基于数据挖掘和实验验证探讨舒筋健腰丸干预黄韧带肥厚腰椎管狭窄症的作用机制

The Mechanism of Shujin Jianyao Pills in Inhibiting Hypertrophy of the Ligamentum Flavum Based on Data Mining and Experimental Validation

目的结合数据挖掘和实验验证,探讨舒筋健腰丸抑制黄韧带肥厚的作用机制。方法借助TCMSP和TCMID数据平台筛选舒筋健腰丸的有效活性成分和靶点,利用GEOquery包获取GEO数据库中黄韧带肥厚腰椎管狭窄症的差异表达蛋白芯片数据,提取药物与疾病的共同靶点,利用STRING数据库构建蛋白质相互作用网络,利用Cytoscape 3.7.1软件对关键靶点进行网络拓扑学分析。再利用DAVID在线工具对核心靶点进行GO富集分析和KEGG信号通路分析,以探究舒筋健腰丸干预黄韧带肥厚腰椎管狭窄症可能的分子机制和关键靶点。建立大鼠黄韧带肥厚模型,随机将大鼠分为正常组、模型组、氯磷酸盐组(阳性药物组,Clodronate Liposomes,5 mg·mL^(-1),每次0.5 mL)和中药组(60 kg人用药等效剂量,1.5 g·kg^(-1)·d^(-1)),采取预防性灌胃给药策略,于造模8周后取材。利用HE和Masson染色从组织病理学角度评估对舒筋健腰丸黄韧带肥厚的干预作用,利用免疫组化法检测PPI网络中关键蛋白表达。结果网络药理学方法筛选出舒筋健腰丸干预黄韧带肥厚的靶点77个,利用网络拓扑参数筛选出关键蛋白TGF-β_(1)、LOXL2、TNF-α、IL-6和IL-1β。GO富集和KEGG通路分析表明该药可能通过炎症因子的调控、细胞外基质调节和胶原蛋白的分泌等发挥作用。通过HE和Masson染色,与模型组相比,中药组显著抑制大鼠肥厚黄韧带的病理产物堆积,抑制其胶原容积分数增高(P<0.05),但其抑制作用不及氯磷酸盐组(P<0.05);通过免疫组化分析,与模型组相比,中药组能够显著抑制关键蛋白TGF-β_(1)、LOXL2、TNF-α、IL-6和IL-1β的表达(P<0.05)。结论舒筋健腰丸可能通过抑制TGF-β_(1)、LOXL2、TNF-α、IL-6和IL-1β蛋白的表达发挥干预黄韧带肥厚腰椎管狭窄症的作用。

Objective Study on the mechanism of Shujin Jianyao Pills in inhibiting hypertrophy of the ligamentum flavum based on data mining and experimental validation.Methods Screening the active ingredients and targets of Shujin Jianyao pills with the help of tcmsp and tcmid data platforms.Differential expression protein microarray data of hypertrophic ligamentum flavum in GEO database were accessed using the GEO query package.Extracting common targets of TCMs and diseases by research teams.The STRING database was used to construct the protein interaction network,and the software Cytoscape 3.7.1 was used to topologically analyze the key targets.Go enrichment analysis and KEGG signaling pathway analysis of the core targets were performed using the David online tool to explore the possible molecular mechanisms and key targets of the intervention of Shujin Jianyao Pills on hypertrophy of the ligamentum flavum.Using a rat model of hypertrophy of the ligamentum flavum,the research team randomly divided the rats into four groups:normal,model,clodronate liposomes(5 mg·mL,0.5 mL),and TCM(1.5 g·kg^(-1)·d^(-1)).Rats were sacrificed after eight weeks of modeling using a prophylactic intragastric administration strategy.Histopathological evaluation of the intervention effect of Shujin Jianyao Pills on hypertrophy of the ligamentum flavum using HE and Masson staining.Immunohistochemistry was used to detect the key protein expression in the PPI network.Results 77 targets of Shujin Jianyao pills for intervention of hypertrophic ligamentum flavum were identified.The key protein TGF-β_(1),LOXL2,TNF-α,IL-6 and IL-1βwere also identified.Go enrichment and KEGG pathway analysis indicated that the drug might act through the regulation of inflammatory factors,extracellular matrix regulation,and collagen secretion.By HE and Masson staining,compared with the model group,the TCM group significantly inhibited the accumulation of pathological products and inhibited the increase of collagen volume fraction in the hypertrophic ligamentum flavum of rats(P<0.05),but its inhibitory effect was less pronounced than that in the Chlorophosphate group(P<0.05);By immunohistochemical analysis,compared with the model group,the TCM group was able to significantly inhibit TGF-β_(1),LOXL2,TNF-α,IL-6 and IL-1β(P<0.05).Conclusion Shujin Jianyao pills may play a role in the treatment of hypertrophic lumbar spinal stenosis through inhibiting the expression of TGF-β_(1),LOXL2,TNF-α,IL-6 and IL-1β.