亚硫酸钠对HL-7702细胞极低密度脂蛋白组装分泌和内质网应激的影响

目的:研究亚硫酸钠(Na₂SO₃)是否通过抑制极低密度脂蛋白(VLDL)组装分泌和诱导内质网应激引起肝细胞脂肪变性。方法:分别经不同浓度(0.1、0.5、2.5、10 mmol/L)Na₂SO₃染毒人正常HL-7702肝细胞24和48 h后,采用酶联免疫吸附法(ELISA)和Western blot测定细胞外VLDL水平;采用荧光定量PCR法(qPCR)检测VLDL组装相关因子微粒体载脂蛋白B100(apoB100)、甘油三酯转移蛋白(MTP)、二脂酰甘油酰基转移酶2(DGAT2)、甘油三酯水解酶(TGH)的mRNA表达水平;采用Western blot法检测内质网应激相关重链结合蛋白(BiP)和钙连蛋白(Calnexin)的表达水平。结果:ELISA和Western blot结果显示染毒24 h后,与阴性对照组相比,各Na₂SO₃处理组VLDL含量均明显增加,差异均有统计学意义(P均<0.05);而作用48 h后,各Na₂SO₃处理组VLDL含量与阴性对照组相比均无明显变化(P均>0.05)。qPCR检测结果显示,与阴性对照组相比,染毒24 h,10 mmol/L Na₂SO₃组肝细胞内apoB100 mRNA表达升高(P<0.05);2.5和10 mmol/L Na₂SO₃可使肝细胞内TGH mRNA表达升高(P<0.05);染毒48 h,0.5和2.5 mmol/L Na₂SO₃引起肝细胞内TGH mRNA表达量明显升高(P<0.05)。Western blot结果显示染毒24 h和48 h后,不同浓度Na₂SO₃对细胞内BiP蛋白表达水平均无明显影响,但可以明显增加Calnexin蛋白表达(P<0.05)。结论:Na₂SO₃不能抑制VLDL组装分泌,但可能对肝细胞糖蛋白的正确折叠有一定干扰作用。

Effects of sodium sulfite on very low density lipoprotein assembly and secretion,and endoplasmic reticulum stress in HL-7702 liver cells

OBJECTIVE:To investigate whether sodium sulfite (Na₂SO₃) induces lipid accumulation by inhibition of very low density lipoprotein (VLDL) assembly and activation of endoplasmic reticulum stress. METHODS:Human diploid liver cells (HL-7702) were cultured in DMEM medium containing 10% fetal bovine serum. Cells were exposed to different concentrations (0.1, 0.5, 2.5, 10 mmol/L) of Na₂SO₃ for 24 h and 48 h, and positive controls were used. VLDL secretion was detected by ELISA and Western blot, mRNA levels of VLDL assembly related factors were detected by quantitative real-time PCR (qPCR) and protein expressions of endoplasmic reticulum stress were detected by Western blot. RESULTS:ELISA and Western blot results showed that after exposure to Na₂SO₃ for 24 h, VLDL contents were significantly increased compared to the controls (P<0.05). After 48 hours, the difference became non-significant (P>0.05). QPCR test results showed that, compared with the negative control group, exposure to 10 mmol/L Na₂SO₃ for 24 h increased the expression of apoB100 mRNA significantly (P<0.05). In addition, the mRNA level of TGH in 10 mmol/L Na₂SO₃ and 2.5 mmol/L Na₂SO₃ groups were significantly increased (P<0.05). After exposure for 48 h, the mRNA levels of TGH in 2.5 mmol/L Na₂SO₃ and 0.5 mmol/L Na₂SO₃ groups were increased significantly (P<0.05). Protein expression of Calnexin was significantly increased (P<0.05) after Na₂SO₃ exposure. CONCLUSION:Na₂SO₃ may increase VLDL assembly and interfere with the correct folding of glycoprotein in hepatocytes.