电针对自发性高血压大鼠肾组织转化生长因子-β1及肾小管上皮间充质转化的影响

Effect of electroacupuncture on transforming growth factor-β1 and renal tubular epithelial mesenchymal transformation in renal tissue of spontaneously hypertensive rats

目的:观察电针对自发性高血压大鼠(SHR)肾损害纤维化及肾组织转化生长因子-β1(TGF-β1)与上皮间充质转化(EMT)标志蛋白表达的影响,探讨电针减缓高血压肾损害的可能机制。方法:SHR随机分为模型组、氯沙坦组及针刺组,每组8只;Wistar-Kyoto大鼠8只为正常组。氯沙坦组予氯沙坦钾溶液(30 mg·kg-1·d-1)灌胃,针刺组电针双侧“肾俞”“膈俞”,每次15 min,治疗均隔日1次,治疗12周。治疗前及治疗4、8、12周末测量各组大鼠尾动脉收缩压;治疗前及治疗6、12周末测量各组大鼠24 h尿蛋白定量;HE染色法观察肾组织病理形态变化,PAS染色法观察肾小球与肾小管基底膜等细胞外基质,MASSON染色法观察肾组织基底膜及胶原纤维并计算胶原容积分数;实时荧光定量PCR法测定肾组织TGF-β1 mRNA表达;免疫组织化学法测定肾组织TGF-β1及EMT标志物钙黏蛋白E(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、纤连蛋白(FN)表达。结果:治疗前与治疗4、8、12周,模型组大鼠尾动脉收缩压显著高于正常组(P<0.01);治疗4、8、12周,氯沙坦组与针刺组尾动脉收缩压显著低于模型组(P<0.01,P<0.05),治疗4周氯沙坦组尾动脉收缩压显著低于针刺组(P<0.01)。治疗前与治疗6、12周,模型组24 h尿蛋白定量显著高于正常组(P<0.01);治疗6、12周,氯沙坦组与针刺组24 h尿蛋白定量均显著低于模型组(P<0.01)。肾组织病理染色显示,模型组肾组织细胞排列较不规则,肾小管萎缩、管腔狭窄,细胞外基质增生沉积,肾小管间质胶原容积分数显著升高(P<0.01),瘢痕硬化形成;与模型组比较,氯沙坦组与针刺组肾小管萎缩、管腔狭窄减轻,细胞外基质增生沉积减少,肾小管间质胶原容积分数显著降低(P<0.01),瘢痕硬化改善。模型组肾组织TGF-β1 mRNA表达显著高于正常组(P<0.01);氯沙坦组与针刺组TGF-β1 mRNA表达显著低于模型组(P<0.05)。与正常组相比,模型组肾组织TGF-β1、α-SMA、FN阳性面积百分比显著升高(P<0.01),E-cadherin阳性面积百分比显著降低(P<0.01);与模型组相比,氯沙坦组与针刺组肾组织TGF-β1、α-SMA、FN阳性面积百分比显著降低(P<0.05,P<0.01),E-cadherin阳性面积百分比显著升高(P<0.01);与氯沙坦组相比,针刺组E-cadherin阳性面积百分比显著升高(P<0.01),α-SMA阳性面积百分比显著降低(P<0.01)。结论:电针可有效减缓SHR高血压及肾间质纤维化,其机制可能与降低肾组织中TGF-β1的表达、抑制EMT过程有关。

Objective To observe the effects of electroacupuncture(EA)on renal fibrosis,the expression of transforming growth factor-β1(TGF-β1)and epithelial mesenchymal transition(EMT)marker proteins in renal tissue of spontaneously hypertensive rats(SHR),so as to explore the underlying mechanism on EA alleviating hypertensive renal impairment.Methods Twenty-four male SHR were randomly divided into model group,losartan group and EA group,with 8 rats in each group,and eight male Wistar-Kyoto rats were taken as the normal group.Rats in the losartan group received gavage of losartan potassium solution(3 mg/mL,30 mg·kg-1·d-1),once every other day for 12 weeks.Rats in the EA group received EA stimulation at bilateral“Shenshu”(BL23)and“Geshu”(BL17)(2 Hz/15 Hz,1.0 mA),15 min each time,once every other day for 12 weeks.The systolic blood pressure of caudal artery was measured before,and 4,8 and 12 weeks after the intervention.The 24-hour urinary protein was measured before,and 6 and 12 weeks after the intervention.Histopathological changes of the left renal tissue were observed under light mircoscope after H.E.stain.Extracellular matrix(ECM)in renal tissues was observed after periodate Schiff staining.Basement membrane and collagen fibers were observed after Masson staining with collagen volume fraction(CVF)evaluated.The expression of TGF-β1 mRNA in the right renal was detected by real-time fluorescence quantitative PCR.The expression of TGF-β1 and EMT marker E-cadherin,α-SMA and fibronectin(FN)proteins in the left renal tissue was measured by immunohistochemistry.Results In the model group,irregular arrangement of nephrocytes,renal tubule atrophy,lumen stenosis,ECM hyperplasia and deposition,scar and sclerosis were observed,which were relatively milder in the EA and losartan groups.Compared with the normal group,tubulointerstitium CVF,systolic blood pressure of caudal artery before,and at 4,8 and 12 weeks after the intervention,24-hour urinary protein before,and at 6 and 12 weeks after the intervention,the expression of TGF-β1 mRNA,area of TGF-β1,α-SMA and FN positive staining in renal tissues were significantly increased(P<0.01),while the area of E-cadherin positive staining was significantly decreased(P<0.01)in the model group.Compared with the model group,tubulointerstitium CVF,systolic blood pressure of caudal artery at 4,8 and 12 weeks after the intervention,24-hour urinary protein at 6 and 12 weeks after the intervention,the expression of TGF-β1 mRNA,area of TGF-β1,α-SMA and FN positive staining in renal tissues were significantly decreased(P<0.01,P<0.05),while area of E-cadherin positive staining was significantly increased(P<0.01)in the losartan and EA groups.Compared with the losartan group,the area of E-cadherin was conside-rately increased(P<0.01),while the area ofα-SMA protein decreased(P<0.01)in the EA group.Conclusion EA could effectively alleviate hypertension and renal interstitial fibrosis in SHR,the mechanism of which may be related to its function in reducing the expression of TGF-β1 and inhibiting EMT in renal tissue.