小檗碱通过抑制内质网应激反应发挥对糖尿病肾病大鼠的保护作用

Berberine protects diabetic nephropathy rats by inhibiting endoplasmic reticulum stress

目的:探讨小檗碱(Berberine,BBR)对糖尿病肾病(diabetic nephropathy,DN)肾脏组织中内质网应激(endoplasmic reticulum stress,ERS)的影响。方法:高糖高脂饮食联合腹腔注射链脲佐菌素(STZ)复制DN大鼠模型。实验共分为3组,正常组(NC组)、模型组(DN组)和小檗碱干预组(DN+BBR组)(n=6)。DN+BBR组在DN模型基础上予BBR 200 mg·kg^(-1)·d^(-1)灌胃治疗,NC组及DN组则予等计量羧甲基纤维素钠灌胃处理。给药6周后,检测并记录大鼠体重、肾指数(KI=肾重/体重)、空腹血糖(FBG)、血肌酐(Scr)、尿素氮(BUN)及24 h尿蛋白(24 h UPro)水平。采用HE、PAS、MASSON染色观察肾脏组织病理改变。透射电镜观察肾小球及肾间质变化。免疫组织化学染色观察肾组织ERS标志物PERK、IRE1、ATF6、CHOP及凋亡蛋白Caspase3的表达。结果:(1)DN组较NC组Scr、BUN、FBG及24 h Upro均显著上升,肾功能损伤严重;经小檗碱干预后,DN+BBR组较DN组肾功能损伤情况改善,各组大鼠Scr、BUN、FBG及24 h Upro差异均具有统计学意义(P<0.05)。(2)HE、PAS及Masson染色结果显示DN组较NC组肾小球形态不规则,体积增大,肾小球囊腔缩窄,弥漫性系膜基质增多,肾小管水肿,肾间质紫红色糖原沉积物增多,蓝染胶原纤维堆积,炎性细胞浸润;经小檗碱干预后,DN+BBR组肾小球状况明显改善,肾小管水肿减轻,糖原沉积减少,胶原纤维堆积相对较少。(3)透射电镜结果显示DN组较NC组大鼠足细胞不规则排列,大量足突融合、断裂,基底膜不均匀增厚;经小檗碱干预后,DN+BBR组足细胞排列较不规则,可见部分足突融合、断裂,基底膜略增厚。(4)免疫组化显示DN组CHOP、PERK、IRE1、ATF6及Caspase3蛋白表达均显著升高;经小檗碱干预后,DN+BBR组大鼠各蛋白表达情况均显著下降,蛋白表达差异均具有统计学意义(P<0.05)。结论:BBR能显著改善DN大鼠肾脏的结构和功能,其机制可能是通过抑制DN大鼠肾脏组织内质网应激反应,减轻肾脏细胞的凋亡,从而延缓DN的发生、发展,保护肾脏组织。

Objective:To investigate the effects of Berberine(BBR)on Endoplasmic Reticulum Stress(ERS)in renal tissue of Diabetic Nephropathy(DN).Methods:The DN RAT model was induced by high glucose and high fat diet combined with intraperitoneal injection of Streptozocin.The experiment was divided into 3 groups:normal group(NC group),model group(DN group)and berberine intervention group(DN+BBR group)(n=6).DN+BBR group was treated with 200 mg·kg^(-1)·d^(-1))on the basis of the model of DN.NC group and DN group were treated with the same dose of sodium carboxymethyl cellulose.After 6 weeks treatment measured various indicators(include body weight,renal index(KI=kidney weight/body weight),fasting blood glucose,blood creatinine,urea nitrogen and 24h urine protein(24 h UPro).The pathological changes of kidney were observed by HE,PAS and Masson staining.The changes of glomerulus and renal interstitium were observed by transmission electron microscope.The expression of PERK,IRE1,ATF6,CHOP and Caspase3 were detected by immunohis to chemical staining.Results:(1)SCR,Bun,FBG and 24 h Upro in DN group were significantly higher than those in NC group and the renal function of DN group severely impaired.Compare with DN group the renal function of DN+BBR group was significant improved.There were significant differences in SCR,Bun,FBG and 24 h Upro in each group(P<0.05).(2)The results of HE,PAS and Masson staining showed that the glomerulus in DN group was more irregular and larger than that in NC group;the lumen of glomerulus became narrowed,diffuse mesangial matrix was increased and renal tubule was edematous.The glycogen deposits and collagen fibers in the renal interstitium were increased and inflammatory cells were infiltrated.After the berberine rescued,the glomerular condition of DN+BBR group was obviously improved;the edema of renal tubules was alleviated;the deposition of glycogen was decreased and the collagenous fibers accumulation is relatively reduced.(3)The results of transmission electron microscope showed that the podocytes of DN Group were irregular and a large number of podocytes fused and broken.The basement membrane was inhomogeneous and thickened.Whereas,the morphological function of podocyte of DN+BBR Group was improved.And the basement membrane was slightly thickened.(4)Immunohistochemistry shows that the expression of CHOP,PERK,IRE1,ATF6 and Caspase3 in DN group was significantly increased and the DN+BBR group was contrarily.The difference of protein expression was statistically significant(P<0.05).Conclusion:These findings provide insights into the BBR can significantly improve the structure and function of kidney in DN rats.The hypothesis is that BBR can suppress endoplasmic reticulum stres and reduce the apoptosis of kidney cells,thus prevent the progress of DN and protect the kidney tissue.