白桦脂酸对免疫性肝损伤小鼠细胞因子及Bcl-2、activated-Caspase-3表达的影响

The Effects of Betulinic Acid on the Expression of Cytokines and Apoptosis-related Proteins in Immune Liver Injury Induced by Concanavalin A

目的研究白桦脂酸(BA)对刀豆蛋白(Con A)诱导的急性免疫性肝损伤小鼠细胞因子及凋亡相关蛋白Bcl-2、activated-Caspase-3表达量的影响。方法将60只雄性KM小鼠随机分为6组,包括正常对照组,肝损伤模型组,联苯双酯(BIF)阳性对照组,BA高、中、低剂量组(H-BA、M-BA、L-BA组剂量分别为30 mg·kg^(-1)、15 mg·kg^(-1)、7.5 mg·kg^(-1))。BIF阳性对照组和BA高、中、低剂量组预防性给药15 d后,尾静脉注射20 mg·kg^(-1)Con A构建小鼠急性免疫性肝损伤模型。采用自动生化分析仪测定血清肝功能指标:谷丙转氨酶(ALT)、谷草转氨酶(AST)含量;ELISA法测定血清炎性细胞因子IL-2、IL-4、IL-10、TNF-α、IFN-γ水平;Western blot法检测肝组织凋亡相关蛋白Bcl-2、activated-Caspase-3表达量的变化。结果与正常对照组比较,肝损伤模型组血清ALT和AST含量明显降低,IL-2、IL-4、IL-10和肿瘤坏死因子(TNF-α)、γ干扰素(IFN-γ)水平明显升高,Bcl-2表达量下降而activated-Caspase-3表达量升高,差异均有统计学意义(P<0.05或P<0.01);与肝损伤模型组比较,BA不同剂量组可显著降低Con A所致急性肝损伤小鼠血清ALT和AST含量,使小鼠血清中IL-2、IL-4、TNF-α、IFN-γ水平显著降低,而IL-10水平显著升高,尤其是H-BA组、M-BA组,同时使Bcl-2表达量升高、activated-Caspase-3表达量下降,差异均有统计学意义(P<0.05或P<0.01)。结论 BA对由Con A诱导的小鼠急性免疫性肝损伤具有拮抗作用。其机制可能与抗凋亡及降低炎性细胞因子水平和提高抗炎细胞因子水平、减轻T淋巴细胞毒性作用有关。

Objective To study the effects of betulinic acid（ BA） on the expression of cytokines and apoptosis-related proteins in acute immune liver injury induced by concanavalin A（ Con A）. Methods A total of 60 male KM mice were divided into six groups randomly,including control group,Con A model group,bifendate（ BIF） group and three BA groups with gradient concentrations. Saline diluted BA（ 30 mg·kg^（-1）,15 mg·kg^（-1）,7. 5 mg·kg^（-1）） was given orally to mice. After 15days＇ BA pretreatment,mice were then injected with Con A（ 20 mg·kg^（-1）） to establish the mouse model with acute immune liver injury. Automatic biochemical analyzer was conducted to determine serum liver function indices such as plasma alanine aminotransferase（ ALT） and aspartate aminotransferase（ AST）; ELISA was used to determine the levels of serum inflammatory cytokines（ IL-2,IL-4,IL-10,TNF-α,IFN-γ）; Western blot was used to detect the expression of liver tissue apoptosis related proteins（ Bcl-2 and activated-Caspase-3）. Results Compared with the control group,Con A treated mice showed significantly lower serum ALT and AST levels,while the levels of inflammatory cytokines（ IL-2,IL-4,IL-10,TNF-α,IFN-γ）increased significantly. The expression of Bcl-2 was significantly decreased（ P〈0. 05）,whereas the expression of activated-Caspase-3 was significantly increased（ P〈0. 01）. Compared with the Con A model group,the mice that treated with different doses of BA,especially the middle and high dose groups,showed significantly reduced serum ALT and AST levels,as well as significantly decreased serum IL-2,IL-4,TNF-α and IFN-γ levels but increased IL-10. The apoptosis related proteins Bcl-2 and activated-Caspase-3 also showed contrary expression changes： the former increased while the latter decreased. Conclusion These findings suggested that BA had antagonistic action on the mouse with acute immune liver injury induced by Con A to some extent,and the mechanism of this process may be related with the anti-apoptosis and anti-inflammatory functions of BA to reduce the toxic effects of T lymphocytes.