| Knee osteoarthritis(KOA)is an irreversible degenerative bone and joint disease with the main clinical manifestations of knee pain,swelling,stiffness and limited joint activity.At present,there are still many problems in the initial and early clinical research and clinical diagnosis and treatment of KOA.In terms of clinical research,there is a lack of high-quality randomized controlled clinical trials of drug intervention for KOA with x-grade Ⅰ～Ⅱ and early stage;In terms of clinical diagnosis and treatment,it is impossible to determine which KOA groups can benefit from exercise therapy for a long time.The curative effect of physical therapy cannot be guaranteed due to the lack of unified operation standards.Western medicine mainly improves clinical symptoms and has different types of toxic and side effects.Tibetan medicine Ruyi Zhenbao pill has a history of more than 1000 years.It belongs to the category of classic prescriptions of Tibetan medicine and belongs to class B variety of national medical insurance.It can dry yellow water and dredge muscles and collaterals for KOA and can prevent and treat the characteristics of "Xiazhen"(meat arthralgia),"Ruizhen"(bone arthralgia),"Zazhen"(pulse arthralgia)and "Quzhen"(muscle arthralgia)caused by the inward and outward diffusion of "wood cloth".This study evaluated the clinical efficacy of Ruyi Zhenbao Pill on initial and early KOA through multicenter,randomized,double-blind,placebo and parallel controlled clinical trial,filled the lack of high-quality evidence-based evidence of classic and famous prescriptions of national medicine in the treatment of KOA,and further explored the biological mechanism of "multi-component,multi-target and multi-channel" of Ruyi Zhenbao Pill on initial and early KOA through mRNA transcriptomics,provided demonstration research for the modern inheritance and development of classic prescriptions of ethnic medicine.ObjectiveThe purpose of this study is to evaluate the clinical efficacy of Ruyi Zhenbao pill in the treatment of initial and early KOA through a prospective multicenter,randomized,double-blind,placebo and parallel control study,to provide evidence for the stepped diagnosis and treatment scheme of initial and early KOA.In addition,through mRNA transcriptomics,further explore Ruyi Zhenbao pill,and elaborate its effect mechanism by regulating related genes and their corresponding signal pathways and biological processes.Method1.A multicenter,randomized,double-blind,placebo-controlled clinical trial of Ruyi Zhenbao pill,a classic and famous Tibetan medicine,in the treatment of KOA:This study was conducted from October 13,2021 to December 25,2021 at Wangjing Hospital of China Academy of Traditional Chinese Medicine,the Third Affiliated Hospital of Beijing University of Chinese Medicine,Xiyuan Hospital of China Academy of Traditional Chinese Medicine,Affiliated Hospital of Traditional Chinese Medicine of Southwest Medical University,Beijing Traditional Chinese Medicine Hospital Affiliated to Capital Medical University,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Guangdong Provincial Hospital of Traditional Chinese Medicine,and 15 sub-centers included a total of KOA subjects,which were randomly divided into experimental group and control group,each group with 120 participants.The intervention measures of the participants in the experimental group were Ruyi Zhenbao Pills+health education;the intervention measures of the control group were Ruyi Zhenbao Pills placebo+health education.The intervention period of both groups of participants were 4 weeks,followed up after the intervention were 4 weeks,and the total test period were 8 weeks.The main outcome indicator was the response rate of VAS pain score,and the secondary outcome indicators were WOMAC total points,WOMAC subproject points(joint pain,joint stiffness,joint function),quality of life concise version score(SF-12),and traditional Chinese medicine syndrome score.The participants were interviewed and evaluated after 2 weeks,4 weeks,6 weeks and 8 weeks respectively,and the safety of the experimental drugs was evaluated during the intervention.The missing data was interpolated by carrying forward the last measurement data.In the treatment of confounding factors,the longitudinal model was used to control the confounding factors and evaluate the clinical efficacy.The research results were reported according to the consort specification of randomized controlled trials.2.Study on the effect mechanism of Ruyi Zhenbao Pills in the treatment of KOA based on mRNA transcriptomics:the blood samples used in the experiment came from the whole blood of the clinical trial taken from the Ruyi Zhenbao Pill Group and the placebo group before and 4 weeks after intervention,and all RNA blood samples are PAXgene Blood RNA Tube collects blood vessels.The standard blood collection amount of each blood vessel is 2.5mL,of which there is 6.9mL RNA protection reagent in