内质网应激在酸诱导的人椎间盘髓核细胞损伤中的作用机制研究

A study of the effect of the endoplasmic reticulum stress in protection the human nucleus pulposus cells from the acid-induced injury

目的 :模拟人椎间盘髓核(nucleus pulposus,NP)酸性环境,研究酸诱导的内质网应激活化以及内质网应激在酸诱导的髓核细胞损伤中的作用机制。方法:体外单层培养人正常髓核细胞(nucleus pulposus cells,NPCs)系,以p H值7.4为对照,p H值7.0和6.5分别模拟正常和退变椎间盘酸性环境,培养12~72h,建立酸诱导的髓核细胞损伤模型。采用CCK8法检测髓核细胞增殖情况,透射电镜检测髓核细胞中内质网应激活化情况,免疫荧光检测内质网应激特异性标志物糖调节蛋白78(78 k Da glucose-regulated protein,GRP78)和C/EBP同源蛋白(C/EBP homologous protein,CHOP)表达。应用4-PBA(内质网应激阻断剂)阻断内质网应激后,流式细胞术检测细胞凋亡和细胞周期;β半乳糖苷酶染色检测细胞老化。Western blot检测LC3、GATA4、p53、p21、p16、Bax、Bcl-2和Caspase3蛋白变化情况。结果 :与对照组相比,酸刺激下(p H6.5组)髓核细胞整体增殖力较对照组明显下降;透射电镜下可见内质网扩张明显,膜表面积增加,内质网线粒体膜结构形成,伴线粒体肿胀;细胞免疫荧光检测提示GRP78和CHOP表达明显增加(P<0.05)。应用4-PBA后,细胞凋亡率增加,且能够显著增加酸诱导的G1期停滞及β半乳糖苷酶阳性染色率(P<0.05)。Western blot检测发现,酸刺激下,髓核细胞LC3、GATA4、p53、p21、p16、Bax和Caspase3表达增高(P<0.05),Bcl-2表达降低(P<0.05);应用4-PBA后可降低LC3比值和Bcl-2表达水平,并显著升高GATA4、p53、p21、p16、Bax和Caspase3(P<0.05)。结论:酸性微环境能够活化内质网应激,在酸诱导的人髓核细胞急性损伤中起保护作用。

Objectives： To investigate the role of endoplasmic reticulum（ER） stress played in acid-induced injury of nucleus pulposus cells. Methods： Normal human nucleus pulposus cells（NPCs） were cultured in vitro, pH 7.4 was used as the control, pH 7.0 and 6.5 were used to simulate the normal and degenerative intervertebral disc acidic environment. All cells were cultured for 12-72h, acid-induced damage model of NPCs was established. CCK8 was used to detect the proliferation of NPCs and transmission electron microscopy（TEM） was used to detect the stress activation of endoplasmic reticulum in NPCs. Immunofluorescence assay was performed to detect the expressions of glycoregulin 78（78 kDa glucose-regulated protein, GRP78） and C/EBP homologous protein（CHOP）, the stress specific markers of endoplasmic reticulum. Apoptosis and cell cycle were detected by flow cytometry after 4-PBA（endoplasmic reticulum stress blocker） was used to block endoplasmic reticulum stress. SA-β-gal staining kit was used to observe aging cells. Acidity-induced changes in autophagy-, aging- and apoptosis-related markers were studied by using Western blotting analysis, including LC3, GATA4, p53, p21, p16, Bax, Bcl-2 and Caspase3. Results： Compared with the control group, the overall proliferation capacity of myeloid nucleus cells under acid stimulation （pH6.5 group） was significantly lower than that of the control group. Under TEM, the endoplasmic reticulum expanded significantly, the membrane surface area increased, and the mitochondrial membrane structure of the endoplasmic reticulum was formed, accompanied by mitochondrial swelling. Immunofluorescence detection of cells indicated that GRP78 and CHOP expression increased significantly（P〈0.05）. After 4-PBA was applied, the apoptosis rate of cells increased, and the acid induced G1 stagnate and transglutaminase positive staining rate significantly increased（P〈0.05）. Western blot assay showed that the expressions of LC3, GATA4, p53, p21, p16, Bax and Caspase3 increased under acid stimulation（P〈0.05）, while Bcl-2 expression decreased（P〈0.05）. After applying 4-PBA, LC3 ratio and bcl-2 expression level decreased, and GATA4, p53, p21, p16, Bax and Caspase3 significantly increased（P〈0.05）. Conclusions： Acidic microenvironment can activate endoplasmic reticulum stress and play a protective role in acute injury of human medullary nucleus cells induced by acid.