甘露消毒饮对手足口病模型乳鼠的干预效果及对TLR4/MAPKs/NF-κB信号通路影响

Effect of Ganlu Xiaodu Decoction on Suckling Mice with Hand-Foot-Mouth Disease Model and Influence on TLR4/MAPKs/NF-κB Signaling Pathway

分析甘露消毒饮对手足口病(HFMD)模型乳鼠的干预效果及对TLR4/MAPKs/NF-κB信号通路影响。采用颅内接种EV71病毒建立18只HFMD乳鼠模型,随机分为模型组(n=6)、甘露消毒饮组(n=6)、TLR4信号通路抑制剂组(抑制剂组)(n=6),同时另取6只同龄健康乳鼠为正常组。甘露消毒饮组给予灌胃甘露消毒饮治疗(24.4 g/kg),抑制剂组给予腹腔注射TLR4抑制剂TAK-242(0.3 mg/kg),其余组给予生理盐水治疗,干预后12 h脱颈处死各组乳鼠,取腹主动脉血,并分离脑、骨骼肌、心脏、肺、小肠组织。HE染色观察病理组织,免疫荧光染色各组织EV71病毒抗原的表达。ELISA检测4组乳鼠血清MBP、LDH、CK、NSE、IL-6、IL-1β、IL-12、TNF-α含量,Western blot法检测各组乳鼠脑组织中TLR4/MAPKs/NF-κB信号通路相关蛋白的表达水平。HE染色和免疫荧光染色结果表明,正常组各组织正常,且各组织无EV71病毒抗原的表达。模型组乳鼠各组织中均观察到病理损伤和炎症反应,且各组织有EV71病毒抗原的表达。甘露消毒饮组和抑制剂组乳鼠各组织病理损伤均得到改善,且各组织EV71病毒抗原表达降低。与正常组比较,模型组乳鼠血清MBP、LDH、CK、NSE、IL-6、IL-1β、IL-12、TNF-α含量均显著升高(P<0.05)。与模型组相比,甘露消毒饮组和抑制剂组乳鼠血清MBP、LDH、CK、NSE、IL-6、IL-1β、IL-12、TNF-α含量均显著降低(P<0.05)。与正常组比较,模型组乳鼠脑组织中TLR4、p-P38MAPK、p-JNK/JNK、p-ERK/ERK、p-NF-κB/NF-κB、p-IKB-α表达水平均显著升高(P<0.05),而P38MAPK、IKB-α表达水平均显著降低(P<0.05)。与模型组相比,甘露消毒饮组和抑制剂组乳鼠脑组织中TLR4、p-P38MAPK、p-JNK/JNK、p-ERK/ERK、p-NF-κB/NF-κB、p-IKB-α表达水平均显著降低(P<0.05),而P38MAPK、IKB-α表达水平均显著升高(P<0.05)。甘露消毒饮可能通过抑制TLR4/MAPKs/NF-κB信号通路治疗乳鼠手足口病。

To analyze the intervention effect of Ganlu Xiaodu Decoction in suckling mice with hand,foot and mouth disease(HFMD)model and its influence on TLR4/MAPKs/NF-κB signaling pathway,the HFMD suckling mice models were established by intracranial inoculation of EV71 virus,and they were randomly divided into model group(n=6),Ganlu Xiaodu Decoction group(n=6),TLR4 signal pathway inhibitor group(inhibitor group)(n=6),and the same number of healthy and same-age suckling mice were taken as normal group.Ganlu Xiaodu Decoction group was given Ganlu Xiaodu Decoction(24.4 g/kg),inhibitor group was given intraperitoneal injection of TLR4 inhibitor TAK-242(0.3 mg/kg),and the rest of the groups were given normal saline treatment.12 h after the intervention,the suckling mice were killed by removing their necks,taking blood from the abdominal aorta,and separating the brain,skeletal muscle,heart,lung,and intestine tissues.The pathological tissue was observed by HE staining,and the expression of EV71 virus antigen in brain and hind limb muscle tissue was detected by Immunofluorescence.ELISA was used to detect serum MBP,LDH,CK,NSE,IL-6,IL-1β,IL-12,and TNF-αlevels in the four groups of suckling mice;Western blot method was used to detect the proteins expression levels of TLR4/MAPKs/NF-κB signaling pathway in the brain tissues of suckling mice.The results of HE staining and immunofluorescence staining showed that the tissues of the normal group were normal,and there was no expression of EV71 virus antigen in each tissue.In the model group,pathological damage and inflammation were observed in all tissues of suckling mice,and there were a large number of EV71 virus antigens in in each tissue.In the Ganlu Xiaodu Decoction group and inhibitor group,the pathological damage of each tissue of the suckling mice was improved,and the expression of EV71 virus antigen in each tissue was reduced.Compared with the normal group,the serum MBP,LDH,CK,NSE,IL-6,IL-1β,IL-12,and TNF-αlevels of the model group were significantly increased(P<0.05).Compared with the model group,the serum MBP,LDH,CK,NSE,IL-6,IL-1β,IL-12,and TNF-αlevels of the suckling mice in the Ganlu Xiaodu Decoction group and inhibitor group were significantly reduced(P<0.05).Compared with the normal group,the expression levels of TLR4,p-P38 MAPK,p-JNK/JNK,p-ERK/ERK,p-NF-κB/NF-κB,and p-IKB-αin the brain tissue of the model group were significantly increased(P<0.05),while the expression levels of P38 MAPK and IKB-αin the brain tissue of suckling mice were significantly reduced(P<0.05).Compared with the model group,the expression levels of TLR4,p-P38 MAPK,p-JNK/JNK,p-ERK/ERK,p-NF-κB/NF-κB,p-IKB in the Ganlu Xiaodu Decoction drink group and the inhibitor group were significantly reduced(P<0.05),while the expression levels of P38 MAPK and IKB-αin the brain tissue of suckling mice were significantly increased(P<0.05).Ganlu Xiaodu Decoction can effectively improve the symptoms of HFMD in suckling mice,and its mechanism may be related to the inhibition of TLR4/MAPKs/NF-κB signaling pathway.