the blood vessels.After blood collection,the blood vessels should be reversed up and down 10-20 times to ensure the full integration of whole blood and protective reagents.After the blood sample is collected,put it into the refrigerator at-80℃ to freeze for detection.In this study,the selection of blood samples of experimental group will be sorted from large to small according to the main outcome indicators VAS scoring improvement before and after intervention.After strict screening,the experimental group selected 17 subjects with the largest VAS score improvement.In the control group,10 subjects with 0 VAS score improvement were selected.A total of 54 blood samples were selected before and after treatment.This study passed the extraction of total RNA,sample detection of total RNA,library construction and on-machine sequencing,sequencing data filtering,reference genomic comparison,gene expression analysis,hierarchical clustering analysis of differential expression genes,GO function analysis of differential expression genes,and Pathway function of differential expression genes.Analyzing and other processes,the differential expression genes of Ruyi Zhenbao Pill Group and Placebo Group on KOA subjects were screened respectively,and the differential expression genes of Ruyi Zhenbao Pill Group relative to the placebo group were screened out,and the differential expression genes were enriched and analyzed by GO and KEGG Pathway.The signaling pathways and biological processes related to KOA further explore the therapeutic mechanism of Ruyi Zhenbao Pills in the treatment of KOA.Results1.A multicenter,randomized,double-blind,placebo-controlled,parallel controlled,clinical trial of Ruyi Zhenbao pill in the treatment of knee osteoarthritis:(1)A total of 240 participants who met the acceptance criteria and signed informed consent were included.During the visit in the second week,one participant was excluded because he did not meet the acceptance criteria,and two participants lost the visit(there were no drug records and efficacy evaluation records);During the visit in the fourth week,4 participants were lost(there were no medication records and efficacy evaluation records in the third to fourth weeks).237 participants were included in the Full analysis set,including 120 participants in the experimental group and 117 participants in the control group;(2)In terms of baseline,there had imbalance in age and systolic blood pressure between the two groups,P<0.05;(3)In terms of efficacy evaluation,temporary standby painkillers belong to confounding factors.Considering that the baseline measurement values of confounding factors and effectiveness outcome indicators have an impact on the outcome indicators,these factors and their interaction with time are included in the longitudinal data margin model to eliminate the impact of other confounding factors other than treatment methods on the outcomes;After repeated measurement of the expected structural variance of the three covariates,it is composed of the correlation between the observation conditions and the covariates.The first two parts are exactly the same as the generalized linear model except that they do not assume the distribution form of response variables.The third part of the model reflects the characteristics of correlation between observations from the same individual by defining the correlation form between individual repeated observations.Therefore,the generalized linear model is extended to the case of longitudinal data.(4)In the main analysis group,in terms of improving the total score of WOMAC in the treatment of knee osteoarthritis,the marginal model results showed that the coefficient P value of group*time variable was 0.0265<0.05.There was a significant difference between the two groups in the total score of WOMAC over time,which showed that the advantage of the experimental group over the control group would increase with time.In terms of improving WOMAC pain score in the treatment of knee osteoarthritis with Ruyi Zhenbao pill,the marginal model results show that the coefficient P value of group*time was 0.0305<0.05,and there is a significant difference in the change of WOMAC pain score between the two groups over time,which shows that the advantage of the experimental group over the control group will increase with the increase of time.In terms of improving the response rate of VAS score in the treatment of knee osteoarthritis with Ruyi Zhenbao pill,the marginal model results show that there is no significant difference between the two groups,but the response rate odds ratio of the experimental group relative to the control group is greater than 1 between 4 and 8 weeks,indicating that the response rate advantage of VAS score of the experimental group is better than that of the control group during this period,and the response rate odds ratio of VAS score of the two groups reaches the peak in 6 weeks.In terms of improving WOMAC function score,WOMAC stiffness score,SF12-PCS score,SF12-MCS score and TCM syndrome score in the treatment of knee osteoarthritis with Ruyi Zhenbao pill,the marginal model results show that there is no significant difference between the two groups.