摘要
目的:探讨微小核糖核酸126(miRNA126)调控磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路在慢性肾脏病合并动脉粥样硬化(CKD AS)中的作用,以及肾衰泄浊汤干预5/6肾切除术联合高脂饲养的CKD AS大鼠的作用机制。方法:将60只SD大鼠随机分为假手术组、模型组、氯沙坦组、肾衰泄浊汤低、中、高剂量组。通过5/6肾切除术联合高脂饲养10周建立大鼠CKD AS模型,肾衰泄浊汤低、中、高剂量组(6.0、12.0、24.0 g·kg^(-1)·d^(-1)),氯沙坦组(20 mg·kg^(-1)·d^(-1))剂量灌胃,进行相应的干预8周,然后处死大鼠,取材进行相应检测。全自动生化分析仪器检测大鼠肾功能和血脂:血肌酐(SCr)、尿素氮(BUN)、总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)水平;苏木素-伊红(HE)、马松(Masson)染色主动脉组织并在光学显微镜下进行病理学观察;通过透射电子显微镜观察自噬体和自噬溶酶体;实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠miRNA126、PI3K、Akt、mTOR mRNA水平;蛋白免疫印迹法(Western blot)检测大鼠磷酸化(p)-PI3K、PI3K、p-Akt、Akt、p-mTOR、mTOR、苄氯素-1(Beclin-1)、微管相关蛋白1轻链3Ⅱ/Ⅰ(LC3Ⅱ/LC3Ⅰ)蛋白表达。结果:与假手术组比较,模型组大鼠血清SCr、BUN、TC、TG、LDL-C均显著升高(P<0.01);与模型组比较,氯沙坦组与肾衰泄浊汤低、中、高剂量组大鼠SCr、BUN、TC、TG、LDL-C明显降低(P<0.05);与假手术组比较,模型组大鼠内膜可见增厚斑块形成,单核巨噬细胞浸润,少量泡沫细胞,中膜平滑肌纤维排列紊乱,胶原纤维增多,氯沙坦组与肾衰泄浊汤各组较模型组病变减轻;与模型组比较,肾衰泄浊汤中、高剂量组中电镜的自噬小体及自噬溶酶体的数量增多;与假手术组比较,模型组大鼠主动脉组织miRNA126表达显著下降(P<0.01),PI3K、Akt、mTOR mRNA表达显著升高(P<0.01)。与模型组比较,肾衰泄浊汤高、中、低剂量组及氯沙坦组大鼠主动脉组织miRNA126表达显著升高(P<0.01),PI3K、Akt、mTOR mRNA表达显著降低(P<0.01)。与假手术组比较,模型组p-PI3K、PI3K、p-Akt、Akt、p-mTOR、mTOR蛋白表达显著升高(P<0.01),Beclin-1、LC3Ⅰ、LC3Ⅱ蛋白水平显著降低(P<0.01)。与模型组比较,氯沙坦组和肾衰泄浊汤低、中、高剂量组的p-PI3K、PI3K、p-Akt、Akt、p-mTOR、mTOR蛋白表达降低(P<0.05),Beclin-1、LC3Ⅱ/LC3Ⅰ蛋白水平升高(P<0.05)。结论:CKD AS大鼠主动脉组织miRNA126表达降低,激活PI3K/Akt/mTOR通路抑制自噬通量;肾衰泄浊汤通过miRNA126调控PI3K/Akt/mTOR信号通路,恢复主动脉内皮细胞的自噬,保护CKD血管损伤,减少As斑块的形成,减缓心血管并发症的发展。
Objective:Exploring the role of microRNA126(miRNA126)in chronic kidney disease combined with atherosclerosis(CKD AS)by regulating the phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway and the mechanism of Shenshuai Xiezhuo decoction in the intervention of CKD AS rats with 5/6 nephrectomy combined with high-fat feeding.M ethod:A total of 60 SD rats were randomly divided into sham operation group,model group,losartan group,and low,medium,and high dose groups of Shenshuai Xiezhuo decoction.The CKD AS rat model was established by 5/6nephrectomy combined with high-fat feeding for 10 weeks.The low,medium,and high dose groups(6.0,12.0,24.0 g·kg^(-1)·d^(-1))of Shenshuai Xiezhuo decoction and the losartan group(20 mg·kg^(-1)·d^(-1))were gavaged,and the corresponding intervention was carried out for eight weeks.Then,the rats were killed,and samples were collected for corresponding detection.Fully automated biochemical analyzers were used to detect kidney function and blood lipids in rats:blood creatinine(SCr),blood urea nitrogen(BUN),total cholesterol(TC),triglyceride(TG),and low-density lipoprotein cholesterol(LDL-C)levels.Hematoxylin-eosin(HE)and Masson staining of aortic tissue and pathological observation under a light microscope were carried out,and autophagosomes and autophagy lysosomes were observed by transmission electron microscopy.