| Background: Tuberculosis is one of the diseases that seriously endanger human health.According to the latest data provided by the WHO,there were approximately 9-11 million new tuberculosis patients in the global population in 2018.Tuberculosis is still one of the top ten causes of death worldwide and is the main cause of death for single infectious agents(Global tuberculosis report 2019,WHO).Mycobacterium is one of the three major infectious diseases that cause human death worldwide,invading the innate and adaptive immune systems of the body.Mycobacterium tuberculosis(M.tb)in the body is the pathogen of tuberculosis.Macrophages are the main target cells of M.tuberculosis invading the innate immune system.The complex reactions of macrophages and tuberculosis are closely related to oxidative stress and autophagy.The role of transcription factor NF-E2 related factor 2(NRF2)and its mediated signaling pathways in resisting microbial infection and inflammatory damage is attracting more and more attention.As a central regulator of the antioxidant system,NRF2 is associated with aging associated with oxidative stress.We found in previous studies that the expression level of NRF2 in TB patients was significantly higher than that in healthy controls group.Therefore,the purpose of this study was to investigate the role and mechanism of the transcription factor NRF2 in the susceptibility of Mycobacterium tuberculosis to macrophages in aged rats.Methods:(1)RNA-seq technology was used to compare the expression levels of NRF2 gene linear RNA in peripheral blood mononuclear cell(PBMC)of TB patients in the young and the elderly group,analyze its correlation with age.(2)Using real-time q PCR(q-PCR)technology,the peripheral blood PBMC NRF2 expression levels in young SD rats and old SD rats were detected.(3)The transcription expression levels of NRF2,SOD-1,NOX-1,NOX-2,IL-12P35 and IL-12P40 gene in M.tb infected alveolar macrophages of young SD rats and old SD rats by q-PCR.(4)Western Blot was used to detect the expression levels of NRF2 and LC3-? in macrophages of young SD rats and old SD rats infected with M.tb.(5)The difference of M.tb phagocytosis rate and apoptosis rate in macrophages of young SD rats and old SD rats was detected by flow cytometry.(6)Flow cytometry was used to detect the changes of reactive oxygen species(ROS)in alveolar macrophages of young SD rats and old rats infected with M.tb.(7)The effect of NRF2 and P62 protein levels in macrophages of young SD rats and old SD rats were infected by M.tb after pretreatment with NRF2 inhibitor Brusatol using WB technology.Results:(1)RNA-seq results showed that the expression of NRF2 linear RNA gene in PBMC of healthy control group and tuberculosis patients in each groupwas significantly correlated with age(P < 0.05),but no significant correlation was found between age and tuberculosis.(2)There was a statistically significant difference in the expression level of NRF2 in peripheral blood PBMCs between young SD rats and old SD rats(P < 0.05,n = 3).NRF2 expression in young SD rats was lower than that in old SD rats at both transcription level and protein level.(3)After M.tb infection of macrophages in young SD rats and old SD rats,the NRF2 m RNA level was significantly reduced compared with the control group(P < 0.05,n = 3),Significantly higher than the aging group(P < 0.05,n = 3);the NRF2 protein level in young rats after M.tb infection was higher than that in the aging group(P < 0.05,n = 3).(4)After 6 h of M.tb infection of alveolar macrophages in the young and old SD rats,the expression levels of SOD-1 m RNA,NOX-1 m RNA,NOX-2 m RNA,IL-12P35 m RNA,IL-12P40 m RNA in the old and young groups were compared with the control The level of the group was significantly increased,and the expression level of NOX-4 m RNA was decreased(P < 0.05,n = 3);the expression level of SOD-1 m RNA,NOX-4 m RNA,IL-12P35 m RNA in the elderly group was lower than that in the young group;The expression levels of-12P40 m RNA and NOX-1 m RNA were higher than those in the young group(P < 0.05,n= 3).(5)After M.tb infected alveolar macrophages in young SD rats and old SD rats,the levels of autophagy protein LC3-in young SD rats w? as higher than the old SD rats(P < 0.05,n = 3),and the uninfected group in the young group was significantly higher than the uninfected group in the old group(P < 0.05,n = 3).(6)The phagocytic rate of M.tb-FITC in alveolar macrophages of in the old SD rats group was greater than that of in the young SD rats group at 2 h,4 hand 6 h,and the phagocytic rate increased with time.Brusatol pretreatment reduced alveolar macrophage phagocytosis(P < 0.05,n = 3).(7)The apoptosis rate of alveolar macrophages in aged SD rats M.tb at 2 h,4 h,and 6 h was significantly higher than that in young SD rats,and the apoptosis rate increased with time(P < 0.05,n = 3).(8)After 30 minutes of M.tb infection,the ROS levels of alveolar macrophages in young SD rats and old SD rats were significantly higher than those of the control group(P < 0.05,n = 3),and the levelsof the old SD group were significantly higher than young SD rats(P < 0.01,n = 3).(9)Compared with the control group,the expression of P62 protein in M.tb-infected alveolar macrophages was completely inhibited after Brusatol pretreatment.After M.tb infection,the level of P62 protein in young SD rats was significantly higher than that in old SD rats.(P < 0.05,n = 3);NRF2 protein level in young SD rats was significantly lower than that in old SD rats(P < 0.05,n = 3).Conclusions:(1)Decreased NRF2 level of alveolar macrophages of aged rats related to their susceptibility to M.tb.(2)NRF2 enhances the susceptibility of macrophages to M.tb by regulating intracellular oxidative stress levels and the autophagy receptor P62. |
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