Clinical And Experimental Study Of Buzheng Xugu Pills In The Treatment Of Steroid-induced Necrosis Of The Femoral Head Based On TLR4/MyD88/NF-κB Signaling Pathway

Purpose:1.To observe the clinical effect of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head and provide intuitive and quantitative basis for its clinical application;2.To explore the potential active ingredients and specific pharmacological mechanism of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head from the molecular level based on network pharmacology;3.To explore the mechanism of Buzhengxuguwan in the intervention of steroid induced necrosis of the femoral head in rats based on TLR4/MyD88/NF-κB signaling pathway.Material and method:1.The clinical effect of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head78 patients with steroid induced necrosis of the femoral head admitted to the Department of orthopedics II,Affiliated Hospital of Liaoning University of traditional Chinese medicine were selected as the research objects.The research period was from September 2018 to October 2019.The patients who met the requirements were randomly divided into the observation group and the control group,with 39 cases in each group.The control group was given Xianlinggubao capsule,and the observation group was given Buzhengxuguwan.Two groups of patients were treated for 3 months.Harris hip function score,VAS score,SF-36 score,hip imaging indexes and adverse reactions were observed before and after treatment in the two groups.The changes of blood routine,liver function and renal function indexes were detected before and after treatment in the two groups.2.The molecular mechanism of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head based on network pharmacologyBATMAN-TCM analysis tool was used to search the active compounds and predicted targets of Buzhengxuguwan.Dig See database,OMIM database and Gene Cards database were selected to predict the targets of steroid induced necrosis of the femoral head.Draw Venn Diagram was used to predict the core target of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head.The PPI network was constructed by using STRING database,and the compound-disease-target regulatory network of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head was constructed by using Cytoscape3.7.1.The GO function enrichment analysis and KEGG pathway enrichment analysis of the core target were carried out based on DAVID database.3.The therapeutic mechanism of Buzhengxuguwan in the intervention of steroid induced necrosis of the femoral head in rats based on TLR4/MyD88/NF-κB signaling pathwayAdult male SD rats were randomly divided into blank group,model group,western medicine group,Buzhengxuguwan low dose group(TCM low dose group),Buzhengxuguwan medium dose group(TCM medium dose group)and Buzhengxuguwan high dose group(TCM high dose group),with 12 rats in each group.Steroid induced necrosis of the femoral head was induced by methylprednisolone sodium succinate for injection combined with lipopolysaccharide.After successful modeling,rats in the blank group and the model group were given the same amount of physiological saline twice a day,rats in the western medicine group were given 0.9mg/kg alendronate sodium tablets suspension once a day,rats in the TCM low-dose group were given 0.045g/kg Buzhengxuguwan suspension twice a day,rats in the TCM medium dose group were given 0.09g/kg Buzhengxuguwan suspension twice a day,rats in the TCM high-dose group were given 0.18g/kg Buzhengxuguwan suspension twice a day.The pathological changes of femoral head were observed by HE staining;the expression levels of IL-1β,IL-6 and TNF-α in serum were detected by ELISA;the expression levels of TLR4,MyD88 and NF-κB p65 protein in femoral head were detected by Western-blotting;the expression levels of TLR4,MyD88 and NF-κB p65 protein in femoral head were detected by immunohistochemistry;the expression levels of TLR4,MyD88 and NF-κB p65 mRNA were detected by qRT-PCR.Results:1.The clinical effect of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral headThere was no significant difference between the two groups in gender,age,course of disease,ARCO stage,disease location and other aspects of demographic data and clinical characteristics(P>0.05).After treatment,the clinical effective rate of the observation group was significantly higher than that of the control group(P<0.05).After treatment,Harris hip function score and SF-36 score of the two groups were significantly higher than those before treatment(P<0.05),and the increase degree of Harris hip function score and SF-36 score of the observation group was significantly better than those of the control group(P<0.05).After treatment,VAS scores of the two groups were significantly lower than that before treatment(P<0.05),and the decrease degree of VAS scores of the observation group were significantly better than that of the control group(P<0.05).After treatment,the effective rate of hip joint imaging index results in the observation group was significantly higher than that in the control group(P<0.05).The incidence of adverse reactions in the observation group was 2.56%(1/39),and that in the control group was 7.69%(3/39).There was no significant difference in the incidence of adverse reactions between the two groups(P>0.05).2.The molecular mechanism of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head based on network pharmacology75 kinds of active compounds and 548 corresponding targets of Buzhengxuguwan were retrieved through BATMAN-TCM analysis tool.427 targets related to the occurrence and development of steroid induced necrosis of the femoral head were finally included through Dig See database,OMIM database and Gene Cards database.548 active targets of Buzhengxuguwan and 427 targets of steroid induced necrosis of the femoral head were mapped by using Draw Venn Diagram.30 core targets of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head were screened through constructing PPI network by using STRING database.The compound-disease-target regulatory network of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head by using Cytoscape 3.7.1,21 core targets can be connected with no less than two active compounds,and 41 active compounds can be connected with no less than two core targets.Using DAVID6.8 to analyze the GO function of 30 core targets of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head,we