The Role And Mechanism Of Andrographolide On Mycobacterium Tuberculosis Induced Inflammation In Macrophage And Lung Epithelial Cell

[Objective] Tuberculosis is still a lethal infectious disease that seriously threatens human health worldwide.Tuberculosis precention and therapy is facing great challgenges due to the emergence of drug-resistant Mycobacterium tuberculosis(Mtb)strainsand multiple drug-resistant Mtb strains.During the course of tuberculosis,pathological damage and lesions caused by excessive inflammation is also an important issue needs to be solved.There are abundant resources of traditional Chinese medicine(TCM)in our country,and researches have confirmed that many TCM have substantial functions of regulating our immune response.Andrographolide(Andro)is the major active component from Andrographis paniculata,which is a TCM commonly used in the treatment of infection in upper respiratory tract,fever and the inflammation of intestine.This study intended to evaluate the antimycobacterial and anti-inflammatory effects of andrographolideon macrophages and lung epithelial cells and to explore its underlying molecular mechanisms.It provides a theoretical basis and experimental evidence for andrographolide as a drug candidate for controlling excessive inflammatory response during the course of tuberculosis.[Method](1)ELISA was used to observe the effects of Andrographolide on the secretion of inflammatory cytokine of Mtb-infected macrophages.(2)Co-immunoprecipitation,Western blot and immunofluorescence assays were used to observe the activation of NLRP3 inflammasome in Mtb-infected macrophages with the treatment of Andrographolide,and the results were verified both in THP-1 cells and mouse primary peritoneal macrophages.(3)Western blot was applied to detect the expression of autophagic marker protein of Mtb-infected macrophages with the treatment of different doses of Andrographolide.(4)The Colony Forming Units counting method(CFU)was utilized to evaluate the killing ability of Andrographolide on intracellular Mtb.(5)Western blot and immunofluorescence assays were used to explore the possible molecular mechanisms of Andrographolide,which were focused on the regulatory pathways of autophagy,including: PI3K/Akt/m OR and MAPKs,and NF-κB pathways.The results were verified both in THP-1 cells and mouse primary peritoneal macrophages.(6)Establish Mtb-infected co-culture system of macrophages and lung epithelial cells,and the gene expression of chemokines and matrix metalloproteinases were detected.(7)In order to block the biologic effects of IL-1β,the neutralizing antibody αIL-1β and its control antibody m Ig G1 were added to the co-culture system to verify the possible mechism of Andrographolide.[Results](1)Andrographolide decreased the secretion of Mtb-induced inflammatory cytokines(IL-1β,IL-6 and TNF-α)in supernatant of macrophage.(2)The results of Western blot assays indicated that Andro decreased the expression of NLRP3,pro-IL-1β,caspase-1 p20 and pro-IL-1β.(3)The results of Western blot assays indicated that Andro increased the expression of LC3 II and decreased the expression of p62 in a dose-dependent manner.(4)The Colony Forming Units counting method(CFU)showed that Andro increased killing ability of intracellular Mtb.(5)immunofluorescence studies demonstrated that Andro promoted the co-localization of inflammasome with autophagosome.(6)Andro decreased the phosphorylation of Akt and m TOR at Ser473 and Ser2448 separately both in THP-1 and mouse primary peritoneal macrophages.(7)Andro exerted inhibitory effects on the gene expression of chemokine and MMP in co-culture system infected with Mtb.(8)IL-1β neutralizing antibody exerted inhibitory effects on the gene expression of chemokine and MMP in co-culture system infected with Mtb.[Conclusion] In conclusion,Andrographolide inhibited the inflammation in Mtb-infected macrophages through decreasing cytokines secretion such as IL-1β.The down-regulation of IL-1β reduced the gene expression of chemokine and MMP in lung epithelial cell.Andro induced PI3K/Akt/m TOR and NF-κB inhibition contribute to inhibit inflammasome activation and subsequently down-regulatecytokine secretion.These findings collectively implicated an anti-inflammatory role and tissue protection role of Andro on Mtb infection,thereby providing evidence of Andro as an efficacious adjunct treatment during mycobacterial infection.