| Taurochenodeoxycholic acid(TCDCA)is one of the main active components of bile acids(BAs),which can regulate inflammation and immune function through various signaling pathways.However,it is unclear whether TCDCA exerts anti-inflammatory and immunomodulatory effects through the TGR5 receptor-mediated cAMP-PKA-CREB and Raf-1-CREB signaling pathways.Therefore,first things first,TGR5-knockdown H1299 cells(TGR5-KD H1299)was constructed by biotechnology of the CRISPR/Cas9 in this study,used to research the effects of TCDCA on expression of cAMP,PKA and CREB gene and protein.The aim of the study is to reveal whether TCDCA could activate the TGR5 receptor and in turn produce cAMP,PKA and CREB signaling molecules.NR8383 cell lines were subjected to researching for demonstrating that whether TNF-?,IL-1?,IL-6,IL-8 and IL-12 secret from NR8383 cells via cAMP-PKA-CREB and Raf-1-CREB signaling pathways;meanwhile,whether TCDCA plays important roles of anti-inflammation and immunomodulatory effects through the cAMP-PKA-CREB and Raf-1-CREB signaling pathways mediated by TGR5.The detailed research contents are as follows:(1)TCDCA can activate TGR5 receptorH1299 cell lines were subjected to editing TGR5 gene using CRISPR/Cas9;the expression of TGR5 gene and the effects of TCDCA on the expression of PKA and CREB genes in H1299 cells and TGR5-KD H1299 cells were detected by qPCR.The expression of TGR5 protein cells as well as the effect of TCDCA on the expression of PKA and CREB protein in H1299 cells and TGR5-KD H1299 cells were detected by Western blot.The effect of TCDCA on cAMP content in H1299 cells and TGR5-KD H1299 cells was detected by ELISA.Results are as follows:The results of TGR5 gene editing showed that the knockout rate of TGR5 gene was 96.91%in TGR5-KD H1299 cells,indicating that the TGR5 gene was successfully edited in H1299 cells.Both low and high concentration of TCDCA significantly decreased cAMP content in TGR5-KD H1299 cells compared to H1299 cells(P<0.05);however,the medium concentration of TCDCA in TGR5-KD H1299 cells extremely significant decreased cAMP content compared to H1299 cells(P<0.01).TCDCA was extremely significant decreased cAMP content at 10 min,15 min and 20 min in TGR5-KD H1299 cells compared to H1299 cells(P<0.01),and decreased cAMP at 15 min was more than other time points;cAMP content was gradually decreased in H1299 cells over time.Expression of PKA gene and CREB protein were extremely significant decreased(P<0.01)and expression of CREB gene was significantly decreased(P<0.05)in TGR5-KD H1299 cells treated with TCDCA for 3 h;expression of PKA gene and protein as well as CREB protein were extremely significant decreased(P<0.01)in TGR5-KD H1299 cells treated with TCDCA for 6 h.(2)The effect of TCDCA on cAMP-PKA-CREB signaling pathway in NR8383 cellsAdenylate cyclase(AC)specific inhibitor SQ22536,protein kinase A(PKA)specific inhibitor H89 and Raf specific inhibitor Dabrafenib were used to assay expression of gene and protein of PKA and CREB;and qPCR was used to assay expression of gene of PKA and CREB in NR8383 cells and NR8383-TGR5 cells;Western blot was used to assay expression of protein of PKA and CREB in NR83 83 cells and NR83 83-TGR5 cells.The results that effects of TCDCA on gene and protein of PKA in NR8383 cells treated with SQ22536 show that expression of gene and protein of the PKA in both groups of NR8383 and NR8383-TGR5 treated with TCDCA were extremely significant increased(P<0.01)compared to groups of NR8383 cells and NR8383-TGR5 cells;expression of gene and protein of PKA in NR8383-TGR5 cells treated with TCDCA were significantly increased(P<0.05)or extremely significant increased(P<0.01)compared to NR8383 cells treated with TCDCA;expression of protein of PKA in NR8383 cells treated with TCDCA was significantly increased compared to NR8383 cells treated with TCDCA and SQ22536(P<0.05);expression of gene and protein of PKA NR8383-TGR5 cells treated with TCDCA were significantly increased(P<0.05)or extremely significant increased(P<0.01)compared to NR8383-TGR5 cells treated with TCDCAand SQ22536.The results that effects of TCDCA on expression of gene and protein of CREB in NR8383 cells treated with H89 show that expression of protein of CREB in NR8383 cells treated with TCDCA was significantly increased compared to NR8383 cells(P<0.05);expression of gene and protein of CREB in NR8383-TGR5 cells treated with TCDCA were extremely significant increased compared to NR8383-TGR5 cells(P<0.01);expression of gene and protein of CREB in NR8383-TGR5 cells treated with TCDCA were extremely significant increased compared to NR8383 cells treated with TCDCA(P<0.01);expression of protein of CREB in NR8383 cells treated with TCDCA was significantly increased compared to NR8383 cells treated with H89 and TCDCA(P<0.05);expression of protein of CREB in NR8383-TGR5 cells treated with TCDCA was significantly increased compared to NR8383-TGR5 cells treated with H89 and TCDCA(P<0.05).The results that effects of TCDCA on expression of gene and protein of CREB in NR8383 cells treated with dabrafenib show that expression