Effect Of Curcumin On Proliferation And Apoptosis Of Hepatic Stellate Cells

Objective1. To improve the method of HSC isolation.2. To investigate the effect of curcumin on proliferation and apoptosis of HSC in vitro.MethodsSD rats were treated with Cl2MDP intravenously. HSCs of these rats were isolated with collagenase perfusion in situ and density gradient centrifugation. The cell viability was determined by trypan blue exclusion staining. The purity of HSC was identified by the expression of Desmin using immunocytochemistry method. Endogenous peroxidase staining was used to detect kupffer cells.HSC-T6 was incubated with different concentrations of curcumin. The effect of curcumin on cell proliferation was observed by MTT colormetric assay. HSC cell cycle was analyzed by flow cytometry (FCM). HSC apoptosis was detected by FCM, transmission electron microscopy (TEM) and agarose gel electrophoresis.ResultsThe yield rate of HSC was 2X 107 -3 X 107 per rat. The cell viability was more than 95%. HSC emitted a characteristic rapidly fading bluegreen fluorescence at an excitation wavelength of 328 nm. The desmin positive cell rate was 90%. No endogenous peroxidase positive cells were detected.Administration of 20~100umol/L curcumin could significantly inhibit HSC proliferation in dose-dependent manner compared with the control group (p<0.05). After treated with curcumin at concentrations of 20, 40, 60 mol/L for 24h, the number of HSC in G2/M phase increased and the number of HSC in S phase decreased (p<0.05). Apoptosis index (%) were 15.3+1.88, 26.7 + 2.79, 37.6 + 4.38respectively, which were significantly higher than that of control group (1.9 0.64, P<0.01). Cell shrinkage, chromatin condensation and (or) ranked along inside of nuclear membrane and apoptosis body could be found by TEM. An oligonucleosomal DNA ladder of curcumin-treated cells was demonstrated. Incubated for 12h,24h,36h and 48h at the same concentration of 40 u mol/L, apoptosis index (%) were 12.0 + 2.35,26.7 + 3.47,33.8+1.78 and 49.3+1.56 respectively, which were significantly higher than that of control groups (P<0.01).Conclusions1. An improved method for the isolation of HSC was developed without kupffer cells confusion.2. Curcumin could make HSC cell cycle arrest in G2/M phase.3. Curcumin significantly inhibited HSC proliferation and induced apoptosis in dose and time-dependent manner.