Uric Acid Promotes Collagen Synthesis And Inflammatory Secretion Of Cardiac Fibroblasts Through MicroRNA-155

Objective: By observing the effects of different concentrations of uric acid on proliferation,collagen synthesis and inflammatory secretion in primary cultured neonatal rat cardiac fibroblasts(CFs),and the role of micro RNA-155(mi R-155)in it.Also to explore the molecular mechanism of myocardial fibrosis induced by uric acid and provide a new targeted therapy for myocardial fibrosis.Methods:(1)Separation and culture of CFs: Took out the hearts of Wistar rats aged 2-3days in sterile environment,and cut the ventricle into pieces of about 1mm3 in size.Tissue fragments were digested by trypsin and blown into cell suspension.CFs were obtained by differential attachment method in cell culture box.(2)MTT assay: primary CFs were cultured in 96-well plate for 24 hours,then stimulated with different concentrations of uric acid(0,200,400,600,800mol/L)for 24 hours.MTT assay was used to detect the effects of different concentrations of uric acid on the proliferation of CFs.(3)Hydroxyproline test: When primary CFs were cultured in good condition,they were inoculated in 6-well plate for 24 hours,and stimulated by uric acid(0,200,400,600,800mol/L)at different concentrations for 24 hours.Collagen content was measured by hydroxyproline test.(4)ELISA assay: When primary CFs were cultured in good condition,they were inoculated in 6-well plate for 24 hours,and stimulated by uric acid(0,200,400,600,800mol/L)at different concentrations for 24 hours.The contents of IL-6,IL-1β and TNF-α in the supernatant of cultured cells were detected by ELISA.(5)Cell transfection:Transfected with mi R-155 inhibitor to CFs.Selected the above-mentioned experimental results of stable uric acid concentration(600μmol/L)to stimulate CFs,The experiment was divided into uric acid group,uric acid + negative control group and uric acid +inhibitor group.The expression of mi R-155 in CFs was detected by fluorescent quantitative PCR.(6)The expressions of IL-6,IL-1β,TNF-α,Col-I and Col-III m RNA in CFs of uric acid group,uric acid + negative control group and uric acid + inhibitor group were detected by fluorescence quantitative PCR.(7)Western blot was used to detect the expression levels of IL-6,IL-1β,TNF-α,Col-I and Col-III proteins in CFs of uric acid group,uric acid + negative control group and uric acid + inhibitor group.Results:(1)The morphology of CFs was observed by inverted microscope.After 3 to 5days,CFs grew into a fusion state.The cells were large and thin,long spindle or polygonal,slightly transparent,closely arranged,overlapping and growing without spontaneous pulsation.(2)There was no significant difference in the proliferation ability of CFs between 200mol/L uric acid group and control group,but 400mol/L,600mol/L and 800mol/L increased significantly compared with normal group(P < 0.05).(3)There was no significant difference in collagen content of CFs between 200mol/L uric acid group and control group.OD value of 400mol/L,600mol/L and 800mol/L increased significantly compared with normal group(P < 0.05).(4)Compared with the control group,the levels of IL-6,IL-1β and TNF-α in other groups increased significantly(P <0.05).(5)After transfection,uric acid(600mol/L)stimulated the cells for 24 hours.The results of PCR showed that the relative expression of mi R-155 in uric acid + inhibitor group was significantly higher than that in uric acid group and uric acid + negative control group(P < 0.05).(6)The results of PCR showed that the inhibitor of mi R-155 partially inhibited the m RNA expression of IL-6,IL-1β,TNF-α,Col-I and Col-III induced by uric acid(600mol/L)(P < 0.05).(7)Western blot results showed that the inhibitor of mi R-155 partially inhibited the increase of IL-6,IL-1β,TNF-α,Col-I and Col-III protein expression induced by uric acid(600mol/L)(P < 0.05).Conclusion: The results showed that high concentration of uric acid could induce the proliferation of CFs,increase the synthesis of Col-I and Col-III collagen,increase the inflammatory secretion of IL-6,IL-1β and TNF-α,and mi R-155 participated in this process and played a role.Inhibition of mi R-155 could partly inhibit the effect of uric acid on CFs.