Toll样受体4在对乙酰氨基酚致小鼠肝损伤过程中对肝脏再生的影响

Effect of Toll-like receptor 4 on liver regeneration during acetaminophen-induced liver injury in mice

目的观察抑制Toll样受体4(TLR4)是否影响对乙酰氨基酚(APAP)致小鼠肝损伤过程中的肝脏再生,初步探讨TLR4参与肝脏再生的机制。方法将78只雄性CD-1小鼠采用随机数字表法分为9组,其中对照组(正常对照组、溶剂对照组、抑制剂对照组)每组6只,实验组(APAP 24 h组、TAK-242+APAP 24 h组、APAP 48 h组、TAK-242+APAP 48 h组、APAP 72 h组、TAK-242+APAP 72 h组)每组10只。实验组小鼠给予单剂量腹腔注射APAP(300 mg/kg),TAK-242在APAP注射前3 h以3 mg/kg剂量腹腔注射。在不同时间点收集各组小鼠血清和肝脏组织。采用生化方法检测小鼠血清ALT水平,HE染色检测肝脏组织病理改变。RT-PCR、Western blot、免疫组化方法检测Cyclin D1、PCNA、Ki-67、STAT3、p-STAT3的表达。正态分布的计量资料两组间比较采用成组t检验;多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。非正态分布的计量资料两组间比较采用Mann-Whitney U检验;多组间比较及进一步两两比较均采用Kruskal-Wallis H检验。结果与正常对照组相比,APAP 24 h组和APAP 48 h组的血清ALT水平均明显较高(P值均<0.05);TAK-242+APAP 24 h和48 h组的血清ALT水平均明显高于同时间点APAP组(P值均<0.05)。HE染色结果显示,APAP处理的小鼠肝脏可见典型的小叶中心性坏死,TAK-242+APAP 24 h和48 h组小鼠肝脏的坏死面积均显著大于同时间点APAP组(P值均<0.05)。RT-PCR、Western blot、免疫组化结果显示,TAK-242+APAP 24 h、48 h和72 h组Cyclin D1 mRNA和蛋白表达水平均明显低于同时间点的APAP组(P值均<0.05);TAK-242+APAP 24 h、48 h和72 h组PCNA mRNA表达水平均明显低于同时间点的APAP组(P值均<0.05),TAK-242+APAP 24 h和48 h组PCNA蛋白表达水平均明显低于同时间点的APAP组(P值均<0.05);TAK-242+APAP 24 h和72 h组Ki-67 mRNA表达水平均明显低于同时间点的APAP组(P值均<0.05),TAK-242+APAP 24 h、48 h和72 h组Ki-67蛋白表达水平均明显低于同时间点的APAP组(P值均<0.05)。此外,TAK-242+APAP 24 h和48 h组STAT3磷酸化水平均显著低于同时点的APAP组(P值均<0.05)。结论TLR4可能通过提高STAT3磷酸化水平促进APAP诱导的小鼠肝损伤过程中的肝脏再生。

Objective To investigate whether Toll-like receptor 4(TLR4)inhibition affects liver regeneration during acetaminophen(APAP)-induced liver injury in mice,as well as the mechanism of TLR4 involved in liver regeneration.Methods A total of 78 male CD-1 mice were divided into nine groups using a random number table,i.e.,three control groups(normal control group,solvent control group,inhibitor control group)with 6 mice in each group and six experimental groups(APAP 24-hour group,TAK-242+APAP 24-hour group,APAP 48-hour group,TAK-242+APAP 48-hour group,APAP 72-hour group,TAK-242+APAP 72-hour group)with 10 mice in each group.The mice in the experimental groups were given a single dose of intraperitoneally injected APAP(300 mg/kg),and TAK-242 was intraperitoneally injected at a dose of 3 mg/kg at 3 hours before APAP administration.Serum and liver tissue samples were collected at different time points.The biochemical method was used to measure the serum level of alanine aminotransferase(ALT);HE staining was used to observe liver pathological changes;RT-PCR,Western blot,and immunohistochemistry were used to measure the expression levels of Cyclin D1,PCNA,Ki-67,STAT3,and p-STAT3.The t-test was used for comparison of normally distributed continuous data between two groups;a one-way analysis of variance was used for comparison between multiple groups,and the least significant difference t-test was used for further comparison between two groups.The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups,and the Kruskal-Wallis H test was used for comparison between multiple groups and further comparison between two groups.Results Compared with the normal control group,the APAP 24-hour and 48-hour groups had a significantly higher serum level of ALT(both P<0.05),and the TAK-242+APAP 24-hour and 48-hour groups had a significantly higher serum level of ALT than the APAP group at the same time point(both P<0.05).HE staining showed typical central lobular necrosis in the liver of APAP-treated mice,and the TAK-242+APAP 24-hour and 48-hour groups had a significantly larger necrotic area than the APAP group at the same time point(both P<0.05).RT-PCR,Western blot,and immunohistochemistry showed that the TAK-242+APAP 24-hour,48-hour,and 72-hour groups had significantly lower mRNA and protein expression levels of Cyclin D1 than the APAP group at the same time point(all P<0.05);the TAK-242+APAP 24-hour,48-hour,and 72-hour groups had a significantly lower mRNA expression level of PCNA than the APAP group at the same time point(all P<0.05),and the TAK-242+APAP 24-hour and 48-hour groups had a significantly lower protein expression level of PCNA than the APAP group at the same time point(all P<0.05);the TAK-242+APAP 24-hour and 72-hour groups had a significantly lower mRNA expression level of Ki-67 than the APAP group at the same time point(all P<0.05),and the TAK-242+APAP 24-hour,48-hour,and 72-hour groups had a significantly lower protein expression level of Ki-67 than the APAP group at the same time point(all P<0.05).In addition,the TAK-242+APAP 24-hour and 48-hour groups had a significantly lower phosphorylation level of STAT3 than the APAP group at the same time point(both P<0.05).Conclusion TLR4 may promote liver regeneration by increasing the phosphorylation level of STAT3 during APAP-induced liver injury in mice.