姜黄素保护急性肝损伤的作用及机制研究

Protective effect of curcumin on acute liver injury and its mechanisms

目的基于线粒体自噬-核酸结合寡聚结构域样受体家族3(NLRP3)炎症小体通路研究姜黄素对急性肝损伤的可能保护机制,为其用于急性肝损伤的治疗提供理论依据。方法30只雄性SD大鼠随机分为对照组、肝损伤组、姜黄素组、3-甲基腺嘌呤(3MA)组和姜黄素+3MA组,每组6只。除对照组外,其余4组腹腔注射脂多糖(LPS)/D-氨基半乳糖(D-GalN)建立肝脏损伤模型,对照组腹腔注射等体积的0.9%氯化钠注射液。建模前5天,姜黄素组和姜黄素+3MA组大鼠按5 mL/kg灌饲姜黄素药液,1次/d;对照组、肝损伤组和3MA组灌饲5 mL/kg的0.5%羧甲基纤维素钠(CMC-Na)溶液。建模前2 h,3MA组和姜黄素+3MA组大鼠腹腔注射3.35 mL/kg的3MA,其余各组腹腔注射3.35 mL/kg的二甲基亚砜。造模后12 h,颈椎脱臼法处死大鼠并收取血液和肝脏组织。采用HE染色观察各组大鼠肝组织病理变化,采用流式细胞仪测量肝细胞线粒体膜电位,采用ATP试剂盒检测肝细胞线粒体ATP的水平,通过透射电镜观察肝细胞线粒体形态和自噬小体,采用免疫组化检测肝组织IL-1β表达情况,采用ELISA法检测血清IL-1β水平,采用Western blot法检测线粒体自噬相关蛋白Parkin、PTEN诱导假定激酶1(PINK1)以及炎症相关蛋白NLRP3、半胱氨酸蛋白酶-1(Caspase-1)和IL-1β的表达情况。结果与肝损伤组相比,姜黄素组肝组织肿胀及点状坏死显著减少,炎症细胞浸润相对较轻,线粒体膜电位和ATP水平显著增高,线粒体损伤明显减轻,自噬体数量增加,肝组织IL-1β阳性细胞数及相应吸光度值显著下降,血清IL-1β含量显著下降,肝组织Parkin、PINK1蛋白灰度值显著上调,NLRP3、Caspase-1和IL-1β蛋白灰度值显著下降,差异均有统计学意义(均P<0.05)。与姜黄素组相比,3MA组和姜黄素+3MA组肝组织严重充血,可见点状坏死,肝细胞线粒体损伤加重,自噬体数量减少,IL-1β阳性细胞数及相应吸光度值显著上升,血清IL-1β含量显著增高,肝组织Parkin、PINK1蛋白灰度值显著下降,NLRP3、Caspase-1和IL-1β蛋白灰度值显著上调,差异均有统计学意义(均P<0.05)。结论姜黄素对LPS/D-GalN诱导的急性肝损伤具有保护作用,其机制可能与激活线粒体自噬-抑制NLRP3炎性小体介导的炎症反应相关。

Objective To investigate the effect of curcumin on acute liver injury and its mechanism.Methods Thirty male SD rats were randomly divided into five groups:control group,acute liver injury(AHI)group,curcumin(CUR)group,3-methyladenine(3MA)group,and curcumin+3MA(CUR+3MA)group,with six rats in each group.The liver injury was induced by intraperitoneal injection of lipopolysaccharide(LPS)and D-galactosamine(D-GalN)in the later 4 groups.Five days before modeling,rats in the CUR and CUR+3MA groups were administered curcumin solution once a day at a dose of 5.00 mL/kg for five consecutive days.The control,AHI,and 3MA groups received intragastric administration of 5.00 mL/kg of 0.5%carboxymethyl cellulose sodium(CMC-Na)solution.Two hours before modeling,rats in the 3MA and CUR+3MA groups received an intraperitoneal injection of 3MA solution at a dose of 3.35 mL/kg,while the other groups received an intraperitoneal kiinjection of 3.35 mL/kg dimethyl sulfoxide(DMSO)solution.After 12 h of modeling,rats were sacrificed by cervical dislocation,and blood and liver tissue samples were collected.Hepatic pathological changes were observed by hematoxylin and eosin(HE)staining,mitochondrial membrane potential was measured by flow cytometry,mitochondrial ATP level was detected using an ATP assay kit,mitochondrial morphology and autophagosomes were observed by transmission electron microscopy,IL-1βexpression in liver tissue was examined by immunohistochemistry,serum IL-1βlevel was measured by ELISA,and the expression of mitochondrial autophagy-related protein Parkin,PTEN-induced putative kinase 1(PINK1),inflammatory proteins NLRP3,Caspase-1,and IL-1βin liver tissue was evaluated by Western blot.Results Compared with AHI group,CUR group exhibited significantly reduced liver cell swelling and focal necrosis,milder inflammatory cell infiltration,increased mitochondrial membrane potential and ATP levels,attenuated mitochondrial damage,and increased autophagosome numbers.Immunohistochemistry of liver tissues showed a significant decrease in the number of IL-1β-positive cells and optical density values in CUR group,as well as a significant decrease in serum IL-1βlevels.Western blot analysis revealed elevated levels of Parkin and PINK1 proteins and reduced levels of NLRP3,Caspase-1,and IL-1βproteins in CUR group compared to AHI group(all P<0.05).In contrast,the 3MA and CUR+3MA groups displayed severe liver cell congestion,focal necrosis,aggravated mitochondrial damage,decreased autophagosome numbers,increased IL-1β-positive cells,elevated serum IL-1βlevels,decreased Parkin and PINK1 protein levels,and elevated NLRP3,Caspase-1,and IL-1βprotein levels compared to the curcumin group(all P<0.05).Conclusion Curcumin exerts a protective effect against LPS/D-GalN-induced acute liver injury,which may be associated with the activation of mitochondrial autophagy and inhibition of the NLRP3 inflammasome-mediated inflammatory response.