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目的:探讨绿原酸(CGA)对过氧化氢(H2O2)诱导的大鼠髓核细胞凋亡的作用机制。方法:采用体外培养大鼠髓核细胞,传代后取第3代细胞进行实验。随机分成5组:正常对照组、H2O2处理组、CGA+H2O2组、CGA组和CGA+PI3K/Akt抑制剂(LY294002)干预组。流式细胞术检测各组的细胞凋亡率及细胞内活性氧水平;Western blot检测各组髓核细胞中Akt,p-Akt,BCL-2的表达。结果:与正常对照组比较,H2O2组诱导的髓核细胞凋亡数显著增高,细胞内ROS含量显著增高;CGA预处理后显著抑制了髓核细胞凋亡和ROS产生,增加了细胞内p-Akt和BCL-2的表达,但LY294002抑制剂显著抑制了CGA对氧化应激后髓核细胞的上述功能。结论:绿原酸可通过激活PI3K-Akt信号途径抑制细胞内活性氧的产生、上调抗凋亡BCL-2蛋白水平的表达来保护髓核细胞免受氧化应激引起的损伤。
Objective: To investigate the mechanism of chlorogenic acid (CGA) on hydrogen peroxide (H2O2) induced apoptosis in rat nucleus pulposus cells. Methods: The rat nucleus pulposus cells were cultured in vitro. The passage 3 cells were passaged for experiments. The rats were randomly divided into 5 groups: normal control group, H2O2 treatment group, CGA + H2O2 group, CGA group and CGA + PI3K / Akt inhibitor (LY294002) intervention group. Flow cytometry was used to detect the apoptotic rate and intracellular reactive oxygen species (ROS) levels in each group. Western blot was used to detect the expression of Akt, p-Akt and BCL-2 in each group. Results: Compared with the normal control group, the apoptosis of nucleus pulposus cells induced by H2O2 group was significantly increased and the intracellular ROS content was significantly increased. CGA pretreatment significantly inhibited the apoptosis of nucleus pulposus cells and ROS production, increased the intracellular p- Akt and BCL-2 expression, but LY294002 inhibitor significantly inhibited the CGA on oxidative stress after the above-mentioned function of nucleus pulposus cells. CONCLUSION: Chlorogenic acid can protect the nucleus pulposus cells against oxidative stress by activating the PI3K-Akt signaling pathway to inhibit the production of reactive oxygen species (ROS) and up-regulating the expression of anti-apoptotic BCL-2 protein.