[1]陶　涛,周　全,周　嘉.ω-3多不饱和脂肪酸对对乙酰氨基酚诱导小鼠急性肝损伤的保护作用[J].新乡医学院学报,2019,36(6):506-510.[doi:10.7683/xxyxyxb.2019.06.002]
　TAO Tao,ZHOU Quan,ZHOU Jia.Protective effects of ω-3 polyunsaturated fatty acids on acute liver injury induced by acetaminophen in mice[J].Journal of Xinxiang Medical University,2019,36(6):506-510.[doi:10.7683/xxyxyxb.2019.06.002]
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ω-3多不饱和脂肪酸对对乙酰氨基酚诱导小鼠急性肝损伤的保护作用
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
36
期数:
2019年6
页码:
506-510
栏目:
基础研究
出版日期:
2019-06-05

文章信息/Info

Title:
Protective effects of ω-3 polyunsaturated fatty acids on acute liver injury induced by acetaminophen in mice
作者:
陶　涛1; 周　全1; 周　嘉2
（1.湛江中心人民医院麻醉科，广东　湛江　524045；2.南方医科大学基础医学院免疫学教研室，广东　广州　510515）
Author(s):
TAO Tao1; ZHOU Quan1; ZHOU Jia2
（1.Department of Anesthesiology，Central People′s Hospital of Zhanjiang，Zhanjiang 524045，Guangdong Province，China；2.Department of Immunology，School of Basic Medical Sciences，Southern Medical University，Guangzhou 510515，Guangdong Province，China）
关键词:
多不饱和脂肪酸; 对乙酰氨基酚; 急性肝损伤; 腺苷酸激活蛋白激酶
Keywords:
polyunsaturated fatty acid; acetaminophen; acute liver injury; adenosine monophosphate activated protein kinase
分类号:
R965
DOI:
10.7683/xxyxyxb.2019.06.002
文献标志码:
A
摘要:
目的　探讨ω-3多不饱和脂肪酸（ω-3PUFA）对对乙酰氨基酚（APAP）诱导的小鼠急性肝损伤的保护作用及机制。方法　选取健康C57BL/6雌性野生型小鼠（WT组）和Fat-1转基因雌性小鼠（Fat-1组）各24只，采用腹腔注射APAP法建立小鼠急性肝损伤模型，分别于APAP腹腔注射后0、2、6、24 h应用CO2法每组处死6只小鼠，采集小鼠肝组织和静脉血。应用丙氨酸转氨酶（ALT）检测试剂盒测定小鼠血清ALT水平，苏木精-伊红（HE）染色观察小鼠肝组织形态学变化，Western blot法检测小鼠肝组织中腺苷酸活化蛋白激酶（AMPK）和磷酸化AMPK（p-AMPK）水平。取HepaRG细胞，应用APAP孵育6 h诱导HepaRG细胞凋亡，换培养液后将细胞分为APAP+二十二碳六烯酸（DHA）组和APAP+二甲基亚砜（DMSO）组，APAP+DHA组在培养基中加入DHA，APAP+DMSO组在培养基中加入DMSO；分别于处理0、3、6 h时收集HepaRG细胞，采用Western blot法检测HepaRG细胞中AMPK和p-AMPK表达水平。取HepaRG细胞并随机分为磷酸盐缓冲液（PBS）+DHA组、PBS+DMSO组、APAP+DHA组和APAP+DMSO组，APAP+DHA组和APAP+DMSO组细胞应用APAP孵育6 h诱导HepaRG细胞凋亡，PBS+DHA组和PBS+DMSO组细胞加入PBS；换培养液后，APAP+DHA组和PBS+DHA组在培养基中加入DHA，APAP+DMSO组和PBS+DMSO组在培养基中加入DMSO；各组细胞继续温箱孵育6 h，收集细胞，采用流式细胞术检测各组HepaRG细胞凋亡情况。结果　2组小鼠腹腔注射APAP后2、6、24 h时血清ALT水平显著高于0 h时（P<0.05）；腹腔注射APAP后2、6、24 h时，Fat-1组小鼠血清ALT水平显著低于WT组（P<0.05）。HE染色结果显示，腹腔注射APAP后24 h WT组小鼠肝细胞呈现不同程度的变性或坏死，细胞核溶解或固缩，肝细胞索正常形态被破坏并伴有明显的炎性细胞浸润；而Fat-1组小鼠肝脏组织病变较轻。腹腔注射APAP后2、6 h时Fat-1组小鼠肝组织中p-AMPK相对表达量显著高于WT组（P<0.05）；WT组小鼠腹腔注射APAP后2、6 h时肝组织中p-AMPK相对表达量显著低于0 h时（P<0.05），Fat-1组小鼠腹腔注射APAP后0、2、6 h时肝组织中p-AMPK相对表达量比较差异无统计学意义（P>0.05）。腹腔注射APAP后0、2、6 h时2组小鼠肝组织中AMPK相对表达量比较差异均无统计学意义（P>0.05）。APAP+DHA组HepaRG细胞处理3、6 h时p-AMPK相对表达量显著高于APAP+DMSO组（P<0.05），APAP+DHA组和APAP+DMSO组HepaRG细胞处理3、6 h时p-AMPK相对表达量显著低于0 h时（P<0.05）；APAP+DHA组和APAP+DMSO组HepaRG细胞处理0、3、6 h时AMPK相对表达量比较差异均无统计学意义（P>0.05）。APAP+DMSO组、APAP+DHA组HepaRG细胞凋亡率均显著高于PBS+DMSO组和PBS+DHA组（P<0.05），APAP+DHA组HepaRG细胞凋亡率显著低于APAP+DMSO组（P<0.05），PBS+DMSO组与PBS+DHA组HepaRG细胞凋亡率比较差异无统计学意义（P>0.05）。结论　ω-3PUFA对APAP诱导的小鼠急性肝损伤有保护作用，其机制可能与ω-3PUFA促进AMPK通路活化有关。
Abstract:
Objective　To investigate the protective effect and mechanism of ω-3 polyunsaturated fatty acid （ω-3 PUFA） on acute liver injury induced by acetaminophen （APAP） in mice.Methods　Twenty-four healthy C57BL/6 female wild-type mice （WT group） and 24 Fat-1 transgenic female mice （Fat-1 group） were selected as subjects.The acute liver injury model of mice was established by intraperitoneal injection of APAP.At 0，2，6 and 24 hours after intraperitoneal injection of APAP，six mice in each group were executed by CO2 method，and the liver tissues and venous blood were collected for subsequent experiments.The level of serum alanine aminotransferase （ALT） of the mice was detected by ALT kit，The morphological change of liver tissues of the mice was observed by hematoxylin-eosin （HE） staining，and the expression of adenosine monophosphate activated protein kinase（AMPK） and phosphorylated AMPK （p-AMPK） in liver tissues of the mice was detected by Western