三黄益肾胶囊抑制细胞焦亡保护糖尿病肾病小鼠肾脏作用研究

Sanhuang Yishen Capsule protects renal function in diabetic kidney disease mice by inhibiting podocyte pyroptosis

关键词:  三黄益肾胶囊  细胞焦亡  肾脏功能  糖尿病肾病

Key words:  Sanhuang Yishen Capsule  pyroptosis  renal function  diabetic kidney disease

摘要:

[目的] 构建糖尿病肾病（DKD）小鼠模型，探讨三黄益肾胶囊对DKD小鼠肾脏足细胞的影响及作用机制，同时观察DKD小鼠肾脏功能的变化情况。[方法] 选取C57BL/6雄性小鼠（SPF级）32只，适应性喂养1周。除正常组外，其余各组均通过腹腔注射链脲佐菌素（STZ）以及喂养高脂饲料构建DKD小鼠模型。将造模成功的小鼠分为模型组、三黄益肾胶囊组及卡托普利组。造模成功后稳定1周，三黄益肾胶囊组和卡托普利组开始定时灌胃治疗，正常组和模型组给予等量生理盐水灌胃，疗程8周，同时记录小鼠体质量、血糖。检测小鼠血肌酐、血尿素氮、24 h尿蛋白、三酰甘油、总胆固醇、低密度脂蛋白；苏木精-伊红（HE）、过碘酸雪夫（PAS）染色后观察不同组小鼠肾脏病理的差异；免疫荧光法观察肾脏细胞焦亡程度及半胱氨酸天冬氨酸蛋白酶1（Caspase-1）表达水平；蛋白免疫印迹（Western blot）法检测小鼠肾脏组织中突触足蛋白（Synaptopodin）、Caspase-1蛋白因子表达情况。[结果] 与正常组比较，模型组小鼠体质量、血糖升高（P＜0.05）；治疗8周后，与模型组比较，三黄益肾胶囊组、卡托普利组小鼠体质量、血糖降低（P＜0.05或P＜0.01）。肾功能：与正常组比较，模型组小鼠24 h尿蛋白、血肌酐升高（P＜0.05或P＜0.01）；治疗8周后，与模型组比较，三黄益肾胶囊组、卡托普利组小鼠24 h尿蛋白、血肌酐降低（P＜0.01）；小鼠血尿素氮未见明显变化，差异无统计学意义（P＞0.05）。血脂水平：与正常组比较，模型组小鼠总胆固醇、低密度脂蛋白水平升高（P＜0.05）；治疗8周后，与模型组比较，三黄益肾胶囊组小鼠总胆固醇、低密度脂蛋白水平降低（P＜0.05），卡托普利组小鼠总胆固醇水平降低（P＜0.05），低密度脂蛋白水平无显著变化，差异无统计学意义（P＞0.05）；小鼠三酰甘油未见显著变化，差异无统计学意义（P＞0.05）。HE、PAS染色结果显示：与正常组比较，模型组小鼠肾脏损伤明显，肾脏出现异常；与模型组比较，三黄益肾胶囊组、卡托普利组小鼠肾脏损伤较轻，病理损伤有所好转。免疫荧光染色结果显示，与正常组比较，模型组小鼠Caspase-1表达增加（P＜0.01）；与模型组比较，三黄益肾胶囊组、卡托普利组小鼠Caspase-1表达降低（P＜0.01）。Western blot结果显示：与正常组比较，模型组Synaptopodin蛋白表达降低（P＜0.05），Caspase-1蛋白表达升高（P＜0.05）；与模型组比较，三黄益肾胶囊组、卡托普利组Synaptopodin蛋白表达升高（P＜0.05或P＜0.01），Caspase-1蛋白表达降低（P＜0.01）。[结论] 三黄益肾胶囊能够降低DKD小鼠的24 h尿蛋白、血肌酐水平，保护肾脏功能，延缓DKD的微炎症状态及细胞焦亡进程。

Abstract:

[Objective] To establish a diabetic kidney disease（DKD） mouse model and investigate the effects of Sanhuang Yishen Capsule（SHYS） on renal podocytes and its mechanism，while observing changes in renal function. [Methods] Thirty-two SPF-grade male C57BL/6 mice were adaptively fed for 1 week. Except for the normal group，DKD models were induced by intraperitoneal streptozotocin（STZ） injection and high-fat diet. Successfully modeled mice were divided into the model group，SHYS group，and captopril group. After 1 week of stabilization，SHYS and captopril groups received daily gavage for 8 weeks，while the normal and model groups received saline. Body weight and blood glucose were monitored. Serum creatinine（Scr），blood urea nitrogen（BUN），24-hour urinary protein（24 h-UP），triglycerides（TG），total cholesterol（TC），and low-density lipoprotein（LDL） were measured. Renal pathology was assessed via hematoxylin-eosin（HE） and periodic acid-Schiff（PAS） staining. Caspase-1 expression and pyroptosis were detected by immunofluorescence. Synaptopodin and Caspase-1 protein levels were analyzed by Western blot. [Results] Compared with the normal group，the model group showed increased body weight，blood glucose，24 h-UP，Scr，TC，and LDL（P<0.05 or P<0.01）. After treatment，SHYS and captopril groups exhibited reduced body weight，blood glucose，24 h-UP，Scr，and TC（P<0.05 or P<0.01），with no significant changes in BUN or TG（P>0.05）. HE/PAS staining revealed severe renal injury in the model group，which was alleviated by SHYS and captopril. Immunofluorescence and Western blot confirmed that SHYS downregulated Caspase-1（P<0.01） and upregulated Synaptopodin（P<0.05） compared to the model group. [Conclusion] SHYS ameliorates renal dysfunction in DKD mice by reducing proteinuria，improving lipid metabolism，and suppressing podocyte pyroptosis via Caspase-1 inhibition.