| Objective: Tuberculosis is one of the major infectious diseases in humans caused by mycobacterium tuberculosis Mtb infection.The mouse intracellular pathogen resistance 1(Ipr1)gene,located within the hypersensitivity site 1(sst1)of tuberculosis,has been identified to inhibit the proliferation of TUBERCULO bacteria within host cells,mediating macrophage resistance to pathogens.SP110 is a homologous gene of Ipr1 humans,and SP110 has been shown to be localized in the nucleus and may act as a transcriptional activator,and its single nucleotide polymorphism is closely related to host tuberculosis susceptibility(12).However,it is not clear how SP110 regulates the tb resistance mechanism of host cells,so studying the regulation of SP110 in the cellular immune response has an important role in understanding the body’s fight against intracellular pathogens.Methods:(1)THP-1 cells were infected with BCG,the mRNA and total protein of macrophages of 0h,6h,12 h,24h,and 48 h were collected,and the SP110,NF-κB signaling pathway-related genes(IKBα,p65,P-IKBα,P-p65),pro-inflammatory factors(IL-1,IL-6,TNF-α)were detected in real-time PCR and Western Blot.Expression of apoptosis-related genes BCL-2 and BAX;(2)Design and synthesis of si RNA-SP110 and construction of PCDNA 3.1-SP110 overexpression vector,establishment of SP110 gene expression knockdown and gene overexpression cell model,cell immunofluorescence to verify SP110 nuclear localization.(3)To explore whether SP110 is involved in the inflammatory response and apoptosis induced by macrophages infected by BCG in vitro,BCG infects TTP-1 cells under the interference and overexpression state of SP110,collects 24 h cell total mRNA and protein,and quantitatively detects pro-inflammatory factors(IL-1,IL-6,TNF-α),NF-κB signaling pathway key proteins(p65,P-p65),apoptosis-related genes BAX,BCL-2,At the same time,the effects of different expression levels of SP110 on cell viability,apoptosis and intracellular viable bacteria under BCG infection with macrophages were also explored.Results:(1)After BCG infection,the expression of NF-κB signaling pathway-related molecules(IKBα,p65,P-IKBα,P-p65)was significantly increased(P<0.001),the expression of pro-inflammatory factors(IL-1,IL-6,TNFα)was significantly increased(P<0.001),the expression of BAX,the signature molecule of apoptosis(P<0.001),the expression of BCL-2 of the apoptosis marker molecule was significantly reduced(P<0.001),and the expression of SP110 was significantly increased(P <0.001),the trend was most obvious at 24 hours.(2)the successful design of SP110 small interfering RNAs,si SP110-1,si SP110-2,si SP110-3,and si RNA-SP110-3 interference efficiency of 70%(P<0.001);Successfully constructed PCDNA 3.1-SP110 overexpression vector with an overexpression efficiency of about 5 times(P<0.001),and successfully established SP110 gene interference and gene overexpression cell regulation model.(3)During BCG infection,interference with SP110 effectively reduced the expression of pro-inflammatory factors(IL-1,IL-6,TNFα)(P<0.001),significantly decreased the expression levels of NF-κB p65 protein and its phosphorylated proteins,decreased the expression of the apoptosis signature molecule BAX(P<0.001),increased the inhibition of the apoptosis-related molecule BCL-2 expression(P<0.001),reduced the apoptosis rate of host cells after BCG infestation(P<0.001)and increased the number of BCG intracellular survivals(P<0.001).During BCG infection,overexpression of SP110 effectively promoted the expression of pro-inflammatory factors(IL-1,IL-6,TNFα)(P<0.001)and significantly increased the expression levels of NF-κB p65 protein and its phosphorylated protein,which increased the apoptotic efficiency of host cells after BCG infestation and decreased the number of BCG intracellular survivors.Conclusion: During BCG infection with macrophages,SP110 can activate the NF-κB signaling pathway,cause pro-inflammatory factor release,induce macrophage apoptosis,and reduce intracellular activity of BCG. |
PDF Full Text Request