatment group was intragastrically administered with water extract of Rongjin Niantong Fang,and the control group was intragastrically administered with glucosamine hydrochloride capsules for 12 weeks.After intervention,the gross morphology,microstructure and ultrastructure of cartilage tissue were observed with the naked eye,optical microscope and transmission electron microscope.The content of SDF-1 in synovial membrane was detected by ELISA.The protein expression of CXCR4,MMP-3,MMP-9,MMP-13 in cartilage was detected by immunohistochemistry.Western blot was used to detect the phosphorylation expression of P38,ERK1/2,JNK.3.In this experiment,30 male 4-week-old SPF SD rats were used.Rat chondrocytes were isolated and extracted by mechanical-type Ⅱ collagenase digestion.Chondrocytes were identified by immunocytochemical staining of type Ⅱ and toluidine blue staining,and in vitro culture of rat chondrocytes was established.The second-generation chondrocytes were used in subsequent experiments.ELISA detection method was used to determine the intervention dose and time of SDF-1-induced chondrocytes,and the intervention concentration of Rongjin Niantong Fang.Chondrocytes were randomly divided into blank group,model group(SDF-1),Rongjin Niantong Fang(SDF-1 + Rongjin Niantong Fang),and inhibitor group(SDF-1 +AMD3100).After intervention,laser confocal microscopy was used to observe the protein expression of CXCR4 in chondrocytes.Western blot was used to detect CXCR4,MMP-3,MMP-9,MMP-13 protein expressions,P38,ERK1/2,JNK phosphorylation expressions in chondrocytes of each group.Results1.1787 Rongjin Niantong Fang traditional Chinese medicine compound binding targets were obtained by TCMSP,BATMAN-TCM,and ETCM.226 genes were differentially expressed in cartilage of osteoarthritis rats,of which 167 genes were up-regulated and 59 genes were down-regulated.The core genes in the PPI network are CXCR4,CCL2,CD44,IGF1,COL1A1,etc.There are 42 target molecules of Rongjin Niantong Fang on osteoarthritis cartilage degeneration,including CXCR4,CCL2,etc.Rongjin Niantong Fang acts on osteoarthritis cartilage degeneration through the interaction of cytokines and cytokine receptors,which including SDF-1/CXCR4 signal axis,MAPK pathway,et al.2.In animal experiments,general observation showed that the rat cartilage in blank group was smooth,pink and shiny.The rat cartilage in model group was abnormal color,cartilage defects,and increased osteophyte.The rat cartilage in treatment group and control group was between the blank group and the model group.The light microscopeic observation showed that the rat articular cartilage surface in blank group was smooth and flat,and no chondrocytes showed disorder,and the matrix was uniformly stained and unstained.In model group,the rat articular cartilage surface was rough,cracked,chondrocytes clustered,hypertrophic,and the matrix was severely stained.In control group and treatment group,the rat articular cartilage surface was slightly uneven,with chondrocytes clustered and matrix staining.Electron microscopy showed that in blank group,the structure of rat chondrocytes was relatively complete,and the collagen fibers were fine.In model group,the rat chondrocytes were damaged,the number of organelles decreased,the endoplasmic reticulum protein synthesis function was abnormal,and the collagen fibers were thick and loose.The morphological structure of chondrocytes and collagen fibers in treatment group and control group was between blank group and model group.ELISA,immunohistochemistry and western blot showed that the expression of SDF-1,CXCR4,MMP-3,MMP-9,MMP-13 protein and P38,ERK1/2 phosphorylation in model group was higher than that in blank group(P < 0.05 or P < 0.01).The expression of SDF-1,CXCR4,MMP-3,MMP-9,MMP-13 protein and P38,ERK1/2 phosphorylation in treatment group and control group were lower than that in model group(P < 0.05 or P < 0.01).3.In cell experiments,immunocytochemical staining of type Ⅱ collagen and toluidine blue staining showed that the cytoplasm of the extracted cells was brownish yellow and blue-purple,with the typical characteristics of chondrocytes.ELISA showed that the effective concentration and time of SDF-1-induced chondrocytes were 100 ng/m L,12 h,and the effective intervention concentration of Rongjin Niantong Fang was 300 μg/m L The laser confocal microscope observation showed that the CXCR4 fluorescence intensity in model group was higher than that in blank group,and the CXCR4 fluorescence intensity in Rongjin Niantong Fang group and inhibitor group was lower than that in model group.Western blot showed that the expression of CXCR4,MMP-3,MMP-9,MMP-13 protein and P38 phosphorylation in model group was higher than that in blank group(P < 0.05 or P <0.01).The expression of CXCR4,MMP-3,MMP-9,MMP-13 protein and P38 phosphorylation in Rongjin Niantong Fang group was lower than that in model group(P < 0.05 or P <0.01).Conclusions1.The mechanism of Rongjin Niantong Fang on the degeneration of osteoarthritis cartilage is related to the inflammatory response,and its regulatory effect fits the pathological mechanism of osteoarthritis cartilage.It can act on osteoarthritis cartilage degeneration through SDF-1/CXCR4 signal axis and MAPK pathway2.Rongjin Niantong Fang can improve the morphological structure of cartilage tissue in knee osteoarthritis rats,inhibits the protein expression of SDF-1 in synovial and CXCR4 in cartilage,inhibits the phosphorylation expression of P38,ERK1/2 in the MAPK pathway and reduces MMP-3,MMP-9,MMP-13 protein expression,to delay cartilage degeneration in knee osteoarthritis rats.3.After intervention of SDF-1-induced chondrocytes in vitro,Rongjin Niantong Fang inhibited the protein expression of CXCR4 and the phosphorylation expression of P38 in the MAPK pathway,reduce the synthesis of MMP-3,MMP-9,MMP-13 protein in chondrocyte,to delay the degeneration of osteoarthritis cartilage. |
PDF Full Text Request