miR-135a 通过 Notch 通路调控糖尿病肾病大鼠肾脏纤维化。
MiR-135a regulates renal fibrosis in rats with diabetic kidney disease through the Notch pathway.
机构信息
Department of Nephrology, The Second Hospital of Shanxi Medical University, Taiyuan, China.
出版信息
Eur Rev Med Pharmacol Sci. 2020 Feb;24(4):1979-1987. doi: 10.26355/eurrev_202002_20375.
OBJECTIVE
To explore the influence of micro ribonucleic acid (miR)-135a on the renal fibrosis in rats with diabetic kidney disease (DKD) through the Notch signaling pathway.
MATERIALS AND METHODS
A total of 30 male Wistar rats weighing 200-220 g were selected and randomly divided into Control group (n=10), diabetes mellitus (DM) group (n=10), and miR-13a inhibitor group (n=10). Streptozotocin (STZ) was intraperitoneally injected daily to establish the DM model in rats of both DM group and miR-135a group, while normal saline was given daily through intraperitoneal injection in rats of Control group. After 4 weeks, the rats in miR-135a inhibitor group were intraperitoneally injected with miR-135a inhibitor, and those in Control and DM groups were administrated with an equal amount of normal saline. Changes in the blood glucose (BG), glycated hemoglobin (GHb), serum creatinine (Scr), triglyceride (TG), and total cholesterol (TC) of rats were evaluated, and the pathological changes in the renal tissues of DM rats were observed via hematoxylin-eosin (HE) staining. Sirius red staining was performed to observe the changes in collagen fibers in the kidney of all groups of rats. The expressions of Notch and Hes1 in the renal tissues of rats in each group were detected using immunohistochemistry. Immunofluorescence assay was employed to detect the positive expression of Notch in the renal tissues of rats. The mRNA expressions of Notch and miR-135a were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Finally, Western blotting was conducted to detect the protein expressions of Notch, Notch intracellular domain (NICD) and Hes1.
RESULTS
Compared with Control group, rats in DM group had substantially raised levels of BG, GHb, Scr, TG, and TC (p<0.05). HE staining showed that the rats in Control group had renal tubular cells with normal morphology and well-defined structure, while those in DM group exhibited evident cavitation in the renal tubular epithelium. Sirius red staining results manifested that the red collagen fibers were evenly distributed with light staining in the glomeruli and renal tubules of rats in Control group. In contrast, the collagen fibers of the glomeruli and renal tubules of rats in DM group exhibited deep and evident red staining. Moreover, compared with DM group, rats in miR-135a inhibitor group had notably faded red staining in the glomeruli and renal tubules of rats, evenly distributed collagen and remarkably decreased fibrotic nodules. According to immunohistochemistry detection results, the protein levels of Notch and Hes1 in the renal tubulointerstitial cells and renal tubular epithelial cells of rats in DM group were markedly higher than those in Control group. Compared with those in DM group, their protein levels were remarkably lowered in miR-135a inhibitor group (p<0.05). Immunofluorescence assay results revealed that the protein level of Notch in the renal tissues of rats in DM group was considerably higher than that in Control group (p<0.05), while its protein level in miR-135a inhibitor group was significantly lower than that in DM group. According to qRT-PCR results, compared with those in Control group, mRNA expressions of Notch mRNA and miR-135a in the rat kidney tissues were substantially raised in DM group (p<0.05), and they were notably lowered in miR-13a inhibitor group compared with those in DM group (p<0.05). Finally, Western blotting results manifested that the protein levels of Notch, NIC, and Hes1 in the renal tissues of rats in DM group were considerably higher than those in Control group (p<0.05), and that their protein expression levels in miR-135a inhibitor group were markedly lower than those in DM group (p<0.05).
CONCLUSIONS
Inhibition of miR-135a can reduce the renal fibrosis in DKD rats through the Notch pathway.
目的
通过 Notch 信号通路探讨微小 RNA-135a（miR-135a）对糖尿病肾病（DKD）大鼠肾纤维化的影响。
材料与方法
选择 30 只雄性 Wistar 大鼠，体重 200-220 g，随机分为对照组（n=10）、糖尿病组（DM 组，n=10）和 miR-135a 抑制剂组（n=10）。DM 组和 miR-135a 抑制剂组大鼠每天腹腔注射链脲佐菌素（STZ）建立糖尿病模型，对照组大鼠每天腹腔注射生理盐水。4 周后，miR-135a 抑制剂组大鼠腹腔内注射 miR-135a 抑制剂，对照组和 DM 组大鼠腹腔内注射等量生理盐水。评估大鼠血糖（BG）、糖化血红蛋白（GHb）、血清肌酐（Scr）、甘油三酯（TG）和总胆固醇（TC）的变化，通过苏木精-伊红（HE）染色观察 DM 大鼠肾组织的病理变化。采用天狼猩红染色观察各组大鼠肾脏胶原纤维的变化。采用免疫组织化学法检测各组大鼠肾组织 Notch 和 Hes1 的表达。采用免疫荧光法检测大鼠肾组织 Notch 的阳性表达。采用实时荧光定量聚合酶链反应（qRT-PCR）检测 Notch 和 miR-135a 的 mRNA 表达。最后，采用 Western blot 检测 Notch、Notch 胞内结构域（NICD）和 Hes1 的蛋白表达。
结果
与对照组相比，DM 组大鼠 BG、GHb、Scr、TG 和 TC 水平显著升高（p<0.05）。HE 染色显示，对照组大鼠肾小管细胞形态正常，结构清晰，DM 组大鼠肾小管上皮细胞空泡明显。天狼猩红染色结果显示，对照组大鼠肾小球和肾小管内胶原纤维呈均匀分布，染色较浅。相反，DM 组大鼠肾小球和肾小管的胶原纤维呈深而明显的红色染色。此外，与 DM 组相比，miR-135a 抑制剂组大鼠肾小球和肾小管的红色染色明显变淡，胶原纤维均匀分布，纤维化结节明显减少。免疫组织化学检测结果显示，DM 组大鼠肾小管间质细胞和肾小管上皮细胞 Notch 和 Hes1 蛋白水平明显高于对照组。与 DM 组相比，miR-135a 抑制剂组大鼠的蛋白水平明显降低（p<0.05）。免疫荧光检测结果显示，DM 组大鼠肾组织 Notch 蛋白水平明显高于对照组（p<0.05），而 miR-135a 抑制剂组大鼠肾组织 Notch 蛋白水平明显低于 DM 组。qRT-PCR 结果显示，与对照组相比，DM 组大鼠肾脏组织 Notch mRNA 和 miR-135a 的 mRNA 表达明显升高（p<0.05），而 miR-13a 抑制剂组大鼠肾脏组织 Notch mRNA 和 miR-135a 的 mRNA 表达明显低于 DM 组（p<0.05）。最后，Western blot 结果显示，DM 组大鼠肾组织 Notch、NIC 和 Hes1 蛋白水平明显高于对照组（p<0.05），而 miR-135a 抑制剂组大鼠肾组织 Notch、NIC 和 Hes1 蛋白水平明显低于 DM 组（p<0.05）。
结论
抑制 miR-135a 可通过 Notch 通路减少 DKD 大鼠的肾纤维化。
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