Zhao Wang,1 Juan Yang,2 Deng-Er Zhang,3 Xia Qiao,4 Shu-Long Yang,5 Zhen-Hai Wang,2,6 Qian Yang1 

1Department of Experimental Surgery, The Second Affiliated Hospital of Air Force Medical University, Xi’an, People’s Republic of China; 2Neurology Center, The General Hospital of Ningxia Medical University, Yinchuan, People’s Republic of China; 3The First Clinical Medical School, Ningxia Medical University, Yinchuan, People’s Republic of China; 4Institute of Medical Science, The General Hospital of Ningxia Medical University, Yinchuan, People’s Republic of China; 5Department of Orthopedics, The People’s Hospital of Wuhai, Wuhai, People’s Republic of China; 6Diagnosis and Treatment Engineering Technology Research Center of Nervous System Diseases of Ningxia Hui Autonomous Region, The General Hospital of Ningxia Medical University, Yinchuan, People’s Republic of China

Correspondence: Qian Yang, Department of Experimental Surgery, The Second Affiliated Hospital of Air Force Medical University, No. 569 of Xinsi Road, Baqiao District, Xi’an, 710032, People’s Republic of China, Tel +86029-84777007, Email yangqianyqq@126.com Zhen-Hai Wang, Neurology Center, The General Hospital of Ningxia Medical University, No. 804 of Shengli Street, Xingqing District, Yinchuan, 750003, People’s Republic of China, Tel +860951-6744025, Email wangzhenhaiwzh01@126.com

Objective: This study aims to elucidate the apoptotic mechanism induced by doxycycline (Dox) in human microglial clone 3 (HMC3) cells infected with the Brucella suis S2 strain, with the goal of identifying potential therapeutic targets for neurobrucellosis.
Methods: 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 B. suis S2-infected HMC3 cells following treatment with 160 μM Dox.
Results: 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 B. suis S2-infected HMC3 cells following Dox treatment.
Conclusion: Dox promotes apoptosis in B. suis 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.

Keywords: apoptosis, Brucella suis S2 strain, doxycycline, HMC3, IRE1/Caspase-12/Caspase-3 pathway