Effect Of Noredrenaline And Phentolamine In The Hepatic Stellate Cells Of Rat

OBJECTIVE: Through in vitro observation of norepinephrine (NA) and phentolamine ( PTL) on the impact of the activation of HSC-T6 cells, the article was designed to exp- lore the effect and mechanisms of NA and PTL in the rat hepatic stellate cells.METHODS: After HSC-T6 cells were recoved and cultured ,their proliferation was evaluated by MTT colorimetric assay. The levels of HA and PCIII released from the cultured rat hepatic stellate cells (HSC-T6) was determined by radioimmunoassay (RIA); the levels of cAMP of HSC-T6 cells was determined by enzyme linked immuno- sorbent assay(ELISA); the apoptosis of HSC-T6 cells was determined by flow cytometric analysis (FCM) ; The expression of TIMP-1,MMP-13 and NF-KBp65 protein were detected by immunocytochemical technique.RESULTS:1. Effect of noredrenaline and phentolamine in the proliferation and extracellular matrix of rat hepatic stellate cellsIn vitro models for proliferation and extracellular matrix of a cell strain named HSC-T6 cells stimulated with serum and cytokines(PDGF-BB,TGF-β1)respectively were established, which were measured by MTT and RIA . The dose-effect relationsh- ip of PTL and NA on proliferation,extracellular matrix and cAMP stimulated with serum and cytokines(PDGF-BB,TGF-β1)respectively were studied in vitro. the results were showed as the following(1)PTL(10-5,10-7,10-9mol/L)inhibited the proliferation and extracellular matrix production of HSC-T6 cells stimulated by 10%NBS, concen- tration dependently , on the contrary to NA; (2)PTL(10-5,10-7,10-9mol/L)concen- tration-dependently inhibited PDGF-BB(10μg.L-1)-driven proliferation and increased the contents of cAMP of HSC-T6 cells; PTL(10-5,10-7,10-9mol/L)concentration -dependently inhibited TGF-β1(2μg.L-1)-driven extracellular matrix production and in- creased the contents of cAMP of HSC-T6 cells, NA has opposite effect .2. Effect of PTL and NA on apoptosis of HSC-T6After HSC-T6 cells had co-cutured with drugs for 48h, compared to the control group, PTL increased the apoptosis of HSC-T6 cells, on the contrary to the NA.3. PTL and NA regulates the expression of MMP-13 and its inhibitor TIMP-1 in HSC-T6In vitro, PTL(10-5,10-7,10-9mol/L)enhanced MMP-13 expression but reduced TIMP-1 expression in HSC-T6 cells activated by serum,PDGF-BB and TGF-β1 as detected by immunostaining, NA has opposite effect.4. Effect of PTL and NA on NF-KBp65 in HSC-T6ICC showed that strong positive immunostaining for NF-KBp65 was located in the cytoplasm and nucleus of HSC-T6 cells. PTL decreased the immunostaining intensity in the cytoplasm and nucleus of HSC-T6 cells, as opposed to NA.CONCLUSIONS:1. PTL can suppress HSC-T6 cells proliferation and extracellular matrix production , the inhibitory effect of PTL on HSC-T6 cells function are related to its direct promotion on contents of cAMP and inhibition on PDGF-driven proliferation and TGF-driven extracellular matrix production throughα-AR.2. PTL can induce the apoptosis of HSC-T6 cells throughα-AR.3. PTL can regulate the abnormal expression of MMP-13 and TIMP-1 in HSC-T6 cells throughα-AR, and these may be one of the mechanisms of anti-hepatofibrotic effect of PTL.4. PTL can inhibit both content and activity of NF-KBp65 in HSC-T6 cells throughα-AR. the inhibitory effect of PTL on NF-KBp65 in HSC-T6 cells may suppress the proliferation and activation of HSC-T6 cells and induce its apoptosis.