Doxycycline-Induced Apoptosis in S2-Infected HMC3 Cells via Calreticulin Suppression and Activation of the IRE1/Caspase-3 Signaling Pathway.

OBJECTIVE

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.

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 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 S2-infected HMC3 cells following Dox treatment.

CONCLUSION

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.