柚皮苷通过抑制Kupffer细胞的焦亡改善脂多糖诱导的肝损伤

Naringin Ameliorates Lipopolysaccharides-Induced Liver Injury by Inhibiting Pyroptosis of Kupffer Cells

目的探究柚皮苷(naringin,Nar)在脂多糖(LPS)诱导的肝损伤中的作用及相关作用机制。方法60只C47BL/6野生型雄性小鼠随机分为4组(每组15只):对照组(Control),柚皮苷组(Nar),脂多糖模型组(lipopolysaccharide,LPS)与柚皮苷组处理的LPS模型组(Nar+LPS),其中Nar组与Nar+LPS组小鼠予以100 mg·kg^(-1)·d^(-1)的柚皮苷持续灌胃10天,而LPS与LPS+Nar组小鼠经腹注射5 mg·kg-1LPS进行肝损伤造模,6 h后脱颈处死各组小鼠;试剂盒检测各组小鼠外周血中谷丙转氨酶(alanine transaminase,ALT)、谷草转氨酶(aspertate aminotransferase,AST)含量,ELISA检测炎症因子白介素-6(interlukin-6,IL-6)、白介素-1β(interlukin-1β,IL-1β)与肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的水平;取小鼠肝脏组织,苏木精-伊红(hematoxylin-eosin,HE)与脱氧核糖核酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transferase-mediated nick end labeling,TUNEL)观察各组小鼠肝组织病理改变与细胞焦亡水平,反转录酶-聚合酶链锁反应(reverse transcription-polymerase chain reaction,RT-PCR)检测肝组织中IL-6、IL-1β、TNF-α的mRNA表达,试剂盒检测氧化应激标志物丙二醛(malondialdehyde,MDA)与谷胱甘肽(glutathione,GSH)水平;体外培养小鼠Kupffer细胞,CCK-8筛选LPS与Nar的最佳作用浓度后,将细胞同样分为Control组、Nar组、LPS组与Nar+LPS组;ELSIA检测各组细胞上清液中IL-6、IL-1β、TNF-α的表达;试剂盒检测细胞中乳酸脱氢酶(lactate dehydrogenase,LDH)、MDA与GSH水平;cleaved Caspase-1/TUNEL染色检测各组细胞的焦亡情况,Western blot检测细胞中焦亡相关蛋白,核苷酸结合寡聚化结构域样受体蛋白3(nucleotidebinding oligomerization domain-like receptor protein 3,NLRP3)、含半胱氨酸的天冬氨酸蛋白水解酶1裂解片段(cleaved-cysteinyl aspartate specific proteinase-1,cleaved Caspase-1)、gasdermin D的N端寡聚化(Gasdermin D N-terminal oligomerization,GSDMD-N)、IL-1β的表达水平和核因子-κB(nuclear factor kappaB,NF-κB)的磷酸化水平(p-NF-κB/NF-κB)。结果与Control组相比,LPS组与Nar+LPS组的血清中ALT、AST与IL-6、IL-1β、TNF-α水平均显著增加(P<0.05或P<0.01),肝组织病理损伤加重,肝细胞焦亡率升高,细胞中IL-6、IL-1β、TNF-α的mRNAs水平与MDA的含量均显著升高(P<0.05),而GSH明显降低(P<0.05),Nar组无明显改变(P>0.05),但较LPS组相比,Nar+LPS组中除肝组织中GSH含量明显升高外,上述其他检测指标均明显降低(P<0.05);体外细胞实验中筛选出100 ng·mL-1的LPS与200μmol·L-1的Nar为最佳作用浓度;与Control组Kupffer细胞相比,LPS组与LPS+Nar组的IL-6、IL-1β、TNF-α与MDA、LDH含量和细胞焦亡水平及焦亡相关蛋白NLRP3、cleaved Caspase-1、GSDMD-N、IL-1β的表达均明显升高(P<0.05),NF-κB的磷酸化水平(p-NF-κB/NF-κB)亦显著升高,而GSH含量明显降低(P<0.05),但与LPS组相比,LPS+Nar组除GSH明显升高外,其他检测指标均显著降低(P<0.05)。结论柚皮苷可在体内外降低炎症反应与氧化应激来减轻LPS诱导的肝损伤与功能障碍,这可能与其能通过NF-κB途径抑制Kupffer细胞焦亡的机制相关。

