左旋紫草素抑制NF-κB信号通路保护LPS/D-GalN诱导的小鼠急性肝损伤

The protective effect of L-Shikonin on LPS/D-GalN-induced acute liver injury in mice via inhibiting NF-κB inflammatory signaling pathway

目的探讨左旋紫草素(L-Shikonin,L-SK)对脂多糖(lipopolysaccharide,LPS)诱导的RAW264.7巨噬细胞的体外抗炎作用及其对LPS/D-GalN诱导的急性肝损伤的保护作用。方法体内实验采用LPS/D-GalN建立小鼠急性肝损伤模型,考察存活率和各组小鼠的肝脾指数变化,测定血清中AST、ALT、AKP与肝组织匀浆中的NO含量、超氧化物歧化酶(SOD)、丙二醛(MDA)水平,HE染色观察各组小鼠肝组织病理切片。体外实验采用MTT法检测细胞存活率、Griess法检测NO含量、RT-PCR和Western blot检测分别考察L-SK对LPS诱导的RAW264.7细胞中促炎因子的mRNA和相关通路蛋白表达水平的影响。结果体内实验结果表明,L-SK可明显改善急性肝损伤小鼠的肝脾指数,血清中AST、ALT和AKP水平明显下降,肝匀浆中的NO和MDA含量明显下降,SOD活性提高,能明显改善小鼠肝组织病理性损伤。体外实验结果表明,L-SK能明显抑制LPS诱导的RAW264.7细胞中的INOS、COX2、IFN-β以及促炎因子IL-6、TNF-α和IL-1β的mRNA表达,并且明显抑制INOS、COX2蛋白表达以及NF-κB信号通路蛋白表达。结论L-SK在LPS诱导的RAW264.7细胞的体外炎症中具有良好的抗炎作用,通过抑制NF-κB信号通路中的磷酸化p65以及IKKα/β的蛋白表达,从而发挥体外抗炎作用,同时L-SK对小鼠急性肝损伤具有保护作用,其作用机制可能与抗炎作用相关。

Aim To investigate the anti-inflammatory effect of L-Shikonin(SK)on lipopolysaccharide(LPS)-induced RAW264.7 macrophages in vitro and its protective effect on LPS/D-GalN-induced acute liver injury.Methods The mouse model of acute liver injury was established in vivo experiments by LPS/D-GalN.The survival rate of the mice and the changes of liver and spleen indices in each group were examined.The levels of AST,ALT and AKP in serum and NO,superoxide dismutase(SOD)and malondialdehyde(MDA)in liver tissue homogenate were measured,and the histopathological sections of the liver of each group were observed by H&E staining.MTT colorimetric assay was used for cell viability in vitro experiments,Griess method for the detection of NO content,RT-PCR assay and Western blot assay for examining the effect of levulinic acid on the expression levels of mRNA and related pathway proteins of pro-inflammatory factors in LPS-induced RAW264.7 cells.Results The results of in vivo experiments showed that L-SK significantly improved the liver and spleen indices,decreased AST,ALT and AKP levels in serum,decreased NO and MDA in liver homogenate,and increased SOD activity in mice with acute liver injury.The results of in vitro experiments showed that L-SK significantly inhibited the mRNA expression of INOS,COX2,IFN-βand pro-inflammatory factors IL-6,TNF-αand IL-1βin LPS-induced RAW264.7 cells,and significantly inhibited the protein expression of INOS,COX2 and the NF-κB signaling pathway.Conclusions L-SK has good anti-inflammatory effects in LPS-induced inflammation in RAW264.7 cells in vitro.It inhibits the protein expression of phosphorylated P65 and IKKα/βin the NF-κB signaling pathway,thereby suppressing the anti-inflammatory effects in vitro and L-Shikonin has protective effects against acute liver injury in mice.