Tauroursodeoxycholic Acid Facilitates Burkholderia Pseudomallei Clearance By Inhibiting Endoplasmic Reticulum Stress-induced Apoptosis

Burkholderia pseudomallei,a kind of gram-negative opportunistic bacterium,is the causative agent of melioidosis.As a tropical medical disease,melioidosis is widely found in Southeast Asia and northern Australia,as well as in Hainan,Taiwan and so on.The clinical presentation of melioidosis are various,including local organ abscess,pneumonia,acute septicemia.There are about 89000 people die every year in the world,and the mortality rate is as high as 40%.The US Centers for Disease Control and Prevention(CDC)have classified B.pseudomallei as tier 1 select agent due to its potential biothreat.Currently,no effective vaccine is available,and the drug resistance is a major problem in the treatment of melioidosis.Therefore,finding new clinical treatment strategies in melioidosis is extremely urgent.The pathogen-host interaction is the premise of the infection and pathogenesis.Further revealing the pathogenic mechanism of B.pseudomallei will provide new targets and theoretical guidance for its prevention and control.Our research group has been committed to the study of the immune mechanism of the infection of B.pseudomallei for a long time.In previous study,we observed that B.pseudomallei can induce endoplasmic reticulum stress(ERS)and apoptosis.Its biological significance and molecular mechanism naturally aroused our interest,and became the subject of this paper.The results showed that Shigella flexneri,Salmonella typhimurium,Yersinia enterocolitica and other bacteria can promote macrophage apoptosis,protect bacteria from phagocytosis and promote bacterial survival,suggesting that inhibition of macrophage apoptosis may be beneficial to the clearance of intracellular bacteria.It has been reported that B.pseudomallei can induce the expression of apoptosis gene in mouse macrophages.Previous studies found that B.pseudomallei can induce ERS and apoptosis.Is it possible to promote the clearance of B.pseudomallei by inhibiting ER stress-induced apoptosis? Tauroursodeoxycholic acid(TUDCA)is a hydrophilic bile acid that is normally produced endogenously in humans in the liver,by conjugation of taurine to ursodeoxycholic acid(UDCA).Recent studies have revealed that TUDCA,as an ERS inhibitor,protects cells by improving the ability of cells to process unfolded proteins and reducing apoptosis.TUDCA is a drug that has been used clinically in hepatobiliary diseases,and has an ameliorating effect on several diseases,including influenza A,chronic hepatitis C,osteoarthritis,kidney disease,diabetes and so on.Therefore,this study observes the clearance effect of TUDCA on B.pseudomallei in vitro and in vivo by B.pseudomallei infected C57BJ/6 mice model and RAW264.7 cell model.It is preliminarily revealed that the mechanism of TUDCA by inhibiting ERS to reduce B.pseudomallei-induced apoptosis using transmission electron microscopy,laser scanning microscope,RT-q PCR,flow cytometry,western blot and si RNA interference.First,we studied in melioidosis mice,and the survival rate of TUDCA-treated group was increased,while the bacterial burdens and inflammation in liver,spleen and lung were significantly decreased.And the intracellular survival of B.pseudomallei in TUDCA-treated group was reduced in vitro.These data suggest that TUDCA facilitates the clearance of B.pseudomallei.Then we analyzed the molecular process of apoptosis induced by B.pseudomallei through ERS in RAW264.7 cell model.It has been reported that apoptosis is related to bacterial clearance.In this study,the proportion of apoptotic cells increased in a time?dependent manner after B.pseudomallei infection in RAW264.7 cells by flow cytometry.And the western blot result indicated that B.pseudomallei infection triggers the expression of caspase?3 and cleaved caspase-3.Next,we explored the relationship between ER stress and B.pseudomallei because TUDCA plays an important role as an ER stress inhibitor in many diseases.We firstly examined the morphologic alteration of ER.The TEM figures showed that ER was obviously swelled in the B.pseudomallei infected group.Meanwhile,calreticulin was dispersive after B.pseudomallei infection.Taken together,these results suggested the shape-structure of ER was changed in B.pseudomallei infected RAW264.7 cells.Secondly,we further verified the relationship between ERS and B.pseudomallei.The results showed that the m RNA and protein expression levels of Bip,CHOP,PERK and XBP1 were consistently increased;whereas ATF6 was decreased by western blot and RT-q PCR.Collectively,the results indicated that B.pseudomallei infection could induce ER stress in RAW264.7 cells.If the ERS is persistent,an apoptotic pathway is triggered.B.pseudomallei infection resulted in the increase of caspase-12 in a time?dependent manner.Moreover,CHOP si RNA group significantly decreased the cell apoptosis.Meanwhile,the CHOP knockdown decreased the protein expression levels of caspase?3,cleaved caspase-3 and caspase-12.Taken together,these results indicate that B.pseudomallei-induced apoptosis is dependent on ERS in RAW264.7 cells.At last,we detected the apoptosis and ERS after TUDCA treatment in RAW264.7 cells.The results of flow cytometry indicated that the proportion of apoptotic B.pseudomallei-induced cells was markedly reduced in the TUDCA treatment group.On the morphology of ER,TUDCA treatment attenuated the swelling of ER,and lessened the dispersion of calreticulin.Furthermore,the m RNA and protein expression levels of Bip,CHOP,PERK and XBP1 were reduced,and ATF6 was increased subsequent to the TUDCA treatment.The results suggest that TUDCA reduce ERS induced by B.pseudomallei in RAW264.7 cells.And the protein expression levels of caspase-3,cleaved caspase-3 and caspase-12 were reduced in the TUDCA treatment group.These results suggest that TUDCA reduce the B.pseudomallei-induced apoptosis by inhibiting ERS.In addition,there was no significant difference in the intracellular survival of B.pseudomallei in RAW264.7 cells between the TUDCA treatment group and PBS control group after CHOP knockdown.The result further indicated that TUDCA promoted B.pseudomallei clearance by inhibiting ERSinduced apoptosis.Briefly,the role and mechanism of TUDCA in the treatment of B.pseudomallei infection were discussed from the perspective of ERS in this study.It was preliminarily elucidated that TUDCA may promote B.pseudomallei clearance by inhibiting ER stress-induced apoptosis.These insights revealed the relationship between the ERS and B.pseudomallei and may provide an alternative treatment for melioidosis.