和血柔肝方对肝纤维化大鼠SphK1/S1P/S1PR信号通路的影响

Effects of Hexue Rougan Prescription on SphK1/S1P/S1PR Signaling Pathway in Liver Fibrosis Rats

目的观察和血柔肝方对肝纤维化大鼠SphK1/S1P/S1PR信号通路相关因子表达的影响,探讨其治疗肝纤维化的作用机制。方法SPF级雄性SD大鼠随机分为对照组和造模组,造模组大鼠每周2次颈背部皮下注射60%CCl4溶液(CCl4∶橄榄油=3∶2),剂量3 mL/kg,连续12周,复制肝纤维化大鼠模型。造模成功后,将造模组大鼠随机分为模型组、秋水仙碱组及和血柔肝方低、中、高剂量组,每组10只。各给药组分别予相应药液灌胃,对照组和模型组予等体积蒸馏水,每日1次,连续4周。给药结束后分别检测大鼠血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST),HE染色观察大鼠肝组织病理变化,RT-PCR检测大鼠肝组织鞘氨醇激酶1(SphK1)、1-磷酸鞘氨醇受体3(S1PR3)、细胞外信号调节激酶1(ERK1)、第10号染色体缺失的磷酸酶和张力蛋白同源物基因(PTEN)、肿瘤坏死因子受体相关因子2(TRAF2)及转化生长因子-β(TGF-β)mRNA表达,Western blot检测大鼠肝组织SphK1、S1PR3、ERK1、PTEN和TGF-β蛋白表达。结果与对照组比较,模型组大鼠体质量、肝质量、肝系数和脾系数显著降低(P<0.01,P<0.05),血清ALT、AST含量显著增加(P<0.01),肝脏呈深褐色、无光泽,肝体缩小,包膜粗糙,散在大小不等的结节,肝小叶结构破坏,汇管区和中央静脉间有明显桥接纤维间隔形成,并伴有少量炎性细胞浸润,肝组织TGF-β、SphK1、TRAF2、PTEN、ERK1和S1PR3 mRNA表达显著升高(P<0.01,P<0.05),TGF-β、SphK1、PTEN、ERK1、S1PR3蛋白表达显著升高(P<0.01,P<0.05);与模型组比较,各给药组大鼠体质量、肝质量、肝系数、脾质量和脾系数差异无统计学意义(P>0.05),和血柔肝方高剂量组大鼠肝脏形态和肝小叶结构改善,纤维间隔明显减少,炎性细胞浸润减少,和血柔肝方各剂量组大鼠肝组织TGF-β、SphK1、TRAF2、PTEN、ERK1和S1PR3 mRNA表达显著降低(P<0.01),TGF-β、SphK1、PTEN、ERK1、S1PR3蛋白表达显著降低(P<0.01)。结论和血柔肝方可减轻CCl4诱导的肝纤维化大鼠肝脏炎症及纤维化状态,尤以和血柔肝方高剂量组疗效显著,其可能通过抑制SphK1/S1P/S1PR信号轴活化状态、减少TGF-β产生,发挥治疗肝纤维化的作用。

Objective To evaluate the effects of Hexue Rougan Prescription on the expressions of factors related to SphK1/S1P/S1PR signaling pathway in rat model with liver fibrosis;To explore its therapeutic mechanism on liver fibrosis.Methods SD rats were randomly divided into control group and modeling group.Rats in the modeling group received subcutaneous injection of 60% CCl_(4) solution(CCl_(4)∶olive oil=3∶2)into the back of the neck twice a week at a dose of 3 mL/kg for 12 weeks to replicate the rat model with liver fibrosis.Then,the rats in the modeling group were randomly divided into model group,colchicine group,and Hexue Rougan Prescription low-,medium-,and high-dosage groups,with 10 rats in each group.Each intervention group was given the corresponding medicinal solution by gavage,and the control group and the model group were given equal volume of distilled water,once a day for 4 consecutive weeks.At the end of these interventions,the contents of alanine aminotransferase(ALT),aspartate aminotransferase(AST)in serum were examined;the histopathological changes of liver tissue were observed by HE staining;the mRNAs expression of Sphingosine kinase 1(SphK1),sphingosine 1-phosphate receptor 3(S1PR3),extracellular signal regulated kinase 1(ERK1),gene of phosphate and tension homology deleted on chromsome ten(PTEN),TNF receptor-associated factor 2(TRAF2),and transforming growth factorβ(TGF-β)in liver tissue were assessed by RT-PCR;the proteins expression of SphK1,S1PR3,ERK1,PTEN and TGF-βin liver tissue were detected by Western blot.Results Compared with the control group,the body mass,liver mass,liver coefficient and spleen coefficient of the rats in model group significantly decreased(P<0.01,P<0.05),the contents of ALT and AST in serum increased(P<0.01).The liver was dark brown,dull,with a reduced size of the hepatic body,a rough capsule,scattered nodules of varying sizes,disruption of the hepatic lobular architecture,marked bridging fibrous septa formation between the confluent zone and central veins,and a few inflammatory cell infiltrates.The expressions of TGF-β,SphK1,TRAF2,PTEN,ERK1 and S1PR3 mRNA significantly increased(P<0.01,P<0.05),the proteins expression of TGF-β,SphK1,PTEN,ERK1 and S1PR3 significantly increased(P<0.01,P<0.05).Compared with the model group,the body mass,liver mass,liver coefficient,spleen mass and spleen coefficient of the rats in each intervention group were with no stastical significance(P>0.05).The morphology of liver and structure of hepatic lobular improved in Hexue Rougan Prescription high-dosage group,with fewer fibrous septa and less inflammatory cell infiltration.The mRNAs expression of TGF-β,SphK1,TRAF2,PTEN,ERK1 and S1PR3 in liver tissue significantly decreased(P<0.01)in each dosage group of Hexue Rougan Prescription,the proteins expression of TGF-β,SphK1,PTEN,ERK1 and S1PR3 significantly decreased(P<0.01).Conclusion Hexue Rougan Prescription can reduce liver inflammation and fibrosis in liver fibrosis rats induced by CCl_(4),especially in Hexue Rougan Prescription high-dosage group.It may play a role in the treatment of liver fibrosis by inhibiting the activation of SphK1/S1P/S1PR signaling axis and reducing the production of TGF-β.