乙型肝炎病毒通过自分泌运动因子/溶血磷脂酸信号损伤对糖稳态的作用及其机制研究  

Hepatitis B virus impairs glucose homeostasis via autotaxin/ lysophosphatidic acid signaling

作　　者：朱政全 梁斌[1] 石明连 谷云艳 周先丽[1] 梁成钦[3] 刘永明[1] 苏何玲[1] Zhu Zheng-quan;Liang Bin;Shi Ming-lian;Gu Yun-yan;Zhou Xian-li;Liang Cheng-qin;Liu Yong-ming;Su He-ling(Key Laboratory of Biochemistry and Molecular Biology,Guilin Medical College,Guilin,Guangxi 541199,China;Department of Pathology,Guilin Medical College,Guilin,Guangxi 541199,China;Department of Medicinal Chemistry,Guilin Medical College,Guilin,Guangxi 541199,China)

机构地区：[1]桂林医学院生物化学与分子生物学重点实验室,广西桂林541199 [2]桂林医学院病理学教研室,广西桂林541199 [3]桂林医学院药物化学教研室,广西桂林541199

出　　处：《中国现代医学杂志》2024年第16期17-25,共9页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:81460320,No:82060787,No:31560100)。

摘　　要：目的探讨乙型肝炎病毒(HBV)通过自分泌运动因子(ATX)/溶血磷脂酸(LPA)信号对糖稳态的损伤作用及相关机制。方法利用基因芯片数据库分析HBV对ATX表达的影响。Western blotting检测HBV细胞株HepG2215、稳定表达HBV反式调节蛋白HBx和PreS2的HBx-HepG2和PreS2-HepG2细胞、对照组HepG2细胞的ATX蛋白表达水平。双萤光素酶报告基因检测HBx和PreS2对ATX启动子作用。构建稳定表达HBx和Pre-S2的小鼠分泌胰岛素细胞HBx-NIT、PreS2-NIT,检测两者对胰岛素分泌的影响。利用rAAV8-1.3HBV经尾静脉注射C57BL/6小鼠复制HBV小鼠模型,NC为注射同体积生理盐水的C57BL/6小鼠。小鼠按2 g/kg腹腔注射葡萄糖进行糖耐量试验(GTT)。采用液相色谱-质谱联用法、酶联免疫吸附试验和血糖分析仪分别检测小鼠血清LPA、血清胰岛素和血糖浓度。结果HepG2215细胞ATX蛋白相对表达量高于HepG2(P<0.05)。PreS2与ATX启动子共转染组萤光素酶活性高于ATX启动子转染组(P<0.05),HBX与ATX启动子共转染组的萤光素酶活性高于ATX启动子转染组(P<0.05)。HBx-HepG2细胞ATX蛋白相对表达量高于HepG2细胞(P<0.05),PreS2-HepG2细胞高于HepG2细胞(P<0.05)。添加不同浓度LPA后NIT细胞胰岛素分泌表达量比较,差异有统计学意义(P<0.05)。1~3μmol/L LPA相对表达量与胰岛素分泌呈负相关(r=-0.990,P<0.05)。HBx-NIT细胞添加Ki16425前的胰岛素水平低于NIT细胞(P<0.05),PreS2-NIT细胞低于NIT细胞(P<0.05)。NIT、HBx-NIT和PreS2-NIT细胞添加Ki16425后的胰岛素水平较添加前高(P<0.05)。实验组血清LPA相对表达量、平均空腹血糖浓度、注射葡萄糖后的60、120 min平均血糖浓度和血糖浓度曲线下面积(AUC)平均值较对照组高(P<0.05)。实验组注射葡萄糖后15 min血清胰岛素浓度、血清胰岛素水平的AUC值较对照组低(P<0.05)。用HBV小鼠GTT血糖AUC绘制的ROC曲线显示,曲线面积为0.770(95%CI:0.556,0.984),特异性为60.00%(95%CI:0.122,0.73Objective To investigate the effects and underlying mechanisms of hepatitis B virus on impairing glucose homeostasis through the autotaxin(ATX)/lysophosphatidic acid(LPA)signaling.Methods Gene expression databases were used to analyze the effect of HBV on the expression of ATX.Western blotting(WB)was performed to detect the protein expressions of ATX in HBV-expressing cell line HepG2215,HBX-HepG2 and PRES2-HepG2 cells stably expressing HBV trans-regulatory proteins HBx and PreS2,and normal control(NC)HepG2 cells.The dual luciferase reporter assay was applied to detect the effects of HBx and PreS2 on the promoter activity of ATX.HBx-NIT and PreS2-NIT,the mice insulin-secreting cells with stable expressions of HBx and pre-S2,were constructed to detect the effects of HBx and pre-S2 on insulin secretion.Mice models of HBV infection were established by injecting C57BL/6 mice with rAAV8-1.3 HBV via tail veins,while C57BL/6 mice injected with the same volume of normal saline were set as NCs.The glucose tolerance test(GTT)was performed by intraperitoneal injection of glucose at a dosage of 2 g/kg body weight of mice.Serum LPA,serum insulin and blood glucose were measured by liquid chromatography-mass spectrometry,ELISA kits and blood glucose analyzer,respectively.Results The protein expression of ATX in HepG2215 cells was higher than that in HepG2 cells(P<0.05).The luciferase activity in those co-transfected with PreS2 and ATX promoter was higher than that in those transfected with ATX promoter alone(P<0.05),and that in those co-transfected with HBX and ATX promoter was also higher compared with that in those transfected with ATX promoter alone(P<0.05).The protein expression of ATX in HBx-HepG2 cells was higher than that in HepG2 cells(P<0.05),and that in PreS2-HepG2 cells was higher than that in HepG2 cells(P<0.05).There were differences in insulin secretion among NIT cells treated with different concentrations of LPA(P<0.05),and the concentration of LPA was negatively correlated with insulin secretion when it was withi

关 键 词：乙型肝炎病毒 反式调节蛋白 自分泌运动因子/溶血磷脂酸信号 糖稳态 

分 类 号：R373.2[医药卫生—病原生物学]