桦褐孔菌提取物对黄曲霉素B1暴露小鼠肝损伤的缓解作用

doi:10.16431/j.cnki.1671-7236.2024.02.042

摘要/Abstract

摘要： 【目的】探究桦褐孔菌提取物(IOE)对黄曲霉毒素B₁(AFB₁)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;AFB₁组第1~7天连续灌胃蒸馏水,第8~10天连续灌胃2 mg/kg AFB₁;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 AFB₁,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AFB₁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AFB₁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,AFB₁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AFB₁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,AFB₁诱导AML12细胞损伤最适条件为10 μg/mL AFB₁作用24 h,IOE防治AFB₁诱导AML12细胞损伤的浓度梯度为1.5、3、6 μg/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AFB₁暴露引起的小鼠肝脏损伤。

关键词: 桦褐孔菌提取物(IOE); 黄曲霉毒素B₁(AFB₁); 肝脏; 凋亡; 炎症

Abstract: 【Objective】 The objective of this study was to investigate the protective effect and mechanism of Inonotus obliquus extract (IOE) on liver injury in aflatoxin B₁ (AFB₁) 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.AFB₁ group received continuous intragastric administration of distilled water from day 1 to day 7, and 2 mg/kg AFB₁ 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 AFB₁ 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 factor α (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 AFB₁ group (P＜0.01).The contents of TNF-α, IL-6 and IL-1β in serum were extremely significantly increased (P＜0.01).The expressions of TNF-α, IL-6, IL-1β, NLRP3 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 AFB₁ 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-1β contents were extremely significantly decreased (P＜0.01).The mRNA expressions of TNF-α, IL-6, IL-1β, NLRP3 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 AFB₁-induced AML12 cell damage was 10 μg/mL AFB₁ for 24 h, and the concentration gradients of IOE for prevention and treatment of AFB₁-induced AML12 cell damage were 1.5, 3 and 6 μg/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 AFB₁ in mice.

Key words: Inonotus obliquus extract (IOE); aflatoxin B₁ (AFB₁); liver; apoptosis; inflammation

中图分类号:

S852.4⁺4

赵成铭, 谢为天, 林红英, 胡颍昕, 马文澳, 李颖, 陈志宝. 桦褐孔菌提取物对黄曲霉素B₁暴露小鼠肝损伤的缓解作用[J]. 中国畜牧兽医, 2024, 51(2): 864-874.

ZHAO Chengming, XIE Weitian, LIN Hongying, HU Yingxin, MA Wenao, LI Ying, CHEN Zhibao. Alleviating Effects of Inonotus obliquus Extract on Liver Injury Induced by AFB₁ in Mice[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 864-874.

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桦褐孔菌提取物对黄曲霉素B₁暴露小鼠肝损伤的缓解作用

Alleviating Effects of Inonotus obliquus Extract on Liver Injury Induced by AFB₁ in Mice

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