Objective To explore the effects of naringin(Nar)on liver injury induced by lipopolysaccharides and its related mechanism.Methods 60 C47BL/6 wild-type male mice were randomly divided into 4 groups(15 mice in each group):Naringin group(Control),naringin group(Nar),lipopolysaccharide modeling group(LPS)and Naringin treated with LPS modeling group,in which Nar group and Nar+LPS group were given naringin 100 mg·kg^(-1)·d^(-1)intragastally for 10 days,while LPS and LPS+Nar group were injected 5 mg·kg-1 LPS through abdomen for modeling,and the mice in each group were killed by neck-decapsulation 6 h later.Alanine aminotransferase(ALT)and aspartate aminotransferase(AST)contents in peripheral blood of mice in each group were detected by kit,and the levels of inflammatory factors interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were detected by ELISA assay.The pathological changes and apoptosis levels of liver tissues were observed by hematoxylin-eosin(HE)and terminal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL)staining.The mRNA expressions of IL-6,IL-1βand TNF-αin liver tissues were detected by reverse transcription-polymerase chain reaction(RT-PCR).The levels of malondialdehyde(MDA)and glutathione(GSH),markers of oxidative stress,were detected by commercial kits.Mice Kupffer cells were cultured in vitro,and the optimal concentrations of LPS and Nar were screened by CCK-8.The cells were also divided into Control group,Nar group,LPS group and Nar+LPS group.The expressions of IL-6,IL-1βand TNF-αin supernatant of each group were detected by ELSIA.The levels of lactate dehydrogenase(LDH),MDA and GSH were detected by the commercial kits.Cleaved Caspase-1/TUNEL staining was used to detect the pyroptosis in each group.Western blot was used to detect the pyrotopic-associated protein of nucleotidebinding oligomerization domain-like receptor protein 3(NLRP3),cleaved-cysteinyl aspartate specific proteinase-1(cleaved caspase-1),Gasdermin D N-terminal oligomerization(GSDMD-N),IL-1βand the phosphorylation of nuclear factor kappa-B(p-NF-κB/NF-κB).Results Compared with Control group,serum levels of ALT,AST,IL-6,IL-1βand TNF-αin LPS group and Nar+LPS group were significantly increased(P<0.05 or P<0.01),liver histopathological injury was aggravated,and hepatocyte apoptosis rates was increased.The mRNAs levels of MDA of IL-6,IL-1βand TNF-αwere significantly increased(P<0.05),while GSH was significantly decreased(P<0.05).No significant changes were observed in Nar group(P>0.05),while except GSH was significantly decreased,other indexes were significantly decreased in Nar+LPS group compared with LPS group(P<0.05).In vitro cell experiments,100 ng·mL-1 LPS and 200μmol·L-1 Nar were selected as the best concentration.Compared with Kupffer cells in the Control group,Il-6,IL-1β,TNF-α,MDA,LDH levels,pyrotopic-associated protein protein NLRP3,cleaved in LPS group and LPS+Nar group.The expressions of Caspase-1,GSDMD-N,IL-1βand NF-κB phosphorylation(p-NF-κB/NF-κB)were significantly increased(P<0.05),while GSH was significantly decreased(P<0.05),other indexes were significantly decreased(P<0.05).Conclusion Naringin alleviates LPS-induced liver injury and dysfunction by reducing inflammatory response and oxidative stress in vitro and in vivo,which may be related to the mechanism of inhibiting pyroptosis of Kupffer cells through NF-κB pathway.