| Objects:Through the observation and comparison of Cangxi Tongbi capsule,Xianling Gubao capsule and glucosamine sulfate in the treatment of KOA,the clinical advantages of Cangxi Tongbi capsule were compared;Using network pharmacology and molecular docking technology,the molecular mechanism of Cangxi Tongbi capsule in the treatment of KOA and the protection of knee joint cartilage was analyzed systematically and scientifically,and the molecular mechanism of Cangxi Tongbi capsule in the treatment of KOA was explained;The pharmacodynamic evaluation and related mechanism of Cangxi Tongbi capsule were explored by cell experiment in vitro.MethodsThe first part is the clinical retrospective study.According to the diagnosis and treatment guidelines of KOA and the actual situation of the hospital,the standards needed for this study were formulated.140 cases of KOA patients with KL stage I-II in our hospital(Affiliated Hospital of Shandong University of traditional Chinese Medicine)from September 2019 to August 2020 were collected and divided into three groups.Group A:Cangxi Tongbi capsule drug group,50 cases;group B: Xianling Gubao capsule drug group,48 people,group C: glucosamine sulfate group,three groups of medication time is 2 months,after determining the treatment method,randomly selected,the selected personnel case information system must have regular follow-up data,according to the pain visual simulation score standard,Lequesne index score standard and treatment efficiency to evaluate the effect,through the system clustering method to study the syndrome type.The second part is the data mining of Cangxi Tongbi capsule related research.Through tcmsp database,we find the effective compounds of the prescription: Duhuo,Weilingxian,Atractylodes,Spatholobus,caoshuei,sangsheng,Drynaria,chuanniuxi and Chuanduan The relative molecular weight of compounds and other general core parameters of pharmacology are used to evaluate drug compounds,construct drug component target disease network,explore the mechanism of drug intervention after target,and scientifically and comprehensively analyze the drug distribution rules and reasons of "junchenzushi".The molecular docking technology was used to further verify the mechanism of its protection of articular cartilage in the treatment of osteoarthritis and its relationship with p38 MAPK signaling pathway,and to further analyze and verify the cooperative treatment of multicomponent,multi-channel and multi-target.The third part is the in vitro cell experiment of rats.In this experiment,the chondrocytes of the articular cartilage of SD rats of 1 week old were identified by toluidine blue staining.The inflammatory factor reagent IL-1?was used(The model of coa chondrocytes was established as the research object.Cell counting kit-8 was used to observe the best concentration of cell activity screening drug after drug intervention;The expression of lcrna hotair in chondrocytes,the changes of lcrna hotair expression after canggentongbi capsule intervention in koa chondrocytes and the expression of the main target gene m RNA in p38 MAPK signal pathway after canggentongbi capsule were detected by q RT-PCR;The apoptosis rate of chondrocytes was detected and the changes were obtained by flow cytometry.The inflammatory factor IL-1?,which was closely related to koa,was detected by ELISA 1L-1??TNF-?The expression level of p38 MAPK signaling pathway was detected by Western blot.ResultsThe first part of the clinical experimental study,1,according to all the data,the three groups of drug treatment have achieved satisfactory results,in which after 2 weeks of treatment,the total effective rate of group A was higher than that of group B,the difference was statistically significant(P<0.05);The longer the treatment period,the smaller the difference.There was no significant difference between the two groups after 1 month and 2months(P > 0.05).2.The VAS score and Lequesne index of the included researchers decreased and showed a downward trend after the treatment according to the doctor's advice,and the effect was more significant.Compared with group B,there were differences in the reference scores of patients in group A after 2 weeks of treatment(P<0.05),and the effect of group A was better than that of group B;There was no significant difference in the study indexes among the three groups in January and February(P>0.05).3.Through clinical trials,the safety level of the three drugs included in this study can reach grade I,and there are no abnormal or adverse reactions.In the second part,the data mining of Cangxi Tongbi capsule showed