| Background: With the development of living conditions, the incidence of cardiovascular disease were rising. Ventricular remodeling plays an important role in the occurrence and development of cardiovascular disease, which results from the participation of multiple damage factors, the inflammatory response is very important, which mediated by NF-kappa B. Heart tissue was composed of heart cells and extracellular matrix, the myocardial fibroblasts are essential for ventricular remodeling. G protein-coupled receptor for bile acids(TGR5) was found expressed in multiple tissues and organs, which is vital in many reaction process, such as energy metabolism, immune regulation etc. Recent research shows that TGR5 activation on the cell membrane in mononcyte-macrophages[1, 2], biliary epithelial cells and the gastric[3, 4], etc, which can reduce the inflammatory response mediated by NF-?B. But there is no related report that shows the effect of the TGR5 activation on myocardial fibroblasts.Objective: To explore the effect of TGR5 activation on the proliferation of neonatal mice cardiac fibroblasts(CFs) induced by Angiotensin ?, and to explore the related mechanisms.Methods: 1. The cultivation and identification of neonatal mice myocardial fibroblasts: CFs were cultured by the way of Trypsin enzymic digestion and different speed adherence; CFs were identification by immunofluorescence. 2. To detect the expression of TGR5 mRNA on the CFs: CFs, heart, liver, spleen tissue were extracted to test the expression of TGR5 mRNA by RT-PCR. 3. To prepare and test the overexpression virus of TGR5. 4. To detect the efficiency of TGR5 virus in CFs, the groups were set as follows:(1)The experimental groups: CFs-v+medium group, CFs-v+Polybrene group, CFs-v+ENi.S group, CFs-v+Polybrene+ ENi.S group(CFs-v means the CFs infect by virus of TGR5, “+” means positive virus, “-” means the negative virus);(2)The control group: CFs+medium group, CFs+Polybrene group, CFs+ENi.S group, CFs+ Polybrene+ENi.S group. 5. To detect the expression of TGR5 mRNA on the CFs that infected by TGR5 virus: CFs, CFs-v(+), heart, liver, spleen tissue were extracted to test the expression of TGR5 mRNA by RT-PCR. 6. To observe the proliferation of the CFs induced by Ang II or the overexpression virus of TGR5.(1)The groups of CFs induced by Ang II were set as follows: CFs+Ang ?(0mol/L)group was control group; CFs+ Ang ?(10-5mol/L)group, CFs+ Ang ?(10-6mol/L)group, CFs+ Ang ?(10-7mol/L)group were experimental groups.(2)The groups of CFs induced by TGR5 virus were set as follows: Ang ?+CFs group was control group; Ang ?+CFs-v(+)group, Ang ?+CFs-v(-)group were experimental groups. The OD value were tested after been interfered by different experimental conditions at specific time, then computed the cell proliferation rate or inhibition rate. 7. To test the affect of CFs induced by TGR5 virus and activation TGR5 receptor, experimental groups were set as follows:(1)CFs group;(2)CFs-v(+) group;(3)Ang ?+CFs-v(+) group;(4)Ang ?+CFs-v(-) group(5)Ang ?+CFs-v(+)+nomilin group(TGR5 agonist) group;(6)Ang ?+CFs-v(-)+nomilin group. The proliferation were tested after been interfered by different experimental conditions at specific time. The expression of TGR5 and P65 in each groups were detect by Western Blot technology. The expression of c AMP, the type I and the type ? of collagen were measured by ELISA.Results: 1. The cultivation of CFs was successful. The fluorescence immunoassay showed that the purity of CFs is more than 90%. 2. The results of RT-PCR: Under normal circumstances, Compare with heart, liver, spleen tissue, the expression of TGR5 mRNA on CFs was quite few(P<0.05); The TGR5 mRNA expression on CFs-v(+) group was increased, compared with the expression of TGR5 mRNA on the CFs group, there was statistical significant(P<0.05). 3. The results of MTT:(1)Different concentrations of Ang ? could promote the proliferation of CFs. The proliferation in Ang ?+CFs group was significant increased than CFs group, and proliferation CFs induced by Ang ?(10-7mol/L) is obvious during 24 hour, compared with Ang II(10-5mol/L) and Ang II (10-6mol/L), P<0.05. Infected by the overexpression virus of TGR5, the inhibition of CFs-v(+) was great than the CFs group, P<0.05. TGR5 activation could strengthen the role of proliferation inhibition: The inhibitory action of Ang ?+CFs-v(+)+Nomilin group was high than Ang ?+CFs-v(+) group, P<0.05; 4. The results of Western Blot:(1)After the CFs been infected by the virus of TGR5, compared with CFs group, the quantity of TGR5 protein expression increased significantly in CFs-v(+) group, Ang ?+CFs-v(+) group and Ang?+CFs-v(+)+Nomilin group, P<0.05;(2)Infected by the virus of TGR5, the quantity of P65 protein expression in CFs-v(+) group was less than CFs group, P<0.05; Activated the TGR5 receptor, the quantity of P65 protein expression in Ang ?+CFs-v(+)+Nomilin group was less than Ang ?+CFs-v(+) group, P<0.05. 5. The results of ELISA:(1)Infected by TGR5 virus, the concentration of c AMP in CFs-v(+) group was great than CFs group; Activation of TGR5, the concentration of c AMP in Ang ?+CFs-v(+)+ Nomilin group was great than Ang ?+CFs-v(+) group, P < 0.05.(2)Induced by Ang II, the secretion of collagen ?/? in Ang ?+CFs-v(+) group was great than CFs-v(+) grop, P<0.05; Infected by TGR5 virus, the secretion of collagen ?/? in CFs-v(+) group was less than CFs grop, P<0.05; Activated the TGR5 receptor, the secretion of collagen ?/? in Ang ?+CFs-v(+)+Nomilin group was decreased significant than Ang ?+CFs-v(+) group, P<0.05.Conclusion:1. The expression of TGR5 mRNA in neonatal mice CFs is quite few. 2. TGR5 activation can increase the concentration of c AMP, which can reduce the translocation of P65 in nuclear. 3. TGR5 activation can suppress the proliferation of CFs and reduce the collagen secretion of CFs induced by Ang ?, the possible mechanism is thro?gh the NF-?B signaling pathway. |
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