桑叶水提物对db/db糖尿病小鼠肾脏的保护作用及其机制

Protective Effect and Mechanism of Mori Folium Extract on Kidney of db/db Diabetic Mice

目的:观察桑叶水提物(MLE)对db/db糖尿病小鼠肾脏的保护作用,并探讨其机制。方法:24只雄性C57BLKS/JGpt-Leprdb/Leprdb(db/db)小鼠按空腹血糖(FBG)值随机分为模型组、二甲双胍组、桑叶水提物低剂量(MLE-L)组和桑叶水提物高剂量(MLE-H)组,每组6只;另设6只C57BLKS/JGpt同窝野生型(m/m)小鼠作为正常组。各给药组小鼠灌胃相应药物,正常组和模型组小鼠均给予等体积去离子水,每日灌胃1次,连续给药8周。测定小鼠体质量、双侧肾脏绝对重量、FBG,并进行口服葡萄糖耐量实验(OGTT),苏木素-伊红染色、过碘酸雪夫染色观察肾脏组织病理变化,检测血清肌酐(SCr)、血清尿素氮(BUN)含量,酶联免疫吸附测定法检测血清肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和尿微量白蛋白(U-mAlb)含量,蛋白免疫印迹法(Western blot)检测肾组织Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核转录因子-κB p65(NF-κB p65)蛋白表达水平。结果:与正常组比较,模型组小鼠体质量、肾脏绝对重量、FBG、OGTT曲线下面积(AUC)显著升高(P<0.01),SCr、BUN、U-mAlb含量显著升高(P<0.01),血清TNF-α、IL-6含量显著升高(P<0.01),肾组织出现肾小球基底膜增厚,炎性细胞浸润明显,肾组织TLR4、MyD88、NF-κB p65蛋白表达水平显著升高(P<0.01)。与模型组比较,各给药组小鼠体质量差异无统计学意义;MLE-H组、二甲双胍组小鼠肾脏绝对重量明显降低(P<0.05,P<0.01);第4~8周二甲双胍组、MLE-L组、MLE-H组FBG明显降低(P<0.05,P<0.01);MLE-H组、二甲双胍组小鼠AUC显著降低(P<0.01),MLE-L组小鼠AUC呈下降趋势,差异无统计学意义;MLE-H组、二甲双胍组小鼠SCr、BUN、U-mAlb含量显著降低(P<0.01),MLE-L组小鼠SCr、U-mAlb含量显著降低(P<0.01);MLE-H组、二甲双胍组小鼠血清TNF-α、IL-6含量显著降低(P<0.01);各给药组小鼠肾组织病变均得到不同程度改善;MLE-H组小鼠肾组织TLR4、MyD88、NF-κB p65蛋白表达水平明显降低(P<0.05,P<0.01)。结论:桑叶水提物可改善db/db糖尿病小鼠肾组织结构和功能,其机制可能与抑制TLR4/MyD88/NF-κB信号通路有关。

Objective:To investigate the protective effects of Mori Folium extract(MLE)on the kidney of db/db diabetic mice and its mechanism.Method:Twenty-four male C57BLKS/JGpt-Leprdb/Leprdb(db/db)mice were randomly divided into model group,metformin group,low-dose group of MLE(MLE-L),and high-dose group of MLE(MLE-H)according to their fasting blood glucose(FBG),with six mice in each group,and other six C57BLKS/JGpt wild littermate(m/m)mice were selected as normal group.The mice in the drug administration groups were given corresponding drugs by gavage,and the mice in the normal group and model group were given the same dose of deionized water by gavage once a day for continuous eight weeks.Body weight,bilateral kidney weight,and FBG were measured,and an oral glucose tolerance test(OGTT)was performed.The pathological changes in the kidney tissue of mice were observed by hematoxylin-eosin(HE)and periodic acid-silver(PAS)staining,and serum creatinine(SCr)and blood urea nitrogen(BUN)levels were detected.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)in serum and urinary microalbumin(U-mAlb)of mice.The expression levels of toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),and nuclear factor-kappa B p65(NF-κB p65)protein in kidney tissue of mice were tested by Western blot.Result:Compared with the normal group,the body weight,absolute renal weight,FBG,and the area under the curve(AUC)of OGTT of mice in the model group were significantly increased(P<0.01),and the levels of SCr,BUN,and U-mAlb,as well as TNF-αand IL-6 in serum were significantly increased(P<0.01).The glomerular basement membrane in the kidney tissue of mice was thicker,with obvious inflammatory cell infiltration.The protein expression levels of TLR4,MyD88,and NF-κB p65 in the kidney tissue of mice were increased significantly(P<0.01).Compared with the model group,there was no statistical difference in the body weight of mice in each drug administration group.The absolute renal weight of mice in the MLE-H and metformin groups was significantly reduced(P<0.05,P<0.01).The FBG levels of mice in the metformin,MLE-L,and MLE-H groups started to decrease after treatment for four to eight weeks(P<0.05,P<0.01).The AUC of mice in the MLE-H and metformin groups was significantly decreased(P<0.01).The levels of SCr,BUN,and U-mAlb of mice in the MLE-H and metformin groups were significantly decreased(P<0.01),and those of SCr and U-mAlb of mice in the MLE-L group were significantly decreased(P<0.01).The levels of TNF-αand IL-6 in the serum of mice in the MLE-H and metformin groups were significantly decreased(P<0.01).The renal tissue pathology of mice in each drug administration group was improved to varying degrees,and the protein expression levels of TLR4,MyD88,and NF-κB p65 in the MLE-H group were decreased significantly(P<0.05,P<0.01).Conclusion:MLE can improve the renal structure and function of db/db diabetic mice,and its mechanism may be related to the inhibition of the TLR4/MyD88/NF-κB signaling pathway.