Study On The Mechanism Of LncRNA TUG1 Regulating Microglial Inflammatory Activation And The Anti-inflammatory Effect Of Aspirin Curcumin Este

Objective: To investigate the mechanism of long chain non-coding RNA taurine upregulation gene 1(LncRNATUG1)regulating inflammatory activation of microglia and the anti-inflammatory effect of curcumin acetylsalicylate(CA).Methods: BV2 microglia inflammatory activation model was established by LPS combined with IFN-γ intervention,the microglia inflammatory activation model was evaluated by the expression levels of inflammatory factors IL-1 β,IL-6 and TNF-α,the changes of LncRNA TUG1 expression level in microglia inflammatory activation were detected by PCR,and the entry of NF-κB into the nucleus was detected by cellular immunofluorescence.The causal relationship between TUG1 and microglia inflammatory activation was verified by si RNA interference and CRISPR/Cas9 knockout,and the mechanism of TUG1 in microglia inflammatory activation was confirmed by transcriptome sequencing.Curcumin acetylsalicylate intervention was used in microglia inflammatory activation model.The best anti-inflammatory dose of CA was determined by detecting the expression level of inflammatory factors.The expression levels of microglial inflammatory phenotypic markers CD16/32,CD68 and CD206 and apoptosis were detected by flow cytometry.PCR was used to detect the expression levels of IL-1β,IL-6 and TNF-α m RNAs and the transcription level of TUG1 after CA intervention.Cellular immunofluorescence assay was used to analyze the nuclear entry level of NF-κ B after CA intervention.Results: LPS combined with IFN-γ successfully induced inflammatory activation of BV2 microglia.The expression levels of IL-1β,IL-6,TNF-α and TUG1 in inflammatory activated BV2 cells were significantly higher than those in normal group.Compared with normal group,the number of transcription factor NF-κ B was increased.After si RNA interference or TUG1 knockout,compared with normal BV2 cells,the expression of IL-1 β,IL-6 and TNF-α m RNA decreased significantly,and the amount of NF-κB into the nucleus decreased,the difference was statistically significant(P <0 05),and the expression of inflammatory cytokines did not increase significantly after LPS combined with IFN-γ intervention.The results of exon sequencing showed that BV2 cells had two genotypes after TUG1 knockout,both of which showed the deletion of fragments on exon 4,indicating that the inflammatory activation site of microglia involved in TUG1 was located in exon 4.The results of GO analysis of differential expression in transcriptional group showed that,compared with the normal group,the differences in biological processes after TUG1 knockout were mainly concentrated in inflammatory reaction and immune response,and the cellular localization was mainly concentrated in the cytoplasm.The results of KEGG analysis of differentially expressed genes showed that the main enriched pathways were TNF signal pathway,type I diabetes and NF-κB signal pathway,which affected the expression of inflammatory factors in microglia,and the pharmacodynamic evaluation showed that 10 μM was the best concentration of CA for anti-inflammation,the expression levels of IL-1 β,IL-6,TNF-α and TUG1 m RNA decreased after drug intervention,and the level of NF-κ B in the nucleus was significantly lower than that in the model group.The results of flow cytometry showed that CA could reduce the expression of pro-inflammatory phenotypic markers and inhibit apoptosis in a dose-dependent manner.Conclusion: LncRNA TUG1 promotes the expression of IL-1 β,IL-6 and TNF-α by activating transcription factor NF-κ B,and participates in the process of inflammatory activation of microglia.The aspirin curcumin ester has anti-inflammatory effect,which may be its potential mechanism by down-regulating the expression of TUG1,inhibiting the activation of NF-κ B and reducing the release of inflammatory factors.