白皮杉醇对糖尿病肾病大鼠ERK1/2通路及CTGF蛋白表达的影响

目的：探究白皮杉醇（PIC）对糖尿病肾病（DN）大鼠细胞外调节蛋白激酶（ERK1/2）通路及其调控的结缔组织生长因子（CTGF）蛋白表达的影响。方法：取健康雄性SD大鼠，采用高脂高糖喂养+腹腔注射链脲霉素建立大鼠DN模型，并将造模成功的50只SD大鼠随机分为模型组（DN组）、PIC低（50 mg·kg^-1）、中（100 mg·kg^-1）、高（200 mg·kg^-1）剂量组，阳性对照组（厄贝沙坦25 mg·kg^-1），每组10只；另取10只大鼠，正常饲养，作为正常对照组。各组均于造模成功后开始给药，PIC低、中、高剂量组及阳性对照组分别灌胃给予相应剂量的PIC及厄贝沙坦溶液，正常对照组和DN组灌胃给予10 mL·kg^-1生理盐水，各组大鼠连续给药4周，每天1次。给药期间观察大鼠饮食状况，于末次给药12 h后，称重、收集大鼠尿液，麻醉处死大鼠，取腹主动脉血和肾脏组织；用酶联免疫吸附（ELISA）试剂盒检测血液肾功能指标血清肌酐（Scr）、血清尿素氮（BUN）、空腹血糖及尿液中24 h尿蛋白量及空腹血糖（FBG）含量；取肾脏组织，称重，并用苏木精-伊红染色法检测大鼠肾脏组织病理形态；用黄嘌呤氧化酶法、硫代巴比妥酸缩合法检测肾组织氧化指标超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量；用蛋白免疫印迹法（Western Blot）检测肾脏组织缺氧诱导因子-1α（HIF-1α）、血红素加氧酶-1（HO-1）、ERK1/2、CTGF蛋白相对表达水平。结果：与正常对照组相比，DN组大鼠精神萎靡、形体消瘦及肾小球基底膜增厚、肾小管扩张、肾间质炎性浸润等病理损伤严重，肾脏/体质指数、FBG、血清Scr和BUN、尿液中24 h尿蛋白量、肾脏组织中MDA含量、HIF-1α、HO-1、p-ERK1/2、CTGF蛋白表达升高（P<0.05），SOD活性明显降低（P<0.05）；与DN组相比，PIC低、中、高剂量组及阳性对照组大鼠精神萎靡、形体消瘦及肾小球基底膜增厚、肾小管扩张、肾间质炎性浸润等病理损伤现象缓解，肾脏/体质指数、FBG、血清Scr和BUN、尿液中24 h尿蛋白量、肾脏组织中MDA含量、HIF-1α、p-ERK1/2、CTGF蛋白表达降低（P<0.05），HO-1、SOD活性明显升高（P<0.05），且PIC各剂量组上述指标呈剂量依赖性。阳性对照组上述指标变化与PIC组相比差异无统计学意义。结论：PIC可抑制ERK1/2-CTGF信号通路激活，升高HO-1表达、降低HIF-1α表达，抑制肾脏氧化应激损伤，改善DN大鼠肾脏损伤

Effect of piceatannol on ERK1/2 pathway and expression of CTGF protein in diabetic nephropathy rats

OBJECTIVE To explore the effect of piceatannol(PIC)on the pathway of extracellular regulatory protein kinase-1/2(ERK1/2)and its regulated expression of connective tissue growth factor(CTGF)protein in diabetic nephropathy(DN)rats.METHODS Healthy male SD rats were selected and DN model was established by feeding high-fat and high-glucose along with an intraperitoneal injection of streptozotocin.Fifty rats were randomly divided into model group(DN),low-dose(50 mg·kg-1),medium-dose(100 mg·kg-1),high-dose(200 mg·kg-1)PIC groups and positive control group(irbesartan 25 mg·kg-1)(n=10 each).Another 10 rats in normal control group were fed normally.After successful modeling, low/middle/high-dose PIC groups received the corresponding doses of PIC and irbesartan solution by gavage while control and DN groups had 10 mL·kg-1 normal saline by gavage.All rats were administrated once daily for 4 weeks.Dietary status was observed during dosing.Rats were weighed and urine was collected at 12 h after the last dosing.After anesthetizing and sacrificing, abdominal aortic blood and renal tissue were collected.Serum contents of creatinine(Scr)and urea nitrogen(BUN),24 h urine protein and fasting blood glucose(FBG)were measured by enzyme-linked immunosorbent assay(ELISA).Renal tissue was harvested and weighed.Renal pathomorphology was detected by hematoxylin-eosin stain.And xanthine oxidase method and thiobarbituric acid condensation were utilized for detecting the activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA).Western blot was employed for detecting the relative protein expression levels of hypoxia inducible factor-1α(HIF-1α),heme oxygenase-1(HO-1),ERK1/2 and CTGF in renal tissues.RESULTS Compared with those in normal control group, pathological damage was serious in DN group, such as mental depression, body emaciation, glomerular basement membrane thickening, renal tubule dilatation and renal interstitial inflammatory infiltration.kidney/body mass index, FBG,serum Scr/BUN,content of 24 h urine protein, content of MDA and protein expression of HIF-1α,HO-1,p-ERK1/2 and CTGF were higher(P<0.05)while the activity of SOD decreased markedly(P<0.05);as compared with those in DN group, pathological damage was relieved in low/medium/high-dose PIC and positive control groups, such as mental depression, body emaciation, glomerular basement membrane thickening, renal tubule dilatation and renal interstitial inflammatory infiltration.Kidney/body mass index, FBG,serum Scr/BUN,content of 24 h urine protein, content of MDA and protein expression of HIF-1α,p-ERK1/2 and CTGF declined(P<0.05)while the activity of SOD/HO-1 became stronger(P<0.05).In addition, the above parameters of PIC groups were dose-dependent.No significant difference existed between positive control and PIC groups.CONCLUSION PIC can inhibit the activation of ERK1/2-CTGF signal pathway, up-regulate the expression of HO-1,down-regulate the expression of HIF-1α,suppress the oxidative stress injury of kidney and improve renal injury of DN rats.