The Role And Mechanism Of FK228 In Renal Interstitial Fibrosis

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FK228 attenuates UUO-induced renal interstitial fibrosis in mice[Objective] To investigate the relationship between HDAC expression and renal fibrosis,and explore the role and mechanism of FK228 in inhibiting the development of renal fibrosis.[Methods] The unilateral ureteral obstruction(UUO)model,which was injected intraperitoneally with FK228,was established by using male C57BL/6 mice.The experiment was divided into three groups: Sham group,UUO group and UUO+FK228 group.The left kidneys were collected on day 7 after the left ureteral ligation.Pathological changes and fibrosis of the kidneys were assessed by HE staining and Sirius red staining.The protein and mRNA expression levels of ?-SMA,Collagen I and Fibronectin were detected by immunohistochemical staining,Western blot and real-time quantitative PCR.The mRNA levels of inflammatory cytokines in mouse kidneys were detected by real-time quantitative PCR.Activation of fibroblasts was evaluated by immunohistochemical staining of F-actin.The mRNA levels of HDAC1 and HDAC2 in mouse kidneys were measured by real-time quantitative PCR,and the protein levels of acetylated histone H3 of kidneys was detected by Western blot.[Results] Compared with the Sham group,the kidneys in UUO group developed obvious interstitial fibrosis,obvious renal tubular expansion and protein canal.The protein levels of ?-SMA,Collagen I,Fibronectin and F-actin of the kidneys significantly increased,and the expression of HDAC1 and HDAC2 also obviously increased.Compared with the UUO group,the treatment of FK228 significantly reduced the kidney damage and deposition of collagen in the interstitium.FK228 significantly reduced the degree of tubular dilatation and protein canal,and inhibited the expression of fibrosis-related proteins such as ?-SMA,Collagen I and Fibronectin in the kidneys(P<0.05).It was shown that the expression of F-actin was downregulated in the UUO+FK228 group,indicating that FK228 inhibited the activation of renal fibroblasts.Furthermore,the treatment of FK228 inhibited the expression of HDAC1 and HDAC2 and upregulated the expression of acetylated histone H3 in the kidneys(P<0.05).[Conclusion] HDAC1 and HDAC2 are highly expressed in UUO-induced renal interstitial fibrosis in mice.Treatment with FK228 inhibits the expression of HDAC1 and HDAC2,and induces histone H3 hyperacetylation.FK228 remarkably attenuates the development of renal fibrosis by inhibiting the expression of inflammatory cytokines,fibrosis-related proteins and the activation of fibroblasts.Part ? The effect and mechanism of FK228 on renal fibroblasts in vitro[Objective] To investigate the effect and mechanism of a type I HDAC inhibitor FK228 in inhibiting renal interstitial fibroblasts,and provide the experimental data for clinical practice.[Methods] Rat renal interstitial fibroblast cell line(NRK-49F)was cultured with 10% fetal bovine serum.Cells were plated in 96-well plate at 104 per well.The effect of different concentrations of FK228 on the proliferation of NRK-49 F cells were detected by CCK8.NRK-49 F cells were plated in six-well plate at 4×105 per well.There were six experimental groups: Control group,TGF-?1 group,TGF-?1+FK228(5n M)group,TGF-?1+FK228(8n M)group,TGF-?1+FK228(10n M)group and TGF-?1+FK228(15n M)group.After overnight incubation,the complete medium was replaced with 0.5% fetal bovine serum medium for 24 h.Then,the cells in the FK228 group were pre-treated with FK228 at varying concentrations(5n M,8n M,10 n M,15 n M)for 40 min.The cells were then treated with recombinant human TGF-?1 for 24 hours.The effect of FK228 on the apoptosis of NRK-49 F cells was analyzed by flow cytometry.The expression of fibrosis-associated proteins,HDAC1,HDAC2,acetylated histone H3 and cell cyclin proteins were detected by Western blot.The phosphorylation levels of Smad2/3,MEK1/2,ERK1/2,and P38 were measured to assess the effect of FK228 on TGF-?1/Smad,ERK1/2 and P38 MAPK signaling pathways in fibroblasts.[Results] After stimulation of NRK-49 F cells with TGF-?1,the expression of fibrosis-associated proteins significantly increased.FK228 significantly inhibited the expression of fibrosis-related proteins(P<0.05).The protein levels of HDAC1 and HDAC2 were significantly increased in TGF-?1-induced NRK-49F(P<0.05).FK228 significantly reduced the expression levels of HDAC1 and HDAC2 in NRK-49 F cells,while significantly induced histone H3 hyperacetylation(P<0.05).In addition,FK228 significantly inhibited the proliferation of NRK-49 F cells and promoted its apoptosis.The administration of FK228 downregulated the expression of Cyclin D1 and upregulated the expression of P21 and P27(P<0.05).Western blot results showed that FK228 significantly inhibited the expression of p-Smad2/3,p-MEK1/2,p-ERK1/2,and p-P38 in NRK-49 F cells(P<0.05).[Conclusion] FK228 promotes the acetylation of histone H3 by inhibiting HDAC1 and HDAC2.The administration of FK228 inhibits the proliferation of fibroblasts,promotes its apoptosis and inhibits the expression of fibrosis-associated proteins.They are regulated through blocking the TGF-?1/Smad,ERK1/2 and P38 MAPK signaling pathways.Part ? The effect and mechanism of FK228 on renal tubular epithelial cells in vitro[Objective] To investigate the effect and mechanism of type I HDAC inhibitor FK228 on renal tubular epithelial cells.[Methods] Rat renal tubular epithelial cell line(NRK-52E)was cultured with 10% fetal bovine serum.NRK-52 E cells were plated in six-well plate at 4×105 per well.There were six experimental groups: Control group,TGF-?1 group,TGF-?1+FK228(5n M)group,TGF-?1+FK228(8n M)group,TGF-?1+FK228(10n M)group and TGF-?1+FK228(15n M)group.After overnight incubation,the complete medium was replaced with 0.5% fetal bovine serum medium for 24 h.Then,the cells in the FK228 group were pre-treated with FK228 at varying concentrations(5n M,8n M,10 n M,15 n M)for 40 min.The cells were then treated with recombinant human TGF-?1 for 24 hours.After treatment with different concentrations of FK228,the expression of fibrosis-associated protein and HDAC1,HDAC2,acetylated histone H3 were measured by Western blot.And the phosphorylated levels of Smad2/3,MEK1/2,ERK1/2 and P38 were measured after the administration of FK228 followed by stimulating with TGF-?1.[Results] The expression levels of fibrosis-associated proteins,HDAC1 and HDAC2 in TGF-?1 group were significantly higher than those in Control group.After the administration of FK228 and TGF-?1,the expression levels of fibrosis-associated proteins were significantly inhibited by FK228 in a dose dependent manner(P<0.05).In addition, FK228 significantly reduced the expression of HDAC1 and HDAC2 in NRK-52 E cells,while induced the acetylation of histone H3(P<0.05).Furthermore,the treatment of FK228 significantly inhibited the expression of p-Smad2/3,p-MEK1/2,p-ERK1/2,and p-P38 in NRK-52 E cells(P<0.05).[Conclusion] FK228 inhibits the production of ECM and EMT of renal tubular epithelial cells by suppressing HDAC1 and HDAC2 production,increasing acetylated histone H3.Furthermore,FK228 inhibits the activation of TGF-?1/Smad,ERK1/2 and P38 MAPK signaling pathways in NRK-52 E.