The Study Of The Effect Of Total Glucosides Of Paeony, Agonist And Antagonist Of α Adenoreceptor On The Rat Hepatic Stellate Cells

OBJECTIVE To investigate the anti-chemical hepatofibrotic effects of total glucosides of paeony (TGP) on CC14-induced hepatic fibrosis model were observed in vivo. Isolate hepatic stellate cell (HSC)from normal rats and model rats. Study the effects of TGP,noradrenaline (NA),Phentolamine on HSC synthesis extracellular matrix (ECM),and apoptosis, Study the effects of TGP,NA on the expression of typeâ… collagen.Study the influence TGP,NA,Phentolamine in HSC,for the further research of exploring the mechanism of hepatic fibrosis.METHODS The model of rat chemical hepatic fibrosis was prepared by back injection of carbon tetrachloride. The serum activities of alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP), albumin(ALB), total protein(TP) were assayed by HITACHI 7060 automatic biochemistry detection instrument. The serum Concentration of hyaluronic and acid (HA),Procollagen Typeâ…¢(PCâ…¢) levels, the levels of HA and PCâ…¢releasing from the cultured hepatic stellate cell (HSC) was determined by radio immunoassay method. HSC isolated from were exposed to TGP and determined by Flow Cytometer, The expression of collagen typeâ… (Colâ… ) mRNA were detected by real time polymerase chain reaction (RT-PCR).Hepatic tissue sections were stained with hematoxylin-eosin and examined under a light microscope, after 48 h of HSC co-cultured with drugs,the morphrage changes of HSC creeped on the microscope slides were observed with light microscope.RESULTS1.Effects of TGP on serum values examination the model of rat chemical hepatic fibrosis induced by carbon tetrachlorideTGP (40 mg·kg^-1,80 mg·kg^-1,160 mg·kg^-1, ig) had obvious protective effects on CCl₄-induced hepatic fibrosis in rats. The results showed that the serum ALT, AST,HA,PCâ…¢remarkably decreased and the level of total protein,A /G ratio increased. TGP (40 mg·kg^-1,80 mg·kg^-1,160 mg·kg^-1, ig)group, hepatic cells degeneration and necrosis was in slight extent, mitochondria membrane and cristedis resumed, dilatation of the endoplasmic reticulum decreased and faciliated the regeneration of the hepatocyte, retard the forming of hepatic fibrosis. These indicate that TGP has protective effect on hepatic cells directly and decrease the ECM production of hepatic fibrosis in rats. TGP (40 mg·kg^-1,80 mg·kg^-1,160 mg·kg^-1, ig) had obvious protective effects on CCl₄-induced hepatic fibrosis in rats.2.Effects of TGP,NA,Phentolamine on the HSC synthesis HA and PCâ…¢in vitro.After 48 h of HSC co-cultured with drugs in vitro, compare to HSC from normal group rats, the levels of HA and PCâ…¢in HSC from model group significantly increased. Compare to model control group, the levels of HA and PCâ…¢in HSC co-cultured with of HSC from model group significantly increased. Compare to model control group, the levels of HA and PCâ…¢of HSC co-cultured with TGP,Phentolamine, TGP combinated Phentolamine decreased significantly,while the NA had significantly increased,TGP combinated NA had no significant difference.3. Effects of TGP,NA,Phentolamine on the apoptosis of HSC in vitro.After 48 h of HSC co-cultured with drugs in vitro, compare to HSC from normal group rats, the apoptosis rate of HSC from model group significantly increased.Compare to model control group, the apoptosis rate of HSC co-cultured with TGP,Phentolamine,TGP combinated Phentolamine,TGP combinated NA decreased significantly,while the apoptosis rate of HSC co-cultured with NA had no significant difference.4.Effects of TGP and NA on the expression of Colâ… mRNA in HSC.The results showed that NA promoted the expression of Colâ… mRNA,and the effects were inhibited by TGP.5. Effects of TGP on liver pathological examination, and effects of TGP,NA,Phentolamine on the morphrage changes of HSC.In TGP (40 mg·kg^-1,80 mg·kg^-1,160 mg·kg^-1, ig)group, hepatic cells degeneration and necrosis was in slight extent, mitochondria membrane and cristedis resumed, dilatation of the endoplasmic reticulum decreased and faciliated the regeneration of the hepatocyte, retard the forming of hepatic fibrosis. Compare to HSC in the model control group, HSC co-cultured with TGP,Phentolamine,TGP combinated Phentolamine grew rarefaction, and some HSC shrinked,while HSC co-cultured with NA creeped close over the glass slides and grew better, HSC co-cultured wih TGP combinated NA creeped on much area of the glass slides,grew worse,with a little HSC shrinked.CONCLUSIONS1. In our study,TGP (40 mg·kg^-1,80 mg·kg^-1,160 mg·kg^-1, ig)had obvious protective effects on CCl₄-induced hepatic fibrosis in rats. The results showed that the serum ALT, AST,HA,PCâ…¢remarkably decreased and the level of total protein,A /G ratio increased.TGP (40 mg·kg^-1,80 mg·kg^-1,160 mg·kg^-1, ig)group, hepatic cells degeneration and necrosis was in slight extent, mitochondria membrane and cristedis resumed, dilatation of the endoplasmic reticulum decreased and faciliated the regeneration of the hepatocyte, retard the forming of hepatic fibrosis indicate that TGP has protective effect on hepatic cells directly and decrease the ECM production of hepatic fibrosis in rats. TGP (40 mg·kg^-1,80 mg·kg^-1,160 mg·kg^-1, ig) had obvious protective effects on CCl₄-induced hepatic fibrosis in rats.2. In our study, after 48 h of HSC co-cultured with drugs in vitro, compare to HSC from normal group rats, the levels of HA and PCâ…¢in HSC from model group significantly increased. Compare to model control group, the the levers of HA and PCâ…¢of HSC co-cultured with TGP had significantly increased. TGP probabaly via mechanisms of inducing HSC into apoposis,inhibiting the HSC synthesis HA and PCâ…¢to play an anti-fibrogenesis role.3. In our study,NA group can increase the levels of HA and PCâ…¢releasing fromHSC, the levels of HA and PCâ…¢of HSC co-cultured with TGP combinated NA had no significant difference. The levels of HA and PCâ…¢of HSC co-cultured with Phentolamine,TGP combinated Phentolamine decreased significantly.The apoptosis rate of HSC co-cultured with Phentolamine,TGP combinated Phentolamine,TGP combinated NA decreased significantly,while the apoptosis rate of HSC co-cultured with NA had no significant difference. NA can increase HSC expression Colâ… mRNA. These effects show that TGP combinated NA dose dependent on inhibit HSC expression Colâ… mRNA. Theαadenoreceptor agonist NA can increase HSC synthesize ECM, increase HSC expression Colâ… mRNA. Theαadenoreceptor antagonist Phentolamine can decrease HSC synthesize ECM, increase HSC expression Colâ… mRNA. TGP combinated NA can decrease HSC synthesize ECM that caused by NA. The effects of TGP combinated Phentolamine had less difference than TGP and NA. Theαadenoreceptor (αAR) may participate in the mechanisms of modulating the apoptosis, HSC synthesis HA and PCâ…¢ECMs.