从外周TLR4/NF-κB信号通路至中枢正负调节介质探究退热六法对发热幼兔的退热效果及机制研究

The primary conclusions of preliminary research are as follows: ① infant tuina for the treatment of fever is safe and effective; ② the effectiveness and the mechanism of infant tuina for the use of two different types (bacterial and viral), three different age groups (Infants, toddlers, preschool age), and three different degrees of fever (low, medium, high) are urgently in need of elucidation. Based on the above, this experiment is divided into an evaluation of effectiveness and research of the mechanism. In the evaluation of the effectiveness, this research project will use ‘reducing fever six methods’ technique on New Zealand infant rabbits as the intervention. Through standard blood analysis combined with flow cytometry, the morphological changes of blood cells will be detected; ethological and morphological methods will be combined to evaluate the therapeutic effectiveness, and to evaluate the effectiveness of using infant tuina to treat fever in an experimental setting. To study the mechanism, the body temperature regulation pathway (serum→cerebrospinal fluid→hypothalamus) from the periphery to the central region will be studied to explore the mechanism of the therapeutic effectiveness. Integrated molecular biology will be used to detect the expression of peripheral TLR4/NF-κB signaling pathway key indicators (TLR4、NF-κB、IL-1β、IL-6、TNF-α、IFN, etc.), the role of central POAH area in adjusting the medium (PGE2, cAMP, etc.), and the negative regulation of the ventral septal area (AVP, alpha MSH, beta EP, etc.). A comprehensive analysis of the results will be carried out to clarify the antipyretic effect and mechanism of‘reducing fever six methods’for the use of two different types，three different age groups，and three different degrees of fever, as well as provide scientific evidence on the safety of using infant tuina to reduce fever.

前期研究主要结论如下：①小儿推拿退热安全、有效；②小儿推拿对两种类型（细菌和病毒）、三个年龄段（婴儿、幼儿、学龄前）、三种热度（低、中、高）的疗效亟待评价、机制亟须阐述。基于以上，实验分疗效评价和机制研究。在疗效评价中，拟采用退热六法对发热幼兔进行干预，通过血常规联合流式细胞术检测血细胞的形态学变化，行为学联合形态学作为疗效评价指标，评价退热六法的退热效果。在机制研究中，拟从外周至中枢的体温调节通路（血清→脑脊液→下丘脑），采用分子生物学技术检测外周TLR4/NF-κB信号通路关键指标（TLR4、NF-κB、IL-1β、IL-6、TNF-α、IFN等）、中枢POAH区正调节介质（PGE2、cAMP等）、腹中隔区负调节介质（AVP、α-MSH、β-EP等）的表达。对结果综合分析，得出退热六法对于两种致热类型、三个年龄段、三种热度的退热疗效及机制，为小儿推拿治疗发热提供科学依据。

背景：小儿推拿退热历史悠久、安全，但小儿推拿的疗效亟待评价、机制亟须阐述。.主要研究内容：选用新西兰兔为实验动物，退热六法（开天门、推坎宫、揉太阳、揉耳后高骨、清天河水、推脊）作为干预方式，以1月、2月、3月龄发热幼兔分别模拟临床婴儿期、幼儿期、学龄前期发热，以低热、中热、高热的发热幼兔分别模拟临床小儿的低热、中热、高热，评价小儿推拿对3个年龄段和3种发热程度的退热效果；机制研究通过检测血常规变化，通过分子实验ELISA、WB、RT-PCR检测外周血浆、肝、肺组织、中枢下丘脑TLR4/NF-κB信号通路关键指标及中枢正、负调节介质的表达，通过代谢组学检测血浆和下丘脑的代谢物，综合分析验证退热六法的外周和中枢机制。.重要结果及关键数据：①退热六法对1月龄幼兔降温幅度最大，2月龄降温最稳定；②对低热幼兔效果最明显；③能降低外周血中性粒细胞比率；④通过影响外周、中枢TLR4/NF-κB信号通路以及体温调节介质PGE2、cAMP、AVP、α-MSH、NT、β-EP发挥退热作用；⑤退热六法影响了外周血浆的主要代谢通路为：甘氨酸、丝氨酸和苏氨酸代谢、精氨酸和脯氨酸代谢、卟啉与叶绿素代谢、嘧啶代谢、初级胆汁酸生物合成以及氰基氨基酸代谢；⑥退热六法与调节中枢下丘脑 cGMP-PKG、MAPK、PI3K-Akt、HIF-1、cAMP等以代谢信号转导通路有关；与上调TRIM32、TRPV2、STAT5等蛋白表达，下调S100A4、VIM、SMAD2、NT5E等蛋白表达有关。.科学意义：明确小儿推拿退热的疗效及机制，为临床推拿退热提供基础实验数据支持，指导患儿家长参考患儿发热的年龄、热度，确定是否选择小儿推拿为孩子退热，为保证治疗发热在内的小儿推拿事业健康、有序地发展提供基础研究数据；为中医药事业的普及、应用做出贡献。