| Objects: The object of the study is to explore the clinical efficacy of Cangxitongbi capsule in the treatment of knee osteoarthritis by comparing the difference of Cangxitongbi capsule and Biqi capsule in the treatment of KOA through clinical experiments.Futhermore,the pathogenesis of KOA and the mechanism of Cangxitongbi capsule were discussed through in vivo and in vitro experiments.To clarify the relationship between KOA and p38 MAPK signaling pathway and if Cangxitongbi capsule can protect articular cartilage via inhibiting p38 MAPK signaling pathway.Methods: The first part of clinical research: According to the diagnostic criteria and rejection criteria of this study,60 patients with David stage I-II KOA admitted to our hospital from September 2017 to September 2018 were randomly divided into Cangxitongbi capsule group(group A)and Biqi capsule group(group B),with 30 cases in each group.They were treated with the corresponding medication for 2 months.Periodic follow-up was carried out.The patients were scored according to pain visual analogue score,bone and joint index WOMAC score and Lequesne index score pretreatment and two weeks,one month and two months after treatment.The second part of experiment in vitro: Chondrocyte of knee joint of 1 week old SD rats was isolated and extracted by two-step enzyme digestion method,identified by toluidine blue staining and type II collagen immunohistochemical method,cultured to the second generation,and induced to be degenerative chondrocyte by IL-1 beta(10 ng/ml)for 24 hours.The content of Osthol in the dry ointment of Cangxitongbi capsule was determined by high performance liquid chromatography and the optimum concentration was screened by CCK-8 method.The cells were divided into blank group(KB),model group(MX),DMSO group(DM),Cangxitongbi capsule group(CX),SB203580 group(SB),Cangxitongbi capsule and SB203580 co-culture group(CS).After successful modeling,KB group and MX group were cultured normally,DM group was cultured in complete medium containing 0.1% DMSO,CX group was cultured in complete medium containing 100 ?g/ml Cangxitongbi capsule,SB group was cultured in complete medium containing 10 ?M SB203580,CS group was cultured in complete medium containing 100 ?g/ml Cangxitongbi capsule and 10 ?M SB203580.Cells and supernatants were collected after 24 hours of intervention.Flow cytometry was used to detect apoptosis,Elisa was used to detect the expression of IL-1beta and TNF-alpha,Western Blot was used to detect the expression of p38,p-p38,MMP13 and Collagen II in p38 MAPK signaling pathway,RT-PCR was used to detect the expression of p38 mRNA,MMP13 mRNA and Collagen II mRNA in p38 MAPK signaling pathway,and immunohistochemistry was used to detect the expression of p-p38.The third part of experiment in vivo: Sixty healthy male SD rats aged 4 weeks were randomly divided into six groups: blank group(KB),model group(MX),DMSO group(DM),Cangxitongbi capsule group(CX),SB203580 group(SB)and Cangxitongbi capsule combined with SB203580 group(CS),with 10 rats in each group.Except for the blank group,KOA model was established by Hulth method in other groups.The dosage was calculated according to Meeh-Rubner body surface area calculation formula.CX group was given 35 mg/ml Cangxitongbi capsule solution,SB group was given 2 mg/ml SB203580 solution,CS group was given mixed solution containing 2 mg/ml SB203580 and 35 mg/ml Cangxitongbi capsule,DM group was given 1% DMSO solution,and the MX and KB groups were given normal saline.The dosage of gastric lavage was 3 mland the frequency was once a day.After 4 weeks of medication intervention,the rats were executed and sampled.HE staining was used to observe the histomorphological changes of articular cartilage.Elisa method was used to detect the expression of IL-1beta and TNF-alpha in peripheral blood supernatant.Western Blot method was used to detect the expression levels of p38,p-p38,MMP13 and Collagen II in articular cartilage.RT-PCR was used to detect the expression levels of p38 mRNA,MMP13 mRNA and Collagen II mRNA in p38 MAPK signaling pathway.The expression of p-p38 was detected by immunohistochemistry.Results: The first part of clinical research: 1.After two weeks,one month and two months of treatment,satisfactory results were achieved in both groups.The total effective rate of group A was 16.63% higher than that of group B after two weeks of treatment with significant difference(P < 0.05);At the same time,the total effective rate of group A was 6.70% and 3.37% higher than that of group B after one month and two months of treatment,but there was no significant difference between the two groups(P > 0.05).2.Moreover,compared with those before treatment,the results of VAS score,Lequesne index and WOMAC score were significantly lower(P < 0.05)after two weeks,one month and two months of treatment.Meanwhile,the results of VAS score,Lequesne index and WOMAC score in group A were better than those in group B after two weeks of treatment with significant difference(P < 0.05).But the difference was not statistically significant after one month and two months of treatment(P > 0.05).3.There were no adverse reactions such as allergy,rash,nausea and vomiting in the two groups,and the safety level is level I.The second part of experiment in vitro: 1.The content of Osthol in the dry extract of Cangxitongbi capsule is 0.1404 mg/g.2.The optimum concentration of Cangxitongbi capsule on degenerative chondrocytes induced by IL-1 beta(10 ng/ml)is 100 ?g/ml.3.The apoptotic rate of MX group was significantly higher than that of KB group(P < 0.05).The apoptotic rate of DM group was almost the same as that of MX group(P > 0.05).But the apoptotic rate of CX group,SB group and CS group was significantly lower(P < 0.05).4.The expression levels of inflammatory factors IL-1beta and TNF-alpha in KB group were the lowest and the expression level of