5‐HT2 and 5‐HT2B Receptor Antagonism Regulates Fibrotic Potential of Peritoneal Fibroblasts in CAPD by Targeting TGF‐β1‐Src‐ERK1/2‐STAT3 Pathway

Abstract

Peritoneal fibrosis (PF) results in ultrafiltration failure in continuous ambulatory peritoneal dialysis (CAPD) patients. 5‐hydroxytryptamine (5‐HT; serotonin) induces extracellular matrix (ECM) synthesis in peritoneal fibroblasts in a transforming growth factor beta 1 (TGF‐β1) dependent manner. We evaluated anti‐fibrotic role of inhibitors of 5‐HT2 (Terguride) and 5‐HT2B (SB204741) in human peritoneal fibroblasts (HPFB) of CAPD patients. Peritoneal fibroblasts isolated fromCAPD patients (n=6) and controls (n=8), were incubated with 5‐HT (1μM)/TGF‐β1 (10ng/ml) for 1 hour and later with 5‐HT (1μM)/TGF‐β1 (10ng/ml) and terguride or SB204741 (1μM, each) for 24 hours (Post‐treatment strategy). In pre‐treatment strategy, cells were pre‐treated with terguride or SB204741 (1μM, each) for 1 hour and later with only 5‐HT (1μM)/TGF‐β1 (10ng/ml) for 24 hours. Real time PCR for pro‐fibrotic TGFB1, Type 1 collagen (COL1A1), Type 2 collagen (COL1A2), Actin alpha 2 (ACTA2), Connective tissue Growth factor (CTGF) and Fibronectin 1 (FN1) and anti‐fibrotic genes Matrix mettaloproteinase‐2/Tissue inhibitor of metalloproteinase (MMP2/TIMP1) expression was performed. Type I collagen and α‐SMA, phosphorylation status of Smad‐3, ERK1/2, Src and STAT‐3 was examined by western blotting.In 5‐HT/TGF‐β1stimulated HPFB, upregulated pro‐fibrotic gene expressionwas observed, which significantly reduced on co‐culture with 5‐HT2/5‐HT2B inhibitors, with no effect on anti‐fibrotic genes mRNA expression. In 5‐HT stimulated HPFB, treatment with both 5‐HT inhibitors decreased type 1 collagen and α‐SMA with reduced ERK1/2 phosphorylation (figure 1), however, Smad‐3 phosphorylation remain unaltered (figure 2). In 5‐HT/TGF‐β1 simulated HPFB, 5‐HT inhibitors decreased STAT3 phosphorylation, without affecting Src phosphorylation. 5‐HT receptor antagonists attenuate 5‐HT/TGF‐β1 mediated pro‐fibrotic potential of HPFB by suppressingTGF‐β1‐Src‐ERK1/2‐STAT3 pathway.Support or Funding InformationThis project received funding from intramural grant(Grant No ‐ 2017‐1‐IMP‐95) of Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India.