| Objective:1.Network pharmacology analysis:Through the network Pharmacology Method to analyze the components and targets of all the drugs in Jingu Tongxiao pill,to screen the targets of KOA,and then match the targets of the two,to find the main pathway and target of Jingu Tongxiao Pill on KOA.2.Animal experiments : After the establishment of KOA model in rats,Jingu Tongxiao pill was given by gavage.Combined with the results of network pharmacology analysis,the possible mechanism of Jingu Tongxiao pill in the intervention of KOA was explored..3.Clinical research:We used randomized controlled trials to verify the clinical efficacy and safety of Jingu Tongxiao Pill in the treatment of KOA of qi stagnation and blood stasis type.Then we test and analyze the related laboratory indexes in the peripheral serum of patients,and explore the possible mechanism of Jingu Tongxiao Pill in the treatment of KOA.Methods:1.Network pharmacology analysis:The chemical constituents and targets of Jingu Tongxiao pills were searched by TCMSP.TCMS database,Uni Prot KB network platform and Genecards database were used to obtain the corresponding target proteins and their corresponding compounds.Genecards,NCBI gene database and OMIM database were used to obtain KOA disease targets,and then the target of Jingu Tongxiao Pill and KOA related targets were docking,and the visual analysis was carried out by using Wayne diagram operation software.The network of PPI was constructed and analyzed by using STRING data.On this basis,topology analysis and MCODE cluster analysis were carried out by using Cytoscape.Go analysis and KEGG enrichment pathway analysis of key target proteins were performed by String data platform.After summarizing and classifying,the traditional Chinese medicine composition disease pathway target network diagram of Jingu Tongxiao Pill and KOA was formed with the help of Cytoscape.2.Animal experiments : Fifty rats were randomly divided into 5 groups: blank group,model group,experimental group,positive control group and inhibitor group.Except for the blank group,the other four groups were injected with papain to make KOA model.After successful modeling,rats in model group and blank group were given 2ml / 100 g distilled water by gavage.The rats in the experimental group were given Jingutongxiao pharmaceutical liquid by gavage at the rate of 1.234 g / kg.The rats in the positive control group were given 2.725mg/kg diacerein capsule diluent by gavage.The inhibitor group was given SIRT1 inhibitor ex527 intraperitoneal injection according to the intervention method of the experimental group.After 6 weeks of intervention,all animals were killed and knee joint fluid and cartilage samples were collected.The levels of MMP3,IL-17 and TNF-α in synovial fluid were detected by ELISA.The expression of SIRT1,TLR4 and NF-κB mRNA in articular cartilage was detected by real-time reverse transcription PCR(RT qPCR),and the protein expression of SIRT1,TLR4 and NF-κB in articular cartilage was detected by Western blot.3.Clinical research:The sample size of this study was estimated based on the total effective rate of pre-test.62 patients with KOA of qi stagnation and blood stasis type were divided into experimental group and control group(according to random number table).The control group was treated with Pingle bone setting manipulation,acupuncture and functional exercise.On the basis of the treatment of patients in the control group,the patients in the experimental group took Jingu Tongxiao Pill orally,1bag each time,twice a day.All patients were treated for 3 weeks.Lysholm knee function score,VAS pain score,Young’s modulus of biceps femoris and quadriceps femoris muscle group and joint flexion degree were compared between the two groups before and after treatment,and the clinical efficacy of the two groups was evaluated.SIRT1,MMP3,TNF-α and IL-17 were measured by ELISA in the serum of the two groups.Results:1.Network pharmacology analysis1.1 The effective components of 11 drugs in Jingu Tongxiao Pill were searched from tcmsp database.After ob ≥ 30% and DL ≥ 0.18 screening,246 components were found and 1295 targets were summarized.1.2 With the help of Cytoscape software,the composition target network diagram of Jingu Tongxiao pills was constructed.Network diagram shows that the key components of Jingu Tongxiao pill are quercetin,luteolin,kaempferol,naringenin,beta sitosterol,etc.1.3 Through genecards,NCBI gene database and OMIM database,a total of 2065 Kao related genes were retrieved,and 134 common targets were obtained.1.4 Go analysis and KEGG pathway enrichment analysis were performed