Preliminary Study Of The Effects Of Nerve Growth Factor On Hepatic Stellate Cells

Objective1. To investigate the effect of NGF on proliferation and apoptosis of HSC in vitro and explore the possible mechanism.2. To study the expressions of NGF and p75NTR proteins and apoptosis-regulating proteins Caspase-3, P53 and Bcl-2 of HSC after treated with NGF.3. To investigate the effect of NGF on the synthesis of collagen I and III by HSC.MethodsHSC was incubated with different concentrations of NGF. The effect of NGF on cell proliferation was observed by XTT colormetric assay. HSC cell cycle was analyzed by flow cytometry (FCM). HSC apoptosis was identified by FCM, acridine orange staining and transmission electron microscopy (TEM). Expressions of NGF and p75NTR were detected by immunofluorescence. The expressions of apoptosis-regulating proteins Caspase-3, P53 and Bcl-2 of HSC after apoptosis induced by NGF were examined by immunohistochemical staining. Collagen I and III in the supernate of medium secreted by HSC were detected by enzyme-linked immunosorbent assay.Results(100,200,400)ng/mL NGF incubated with HSC for 24h could inhibit proliferation of HSC(P<0.05) without concentration-dependent(P>0.05):XTT colormetric assay showed that optical density of NGF groups were 0.66±0.03, 0.69±0.03 and 0.66±0.03, respectively, but in control group it was 0.73±0.01. After treating HSC with NGF at the concentration of 100,200,400ng/mL, the number of HSC in G2 phase were (14.83±5.41)%, (14.73±2.50)% and (14.87±2.06)%, respectively. They were significantly higher than control group in which the number of HSC in G2 phase was (7.47±4.39)% (P<0.05). Apoptosis index of HSC was higher than that of control group [(22.36±9.51)% vs (5.88±1.36)%] afer theated with NGF (100ng/mL) detected by flow cytometry (P<0.05). Acridine orange staining showed fluorescence enhancement, dense concentration of dark green or yellowish green fluorescence, the shape of HSC became shrinkage and irregular in NGF group. Cell shrinkage, chromatin condensation and (or) ranked along inside of nuclear membrane and apoptosis body could be found by transmission electron microscope. When HSC was stimulated with 100ng/mL NGF for 24h, immunofluorescence assay showed that the average optical density of NGF increased significantly compared with the control group (6.53±1.40 vs 1.77±0.17) (P<0.05), while the expression of p75NTR was not significantly changed (3.52±0.36 vs 4.24±0.38) (P>0.05). After incubating with 100ng/mL NGF for 24h, the positive expression rates of P53 and Caspase-3 of HSC increased significantly compared with those of control group:(78.41±4.00)% vs (34.96±3.84)%, (39.26±1.57)% vs (9.27±1.01)%, respectively (P<0.05). The positive expression rate of Bcl-2 protein of HSC decreased significantly compared with that of control group [(18.12±1.38)% vs (91.53±2.98)%] (P<0.05). When HSC was incubated with (100,200,400)ng/mL NGF for 24h, the contents of collagen I in the supernate of medium were (3.27±0.41)ng/mL, (3.45±0.17) ng/mL and (3.88±0.71) ng/mL, respectively, which were less than that of the control group [(6.28±0.81)ng/mL](P<0.05); as well as collagen I in NGF groups the contents of collagen III were (3.05±0.52) ng/mL, (3.06±0.41) ng/mL and (2.98±0.48) ng/mL, respectively, which were also significantly less than that of the control group [(3.89±0.29) ng/mL] (P<0.05).Conclusions1. NGF could significantly inhibit the proliferation and induce the apoptosis of HSC.2. NGF could make HSC cell cycle arrest in G2 phase, it maybe the mechanism of NGF inhibited the proliferation of HSC.3. NGF could be used as an initiating factor and effect factor to increase the expression of NGF on HSC, but no significant effect on p75NTR expression.4. The mechanism of NGF to induce HSC apoptosis may be associted with the up-regulating expression of Caspase-3, P53 and down-regulating expression of Bcl-2 on HSC. 5. NGF could inhibit HSC from synthesizing collagenⅠand collagenⅢ.