荣肝合剂对ConA诱导急性免疫性肝损伤小鼠免疫调节及肝细胞凋亡相关因子的影响

Effect of Ronggan Mixture on Immunoregulation and Hepatocyte Apoptosis-related Factors in Concanavalin A Induced Acute Immunological Liver Injury Mice

目的探讨荣肝合剂对刀豆蛋白A(concanavalin A,ConA)介导的急性免疫性肝损伤小鼠免疫调节因子及细胞凋亡相关基因的影响。方法乙肝病毒(hepatitis B virus,HBV)转基因小鼠60只,随机分为6组,即空白组、模型组、荣肝合剂组、茵陈蒿汤组、茵陈组、联苯双酯组,每组10只。采用ConA尾静脉注射制备急性免疫性肝损伤模型。造模前14天,空白组、模型组给予生理盐水灌胃,其余各组分别给予:荣肝合剂、茵陈蒿汤、单味茵陈煎液、联苯双酯溶液,每日灌胃给药干预。末次灌胃给药后1 h,空白组给予磷酸盐缓冲液(PBS)尾静脉注射,其余各组按照ConA 3μg/g体重尾静脉注射造模。造模给药后8 h处死动物取血或组织标本检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、总胆红素(TBil)、肿瘤坏死因子α(TNF-α)、干扰素γ(INF-γ)、白介素-4(IL-4)、白介素-10(IL-10)及凋亡相关基因(Fas、FasL、Bax、bcl-2)等指标。结果模型组各指标与空白组比较,差异均有统计学意义(P<0.05,P<0.01)。与模型组比较,荣肝合剂组、联苯双酯组小鼠ALT、AST及荣肝合剂组TBil水平显著降低(P<0.01);荣肝合剂组小鼠肝组织TNF-α的水平降低(P<0.05),荣肝合剂组、茵陈蒿汤组小鼠肝组织IFN-γ的表达水平均降低(P<0.05),各干预药组小鼠肝组织IL-4的表达提高(P<0.05),荣肝合剂组小鼠肝组织IL-10的表达提高(P<0.05);荣肝合剂组、茵陈蒿汤组小鼠肝组织Fas的表达下降(P<0.05),荣肝合剂组小鼠肝组织FasL的表达下降(P<0.05),bcl-2基因的表达升高(P<0.05),荣肝合剂、茵陈组小鼠肝组织Bax基因的表达下调(P<0.05),荣肝合剂组bcl-2/Bax比值上调。同时,荣肝合剂组降低ALT、AST作用优于茵陈组(P<0.05);荣肝合剂组降低TNF-α表达水平优于联苯双酯、茵陈组(P<0.05);荣肝合剂组提高IL-10表达水平作用优于茵陈蒿汤组(P<0.01);荣肝合剂组降低Fas、FasL表达作用优于茵陈蒿汤、茵陈组及联苯双酯组(P<0.05);荣肝合剂组提高肝组织bcl-2基因的表达作用优于茵陈组(P<0.05)。结论荣肝合剂对ConA所致转基因小鼠急性免疫性肝损伤具有保护作用,其保护作用是通过改变Th1/Th2因子平衡(降低TNF-α、IFN-γ的表达,提高IL-10、IL-4表达)和调控肝细胞凋亡相关因子(下调Fas、FasL、Bax基因表达,上调bcl-2基因表达,上调bcl-2/Bax比值)实现的。

Objective To explore the effect of Ronggan Mixture （RM） on immunoregulation and hepatocyte apoptosis-related factors in concanavalin A （Con A） induced acute immunological liver injury mice. Methods Totally 60 hepatitis B virus （HBV） transgenic mice were randomly divided into 6 groups, i.e., the blank control group, the model group, the RM group, theHerba Artemisiae Scopariae （HAS） group, the Yinchenhao Decoction （YD） group, and the Bifendate group, 10 mice in each group. The a- cute immunological liver injury model was established by tail vein injection of ConA. Fourteen days before modeling, normal saline was administered to mice in the blank control group and the model group. RM, YD, HAS decoction, and Bifendate solution was respectively given to mice in the RM group, the YD group, the HAS group, and the Bifendate group. The medication was performed once daily. One h after the last gastrogavage, phosphate buffer solution （PBS） was injected to mice in the blank control group from the tail vein. Modeling was conducted by injecting Con A at 3 t^g/g body weight from the tail vein. Mice were sacrificed 8 h after modeling. Blood or tissue samples were collected to detect lab indicators such as alanine aminotransferase （ALT）, aspartate aminotransferase CAST）, total bilirubin （TBil）, tumor necrosis factor （x （TNF-o~）, interferon ~/ （ INF-~/）, IL- 4, IL-10, Fas, FasL, Bax, and bcl-2. Results There was significant difference in all lab indicators between the normal group and the blank control group （P 〈0.05, P 〈0.01 ）. Compared with the model group, ALT and AST levels were significantly lower in the RM group and the Bifendate group （P 〈0.01 ） ; TBil significantly decreased in the RM group （P 〈0.01 ）. The expression level of TNF-e~ decreased in the RM group （P 〈0.05）. The expression level of IFN--y de- creased in the RM group and the YD group （P 〈0.05）. The expression level of IL-4 could be elevated in all medicated groups （P 〈0.05）. RM could elevate the expression level of IL-10 （P 〈0.05）. The expres- sion level of Fas in the liver tissue decreased in the RM group and the YD group （P 〈0.05）. The expres- sion level of FasL decreased and the expression of bcl-2 gene increased in the RM group （both P 〈0.05）. The expression level of Bax was down-regulated in the RM group and the YD group （P 〈0.05）. The ratio of bcl-2/Bax was up-regulated in the RM group （P 〈0.05）. Meanwhile, RM showed better effect in decrea- sing expressions of ALT and AST than HAS （P 〈0.05）. The effect of increasing IL-10 expression levels was better in the RM group than in the YD group （P 〈0.01 ）. The effect of decreasing expressions of Fas and FasL was better in the RM group than in the HAS group, the YD group, and the Bifendate group （P 〈 0.05）. The effect of enhancing the expression of IL-10 in the liver tissue was better in the RM group than in the HAS group （P 〈0.05）. Conclusion RM had protective effect on Con A induced acute immunologi- cal liver injury mice, which might be achieved by changing the immunological balance of Thl/Th2 factors （decreasing expressions of TNF-α and IFN-γ, elevating expressions of IL-10 and IL-4） and regulating hepatocyte apoptosis-related factors （down-regulating gene expressions of Fas, FasL, and Bax; up-reg- ulating bcl-2 gene expression, and up-regulating the bcl-2/Bax ratio）.