can see that the functional pathways mainly involve steroid hormone receptor activity,oxidoreductase activity,lipid response,steroid hormone response,cell proliferation regulation,reactive oxygen species metabolism process,intimal system,mitochondria,organelles and so on.Using DAVID 6.8 to analyze the KEGG pathway of 30 core targets of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head,we can see that the pathway mainly involves HIF-1 signaling pathway,NF-κB signaling pathway,osteoclast differentiation,TGF-β signaling pathway,estrogen signaling pathway,TNF signaling pathway,apoptosis and so on.3.The therapeutic mechanism of Buzhengxuguwan in the intervention of steroid induced necrosis of the femoral head in rats based on TLR4/MyD88/NF-κB signaling pathwayThe pathomorphological observation of femoral head of rats in each group showed that,the bone trabecula was intact,normal in shape and regular in arrangement,with less empty bone lacunae,thicker cartilage layer and less fat vacuoles in the blank group;the bone trabecula were obviously sparse and thin,arranged disorderly,and the continuity was interrupted,a large number of empty bone lacunae appeared in the bone trabecula,the cartilage layer became thinner,the adipocytes increased significantly,increased or fused with each other to form large fat vacuoles in the model group;the bone trabecula became thinner,arranged regularly and interrupted occasionally,empty bone lacunae were found in the bone trabecula,cartilage layer was relatively thick,fat cells accumulated,and fat vacuoles were common in the western medicine group;the bone trabecula was slightly thinner,arranged disorderly,and partially interrupted,with more empty bone lacunae in the bone trabecula,thinner cartilage layer,and more fat vacuoles in the TCM low dose group;the bone trabecula became thinner,arranged regularly and interrupted occasionally,empty bone lacunae were found in the bone trabecula,cartilage layer was relatively thick,fat cells accumulated,and fat vacuoles were common in the TCM medium dose group;the bone trabecula was relatively complete and arranged regularly,with empty bone lacunae,relatively thick cartilage layer and fat vacuoles in the TCM high dose group.The results of empty bone lacuna rate of femoral head tissue showed that compared with the blank group,the empty bone lacuna rate of other groups was significantly increased(P<0.01);compared with the model group,the empty bone lacuna rate of the western medicine group and TCM other dose group was significantly decreased(P<0.01);compared with the western medicine group,the empty bone lacuna rate of the TCM low dose group was significantly increased(P<0.01),and of the TCM high dose group was significantly decreased(P<0.01).The expression levels of IL-1β,IL-6 and TNF-α in serum of rats in each group showed that,compared with the blank group,the expression levels of IL-1β,IL-6 and TNF-α in serum of other groups were significantly increased(P<0.01);compared with the model group,the expression levels of IL-1β,IL-6 and TNF-α in serum of the western medicine group and TCM other dose group were significantly decreased(P<0.01);compared with the western medicine group,the expression levels of IL-1β,IL-6 and TNF-α in serum of the TCM low dose group was significantly increased(P<0.01),and of the TCM high dose group was significantly decreased(P<0.01).The protein expression levels of TLR4,MyD88 and NF-κB p65 in femoral head of rats in each group showed that,compared with the blank group,the protein expression levels of TLR4,MyD88 and NF-κB p65 in femoral head of other groups were significantly increased(P<0.01);compared with the model group,the protein expression levels of TLR4 and MyD88 in femoral head of the western medicine group,TCM medium dose group and TCM high dose group were significantly decreased(P<0.01),and the protein expression levels of NF-κB p65 in femoral head the western medicine group and TCM other dose group were significantly decreased(P<0.05 or P<0.01);compared with the western medicine group,the protein expression levels of TLR4,MyD88 and NF-κB p65 in femoral head of the TCM low dose group was significantly increased(P<0.01),the protein expression levels of MyD88 and NF-κB p65 in femoral head of the TCM high dose group was significantly decreased(P<0.05 or P<0.01);compared with the TCM medium dose group,the protein expression levels of TLR4 in femoral head of the TCM high dose group was significantly decreased(P<0.05).The mRNA expression levels of TLR4,MyD88 and NF-κB p65 in femoral head of rats in each group showed that,compared with the blank group,the mRNA expression levels of TLR4,MyD88 and NF-κB p65 in femoral head of other groups were significantly increased(P<0.05 or P<0.01);compared with the model group,the mRNA expression levels of TLR4,MyD88 and NF-κB p65 in femoral head of the western medicine group and TCM other dose group were significantly decreased(P<0.01);compared with the western medicine group,the mRNA expression levels of TLR4,MyD88 and NF-κB p65 in femoral head of the TCM low dose group was significantly increased(P<0.01).Conclusion:1.Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head is effective,safe and reliable.It can effectively improve the hip joint function of steroid induced necrosis of the femoral head patients,relieve the pain symptoms of steroid induced necrosis of the femoral head patients,and improve the quality of life of steroid induced necrosis of the femoral head patients,which is worthy of further clinical application.2.The overall regulation of using Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head multi-component,multi-target and multi-pathway was preliminarily discussed and verified based on network pharmacology,and predicted that TLR4/MyD88/NF-κB signaling pathway regulating the inflammatory process may be the potential mechanism of Buzhengxuguwan in the treatment of steroid induced necrosis of the femoral head.3.Buzhengxuguwan in the intervention of steroid induced necrosis of the femoral head in rats can significantly improve the pathological morphology of femoral head and reduce the empty bone lacuna rate,decrease the expression levels of serum IL-1β,IL-6 and TNF-α,protein and mRNA TLR4,MyD88 and NF-κB p65 in femoral head.Its mechanism of action is through the inhibition of TLR4/MyD88/NF-κB signaling pathway,thereby reducing the inflammatory reaction and play a preventive role.