苓桂术甘汤改善高糖诱导的足细胞的自噬

doi:10.24920/004330

摘要/Abstract

摘要： 目的探讨苓桂术甘汤（Linggui Zhugan Decoction (LGZGD,LGZGD）对高糖诱导的足细胞自噬的影响。
方法分别给SD大鼠胃内灌注4.2 g·kg^-1（低剂量）、8.4 g·kg^-1（中剂量）和12.6 g·kg^-1（高剂量）LGZGD,制备含血清LGZGD。用60 mmol/L葡萄糖分别处理足细胞MPC5和AB8/13,在体外建立糖尿病肾病足细胞模型。将足细胞分为对照组、高糖组、低剂量LGZGD组、中剂量LGZGD组、高剂量LGZGD组。对于3个LGZGD组,在LGZGD干预前,先用60 mmol/L葡萄糖处理足细胞3天。在含LGZGD的血清处理后,收集细胞,用Transwell法分析细胞迁移情况,用CCK8法分析细胞增殖情况,用流式细胞术分析细胞凋亡和细胞周期,用透射电镜分析自噬体的形成,用western blot分析Beclin-1、Atg5、LC3II/I和P62蛋白的表达水平。
结果与对照组相比,高糖组MPC5和AB8.13细胞的增殖和迁移能力略有下降,而在低、中浓度LGZGD干预后,这些指标均恢复正常,以中剂量LGZGD的效果最好。流式细胞术分析显示,与高剂量组比较,中剂量LGZGD组的细胞凋亡率较低（P < 0.05）,存活率较高（P > 0.05）。高糖使足细胞阻滞在G1期,而LGZGD则使足细胞从G1期进入G2期。高剂量LGZGD显著降低了足细胞因高糖作用而增加的自噬体形成（P < 0.05)。Western blot分析显示,高糖处理的MPC5细胞中Beclin-1、Atg5、LC3Ⅱ/Ⅰ和P62表达增加,而给予低剂量和中剂量的LGZGD后,这些蛋白的表达被逆转（P < 0.05)。
结论 LGZGD通过调节Beclin-1/LC3II/I/Atg5的表达,减少了高糖处理的足细胞的凋亡,并增加了细胞自噬。

关键词: 糖尿病肾病, 足细胞, 苓桂术甘汤, 凋亡, 自噬

Abstract: Objective To explore the influence of Linggui Zhugan Decoction (LGZGD) on high glucose induced podocyte autophagy
Methods LGZGD containing serum were prepared by intragastric administation of 4.2 g·kg^{-1 (}low dose⁾, 8.4 g·kg^-1(medium dose), and 12.6 g·kg^-1(high dose) LGZGD into SD rats respectively. MPC5 and AB8/13 cells were treated with 60 mmol/L glucose to establish diabetic nephropathy podocyte model in vitro. Podocytes, MPC5 and AB8.13, were divided into control group, high glucose group, low dose LGZGD group, medium dose LGZGD group, and high dose LGZGD group, respectively. For the three LGZGD groups, before LGZGD intervention, podocytes were treated with 60 mmol/L glucose for 3 days. After treated with LGZGD containing serum, cells were collected to analyze cell migration using Transwell assay, proliferation using CCK8, apoptosis and cell cycle using flow cytometry,, autophagosome formation using transmission electron microscopy, and expression levels of Beclin-1, Atg5, LC3II/I, and P62 proteins using western blot.
Results Compared with the control group, the proliferation and migration of MPC5 and AB8.13 cells in high glucose group showed slightly decreased, whereas these parameters restored after intervention with low and medium concentrations of LGZGD, with the medium dose LGZGD having the best effect. Flow cytometry analysis showed that the medium dose LGZGD group had a lower apoptosis rate (P < 0.05) and higher survival rate (P > 0.05) compared to the high dose group. High glucose arrested podocytes in G1 phase, whereas LGZGD shifted podocytes from being predominant in G1 phase to increasing into G2. High dose LGZGD significanly reduced increased autophagosome formation due to high glucose in both podocytes (P < 0.05). Western blot analysis showed that Beclin-1, Atg5, LC3Ⅱ/Ⅰ, and P62 expressions were increased in MPC5 cells treated with high glucose, and reversed after adminstration of low and medium doses of LGZGD (P < 0.05).
Conclusion LGZGD reduced apoptosis and enhanced autophagy in high glucose treated podocytes via regulating Beclin-1/LC3II/I/Atg5 expression.

Key words: diabetic nephropathy, podocyte, Linggui Zhugan decoction, apoptosis, autophagy

Yuan-Yuan Wang, Jian Qin, Ru-Lan Zhang, Min-Ji Yu, Ming-Hua Jin. Linggui Zhugan Decoction Improves High Glucose-Induced Autophagy in Podocytes[J/OL].Chinese Medical Sciences Journal, .DOI:10.24920/004330.[Epub ahead of print].

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