桦褐孔菌提取物对黄曲霉素B_(1)暴露小鼠肝损伤的缓解作用

Alleviating Effects of Inonotus obliquus Extract on Liver Injury Induced by AFB_(1) in Mice

[目的]探究桦褐孔菌提取物(IOE)对黄曲霉毒素B1(AFB_(1))暴露小鼠肝损伤的保护作用及机制。[方法]选取30只6周龄、体重相近的SPF级雄性C57BL/6小鼠,随机分为5组,对照组连续灌胃蒸馏水10 d;AFB_(1)组第1~7天连续灌胃蒸馏水,第8~10天连续灌胃2 mg/kg AFB_(1);不同浓度IOE组第1~7天分别连续灌胃25、50和100 mg/kg IOE,第8~10天各组均连续灌胃2 mg/kg AFB_(1),灌胃剂量均为0.2 mL,每天1次。试验结束后对小鼠称重并采集血液、肝脏组织,统计肝脏指数;通过苏木精-伊红(HE)染色观察肝脏组织病理变化;利用流式细胞仪分析肝脏细胞凋亡情况。通过细胞毒性试验,筛选AFB_(1)诱导AML12细胞损伤最适条件,并以此建立肝损伤模型,给予不同浓度IOE进行干预,利用CCK-8法测定细胞活力,确定IOE防治AFB_(1)诱导AML12细胞损伤的剂量范围。使用ELISA法检测血清和细胞中炎性因子白细胞介素-1β(IL-1β)、IL-6和肿瘤坏死因子α(TNF-α)含量;利用实时荧光定量PCR检测肝脏和AML12细胞中TNF-α、IL-6、IL-1β、NOD样受体热蛋白结构域相关蛋白3(NLRP3)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)基因mRNA表达量;采用Western blotting检测肝脏和AML12细胞中NLRP3、Bax和Bcl-2蛋白表达量。[结果]与对照组相比,AFB_(1)组肝细胞排列紊乱、细胞肿胀,细胞凋亡率极显著升高(P<0.01);血清中TNF-α、IL-6、IL-1β含量均极显著升高(P<0.01);肝脏中TNF-α、IL-6、IL-1β、NLRP3和Bax基因表达量均极显著升高,Bcl-2基因表达量极显著降低(P<0.01);NLRP3和Bax蛋白水平极显著升高,Bcl-2蛋白水平极显著降低(P<0.01)。与AFB_(1)组相比,不同浓度IOE组小鼠炎性细胞浸润减少,细胞排列整齐,细胞凋亡率极显著降低(P<0.01);血清TNF-α、IL-6、IL-1β含量极显著降低(P<0.01);肝脏TNF-α、IL-6、IL-1β、NLRP3和Bax基因mRNA表达量极显著降低,Bcl-2基因表达量极显著升高(P<0.01);NLRP3和Bax蛋白水平均极显著降低,Bcl-2蛋白水平极显著升高(P<0.01)。细胞毒性试验结果显示,AFB_(1)诱导AML12细胞损伤最适条件为10μg/mL AFB_(1)作用24 h, IOE防治AFB_(1)诱导AML12细胞损伤的浓度梯度为1.5、3、6μg/mL。体外试验结果显示,AML12细胞中各炎性因子分泌、凋亡相关基因及蛋白表达量变化趋势与体内试验结果均一致。[结论]IOE可通过调节NLRP3、Bax和Bcl-2表达抑制炎性因子分泌,降低细胞凋亡,进而缓解AFB_(1)暴露引起的小鼠肝脏损伤。

[Objective]The objective of this study was to investigate the protective effect and mechanism of Inonotus obliquus extract(IOE)on liver injury in aflatoxin B 1(AFB_(1))exposed mice.[Method]Thirty 6-week-old SPF male C57BL/6 mice with similar body weight were randomly divided into 5 groups.The control group was continuously gavage with distilled water for 10 days.AFB_(1) group received continuous intragastric administration of distilled water from day 1 to day 7,and 2 mg/kg AFB_(1) from day 8 to 10.Groups with different concentrations of IOE were given continuous intragastric administration of 25,50 and 100 mg/kg IOE on days 1 to 7,and 2 mg/kg AFB_(1) on days 8 to 10,respectively.The intragastric dose was 0.2 mL,once a day.After the experiment,the mice were weighed,blood and liver tissues were collected,and liver index was calculated.The pathological changes of liver were observed by hematoxylin-eosin(HE)staining.The apoptosis of liver cells was analyzed by flow cytometry.The contentss of inflammatory factors interleukin-1β(IL-1β),IL-6 and tumor necrosis factorα(TNF-α)in serum and cells were detected by ELISA.The mRNA expression of TNF-α,IL-6,IL-1β,NOD-like receptor thermal protein domain associated protein 3(NLRP3),B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax)genes in liver and AML12 cells were detected by Real-time quantitative PCR.The expression of NLRP3,Bax and Bcl-2 proteins in liver and AML12 cells were detected by Western blotting.[Result]Compared with control group,hepatocyte arrangement was disordered,cell swelling and apoptosis rate were extremely significantly increased in AFB_(1) group(P<0.01).The contents of TNF-α,IL-6 and IL-1βin serum were extremely significantly increased(P<0.01).The expressions of TNF-α,IL-6,IL-1β,NLRP 3 and Bax genes in liver were extremely significantly increased,and the expression of Bcl-2 gene was extremely significantly decreased(P<0.01).The protein levels of NLRP3 and Bax were extremely significantly increased,and the level of Bcl-2 protein was extremely significantly decreased(P<0.01).Compared with AFB_(1) group,inflammatory cell infiltration was decreased,cells were arranged neatly,and cell apoptosis rate was extremely significantly decreased in IOE groups(P<0.01).Serum TNF-α,IL-6 and IL-1βcontents were extremely significantly decreased(P<0.01).The mRNA expressions of TNF-α,IL-6,IL-1β,NLRP 3 and Bax genes in liver were extremely significantly decreased,and the expression of Bcl-2 gene was extremely significantly increased(P<0.01).NLRP3 and Bax proteins levels were extremely significantly decreased,while Bcl-2 protein levels was extremely significantly increased(P<0.01).The results of cytotoxicity test showed that the optimal condition for AFB_(1)-induced AML12 cell damage was 10μg/mL AFB_(1) for 24 h,and the concentration gradients of IOE for prevention and treatment of AFB_(1)-induced AML12 cell damage were 1.5,3 and 6μg/mL,respectively.The results of in vitro test showed that the secretion of inflammatory factors,apoptosis-related genes and protein expression of AML12 cells were consistent with the results of in vivo test.[Conclusion]IOE could inhibit the secretion of inflammatory factors and reduce apoptosis by regulating the expression of NLRP3,Bax and Bcl-2,thus alleviating the liver injury induced by AFB_(1) in mice.