Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, 100730, China.
Chin J Integr Med. 2020 Mar;26(3):197-204. doi: 10.1007/s11655-019-2712-8. Epub 2020 Mar 16.
To investigate protective effects of hirudin on oxidative stress and apoptosis of spinal dorsal root ganglion cells in high-glucose rats at the cellular and molecular level.
Dorsal root ganglion neurons (DRGn) were harvested from embryonic day in 15 SD rats, purified and identificated after primary culture. They were divided into the normal control group, high-glucose (HG) group, positive control (alpha-lipoic acid, ALA) group, low-dose hirudin group (H1), medium-dose hirudin group (H2) and high-dose hirudin group (H3). The control group was cultured by neuron specific culture medium, while the HG group was cultured by neuron specific culture medium and 20 mmol/L glucose (HG medium). The hirudin groups were cultured by HG medium+0.25 IU/mL hirudin (H1), HG medium+0.5 IU/mL hirudin (H2) and HG medium+1 IU/mL hirudin (H3). The ALA group was cultured by HG medium+100 µ mol/L ALA. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylt etrazolium bromide (MTT) assay was used to explore the optimum concentration and intervention time. Flow cytometry assay was used to detect the level of reactive oxygen series (ROS). Western blot and quantificational realtime polymerase chain reaction (qRT-PCR) were used to detect the expression of protein and mRNA of nuclear factor erythroid 2-related factor 2 (Nrf-2), hemeoxygence-1 (HO-1), nuclear factor-κ B (NF-κ B) and Caspase-3. TUNEL assay was used to test the apoptosis rate of different groups.
After 24 h of culture, the cell activity of hirudin and ALA groups were higher than that of HG group, and there was a statistical difference between the H1 group and HG group (P<0.05). In hirudin groups, the apoptosis rate of cells, the expression of activated Caspase-3 protein and Caspase-3 mRNA were lower than those of HG group (P<0.01), higher than those of ALA group (P<0.01 or P<0.05). The ROS level of hirudin groups was higher than that of ALA group (P<0.01), lower than that of HG group (P<0.01 or P<0.05). The expression of NF-κ B (P65) protein in H3 group were lower than those of HG group (P<0.05). The expression of Nrf-2 protein in hirudin groups was higher than that of HG group (P<0.01), lower than that of ALA group (P<0.01 or P<0.05). The expression of HO-1 protein in hirudin groups was lower than that of ALA group (P<0.01 or P<0.05), higher than that of HG group (P<0.01 or P<0.05).
The activity of DRGn cells can be promoted by hirudin under HG conditions. The effects of hirudin on the inhibition of HG on DRGn cells damage mainly include scavenging ROS, up-regulating Nrf-2/HO-1 pathway, inhibiting activation of NF-κ B pathway, down-regulating the expression of and Caspase-3 and reducing DRGn cell apoptosis.
在细胞和分子水平上研究水蛭素对高糖诱导的大鼠背根神经节细胞氧化应激和细胞凋亡的保护作用。
取 15 日龄 SD 大鼠胚胎背根神经节神经元（DRGn），原代培养后鉴定。分为正常对照组、高糖组（HG 组）、阳性对照组（α-硫辛酸，ALA）、低剂量水蛭素组（H1 组）、中剂量水蛭素组（H2 组）和高剂量水蛭素组（H3 组）。对照组用神经元专用培养液培养，HG 组用神经元专用培养液和 20 mmol/L 葡萄糖（HG 培养基）培养。水蛭素组用 HG 培养基+0.25 IU/mL 水蛭素（H1 组）、HG 培养基+0.5 IU/mL 水蛭素（H2 组）和 HG 培养基+1 IU/mL 水蛭素（H3 组）培养。ALA 组用 HG 培养基+100 μmol/L ALA 培养。噻唑蓝（MTT）法探索最佳浓度和干预时间。流式细胞术检测活性氧（ROS）水平。Western blot 和实时定量聚合酶链反应（qRT-PCR）检测核因子红细胞 2 相关因子 2（Nrf-2）、血红素加氧酶-1（HO-1）、核因子-κB（NF-κB）和 Caspase-3 蛋白和 mRNA 的表达。TUNEL 检测各组细胞凋亡率。
培养 24 h 后，水蛭素和 ALA 组细胞活性高于 HG 组，H1 组与 HG 组比较有统计学差异（P<0.05）。水蛭素组细胞凋亡率、活化 Caspase-3 蛋白和 Caspase-3 mRNA 的表达均低于 HG 组（P<0.01），高于 ALA 组（P<0.01 或 P<0.05）。水蛭素组 ROS 水平高于 ALA 组（P<0.01），低于 HG 组（P<0.01 或 P<0.05）。H3 组 NF-κB（P65）蛋白表达低于 HG 组（P<0.05）。水蛭素组 Nrf-2 蛋白表达高于 HG 组（P<0.01），低于 ALA 组（P<0.01 或 P<0.05）。水蛭素组 HO-1 蛋白表达低于 ALA 组（P<0.01 或 P<0.05），高于 HG 组（P<0.01 或 P<0.05）。
在高糖条件下，DRGn 细胞的活性可被水蛭素促进。水蛭素对抑制高糖诱导的 DRGn 细胞损伤的作用主要包括清除 ROS、上调 Nrf-2/HO-1 通路、抑制 NF-κB 通路激活、下调 Caspase-3 表达和减少 DRGn 细胞凋亡。
