目的探讨灵桂术甘汤(LGZGD)对高糖诱导足细胞自噬的影响。方法用4.2g·kg-1(低剂量)灌胃制备LGZGD含药血清,8.4g·kg-1(中等剂量),和12.6g·kg-1(高剂量)LGZGD分别进入SD大鼠。用60mmol/L葡萄糖处理MPC5和AB8/13细胞,建立糖尿病肾病足细胞体外模型。足细胞,MPC5和AB8.13,分为对照组,高糖组,低剂量LGZGD组,中剂量LGZGD组,和高剂量LGZGD组,分别。对于三个LGZGD组,在LGZGD干预之前,足细胞用60mmol/L葡萄糖处理3天。用含LGZGD的血清处理后,收集细胞以使用Transwell测定法分析细胞迁移,使用CCK8的增殖,使用流式细胞术的凋亡和细胞周期,,使用透射电子显微镜形成自噬体,和Beclin-1,Atg5,LC3II/I的表达水平,和使用蛋白质印迹的P62蛋白。结果与对照组比较,高糖组MPC5和AB8.13细胞的增殖和迁移能力略有下降,而这些参数在低和中等浓度的LGZGD干预后恢复,中剂量LGZGD效果最好。流式细胞术分析显示,中剂量LGZGD组较高剂量组细胞凋亡率更低(P<0.05),存活率更高(P>0.05)。高糖在G1期抑制足细胞,而LGZGD使足细胞从G1期占优势转变为增加到G2期。高剂量LGZGD显著降低了两个足细胞中由于高糖引起的自噬小体形成的增加(P<0.05)。Westernblot分析显示Beclin-1,Atg5,LC3Ⅱ/Ⅰ,在高糖处理的MPC5细胞中,P62表达增加,低剂量和中剂量LGZGD后逆转(P<0.05)。结论LGZGD通过调节Beclin-1/LC3II/I/Atg5的表达减少高糖处理足细胞的凋亡并增强自噬。
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.