| [Objective] The prevention and treatment of tuberculosis is a huge challenge worldwide due to its long period of treatment cycle,high adverse reaction of the existing first-line anti-tuberculosis drugs clinically,the occurrence of increased multi-drug resistant tuberculosis,together with the severe inflammatory pathological lesions in the course of tuberculosis.Recently the development of anti-tuberculosis drugs proceeded slowly on account of merely targeting on Mycobacterium tuberculosis(Mtb).Therefore,it is indispensable to develop novel anti-tuberculosis drugs and explore combined therapy method.Based on this,the host-directed therapy(HDT),an adjuvant therapy for eliminating Mycobacterium tuberculosis,treating or alleviating tuberculosis by regulating the immune function to enhance the antibacterial ability of the host becomes a novel effective strategy,which has gradually been applied in vivo and in vitro studies.Traditional Chinese medicine focuses on the overall adjustment of the human body,which coincides with the concept of HDT.The anti-TB efficacy of the clinical decomposed prescription “Qinbudan” is relatively excellent,but the research on its mechanism has been rarely reported.Our research group found that the anti-TB efficacy of Salvia miltiorrhiza was prominent on account of the previous analysis and study of the decomposed recipes of “Qinbudan”.Tanshinone ⅡA(Tan)is one of the main active ingredients of Salvia miltiorrhiza,and this study was designed to evaluate the anti-TB effect of Tan on macrophages infected with Mycobacterium tuberculosis and explore its molecular regulatory mechanism to provide the experimental foundation for “Qinbudan” and further study on its regulatory mechanism.[Method](1)Western blot and lactate dehydrogenase(LDH)activity assays were used to detect the effects of Tan on cell pyroptosis of macrophages infected with Mycobacterium tuberculosis;(2)Western blot was applied to observe the effect of Tan on the activation of NLRP3 inflammasome in Mtb-infected Raw264.7 cells and THP-1cells simultaneously;(3)Western blot,Co-immunoprecipitation and Immunofluorescence assays were used to observe the effect of Tan on the expression level of TXNIP in Mtb-infected Raw264.7 cells and THP-1 cells simultaneously.(4)Western blot and Immunofluorescence assays were applied to explore the effect of Tan on mitochondrial damage and mt ROS in Mtb-infected macrophages;(5)Western blot and agarose gel electrophoresis were adopted to observe the effect of Tan on endoplasmic reticulum stress in Mtb-infected macrophages;(6)The si RNA technique was applied to knockdown the expression levels of PERK/pe IF2α,IRE1α and ATF6 which were the endoplasmic reticulum stress pathway proteins,and Western blot assay was employed to explore the NLRP3 inflammasome activation and possible molecular regulation mechanism of Tan to identify its therapeutic target.[Results] The results of Western blot assay for detecting the marker protein of pyroptosis showed that Tan significantly reduced the activation of GSDMD-N,and the results of LDH activity assay indicated that Tan significantly reduced the release of LDH in Mtb-infected macrophages;(2)The results of Western blot assay showed that Tan could significantly suppress the expression levels of intracellular NLRP3,pro-IL-1βand IL-1β in supernatant in Mtb-infected macrophages;(3)Western blot results showed that Tan significantly inhibited the expression of TXNIP in Mtb-infected infected macrophages in a concentration-dependent and time-dependent manner,Co-immunoprecipitation assay results showed that Tan inhibited the interaction between TXNIP and NLRP3 protein,Immunofluorescence assay results showed that Tan oppressed the co-localization of TXNIP and NLRP3;(4)Immunofluorescence assay results displayed that Tan significantly reatrained the production of mt ROS.Western blot results revealed that Tan significantly suppressed the expression levels of PINK1,Parkin and Cyto C.Immunofluorescence assay results indicated that Tan significantly inhibited the co-localization of Mitotracker with NLRP3 and Mitotracker with TXNIP in Mtb-infected Raw264.7 cells;(5)The results of Western blot assay showed that Tan significantly inhibited the expression levels of Bip,CHOP,p IRE1α,pe IF2α and ATF6 of endoplasmic reticulum stress-related proteins.The agarose gel electrophoresis detection indicated that XBP1 m RNA spliced in Mtb-infected Raw264.7 cells,but Tan had no obvious regulatory effect on it.The results of Western blot assay showed that Tan significantly inhibited the protein expression levels of p JNK,pp38,pp65 and NLRP3 which belonged to the downstream of the IRE1α pathway of endoplasmic reticulum stress;(6)Western blot results showed that the protein expressions of PERK/pe IF2α,IRE1α and ATF6 decreased after interferenced,which indicated the knockdown was effective.Western blot assay results displayed that the expression of mature IL-1β reduced in si PERK/pe IF2α group compared with Mtb group,meanwhile,the expression of caspase-1(p20)and HMGB1 showed the similar tendency.Whereas the expression of mature IL-1β remained basically unchanged in si IRE1α group compared with Mtb group,and the expression of caspase-1(p20)and HMGB1 decreased in si IRE1α group compared with Mtb group.For another knockdown protein(si ATF6 group),the changes among IL-1β,caspase-1(p20)and HMGB1 did not show obvious decreasing trend compared with Mtb group.Whereas the combination of Tan with si PERK/si IRE1α/si ATF6 could decrease the protein expression levels of IL-1β,caspase-1(p20)and HMGB1.[Conclusion] The present study shows that Tan targets on endoplasmic reticulum stress(ERS)to inhibit activation of NLRP3 inflammasome as well as subsequent cell pyroptosis to exert the anti-inflammatory effect in Mycobacterium tuberculosis infected macrophages.The overall regulation of Tan on the inflammatory response of macrophages infected by Mycobacterium tuberculosis provides a strong experimental basis for the clinical application. |
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