从cAMP-Rap2B-PLCε-IP3R/Ca2+信号通路介导的B细胞自噬途径探讨纳米雄黄治疗SLE的分子机制

Clinically, there is a lack of ideal drugs to treat SLE, so it is necessary to explore the treatment of SLE.The latest study found that B cells in SLE autophagy increases, while the cAMP - Rap2B - PLCε - IP3R/Ca2+ signaling pathways can regulate autophagy B cells, and to the key proteins of autophagy and its related signaling pathways for the targets for new drug research is one of the current hot spot.By previous research, we find that nano realgar can rise through called simply MRL/LPR mice B fine Ca2+ concentration to increase its apoptosis, and can reduce the production of autoantibodies, such as ds-DNA by reducing its surface expression of CD40 and its activation, thus and play a role in the treatment of SLE.However, it is not clear that the effect of B cell autophagy and related signaling pathways is closely related to the hyperactivation of B cells.On this basis, we put forward the following scientific questions: Can realgar nanoparticles reduce the level of B cells autophagy in SLE?Can realgar nanoparticles inhibit the excessive autophagy of B cells by activating cAMP - Rap2B - PLCε - IP3R/Ca2+ signaling pathways , so as to play a role in the treatment of SLE.In this study, the effects of nano-realgar on the autophagy of B cells and the expression of the related proteins in the cAMP-Rap2B-PLCε-IP3R/Ca2+ signaling pathway were investigated in order to answer the above scientific questions.Thus, the molecular pharmacological mechanism of nano-realgar in the treatment of SLE is revealed, which provides a new experimental basis for its clinical application.

临床上尚缺乏较为理想的治疗SLE的药物，因此探索治疗SLE的药物成为必然。最新的研究发现:SLE中B细胞自噬增加直接导致B细胞过度增殖分化而cAMP-Rap2B-PLCε-IP3R/Ca2+信号通路能调控B细胞自噬。我们前期的研究发现：纳米雄黄能通过升高MRL/lpr小鼠B细内Ca2+浓度增加其凋亡，从而减少抗ds-DNA等自身抗体的产生，而起到治疗SLE的作用。但其对与B细胞过度增殖分化密切相关的B细胞自噬及相关的信号通路的影响尚不清楚。因此我们提出以下科学问题：纳米雄黄能否通过激活cAMP-Rap2B-PLCε-IP3R/Ca2+信号通路而减少B细胞自噬水平，从而抑制B细胞过度增殖分化而起到治疗SLE的作用。本实验研究拟通过观察纳米雄黄对MRL/lpr小鼠B细胞自噬及对cAMP-Rap2B-PLCε-IP3R信号通路中相关蛋白表达的影响，以揭示其机制，为其临床应为提供新的依据。

目的：探索纳米雄黄对MＲL/lpr小鼠B细胞的增值\自噬及其潜在的分子机制。方法: 分别于饲养16周后,断髓处死各组小鼠,采集脾脏,分离纯化并培养B淋巴细胞。采用透射电镜直接观察B淋巴细胞自噬体;采用GFP-LC3融合蛋白在荧光显微镜下示踪自噬形成;利用Western Blot检测LC3-II/I比值的变化以评价B淋巴细胞自噬形成;采用荧光定量PCR测定B细胞中cAMP、Rap2B、IP3R、Atg5mRNA表达丰度;采用Western blot检测B细胞中cAMP、Rap2B、IP3R、Atg5表达水平。结果: 获得的B细胞纯度高达95%。与模型组比较,纳米雄黄高、中、低三个剂量治疗组、水飞雄黄组各组B细胞中自噬体数低于模型对照组；B细胞中GFP-LC3融合蛋白荧光强度低于模型对照组P<0.05；B细胞中LC3-II/I比值低于模型对照组P<0.05；与模型组比较，纳米雄黄中、低二个剂量治疗组、水飞雄黄组各组B细胞中 cAMP、Rap2B、IP3RmRNA表达丰度升高而Atg5mRNA表达丰度降低P<0.05；与模型组比较，纳米雄黄中、低二个剂量治疗组、水飞雄黄组各组B细胞中cAMP、Rap2B、IP3R表达水平升高而Atg5的表达水平降低P<0.05。结论：纳米雄黄能够通过激活cAMP-Rap2B-PLCε-IP3R信号通路而升高细胞内Ca2+浓度而降低ATG5的水平，从而减少SLE中B细胞自噬水平，进而抑制B细胞的过度增殖分化而起到治疗SLE的作用，为其治疗SLE的临床应用提供新的理论依据。