| Objective:To explore the influence and significance of Nedd4 L gene on TGF-?1/Smad signaling pathway in rat hepatic stellate cells. Methods: 1. Optimal concentrations of plasmidsDifferent concentrations(50nmol/L,30nmol/L,10nmol/L,0nmol/L)of negative plasmid p EGFP-N1 or FAM-Negative si RNA with fluorescence were transfected into HSC-T6 via lipofectamine TM 2000 for 24 hours. The proportion of green fluorescence cells were observed and calculated under the inverted fluorescence microscope, and the appropriate concentration of plasmid was determined. 2. Effect of TGF-?1on Nedd4 L expression in HSC-T6First, different concentrations of TGF-?1(0 ng/ml, 1 ng/ ml, 2.5 ng/ml, 5 ng/ml) stimulate HSC-T6 for 24 hours, and then the relative expression of Collagen-?m RNA was assessed by RT-PCR.Then select the appropriate concentration of TGF-?1 to stimulate HSC-T6 for 0 hours, 12 hours, 24 hours, 48 hours, the relative expression of Collagen-?m RNA was detected by RT-PCR. To make sure that the best model of liver fibrosis in vitro was established.And at the same time, the Nedd4 L m RNA expression was detected by RT-PCR in different processing conditions. 3. Effect on TGF-?1/Smad signaling pathway in HSC-T6 by gene overexpression of Nedd4LThe HSC-T6 were divided into three groups: control group(TGF-?1 + liposome + equal amounts of serum-free and antibiotic-free DMEM); negative plasmid group(TGF-?1 + +negative plasmid liposome + DMEM medium); Nedd4 L overexpression group(TGF-?1 + liposome + Nedd4 L overexpression plasmid + DMEM culture medium),m RNA and protein expression of Nedd4 L protein,Collagen-?, Smad3, p-Smad2/3 were detected by RT-PCR and Western Blot assay after 24 hours of transfection. 4. Effect on TGF-?1/Smad signaling pathway in HSC-T6 by Si RNA of Nedd4LThe HSC-T6 were divided into three groups: control group(TGF-?1 + liposome + equal amounts of serum-free and antibiotic-free DMEM); negative plasmid group(TGF-?1 + liposome + FAM-Negative si RNA + DMEM culture liquid); Nedd4 L si RNA interference group(TGF-?1 + liposome + + Nedd4 L si RNA + DMEM culture medium), m RNA and protein expression of Nedd4 L protein,Collagen-?, Smad3, p-Smad2/3 were detected by RT-PCR and Western Blot assay after 24 hours of transfection. Results: 1. Detection of Transfection EfficiencyAfter 24 hours of transfection, green fluorescent signals were seen in group of 50,30,10nmol/L of negative plasmid p EGFP-N1 or FAM-Negative si RNA,no fluorescent signal was detected in the control group. Plasmid transfection efficiency in group of 50mmol/L, of 30mmol/L p EGFP-N1 or FAM-Negative si RNA was significantly higher than 10nmol/L plasmid(38% vs 34% vs 19%; 33% vs 35% vs 23%),significant difference between group of 50 mmol/L and group of 30 nmol/L was not displayed in therms of transfection efficiency, however, part death of the cells were observed in group of 50 mmol/L. Therefore, the optimal transfection concentration of p EGFP-N1 or FAM-Negative si RNA was 30nmol/L. 2. Effect of TGF-?1on Nedd4 L expression in HSC-T6(1) Relative expression levels of Collagen-?m RNA increases in a dose-based manner after 24 hours of 0ng/ml,1ng/ml,2.5ng/ml,5ng/ml of TGF-?1 stimulation on HSC-T6,and by pairwise's comparison, there were significant differences(P<0.05)besides that between 2.5 ng/ ml group and 5ng/ml group.(2) Relative expression levels of Collagen-?m RNA increases in a time-based manner after 2.5ng/ml TGF-?1 stimulation on HSC-T6 for 0 hours, 12 hours, 24 hours, 48 hours,and by pairwise's comparison, there were significant differences(P<0.05) besides that between 24 hours group and 48 hours group.