百草枯致小鼠心肌损伤中Toll样受体4表达及意义

Influence of Paraquat Poisoning on the Expression of Toll-like Receptor 4 in Myocardial Damage Following in Mice and Its Significance

目的明确Toll样受体4(TLR4)在百草枯(PQ)所致的心肌损伤是否具有作用,并分析其相关意义。方法雄性C57BL/6J野生(WT)小鼠和TLR4基因敲除(TLR4-ko)小鼠,分为4组:对照组(WT小鼠,n=6)、TLR4-ko组(TLR4-ko小鼠,n=6)、WT+PQ组(WT小鼠,n=30)、TLR4-ko+PQ组(TLR4-ko小鼠,n=30)。对照组和TLR4-ko组小鼠腹腔注射生理盐水,WT+PQ组和TLR4-ko+PQ组腹腔注射PQ(75 mg/kg)。染毒后2,4,8,16,24 h,分别选取6只WT+PQ组和TLR4-ko+PQ组小鼠处死,留取心肌组织标本用于组织病理学分析,WT+PQ组小鼠检测TLR4 m RNA表达。染毒2、8、24 h后,2组小鼠检测心肌组织中肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)表达,Western blot检测WT+PQ组小鼠心肌中TLR4蛋白表达。对照组和TLR4-ko组小鼠在生理盐水处理后8 h,以同样的方法留取标本并进行相应检测。此外,在接受PQ或生理盐水处理后8 h,4组小鼠均使用2维超声M模式超声心动图仪评价心脏形态和心功能。结果 WT+PQ组小鼠在PQ致心肌损伤过程中,心肌组织出现了显著的病理损伤改变,心功能明显下降,心肌组织中TNF-α和IL-1β表达增加。与WT+PQ组小鼠比较,TLR4-ko组小鼠心肌功能下降程度和病理损伤程度减轻,相应的细胞因子TNF-α和IL-1β表达减少。结论 TLR4基因表达与PQ中毒后心脏功能下降和病理损伤之间存在密切联系,其作用机制涉及到下游信号细胞因子TNF-α和IL-1β。TLR4信号系统在PQ所致的心肌损伤中具有重要的作用。

Objective To clarify the role and significance of Toll-like receptor 4（TLR4）in myocardial damage following paraquat（PQ）poisoning in mice. Methods Male wild type C57BL/6J mice（WT）and male TLR4 deficient mice（TLR4-ko）were divided into four groups in the study：（1）control group（WT mice,n =6）;（2）TLR4-ko group（TLR4-ko mice,n =6）;（3）WT＋PQ group（WT mice,n =30）;（4）TLR4-ko＋PQ group（TLR4-ko mice,n =30）. The mice in group 1 and group 2 were injected intraperitoneally with saline;mice in group 3 and group 4 were injected intraperitoneally with 75 mg/kg of PQ. At 2 h,4 h,8 h,16 h and 24 h after PQ administration,6 mice of WT＋PQ group and TLR4-ko＋PQ group were euthanized and the heart tissue specimens were harvested. All the specimens were analysed by histology,while the expression of TLR4 m RNA was only detected in samples of WT＋PQ mice. The specimens at 2 h,8 h and 24 h after PQ administration were used for cytokines detection for WT＋PQ group and TLR4-ko＋PQ group;in addition,Western blot analysis was performed for WT＋PQ group. At 8 h after treatment,control mice and TLR4-ko mice were euthanized by the same method. Mice were anesthetized for cardiac geometry and functional assessment using a 2-D guide M-mode echocardiography at 8 h following injection of either PQ or saline. Results During myocardial damage due to PQ exposure in WT＋PQ mice,obvious histopathological changes were observed,as well as a noticeable decrease of heart function and increased expressions of TNF-αand IL-1β. Compared with the WT mice,TNF-α and IL-1β protein levels,changes in heart function and histopathological changes were significantly attenuated following PQ exposure in myocardial damage in TLR4-ko mice. Conclusion The TLR4 gene is involved with in heart functional injury and histopathological changes in myocardial damage following PQ poisoning in mice,which may through the regulation of TNF-α and IL-1β expression. Our findings indicate that TLR4 plays an important role in mediating myocardial injury due to PQ.