Effects Of Taurocholic Acid On TNF-α And IL-1β Related Signaling Pathway Via TGR5 In NR8383 Cells

TCA, as one of the main components of bile acids, had anti-inflammatory immune regulatory actions and other different pharmacological effects via different signaling pathways. The aim of this study was to further investigate the interaction between TCA and TGR5 receptor, and the actions of TCA on the secretion of TNF-α and IL-1β mediated by TGR5. The secretion of TNF-α and IL-1β of NR8383 cells treated with TCA were associated with Gɑq-PKC-ERK signaling transduction pathway. Contents of the researches are as follows:(1) Determining the interaction of TCA and TGR5Eukaryotic expression vector containing human TGR5 were transiently transfected into HEK-293 T cells(expression eukaryotic vector pCMV-EGFP-hTGR5 and pCMV-hTGR5 containing human TGR5 was constructed and identified in this laboratory), and fusion expression human TGR5 with green fluorescence tag located on the cell membrane of HEK-293 T cells. TGR5 were prompted internalized with different treatments of TCA under fluorescence microscopy; HEK-293 T cells were administrated to different concentrations of 10000μM,1000μM, 100μM, 10μM, 1μM, 0.1μM TCA, and the results showed that the inhibition rate of 10000μM TCA was 85%, and the dose ranges of TCA should be≤1000 μM determined by MTT toxicity tests; cAMP significantly elevated in HEK-293 T cells transfected with expression vector containing hTGR5 treated with different concentrations of TCA or TLCA. Logistic dose-response curve of TCA in dose-dependent maner determined with ELISA method(P<0.01) was fit with a nonlinear function(four parameter logistic equation) using GraphPad Prism package version 6.0. The effective concentration 50%(EC50) of TCA was 502 μM in HEK-293 T cell. Meanwhile TCA could activate TGR5 receptor, but the efficiency and potency of TCA in increasing cAMP content were lower than lithocholic acid(TLCA).(2) Effects of TCA on TNF-α or IL-1β in NR8383 cells or NR8383-TGR5 cellsThe gene and protein levels TGR5 had no changes treated with different concentrations of 1000 μM、500 μM、100μM、50 μM、10μM TCA determined by RT-qPCR technology and western blotting in NR8383 cells(P>0.05). But TGR5 were low expression in NR8383 cells overall. NR8383 cells transfected with hTGR5-PCMV expression vectors were screened with G418, and then established NR8383-TGR5 stable expression cell lines(NR8383-TGR5 stable expression cell lines had been built and identified in this laboratory). Compared with the empty NR8383 cells, cAMP content significantly increased in NR838-TGR5 cells with the treatment of 10 μM TCA(P<0.01) using ELISA method,, which suggested hTGR5 was expressed in NR8383-TGR5 cells. The TGR5 receptor was observed under confocal microscope in NR8383 cells. Results showed that hTGR5 located in the plasma membrane was consistent with the characteristics of membrane receptor. NR8383 cells and NR8383-TGR5 cells were controlled by each other, which was helpful to study the function of TGR5 in NR8383 cells, and which was the basis for the subsequent application of NR8383-TGR5 cells.TNF-α and IL-1βwere inflammatory and immune related cytokines, and which would be the experimental basis for further exploration of anti-inflammatory and immune regulation of TCA in NR8383 cells. TNF-α and IL-1βtreated with different concentrations of 1000 μM, 100 μM, 10 μM TCA were determined by RT-qPCR and ELISA in NR8383 cells and NR8383-TGR5 cells. TNF-α content and IL-1β mRNA increased induced by LPS(P<0.05). Levels of TNF-α gene and protein stimulated by LPS in NR8383 cells and NR8383-TGR5 cells significantly decreased with the treatment of 1000 μM TCA(P<0.01), while the content of TNF-α increased with the increasement of TGR5 compared within untreated NR8383 cells(P<0.05). After LPS stimulation, IL-1βcell protein content increased with the treatment of 1000 μM TCA in NR8383 cells(P<0.05); IL-1βprotein significantly decreased stimulated by LPS with the treatment of 100 μM TCA(P<0.01). Compared with NR8383-TGR5 cell as blank control group, IL-1β protein significantly increased with the treatments of 1000 μM and 10 μM TCA in NR8383-TGR5 cells(P< 0.05). IL-1βmRNA and protein content reduced stimulated by LPS with the treatments of 100 μM and 10 μM TCA in NR8383-TGR5 cells(P<0.05).