α-乳香酸灌胃对小鼠肾间质纤维化的防治作用及其机制探讨

摘要

Objective To investigate the preventive and therapeutic effects of α-boswellic acid on renal interstitial fi-brosis in mice and its mechanism.Methods A total of 52 male C57/BL mice were divided into three groups: sham group,model group,and boswellic acid group.The unilateral ureter separation was performed in mice of the sham group, and mice in the other two groups underwent unilateral ureteral obstruction(UUO)to induce renal interstitial fibrosis.The mice in the boswellic acid group were administered orally 60 mg/kg α-boswellic acid after surgery for 21 days.The blood and kidney tissues in each mouse were collected after anesthesia.The levels of urea nitrogen(BUN), creatinine(Scr), malondialdehyde(MDA),and total superoxide dismutase(t-SOD)in serum were detected.The pathological changes of kidney were observed by HE and MASSON staining.Western blotting was used to detect the protein expression of α-SMA, TGF-β1,Smad2/3,and Smad7 in the renal tissues.qPCR was used to detect the mRNA expression of α-SMA,TGF-β1, Smad2/3,and Smad7 in the renal tissues.HK-2 cells were cultured and then were divided into the control group,the ex-perimental group 1 and experimental group 2.Cells in the group 1 and group 2 were cultured under hypoxia condition for 48 h to make the cell models of renal interstitial fibrosis.Before hypoxia treatment,25 μg/mL α-boswellic acid was added in the group 2.Western blotting was used to detect the protein expression of α-SMA,TGF-β1,Smad2/3,and Smad7 in each group at 48 h after culturing.Results The levels of serum BUN, Scr, and MDA in the model group were higher than those of the sham group,and the level of SOD was lower than that of the sham group(all P<0.01).The levels of serum BUN,Scr,and MDA in the boswellic acid group were lower than those of the sham group,and the level of SOD was higher than that of the model group(all P<0.05).The pathological changes indicated that the pathological injuries and degree of renal tubulointerstitial fibrosis in the boswellic acid group significantly relieved as compared with that of the model group. The mRNA and protein expression levels of TGF-β1,Smad2,Smad3,α-SMA in the kidney tissues of the model group were higher than those of the sham group, and the mRNA and protein expression of Smad 7 was lower than that of the sham group;the mRNA and protein expression levels of TGF-β1,Smad2,Smad3,and α-SMA in the boswellic acid group were lower than those of the model group, and the mRNA and protein expression of Smad 7 was higher than that of the model group(all P<0.05).The relative expression of TGF-β1,Smad2,Smad3,and α-SMA in the group 1 was higher than that of the control group,and the relative expression of Smad7 was lower than that of the control group;the relative expression of TGF-β1,Smad2,Smad3,and α-SMA in the group 2 was lower than that of the group 1,and the relative expression of Smad7 was higher than that of the group 1(all P<0.05).Conclusion The α-boswellic acid has preventive and thera-peutic effects on the renal interstitial fi-brosis in mice,and the mechanism may be involved in the down-regulation of TGF-β1,Smad2,Smad3,α-SMA,and the up-regulation of Smad7.%目的 观察α-乳香酸灌胃对小鼠肾间质纤维化的防治作用,并探讨其机制.方法 雄性C57/BL小鼠52只,分为假手术组、模型组和乳香酸组.假手术组行单侧输尿管分离术,其余两组行单侧输尿管结扎术诱导肾间质纤维化.乳香酸组术后第1天开始给予α-乳香酸60 mg/kg灌胃,连续21 d.将各组小鼠麻醉后取血液、肾脏组织.检测血清尿素氮(BUN)、肌酐(Scr)、丙二醛(MDA)和总超氧化物歧化酶(t-SOD);采用HE染色和MASSON染色观察各组小鼠肾脏组织病理变化;采用Western blotting 法检测各组小鼠肾脏组织中的TGF-β1、Smad2、Smad3、Smad7、α-SMA蛋白,采用qPCR法检测TGF-β1、Smad2、Smad3、Smad7、α-SMA mRNA.培养HK-2细胞并分为对照组、实验1组、实验2组,实验1、2组下缺氧条件下培养48 h制作肾间质纤维化细胞模型.实验2组缺氧处理前加入25 μg/mL的α-乳香酸.培养48 h后采用Western blotting 法检测各组细胞中的TGF-β1、Smad2、Smad3、Smad7、α-SMA蛋白.结果 模型组小鼠血清BUN、Scr、MDA水平高于假手术组,SOD水平低于假手术组(P均<0.01).乳香酸组小鼠血清BUN、Scr、MDA水平低于模型组,SOD水平高于模型组(P均<0.05).肾脏组织病理观察结果显示,乳香酸组病理改变及肾小管间质纤维化程度较模型组明显减轻.模型组小鼠肾脏组织中TGF-β1、Smad2、Smad3、α-SMA mRNA及蛋白相对表达量高于假手术组,Smad7 mRNA及蛋白相对表达量低于假手术组;乳香酸组小鼠肾脏组织中TGF-β1、Smad2、Smad3、α-SMA mRNA及蛋白相对表达量低于模型组,Smad7 mRNA及蛋白相对表达量高于模型组(P均<0.05).实验1组细胞中TGF-β1、Smad2、Smad3、α-SMA相对表达量高于对照组,Smad7相对表达量低于对照组;实验2组TGF-β1、Smad2、Smad3、α-SMA相对表达量低于实验1组,Smad7相对表达量高于实验1组(P均<0.05).结论 α-乳香酸对小鼠肾间质纤维化有防治作用,作用机制可能与TGF-β1、Smad2、Smad3、α-SMA表达下调及Smad7表达上调有关.