番石榴叶总三萜调控mTOR/S6K1通路减轻糖尿病肾病大鼠肾损害的实验研究-现代医学

番石榴叶总三萜调控mTOR/S6K1通路减轻糖尿病肾病大鼠肾损害的实验研究

单位：1. 苏北人民医院药剂科, 江苏扬州 225001;
2. 苏北人民医院肾脏科, 江苏扬州 225001

关键词：番石榴叶总三萜糖尿病肾病肾损害哺乳动物雷帕霉素靶蛋白核糖体蛋白S6激酶1

分类号：R286

出版年·卷·期（页码）：2021·49·第十一期（1282-1290）

摘要：

目的：观察番石榴叶总三萜（TTPGL）对糖尿病肾病（DN）大鼠肾损害的治疗作用，并探讨其对哺乳动物雷帕霉素靶蛋白（mTOR）/核糖体蛋白S6激酶1（S6K1）通路的调控作用。方法：采用醋酸脱氧皮质酮-盐制剂皮下注射联合高脂饲料喂养对50只SD大鼠建立糖尿病肾病（DN）模型，建模成功后采用随机数字表分为缬沙坦组，TTPGL低、中、高剂量组和模型组，另取8只SD大鼠记为正常对照组。缬沙坦组予以缬沙坦10 mg·kg^-1溶于1 ml·100 g体质量^-1生理盐水中灌胃，TTPGL低、中、高剂量组分别予以TTPGL 30、60、120 mg·kg^-1溶于1 ml·100 g体质量^-1生理盐水中灌胃，模型组与正常对照组均予以等量生理盐水灌胃，每天1次，持续8周。分别用血糖仪、放射免疫法、酶联免疫法检测干预后空腹血糖（FBG）、24 h尿蛋白、血尿素氮（BUN）、血肌酐（Scr）；苏木素-伊红（HE）染色观察肾脏病理改变，透射电镜下观察亚细胞形态结构改变；实时-逆转录荧光定量聚合酶链反应（RT-qPCR）检测大鼠肾组织mTOR、S6K1、自噬相关基因Beclin1、LC3、人肾病蛋白（Nephrin）mRNA表达；蛋白质免疫印迹法（WB）检测大鼠肾组织mTOR、S6K1、Beclin1、LC3Ⅱ/Ⅰ、Nephrin蛋白表达及磷酸化mTOR/mTOR（p-mTOR/mTOR）、磷酸化S6K1/S6K1（p-S6K1/S6K1）水平。结果：DN建模成功率为92.00%；模型组FBG、24 h尿蛋白、BUN、Scr，肾组织mTOR、S6K1、Nephrin mRNA表达，肾组织p-mTOR/mTOR、p-S6K1/S6K1水平均高于正常对照组（P<0.05），缬沙坦组和TTPGL 3个剂量组均低于模型组（P<0.05）；模型组Beclin1、LC3、Nephrin mRNA与蛋白相对表达量均低于正常对照组（P<0.05），缬沙坦组和TTPGL 3个剂量组均高于模型组（P<0.05）；TTPGL的作用均呈剂量依赖性，且缬沙坦组与TTPGL中剂量组上述指标差异均无统计学意义（P>0.05）；正常对照组大鼠可见肾小球及肾小管形态正常，结构清晰，亚细胞结构正常，模型组可见肾小球基底膜显著增厚，囊腔轮廓严重不规则且显著增大，细胞分布严重不均且多见脂肪变性，肾小管可见严重萎缩塌陷，管壁细胞可见严重不规则，亚细胞结构显著改变，缬沙坦组和TTPGL 3个剂量组均改善，且TTPGL高剂量组与正常对照组最为接近。结论：TTPGL可减轻DN大鼠肾组织病理改变，增强肾功能，推测是通过抑制mTOR/S6K1通路，降低mTOR、S6K1表达和p-mTOR/mTOR、p-S6K1/S6K1水平，上调Beclin1、LC3、Nephrin表达发挥效应的。

Objective: To observe the therapeutic effect of total triterpenoids of psidium guajava leaves (TTPGL) on diabetic nephropathy (DN) rats, and to explore its regulatory effect on mammalian target of rapamycin (mTOR)/ S6 kinase 1 (S6K1) pathway. Methods: 50 SD rats were established diabetic nephropathy (DN) models by subcutaneous injection of deoxycorticosterone acetate salt combined with high-fat diet. After successful modeling, the rats were randomly divided into Valsartan group; TTPGL low, medium and high dose groups and modelgroup. and 8 SD rats were taken as normal control group. Valsartan group was given valsartan 10 mg·kg^-1dissolved in 1 ml·100 g^-1 body weight normal saline by gavage, and TTPGL low, medium and high dose groups were given TTPGL 30, 60 and 120 mg·kg^-1 dissolved in 1 ml·100 g^-1 body weight normal saline by gavage, while the model group and the normal control group were given the same amount of normal saline, once a day for 8 weeks. Fasting blood glucose (FBG), 24-hour urine protein, blood urea nitrogen (BUN) and serum creatinine (Scr) were detected by blood glucose meter, radioimmunoassay and enzyme-linked immunosorbent assay. The pathological changes of kidney were observed by hematoxylin eosin (HE) staining, and the changes of subcellular morphology were observed by transmission electron microscope. The mRNA expressions of mTOR, S6K1, Beclin1, LC3, Nephrin were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The expressions of mTOR, S6K1, Beclin1, LC3Ⅱ/Ⅰ, Nephrin and phosphorylated mTOR/mTOR (p-mTOR/mTOR) and phosphorylated S6K1/S6K1 (p-S6K1/S6K1) were detected by Western blot (WB). Results: The success rate of DN modeling was 92.00%. After intervention, FBG, 24 h urine protein, BUN, SCR, mTOR, S6K1 mRNA and protein expressions in renal tissues, p-mTOR/mTOR, p-S6K1/S6K1 levels in renal tissues of model group were higher than those of the normal control group (P<0.05), of which the Valsartan group and TTPGL 3-dose groups were lower than those of the model group (P<0.05). After intervention, the relative expressions of Beclin1, LC3 II/I, Nephrin mRNA and proteins in the model group were lower than those in the normal control group (P<0.05), of which the Valsartan group and TTPGL 3 dose group were higher than those in the model group (P<0.05). The effect of TTPGL was dose-dependent, and there were no significant differences in the above indexes between Valsartan group and TTPGL medium dose group (P>0.05). In the normal control group, the morphology of glomerulus and renal tubules was normal, the structure was clear, and the subcellular structure was normal. In the model group, the basement membrane of glomerulus was significantly thickened, the contour of capsule cavity was seriously irregular and significantly increased, the distribution of cells was seriously uneven and fatty degeneration was common. The renal tubules were severely atrophied and collapsed, and the wall cells were severely irregular, and the subcellular structure was significantly changed. The improvement was observed in Valsartan group and TTPGL 3-dose groups, and the TTPGL high dose group was the closest to the normal control group.Conclusion: TTPGL can reduce the pathological changes of renal tissues, enhance renal function, speculate and inhibit the mTOR/S6K1 pathway, reduce the expressions of mTOR and S6K1 and the levels of p-mTOR/mTOR and p-S6K1/S6K1, and upregulate the expressions of Beclin1, LC3 and Nephrin.

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