车叶草苷对非酒精性脂肪性肝病大鼠肝纤维化的影响及机制

Effect and mechanism of asperuloside on liver fibrosis in non-alcoholic fatty liver rats

目的探究车叶草苷调节鞘氨醇激酶1(SphK1)/1-磷酸鞘氨醇(S1P)信号通路对非酒精性脂肪性肝病(NAFLD)大鼠肝纤维化的影响及机制。方法以高脂饲料饲养的方法建立大鼠NAFLD模型,将造模成功的大鼠随机分为模型组、车叶草苷低剂量组(14 mg/kg,灌胃,下同)、车叶草苷高剂量组(28 mg/kg)、车叶草苷高剂量(28 mg/kg)+pc-NC(空载质粒,50µg,尾静脉注射,下同)组、车叶草苷高剂量(28 mg/kg)+pc-SphK1(SphK1过表达质粒,50µg)组,每组12只。另取12只大鼠以正常饲料饲养作为对照组。各组大鼠灌胃或尾静脉注射相应药物或质粒,灌胃处理每天1次,尾静脉注射处理每周2次,均连续3周。末次给药后,检测各组大鼠血脂指标[总胆固醇(TC)、甘油三酯(TG)、游离脂肪酸(FFA)]与肝功能指标[天冬氨酸转氨酶(AST)和丙氨酸转氨酶(ALT)]水平;观察大鼠肝组织病理变化以及肝纤维化情况;检测大鼠血清纤维化相关因子[Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(Ⅳ-Col)、层粘连蛋白(LN)]、促纤维化因子[转化生长因子β(1 TGF-β1)]与促炎因子[白细胞介素1β(IL-1β)、诱导型一氧化氮合酶(iNOS)、IL-6]水平;检测大鼠肝组织胶原形成相关蛋白(Ⅰ-Col、Ⅳ-Col)与SphK1/S1P信号通路相关蛋白表达。结果与对照组比较,模型组大鼠肝组织呈现明显病理损伤症状;NAFLD活动度评分、肝组织胶原容积分数(CVF)和血清中TC、TG、FFA、AST、ALT、PCⅢ、Ⅳ-Col、LN、TGF-β1、IL-1β、iNOS、IL-6水平以及肝组织中Ⅰ-Col、Ⅳ-Col、SphK1、S1P蛋白表达水平均显著升高(P<0.05)。与模型组比较,车叶草苷低、高剂量组大鼠肝组织病理损伤症状均减轻,上述指标水平均显著降低(P<0.05),且高剂量组降低效果更好(P<0.05)。与车叶草苷高剂量组和车叶草苷高剂量+pc-NC组比较,车叶草苷高剂量+pc-SphK1组大鼠肝组织病理损伤症状加重,上述指标水平均显著升高(P<0.05)。结论车叶草苷可通过抑制SphK1/S1P信号通路活性,降低促纤维化因子、促炎因子以及胶原形成蛋白表达,从而减轻NAFLD大鼠肝纤维化。

OBJECTIVE To investigate the effect and mechanism of asperuloside on liver fibrosis in non-alcoholic fatty liver disease(NAFLD)rats by regulating the sphingosine kinase 1(SphK1)/sphingosine-1-phosphate(S1P)signaling pathway.METHODS SD rats were fed with a high-fat diet to establish a NAFLD model.They were randomly separated into model group,asperuloside low-dose group(14 mg/kg,i.g.,similarly hereinafter),asperuloside high-dose group(28 mg/kg),high dose of asperuloside(28 mg/kg)+pc-NC(empty plasmid,50µg,via tail vein,similarly hereinafter)group,and high dose of asperuloside(28 mg/kg)+pc-SphK1(SphK1 overexpression plasmid,50µg)group,with 12 rats in each group.Another 12 rats were fed with a normal diet as control group.Each group was given relevant medicine or plasmid intragastrically once a day or via tail vein twice a week,for 3 consecutive weeks.After the last medication,the levels of blood lipid indexes[total cholesterol(TC),triglyceride(TG),and free fatty acid(FFA)]and liver function indexes[aspartate transaminase(AST)and alanine transaminase(ALT)]were detected in each group.The pathological changes of liver tissue and liver fibrosis in rats were also observed in each group.The levels of serum fibrosis-related factors[procollagen typeⅢ(PCⅢ),collagen typeⅣ(Ⅳ-Col),laminin(LN)],pro-fibrotic factor[transforming growth factor-β1(TGF-β1)],and pro-inflammatory factors[interleukin-1β(IL-1β),inducible nitric oxide synthase(iNOS),IL-6]of rats were determined in each group.The expressions of collagen formation-related proteins(Ⅰ-Col,Ⅳ-Col)and SphK1/S1P pathway-related proteins in the liver tissues of rats were detected in each group.RESULTS Compared with control group,the liver tissue of rats in model group showed significant pathological damage;the NAFLD activity score,liver tissue collagen volume fraction,serum levels of TC,TG,FFA,AST,ALT,PCⅢ,Ⅳ-Col,LN,TGF-β1,IL-1β,iNOS and IL-6,and protein expressions ofⅠ-Col,Ⅳ-Col,SphK1 and S1P in liver tissue were greatly increased(P<0.05).Compared with the model group,the liver tissue pathological damage symptoms of rats in asperuloside low-dose and high-dose groups were improved,and the above indexes were all reduced significantly(P<0.05);moreover,the high-dose group had a better effect(P<0.05).Compared to asperuloside high-dose group,high dose of asperuloside+pc-NC group,the pathological damage of liver tissue symptoms in rats were aggravated in high dose of asperuloside+pc-SphK1 group,and the above indexes were all increased significantly(P<0.05).CONCLUSIONS Asperuloside can reduce the expressions of pro-fibrotic factor,pro-inflammatory factors and collagen formation-related proteins by inhibiting the activity of SphK1/S1P signaling pathway,thus alleviating liver fibrosis in NAFLD rats.