by common targets of drug diseases.Combined with the analysis of pathways,targets and literature data,it is concluded that Jingu Tongxiao Pill may play a role in KOA mainly by inhibiting the related inflammatory pathways,inhibiting the release of inflammatory factors in cartilage,synovium and other tissues,so as to alleviate the degradation of extracellular matrix and affect the metabolism of articular cartilage.2.Animal experiments2.1 ELISA results showed that: compared with the model group,the contents of IL-17,MMP3 and TNF-α in the joint fluid of the positive control group and the experimental group were lower than those of the model group(P < 0.05);compared with the experimental group,the content of MMP3 in the joint fluid of the experimental group was lower than that of the positive control group(P < 0.05),but there was no significant difference in the contents of IL-17 and TNF-α(P < 0.05)Compared with the inhibitor group,the levels of IL-17 and MMP3 in synovial fluid were lower in the experimental group(P < 0.05).2.2 RT-qPCR results showed that:SIRT1,TLR4 and NF-κB mRNA were expressed in cartilage tissue of rats in each group.Compared with the model group,the expression of SIRT1 mRNA in the positive control group,experimental group and inhibitor group was significantly increased,while the expression of TLR4 and NF-κB mRNA was significantly decreased(P < 0.05);compared with the positive control group,the expression of SIRT1 mRNA in the experimental group was higher(P <0.05),but there was no difference in the expression of TLR4 and NF-κB mRNA between the two groups(P > 0.05);compared with the inhibitor group,the expression of SIRT1 mRNA in the experimental group was higher(P < 0.05)The expression of NF-κB mRNA was higher than that of NF-κB mRNA(P < 0.05).2.3 Western blot results showed that SIRT1,NF-κB and TLR4 were expressed in rat cartilage.The protein expression of SIRT1 in experimental group,positive control group and inhibitor group was higher than that in model group,and the protein expression of NF-κB and TLR4 was lower than that in model group(P < 0.05).Compared with the positive control group,the expression of SIRT1 protein in the experimental group was higher than that in the positive control group,and the expression of NF-κB and TLR4 The expression of SIRT1 protein in the experimental group was higher than that in the inhibitor group,and the expression of NF-κB and TLR4 Protein in the experimental group was lower than that in the inhibitor group(P <0.05).3.Clinical researchThere was no difference in sex ratio,age and course of disease between the two groups.There were no statistically significant differences in VSA score,Lysholm joint function score,knee flexion range of motion,biceps femoris,quadriceps femoris muscle stiffness,serum SIRT1,MMP3,TNF-α,IL-17 indexes between the experimental group and the control group before treatment(P > 0.05).After treatment,the total effective rate of the experimental group was 90.32%,and that of the control group was 83.87%,the difference was statistically significant(P = 0.015).In the same group,the VSA score,Lysholm joint function score,knee flexion range of motion,biceps femoris and quadriceps femoris muscle stiffness were significantly improved(P< 0.05).The levels of SIRT1,MMP3,TNF-α and IL-17 in peripheral serum of patients before and after treatment were also statistically significant(P < 0.05).After treatment,there were significant differences in VSA score,Lysholm joint function score,knee flexion range of motion,serum SIRT1 and IL-17 levels between the two groups(P <0.05).There was no significant difference in other indexes between the two groups after treatment(P > 0.05).Conclusion:1.Network pharmacology analysis:The treatment of KOA with Jinggu Tongxiao pill is a multi-component,multi-target and multi-channel synergistic effect.TLR4 / nfkappa B signaling pathway may play a key role in the treatment of KOA with Jinggu Tongxiao pill.2.Animal experiments:Jingu Tongxiao pill can effectively reduce the levels of1l-17,TNF-α and MMP3 in the synovial fluid of KOA model rats,and may inhibit the downstream TLR4/NF-κB signal pathway mediated by SIRT1 through enhancing the expression of SIRT1,so as to inhibit the occurrence and development of inflammatory response and play a protective role on cartilage.3.Clinical research : Jingu Tongxiao pill combined with acupuncture and manipulation is effective and safe in the treatment of KOA with qi stagnation and blood stasis.SIRT1 and IL-17 may be the targets of Jingu Tongxiao pill in the treatment of KOA. |
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