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to determine the mRNA levels of miRNA126,PI3K,Akt,and mTOR in rats,and Western blot was used to determine the protein expression levels of phosphorylated(p)-PI3K,PI3K,p-Akt,Akt,p-mTOR,mTOR,benzyl chloride 1(Beclin-1),and microtubule-associated protein light chain 3II/I(LC3II/LC3I).Result:Compared with the sham operation group,the serum SCr,BUN,TC,TG,and LDL-C in the model group were significantly increased(P<0.01).Compared with the model group,the SCr,BUN,TC,TG,and LDL-C were decreased in the losartan group and low,medium,and high dose groups of Shenshuai Xiezhuo decoction(P<0.05).Compared with the sham operation group,thickening plaques,infiltration of mononuclear macrophages,a small number of foam cells,disordered arrangement of smooth muscle fibers in the tunica media,and increased collagen fibers were observed in the model group,and the lesions in the losartan group and Shenshuai Xiezhuo decoction groups were alleviated compared with those in the model group.Compared with the model group,the number of autophagosomes and autophagy lysosomes increased in the medium and high dose groups of Shenshuai Xiezhuo decoction.Compared with the sham operation group,the expression of miRNA126 in the aortic tissue of the model group was significantly decreased(P<0.01),and the mRNA expressions of PI3K,Akt,and mTOR were significantly increased(P<0.01).Compared with the model group,the expression of miRNA126 in the aortic tissue of rats in high,medium,and low dose groups of Shenshuai Xiezhuo decoction and losartan group was significantly increased(P<0.01),while the mRNA expressions of PI3K,Akt,and mTOR were significantly decreased(P<0.01).Compared with the sham operation group,the protein expressions of p-PI3K,PI3K,p-Akt,Akt,p-mTOR,and mTOR in the model group were significantly increased(P<0.01),while the protein levels of Beclin-1,LC3I,and LC3II were significantly decreased(P<0.01).Compared with the model group,the protein expressions of p-PI3K,PI3K,p-Akt,Akt,p-mTOR,and mTOR in the losartan group and low,medium,and high dose groups of Shenshuai Xiezhuo decoction were decreased(P<0.05),while the protein levels of Beclin-1 and LC3II/LC3I were increased(P<0.05).Conclusion:The expression of miRNA126 is decreased in the aortic tissue of CKD AS rats,and the PI3K/Akt/mTOR pathway is activated to inhibit autophagy flux.Shenshuai Xiezhuo decoction regulates the PI3K/Akt/mTOR signaling pathway through miRNA126,restores the autophagy of aortic endothelial cells,protects the damage of CKD vessels,reduces the formation of As plaques,and slows the development of cardiovascular complications.
作者
刘庚鑫
张格第
袁文萋
李福生
郭敏
罗富里
晏子友
LIU Gengxin;ZHANG Gedi;YUAN Wenqi;LI Fusheng;GUO Min;LUO Fuli;YAN Ziyou(Jiangxi University of Traditional Chinese Medicine(TCM),Nanchang 330004,China;Affiliated Hospital of Jiangxi University of TCM,Nanchang 330000,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第9期48-55,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(82260908)。
作者简介
第一作者:刘庚鑫,在读博士,从事中医肾病临床与实验研究,E-mail:614289274@qq.com;通信作者:晏子友,博士,教授,博士生导师,从事中医肾病临床与实验研究,E-mail:zysbkfsyy@163.com。
