Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-beta signaling.
OBJECTIVE
To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-beta (TGF-beta) signaling in vitro.


METHODS
HSCs were isolated from rats by in situ perfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 h with DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1, 2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscle-alpha-actin (alpha-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type III procollagen (PCIII) in supernatants were determined by radioimmunoassay. TGF-beta1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively.


RESULTS
Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while alpha-SMA, LN and PCIII expressions were inhibited. As estimated by gray values, the expression of alpha-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2% (control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P<0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCIII to 84.6%, 81.5%, 75.7% or 80.7% respectively (P<0.05 vs control), with no significant difference among tetrandrine groups. RT-PCR showed that TGF-beta1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-beta1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r=-0.755, P<0.01), and both were significantly correlated with alpha-SMA protein expression (r=-0.938, P<0.01; r=0.938, P<0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L) was confirmed by Western blotting as well.


CONCLUSIONS
Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-beta1 expression and signaling.
Chen
Yuan-Wen
YW
Digestive Disease Laboratory, Xinhua Hospital, Shanghai Second Medical University, 1665 Kongjiang Road, Shanghai 200092, China.
Wu
Jian-Xin
JX
Chen
Ying-Wei
YW
Li
Ding-Guo
DG
Lu
Han-Ming
HM
D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D044182:Benzylisoquinolines / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D051906:Smad7 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
D000470:Alkaloids; D000972:Antineoplastic Agents, Phytogenic; D044182:Benzylisoquinolines; D004268:DNA-Binding Proteins; D051906:Smad7 Protein; C497808:Smad7 protein, rat; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; C009438:tetrandrine