内质网应激介导的Kupffer细胞源性TNF-α经TNFR/caspase8途径诱导肝星状细胞凋亡

Endoplasmic reticulum stress enhances tumor necrosis factor-αexpression in rat Kupffer cells to trigger hepatic stellate apoptosis cell through TNFR/caspase-8 pathway

目的探讨Kupffer细胞(KCs)内质网应激以及KCs细胞源性肿瘤坏死因子-α(TNF-α)在肝星状细胞凋亡中的作用。方法建立大鼠肝纤维化模型,按随机数字表法分为4组,15只/组:对照组:腹腔注射生理盐水(2 mg/kg);肝纤维化组:腹腔注射40%CCl4溶液(花生油制成,2 mg/kg);内质网应激组:腹腔注射40%CCl4溶液(2 mg/kg)和衣霉素(1 mg/kg);KCs封闭组:腹腔注射40%CCl4溶液(2 mg/kg)和衣霉素(1 mg/kg)后,经腹腔注射氯膦酸脂质体(50 mg/kg)。以上4组注射处理均为2次/周,共8周。在动物模型基础上,建立KCs、LX2共培养细胞系,随机分为4组:对照组:分离培养对照组大鼠肝脏KCs,与LX2细胞共培养;肝纤维化组:分离培养肝纤维化组大鼠肝脏KCs,与LX2细胞共培养;ER-stress组:分离培养ER-stress组大鼠肝脏KCs,与LX2细胞共培养;肿瘤坏死因子受体(TNFR)阻断组:在ER-stress大鼠基础上,经门静脉注射anti-rat TNFR mAb(0.35 mg/kg)后,按照分离大鼠肝脏KCs,与LX2细胞共培养,收集细胞和上清液。运用肝功能检测、天狼星红染色、ELISA、免疫荧光、RTPCR,检测各组大鼠肝功能水平、肝纤维化程度、KCs极性、炎症因子表达以及活化肝星状细胞数量。运用ELISA、RT-PCR、Western blot,检测各组细胞极性、炎症因子表达、LX2凋亡程度、TNFR/caspase 8通路蛋白表达。结果体内实验结果显示,与对照组相比,肝纤维化组的谷氨酸转氨酶(AST)、天冬氨酸转氨酶(ALT)水平、天狼星红染色阳性区域、结蛋白阳性细胞(活化肝星状细胞)数量均显著增多(P<0.05),CD16阳性细胞、TNF-α表达和mRNA水平显著下降(P<0.05);与肝纤维化组相比,ER-stress组ALT和AST水平、天狼星红染色阳性区域、结蛋白阳性细胞数量均明显降低(P<0.05),CD16阳性细胞、TNF-α表达和mRNA水平均明显增多(P<0.05);与ER-stress组相比,KCs封闭组ALT和AST水平、天狼星红染色阳性区域、结蛋白阳性细胞数量均显著增多(P<0.05),CD16阳性细胞、TNF-α表达和mRNA水平均显著下降(P<0.05)。体外实验结果显示,与对照组相比,肝纤维化组内TUNEL阳性的LX2细胞、CD16阳性细胞、KCs内TNFR、cleaved-caspase 8、cleaved-caspase 3的表达均明显降低(P<0.05),但结蛋白阳性的LX2细胞显著增加(P<0.05);与肝纤维化组相比,ER-stress组内TUNEL阳性的LX2细胞、CD16阳性细胞、KCs内TNFR、cleaved-caspase 8、cleaved-caspase 3的表达均明显增加(P<0.05),但结蛋白阳性的LX2细胞显著减少(P<0.05);同时,在阻断TNFR后,与ER-stress组相比,虽然TNFR表达未见明显变化,但下游cleaved-caspase 8、cleaved-caspase 3的表达均显著下降(P<0.05)。结论KCs内质网应激促进了其M1型极化,并增加了KCs源性TNF-α的表达。KCs源性TNF-α经TNFR/caspase 8途径诱导了肝星状细胞的凋亡。

Objective To investigate the role of endoplasmic reticulum(ER)-stress of Kupffer cells(KCs)and KCs-derived tumor necrosis factor-α(TNF-α)in medicating apoptosis of hepatic stellate cell(HSC).Methods Sixty male SD rats were randomized into control group,model group,ER-stress group,depletion group and KCs block group(n=15).The 4 groups of rats were given intraperitoneal injections(twice a week for 8 weeks)of normal saline(2 mg/kg);40%CCl4 solution(in peanut oil,2 mg/kg);40%CCl4 solution(2 mg/kg)and tunicamycin(1 mg/kg);and 40%CCl4 solution(2 mg/kg)and tunicamycin(1 mg/kg)followed by clodronate liposomes(50 mg/kg),respectively.After the treatments,samples of the liver tissue and serum were collected from the rats from the 4 groups to isolate KC cells,which were co-cultured with LX2 cells.In the depletion group,the rats were injected with anti-rat TNFR mAb(0.35 mg/kg)via the portal vein before isolating the KCs.Liver function examination,Eirius red staining,ELISA,immuno-histochemical staining,and RT-PCR were performed to assess the liver function,liver fibrosis,KC phenotypes,expression of the in fl ammatory factors,and the number of active HSC was detected.The isolated KCs were treated with tunicamycin before co-culture with LX2 cells,and ELISA,RT-PCR and Western blot were performed to examine KC phenotypes,in fl ammatory factors,LX2 cell apoptosis and TNFR/caspase8 pathway activity.Results Compared with the rats in the control group,the rats in the model group had significantly increased ALT and AST levels,Sirius red staining-positive area,and Desmin-positive cells(activated HSCs)(P<0.05)with significantly lowered number of CD16-positive KCs(M1),and TNF-αprotein and mRNA expression(P<0.05).Compared with those in the model group,the rats in ER-stress group showed significantly decreased ALT and AST levels,Sirius red staining positivity and Desmin-positive cells(P<0.05)and increased number of CD16-positive KCs and TNF-αexpressions(P<0.05).In the depletion group,compared with the ER-stress group,the rats had significantly increased ALT and AST levels of,Sirius red staining positivity and Desmin-positive cells(P<0.05)and reduced CD16-positive KCs and TNF-αexpressions(P<0.05).In the cell co-culture experiment,the model group showed significantly reduced TUNEL-positive LX2 cells,CD16-positive cells,and expressions of TNFR1,cleaved caspase-8 and cleaved caspase-3 in the KCs(P<0.05)with increased Desmin-positive LX2 cells(P<0.05).Compared with the model group,the ER-stress group exhibited significantly increased TUNEL-positive LX2 cells,CD16-positive cells and expressions of TNFR,cleaved caspase-8 and cleaved caspase-3 in the KCs(P<0.05)and decreased Desmin-positive LX2 cells(P<0.05).In the depletion group,blocking TNFR resulted in significantly decreased expressions of cleaved caspase-8 and cleaved caspase-3 compared with those in ER-stress group(P<0.05)although there was no significant changed in TNFR expression.Conclusion ER stress of KCs promotes the transformation of KCs towards M1 phenotype and increases the expression of TNF-α,which triggers the apoptosis of HSCs through the TNFR/caspase-8 pathway.