Study On The Mechanism Of Doxycycline Induces Brucella Suis S2 Strain-infected Human Microglia Apoptosis

Backgroud Neurobrucellosis is a life-threatening complication of brucellosis.Brucella replicates and proliferates in the endoplasmic reticulum(ER)of microglia,which is the cause of the patient’s persistent chronic infection.Intracellular bacteria can further inhibit the endoplasmic reticulum-related apoptosis of host cells by inhibiting Endoplasmic Reticulum stress(ERS).Therefore,induction of apoptosis in infected cells may limit the survival of bacteria.Doxycycline(Dox)is an antibiotic that induce cell apoptosis by activating ERS,but its underlying mechanism is unknown.Objectives First,Brucella suis S2 strain was used to infect Human Microglia Clone 3(HMC3)to clarify the mechanism of B.suis S2 inhibiting HMC3 cell apoptosis.On this basis,Dox was used to interfere with HMC3 cells,which were infected by B.suis S2 to further clarify the mechanism of Dox-induced apoptosis of HMC3 cells,in order to find new targets for the treatment of neurobrucellosis.Methods First,B.suis S2 was used to infect HMC3 cells at different MOIs for different times.Protein levels of p-IRE1,p-JNK,cleaved Caspase-12,p-p53 and cleaved Caspase-3 in HMC3 cells were evaluated using Western blot;And RT-q PCR was performed to measure the expression levels of IRE1,JNK,Caspase-12,p53 and Caspase-3 m RNA in HMC3 cells;Apoptosis of HMC3 cells were detected via flow cytometry methods.On this basis,we confirmed the significant downregulation in the levels of ubiquitination of calreticulin(CALR)by ubiquitin proteomics technology.Next,the CALR overexpression cell line HMC3-CALR and the CALR knockdown cell line HMC3-sh-CALR were successfully constructed through lentivirus-mediated technology.Then,each HMC3 cell line was infected with B.suis S2.Both the protein and m RNA levels of CALR and crucial molecules in the JNK/p53 or IRE1/Caspase-3 signaling pathway were respectively analyzed through western blot analysis and RT-q PCR;Morphology of ER was analyzed via ER staining;Immunofluorescence techniques were used to analyze p-JNK nuclear foci formation;And a flow cytometer was used to detect the levels of apoptosis.After this,we investigated the effects of Dox on the CALR,IRE1/Caspase-3 and JNK/p53 signaling pathways of the infected and uninfected HMC3 cells with the same methods described above.Results(1)B.suis S2 infected HMC3 cells at a MOI of 50 for 2 h could reduce the levels of p-IRE1,p-JNK,cleaved Caspase-12,p-p53 and cleaved Caspase-3 proteins.Meanwhile,the m RNA expression levels of IRE1,JNK,Caspase-12,p53 and Caspase-3 were remarkably decreased,while CALR m RNA and protein levels were measurably increased.And HMC3 cells apoptosis rate was decreased.(2)CALR reduced the protein levels of p-IRE1,p-JNK,cleaved Caspase-12,p-p53 and cleaved Caspase-3,and inhibited the nuclear transport of p-JNK protein,thereby reducing the apoptosis rate of HMC3 cells.(3)B.suis S2 further reduced the protein levels of p-IRE1,cleaved Caspase-12,cleaved Caspase-3,p-JNK and p-p53 by increasing the levels of CALR protein and m RNA.This bacterium also inhibited IRE1,Caspase-12,Caspase-3,JNK and p53 m RNA expression and the translocation of p-JNK to nuclear,thereby inhibiting HMC3 cell apoptosis.(4)The effects of Dox on the infected and uninfected HMC3 cells were contrary to the effect of B.suis S2 on HMC3 cells.Dox could induce the ER damage in both B.suis S2-infected and-uninfected HMC3 cells and increase the intracellular calcium ion concentration.And then Dox treatment increased the apoptosis rate of the infected and uninfected HMC3 cells.Conclusions(1)CALR reduced ERS and inhibited HMC3 cells apoptosis via inhibiting the levels of p-IRE1,cleaved Caspase-12,cleaved Caspase-3,p-JNK and p-p53 proteins.(2)B.suis S2 inhibited the IRE1/Caspase-3 and JNK/p53 signaling pathways and reduced the ERS to inhibit the apoptosis of HMC3 cells by increasing the level of CALR.(3)Dox activated the IRE1/Caspase-3 and JNK/p53 signaling pathways and promoted the ERS to induce the apoptosis of B.suis S2-infected or-uninfected HMC3 cells by decreasing the level of CALR.(4)CALR,IRE1/Caspase-3 and JNK/p53 signaling pathways may be considered as promising targets for drug therapy of neurobrucellosis.