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. 2018 Jan 15;32(1):69-74.
doi: 10.7507/1002-1892.201709043.

[Effect of heme oxygenase 1 on the apoptosis of human degenerated nucleus pulposus cells induced by tumor necrosis factor α]

[Article in Chinese]
Xiaozhong Hou  1 , Linfei Xu  2 , Jieliang Shen  3 , Zhenming Hu  4
Affiliations

[Effect of heme oxygenase 1 on the apoptosis of human degenerated nucleus pulposus cells induced by tumor necrosis factor α]

[Article in Chinese]
Xiaozhong Hou et al. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. .

Abstract
in English, Chinese

Objective: To investigate the effect of heme oxygenase 1 (HO-1) on the apoptosis of human degenerated nucleus pulposus (NP) cells induced by tumor necrosis factor α (TNF-α), and explore its possible molecular mechanism.

Methods: The intervertebral disc tissues were derived from patients with lumbar intervertebral disc herniation. Then, the NP cells were cultured in vitro and the third generation of NP cells were used for subsequent experiments. Cell counting kit 8 (CCK-8) method was used to observe the proliferative effect of TNF-α on the NP cells in vitro at the concentration of 10, 20, 50, 100, and 200 ng/mL. The most apropriate concentration was selected according to the result of CCK-8. The NP cells were cultured with basal medium (control group), TNF-α (TNF-α group), TNF-α and CoPP 10 μmol/L (CoPP group), and TNF-α and ZnPP 15 μmol/L (ZnPP group), respectively. After cultured, the cell poptosis was detected by Hoechst staining and flow cytometry; the expression of cleaved Caspase-3, epithelial membrane protein 1 (EMP-1), HO-1, and p-P65 proteins were detected by Western blot. In order to further explore the potential molecular mechanisms of HO-1 for cell apoptosis, the NP cells were cultured with TNF-α (TNF-α stimulated group), TNF-α and pyrrolidine dithiocarbamate (PDTC) 5 μmol/L (TNF-α+PDTC stimulated group), respectively. Then the cell apoptosis rate was measured by flow cytometry at 24 hours after cultured.

Results: The optimal concentration of TNF-α was 100 ng/mL. Hoechst staining showed that a few apoptotic cells could be observed in control group and CoPP group; the apoptosis-like nucleis were observed in TNF-α group and ZnPP group, which was the most significant in ZnPP group. Flow cytometry showed that the cell apoptosis rates of TNF-α group, CoPP group, and ZnPP group were significantly increased when compared with the control group ( P<0.05). Compared with TNF-α group, the cell apoptosis rate in CoPP group decreased ( P<0.05), while in ZnPP group it increased ( P<0.05). Western blot showed that the expression of HO-1 protein in TNF-α group was decreased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were increased when compared with the control group ( P<0.05). Compared with TNF-α group, the expression of HO-1 protein in CoPP group increased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were reduced ( P<0.05); the expression of HO-1 protein in ZnPP group decreased ( P<0.05), the expressions of cleaved Caspase-3 and EMP-1 proteins increased ( P<0.05), and the expression of p-P65 protein was not significantly changed ( P>0.05). Compared with TNF-α stimulated group, the cell apoptosis rate in TNF-α+PDTC stimulated group was significantly reduced ( t=3.076, P=0.031).

Conclusion: HO-1 can inhibit the apoptosis of degerated NP cells induced by TNF-α, and its mechanism effect is by inhibiting the nuclear factor кB signaling pathway.

目的: 探讨血红素氧合酶 1（heme oxygenase 1，HO-1）对 TNF-α 诱导的人退变椎间盘髓核细胞凋亡的作用及其可能的分子机制。.

方法: 取腰椎间盘突出症患者自愿捐赠的椎间盘组织，体外培养人退变髓核细胞并传代，取第 3 代细胞进行实验。采用细胞计数试剂盒 8 测定不同浓度（10、20、50、100 和 200 ng/mL）TNF-α 对髓核细胞活性的影响，筛选最佳刺激浓度进行下一步实验。将髓核细胞分成 4 组，分别采用单纯基础培养液（对照组）、TNF-α 刺激（TNF-α 组）、TNF-α 和 CoPP 10 μmol/L 刺激（CoPP 组）、TNF-α 和 ZnPP 15 μmol/L 刺激（ZnPP 组）培养，24 h 后采用 Hoechst 染色以及流式细胞仪检测细胞凋亡；Western blot 检测凋亡相关蛋白活化型 Caspase-3（cleaved Caspase-3）、上皮膜蛋白 1（epithelial membrane protein 1，EMP-1）以及 HO-1、p-P65 的表达。为进一步探讨 HO-1 对髓核细胞凋亡的潜在分子机制，取髓核细胞分别采用 TNF-α 刺激（TNF-α 刺激组）以及 TNF-α 和吡咯烷二硫基甲酸（pyrrolidine dithiocarbamate，PDTC）5 μmol/L 刺激（TNF-α+PDTC 刺激组）培养 24 h，采用流式细胞仪检测髓核细胞凋亡率。.

结果: 经检测确定 TNF-α 最佳抑制浓度为 100 ng/mL。Hoechst 染色示，对照组和 CoPP 组凋亡细胞较少；TNF-α 组和 ZnPP 组可见凋亡样改变细胞核，其中 ZnPP 组最显著。流式细胞仪检测示，与对照组相比，TNF-α 组、CoPP 组及 ZnPP 组细胞凋亡率均显著提高（ P<0.05）；与 TNF-α 组相比，CoPP 组细胞凋亡率显著降低（ P<0.05），而 ZnPP 组显著提高（ P<0.05）。Western blot 检测示，与对照组相比，TNF-α 组 HO-1 蛋白表达降低，cleaved Caspase-3、EMP-1 及 p-P65 蛋白表达升高（ P<0.05）。与 TNF-α 组相比，CoPP 组 HO-1 蛋白表达明显升高，cleaved Caspase-3、EMP-1、p-P65 蛋白表达明显降低（ P<0.05）；而 ZnPP 组 HO-1 蛋白表达明显降低（ P<0.05），cleaved Caspase-3、EMP-1 蛋白表达增高（ P<0.05），p-P65 蛋白表达无明显变化（ P>0.05）。与 TNF-α 刺激组相比，TNF-α+PDTC 刺激组细胞凋亡率明显降低（ t=3.076， P=0.031）。.

结论: HO-1 能够抑制 TNF-α 诱导的人椎间盘髓核细胞凋亡，其机制可能是通过调控 NF-кB 通路得以实现。.

Keywords: Heme oxygenase 1; apoptosis; nucleus pulposus cells; tumor necrosis factor α.

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Figures

图 1
图 1
Morphological observation of primary degenerated NP cells (×200) 原代髓核细胞形态学观察（×200）
图 2
图 2
Hoechst staining observation of cell apoptosis in each group (Fluorescence microscope×200) Hoechst 染色观察各组髓核细胞凋亡（荧光显微镜×200）
图 3
图 3
Cell apoptosis detection in each group by flow cytometry a. Control group; b. TNF-α group; c. CoPP group; d. ZnPP group 流式细胞仪检测各组细胞凋亡
图 4
图 4
Expressions of cleaved Caspase-3, EMP-1, and HO-1 proteins by Western blot Western blot 检测髓核细胞凋亡相关蛋白及 HO-1 的表达
图 5
图 5
Expression of p-P65 protein by Western blot Western blot 检测髓核细胞 p-P65 蛋白表达
图 6
图 6
Cells apoptosis rate after PDTC treatment by flow cytometry 流式细胞仪检测 PDTC 对髓核细胞凋亡的影响
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