(3) Relative expression levels of Nedd4 L m RNA decreases in a dose-based manner after 24 hours of 1ng/ml,2.5ng/ml,5ng/ml of TGF-?1 stimulation on HSC-T6,and there were significant differences between different concentrations and control group(P<0.05).(4) Relative expression levels of Nedd4 L m RNA decreases in a time-based manner after 2.5ng/ml TGF-?1 stimulation on HSC-T6 for 12 hours, 24 hours, 48 hours, and there were significant differences between different times and control group(P<0.05). 3. Effect on TGF-?1/Smad signaling pathway in HSC-T6 by gene overexpression of Nedd4L(1) RT-PCR results and Western Blot results were shown below: Relative expression level of Nedd4 L m RNA and protein in Nedd4 L overexpression group was significantly higher than those in the control group(28.65±0.94 vs 1;0.69±0.06 vs 0.36±0.01), and statistical differences were displayed(P<0.05);while no statistical difference were showed between negative plasmid group and control group( 0.83±0.16 vs 1; 0.38±0.03 vs 0.36±0.01).(2) RT-PCR results and Western Blot results were shown below: Relative expression level of Collagen-?m RNA and protein in Nedd4 L overexpression group was significantly lower than those in the control group(0.41±0.25 vs 1; 0.35±0.16 vs 0.68±0.04), and statistical differences were displayed(P<0.05);while no statistical difference were showed between negative plasmid group and control group(1.19±0.07 vs 1; 0.71±0.02 vs 0.68±0.04).(3) Western Blot results were shown below:Relative expression level of Smad3 protein among the three groups(0.79±0.03,0.75±0.03,0.78±0.04) were not significantly different(P>0.05);relative expression level of p-smad2/3 protein in Nedd4 L overexpression group(0.17±0.08)was significantly lower than that in control group(0.28±0.04),and statistical differences were displayed(P<0.05);while no statistical difference were showed between negative plasmid group and control group(0.27±0.01 vs 0.28±0.04). 4. Effect on TGF-?1/Smad signaling pathway in HSC-T6 by Si RNA of Nedd4L(1) RT-PCR results and Western Blot results were shown below: Relative expression level of Nedd4 L m RNA and protein in Si RNA Nedd4 L group was significantly slower than those in the control group(0.29±0.05 vs 1; 0.25±0.05 vs 0.36±0.01), and statistical differences were displayed(P<0.05);while no statistical difference were showed between negative plasmid group and control group(0.83±0.16 vs 1;0.38±0.03 vs 0.36±0.01).(2) RT-PCR results and Western Blot results were shown below: Relative expression level of Collagen-?m RNA and protein in Si RNA Nedd4 L group was significantly higher than those in the control group(3.32±0.39 vs 1;1.39±0.07 vs 0.68±0.04), and statistical differences were displayed(P<0.05);while no statistical difference were showed between negative plasmid group and control group(1.19±0.07 vs 1; 0.71±0.02 vs 0.68±0.04).(3) Western Blot results were shown below:Relative expression level of Smad3 protein among the three groups(0.77±0.01?0.75±0.03 ?0.78±0.04)were not significantly different(P>0.05);relative expression level of p-smad2/3 protein in Si RNA Nedd4 L group(0.35±0.02)was significantly higher than that in control group(0.28±0.04),and statistical differences were displayed(P<0.05);while no statistical difference were showed between negative plasmid group and control group(0.27±0.01 vs 0.28±0.04). Conclusions:1.Exogenous stimulation of TGF-?1 may reduce expression of endogenous Nedd4L-m RNA, the mechanism is worthy of further studying.2.Nedd4 L can regulate TGF-?1 signaling pathway by degrading p-Smad2/3 protein.3.Nedd4 L gene can induce decreased synthesis of Collagen-?by inhibiting phosphorylated Smad2/3 in HSC-T6, suggesting that Nedd4 L could serve as a target for reversing liver fibrosis. |
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