DUSP1 Mediates BCG Induced Apoptosis And Inflammatory Response Via MAPKs/NF-κB Signaling Pathway

Tuberculosis(TB),caused by Mycobacterium tuberculosis(Mtb)infection,remains one of the world’s deadliest infectious diseases.Apoptosis and inflammatory responses caused by the interaction between the host and the pathogen play an important role in the pathogenesis of TB.Dual-specificity phosphatase 1(DUSP1)plays an important role in the regulation of the host immune response.However,the role of DUSP1 in the regulation of apoptosis and inflammatory response in THP-1 macrophages induced by attenuated Bacillus Calmette Guerin(BCG)infection is still unclear,therefore,this study will investigate the role of DUSP1 in the regulation of macrophage apoptosis and inflammatory response during BCG infection of human monocyte macrophages.This study will provide a theoretical basis for the prevention and treatment of TB.In this study,we used BCG as the study target and established interfering DUSP1 combined with BCG-infected THP-1 macrophages and C57BL/6J mouse model.We used Western Blotting,RT-qPCR,immunofluorescence,transmission electron microscopy,flow cytometry,ELISA,HE and IHC to detect macrophage apoptosis and inflammatory response,and obtained the following research Results.(1)BCG infection induced apoptosis in THP-1 macrophages and promoted the expression of DUSP1,TNF-α and IL-1β.The designed and synthesized DUSP1 small interfering RNA significantly reduced(P<0.01)the expression of DUSP1 and successfully constructed a model of interfering DUSP1 combined with BCG infection in THP-1 cells.(2)During BCG infection,siRNA-DUSP1 significantly reduced(P<0.01)the expression of apoptosis key proteins Cleaved-Caspase3,Cleaved-PARP 1,Cleaved-Caspase9,Apaf-1,Bax,Cytochrome C;significantly increased(P<0.01)the expression of apoptosis inhibitory The expression of the protein Bcl-2 was significantly increased,and the results of mitochondrial membrane potential detection by flow cytometry showed that interference with DUSP1 significantly decreased mitochondrial depolarization in macrophages.(3)During BCG infection,siRNA-DUSP1 significantly reduced(P<0.001)the apoptosis rate of macrophages.TEM results showed that compared with the BCG group,the BCG+siRNA-DUSP1 group only experienced nucleus consolidation into a star-moon shape and fewer intracellular vacuoles,indicating that interfering with DUSP1 alleviated the occurrence of apoptosis in THP-1 cells.(4)During BCG infection,siRNA-DUSP1 significantly reversed(P<0.01)the increase in phosphorylation levels of p3 8,ERK and JNK induced by BCG,but had no effect on the expression of p38,JNK and ERK in THP-1 cells.These results suggest that interference with DUSP1 inhibits BCGinduced MAPKs signaling in THP-1 cells,which further inhibits increased apoptosis in THP-1 cells.(5)During BCG infection,siRNA-DUSP1 significantly downregulated(P<0.001)the expression of TNF-α,IL-1β and p-NF-κB p65,suggesting that interfering with DUSP1 inhibited BCG-induced release of pro-inflammatory cytokines TNF-α and IL-1β via the NF-κB signaling pathway.(6)The ELISA results showed that siRNA-DUSP1 significantly reduced(P<0.01)the expression of BCG-induced TNF-α and IL-1β,and the HE results showed that siRNA-DUSP1 reduced the inflammatory damage in the lung tissue of C57BL/6J mice.IHC results showed that siRNA-DUSP1 decreased BCG-induced activation of MAPKs/NF-κB signaling pathway,but siRNA-DUSP1 had no effect on the change of lung tissue bacterial load in C57BL/6J mice.(7)LPS promoted DUSP1 and Cleaved-Caspase3 protein expression(P<0.05),siRNA-DUSP1 decreased LPS-induced DUSP1 expression compared to the LPS-infected group(P<0.01).In contrast,siRNA-DUSP1 significantly increased the expression of Cleaved-Caspase3 protein(P<0.05),suggesting that siRNA-DUSP1 could effectively inhibit DUSP1 expression and act as a negative regulator in LPS-induced apoptosis.In conclusion,BCG infection induced apoptosis in THP-1 macrophages and induced the expression of DUSP1,TNF-α and IL-1β.siRNA-DUSP1 significantly reduced BCG-induced apoptosis and inflammatory response in THP-1 macrophages and inhibited the activation of MAPKs/NF-κB signaling pathway.In addition,siRNA-DUSP1 inhibited the lung inflammatory response induced by BCG infection in C57BL/6J mice.The present study suggests a novel role for DUSP1 as a regulator of MAPKs/NF-κB signaling pathway in BCG-induced macrophage apoptosis and inflammatory response.