| Objective:1 clinical study to verify the clinical efficacy and safety of Sanqi Huoxue Qutan Fang in treating knee osteoarthritis with phlegm-stasis Randomized controlled trial.2 network pharmacological analysis and in vivo experimental verification:the network pharmacological method was used to analyze the components and corresponding targets of all the drugs of Panax Notoginseng Huoxue Qutan recipe,to screen the related targets of Knee Osteoarthritis Knee Osteoarthritis and chromatin regulatory factors,and then to find out the mechanism of Sanqi Huoxue Qutan Fang on knee osteoarthritis,the related laboratory indexes in peripheral serum were detected and analyzed to verify the conclusion of network pharmacological analysis.Methods:1 clinical study The sample size of this study was estimated according to the total effective rate of the pre-trial,and then 66 knee osteoarthritis patients were divided into the control group and the experimental group(according to the method of random number table).The control group was treated with diclofenac sustained-release capsules.The experimental group was treated with Panax Notoginseng blood-activating and phlegm-dispelling decoction.All subjects were treated for 4 weeks.Before and after treatment,Lysholm knee function score,Vas pain score and Womac score were compared between the two groups,and the clinical efficacy of the two groups was evaluated2 Network pharmacological analysis and in vivo experimental verification The eligible osteoarthritis-related chips were retrieved through GEO database and standardized.The genes related to chromatin regulators were extracted and quantified based on the gene expression matrix after the standardized processing,machine learning was used to screen disease-specific genes,and then RT-qPCR was used to verify the selected disease-specific genes.Then,random forest(RF)model,Support Vector Machines(SVM)model and Support Vector machine graph model were used to construct disease prediction model to predict the risk of osteoarthritis.Then,consensus clustering algorithm,principal component analysis(PCA),single-sample genome enrichment analysis(SSGSEA)and immune infiltration were used to analyze the relationship between the molecular subtypes of chromatin regulators and immune microenvironment and inflammatory factors.The peripheral venous blood of patients with knee osteoarthritis were detected before and after treatment.The expressions of EED,TP53,PARP1,TDRD7 and TOP2A mRNA in peripheral blood of the two groups were detected by RT-qPCR,and the contents of IL-4 and IL-7 in peripheral blood of the two groups were detected by enzyme linked immunosorbent assay(Elisa).Results:1 clinical study The baseline data of sex ratio,age and course of disease were compared between the two groups,and the VS A scores of the experimental group and the control group before treatment were statistically analyzed,there was no significant difference in Lysholm joint function score and WOMAC score(P>0.05).After 4 weeks of treatment,the total effective rate was 90.32%in the experimental group and 83.87%in the control group,the difference was statistically significant(p=0.015),there was significant difference between the two groups(p<0.05)in VSA score,Lysholm score and Womac score,but there was significant difference in VS A score,Lysholm score and Womac score between the two groups(p<0.05),the difference was statistically significant(p<0.05).2 Network pharmacological analysis and in vivo experimental verification2.1 the active ingredients of Panax Notoginseng Huoxue Qutan recipe were retrieved from TCMSP database.After screening with OB≥30%and DL≥0.18,59 ingredients were found,including 880 targets.2.2 according to OB value and DL value,the active components of Panax Notoginseng Huoxue Qutan recipe were selected from TCMSP database and imported into cytoscape 3.8.0 to draw the component-disease-target network.The 5 characteristic genes and their corresponding components of traditional Chinese medicine were verified by molecular docking.The results of molecular docking showed that the affinity of the interaction of the five characteristic genes of the Chinese herbal components corresponding to the characteristic genes in the prescription was far less than-5.0 kJ/mol,the five ingredients with the best affinity are Ginsenoside RG1,Luteolin,Kaempferol,Paeoniflorin and Lupinus luteus.It is concluded that the prescription of Panax Notoginseng for promoting blood circulation and expelling phlegm has a good binding activity with the key targets of knee osteoarthritis,which proves that the prediction of this study is reliable.2.3 The Gene expression profiling datasets of osteoarthritis,GSE48556 and GSE63359,were obtained from the comprehensive gene expression database,to extract the intersecting genes between Panax Notoginseng Huoxue Qutan recipe,chromatin regulatory factors and knee osteoarthritis related targets,then,five chromatin regulator signature genes(EED,TP53,PARP1,TDRD7 and Top2a)were screened by machine learning and verified by PCR.A nomogram model was established based on 5 chromatin regulator characteristic genes.Decision curve analysis showed that patients could benefit from the nomogram model.The patients with osteoarthritis were divided into two patterns of chromatin regulators(cluster A and cluster B)by consensus clustering.A principal component analysis algorithm was constructed to calculate the chromatin regulatory factor fraction for each sample to quantify the chromatin regulatory factor pattern.The score of chromatin regulatory factor in group A was higher than that in Group B.In addition,IL-4 and IL-7 were also associated with Cluster B.Therefore,Panax Notoginseng blood-activating and phlegm-dispelling formula may be effective in treating knee osteoarthritis patients by regulating the release of neutrophil related inflammatory factors mediated by chromatin regulatory factors.2.4 The levels of EED,TP53,PARP1,TDRD7 and TOP2A mRNA in peripheral serum and the levels of IL-4 and IL-7 in peripheral serum were also significantly different between pre-treatment and post-treatment neutrophil(p<0.05).The results showed that "Sanqi Huoxue Qutan Fang may be effective in treating knee osteoarthritis patients by regulating the release of neutrophil related inflammatory factors mediated by chromatin regulatory factors,"which was predicted by network pharmacological studies.Conclusion(s):1 clinical study Panax notoginseng decoction for promoting blood circulation and expelling phlegm is effective and safe in the treatment of phlegm-stasis type of knee osteoarthritis.2 network pharmacological analysis and in vivo experimental verification In this study,we established a risk prediction model based on the genes of chromatin regulatory factors,which can better predict the risk of osteoarthritis.It was also found that patients with osteoarthritis could be classified into two completely different molecular subtypes of chromatin regulatory factors(Clustera and clusterB).clusterB was highly associated with neutrophil cell dysregulation and had higher expression levels of IL-4 and IL-7.In addition,it was preliminarily verified that Sanqi Huoxue Qutan prescription may treat knee osteoarthritis through chromatin regulatory factor-mediated immune inflammation. |
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