| Objective: 1. To investigate the impact of recombinant human interleukin-37 on proliferation and apoptosis of hepatic stellate cells(LX2-HSC) activated by transforming growth factor-beta 1(TGF-β1). 2. To investigate the impact of IL-37 on collagen I(Col-I) and collagen III(Col-III) secreted by LX2-HSC. 3. To investigate the anti-fibrotic mechanisms of IL-37.Methods:1. Cell culture: culturing LX2-HSC in the RPMI-1640 medium. 2. Grouping:(1) Blank control group(group A): cells cultured by RPMI-1640 medium only;(2) Experimental group(group B): cells cultured by TGF-β1(5ng/ml), without IL-37;(3)Experimental group(group C): cells cultured by TGF-β1(5ng/ml) and IL-37(100ng/ml);(4) Experimental group(group D): cells cultured by TGF-β1(5ng/ml) and IL-37 200ng/ml;(5)Experimental group(group E): cells cultured by TGF-β1(5ng/ml) and IL-37(400ng/ml);3. Measuring the absorbancy of the LX2-HSC to calculate proliferation inhibition rate. Measuring apoptosis rate. Detecting the expression of Col-I and Col-III in culture supernatants. The detection of proliferation was carried out by MTT assay when cells were cultured for 12 h, 24 h and 48h; The detection of apoptosis was carried out by flow cytometry technique; Using the same method to culture LX2-HSC, then the culture supernatants were harvested after the drug treatment, and using ELISA to analyze the content of Col-I and Col-III. 4. Statistical analysis: Using t test to compare the means of two independent samples; the differences among groups were compared by one-way ANOVA; the differences of any two groups were compared by LSD. The differences of result were statistically significant with P<0.05. Using SPSS17.0 to analyze all the data.Results: 1. Effect on the anti-proliferative of LX2-HSC: LX2-HSC cells were cultured by Human recombinant IL-37 and TGF-β1 after 12 h, 24 h and 48 h, the differences of absorbance between group A and group B were statistically significance at each time(P<0.05), the absorbance of group B was higher than group A; the absorbance of group C, group D and group E was lower than group B, the differences of absorbance between each group had statistically significance(P<0.05), and the absorbance of each group decreased and the anti-proliferative rate of each group increased with the increase of Human recombinant IL-37; When group C compared with group D and group E, the differences of absorbance and anti-proliferative rate had statistically significance at 12 h and 24h(P<0.05), but the differences of absorbance and anti-proliferative rate had no statistically significance at 48h(P>0.05). The differences of absorbance and anti-proliferative rate which in the same group were statistically significance at each time(P<0.05), and the absorbance decreased and the anti-proliferative rate increased with the increase of cultured time. 2. Effect on the cell apoptosis rate of LX2-HSC:The differences of cell apoptosis rate between group A and group B were statistically significance at each time(P<0.05), and the apoptosis rate of group B is lower than group A; the cell apoptosis rates of group C and group D and group E were higher than group B, the differences of cell apoptosis rate between each group were statistically significance(P<0.05), and the cell apoptosis rates of each group increased with the increase of Human recombinant IL-37; When group C compared with group D and group E, the differences of cell apoptosis rate were statistically significance at 12 h and 24h(P<0.05), but the differences of cell apoptosis rate had no statistically significance at 48h(P>0.05). The differences of cell apoptosis rate which in the same group had statistically significance at each time(P<0.05), and the cell apoptosis rate increased with the increase of cultured time. 3. Effect on the expression of Col-I and Col-III: The differences of expression between group A and group B had statistically significance at each time(P<0.05), and the expression of group B was more than group A; When group B compared with group C, group D and group E, the differences of expression had statistically significance(P<0.05), and the expression of Col-I and Col-III decreased with the increase of Human recombinant IL-37, but had no statistically significance in group C, group D and group E(P>0.05). The differences of expression among the groups had no statistically significance at each time(P>0.05).Conclusions:1. TGF-β1(5ng/ml) can promote the proliferation of LX2-HSC and increase the expression of Col-I and Col-III in culture supernatants.2. Recombinant human IL-37 can suppress the proliferation of LX2-HSC which is activated by TGF-β1.3. Recombinant human IL-37 can increase the apoptosis rate of LX2-HSC which is activated by TGF-β1.4. Recombinant human IL-37 can suppress the expression of Col-I and Col-III in LX2-HSC which is activated by TGF-β1. |
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