Effects And Mechanisms Of NS-398 On The Proliferation And Apoptosis Of Hepatic Stellate Cell

Objective1. To investigate the effect ofNS-398 on proliferation and apoptosis of HSC invitro.2. To explore the possible mechanism of NS-398 in inhibiting HSCproliferation and inducing apoptosis of HSC by studying the expressions ofCOX-2 and PCNA proteins and apoptosis-regulating proteins P53 and Bcl-2on HSC.3. To investigate the effect of NS-398 on the synthesis of collagen I of HSC.MethodsHSC was incubated with different concentrations of NS-398. The effect ofNS-398 on cell proliferation was observed by MTT colormetric assay. HSC cell cyclewas analyzed by flow cytometry (FCM). HSC apoptosis was identified by FCM andtransmission electron microscopy(TEM). Expressions of COX-2 and PCNA proteins,apoptosis-regulating proteins P53 and Bcl-2 on HSC after apoptosis induced byNS-398 were examined by immunohistochemical staining, collagen I in the culture ofHSC was detected by ELISA kit.ResultsAdministration of 20～160μmol/L NS-398 incubated with HSC could inhibitHSC proliferation significantly in dose-dependent manner compared with that ofcontrol group (P＜0.05). After treating HSC with NS-398 at concentration of 90, 120,150μmol/L for 48h, the number of HSC in G₂/M phase increased (P＜0.05); Apoptosisindex(%) of HSC were (10.5±0.015) %, (13.2±0.010) %, (17.3±0.012)% respect-ively, which were higher than that of control group(4.3±0.006)% (P＜0.01). Cellshrinkage, chromatin condensation and (or) ranked along inside of nuclear membraneand apoptosis body could be found by TEM; Incubated HSC with NS-398 120μmol/L for 48h, the positive expression rates of PCNA, P53 and Bcl-2 were significantdifferent from that of control group: (28.91±0.11)%vs (85.99±0.13)%, (82.06±0.14)% vs (14.06±0.24)%, (34.20±0.77) % vs (96.66±0.79) % respectively (P＜0.01).The positive expression rate of COX-2 protein was (13.80±0.43) %, there was nostatistic significance compared with that of control group (14.07±0.59) % (P＞0.05).The collagen I in the culture of HSC was decreased obviously compared with that ofcontrol group (P＜0.01)Conclusions1. NS-398 could significantly inhibit HSC proliferation and induce apoptosis indose manner.2. NS-398 could make HSC cell cycle arrest in G₂/M phase and decrease theexpression of PCNA protein on HSC, it was the possible mechanism ofNS-398 inhibited the proliferation of HSC.3. The mechanism of NS-398 to induce HSC apoptosis may be associted withthe up-regulating expression of P53 and down-regulating expression of Bcl-2on HSC.4. NS-398 could inhibit the synthesis of collagen I.