PI3K/Akt和JAK/STAT3信号通路在弓形虫排泄分泌抗原抑制巨噬细胞凋亡中的作用

Toxoplasma gondii can inhibit apoptosis and release excretory/secretory antigens (ESAs) during invasion into host cells. To date in vitro cell infection models have been mostly used to study T. gondii-induced antiapoptosis. However, whether ESAs inhibit host cell apoptosis and its antiapoptotic pathways remain unknown. In previous study, we found that ESAs can induce the phosphorylation of STAT3 and Akt in macrophage cells, followed by overexpression of antiapoptotic protein Mcl-1 and downexpression of proapoptotic proteins. The above results indicate that ESAs have antiapoptotic effect, but the regulatory mechanism is unclear. We speculate that ESAs activate PI3K/Akt and JAK/STAT3 signal pathways, and then upregulate antiapoptotic protein Mcl-1 and downregulate pro-apoptotic proteins in Bcl-2 family, thus inhibiting staurousporine-induced macrophage apoptosis. This project intends to establish stably transfected Raw264.7 cell lines with overexpression of ESAs (MIC3, MIC13, GRA7 and GRA8). The goal is to investigate the role of PI3K/Akt and JAK/STAT3 signal pathways in the inhibition of macrophage apoptosis induced by ESAs and to screen the ESAs with optimal antiapoptotic effect. Our results will not only help uncover the pathogenesis of toxoplasmosis, but also lay a solid foundation for the development of T. gondii vaccines or antiapoptotic drugs.

弓形虫入侵宿主细胞过程中释放排泄分泌抗原（ESA）并抑制细胞凋亡。弓形虫抗凋亡研究多采用体外细胞感染模型，但ESA是否抑制宿主细胞凋亡及其抗凋亡途径目前还不明确。我们发现ESA混合抗原可使巨噬细胞中STAT3及Akt磷酸化，抗凋亡蛋白Mcl-1表达增加，促凋亡蛋白表达减少，具有抗凋亡作用，但调控机制不明。我们提出了"弓形虫ESA可激活PI3K/Akt和JAK/STAT3信号通路，诱导Bcl-2家族中抗凋亡蛋白Mcl-1表达上调并抑制促凋亡蛋白表达，进而抑制星形胞菌素诱导细胞凋亡"的假说。本项目拟建立过表达ESA（MIC3、MIC13、GRA7、GRA8）的稳定转染Raw264.7巨噬细胞株，探究PI3K/Akt和JAK/STAT3信号通路在ESA抑制巨噬细胞凋亡中的作用，并筛选出抗凋亡作用最强的ESA。研究结果不仅有助于阐明弓形虫病发病机制，而且为弓形虫疫苗或抗凋亡药物研制打下坚实基础。

刚地弓形虫（Toxoplasma gondii）是一种专性细胞内寄生性原虫，可引起人兽共患的弓形虫病，尤其对孕妇、免疫抑制和免疫缺陷患者的影响更为严重。近年来弓形虫病发病率有上升趋势，已成为严重的公共卫生问题。弓形虫入侵宿主细胞过程中释放排泄分泌抗原（ESA）并抑制细胞凋亡。弓形虫抗凋亡研究多采用体外细胞感染模型，但ESA是否抑制宿主细胞凋亡及其抗凋亡途径尚未明确。本研究中我们成功构建ESA（MIC3、MIC13、GRA7、GRA8）基因重组表达质粒，建立了稳定表达弓形虫ESA的Raw264.7细胞系。利用星形孢菌素诱导的Raw264.7巨噬细胞凋亡模型，检测弓形虫ESA对细胞凋亡模型的细胞因子、Bcl-2家族蛋白表达以及PI3K/Akt与JAK/STAT3信号通路的影响，结果显示弓形虫ESA一方面激活磷酸化AKT并上调Mcl-1蛋白表达，另一方面活化STAT3并使活化的pSTAT3从胞浆转移至胞核，明确STAT3活化在弓形虫ESA抑制星形孢菌素诱导的Raw264.7细胞凋亡中起关键作用。此外，研究还发现PI3K/Akt为JAK/STAT3的上游信号通路，且弓形虫ESA通过激活STAT3/Mcl-1信号通路抑制星形孢菌素诱导的Raw264.7细胞凋亡。最后，我们确认弓形虫ESA中MIC13和GRA7抑制细胞凋亡效果最显著，明确了MIC13和GRA7在PI3K/Akt和JAK/STAT3/Mcl-1信号通路中的调节作用。研究结果为揭示弓形虫入侵宿主细胞过程中抑制巨噬细胞凋亡机制奠定了坚实的基础，并为弓形虫病防治及抗凋亡药物研制提供理论借鉴。