title: Aldosterone and TGF-β₁ synergistically increase PAI-1 expression in hepatic stellate cells of rats.
abstract: OBJECTIVE
  Aldosterone is related to the fibrosis of several organs, but the specific mechanism underlying the aldosterone induced hepatic fibrosis is still unclear.
  
  
  METHODS
  Separation, culture and identification of primary hepatic stellate cells (HSCs): The fluids and digestives used in the present study were able to completely remove blood cells, digest hepatocytes and matrix, and effectively separate HSCs. The in situ perfusion was performed at 2 steps: in situ perfusion with pre-perfusion fluid and ex vivo perfusion with enzyme-containing perfusion fluid. Influence of Ald on PAI-1 and Smad expressions in HSCs: cells were divided into control group, Ald group (10(-6) M), spironolactone (SPI) group and Ald+SPI group, and the mRNA and protein expressions of PAI-1 and Smad were detected. Ald induced type I collagen expression in HSCs: Immunohistochemistry was performed to detect type I collagen expression in the supernatant of control group, Ald group (10(-6) M), TGF-β1 group, and Ald+TGF-β1 group. Influence of Ald and TGF-β1 on PAI-1 expression in HSCs: cells were divided into control group, Ald group (10(-6) M), TGF-β1 group, and Ald+TGF-β1 group, and the mRNA and protein expressions of PAI-1 were determined by RT-PCR and Western blot assay, respectively. Synergistic effect of Ald and TGF-β1 on PAI-1 expression in HSCs: cells were divided into control group, Ald group (10(-6)), TGF-β1 group, Ald (10(-6) M)+TGF-β1 group, Ald (10(-7) M)+TGF-β1 group and Ald (10(-8) M)+TGF-β1 group, and the mRNA and protein expressions of PAI-1 were detected by RT-PCR and Western blot assay, respectively.
  
  
  RESULTS
  The survival rate, purity, markers and activation of HSCs were determined after separation. Influence of Ald on PAI-1 expression in HSCs: PAI-1 expression increased in HSCs of Ald group, SPI group and Ald+API group, and the PAI-1 expression in Ald group and Ald+SPI group was significantly higher than in control group (P<0.01). Influence of Ald on Smad expression in HSCs: Smad expression in Ald group, TGF-β1 group and ALD+TGF-β1 group was markedly higher than in control group (P<0.05). Smad expression in ALD+TGF-β1 group increased significantly when compared with Ald group (P<0.01). Ald induced type I collagen expression in HSCs: type I collagen expression in Ald group, TGF-β1 group and ALd+TGF-β1 group was dramatically higher than in control group (P<0.05), and it in ALd+TGF-β1 group was also significantly different from that in Ald group and TGF-β1 group (P<0.01). Synergistic effects of Ald and TGF-β1 on PAI expression in HSCs: PAI-1 expression in treated cells was markedly higher than in control group (P<0.01). PAI-1 expression in 10(-6) M Ald+5 ng/ml TGF-β1 group increased dramatically as compared to Ald group and TGF-β1 group (P<0.01), but the increased PAI-1 expression reduced after SPI treatment. Ald at different concentrations exerts synergistic effect with TGF-β1 to increase PAI-1 expression in HSCs: PAI-1 expression in HSCs after different treatments increased markedly as compared to control group (P<0.01). Significant difference in PAI-1 expression was observed in 10(-6) M Ald+50 pg/ml TGF-β1 group and 10(-6) M Ald group (P<0.01), PAI-1 expression in 10(-7) M Ald+50 pg/ml TGF-β1 group was significantly higher than in 50 pg/ml TGF-β1 group (P<0.01), but the PAI-1 expression in 10(-7) M Ald+50 pg/ml TGF-β1 group was similar to that in 10(-6) M Ald group (P>0.05).
  
  
  CONCLUSIONS
  Aldosterone is able to activate HSCs and increase PAI-1 expression during hepatic fibrosis, which may be inhibited by spironolactone. Aldosterone and TGF-β1 may synergistically act on HSCs to increase PAI-1 expression as compared to treatment with aldosterone or TGF-β1 alone. Aldosterone or TGF-β1 alone may slightly increase PAI-1 expression in HSCs, which can be inhibited by spironolactone.
authors.affiliation: Division of Gastroenterology and Institute of Digestive Disease, Tongji Hospital, Tongji University School of Medicine Shanghai 200065, China.
authors.forename: Sheng-Lan
authors.initials: SL
authors.lastname: Wang
authors.affiliation: Department of General Surgery, Tongji Hospital, Tongji University School of Medicine Shanghai 200065, China.
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Wu
authors.affiliation: Division of Gastroenterology and Institute of Digestive Disease, Tongji Hospital, Tongji University School of Medicine Shanghai 200065, China.
authors.forename: Cheng-Zhi
authors.initials: CZ
authors.lastname: He
authors.affiliation: Division of Gastroenterology and Institute of Digestive Disease, Tongji Hospital, Tongji University School of Medicine Shanghai 200065, China.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.affiliation: Division of Gastroenterology and Institute of Digestive Disease, Tongji Hospital, Tongji University School of Medicine Shanghai 200065, China.
authors.forename: Heng-Jun
authors.initials: HJ
authors.lastname: Gao
authors.affiliation: Division of Gastroenterology and Institute of Digestive Disease, Tongji Hospital, Tongji University School of Medicine Shanghai 200065, China.
authors.forename: Chang-Qing
authors.initials: CQ
authors.lastname: Yang
mesh_terms: D000450:Aldosterone / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004195:Disease Models, Animal; D055166:Hepatic Stellate Cells / Q000378:metabolism*; D007150:Immunohistochemistry; D008103:Liver Cirrhosis / Q000378:metabolism*; D008297:Male; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D053773:Transforming Growth Factor beta1; D000450:Aldosterone
keywords: Aldosterone; PAI-1; TGF-β1; hepatic stellate cells; spironolactone