(3) Gɑq-PKC-ERK signaling pathways mediated effects of TCA on TNF-α and IL-1β expressionEffects of TCA on TNF-α and IL-1βrelated signaling pathways via TGR5 receptor in NR8383 cells should reseach the relationship between TGR5 receptor(guanylic acid protein coupled receptor, GRCP) and Gɑq(a type of guanylic acid protein subtype), and then related signaling pathway.Expression of Gaq markedly decreased with the treatment of 1000 μM、100 μM、10μM TCA determined by RT-qPCR and western blotting technology in NR8383 cells and NR8383-TGR5 cells. Results showed that contents of Gaq significantly decreased in both NR8383 cells and NR8383-TGR5 cells treated with different concentrations of TCA(P<0.05), but there were no significant difference contrasted within them(P>0.05), which suggested that Gaq decreased treated with TCA but not coupled with TGR5(P>0.05).PKC were determined by RT-qPCR and western blotting along with blocking Gɑq. Expressions of gene and protein of PKC and pPKC were regulated by1000 μM、500 μM、10μM TCA in NR8383 cells. PLC inhibitors U73122 markedly decreased mRNA of PKC and protein of PKC and pPKC compared with the blank control group. The protein of PKC and pPKC with the treatments of LPS and U73122 decreased treated with 500 μM TCA, but it had the contrary results treated with 500 μM TCA. These results showed that TCA regulated of the content of PKC and pPKC via Gaq.G? 6983, the inhibitor of PKC was used to study the downstream signal ERK, pERK. Gene and protein of ERK and pERK in NR8383 cells were determined by RT-qPCR and western blotting with the treatments of 1000 μM、500 μM、10μM TCA. G? 6983 markedly decreased ERK mRNA, content and pERK content; LPS reduced expressions of ERK, but increased pERK content in NR8383 cells; different concentrations of TCA reduced ERK and pERK content caused by LPS; while TCA significantly decreased content of ERK and pERK treated by PD98059 and LPS compared with PD98059(P<0.01). The results showed that TCA regulated the content of ERK and pERK related to PKC.PD98059, the inhibitor of ERK, was used to study the downstream signal, such as TNF-α and IL-1β. Gene and protein of TNF-α and IL-1βwith the treatments of 1000 μM、500 μM、10μM TCA were determined by RT-qPCR and western blotting technology in NR8383 cells. Compared with blank group, the content of IL-1β and TNF-α significantly increased caused by LPS in NR8383 cells(P<0.01), but TNF-α content and IL-1βmRNA significantly decreased treated with PD98059(P<0.01); Compared with LPS group, the content of TNF-α significantly decreased, but IL-1βsignificantly increased with the treatment of 1000 μM TCA(P<0.01). The content of IL-1βdecreased with the treatment of 100 μM TCA; compared with PD98059 control group, all of different concentrations of TCA had no effects on TNF-α contents and IL-1βmRNA treated with PD98059 and LPS(P>0.05), but IL-1βprotein content significantly increased in NR8383 cells(P<0.01). These results showed that ERK took in effets of TCA on the regulation of TNF-α and IL-1β content. After TGR5 receptors activated by TCA was clearly, the study found that TGR5 indeed mediated TCA regulation TNF-α and Il-1 beta content,Effects of TCA on secretion of contents of TNF-α and Il-1β were related Gaq-PKC-ERK signaling pathway in NR8383 cell. On the basis of TCA activated TGR5 receptor, and TCA could regulate the content of TNF-α and IL-1 beta via TGR5. Therefore, 100 μM TCA regulated TNF-α and IL-1βthrough Gɑq-PKC-ERK signaling pathways. The research provided a new theoretical basis for the further study of the molecular mechanism of anti-inflammatory immune regulation of TCA, and for finding a new target of TCA.