| Myocardial fibrosis(MF),characterized by excessive deposition of extracellular matrix(ECM),is a pathological response to a variety of cardiovascular disease(CVD).Myocardial fibrosis is involved in the development and progression of most CVD,therefore,blocking,inhibition or even reversal of myocardial fibrosis is a major target for the treatment of CVD.Activated cardiac fibroblasts(CFs)have been identified as the major ECM-producing cells,play a key role in myocardial fibrosis.Multiple cytokines and signal transduction pathways are involved in the activation of CFs,but the exact mechanisms are still largely unknown.Recently,scholars have focused on the correlation between nuclear transcription factors and CFs activation in the cardiac fibrogenesis.Recent study showed that orphan nuclear receptor Nur77 can negatively regulate various fibrotic diseases which indicating that Nur77 may be a potential target for anti-cardiac fibrosis.Scutellarin is a flavonoid monomer with antioxidant,anti-tumor,anti-inflammatory and other pharmacological activities.It has been reported that scutellarin can inhibit cardiac fibrosis induced by myocardial infarction in rats through ERK signaling pathway,but the exact cellular and molecular mechanisms have not been fully clarified.Our previous study found that Hyperoside induced the expression of Nur77 in vascular smooth muscle cells through ERK/CREB pathway and inhibited its proliferation.The preliminary experiments showed:(1)Nur77 was highly expressed in cardiac fibrosis tissue and activated primary CFs.(2)Nur77 expression was up-regulated in normal cardiac tissue and quiescent primary CFs upon SCU.Based on above researches and previous reports we hypothesized:(1)The orphan nuclear receptor Nur77 is involved in regulating the function of CFs and the formation of cardiac fibrosis.(2)SCU exhibits an anti-cardiac fibrosis effect by regulating Nur77.Objective:(1)To study the effect of regulating Nur77 on the function of CFs.(2)To investigate the effect of SCU on the function of CFs and cardiac fibrosis and illuminate the potential molecular mechanism.It will provide the new ideas for finding new targets of anti-cardica fibrosis and develop promising therapeutic drug of cardiac fibrosis.Methods:(1)ANG ? induced cardiac fibrosis in mice,and ANG ? sitimulated the primary CFs.Western blot,RT-q PCR,immunohistochemistry and Immunofluorescence detected the protein and m RNA expression of ?-smooth muscle actin(?-SMA),Collagen ?(Col ?),Collegen ?(Col ?)and NR4 A subfamily members in cardiac fibrosis tissuses and CFs.(2)Ad Nur77 adenovirus and Nur77 si RNA were constructed and transfected into CFs,respectively.CCK-8 asssy and cells number counting,transwell and wound-healing assays,western blot,RT-q PCR and flow cytometry were used to detect the proliferation,migration,phenotypic transformation,the level of ?-SMA,collagen synthesis,apoptosis and cell cycle in primary CFs.(3)ANG ? and different dose of SCU stimulated the primary CFs knockdown Nur77 or not.CCK-8 assay,transwell,immunofluorescence and RT-q PCR were used to detect the proliferation,migration,phenotypic transformation and expression of fibrosis-related genes in primary CFs.Western blot was used to determine the level of Nur77 and p-Nur77 protein in the cells.MAPK inhibitor was used to stimulate the cells,and Western blot was used to measure the level of Nur77 and p-Nur77 protein.Western blot analyzed the level of total or phosphorylated ERK1/2 and p38 MAPK in primary CFs or the cells knocking down Nur77.(4)(1)Male C57BL/6 mice were randomly divided into four groups: control group,ANG ? model group,SCU treatment group and SCU control group.Myocardial pathological change and collagen deposition in mice as shown by HE or Masson's trichrome staining.Western blot,RT-qPCR and immunohistochemistry measured the level of Col I,Col ?,a-SMA,Nur77 and p-Nur77 m RNA and protein in cardiac tissue.(2)Injection of Nur77 interfered with lentiviruses or NC-controlled lentiviruses in mice heart,and the ANG ?-induced myocardial fibrosis model was established.Heart tissue paraffin sections were subjected to Masson's trichrome staining analysis.Western blot,immunohistochemistry and RT-q PCR were used to determine the m RNA or protein level of Col ?,Col ?,a-SMA and Nur77 in cardiac tissue.Results:(1)The expression of Nur77 was up-regulated in cardiac fibrosis and primary CFs activated by ANG ?,(2)Overexpression of Nur77 inhibited the proliferation and migration of primary CFs,decreased the protein level of PCNA,Cyclin D1,Col ?,Col ? and a-SMA,there was no significant effect on apoptosis.The Nur77 knockdown in CFs could promote cell proliferation and migration,enhance the expression of fibrosis-related genes Col ?,Col ? and a-SMA m RNA,reduce cell apoptosis,decrease the number of G0-G1 phase cells,and increase the number of S and G2 phase.(3)(1)SCU inhibited the proliferation,migration and phenotypic transformation of primary CFs induced by ANG ?,and suppressed the expression of Col ?,Col ? and a-SMA m RNA.Nur77 knockdown attenuated the above effects.(2)50 ?M SCU stimulated the cells for 12 hr,the Nur77 protein level was the highest;SCU had no effect on p-Nur77 level;(3)SCU could inhibit Nur77 phosphorylation induced by ANG ?;(4)ERK1/2 and p38 MAPK inhibitors could suppress the expression of Nur77 in cells upon ANG ? and SCU;(4)SCU dose-dependently decreased ANG ?-induced phosphorylation of ERK1/2 and p38 MAPK.(4)(1)SCU can alleviate ANG ?-induced fibrosis in mice,inhibit the expression of Col ?,Col ?,a-SMA protein and m RNA expression;Nur77 knockdown,the above effects weakened;(2)SCU decreased the level of p-Nur77 in cardiac fibrosis tissue.(3)SCU inhibited the phosphorylation of ERK1/2 and p38 MAPK in cardiac fibrosis tissue by ANG ?.Conclusion:(1)Nur77 was highly expressed in cardiac fibrosis tissue and activated primary CFs by ANG ? in mice.(2)Nur77 can negatively regulate the function of primary CFs.(3)SCU can inhibit proliferation,migration and phenotypic transformation of primary CFs,and reduce the synthesis of ECM.(4)SCU can inhibit ANG ?-induced cardiac fibrosis in mice.(5)The molecular mechanism is at least through the ERK1/2/p38 MAPK pathway altering the expression of Nur77 and inhibiting the phosphorylation of Nur77.In conclusion,this study preliminarily elucidates the role of Nur77 in the regulation of CFs function,partially clarifies the molecular mechanism of SCU regulating Nur77 inhibiting CFs activation and its anti-fibrotic effects.These data will help to understand the molecular mechanism of cardiac fibrosis comprehensively,and provide a novel idea for developing new anti-cardiac fibrosis drug. |
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