Mechanism And Clinical Research Of Cangxitongbi Capsule Protecting Knee Osteoarthritis Cartilage Through LncRNA HOTAIR/p38MAPK Pathway

Objects:Through clinical research,the therapeutic effects of Cangxitongbi capsule and Tenghuangjiangu capsule on knee osteoarthritis(KOA)were compared to evaluate the therapeutic effect of Cangxitongbi capsule on KOA.Then,the KOA rat models were constructed to explore the protective effects and the underlying mechanism of Cangxitongbi capsule on the articular cartilage of KOA rats in vivo.Methods:The first part of clinical research:One hundred and thirty Kellgren-Lawrence grade I-III KOA patients with liver and kidney deficiency combined with cold dampness syndrome who were admitted to the traumatic orthopedic outpatient clinic of our hospital from October 2019 to October 2021 were included in the study according to the criteria of the relevant references of the Chinese traditional Chinese medicine guidelines for the management and treatment of knee osteoarthritis(2020 Edition).All the patients were randomly divided into the Cangxitongbi capsule group(65 patients)and the Tenghuangjiangu capsule group(65 patients),and they were treated with the corresponding drugs in standard practice for 8 weeks.The patients were evaluated before and after treatment using Lysholm score,WOMAC score,NRS score and TCM syndrome quantification score.The second part of experiment in vivo:50 4-week-old SPF grade SD male rats were randomly divided into blank control group(NC),model control group(Model),Cangxitongbi capsules group(CXTB),Cangxitongbi capsules combined with LncRNA HOTAIR overexpression group(CXTB +AAV-HOTAIR),and Cangxitongbi capsules combined with overexpression virus empty vector control group(CXTB + AAV-control).All groups except the blank control group(NC)were subjected to KOA rat modeling by modified Hulth’s method,driven passive activity for 4 weeks radiographs of the knee joint were taken for demonstrating successful modeling,and then intervened according to the corresponding method of intervention in each group,the rats were sacrificed after 4 weeks of intervention.The morphological changes of cartilage were evaluated by HE staining and OARSI score;Serum IL-1β and TNF-α content was detected by ELISA;The number and morphology of autophagosomes in cartilage were observed by transmission electron microscope;TUNEL method was used to detect chondrocyte apoptosis;The expressions of MMP13,TIMP1,Bcl-2,Bax,LC3-II,p62,p38 and p-p38 proteins were detected by Western blot;The expression of LncRNA HOTAIR and the mRNA expression of p38,MMP13 and TIMP1 were detected by qPCR.Results:The first part of clinical research:1.After 8 weeks of treatment with Cangxitongbi capsule and Tenghuangjiangu Capsule,the Lysholm score,WOMAC score,NRS score and quantitative score of traditional Chinese medicine syndrome of the two groups were better than those before treatment,and the difference was statistically significant(P < 0.05).2.The Lysholm score,WOMAC score,NRS score as well as the TCM syndrome quantification score in the Cangxitongbi capsule group after 8 weeks’ treatment were better than the corresponding scores in the Tenghuangjiangu Capsule group after 8 weeks’ treatment,with significant differences(P < 0.05).3.There were no adverse events such as nausea,rash,hypersensitivity or vomiting after administration in either group.The second part of experiment in vivo:1.The results of LncRNA HOTAIR expression in each group: the expression of LncRNA HOTAIR in the NC group was the lowest,and the expression in the model group was higher,with significant differences(P < 0.05);However,the amount of HOTAIR was decreased after intervention with Cangxitongbi capsules(CXTB vs Model,P < 0.05),whereas it was increased after combined treatment with HOTAIR overexpression adeno-associated virus(CXTB vs CXTB + AAV-HOTAIR,P < 0.05);Compared with the CXTB group,there was no significant difference in HOTAIR expression in the CXTB +AAV-control group(P > 0.05).2.HE staining showed that the surface of cartilage in NC group was smooth and the cells were arranged closely,and the morphological structure of cartilage tissue was normal;The Model group had the worst cartilage condition,with rough cartilage surfaces and appearing concave,fragmented,and longitudinally split,with vessels crossing,and loss of the tideline;The cartilage condition of the CXTB,CXTB + AAV-HOTAIR and CXTB +AAV-control groups improved to different degrees compared with the Model group;Evaluation by OARSI score revealed that Model group had the highest score,which was significantly different(P < 0.05)than NC or CXTB groups;The fraction in the CXTB group was lower than that in the CXTB + AAV-HOTAIR group(P < 0.05),whereas it was not different from that in the CXTB + AAV-control group(P > 0.05).3.TUNEL staining,transmission electron microscopy and examination of Bcl-2,Bax,LC3-II,p62 expression levels found that the Model group had the highest level of apoptosis,the lowest level of autophagy in each group and was significantly different from the NC group and the CXTB group(P < 0.05);The level of autophagy in the CXTB group was higher than that in the CXTB + AAV-HOTAIR group(P < 0.05)but the level of apoptosis was lower(P < 0.05),and the levels of autophagy and apoptosis were not significantly different from those in the CXTB + AAV-control group(P > 0.05).4.Examination of relevant indicators related to cartilage damage and p38 MAPK signaling pathway in each group revealed that MMP13,p38,p-p38,serum IL-1β and TNF-α expressions were significantly increased while TIMP1 expression was decreased after KOA modeling in rats(Model vs NC,P < 0.05);After Cangxitongbi capsule intervention,the expression of MMP13,p38,p-p38,IL-1β and TNF-α decreased,while the expression of TIMP1 increased(CXTB vs Model,P < 0.05);No significant changes in relevant indicators were observed after combined overexpression of viral empty vector intervention compared to drug intervention alone(CXTB + AAV-control vs CXTB,P > 0.05),whereas combined HOTAIR overexpression intervention showed an antagonistic effect,i.e.,decreased TIMP1 expression but increased MMP13,p38,p-p38,IL-1β and TNF-αcontents in vivo(CXTB + AAV-HOTAIR vs CXTB,P < 0.05).Conclusions:1.Cangxitongbi capsule has a high total effective rate in the treatment of Kellgren-Lawrence I-III KOA patients with liver and kidney deficiency combined with cold dampness syndrome.It can alleviate symptoms,improve joint function and improve TCM syndromes,and has therapeutic advantages compared with Tenghuangjiangu capsule.2.Cangxitongbi capsule can inhibit joint inflammation,slow down the degradation of cartilage extracellular matrix,regulate the balance between autophagy and apoptosis,and protect articular cartilage.3.LncRNA HOTAIR/ p38 MAPK pathway is one of the main targets of Cangxitongbi capsule in protecting knee cartilage.