Beraprost sodium attenuates the development of myocardial fibrosis after myocardial infarction by regulating GSK-3β expression in rats

Abstract

Objective: The aim of this study was to elucidate the mechanism of beraprost sodium (BPS) in the intervention of myocardial fibrosis after myocardial infarction (MI) through glycogen synthase kinase-3β (GSK-3β) and to provide new ideas for intervention in myocardial fibrosis.

Materials and methods: MI model rats given BPS and cardiac fibroblasts (CFs) treated with BPS and TGF-β. HE staining and Masson staining were used to detect the pathological changes of myocardial tissue. Fibrotic markers were detected by immunohistochemical staining. The expressions of GSK-3β, cAMP response element binding protein (CREB), and p-CREB were analyzed by qPCR and western blot analysis. EDU staining was used to detect the proliferation of CFs. The promoter activity of GSK-3β was detected by luciferase assay. Chromatin immunoprecipitation assay was used to detect the binding levels of GSK-3β promoter and Y-box binding protein 1 (YBX1). The levels of intracellular cyclic adenosine monophosphate (cAMP) were analyzed by enzyme-linked immunosorbent assay (ELISA).

Results: After operation, BPS improved myocardial fibrosis and upregulated GSK-3β protein expression in male SD rats. BPS can down-regulate α-smooth muscle actin (α-SMA) level and up-regulate GSK-3β protein expression in CFs after TGF-β stimulation. Furthermore, GSK-3β knockdown can reverse the effect of BPS on TGF-β-activated CFs, enhance α-SMA expression, and promote the proliferation of CFs. BPS could regulate GSK-3β expression by promoting the binding of GSK-3β promoter to YBX1. BPS induced upregulation of p-CREB and cAMP, resulting in reduced fibrosis, which was reversed by the knockdown of GSK-3β or prostaglandin receptor (IPR) antagonists.

Conclusion: BPS treatment increased the binding of YBX1 to the GSK-3β promoter, and GSK-3β protein expression was upregulated, which further caused the upregulation of p-CREB and cAMP, and finally inhibited myocardial fibrosis.

Keywords: YBX1/GSK-3β; beraprost sodium; cAMP/p-CREB; myocardial fibrosis; myocardial infarction.

Figure 1

Expression of GSK‐3β and myocardial histomorphology in rats with MI after BPS administration. The rats were divided into sham group, MI group and MI&BPS group. n = 6/group. (A) The pathological changes of myocardial tissue were detected by HE staining. Masson staining was used to detect the degree of myocardial fibrosis. (B) Fibrotic markers α‐SMA and Collagen I were detected by immunohistochemical staining. (C) The protein expressions of GSK‐3β in myocardial tissue were analyzed by Western blotting. *p ＜ .05 versus Sham group or MI group. BPS, beraprost sodium; MI, myocardial infarction.

Figure 2

BPS treatment interferes with GSK‐3β expression in CFs in vitro. (A) CFs were treated with TGF‐β (10 ng/mL) and BPS (0, 5, 10, and 20 μmol/L). Cell viability was detected by CCK‐8 assay. CFs were grouped into blank, TGF‐β, TGF‐β+BPS (10 μmol/L). *p  ＜ .05. (B) α‐SMA level was detected by immunofluorescence staining. (C) EDU was used to detect the proliferation of CFs. CFs were grouped into blank, TGF‐β, TGF‐β+BPS, TGF‐β+BPS+si‐NC, TGF‐β+BPS+si‐GSK‐3β. (D) Western blot was used to detect the expressions of GSK‐3β. (E) α‐SMA level was detected by immunofluorescence staining. (F) EDU was used to detect the proliferation of CFs. BPS, beraprost sodium; CFs, cardiac fibroblasts.

Figure 3

BPS treatment upregulates GSK‐3β promoter transcription in vitro. (A) qPCR was used to detect the mRNA levels of GSK‐3β in the myocardial tissues of sham group, MI group and MI&BPS group. *p ＜ .05 versus Sham group or MI group. (B) qPCR was used to detect the mRNA levels of GSK‐3β in CFs of blank group, TGF‐β group and TGF‐β+BPS group. (C) The promoter fluorescence reporter gene of GSK‐3β was constructed, and the promoter activity of GSK‐3β in CFs of blank, TGF‐β and TGF‐β+BPS groups was detected by luciferase assay. *p  ＜  .05 versus blank group or TGF‐β group. (D&E) The promoter activity of GSK‐3β in CFs transfected with OE‐YBX1, OE‐NC, si‐YBX1, or si‐NC were detected by luciferase assay. *p ＜ .05 versus OE‐NC group or si‐NC group. (E) ChIP assay was used to detect the binding levels of GSK‐3β promoter and YBX1. CFs were grouped into TGF‐β, TGF‐β+BPS, TGF‐β+BPS+si‐NC, TGF‐β+BPS+si‐YBX1. (F) The promoter activity of GSK‐3β was detected by luciferase assay. (G) ChIP assay was used to detect the binding levels of GSK‐3β promoter and YBX1 in CFs. *p ＜ .05 versus TGF‐β group or TGF‐β+BPS+si‐NC. CFs, cardiac fibroblasts; MI, myocardial infarction.

Figure 4

BPS treatment interferes with myocardial fibrosis through activation of prostacyclin receptor. CFs were grouped into TGF‐β, TGF‐β+BPS, TGF‐β+BPS+si‐NC, TGF‐β+BPS+si‐GSK‐3β. (A) Western blotting was used to detect the expressions of p‐CREB and CREB. The relative ratio of p‐CREB/CREB was detected. (B) The levels of intracellular cAMP in each group were analyzed by ELISA. *p  ＜  .05 versus TGF‐β or TGF‐β+BPS+si‐NC. CFs were grouped into blank, TGF‐β, TGF‐β+BPS, TGF‐β+BPS+CAY (1 μM). (C) α‐SMA level was detected by immunofluorescence staining. (D) EDU was used to detect the proliferation of CFs. (E) The levels of intracellular cAMP were analyzed by ELISA. (F) Western blotting was used to detect the expressions of GSK‐3β, p‐CREB, and CREB. The relative ratio of p‐CREB/CREB was detected. *p  ＜  .05 versus blank or TGF‐β+BPS+CAY. (G) Molecular mechanism diagram of BPS regulating the development of myocardial fibrosis after MI. BPS, beraprost sodium; MI, myocardial infarction.