of protein of CREB in NR8383 cells treated with TCDCA was extremely significant increased compared to NR8383 cells(P<0.01);expression of gene and protein of CREB in NR8383-TGR5 cells treated with TCDCA were significantly increased(P<0.05)or extremely significant increased compared to NR8383-TGR5 cells(P<0.01);expression of gene and protein of CREB in NR8383-TGR5 cells treated with TCDCA were significantly increased(P<0.05)or extremely significant increased compared to NR8383 cells treated with TCDCA(P<0.01);expression of protein of CREB in NR8383 cells treated with TCDCA was significantly increased compared to NR83 83 cells treated with dabrafenib and TCDCA(P<0.05);expression of protein of CREB in NR8383-TGR5 cells treated with TCDCA was extremely significant increased compared to NR8383-TGR5 cells treated with dabrafenib and TCDCA(P<0.01).The results that effects of TCDCA on expression of gene and protein of CREB in NR8383 cells treated with H89 and dabrafenib show that expression of protein of CREB in NR8383 cells treated with TCDCA was extremely significant increased compared to NR8383 cells(P<0.01);expression of gene and protein of CREB in NR8383-TGR5 cells treated with TCDCA were extremely significant increased compared to NR8383-TGR5 cells(P<0.01);expression of gene and protein of CREB in NR8383-TGR5 cells treated with TCDCA were significantly increased(P<0.05)or extremely significant increased compared to NR8383 cells treated with TCDCA(P<0.01);expression of protein of CREB in NR8383 cells treated with TCDCA was extremely significant increased compared to NR8383 cells treated with H89,dabrafenib and TCDCA(P<0.01);expression of protein of CREB in NR8383-TGR5 cells treated with TCDCA was extremely significant increased compared to NR8383-TGR5 cells treated with H89,dabrafenib and TCDCA(P<0.01).(3)The effects of TCDCA on anti-inflammatory and immunomodulation related cytokines in NR8383 cellsExpression of gene of NF-?B,TNF-?,IL-1?,IL-6,IL-8 and IL-12 was detected using qPCR,expression of protein of NF-?B was detected using Western blot as well as expression of gene and protein of TNF-?,IL-1?,IL-6,IL-8 and IL-12 was detected by ELISA.Expression of gene of TNF-?,IL-1?,IL-6,IL-8 and IL-12 in NR8383 cells treated with LPS and TCDCA were significantly decreased(P<0.05)or extremely significant decreased(P<0.01)compared to NR8383 cells treated with LPS;expression of gene of NF-?B,TNF-?,IL-1?,IL-6,IL-8 and IL-12 in NR8383-TGR5 cells treated with LPS and TCDCA all were extremely significant decreased compared to NR8383-TGR5 cells treated with LPS(P<0.01);expression of gene of NF-?B,TNF-?,IL-6 and IL-12 in NR8383 cells treated with LPS,H89 and TCDCA were significantly decreased(P<0.05)or extremely significant decreased(P<0.01)compared to NR8383 cells treated with LPS and H89;expression of gene of NF-?B,TNF-?,IL-1?,IL-6,IL-8 and IL-12 in NR8383-TGR5 cells treated with LPS,H89 and TCDCA were extremely significant decreased compared to NR8383-TGR5 cells treated with LPS and H89(P<0.01);expression of gene of NF-?B,TNF-?,IL-1?,IL-6 and IL-8 in NR8383-TGR5 cells treated with LPS and TCDCA were significantly decreased(P<0.05)or extremely significant decreased(P<0.01)compared to NR8383 cells treated with LPS and TCDCA.Expression of protein of NF-?B,TNF-?,IL-6,and IL-12 in NR8383 cells treated with LPS and TCDCA were significantly decreased(P<0.05)or extremely significant decreased(P<0.01)compared to NR83 83 cells treated with LPS(P<0.01);expression of protein of NF-?B,TNF-?,IL-1?,IL-6,IL-8 and IL-12 in NR8383-TGR5 cells treated with LPS and TCDCA were extremely significant decreased compared to NR8383-TGR5 cells treated with LPS(P<0.01);expression of protein of NF-?B,TNF-?,IL-6,and IL-12 in NR8383 cells treated with LPS,H89 and TCDCA were significantly decreased(P<0.05)or extremely significant decreased(P<0.01)extremely significant decreased compared to NR8383 cells treated with LPS and H89;expression of protein of NF-?B,TNF-?,IL-1?,IL-6,IL-8 and IL-12 were extremely significant decreased(P<0.01)or extremely significant increased(P<0.01)in NR8383-TGR5 cells treated with LPS,H89 and TCDCA compared to NR8383-TGR5 cells treated with LPS and H89;expression of protein of NF-?B,TNF-?,IL-1?,IL-6,IL-8 and IL-12 in NR8383-TGR5 cells treated with LPS and TCDCA were significantly decreased(P<0.05)or extremely significant decreased(P<0.01)compared to NR8383-TGR5 cells treated with LPS and TCDCA.The main conclusions are as follows:(1)TCDCA can extremely significant increase cAMP content,expression of PKA and CREB in H1299 cells,indicating TCDCA can activate TGR5 receptor.(2)TCDCA increased phosphorylation level of PKA and CREB protein,however,increased phosphorylation level of PKA and CREB protein were decreased via inhibitors of SQ22536,H89 and Dabrafenib,indicating TCDCA exerts bio-functions via TGR5-mediated signaling pathways cAMP-PKA-CREB and Raf-CREB.(3)TCDCA decreases expression of NF-?B,TNF-?,IL-1?,IL-6,IL-8 and IL-12 gene and protein via signaling pathway of cAMP-PKA-CREB,exerting anti-inflammatory and immune roles. |
PDF Full Text Request