关键词: 热休克蛋白47, 青蒿琥酯, 肝纤维化, 日本血吸虫

Abstract:

Objective To understand the possible mechanism underlying the artesunate’s inhibition on Schistosoma japonicum-induced hepatic fibrosis, the effect of artesunate on the expression of heat shock protein 47 (HSP47) in serum and liver during the early stage of mouse hepatic fibrosis induced by S. japonicum infection was studied. Methods Total 30 female ICR mice were randomly divided into 3 groups: the un-infected control group, the infection group and the treatment group (infection + artesunate treatment), with 10 mice in each group. Each mouse in the infection and treatment groups was infected with 20 ± 1 cercariae of S. japonicum via abdomen skin penetration. All infected mice were treated with 300 mg/kg praziquantel for 2 days to eliminate the adult worms and the mice in treatment group were additionally treated with 60 mg/kg artesunate in 0.3 ml volume by intraperitoneal injection daily for 2 weeks. For the control group, mice were given the same volume of saline. After the artesunate treatment, all mice from 3 groups were sacrificed, blood samples were taken for serum separation, and the levels of HSP47 and transforming growth factor-β1(TGF-β1) in sera were detected by ELISA. The mouse livers and spleens were collected and weighed to calculate their organ index(organ weight/body weight). The livers were fixed with paraformaldehyde and sectioned for hematoxylin eosin (HE) and Masson-trichrome staining to observe the liver pathological changes and collagen proliferation. The hydroxyl-proline level in liver was measured using the alkaline hydrolysis. The mRNA expression levels of liver HSP47 and type Ⅰ collagen (COL1) were determined by real-time quantitative PCR (RT-PCR). Results The liver index in infection group (10.50 ± 0.57) was significantly higher than that in treatment group (8.31 ± 0.52) and in un-infected control group (4.72 ± 0.52) with statistical difference (P < 0.01). The spleen index was significantly higher in infection group (2.41 ± 0.44) than that in un-infected control group (0.38 ± 0.04) (P < 0.01), however it was not significantly different compared to that in treatment group(P > 0.05). Liver pathological damage was significantly reduced in treatment group compared to the infection group, including reduced liver size and egg granuloma. Masson-trichrom staining showed less liver fibrosis in treatment group than in infection group. The area of collagen proliferation in liver in treatment group was (6.87 ± 3.54)%, that is significantly smaller than that in infection group [(25.78 ± 2.61)%] (P < 0.01). The levels of HSP47 and TGF-β1 in sera of mice in the treatment group [(169.81 ± 20.94) pg/ml and (20.82 ± 1.90) ng/ml, respectively] were significantly lower than that in infection group [(203.14 ± 46.29) pg/ml and (27.49 ± 6.81) ng/ml] (P < 0.05), even though the levels of HSP47 and TGF-β1 in both infection and treatment groups were significantly higher than that in the un-infected control group (P < 0.01). The similar results were also observed in the hydroxyproline level and the expression of HSP47 and COL1 in liver tissues. The level of liver hydroxyproline in the treatment group was (0.27 ± 0.08) mg/g, which was significantly lower than that of the infection group [(0.69 ± 0.07) mg/g] (P < 0.01), however, all higher than that in un-infected control group [(0.11 ± 0.04) mg/g] (P < 0.05). The mRNA expression levels of HSP47 and COL1 in the liver of mice of treatment group were 0.49 ± 0.27 and 0.67 ± 0.09, respectively, which are significantly lower than those in the infection group (0.84 ± 0.17 and 0.91 ± 0.11, respectively) (P < 0.05). The expression levels of HSP47 and COL1 in both infection and treatment groups were significantly higher than those in the un-infected control group (0.24 ± 0.04 and 0.24 ± 0.05) (P < 0.01). The serum HSP47 level was positively correlated with the level of TGF-β1 (r = 0.928 0), and serum HSP47 level and TGF-β1 level were positively correlated with the level of hepatic COL1 mRNA expression(r = 0.926 2, 0.872 8). Conclusion Artesunate has protective effects on the early hepatic fibrosis caused by S. japonicum infection and the protection is possibly achieved by down-regulating the expression of HSP47, further suppressing the collagen synthesis so as to alleviate the hepatic fibrosis. HSP47 is expected to be one of the new prognostic markers and treatment targets in S. japonicum infection.

Key words: Heat shock protein 47, Artesunate, Liver fibrosis, Schistosoma japonicum