迷迭香酸活化NRF2负调控ROS/TXNIP/NLRP3信号通路改善急性肝损伤

Rosmarinic acid ameliorates acute liver injury by activating NRF2 and inhibiting ROS/TXNIP/NLRP3 signal pathway

急性肝损伤(acute liver injury,ALI)是临床上常见的重症疾病之一,氧化还原失衡和炎症风暴为主要特点,治疗不及时极易引起肝功能衰竭甚至死亡。迷迭香酸(rosmarinic acid,RA)已被证明具有抗炎抗氧化的活性,但是如何通过抗氧化及抑制炎症反应发挥对ALI的保护作用尚不清楚。因此,本研究通过体内外实验探究RA对ALI的治疗作用和分子机制。在小鼠ALI模型上,研究RA对肝功能和炎性指标水平的影响;HE观察肝脏病理变化;荧光法检测RA对肝脏活性氧(reactive oxygen species,ROS)的影响;免疫组化法检测肝组织F4/80水平;Western blot测定肝脏组织硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein,TXNIP)、NOD-like受体蛋白3(NOD-like receptor protein 3,NLRP3)炎症小体和半胱氨酸天冬氨酸特异性蛋白酶-1(cysteinyl aspartate specific proteinase-1,CASPASE-1)蛋白的水平。动物福利和实验过程均遵循西南民族大学动物伦理委员会的规定。在H2O2诱导的HepG2细胞氧化性损伤模型上,CCK-8法分析RA对细胞活力的影响;试剂盒检测白细胞介素-1β(interleukin-1β,IL-1β)和乳酸脱氢酶(lactatede hydrogenase,LDH)释放量;荧光探针DCFH-DA检测ROS水平;实时荧光定量PCR(quantitative real-time PCR,qPCR)法检测Txnip、Nlrp3、Caspase 1和Il1βmRNA表达水平;Western blot法测定细胞核因子E2相关因子2(nuclear factor erythroid-2 related factor 2,NRF2)、TXNIP、NLRP3和CASPASE-1蛋白的水平。实验结果显示,与模型组比较,RA组小鼠肝肿胀程度明显减轻,组织损伤缓解,肝功能指标和ROS水平显著下降,TXNIP、NLRP3、CASPASE-1蛋白水平均显著降低,F4/80和IL-1β水平明显下降。在细胞水平上,RA能显著降低H2O2诱导的IL-1β、LDH和ROS水平,同时降低了Txnip、Caspase 1、Il1βmRNA表达水平,以及TXNIP、NLRP3、CASPASE-1蛋白水平,但NRF2蛋白表达水平明显上升,而NRF2抑制剂ML385逆转了RA对NRF2、TXNIP、NLRP3、CASPASE-1蛋白表达的作用。结果表明,RA可能通过促进NRF2核转移抑制ROS生成,进而减少TXNIP激活NLRP3炎症小体,减少细胞死亡和炎症反应从而发挥对肝损伤的缓解作用。

Acute liver injury(ALI)is one of the common severe diseases in clinic,which is characterized by redox imbalance and inflammatory storm.Untimely treatment can easily lead to liver failure and even death.Rosmarinic acid(RA)has been proved to have anti-inflammatory and antioxidant activity,but it is not clear how to protect ALI through antioxidation and inhibition of inflammation.Therefore,this study explored the therapeutic effect and molecular mechanism of RA on ALI through in vitro and in vivo experiments.In the mouse ALI model,the effects of RA on liver function and inflammatory indexes were studied,the pathological changes of liver were observed by HE,the effect of RA on reactive oxygen species in liver was detected by fluorescence method,and the level of F4/80 in liver tissue was detected by immunohistochemical method.The levels of thioredoxin interacting protein(TXNIP),NOD-like receptor protein 3(NLRP3)and cysteinyl aspartate specific proteinase-1(CASPASE-1)in liver tissue were measured by Western blot.All animal welfare and experimental procedures follow the rules of the Animal Ethics Committee of Southwest Minzu University.In vitro,human hepatoma cell line HepG2 was used to establish the model of oxidative damage induced by H2O2.The cell viability was detected by CCK-8 method.The level of interleukin-1β(IL-1β)in the supernatant was detected by enzyme linked immunosorbent assay(ELISA),the activity of lactatede hydrogenase(LDH)was detected by LDH kit,and the level of ROS was detected by fluorescence probe DCFH-DA labeling.The mRNA expression of Txnip,Nlrp3,Caspase 1,Il1βwas detected by real-time fluorescence quantitative PCR(qPCR),and the protein levels of nuclear factor erythroid-2 related factor 2(NRF2),TXNIP,NLRP3 and CASPASE-1 were measured by Western blot.The results showed that compared with the model group,the degree of liver swelling,tissue injury,liver function index in RA group were significantly lower than those in model group.And RA significantly attenuated the increases of ROS in liver tissue.The expression levels of TXNIP,NLRP3 and CASPASE-1 in liver tissue were significantly lower than those in model group.Additionally,RA inhibited the expressions of F4/80 and IL-1β.In vitro experiment,compared with model group,RA effectively inhibited the secretion of IL-1βand LDH.The level of ROS also decreased significantly.RA inhibited the mRNA expressions of Txnip,Caspase 1,Il1β.Furthermore,RA significantly increased the expression level of NRF2 protein in nucleus,and decreased the expression level of TXNIP and NLRP3 protein.Specifically,with the addition of ML385,the effect of RA on NRF2,TXNIP,NLRP3,CASPASE-1 protein expression was reversed.Collectively,these findings suggested that RA may inhibit the production of ROS by promoting NRF2 nuclear transfer,and then reduce the activation of NLRP3 inflammatory bodies by TXNIP,reduce cell death and inflammatory response to prevent the liver injury.