当归四逆汤含药血清对高糖培养SD大鼠背根神经元线粒体分裂及RhoA/ROCK通路的影响

Effects of Danggui Sini Decoction containing serum on mitochondrial division and RhoA/ROCK pathway of dorsal root neurons of SD rats cultured with high glucose

  • 摘要:
      目的  探讨当归四逆汤含药血清对高糖培养SD大鼠背根神经元线粒体分裂及Ras同源基因家族成员A(RhoA)/Rho相关卷曲螺旋形成蛋白激酶(ROCK)通路的影响。
      方法  各取6只SD大鼠,分别灌胃生理盐水、当归四逆汤后颈动脉取血分别制备正常血清及含药血清。取正常SD大鼠两侧背根神经元进行体外培养,随机分为对照组、高糖组、当归四逆汤组,高糖组和当归四逆汤组细胞以高糖处理,当归四逆汤组细胞再以10%的含药血清处理,对照组细胞正常培养。经正常血清、含药血清处理后,采用CCK-8实验检测各组细胞活力; 采用试剂盒测定各组细胞活性氧(ROS)水平、线粒体膜电位及释放乳酸脱氢酶(LDH)、白细胞介素-6(IL-6)、白细胞介素-18(IL-18)水平; 采用免疫印迹实验检测各组细胞线粒体分裂蛋白包括动力相关蛋白1(Drp1)与分裂蛋白1(Fis1)、融合蛋白即丝裂原融合蛋白1(MFN1)与丝裂原融合蛋白2(MFN2)、RhoA/ROCK通路蛋白RhoA与ROCK表达。
      结果  与对照组比较,高糖组背根神经元ROS水平及释放的LDH、IL-6、IL-18水平,细胞中Drp1、Fis1、RhoA及ROCK蛋白表达升高,细胞活力、线粒体膜电位、细胞中MFN1及MFN2蛋白表达降低,差异有统计学意义(P < 0.05); 与高糖组比较,当归四逆汤组背根神经元ROS水平及释放的LDH、IL-6、IL-18水平,细胞中Drp1、Fis1、RhoA及ROCK蛋白表达降低,细胞活力、线粒体膜电位、细胞中MFN1及MFN2蛋白表达升高,差异有统计学意义(P < 0.05)。
      结论  当归四逆汤含药血清可下调RhoA/ROCK通路蛋白表达,抑制高糖诱导的ROS和炎性因子表达,减轻炎症反应,减弱线粒体分裂,缓解SD大鼠背根神经元损伤。

     

    Abstract:
      Objective  To investigate the effects of Danggui Sini Decoction containing serum on mitochondrial division and Ras homolog gene family member A(RhoA)/Rho-associated coiled-coil containing kinase(ROCK) pathway of dorsal root neurons of SD rats cultured with high glucose.
      Methods  Six SD rats were given intragastric administration of normal saline, and another six given Danggui Sini Decoction, the blood was taken from the carotid artery to prepare normal serum and drugs containing serum. Bilateral dorsal root neurons of normal SD rats were cultured in vitro and randomly divided into control group, high glucose group, and Danggui Sini Decoction group. The cells in high glucose group and Danggui Sini Decoction group were treated with high glucose, cells in control group were cultured normally, and cells in the Danggui Sini Decoction group were cultured with 10% drug-containing serum. After treatment with normal serum and containing serum, the cell viability of each group was detected with CCK-8 experiment; the levels of cellular reactive oxygen species (ROS), release lactate dehydrogenase (LDH), interleukin-6 (IL-6), and interleukin-18 (IL-18) levels in each group were determined with kits; the expressions of mitochondrial division proteins dynamin-related protein 1(Drp1) and fission protein 1(Fis1), fusion proteins mitofusin 1(MFN1) and mitofusin 2(MFN2) and RhoA/ROCK pathway proteins such as RhoA and ROCK in each group were detected with western blotting.
      Results  Compared with the control group, the levels of ROS, the levels of LDH, IL-6 and IL-18, and the expression of Drp1, Fis1, RhoA and ROCK proteins in the dorsal root neurons of rats in the high glucose group were significantly increased (P < 0.05), while the expressions of cell viability, mitochondrial membrane potential, MFN1 and MFN2 protein in cells were significantly reduced (P < 0.05); compared with the high glucose group, the levels of ROS, the levels of LDH, IL-6 and IL-18, and the expression of Drp1, Fis1, RhoA and ROCK proteins in the dorsal root neurons of rats in the Danggui Sini Decoction group were significantly reduced (P < 0.05), while the expressions of cell viability, mitochondrial membrane potential, MFN1 and MFN2 protein in cells were significantly increased (P < 0.05).
      Conclusion  Danggui Sini Decoction containing serum can down-regulate the expression of RhoA/ROCK pathway proteins, inhibit the expressions of ROS and inflammatory factors induced by high glucose, reduce inflammation reaction, weaken mitochondrial division, and alleviate the damage of dorsal root neurons of SD rats.

     

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