鳖甲煎丸对CCl_(4)诱导小鼠肝纤维化的治疗作用机制研究

Therapeutic mechanism of Biejiajian pills on mice model of liver fibrosis induced by CCl_(4)

目的:研究鳖甲煎丸是否可以通过抑制单核细胞浸润从而减少单核细胞相关的促炎促纤维化细胞因子分泌来缓解肝纤维化。方法:40只小鼠被随机分为五组:空白对照组、四氯化碳(CCl_(4))组、低剂量鳖甲煎丸组、中剂量鳖甲煎丸组和高剂量鳖甲煎丸组。其中CCl_(4)组和鳖甲煎丸组予以CCl_(4)腹腔注射,每周两次,连续4周。高、中、低剂量鳖甲煎丸组小鼠在予以CCl_(4)腹腔注射的同时,分别每日予以鳖甲煎丸灌胃,连续4周,剂量为2.2、1.1、0.55 g/(kg·d)。实验结束后,通过肝酶、HE染色检测肝脏炎症,Masson染色、α-SMA和Col-I免疫组化观察肝脏胶原沉积和HSCs活化,CD11b和F4/80免疫组化、流式细胞术观察肝脏中单核细胞数量,qRT-PCR和WB检测肝脏中单核细胞趋化因子-1(MCP-1)的表达。结果:经4周CCl_(4)腹腔反复注射后,小鼠肝酶谷丙转氨酶(ALT)、谷草转氨酶(AST)明显升高,HE染色显示肝脏肝细胞坏死、炎性细胞浸润、胶原沉积,提示纤维化模型小鼠建立成功。鳖甲煎丸给药后,纤维化模型小鼠肝脏炎症和纤维化明显减少。与CCl_(4)组比较,鳖甲煎丸组小鼠单核细胞的肝内浸润明显减少。同时,鳖甲煎丸显著降低单核细胞相关促炎性和促纤维化细胞因子分泌,与肝内单核细胞数量减少相一致。进一步的研究发现鳖甲煎丸能够降低肝组织损伤时MCP-1分泌,表明鳖甲煎丸可以减少肝纤维化过程中单核细胞的浸润。结论:鳖甲煎丸可通过抑制单核浸润缓解肝纤维化,提示鳖甲煎丸可以作为预防和治疗肝纤维化的候选药物。

Objective:To investigate whether Biejiajian pills(BJJP)can alleviate liver fibrosis by inhibiting monocyte infiltration and thereby reducing monocytes-related pro-inflammatory and pro-fibrotic cytokine secretion.Methods:Forty mice were randomly divided into five groups:blank group,CCl_(4) group,low-dose BJJP group,middle-dose BJJP group and high-dose BJJP group.CCl_(4) group and BJJP group were given intraperitoneal injection of CCl_(4) twice a week for four weeks.Otherwise,the mice in high,middle and low-dose BJJP groups were given BJJP by gavage every day for four consecutive weeks at doses of 2.2,1.1 and 0.55 g/(kg·d).At the end of the experiment,liver inflammation was detected by liver enzyme and HE staining,and liver collagen deposition and HSCs activation were observed by Masson staining,α-SMA and Col-I immunohistochemistry.The number of monocytes in the liver was observed by CD11b and F4/80 immunohistochemistry,flow cytometry.And monocyte chemotactic protein-1(MCP-1)expression in the liver was detected by qRT-PCR and WB.Results:After repeated-CCl_(4) injection intraperitoneally for 4 weeks,liver enzymes ALT and AST in the mice increased significantly.HE staining showed hepatocyte necrosis,leucocytes infiltration and collagen deposition in the liver,suggesting that fibrosis model was successfully established.After BJJP administration,liver inflammation and fibrosis significantly reduced when compared with CCl_(4) group.Meanwhile,the intrahepatic infiltration of monocytes in BJJP group was significantly decreased.BJJP also significantly reduced monocytes-related proinflammatory and fibrogenic cytokines secretion,which was consistent with the decrease of monocytes infiltration.Further study found that BJJP can reduce MCP-1 secretion in liver tissue,which explains why BJJP can reduce monocytes infiltration during the liver fibrosis.Conclusion:BJJP can alleviate liver fibrosis by inhibiting monocytes infiltration.These results suggestted that BJJP can be used as a candidate drug for the prevention and treatment of liver fibrosis.