莲心总碱对脂多糖/D-氨基半乳糖诱导急性肝衰竭小鼠肝脏炎症反应的缓解作用

Total Alkaloids from the Seed Embryo of Nelumbo nucifera Ameliorate Inflammation in the Livers of Mice with Lipopolysaccharide/D-Galactosamine-Induced Acute Liver Failure

目的:观察莲心总碱(total alkaloids from the seed embryo of Nelumbo nucifera,TAENN)对脂多糖(lipopolysaccharide,LPS)/D-氨基半乳糖(D-galactosamine,Gal N)诱导急性肝衰竭小鼠肝脏炎症反应的影响。方法:将昆明小鼠40只随机分为4组(正常对照组、模型组和TAENN高、低剂量组),每组10只。TAENN高、低剂量组的给药剂量分别为100、30 mg/kg,给药途径为灌胃,每日给药1次。正常对照组和模型组灌胃等体积生理盐水。给药结束后,模型组和TAENN高、低剂量组小鼠腹腔注射LPS/Gal N建立小鼠急性肝衰竭模型。用酶联免疫吸附测定法检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、IL-6和前列腺素E2(prostaglandine E2,PGE2)水平。用Griess法检测肝组织中一氧化氮(nitric oxide,NO)的含量。用实时聚合酶链式反应(polymerase chain reaction,PCR)法检测小鼠肝组织中TNF-α、IL-1β、IL-6、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、环氧合酶-2(cyclooxygenase-2,COX-2)的mRNA的表达水平。用免疫印迹法检测小鼠肝组织中iNOS、COX-2蛋白表达和核转录因子-κB(nuclear transcription factor-κB,NF-κB)p65亚基磷酸化水平。结果:与模型组相比,TAENN组小鼠血清与肝脏中炎症因子TNF-α、IL-1β和IL-6水平明显降低(P<0.01)。肝脏中炎症介质NO和PGE2水平亦有极显著下降(P<0.01)。经TAENN处理后,模型小鼠肝脏中TNF-α、IL-1β、IL-6、i NOS、COX-2等的m RNA表达水平明显下降(P<0.01)。此外,iNOS和COX-2的蛋白表达水平明显降低(P<0.01),p65蛋白本身的表达水平不变,但其536位丝氨酸的磷酸化水平亦极显著下降(P<0.01)。结论:TAENN对LPS/Gal N诱导小鼠急性肝衰竭相关的炎症反应具有明显的保护作用,其机制可能与抑制NF-κB信号通路并减少炎症因子和炎症介质相关基因的表达有关。

Objective:To investigate the effect of total alkaloids from the seed embryo of Nelumbo nucifera(TAENN)on inflammation in the livers of mice with acute liver failure(ALF)induced by lipopolysaccharide(LPS)/D-galactosamine(GalN).Methods:Kunming mice were randomly divided into four groups including normal control,ALF model,TAENN low-dosage(30mg/kg)and TAENN high-dosage groups(100mg/kg).The drug was administered by gavage once a day.After2weeks,the model group and two TAENN groups were intraperitoneally injected with LPS and GalN.The contents of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6,and prostaglandine E2(PGE2)were determined by enzyme linked immunosorbent assay(ELISA).NO contents in livers were examined by Griess’method.The transcription of TNF-α,IL-1β,IL-6,inducible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2)in livers were analyzed by real-time PCR.The protein expression levels of iNOS and COX-2,and the phosphorylation levels of nuclear transcription factor-κB(NF-κB)p65subunit at Ser536in mouse livers were detected by immunoblotting.Results:TAENN declined the levels of TNF-α,IL-1βand IL-6(P<0.01)in the sera and livers of ALF model mice.In the livers of ALF model mice,the contents of inflammatory mediators such as NO and PGE2were significantly decreased(P<0.01)in response to TAENN.Moreover,TAENN does-dependently declined the transcription of the encoding genes such as TNF-α,IL-1β,IL-6,iNOS and COX-2(P<0.01)in livers.Consistently,the protein expression levels of iNOS and COX-2were greatly reduced by TAENN(P<0.01).Although the protein expression levels of p65remained unchanged,the phosphorylation of p65at Ser536was remarkably inhibited after TAENN treatment(P<0.01).Conclusion:TAENN inhibited inflammatory responses in the livers of mice with LPS/GalN-induced ALF.This anti-inflammation effect was associated with restrained NF-κB signaling and reduced expression of inflammatory cytokines and mediators.