补肾健脾方通过干预HBX-HMGB1（乙酰化）-Beclin1途径抑制肝细胞自噬治疗慢性乙型肝炎的机制研究

Bushen Jianpi Recipe（BSR） is an effective prescription for the treatment of chronic hepatitis B. Earlier RCT studies have confirmed that the serum negative rates of HBeAg and HBsAg BSR were up-regulated, which is related to the regulation of function of immune cells. The recent studies show that the expression levels of HBsAg, HBeAg, HBx and HMGB1 and the formation of autophagy are inhibited by BSR in cell model. According to the latest report, HDAC1 expression is inhibited by HBx and then HMGB1 acetylation is up-regulated and promotes its cytoplasmic translocation, and autophagy in hepatocytes is induced. The hypothesis is proposed that the expression of HBx is inhibited by BSR, and then HDAC1 expression is increased, and HMGB1 acetylation is decreased, and its cytoplasmic translocation was inhibited, and the complex of HMGB1/Beclin1 is decreased, and autophagy in hepatocytes is inhibited, and HBV replication is decreased, which is the mechanism of BSR playing the role of anti HBV. The following studies will be done: the correlation between the expression of HBx in the liver of patients with chronic hepatitis B and the therapeutic effect of BSR will be analyzed; the mechanism of BSR regulating autophagy against HBV by HBx-HMGB1(acetylation)-Beclin1 will be researched by using HBV transgenic mice; HBx gene is over-expressed in HepG2.2.15 cell, and it is used to screen the effective components of BSR by inhibiting autophagy against HBV. With epigenetics as a breakthrough point, we expect to explore the mechanism of BSR treating HBV by inhibiting autophagy.

补肾健脾方（BSR）是治疗慢乙肝有效验方。前期RCT证实，BSR提高患者血清HBeAg和HBsAg阴转率，与调节免疫细胞功能有关。近期发现，BSR抑制乙肝细胞模型HBsAg、HBeAg、HBx、HMGB1表达和自噬体形成。结合文献“HBx抑制HDAC1表达，促进HMGB1乙酰化后的胞浆转位，增加肝细胞自噬”，提出假说：BSR通过抑制肝细胞HBx表达，促进HDAC1表达，抑制HMGB1乙酰化后的胞浆转位，减少HMGB1/Beclin1复合体，抑制自噬体形成，减少病毒复制，治疗慢乙肝。拟开展：分析慢乙肝患者肝脏HBx表达与BSR疗效的相关性；应用HBV转基因小鼠研究BSR通过HBx-HMGB1（乙酰化）-Beclin1调节自噬抗HBV的机制；应用HBx过表达的HepG2.2.15细胞模型，筛选BSR抑制自噬抗HBV的有效组分。以期从表观遗传学入手阐明中药抑制肝细胞自噬发挥抗HBV作用的机制。

应用ELISA法检测慢乙肝患者治疗前、后血清中HBx和HMGB1的表达水平，补肾健脾方治疗24周和48周HBX和HMGB1均有显著降低。药物血清作用于HepG2.2.15细胞系24和48小时后，HBsAg和HBeAg的表达水平均呈现下降趋势。HDAC1表达被抑制后，药物血清降低HBsAg、HBeAg和HBVDNA表达水平的作用均被抑制。自噬被抑制后，HepG2.2.15细胞系HBsAg和HBeAg的表达水平下降，与复方药物血清干预后结果类似，说明补肾健脾方可以发挥与自噬抑制剂类似的通过抑制肝细胞自噬的发挥抗乙肝病毒的作用。质粒构建乙型肝炎小鼠动物模型后，发现相比于正常C57野生型小鼠，HBx转基因小鼠外周血中HBsAg抗原的表达呈现出显著缓慢下降的表现，而HBeAg抗原则没有显著差异。与此同时，HBX转基因小鼠的ALT和AST的水平也呈现出较慢下降趋势。正常C57野生型小鼠质粒构建乙肝模型后复方灌胃治疗，治疗组的HBsAg和HBeAg的下降速度更快，炎症改善，肝酶ALT也下降的更快，但AST却没有显著差异。治疗组肝组织中HDAC的活性也显著高于对照组。抑制HDAC1后，可以发现补肾健脾方治疗乙肝的疗效减弱。蛋白组学分析复方治疗后的小鼠肝原代细胞和肝脏PBMC，发现中药显著激活了肝细胞的代谢功能，尤其是脂代谢和能量代谢，相反，中药显著抑制免疫细胞的代谢能力，抑制其能量代谢。提示补肾健脾方可以激活肝细胞并抑制免疫细胞，从而改善肝脏炎症。运用细胞毒性实验检测补肾健脾方总多糖、补肾拆方多糖、健脾拆方多糖和单药多糖42个多糖和1个阳性对照ETV给药24h和48h后的细胞活力，结果显示42个多糖对肝癌细胞HepG2.2.15没有毒性。