疏肝健脾方药对NASH大鼠Kupffer细胞NF-κB p65和IKKβ mRNA及蛋白表达的影响

Effects of Shu-gan Jian-pi prescriptions on expression of NF-κB p65 and IKKβ at mRNA and protein levels in Kupffer cells from rats with non-alcoholic steatohepatitis

目的:探讨疏肝健脾方药对非酒精性脂肪性肝炎(NASH)大鼠Kupffer细胞核因子κB(NF-κB)p65和IκB激酶β(IKKβ)mRNA及蛋白表达的影响。方法:采用高脂饲料喂养复制大鼠NASH模型,在施以造模因素的同时分别灌服疏肝方(柴胡疏肝散)、健脾方(参苓白术散)和综合方(柴胡疏肝散和参苓白术散的合方)的高、低剂量进行干预,16周后分离Kupffer细胞,Typan blue染色和流式细胞术(FCM)分别对Kupffer细胞进行活性和纯度鉴定;实时定量PCR法检测Kupffer细胞IKKβ和NF-κB p65 mRNA的表达水平;Western blotting法检测Kupffer细胞IKKβ、p-IKKβ和NF-κB p65蛋白水平。结果:每只大鼠获得纯化的Kupffer细胞(1.5～2.0)×107,Typan blue染色显示这些细胞活力均在95%以上,FCM检测纯度均在90%以上;与正常组比较,模型组Kupffer细胞IKKβmR-NA及蛋白的表达水平均明显升高(P<0 01);与模型组比较,高剂量综合方、健脾方和疏肝方组IKKβmRNA的表达水平下调明显(P<0.01或P<0.05);IKKβ及磷酸化蛋白的表达水平以高剂量健脾方组下降最为显著(P<0.01或P<0.05);与正常组比较,模型组Kupffer细胞NF-κB p65 mRNA及蛋白的表达水平均明显升高(P<0.01),与模型组比较,NF-κB p65 mRNA的表达水平以综合方组及高剂量健脾方组下调明显(P<0.05),NF-κBp65蛋白的表达水平以高剂量健脾方组下降最为显著(P<0.01)。结论:Kupffer细胞IKKβ、NF-κB p65 mRNA及IKKβ、p-IKKβ、NF-κB p65蛋白的高表达可能参与NASH的发病,抑制这些信号分子的表达可能是疏肝健脾方药抗NASH的重要机制之一。

AIM ： To investigate the effects of Shu-gan Jian-pi prescriptions on the expression of nuclear factor kappa B （NF-KB） p65 and I-kappa B kinase beta （IKKJ3） at mRNA and protein levels in Kupffer cells from the rats with non-alcoholic steatohepatitis （NASH）. METHODS： The rat model of NASH was established by feeding a high-fat diet. The Shu-gan Jian-pi prescriptions of traditional Chinese medicine were prepared. The rats were divided into high dose group and low dose group of Shu-gan recipe, Jian-pi recipe and composite recipe. After the rats were intragastrically treated with the recipes for 16 weeks, the Kupffer ceils were isolated. Typan blue staining and flow cytometry （FCM） were applied to test the viability and purity of the cells. The mRNA expression of IKKβ and NF-KB p65 in the Kupffer cells was analyzed by real-time quantitative PCR. The protein levels of IKK[3, p-IKKI3 and NF-KB p65 in the Kupffer cells were examined by ,~ Western blotting. RESULTS： The yields of purified Kupffer cells in each rat were （ 1.5 ~ 2. O） x 107. The viability of the Kupffer cells was higher than 95% with the purity over 90%. The expression of IKK[3 at mRNA and protein levels in model group was higher than that in normal group. Compared with model group~ the mRNA expression of IKKI3 in high dose of composite recipe group, high dose of Jian-pi recipe group and high dose of Shu-gan recipe group was decreased. The pro- tein levels of IKK[~ and p-IKKI3 in high dose of Jian-pi recipe group were decreased. The expression of NF-KB at mRNA and protein levels in model group was higher than that in normal group. Compared with model group, the mRNA expression of NF-KB p65 in high dose of composite recipe group and high dose of Jian-pi recipe group was decreased. The protein level of NF-KB p65 in high dose of Jian-pi recipe group was also decreasesd. CONCLUSION ： The increases in the expression of IKKB/NF-KB at mRNA and protein levels in Kupffer cells play roles in the development of NASH. Reducing the accu- mulation of inflammatory factors and down-regulation of IKKI~ and NF-~B may be the important mechanism of Shu-gan Jian- pi prescriptions to prevent and cure NASH.