鞘氨醇激酶1(Sphk1)抑制剂在肝纤维化大鼠模型中的作用机制

Mechanism of action of sphingosine kinase 1 inhibitor in a rat model of liver fibrosis

目的探讨鞘氨醇激酶1(SphK1)抑制剂在不同肝纤维化大鼠模型中的治疗作用。方法170只SD大鼠随机分为4组:正常组(30只)、HFE组(给予高脂乳剂,45只)、CCl_(4)组(CCl_(4)诱导,45只)、CCl_(4)+HFE组(HFE联合CCl_(4),50只),经病理及实验室检查证实造模成功后,分别给予SphK1抑制剂PF-543,同组别以生理盐水作为对照,分别在第1、7、14天取肝组织进行Masson染色,比较肝纤维化面积占比、透射电镜下观察自噬小体形成情况、Real-time PCR检测肝纤维化及自噬相关标志物mRNA表达水平。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD检验。相关性分析采用Spearman相关分析。结果与正常组(0.57±0.13)相比,CCl_(4)组(6.93±5.81)和HFE+CCl_(4)组(10.89±2.67)肝纤维化面积占比均升高(P值均<0.01)。PF-543干预第7天可显著降低CCl_(4)组及HFE+CCl_(4)组纤维化病变面积(P值均<0.01)。与正常组相比,HFE组、CCl_(4)组和HFE+CCl_(4)组的SphK1表达水平均明显降低(P值均<0.01),HFE+CCl_(4)组的α平滑肌肌动蛋白、转化生长因子-β、α1-Ⅰ型胶原mRNA表达水平均明显升高(P值均<0.01),干预后各指标水平无明显变化(P值均>0.05)。Atg5、Atg12、Becn1的表达水平与肝纤维化病变面积均呈负相关(r分别为-0.715、-0.640、-0.632,P值均<0.01);SphK1的表达水平与Atg5、Atg12、Becn1、Map1lc3a的mRNA表达均呈正相关(r分别为0.603、0.561、0.510、0.498,P值均<0.01)。电镜示:CCl_(4)组肝组织轻微水肿,细胞器丰富,轻度肿胀,以粗面内质网扩张为主。该视野可见9个自噬溶酶体结构。在PF-543干预第7天后,未见典型自噬结构。HFE+CCl_(4)组肝组织轻度水肿,结构不清晰,粗面内质网数量丰富,明显扩张,表面附着核糖体颗粒较少。该视野可见6个自噬溶酶体结构。在PF-543干预第7天后,该视野未见典型自噬结构。结论PF-543对自噬有显著抑制作用,并同时伴有纤维化面积的减少。提示靶向SphK1,可影响到肝脏的自噬水平,并进而缓解两种肝纤维化模型的纤维化状态。

Objective To investigate the therapeutic effect of sphingosine kinase 1(SphK1)inhibitor in different liver fibrosis models.Methods A total of 170 Sprague-Dawley rats were randomly divided into normal group(30 rats),HFE group(45 rats given high-fat emulsion),CCl_(4)group(45 rats induced by CCl_(4)),and CCl_(4)+HFE group(50 rats given HFE and CCl_(4)).After successful modeling confirmed by pathology and laboratory examination,the SphK1 inhibitor PF-543 was given,and the rats in the same group were given normal saline as control.Liver tissue samples were collected on days 1,7,and 14 for Masson staining;the percentage of liver fibrosis area was compared;the formation of autophagosome was observed under a transmission electron microscope;real-time PCR was used to measure the mRNA expression levels of markers associated with liver fibrosis and autophagy.A one-way analysis of variance was used for comparison between multiple groups,and the LSD method was used for further comparison between two groups;a Spearman correlation analysis was also performed.Results Compared with the normal group,the CCl_(4)group and the HFE+CCl_(4)group had a significant increase in the percentage of liver fibrosis area(6.93±5.81/10.89±2.67 vs 0.57±0.13,both P<0.01),the CCl_(4)group and the HFE+CCl_(4)group had a significant reduction in the percentage of liver fibrosis area on day 7(both P<0.01).Compared with the normal group,the HFE group,the CCl_(4)group,and the HFE+CCl_(4)group had a significant reduction in the expression level of SphK1(all P<0.01),and the HFE+CCl_(4)group had significant increases in the mRNA expression levels of alpha-smooth muscle actin,transforming growth factor-β,and collagen type I alpha 1(all P<0.01),while there were no significant changes in these indices after intervention(all P>0.05).The expression levels of Atg5,Atg12,and Becn1 were negatively correlated with the area of liver fibrosis(r=-0.715,-0.640,and-0.632,all P<0.01),and the expression level of SphK1 was positively correlated with the mRNA expression of Atg5,Atg12,Becn1,and Map1lc3a(r=0.603,0.561,0.510,and 0.498,all P<0.01).Electron microscopy showed that the CCL 4 group had slight edema,abundant organelles,and mild swelling in liver tissue,mainly the expansion of rough endoplasmic reticulum,and nine autolysosome(ASS)structures were seen in this field,while no typical ASS structure was observed on day 7 of PF-543 intervention.The HFE+CCL 4 group had mild edema,unclear structure,and abundant rough endoplasmic reticulum with marked expansion and few ribosome particles attached to its surface in liver tissue,and 6 ASS structures were seen in this field,while no typical ASS structure was observed on day 7 of PF-543 intervention.Conclusion PF-543 significantly inhibits autophagy and is associated with a reduction in fibrosis area.It is suggested that targeting SphK1 can affect the level of liver autophagy,thereby alleviating the state of liver fibrosis in two liver fibrosis models.