糖肾灌肠方通过PI3K/AKT/NF-κB信号通路调节糖尿病肾病炎症反应的机制研究

Tangshen Guanchang(糖肾灌肠)Prescription Reduces Inflammation in Diabetic Kidney Disease via Regulating PI3K/AKT/NF-kB Signaling Pathway

目的:糖尿病肾病(Diabetic kidney disease,DKD)是由高血糖引起的慢性肾脏疾病,炎症反应在DKD中起着关键作用,糖肾灌肠方治疗DKD疗效显著,但作用机制尚不清楚。本研究采用体内实验、临床试验、网络药理学及分子对接等方法对糖肾灌肠方治疗DKD的作用机制进行研究。方法:选取60只C57小鼠,随机分为正常对照组、模型对照组、卡格列净0.013 g/kg组、糖肾灌肠方52 g/kg组。高糖高脂饲养并腹腔注射链脲佐菌素(Streptozotocin,STZ)诱导形成DKD模型。造模成功小鼠给予糖肾灌肠方组灌肠,阳性对照组灌胃给予药物,连续28 d。HE染色观察DKD小鼠肾组织病理形态学改变;通过RT-qPCR、Western blot法检测小鼠肾组织Pi3k、Akt、Nfkb、Tnfa、Il6的mRNA及蛋白表达。将40例符合纳入标准的DKD患者随机分为基础治疗对照组和观察组,各20例,另纳入健康体检者20例为正常对照组。基础治疗对照组应用西医基础治疗,观察组在其基础上联合糖肾灌肠方治疗,两组患者连续用药2 w后,比较两组患者尿微量白蛋白(mALB)、尿蛋白/肌酐(UACR)、巨噬细胞M1水平、TNF-α、IL-6含量;以糖肾灌肠方中药物的名称及“糖尿病肾病”为关键词,搜索相关数据库进行网络药理学及分子对接分析。结果:动物实验显示,与正常对照组比较,HE染色显示肾组织病理损伤明显加重,肾组织中Pi3k、Akt、Nfkb、Tnfa、Il6 mRNA及蛋白表达显著上调(P<0.01);与模型对照组相比,糖肾灌肠方52 g/kg组小鼠HE染色显示肾组织病理损伤得到改善,肾组织中Pi3k、Akt、Nfkb、Tnfa、Il6 mRNA及蛋白表达显著下调(P<0.01)。临床研究显示,经治疗,观察组相较于基础治疗组mALB、UACR含量、巨噬细胞M1水平、TNF-α、IL-6含量下降更为明显(P<0.01)。通过网络药理学研究,糖肾灌肠方筛选出活性成分118个,主要包括芦荟大黄素、水黄皮素等,发现核心靶点TNF-α、IL-6等,GO富集在BP中主要包括炎症反应等;MF主要包括蛋白结构域特异性结合等;CC主要包括细胞外空间等;KEGG富集发现PI3K/AKT等信号通路能在糖肾灌肠方治疗DKD中起关键作用。结论:糖肾灌肠方可能通过调控PI3K/AKT/NF-κB信号通路的表达及TNF-α、IL-6等炎症因子的释放,干预DKD炎症反应发挥治疗作用。

Objective:Diabetic kidney disease(DKD)is a chronic kidney disease caused by hyperglycemia,in which inflammation plays a key role.Tangshen Guanchang(糖肾灌肠)Prescription is effective in the treatment of DKD,the mechanism of which,however,remains unclear.To decipher the mechanism of Tangshen Guanchang Prescription in the treatment of DKD by animal experiment,clinical trial,network pharmacology,and molecular docking.Methods:Sixty C57 mice were randomized into normal control,model control,canagliflozin(0.013 g/kg),and Tangshen Guanchang Prescription(52 g/kg)groups.The mouse model of DKD was induced by a high-glucose and high-fat diet combined with intraperitoneal injection of streptozotocin(STZ).The successfully modeled mice were administrated with corresponding drugs for 28 consecutive days.The pathological changes in the renal tissue of DKD mice were observed by hematoxylin-eosin(HE)staining.The mRNA and protein levels of phosphatidylinositol 3-kinase(Pi3k),protein kinase B(Akt),nuclear factor-kappa B(Nfkb),tumor necrosis factor(Tnf)-α,and interleukin(Il)-6 in the renal tissue were determined by RT-qPCR and Western blotting,respectively.Furthermore,40 DKD patients meeting the inclusion criteria were randomized into a control group(n=20)and an observation group(n=20),and other 20 healthy volunteers(physical examination)were included as the normal control group.The control group was treated with Western medicine(routine treatment),and the observation group with Tangshen Guanchang Prescription on the basis of routine treatment.After two weeks of treatment,the levels of microalbumin(mALB),urinary albumin-to-creatinine ratio(UACR),M1 macrophages,TNF-α,and IL-6 were compared between the two groups.With the herbal names in Tangshen Guanchang Prescription and"diabetic kidney disease"as keywords,the relevant databases were searched for network pharmacology and molecular docking.Results:The animal experiments showed that compared with the normal control group,the model control group presented aggravated pathological injuries and up-regulated mRNA and protein levels of Pi3k,Akt,Nfkb,Tnf-α,and Il-6 in the renal tssue(P<0.01).Compared with the model control group,Tangshen Guanchang Prescription(52 g/kg)mitigated the pathological injuries and down-regulated the mRNA and protein levels of Pi3k,Akt,Nfkb,Tnf-α,and Il-6(P<0.01).The clinical trial showed that after treatment,the observation group demonstrated more significant decreases in mALB,UACR,M1 macrophages,TNF-α,and IL-6 than the control group(P<0.01).The network pharmacology predicted 118 active components(including aloe-emodin and karanjin)in Tangshen Guanchang Prescription,and the core targets included TNF-αand IL-6.Gene Ontology(GO)enrichment showcased that the targets involved biological processes such as inflammation,molecular functions such as specific binding to protein domains,and cell components such as extracellular space.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment showed that PI3K/AKT signaling pathways played a key role in the treatment of DKD with Tangshen Guanchang Prescription.Conclusion:Tangshen Guanchang Prescription may reduce inflammation in DKD by regulating the expression of PI3K/AKT/NF-kB signaling pathway and the release of inflammatory cytokines such as TNF-αand IL-6.