that the relative molecular weight of Duhuo and Weilingxian were higher than that of sangsheng,and the lipo water partition coefficient(Alogp)of Duhuo was the highest,which was similar to that of Weilingxian,Spatholobus and sangsheng,but significantly different from other drugs;A total of 472 compounds were found by tcmsp database.According to the threshold requirements of pharmacology parameters such as OB and DL,a total of 27 compounds met the requirements;The network consists of 222 targets,781 edges,27 active ingredients and1 disease name;The results of molecular docking showed that the binding energy of 27 chemical components of Cangxi Tongbi capsule with OA key inflammatory factors was ideal.The third part is the in vitro cell experiment of KOA.Through the preliminary study and the screening of the drug concentration,CCK-8 method was used to screen the drug concentration to obtain canggentongbi capsule in 100 ? The drug concentration was the best in g/ml;The apoptosis rate of lcrna hotair overexpression group was the highest and the normal group was the lowest.The apoptosis rate of over expression group and model group was higher than that of model group(P<0.05),and the apoptosis rate of drug and blocker in the model group and over expression drug group was significantly lower(P<0.05).The expression level of inflammatory factors in over expression group(W3)was significantly higher than that in model group(W2)by ELISA(Compared with model group(W2)and over expression group(W3),the correlation inflammatory factors of KOA showed different degrees of decrease after intervention of drugs and blockers.The data among groups were statistically analyzed(P < 0.05),while W4 group There was no significant difference between W5 group and W6 group and W7 group(P > 0.05);The expression of lncrna hotair was detected by q RT-PCR,compared with that of the normal group(L1),1L-1 ? After dry treatment,lncrna hotair was significantly increased,and 1L-1 was added ? On the basis of intervention cells,lncrna hotair was transfected,and overexpression of adenovirus could significantly improve the expression of lncrna hotair.There were statistical differences and met the statistical significance(P < 0.05),so it indicated that the transfection was successful;The expression of lncrna hotair after intervention of canggentongbi capsule was detected by q RT-PCR.The results showed that the expression of lcrna was in relation to 1L-1 ?Compared with the expression of lncrna hotair in group(H2),CX + 1L-1 ? Group(H3)and cx+1l-1 ? The results showed that the expression of lncrna hotair over expression group(H4)was significantly decreased,P < 0.05,which was statistically significant;Finally,the expression of related proteins and genes in lncrna hotair/p38 mapk signal pathway was detected by q RT-PCR and Western blot respectively.The results showed that the expression of W1 was the highest in collagen II blank group,the lowest in over expression group(W3),the lowest in MMP13 and p38 blank group(W1),and the highest in overexpression group,The statistical analysis had difference and met the statistical significance(P < 0.05);Compared with model group(W2)and over expression group(W3),the expression levels of MMP13 and p38 in chondrocytes were decreased in the drug group(W4)and over expression drug group(W5),while the protein gene expression of collagenii was increased,The difference between the groups was analyzed by statistical data and the differences met the statistical significance(P < 0.05).At the same time,there was no significant difference between W4 group,W5 group and W6 group and W7 group(P>0.05)Conclusion1.Cangxi Tongbi capsule can protect articular cartilage and relieve clinical symptoms in patients with KOA.Through clinical experiments,it is found that the characteristics of Cangxi Tongbi capsule are earlier effective time and more reliable effect,which is also its clinical advantage.2.Through systematic data mining,it is found that the diagnosis and treatment of Cangxi Tongbi capsule for koa involves multiple pathways and targets,and the drug molecules contained in many herbs of Cangxi Tongbi capsule can alleviate the symptoms and pain of KOA,and its symptom relief and corresponding treatment for koa is relatively scientific.3.Through the in vitro cell experiment of rats in this project,we preliminarily believe that Cangxi Tongbi capsule can alleviate the clinical symptoms of KOA patients and protect the articular cartilage of patients by targeting the regulation of lncrna hotair / p38 MAPK signaling pathway. |
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