肝细胞内质网应激对乙型肝炎病毒复制及核苷（酸）类抗病毒药物作用的影响

Endoplasmic reticulum stress （ER stress）of hepatocytes has been observed in a varity of liver diseases. Recent studies have found that hepatitis B virus (HBV) induces hepatocyte ER stress and activates the ER-associated degradation pathway, which, in turn, regulates the secretion of envelope proteins and HBV replication in hepatocytes. However, it is not clear whether the hepatocyte ER stress induced by thapsigargin, saturated fatty acid and glycochenodeoxycholic acid also have similar impacts on HBV replication. The impacts of different intensity of ER stress on HBV replication in hepatocytes have also not been studied yet. Furthermore, the possible influence of hepatocyte ER stress on the antiviral effects of nucleoside/nucleotide analogues is also not known. Therefore, in this study: ① we plan to observe the impacts of different intensity of ER stress induced by thapsigargin, saturated fatty acid and glycochenodeoxycholic acid on HBV replication and the mechanisms involved in HepG2.2.15 cells; ② by determining the intensity of ER stress of hepatocytes obtained from liver biopsy from patients with chronic hepatitis B (CHB), we plan to observe the correlation of different intensity of hepatocyte ER stress with HBV replication and the antiviral effects of nucleoside/nucleotide analogues in vivo; ③ by using tauroursodeoxycholic acid (TUDCA) to alliviate the intensity of hepatocyte ER stress and nucleoside/nucleotide analogues to inhibit HBV replication, we plan to explore the effects of a new treatment strategy of "alliviating ER stress plus antiviral" in the treatment of CHB.

已知内质网应激是多种肝脏疾病常见病理现象,可由多种诱因引起。新近发现：乙型肝炎病毒（HBV）感染可诱导肝细胞内质网应激反应、其信号通路可调节HBV病毒复制及相关抗原分泌。基于此，本研究提出的问题是：其他诱因及不同强度的肝细胞内质网应激对HBV复制是否有影响？是否和抗病毒药物疗效相关？针对这一问题，拟（1）采用HepG2.2.15细胞，用不同剂量毒胡萝卜素、饱和脂肪酸及甘氨鹅去氧胆酸钠诱导不同强度内质网应激，采用定量检测sXBP1mRNA判断内质网应激强度、HBVDNA等观察HBV复制、培养上清HBV抗原浓度观察抗原分泌；（2）采用肝穿样本确定不同强度内质网应激的队列，观察核苷（酸）类抗病毒药物治疗后病毒拷贝数动力学变化差异、肝细胞内病毒复制情况；（3）采用牛磺熊去氧胆酸抑制内质网应激、联合核苷（酸）类抗病毒药物治疗，观察临床受试者疗效。旨在探索"抗病毒药物联合抑制内质网应激"治疗新策略。

摘要:乙型肝炎病毒（HBV）与宿主肝细胞存在复杂的相互作用并对HBV复制有重要影响。内质网应激（ER stress）是多种肝脏疾病中的常见病理现象。有研究证明HBV感染诱导肝细胞ER stress，其信号通路可调节HBV病毒颗粒及相关抗原分泌，可能与HBV在肝细胞内建立持续感染有关，但肝细胞ER stress对HBV复制的影响尚不清楚。本研究首先在体内及体外探讨了HBV DNA复制水平与肝细胞ER stress相关基因表达的相关性，结果发现体内肝细胞ER stress相关基因GRP78 mRNA、ATF6 mRNA表达水平与HBV复制水平呈正相关，与肝脏炎症程度及纤维化程度无明显相关；而在体外用拉米夫定抑制HBV复制后肝细胞ER stress相关基因表达无明显改变。这一结果提示HBV在体内可以诱导肝细胞ER stress。然后我们在体外探讨毒胡萝卜素（TG）及甘氨鹅去氧胆酸钠（GCDCA）诱导的肝细胞ER stress对HBV病毒复制的影响，发现TG及GCDCA诱导的肝细胞ER stress时间及剂量依赖性抑制肝细胞HBsAg、HBeAg分泌及HBV DNA复制；为进一步探讨肝细胞ER stress对HBV DNA复制的影响及其机制，我们建立慢性HBV感染合并脂肪性肝病细胞模型，在体外探讨脂肪酸对HBV复制的影响及其机制，结果发现硬脂酸（stearic acid, SA）、油酸（oleic acid, OA）诱导肝细胞发生脂肪变性及 ER stress, SA及OA通过诱导肝细胞ER stress 抑制HBVDNA及HBsAg、HBeAg分泌，用GRP78 siRNA抑制GRP78表达后，SA对HBsAg分泌及HBV DNA的抑制作用并未减轻，说明脂肪酸可能并不通过诱导GRP78表达抑制HBsAg的分泌和HBV DNA复制。最后我们用salubrinal抑制内质网应激通路eIF2α去磷酸化及应用ISRIB催化eIF2B活化，发现TG对HBVDNA分泌的抑制作用有所减轻，提示肝细胞内质网应激可能通过PERK-eIF2α通路介导其对HBVDNA 的抑制作用。