健脾益气法对HIVgp120损伤肠道黏膜屏障的干预作用研究

Excessive activation of the immune system is an important characteristic of HIV/AIDS, and it’s also closely related to the disease progression of HIV/AIDS. There are a great quantity of HIV copies in intestinal mucosa , they could damage the intestinal mucosal epithelial barrier and cause the Intestinal microbial translocation, which is an important reason for abnormal activation of systemic immune system. The theory above is just coincide with our understanding of HIV/AIDS etiology and pathogenesis of "plague virus invasion, spleen-stomach injuried ".We use the Invigorating the Spleen and Replenishing Qi prescription in the treatment of HIV/AIDS,and has achieved very good clinical results : regulating the immune activation state, improving the immune function and reducing the incidence of opportunistic infections, while the specific mechanism is unclear up to now. Then “protect intestinal mucosal barrier from damage, keep the normal function of the intestinal mucosal barrier, and prevent the occurrence of excessive immune activation” is whether or not related to treatment effect of Invigorating the Spleen and Replenishing Qi prescription on HIV/AIDS? Based on our preliminary works,here we will establish the Caco-2 intestinal mucosa barrier model in vitro by Transwell technology, and induce the model by HIV gp120 to simulate the pathological process of intestinal mucosa damage by HIV in vivo,then explore the protection and intervention mechanism of Invigorating the Spleen and Replenishing Qi prescription by observing changes of permeability and integrity and detecting expression of tight junction proteins of Caco-2 intestinal mucosa barrier model ,at the same time preliminary reveal the modern medicine essence of "plague virus invasion, spleen-stomach injuried ".

免疫系统过度激活是艾滋病的重要特征，与艾滋病的疾病进展密切相关。研究表明，HIV在肠粘膜大量复制，损伤肠粘膜上皮屏障，导致肠道微生物易位，是全身免疫系统异常激活的重要原因，这与本研究团队提出的"疫毒内侵，内伤脾胃"艾滋病中医病因病机学说非常吻合。临床上运用健脾益气方治疗艾滋病取得了较好疗效，其可改善机体免疫激活状态，降低机会性感染的发生，但具体机制尚不明确。那么健脾益气方防治艾滋病的机制是否与抑制肠粘膜屏障损伤、保护肠粘膜屏障完整功能，进而防止免疫过度激活的发生有关呢？本项目以前期研究为基础，采用Transwell 技术建立肠道黏膜体外屏障模型，利用HIV gp120诱导此模型来模拟体内HIV损伤肠粘膜的病理过程，通过通透性、完整性及紧密连接结构及蛋白的改变探讨健脾益气方干预HIV gp120损伤肠粘膜体外屏障的作用及其机制，同时初步明确艾滋病中医“疫毒内侵，内伤脾胃”观点的现代医学本质。

本项目基于黏膜屏障损伤在艾滋病疾病进展中的重要作用和健脾益气方的前期研究应用基础，构建艾滋病肠粘膜损伤体外模型，通过通透性、完整性及紧密连接结构及蛋白的改变探讨健脾益气方干预肠粘膜体外屏障损伤的作用及其机制，验证“健脾益气方防治艾滋病的机制是否与抑制肠粘膜屏障损伤、保护肠粘膜屏障完整功能，进而防止免疫过度激活的发生相关”假说。首先，成功构建易复制、稳定的艾滋病肠粘膜损伤体外研究模型：联合Transwell 技术与Caco-2 细胞培养构建肠粘膜体外屏障，用 HIV gp120 进行诱导，跨膜电阻抗测量、标志物渗漏实验等均证实HIV gp120 诱导后，Caco-2 细胞形成的肠粘膜屏障通透性增加，电镜观察发现HIV gp120对肠粘膜紧密连接结构的完整性有明显的破坏作用，这证实HIV gp120 体外诱导能导致肠粘膜屏障损伤，HIV gp120诱导肠粘膜屏障损伤模型可以作为艾滋病肠粘膜损伤研究的体外平台。第二，初步明确健脾益气方对HIV gp120诱导的肠粘膜损伤的保护作用及其作用机制：健脾益气方含药血清干预后，损伤的肠粘膜屏障通透性和完整性均明显改善，紧密连接结构修复，细胞凋亡减少，紧密连接相关蛋白ZO-1、Claudin-1、Claudin-5、Occludin蛋白及mRNA水平的表达均明显增加，关键蛋白ZO-1及F-actin在细胞膜的分布和表达趋向正常，这些均提示，健脾益气方能改善HIV gp120引起的肠粘膜屏障的通透性增加，维持肠粘膜屏障的完整性，从而保护肠粘膜屏障免受HIV gp120损伤，其保护肠粘膜的机制可能与抑制肠粘膜上皮细胞凋亡、提高紧密连接蛋白ZO-1、Claudin-1、Claudin-5、Occludin表达、维持肠粘膜细胞之间正常紧密连接结构有关。最后，丰富艾滋病中医“疫毒入侵，脾胃内伤”病因病机理论及健脾益气治法的现代科学内涵：脾胃为后天之本，三焦之枢，艾滋病发病及疾病进程与“疫毒入侵，脾胃内伤”密切相关。肠道是中医脾胃系统的重要组成部分，也是HIV在人体复制的重要场所，因此对肠粘膜屏障的保护作用及其机制的揭示，进一步证实了健脾益气治法的科学性及“疫毒入侵，脾胃内伤”病因病机理论的客观性。