Wang Zhao, Yang Juan, Zhang Deng-Er, Qiao Xia, Yang Shu-Long, Wang Zhen-Hai, Yang Qian
Department of Experimental Surgery, The Second Affiliated Hospital of Air Force Medical University, Xi'an, People's Republic of China.
Neurology Center, The General Hospital of Ningxia Medical University, Yinchuan, People's Republic of China.
Infect Drug Resist. 2025 Apr 23;18:2005-2020. doi: 10.2147/IDR.S507193. eCollection 2025.
This study aims to elucidate the apoptotic mechanism induced by doxycycline (Dox) in human microglial clone 3 (HMC3) cells infected with the S2 strain, with the goal of identifying potential therapeutic targets for neurobrucellosis.
The expression of calreticulin (CALR) at both the protein and mRNA levels was assessed using Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively, following exposure of HMC3 cells to varying concentrations and treatment durations of Dox. Apoptosis rates were determined via flow cytometry. To investigate the involvement of the inositol-requiring enzyme-1 (IRE1)/Caspase-12/Caspase-3 pathway, CALR protein levels were analyzed through Western blot after a 12-hour treatment with 160 μM Dox. Endoplasmic reticulum (ER) stress and intracellular calcium (Ca²⁺) concentrations were evaluated using fluorescent staining. The same parameters were measured in S2-infected HMC3 cells following treatment with 160 μM Dox.
Treatment with 160 μM Dox for 12 hours resulted in a reduction in CALR protein levels and the induction of apoptosis in HMC3 cells. The downregulation of CALR activated the IRE1/Caspase-12/Caspase-3 signaling pathway, leading to apoptosis. Similar apoptotic effects were observed in S2-infected HMC3 cells following Dox treatment.
Dox promotes apoptosis in S2-infected HMC3 cells by suppressing CALR expression and activating the IRE1/Caspase-12/Caspase-3 signaling pathway. These findings suggest that CALR regulation may serve as a potential therapeutic target for neurobrucellosis.
本研究旨在阐明强力霉素（Dox）在感染S2菌株的人小胶质细胞克隆3（HMC3）细胞中诱导的凋亡机制，以确定神经布氏杆菌病的潜在治疗靶点。
在HMC3细胞暴露于不同浓度和处理时间的Dox后，分别使用蛋白质印迹分析和逆转录定量聚合酶链反应（RT-qPCR）评估钙网蛋白（CALR）在蛋白质和mRNA水平的表达。通过流式细胞术测定凋亡率。为了研究肌醇需求酶1（IRE1）/半胱天冬酶-12/半胱天冬酶-3信号通路的参与情况，在用160μM Dox处理12小时后，通过蛋白质印迹分析CALR蛋白水平。使用荧光染色评估内质网（ER）应激和细胞内钙（Ca²⁺）浓度。在用160μM Dox处理后的S2感染的HMC3细胞中测量相同参数。
用160μM Dox处理12小时导致HMC3细胞中CALR蛋白水平降低并诱导凋亡。CALR的下调激活了IRE1/半胱天冬酶-12/半胱天冬酶-3信号通路，导致细胞凋亡。在Dox处理后的S2感染的HMC3细胞中观察到类似的凋亡作用。
Dox通过抑制CALR表达并激活IRE1/半胱天冬酶-12/半胱天冬酶-3信号通路促进S2感染的HMC3细胞凋亡。这些发现表明，CALR调节可能是神经布氏杆菌病的潜在治疗靶点。
