基于Kuffer细胞NF-κB信号通路探讨解毒化瘀颗粒拮抗肝功能衰竭的作用机制

Molecular mechanism of Jiedu Huayu Granules antagonizing acute liver failure through NF-κB signaling pathway in Kuffer cell

目的:通过体内、外研究观察解毒化瘀颗粒(JDHY)对Kupffer细胞(KC)中NF-κB信号通路的影响及拮抗肝功能衰竭的作用机制。方法:将45只大鼠随机分为对照组、模型组和JDHY组,各15只。采用D-氨基半乳糖联合脂多糖一次性腹腔注射构建急性肝功能衰竭(ALF)大鼠模型,JDHY组在造模前3天加用JDHY(8.8g/kg)。造模完成后,观察各组大鼠24h存活率,检测各组大鼠ALT、AST、TBIL水平,RT-PCR检测TNF-α、IL-6、IL-1βmRNA,Western blot检测CD14和NF-κB蛋白。另取10只大鼠予JDHY(8.8g/kg)连续灌胃5d,制备含药血清并腹主动脉采血。采用Pronase/Collagenase灌注法分离大鼠KC,并将分离后的KC分为对照组、LPS组及JDHY+LPS组。采用免疫荧光法检测p-NF-κBp65和p-IκBα,RT-PCR检测TNF-α、IL-6、IL-1βmRNA水平。结果:JDHY可提高ALF大鼠存活率(P<0.05),改善肝功能(血清ALT、AST、TBIL)(P<0.05),降低促炎因子(TNF-α、IL-6、IL-1βmRNA)和CD14、NF-κB蛋白水平(P<0.05),改善肝脏病理学状态。与LPS组比较,JDHY可降低LPS诱导KC中p-IκBα、p-NF-κBp65及TNF-α、IL-6、IL-1βmRNA水平(P<0.05)。结论:JDHY可通过调节ALF大鼠KC的NF-κB信号通路,减轻免疫损伤、降低促炎因子释放,从而拮抗ALF。

Objection:To observe the effects of Jiedu Huayu Granules(JDHY)on NF-κB signaling pathway in Kupffer cells(KCs)and the mechanism of antagonizing acute liver failure(ALF)in vivo and in vitro.Methods:A total of 45 rats were randomly divided into control group,model group and JDHY group,15 rats in each group.The model of ALF rats was established by D-Galactosamine(D-Galn)and lipopolysaccharide(LPS),JDHY group added JDHY(8.8 g/kg),3 days before modeling.After the model was completed,observed the survival rate of rats.ALT,AST and TBIL were detected by automatic biochemical analyzer,pathological analysis was performed by HE staining,TNF-α,IL-6 and IL-1βmRNA levels were tested by RT-PCT,and CD14 and NF-κB protein levels were detected by Western blot.Ten rats were continuously treated with JDHY with 8.8 g/kg for 5 d,then collected their blood from abdominal aorta and prepared drug serum.The rat KCs was isolated by the method of Pronase/Collagenase perfusion.The separated KCs were divided into control group,LPS group and LPS+JDHY group.Immunofluorescence was used to detect p-NF-κBp65 and p-IκBα,RT-PCR was used to detect the mRNA levels of TNF-α,IL-6,IL-1β.Results:JDHY group could raise the survival rate of ALF rats(P<0.05),improve the liver function(serum ALT,AST,TBIL)(P<0.05),reduce the mRNA level of pro-inflammatory factor(TNF-α,IL-6,IL-1β)and protein level of CD14,and NF-κB(P<0.05).In addition,JDHY group could improve the liver pathological status of ALF rat.JDHY could reduce the level of p-IκBα,p-NF-κBp65,TNF-α,IL-6 and IL-1βin LPS-induced KCs(P<0.05).Conclusion:JDHY can regulate NF-κB signaling pathway in Kupffer cell of rat with ALF,reduce immune damage,decrease the release of pro-inflammatory factors,and thereby antagonize ALF.