(5)In the subgroup analysis of VAS score response rate,for subjects with baseline VAS score of(4,5],the coefficient P value of group*time was 0.0166<0.05,coefficient P value of group*time2 was 0.0466<0.05,indicating that there is a significant difference between the two groups.During the period of 2-8 weeks,the response rate of the experimental group is always higher than that of the control group,and the difference between the response rates of the two groups first increases and then decreases.At about 4.5 weeks,the difference between the response rates of the two groups reaches the maximum.In terms of interaction analysis,for subjects with baseline VAS score of[3,4]and age group of[50,55],the response rate of VAS score in the experimental group was better than that in the control group;For subjects with baseline VAS score of(4,5],the VAS score response rate of the experimental group also showed an advantage to some extent,especially for older subjects,the advantage of the experimental group was more obvious.(6)In terms of subgroup analysis of WOMAC functional score,subjects with age in years[50,55],coefficient P value of group*time was 0.0428<0.05,coefficient P value of group*time2 was 0.0267<0.05,which was significant between the two groups,indicating that the decrease in WOMAC function total score in the experimental group was always higher than that in the control group,and the difference in the decrease showed a quadratic curve change,and the difference between the two groups reached a maximum at approximately 4.5 weeks;Years at[61,65],coefficient P value of group*time was 0.0221<0.05,which means that the WOMAC functional score in the experimental group decreased more significantly compared with that in the control group;When WOMAC function score was greater than 20 at baseline,coefficient P value of group*time was 0.031<0.05,which means that the WOMAC functional score in the experimental group decreased more significantly compared with that in the control group.In terms of interaction analysis,the experimental group was more advantaged in reducing the WOMAC function score for younger patients and more advantaged for older patients in reducing the WOMAC function score for sicker patients.(7)In terms of subgroup analysis of TCM syndrome scores,subjects with age in years[56,60],coefficient P value of group*time was 0.0439<0.05,indicating that the decrease in the total score of TCM syndromes in the experimental group was better than that in the control group;subjects with age in years[61,65],coefficient P value of group*time was 0.0219<0.05,coefficient P value of group*time2 was 0.0071<0.05,both of which were significant,indicating that the decrease in the total score of TCM syndromes in the experimental group was better than that in the control group;Subjects with baseline TCM syndrome total score greater than 4 points,coefficient P value of group*time was 0.0296<0.05,which was significant between the two groups,and the decrease in the total score of medical syndromes in the experimental group was better than that in the control group.Interaction analysis showed that for subjects with baseline TCM syndrome total scores greater than 4 points,the decrease of TCM syndrome total scores in the experimental group was faster than that of the control group in subjects with age range[50,55]and age range[65,70];For subjects with a total TCM syndrome score greater than 4 at baseline,the decreasing magnitude of the total TCM syndrome score in the experimental group gradually changed over time better than that in the control group in subjects with age ranges of[56,60]and[61,65].2.Study on the effect mechanism of Ruyi Zhenbao Pills in the treatment of KOA based on mRNA transcriptomics:(1)The quality test results of 54 samples are class A,indicating that the quality of all samples meets the requirements of database construction and sequencing,and the current success rate is about 96.18%;(2)In this study,a total of 54 samples were measured using DNBSEQ platform,and each sample produced an average of 6.23G data.The original sequencing data included reads with low quality,joint pollution and high unknown base N content.After filtering,readsclean reads Q20(%)was greater than or equal to 93.75%,and clean reads Q30(%)was greater than or equal to 87.62%.The amount of data between samples was relatively stable without significant difference.The overall sequencing quality was good,and subsequent analysis could be carried out;(3)The results of GO enrichment analysis showed that:① in terms of chondrocyte growth,development and apoptosis,Ruyi Zhenbao pill may be involved in the regulation mechanism of SRP dependent co translation targeted membrane protein