山奈酚对小鼠日本血吸虫肝纤维化α-平滑肌动蛋白、组织金属蛋白酶抑制因子1及胶原表达的影响

Effect of kaempferol on the expression of hepatic α-smooth muscle actin, tissue inhibitor of metalloproteinase 1 and collagen in liver fibrosis of mice infected with schistosomiasis japonicum

目的 研究感染日本血吸虫的小鼠经吡喹酮杀虫治疗后,给予山奈酚治疗对小鼠肝组织虫卵肉芽肿和纤维化的影响. 方法 以日本血吸虫尾蚴感染BALB/c小鼠作为肝纤维化动物模型,将40只健康BALB/c小鼠随机分为6组：正常组和模型组各8只,山奈酚组设5、10、15、20 mg/（kg·d）等4个剂量组,每组6只.除正常组外,其余5组小鼠感染日本血吸虫后6周给予吡喹酮灌胃治疗,剂量为500mg/（kg·d）×2 d.吡喹酮治疗后,山奈酚组小鼠分别给予山奈酚5、10、15、20 mg/（kg·d）灌胃治疗6周,正常组和模型组给予等体积生理盐水灌胃6周.治疗结束后颈椎脱臼处死小鼠,取肝脏.用免疫组织化学法检测各组小鼠肝组织金属蛋白酶抑制因子（tissue inhibitor of metalloproteinase,TIMP）1、α-平滑肌动蛋白（α-smooth muscle actin,α-SMA）、Ⅰ型胶原（type Ⅰ collagen,COLⅠ）、Ⅲ型胶原（typeⅢcollagen,COLⅢ）蛋白的表达;用实时荧光定量RT-PCR检测各组小鼠肝组织α-SMA、TIMP1、COLⅢmRNA的表达. 结果 山奈酚20 mg/（kg·d）组小鼠肝组织α-SMA、TIMP1、COLⅢmRNA的表达量依次为：1.251 7±0.053 8、1.490 1±0.042 9、1.328 3±0.070 3.模型小鼠肝组织α-SMA、TIMP1、COLⅢmRNA的表达量依次为：2.141 7±0.038 6、4.281 7±0.089 1、5.218 3±0.121 6.与模型组相比,山奈酚各剂量组α-SMA、TIMP1、COLⅢ的mRNA的表达量降低,差异均有统计学意义（F=36.93、95.16、48.29,P＜0.05）;山奈酚4个剂量组之间,随用药剂量增大,α-SMA、TIMP1、COLⅢmRNA的表达量下降,差异均有统计学意义（F=70.62、290.51、407.25,P＜0.05）.与模型组相比,山奈酚各剂量组α-SMA、TIMP1、COL Ⅰ、COLⅢ蛋白表达量下降,差异有统计学意义（F=13.46、237.96、191.58、274.32,P＜0.05）;山奈酚4个剂量组之间,随用药剂量增大,α-SMA、TIMP1、COLⅠ、COLⅢ蛋白表达量下降,差异有统计学意义（F=210.92、77.41、186.33、53.18,P＜0.05）. 结论 山奈酚可通过减少α-SMA、TIMP1、COLⅠ、COLⅢ的表达,能显著减轻日本血吸虫引起的小鼠肝纤维化.

Objective To investigate the effect of kaempferol on periovular granuloma and liver fibrosis in mice with schistosomiasis japonicum after treatment with praziquantel.Methods BALB/c mice infected with schistosomiasis japonicum were used for animal model of liver fibrosis.Forty BALB/c mice were randomly divided into a normal group （8 mice）,a model group （8 mice）,and 4 kaempferol groups with different kaempferol dosages [5,10,15,20 mg/（kg·d） respectively,6 mice each group].Besides the normal group,all the mice in the other 5 groups were infected with Schistosoma japonicum,and 6 weeks after the infection,were treated with praziquantel 500 mg/（kg·d） for 2 d.Kaempferol groups were administered intragastrically with kaempferol 5,10,15,20 mg/（kg·d） respectively for 6 weeks.The mice in normal group and model group were administered with normal saline for 6 weeks.All mice were sacrificed at the end of treatment.The protein expression of tissue inhibitors of metalloproteinases（TIMP）1,α-smooth muscle actin（α-SMA）,type Ⅰ collagen （COL Ⅰ）,type Ⅲ collagen （COLⅢ） on tissue microarray sections was detected by immunohistochemistry.The levels of mRNA expression of hepatic α-SMA,TIMP1,CO Ⅲ,mRNA were determined by RT-PCR.Results The relative mRNA expression levels of α-SMA,TIMP1,COL Ⅲ in liver tissues of kaempferol 20 mg/（kg·d） group were 1.251 7±0.053 8,1.490 1±0.042 9 and 1.328 3±0.070 3.The relative mRNA expression levels of α-SMA,TIMP1,COLⅢ in liver tissues of modle group were 2.141 7±0.038 6,4.281 7±0.089 1 and 5.218 3± 0.121 6.The levels of mRNA expression of hepatic α-SMA,TIMP1,type Ⅲ collagen in kaempferol treated groups were lower than those in the modle group.There was significant difference among the five groups （F=36.93,95.16,48.29,P＜0.05）.Increasing with the drug concentration of kaempferol,the mRNA expression levels of α-SMA,TIMP1,type Ⅲ collagen were significantly reduced in four dose groups,there was significant difference among the four groups （F=70.62,290.51,407.25,P＜0.05）,and the protein expression levels of α-SMA,TIMP1,types Ⅰ and Ⅲ collagen were significantly reduced in four dose groups,there was significant difference among the four groups （F=210.92,77.41,186.33,53.18,P＜0.05）.Conclusion By inhibiting the expressions of α-SMA,TIMP1,ypes Ⅰ and Ⅲ collagen,kaempferol can significantly reduce the degree of hepatic fibrosis caused by schistosome eggs.