| As it is known to all that activated hepatic stellate cells (HSCs) play a central role in liver fibrogenesis, Sail et al thought it is hard to be inverted from activated HSCs to the quiescent ones. but apoptosis of activated the critical HSCs might be essential to clear HSCs from injured liver. At present, decreasing HSCs is the main rout to reverse liver fibrosis, therefore, It is a hot topic for HSCs to go in for the mechanism of the hepatic fibrosis and the anti-fibrotic agents researching. Cantharidin is the active ingredient of the blister beetle, and is an active ingredient of the traditional Chinease herbal medicine Mylabris. Norcantharidin (NCTD) is ademethylated from of cantharidin with anti-tumor properties, BEL-7402, human myeloid leukemia K562, HL60, Because of its anti-tumor activity, few side-effects and leukoc- ytosis, NCTD is reported clinically as an anti-tumor drug against esophageal, hepatoma. Meanwhile, NCTD also have anti-fibrotic effect in organ and tissue. NCTD was synthesized by furna and maleic anhydride Diels-Alder reaction. It is effective ininhibiting the proliferation of several tumor cell lines, including human hepatoma HepG2. The study has investgated that NCTD could prevent renal tubulo intertitial. However, No report have investigated the effect on hepatic stellate cells in vitro and in vivo. Therefore, the purpose of our present study was to investigate the effects of NCTD on proliferation and apoptosis of hepatic stellate cells and the possible mechanisim.Objective: The experiment mainly through different concentration of norcantharidin intervention rat HSCs in vivo to investigate the effect on proliferation, apoptosis, cell cycle, and stimutaniously the changes in mitoc- hondrial membrane potential and caspase3 activity.Methods: Proliferation and inhibitory effects of NCTD on activated hepatic stellate cell were measured by MTT test; Nuclear morphological changes were illustrated by staining with Hoechst33342 and were observed by fluorescence microscope; Cell apoptosis rate and cell cycle were analyzed using AnnexinⅤ-FITC/PI double-labled kit and propduim kit by flow cytometry. The changes in mitochondrial membrane potential were determ- ined with JC-1 dye laser confocal microscope and flow cytometry; Caspase3 activity was detected by microplate reader.Results:1 The effect of norcantharidin on HSCs proliferation:①the inhibition effect of different concentrations norcantharidin (0.625μg/ ml, 1.25μg/ml, 2.5μg/ml, 5.0μg/ml, 10.0μg/ml)on HSCs proliferation at different time: The results of MTT assay showed that A490 value overall mean of norcantharidin groups decreased compared control group which had obviously statistically significant differences, (P<0.05), 12h (0.876±0.02, 0.855±0.01, 0.819±0.02, 0.745±0.02, 0.638±0.02 vs 0.949±0.03, P<0.05), 24h(1.068±0.02, 1.007±0.02, 0.949±0.03, 0.881±0.02, 0.633±0.02 vs 1.148±0.01, P<0.05), 36h(1.238±0.02, 1.023±0.01, 0.958±0.02, 0.697±0.02, 0.567±0.01 vs 1.337±0.02, P<0.05). Hence, norcantharidin had inhibition effect on proliferation of HSCs in vitro, The inhibitory effect on HSCs was significantly in a time-and dose-depedent manner.②The result showed that the inhibion rate on HSCs proliferation also had a time-and-dose depedent manner. After different concentrations norcantharidin interval in HSCs for 12h, 24h, 36h, 12h(6.99%±3.77%, 9.85%±3.41%, 13.66%±3.02%,21.53%±2.67%,32.73%±3.72%),24h(6.96%±2.01%,12.64%±2.12%,17.36%±2.92%,23.25%±1.41%,44.85%±1.71%);36h(7.37%±1.64%,23.43%±2.29%,28.44%±2.88%,48.04%±2.85%,57.74%±1.66%),HSCs prolifer- ation was inhibited, the inhibition rate was reached to the peak at 36h; Obviously, HSCs proliferation could be inhibited by norcantharidin.2 The effect of 5.0μg/ml norcantharidin on HSC apoptosis:①The effect of 5.0μg/ml