| Objective: The apoptosis disorder of activated HSC is the main reason for the irreverse of liver fibrosis. Isoquinoline alkaloids have significant cytotoxicity, can significantly induce tumor cells apoptosis. In order to retain the isoquinoline alkaloids pro-apoptotic activity and reduce its cytotoxicity, in collaboration with the Department of Chemistry, Fudan University, we have constructed a novel small molecule isoquinoline compound library. The purpose of this project is to screen out small molecule isoquino line compounds with low cytotoxicity, strong pro-apoptotic activity for anti-hepatic fibrosis from a novel class of small molecules isoquinoline library. Thereby hope to develop novel drugs with new lead compounds for anti-hepatic fibrosis.Methods: Screening small molecule compounds:(1) Classify small molecule compounds into different types by their structure which which constructed by Department of Chemistry, Fudan University.(2) The inhibition of HSCs-T6 propagation was determined by MTT after treated with 7 different concentrations of small molecule compounds for 24 h,then calculate the IC50 of different compounds.(3) Apoptotic bodys were characterized by fluorescence microscope to evaluate the effect on promoting apoptosis of the positive compounds.The explore of 12-4-Y pro-apoptotic mechanisms on HSCs:(1)The inhibition of HSCs-T6 propagation was determined by MTT after treated with 7 different 12-4-Y concentrations for 12h?24h?36h?48h.(2)Cell necrosis was determined by Trypan blue exclusion.(3)Apoptotic bodys were characterized by fluorescence microscope. Apoptosis rate was meas ured by flow cytometry(FCM).(4)Apoptosis key proteins and its active form were measured by western blotting.(5)Real time PCR tested on the expression of apoptotic related genes.Results:(1) MTT results suggested that six positive compounds(12-4-Y 12-1-Y, 09-4-Y, 15-1-Y, 11-2-Y, 09-3-Y) could inhibit the proliferation of HSC-T6 cells significantly and presented concentration dependent phenomenon. IC50 values of these 6 positive compounds are less than 100?g / ml.(2) Hoechst staining suggested that there was one compound showing obvious apoptotic bodies when treats with HSCs, that is 12-4-Y(1- ethyl-8-methoxy-5-phenyl-pyrazolo [5,1-a] isoquinoline).(3) Further,incubation of HSC-T6 with 5 to 80?g/ml 12-4-Y for different times,the cell proliferation rate reduce significantly depend on concentration and time. Trypan blue exclusion shows treating with 40?g/ml 12-4-Y for 12 hours the positive cell rate increased to(0.53±0.04)% significantly higher than that in the control group [(0.98±0.02)%,P<0.01].(4)At the concentration of 20?g/ml 12-4-Y for 24 h flow cytometry analysis showed that the apoptosis rate was( 29.23±1.20) %,signif icantly higher than that in the control group [(5.47±1.39)%,P<0.001] and Hoechst staining shows obvious apoptotic bodies when incubation HSC-T6.(5)Western blotting analys is shows that the expression of cleaved caspase3 of treat groups upregulated comparing with the control group.(6)Real time PCR suggested that incubation HSCs-T6 with 12-4-Y at the concentration of 10?g/ml?20?g/ml, the gene expression of Bax?caspase9 increased significantly which parcipate in mitochondria apoptosis pathway, however, the gene expression of Bcl2 reduced significantly which is inhibitor of apoptosis protein(P<0.05).Conclusions: There are six kinds of positive isoquinoline compounds from the “natural-like products” which can inhibit the proliferation of HSC-T6 cell line. At the same time, in which one kind of isoquinoline skeleton compound(12-4-Y) have significant role in promoting on apoptosis of HSC-T6 cell lines. The new isoquinoline compounds 12-4-Y significantly induced HSC apoptosis by activation of the mitochondrial apoptotic pathway. |
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