blot method.HepaRG cells were incubated with APAP for 6 hours to induce apoptosis.The HepaRG cells were divided into APAP+docosahexenoic acid （DHA） group and APAP+dimethyl sulfoxide （DMSO） group.DHA was added into nutrient medium in APAP+DHA group and DMSO was added into nutrient medium in APAP+DMSO group.HepaRG cells were collected at 0，3 and 6 h of treatment，and the expressions of AMPK and p-AMPK in HepaRG cells were detected by Western blot method.HepaRG cells were randomly divided into phosphate buffered saline（PBS）+DHA group，PBS+DMSO group，APAP+DHA group and APAP+DMSO group.The HepaRG cells in APAP+DHA group and APAP+DMSO group were incubated with APAP for 6 hours to induce apoptosis,and PBS was added in PBS+DHA group and PBS+DMSO group.After changing the medium，DHA was added into nutrient medium in APAP+DHA group and PBS+DHA group，DMSO was added into nutrient medium in APAP+DMSO group and PBS+DMSO group.The cells in each group were collected after incubation in incubator for 6 hours，and the apoptosis of HepaRG cells was detected by flow cytometry.Results　The level of serum ALT of mice at 2，6 and 24 hours after intraperitoneal injection of APAP was significantly higher than that at 0 hour in the two groups （P<0.05）.The level of serum ALT of mice in the Fat-1 group was significantly lower than that in the WT group at 2，6 and 24 hours after intraperitoneal injection of APAP（P<0.05）.Twenty-four hours after APAP injection，the HE staining showed that the hepatocytes of mice in the WT group showed varying degrees of degeneration or necrosis，nucleus dissolution or pyknosis；normal morphology of liver cell cord was destroyed with obvious inflammatory cell infiltration；while the liver tissue lesions of mice in the Fat-1 group were milder.The relative expression of p-AMPK in liver tissues of mice in the Fat-1 group was significantly higher than that in the WT group at 2 and 6 hours after intraperitoneal injection of APAP （P<0.05）.The relative expression of p-AMPK in liver tissues of mice at 2 and 6 hours after intraperitoneal injection of APAP was significantly lower than that at 0 hour in WT group （P<0.05）.There was no significant difference in the relative expression of p-AMPK in liver tissues of mice at 0，2 and 6 hours after intraperitoneal injection of APAP in the Fat-1 group （P>0.05）.There was no significant difference in the relative expression of AMPK between the two groups at 0，2 and 6 hours after intraperitoneal injection of APAP （P>0.05）.The relative expression of p-AMPK in HepaRG cells of the APAP+DHA group was significantly higher than that of the APAP+DMSO group at 3 and 6 hours of intervention （P<0.05），the relative expression of p-AMPK in HepaRG cells at 3 and 6 hours was significantly lower than that at 0 hour in the two groups （P<0.05），but there was no significant difference in the relative expression of AMPK between the two groups at 0，3 and 6 hours （P>0.05）.The apoptotic rate of HepaRG cells in the APAP+DMSO group and the APAP+DHA group was significantly higher than that in the PBS+DMSO group and the PBS+DHA group （P<0.05）.The apoptotic rate of HepaRG cells in the APAP+DHA group was significantly lower than that in the APAP+DMSO group （P<0.05）.There was no significant difference in apoptotic rate of HepaRG cells between the PBS+DMSO group and the PBS+DHA group （P>0.05）.Conclusion　ω-3PUFA has protective effect on acute liver injury induced by APAP in mice，and its mechanism may be related to ω-3PUFA promoting the activation of AMPK pathway.

参考文献/References:

［1］　JAESCHKE H.Acetaminophen：dose-dependent drug hepatotoxicity and acute liver failure in patients［J］.Dig Dis，2015，33（4）：464-471.

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更新日期/Last Update: 2019-06-05