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

Abstract

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: Brucella suis S2 strain; HMC3; IRE1/Caspase-12/Caspase-3 pathway; apoptosis; doxycycline.

Figure 1

Effects of varying Dox treatment durations on CALR expression and HMC3 cell apoptosis. (A) Relative CALR mRNA expression in each group. (B) Flow cytometry analysis of apoptosis in each group. (C) Apoptosis rate (percentage of cells in Q2 and Q3) in each group. Results are presented as mean ± standard deviation.

Figure 2

Effects of varying Dox concentrations on apoptosis in HMC3 cells and CALR expression. (A) RT-qPCR analysis of relative CALR mRNA expression in each group. (B) Apoptotic rate (percentage of cells in Q2 and Q3) in each group. (C) Flow cytometry analysis of apoptosis in different groups. Results are presented as means ± standard deviation.

Figure 3

Dox regulates intracellular Ca²⁺ levels and CALR protein expression. (A) Western blot analysis of CALR protein expression in each group. (B and C) Quantification of CALR mRNA and CALR protein levels. (D) ER tracker fluorescent dye staining of the ER in each group. (E) Relative fluorescence intensity of intracellular Ca²⁺ in each group. Results are presented as mean ± standard deviation. Experiments were performed three times independently.

Figure 4

Dox induces apoptosis in HMC3 cells through activation of the IRE1/Caspase-12/Caspase-3 signaling pathway. (A) Western blot analysis of key proteins in the IRE1/Caspase-12/Caspase-3 signaling pathway. (B–E) Relative protein expression levels of p-IRE1, cleaved Caspase-12, cleaved Caspase-9, and cleaved Caspase-3. (F) Flow cytometry analysis of apoptosis in different groups. (G) Statistical analysis of the apoptosis rate in each group. Results are presented as mean ± standard deviation. Experiments were performed three times independently.

Figure 5

Dox regulates intracellular Ca²⁺ levels and CALR protein expression in B. suis S2-infected HMC3 cells. (A) Western blot analysis of relative CALR protein expression. (B) Densitometric analysis of CALR protein levels. (C) RT-qPCR analysis of CALR mRNA expression. (D) ER fluorescent staining detected by laser confocal microscopy. (E) Relative fluorescence intensity of intracellular Ca²⁺ in each group. Results are presented as mean ± standard deviation of three independent experiments.

Figure 6

Dox induces apoptosis in B. suis S2-infected HMC3 cells through activation of the IRE1/Caspase-12/Caspase-3 signaling pathway. (A) Western blot analysis of key proteins in the IRE1/Caspase-12/Caspase-3 signaling pathway. (B–E) Quantification of p-IRE1, cleaved Caspase-12, cleaved Caspase-9, and cleaved Caspase-3 protein levels. (F) Flow cytometry analysis of apoptosis in different groups. (G) Percentage of apoptotic cells in each group. (H) Colony formation after culturing cell lysates for 3 days (lysates diluted 100-fold). (I) Statistical analysis of intracellular bacterial counts in each group. Results are presented as mean ± standard deviation.