基于TLR4/8相关信号通路研究“补肾生髓”对艾滋病免疫重建的作用及机制

AIDS immune reconstruction is difficult in clinical treatment,and abnormal immune activation is one of the main causations.It has been reported that TLR4/8 mediated signal transduction pathway is associated with HIV/AIDS immune activation.Our previous study confirmed that Diwu Yanggan capsule,which reflects the treatment laws of "tonify kidney producing marrow",could promote the differentiation and proliferation of bone marrow stem cell.It could alsoregulate the expression of immune related cytokine and the immune function,thus effectively improve immune disorder caused by viral infection diseases.We now propose the hypothesis that the treatment laws of "tonify kidney producing marrow" may regulate immune status of specific immune cells to promote the immune reconstruction through affecting the TLR4/8 mediated signaling pathways.On the basis of validation of different population immune status, this project will utilize a combination of flow cytometry, cell culture and RT-PCR to study the effect of "tonify kidney producing marrow" containing serum on the immune response of peripheral blood mononuclear cells from AIDS immune reconstruction patients as well as its effect on monocytes-mediated T lymphocytes activation in molecular, cellular and protein levels.This project will clarify the mechanism by which "tonify kidney producing marrow" method could reconstruct the immune system of AIDS patients, and expand the application of Diwu Yanggan capsule to prevent and control major infectious diseases.

艾滋病免疫重建不全是临床治疗难点，异常的免疫激活是其主要致病机制之一，有研究表明TLR4/8及其信号转导通路与艾滋病的免疫激活机制相关。前期研究证实，体现“补肾生髓”治疗法则的地五养肝胶囊能够促进骨髓干细胞增殖分化，调节免疫相关细胞因子的表达和机体免疫功能，有效改善病毒感染相关性疾病导致的免疫紊乱状态。我们提出“补肾生髓”治疗法则可能通过影响TLR4/8及相关信号通路，调控免疫细胞的免疫状态，促进免疫重建的科学假说。项目应用流式细胞技术，细胞培养技术及RT-PCR技术，在明确不同人群免疫状态的基础上，从分子、细胞、蛋白水平研究“补肾生髓”法含药血清对艾滋病免疫重建不全患者外周血单核细胞免疫反应的调控及经单核细胞激活T淋巴细胞的效应。期望明确“补肾生髓”法重建艾滋病免疫功能的作用及其机制，扩展“补肾生髓”理论和地五养肝胶囊防治重大传染病的新应用。

艾滋病经HAART规范抗病毒治疗后仍有大约20%-30%的患者免疫功能重建不全，是新的科学难题。本项目按计划完成所有研究任务：（1）基于220例艾滋病重建不全患者基线和随访记录数据构建的线性模型，我们发现早期启动HAART和复方新诺明预防性治疗有助于CD4细胞计数恢复，BMI和血清肌酐可作为HAART后免疫重建预后的监测指标。（2）基于TLR4/8介导的相关信号通路探讨“补肾生髓”法对艾滋病HAART后免疫重建不全患者外周血单核细胞免疫反应的调控，研究表明免疫重建不全患者单核细胞TLR4、TLR8表达更高，通路下游细胞因子IL-2、TNF-α表达也呈现高于免疫重建良好患者趋势；且“补肾生髓”法组含药血清能下调艾滋病免疫重建不全患者单核细胞的TLR4、TLR8表达水平，并下调细胞因子IL-2、TNF-α表达水平。结合免疫重建不全患者异常的免疫激活状态，证实体现“补肾生髓”治疗法则的地五养肝胶囊能够通过下调艾滋病免疫重建不全患者外周血单核细胞的TLR4、TLR8的表达及相关信号通路，抑制免疫功能过度激活，达到恢复或重建AIDS患者免疫功能。（3）研究观察发现，艾滋病免疫重建不良患者予“补肾生髓”法的地五养肝胶囊治疗24周后，中药组CD4计数变化值（36.6±126.52）较安慰剂组（14.0±50.51）上升明显（p＜0.0001）；中药组CD4：CD8计数变化值（0.070±0.275）较安慰剂组（0.022±0.046）上升明显（p＜0.0001）；中药组中医证候积分变化值（-1.1±2.74）较安慰剂组（-0.5±1.79）降低明显（p＜0.0001）；中药组生存质量积分变化值（-7.2±4.07）较安慰剂组（-8.6±5.68）下降缓慢（p＜0.0001），这表明地五养肝胶囊联合HAART能更显著提高艾滋病免疫重建不全患者的CD4细胞计数及CD4：CD8计数比值，改善中医症候，抑制生存质量下降，且安全性好。（4）对地五养肝胶囊进行成分分析，共鉴别出210个化学成分，多糖含量为18.30%。本项目资助发表SCI 1篇，核心论文4篇；参与制定共识3部。团队获湖北省科技进步一等奖1项。