norcantharidin on HSC apoptosis rate at different times.We found that the condensed nuclei and the formation of apoptotic body in HSCs after 5.0μg/ml norcantharidin treatment for 12h, 24h, 36h, staining with Hoechest33342 by fluorescence microscopy. The results suggested that norcantharidin had effect on the rate of HSCs apoptosis. Compared control group(2.72%±0.25%) the overall mean of apoptosis rates of 12h, 24h, 36h group (5.31%±0.29%, 18.08%±0.15%, 25.45%±0.51%) had significantly difference(P<0.05), after HSCs treatment with 5.0μg/ml norcantharidin for36h, the apoptosis rate reached the peak.②Different concentration noncantharidin effection on cell cycle and apoptosis rate of HSCs by flow cytometry detecting: the cell cycle results indicated that a decrease in S phase cells and an increase in G2/M phase cells after norcantharidin treatment for 36h, with dose-dependent a decrease in S phase cells(30.76%±0.93%, 28.80%±1.15%, 27.73%±1.20% vs 42.17%±2.00 %, P<0.05) and an increase in G2/M phase cells (19.00%±0.56%, 23.03%±0.35%, 33.33%±0.72% vs 15.97%±0.45%, P<0.05), NCTD induced cell cycle arrest in G2/M phase. Meanwhile, With the same concentration groups and the same time, the apoptotic rates was also increasing in a dose-dependent maner The overall mean of the apoptotic rates with the different concentration norcantharidin have significantly elevated, Which have significantly difference compared with the control group(8.03%±0.50%, 16.10%±1.05%, 25.63%±1.12% vs 5.23%±0.83%, P<0.05), However, compared with NCTD 36h group (25.63%±1.12%), the apoptosis rate decreased in the Ac-EDVE-CHO group(19.60±0.70), (P<0.05). The resuls suggested that Ac-EDVE -CHO could inhibit the apoptosis effection induced by norcantharidin.③The effect of 5.0μg/ml norcantharidin on HSCs mitochondrial membrane potential: The membrane potential were detected by flow cytometry analysis method with JC-1kit. Compared with the negative control group(1.872±0.019). The membrane potential decreased after 5.0μg/ml norcantharidin group treatment for 6h, 12h, 24h?0.891±0.057, 0.591±0.010, 0.714±0.010) and the difference were significant(P<0.05).HSCs treated with 5.0μg/ml norcantharidin for 12h by JC-1 dye confocal laser scanning microscopy detecting mitochondrial membrane potential, Compared with negative control group(1.161±0.077) and positive one (0.795±0.061), the membrane potential declined obviously after 5.0μg/ml norcantharidin group(0.016±0.004) treatment for 12h. the difference of the mitochondrial membrane potential among three groups was significant (P<0.05).④Caspase3 activity analysis Showed that Caspase3 activity overall mean was statistically significant compared with the control group(0.325±0.015), after 5.0μg/ml norcantharidin treatment for 6h, 12h, 24h, 36h(0.657±0.03, 0.785±0.037, 1.771±0.177, 4.806±0.15) (P<0.05), the difference of caspase3 activity overall mean was statistically significant between the control group and norcantharidin group(P<0.05), we found that Caspase3 activity of incubated 36h group was one of the highest in the five groups, however, The caspase3 activity was decrease after Ac-EDVE-CHO(2.763±0.152) and noncantharidin treatment with for 36h compared with noncantharidin group(P<0.05). It was showed that noncantharidin could increase the activity of the HSCs in a time-dependent manner and the effection could be inhibited by Ac-EDVE-CHO.Conclusion:①Cell cultured in vitro studies have shown that, norcantharidin can inhibite HSCs proliferation.②Norcantharidin could induced the activated HSCs apoptosis,inhibited the HSCs growth and had effected on cell cycle.③Norcantharidin induce apoptosis of HSCs through decreasing mitocho- ndrial membrane potential enhance Caspase3 activity.
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