Effects Of The Total Flavone Of Litchi Chinensis Sonn On Expressions Of NF-κB And α-SMA In TGF-β1 Activated Rat Hepatic Stellata Cells

Objective: To investigate the effects of total flavonoids of litchi chinensis sonn(TFL) on cells proliferation and the expressions of Nuclear Factor-kappa B(NF-κB) and α-Smooth Muscle Actin(α-SMA) in rat hepatic stellate cell T6(HSC-T6) activated by Transforming Growth Factor-β1(TGF-β1). Methods:HSC-T6 cells were treated by 0.25% Trypsin-EDTA and then were made to be single cell suspension by DMEM(10% FBS included) which were mixed with TGF-β1(5 ug/L).(1) MTT method was used to detect the proliferation of HSC-T6 cells. Cells were cultured in the 96 well plate and were treated by different groups, including TGF-β1 group, the control group(5‰ DMSO included, the same below), the different concentrations of TFL groups(80、160、320、640、800 mg/L TFL). Each group has three wells. At 24h、48h、72h times after cells treated, absorbance(A) value was measured respectively by enzyme standard meter at the 490 nm wavelength, and then the cell inhibition rate werecalculated; finally the subsequent experimental drug concentration and drug effect time were determined according to the half inhibitory concentration(IC50).(2) The hological changes of apoptosis of HSC-T6 cells were determined by Hoechst33258 fluorescent staining at the time of 48 h.(3) The expressions of Nuclear Factor-kappa B(NF-κB) andα-Smooth Muscle Actin(α-SMA) were detected by semi-quantitative PCR(for m RNA) and Western blot(for protein) assay. Cells were cultured in the 10 cm culture dish and were treated by different groups, including TGF-β1 group, the control group,different concentrations of TFL groups(125、250、500 mg/L TFL). After 48 h,related indicators were measured. Results:(1)The MTT assay showed that at the same treated time, with the increased concentrations of TFL, the cells A values were gradually decreased, and the cell inhibition rate were gradually increased.(2) HSC-T6 cells in the TFL250 group showed apoptosis. And apoptotic bodies appeared. Moreover, the chromatin gradually been splited into pieces.(3) The PCR assay showed that the expression levels of NF-κB m RNA(0.55±0.04) and α-SMA m RNA(0.36±0.05) in the TGF-β1 group and NF-κB m RNA(0.58±0.06) and α-SMA m RNA(0.41±0.03) in the control group were no significant differences. And there were no significant differences in the expression of NF-κB and α-SMA m RNA and protein between TFL125group(NF-κB m RNA(0.53±0.03)、α-SMA m RNA(0.35±0.06)), TGF-β1 group and control group. Compared with the control group, both the TFL250 group NF-κB m RNA(0.23±0.01)、α-SMA m RNA(0.22±0.02) and the TFL500 group(0.14±0.01)、α-SMA m RNA(0.18±0.02) in the NF-κB m RNA level and α-SMA m RNA level respectively had obvious difference(P<0.05).(4) The Western blot assay showed that compared with the control group NF-κB protein(0.46±0.06)and α-SMA protein(0.64±0.13), the TFL250 group both the NF-κB protein(0.28±0.07) and the α-SMA protein(0.38±0.01) respectively had obvious difference(P<0.05). And there were significant differences in the expression of NF-κB protein levels and α-SMA protein levels between the TFL 500 group NF-κB protein(0.19±0.01)、α-SMA protein(0.33±0.13) and the control group.Conclusions: TFL can inhibit TGF-β1-induced HSC-T6 cells proliferation and promote cells apoptosis, which can be involved in the inhibited expressions of NF-κB to anti liver fibrosis effects.