Pathogenesis of pulmonary Mycobacterium bovis infection: basic principles established by the rabbit model

Abstract

Tanshinone IIA (Tan), extracted from Salvia miltiorrhiza Bunge, is a perennial herbal plant widely used as a folk remedy in Asian countries. Several studies have proved that Tanshinone IIA possesses many biological activities, such as anti-inflammatory, free-radical scavenging abilities, antioxidant properties, liver protection, and anti-cancer properties.Aim of the study: The objective of the present study was to examine the anti-inflammatory effects of Tan.The in vitro infection model of Mycobacterium tuberculosis-infected macrophages with the H37Ra strain was established. Murine macrophage Raw 264.7 and human monocyte THP-1 were used for the experiments. Cell viability was determined by the MTT assay. Western blot and lactate dehydrogenase (LDH) activity assays were used to detect the effects of Tan on cell pyroptosis and the level of NLRP3 inflammasome activation. Western blot, Co-immunoprecipitation and Immunofluorescence assays were used to observe the effect of Tan on the expression level of TXNIP. Immunofluorescence assays were applied to explore the effect of Tan on mtROS. Western blot and agarose gel electrophoresis were adopted to observe the effect of Tan on endoplasmic reticulum stress. The siRNA technique was applied to knockdown the expression levels of PERK/peIF2α, IRE1α and ATF6, and Western blot assay was employed to explore the NLRP3 inflammasome activation and possible molecular regulation mechanism of Tan.This study demonstrated that Tan decreased Mtb-induced cell pyroptosis by measuring GSDMD-N and LDH release provoked by NLRP3 inflammasome activation. Additionally, Tan inhibited endoplasmic reticulum stress (ERS), mitochondrial damage, and TXNIP protein expression, all of which acted as upstream signals of NLRP3 inflammasome activation in Mtb-infected macrophages. Significantly, NLRP3 inflammasome activation was suppressed by knocking down ERS pathway proteins, which further clarified that Tan partly targeted ERS to exert anti-inflammatory and immunoregulatory actions.This research confirms Tan's anti-inflammatory and immunoregulatory mechanisms in Mtb-infected macrophages by downregulating NLRP3 inflammasome activation-mediated pyroptosis provoked by ERS. Tan may function as an adjuvant drug to treat TB by adjusting host immune responses.