MX group was the highest,and there was significant difference between them(P<0.05).Compared with MX group,the expression levels of inflammatory factors in DM group were almost the same,and there was no significant difference between them(P>0.05),at the same time,the expression of inflammatory factors in CX group,SB group and CS group decreased significantly(P < 0.05).However,the expression level of inflammatory factors in the CS group decreased the most,and the difference was statistically significant compared with those in the CX group and SB group(P < 0.05).5.The expression level of Collagen II was the highest in KB group,while the expression levels of MMP13,P38 and P-P38 were the lowest.There was significant difference between KB group and MX group(P < 0.05).Compared with MX group,the expression level of Collagen II,MMP13,P38 and P-P38 in DM group was almost the same without significant difference(P > 0.05).Both CX group,SB group and CS group could effectively reduce the expression of MMP13,P38 and P-P38 in degenerative chondrocytes(P < 0.05),while the expression of Collagen II was increased(P < 0.05).But the expression level of Collagen II,MMP13,P38 and P-P38 changed the most in the CS group,the difference was statistically significant compared with those in the CX group and SB group(P<0.05).6.The expression level of Collagen II mRNA was the highest in KB group,while the expression levels of MMP13 mRNA and P38 mRNA were the lowest.There was significant difference between KB group and MX group(P < 0.05).Compared with MX group,the expression level of Collagen II mRNA,MMP13 mRNA and P38 mRNA in DM group was almost the same without significant difference(P > 0.05).Both CX group,SB group and CS group could effectively reduce the expression of MMP13 mRNA and P38 mRNA in degenerative chondrocytes(P < 0.05),while the expression of Collagen II mRNA was increased(P < 0.05).But the expression level of Collagen II mRNA,MMP13 mRNA and P38 mRNA changed the most in the CS group,the difference was statistically significant compared with those in the CX group and SB group(P<0.05).7.Immunohistochemical results showed that the expression level of p-p38 was consistent with western blot.The third part of experiment in vivo: 1.Compared with KB group,the color of articular cartilage in MX and DM group was obviously turbid and rough.HE staining showed that the number of chondrocyte decreased,the arrangement was irregular,the layers were not clear,the structure was disordered,and there were neovascular wings.Although the color of articular cartilage in CX group,SB group and CS group changed,there was no obvious change compared with MX group.HE staining showed that the number of chondrocytes decreased,the arrangement was not regular,the layers were not clear and the structure was a little disordered,but CS group was better than SB group than CX group.2.The expression levels of inflammatory factors IL-1beta and TNF-alpha in KB group were the lowest and the expression level of MX group was the highest,and there was significant difference between them(P<0.05).Compared with MX group,the expression levels of inflammatory factors in DM group were almost the same,and there was no significant difference between them(P>0.05),at the same time,the expression of inflammatory factors in CX group,SB group and CS group decreased significantly(P < 0.05).However,the expression level of inflammatory factors in the CS group decreased the most,and the difference was statistically significant compared with those in CX group and SB group(P < 0.05).3.The expression level of Collagen II was the highest in KB group,while the expression levels of MMP13,P38 and P-P38 were the lowest.There was significant difference between KB group and MX group(P < 0.05).Compared with MX group,the expression level of Collagen II,MMP13,P38 and P-P38 in DM group was almost the same without significant difference(P > 0.05).Both CX group,SB group and CS group could effectively reduce the expression of MMP13,P38 and P-P38 in degenerative chondrocytes(P < 0.05),while the expression of Collagen II was increased(P < 0.05).But the expression level of Collagen II,MMP13,P38 and P-P38 changed the most in the CS group,the difference was statistically significant compared with those in the CX group and SB group(P<0.05).4.The expression level of Collagen II mRNA was the highest in KB group,while the expression levels of MMP13 mRNA and P38 mRNA were the lowest.There was significant difference between KB group and MX group(P < 0.05).Compared with MX group,the expression level of Collagen II mRNA,MMP13 mRNA and P38 mRNA in DM group was almost the same without significant difference(P > 0.05).Both CX group,SB group and CS group could effectively reduce the expression of MMP13 mRNA and P38 mRNA in degenerative chondrocytes(P < 0.05),while the expression of Collagen II mRNA was increased(P < 0.05).But the expression level of Collagen II mRNA,MMP13 mRNA and P38 mRNA changed the most in the CS group,the difference was statistically significant compared with those in the CX group and SB group(P <0.05).5.Immunohistochemical results showed that the expression level of p-p38 was consistent with western blot.Conclusions 1.Cangxitongbi capsule can obviously relieve the pain,swelling and joint dysfunction of KOA patients.And compared with Biqi capsule,Cangxitongbi capsule has the advantages of quick onset and more reliable curative effect,which is worthy of clinical promotion.2.p38 MAPK signaling pathway plays an important regulatory role in the pathological progress of KOA.It is an important pathway in the pathogenesis of KOA and an important target for the prevention and treatment of KOA.3.Cangxitongbi capsule can targeted block p38 MAPK signaling pathway to accelerate the repair of degenerated chondrocyte and protect articular chondrocyte. |
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