through four genes RPL26、RPL31、RPL34、RPL36A,one gene(RPL26)is involved in the positive regulation of endogenous apoptosis signal pathway by p53 mediators,and two genes(COMMD3-BMI 1 and H2AC19)are involved in the regulation of chromatin silencing;One gene(ATP5MF-PTCD 1)is involved in tRNA 3’-terminal processing,and one gene(COMMD-3BMI 1)is involved in the regulation of biological processes such as H2A-K119 ubiquitinated histone;② In delaying the process of KOA bone remodeling and osteophyte formation,Ruyi Zhenbao pill may participate in vascular injury healing through one gene(MCAM),positive regulation of angiogenesis by five genes(CCBE1,FGF2,CXCL8,R1N1 and TEK),and positive regulation of endothelial cell migration by three genes(CCBE1,TEK and ATOH8);③ Ruyi Zhenbao pill may be involved in the development and regulation of muscle fibers through two genes(NEBL and DMD)in repairing periarticular muscle injury and atrophy;④In terms of neuroprotection,Ruyi Zhenbao pill may participate in synaptic formation by regulating two genes(PALM and SHANK1);(4)KEGG pathway enrichment analysis shows that ① in terms of chondrocyte growth and development,Ruyi Zhenbao pill may participate in the translation process of gene information through four genes(RPL26,RPL31,RPL34 and RPL36A),and four genes(RPL6,RPL34,rps4yl and RPS27)participate in the regulation of ribosomes;② In terms of regulating nitrogen metabolism,Ruyi Zhenbao pill may participate in nitrogen metabolism through two genes(CA6 and CA8),regulate nitrogen metabolism,and then regulate the concentration of NO,so as to inhibit its destructive effect on chondrocytes and the occurrence of noninfectious inflammation;③ In terms of nerve protection,Ruyi Zhenbao pill may participate in glycosphingolipid biosynthesis through two genes(ABO,A4GALT)and regulate GSLS through ACTO and neolacto series to promote the growth,proliferation and differentiation of nerve cells;④ In terms of improving inflammation,Ruyi Zhenbao pill may participate in the inflammatory and immune mechanisms related to rheumatoid arthritis through three genes(HLA-DGB,CXCL8 and TEK),which can improve symptoms and delay the course of KOA.Conclusion1.Ruyi Zhenbao pill,a classic and famous Tibetan medicine,has good clinical efficacy in improving VAS score response rate,WOMAC total score,WOMAC pain score,WOMAC function score and SF-12-MCS(quality of life mental health),especially in improving WOMAC total score and WOMAC pain score of patients with KOA.It can fill the lack of evidence for the treatment of KOA with classic and famous prescriptions Ruyi Zhenbao pill of ethnic medicine and made a demonstration study for the inheritance and development of classic and famous prescriptions of ethnic medicine.2.Ruyi Zhenbao pill in the treatment of knee osteoarthritis may participate in the growth,development and apoptosis of chondrocytes by regulating the differential expression of key genes of RPL26,RPL31,RPL34,RPL36A,RPS4y1,RPL27,COMMD3-BMI 1,H2AC19,ATP5MF-PTCD1 and COMMD3-BMI 1;It may delay the process of KOA bone remodeling and osteophyte formation by regulating the differential expression of key genes of MCAM,CCBE1,FGF2,CXCL8,RLN1,TEKCCBE1,TEK and ATOH8;It may repair periarticular muscle injury and atrophy by regulating the differential expression of key genes of NEBL and DMD;It may participate in nerve protection by regulating the differential expression of key genes of PALM,SHANK1,ABO and A4GALT;It may participate in nitrogen metabolism by regulating the differential expression of CA6 and CA8 key genes;It may participate in the inflammatory and immune mechanisms related to rheumatoid arthritis by regulating the differential expression of key genes of HLA-DRB5,CXCL8 and TEK.Innovation1.Based on the randomized controlled trial,this study used the marginal model to control and statistically analyze the longitudinal data and confounding factors in the clinical efficacy evaluation of traditional Chinese medicine for the first time,which confirmed that Ruyi Zhenbao pill can play a better therapeutic role in the clinical treatment of initial and early KOA,and fill in the lack of evidence of classic famous prescriptions Ruyi Zhenbao pill of ethnic medicine in the treatment of KOA.2.Through mRNA transcriptomic study,this study found that Ruyi Zhenbao pill in the treatment of KOA may be mainly involved in the growth,development and apoptosis of chondrocytes,bone remodeling and osteophyte formation,periganglionic muscle injury and atrophy,nerve protection,nitrogen metabolism,inflammation and immunity by regulating key genes such as RPL26、RPL31、RPL34、RPL36A、RPS4Y1、RPS27、COMMD3-BMI1、H2AC19、ATP5MF-PTCD1,etc.This paper preliminarily expounds the relevant "target pathway" and biological process of Ruyi Zhenbao pill in the initial and early stage of KOA,which provides an objective biological basis for the clinical positioning of Ruyi Zhenbao pill.. |
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