Study On The Mechanism Of Endoplasmic Reticulum Stress-mediated Autophagy And Apoptosis During BCG Infection Of Macrophages

Objective:This experiment was conducted to investigate the activation of endoplasmic reticulum stress pathway during tuberculosis infection.To explore the escape mechanism of Mycobacterium tuberculosis based on endoplasmic reticulum stress,and to further reveal the innate immune response of macrophages and the intracellular survival mechanism of mycobacterium tuberculosis.Method:（1）Bioinformatics screening of differentially expressed genes in tuberculosis patients and identification of critical pathways,logging into the GEO online database to download the appropriate m RNA data set of blood samples of tuberculosis patients,Screening of differentially expressed proteins by bioinformatics analysis using volcano maps and heat maps to cluster and visualize differentially expressed genes,and then constructing protein interaction networks.The key biological processes and signaling pathways were identified by GO and KEGG enrichment analysis.（2）To explore the regulation rule of endoplasmic reticulum stress of macrophages after infection with Bacillus Calmette-Guerin（BCG）,Mouse macrophages were infected with RAW264.7 with BCG in vitro,extract total RNA and protein from the cells.Quantitative Real-time PCR（q RT-PCR）and Western Blot were used to detect the activation of key genes of endoplasmic reticulum stress pathway（HSPA5,PERK,P-PERK,ERN1,P-ERN1）.The fluctuation of calcium ions in endoplasmic reticulum was observed by cell immunofluorescence and flow cytometry.And to explore whether BCG activates ERS by Ca²⁺overload through drug intervention experiments.（3）Investigate the activation of apoptosis after BCG infection.Infect RAW264.7 cells with BCG and collect total RNA and protein for 6h,12h,24h and 36h.The ERS signaling pathway related apoptotic genes（Caspase-12 and CHOP）were quantitatively measured by q RT-PCR and Western Blot.The rate of apoptosis was detected by flow cytometry.Meanwhile,the optimal working concentration of PERK pathway blocker salurinal in RAW264.Seven cells were explored.CCK8 was used to evaluate drug toxicity,and Western blot was used to observe the blocking efficiency of PERK pathway（CHOP）at different concentrations of drugs,and the appropriate drug concentration was selected for follow-up experiments.Objective:to investigate the effect of blocking Perk pathway on apoptosis.autophagy and intracellular viable bacteria count in BCG infected RAW264.7 cells.（4）Investigate the activation of autophagy after BCG infection.Total RNA and protein were collected at 3h,6h,12h,24 h after BCG infection in RAW264.7 cells.The m RNA expressions of BECN1 and m TOR were detected by q RT-PCR.The autophagy gene（LC3）protein was quantitatively measured by Western Blot,and the fluorescent spot formation of LC3 was observed by immunofluorescence.At the same time,the optimal working concentration of ERN1 pathway blocker 4μ8C was explored for RAW264.7 cells.CCK8 was used to evaluate drug toxicity,and Western blot was used to observe the blocking efficiency of ERN1 pathway（p-ERN1）in different drug concentrations.The appropriate drug concentration was selected for follow-up experiments.Objective:to investigate the effect of 4μ8C blocking ERN1 pathway on apoptosis.autophagy and intracellular viable bacteria count in RAW264.7 cells were infected with BCG.Results:（1）GSE19435 and GSE83456 were selected as target gene chips.A total of 994 key genes with statistical differences in expression were screened out,and the TOP protein interaction network of key differentially expressed genes was screened out.Meanwhile,Concentration analysis showed that differential genes were mainly involved in antibacterial activity.antiviral,ribosome synthesis,protein synthesis and stress response.（2）m RNA and protein expression levels of ERS related genes（HSPA5,PERK,P-perk,ERN1,p-ERN1）were significantly increased after BCG infection with RAW264.7 cells（P<0.001）,and reached the peak at 12h.Ca²⁺overload in the endoplasmic reticulum reached a peak concentration at 6h.TG treatment effectively eliminated Ca²⁺accumulation in the endoplasmic reticulum,decreased the expression of ERS marker HSPA5（P<0.001）,Decrease the activity of intracellular ERS.（3）During BCG infection,m RNA expression of ERS-related apoptosis genes（Caspase-12 and CHOP）in macrophages was significantly up-regulated（P<0.001）.The protein expression of Caspase-12 gene was also significantly increased（P<0.001）,Significantly increase the apoptosis rate of host cells.At the same time,40u M was selected as the optimal concentration of salurinal,which significantly decreased the expression of the downstream protein of PERK pathway CHOP（P<0.001）.During the period when RAW264 was infected with BCG.7 cells after salurinal treatment,salurinal effectively inhibited the expression of apoptotic gene（Caspase-12）（P<0.001）,but did not affect the expression of autophagy protein（LC3）,promoting the number of BCG survival in macrophages.（4）During BCG infection,the m RNA expression of autophagy gene BECN1 was significantly up-regulated（P<0.001）;Meanwhile,the m RNA expression of anti-autophagy gene（MTOR）was significantly down-regulated（P<0.001）.The expression of autophagy protein LC3 was also significantly increased（P<0.001）.Host cell autophagy fluorescence spots increased significantly;At the same time,10u M was selected as the optimal concentration of 4μ8C,which significantly inhibited the activation of ERN1 pathway（P<0.05）.In RAW264.7 cells after BCG infection with 4μ8C,4μ8C effectively inhibited the expression of autophagy gene LC3（P<0.01）,and slightly promoted the expression of apoptotic gene Caspase-12（P<0.05）,and increased the number of BCG survival in macrophages.Conclusion:BCG activates ERS by inducing ER Ca²⁺overload,and UPR-PERK signaling pathway is involved in the initiation of apoptosis,while UPR-ERN1 signaling pathway is mainly involved in the initiation of autophagy and can slightly inhibit the initiation of apoptosis.Both PERK and ERN1 signaling pathways play important regulatory roles in reducing the survival and reproduction of BCG in the cell...