| Part 1 Effect of melatonin on HIF-1αexpression in human hypertrophic scar fibroblastsObjective To investigate the effect of melatonin on the expression of HIF-1αin human hypertrophic scar fibroblasts.Methods Six cases of human hypertrophic scar tissue were collected.The primary fibroblasts were cultured by tissue block method.The cells were passaged to the fourth generation.The fibroblasts of the third and fourth generation were taken.The proliferation activity of fibroblasts was detected by CCK8.The effects of 1*10-4m M,1*10-3m M,1*10-2m M,1*10-1m M and 1m M melatonin on the proliferation activity of fibroblasts were observed.The fourth passage fibroblasts were digested and transferred to 6-well plate.The Control group was added with 2ml medium,and the experimental group was added with 2ml medium containing 1*10-3m M Melatonin.After 48 hours of culture,the cells were collected.The expression of HIF-1αm RNA in fibroblasts was detected by Real-time Fluorescent Quantitative Nucleic Acid Amplification（QPCR）and Western blotting（Western blot）.The expression of HIF-1αprotein in fibroblasts was detected by flow cytometry.SPSS statistics 24 was used to analyze the results.The results were expressed as mean±standard deviation（x±s）and two sample t test.P<0.05 was regarded as significant difference.Results 1*10-3m M,1*10-2m M,1*10-1m M and 1m M melatonin could inhibit the proliferation of human hypertrophic scar fibroblasts（P<0.05）.The relative expression of HIF-1αm RNA in 1*10-3mm melatonin group was 0.75±0.2,which was lower than 1.01±0.16 in the Control group（P<0.05）.The relative expression of HIF-1αprotein in 1*10-3m M Melatonin group was 1.04±0.34,which was lower than1.50±0.29 in the Control group（P<0.05）.Conclusion 1.Melatonin can inhibit the proliferation of human hypertrophic scar fibroblasts;2.melatonin can reduce the expression of HIF-1αin human hypertrophic scar fibroblasts.Part2 Effect of melatonin on the expression of Smads and CTGF downstream of HIF-1αin human hypertrophic scar fibroblastsObjective To investigate whether melatonin affects the expression of Smads and CTGF downstream of HIF-1αin human hypertrophic scar fibroblasts.Methods 10 cases of human hypertrophic scar tissue were collected.The primary cells were cultured by tissue block method.The third and fourth passage human hypertrophic scar fibroblasts were cultured in simple medium and YC-1 medium.The proliferation activity of fibroblasts was detected by CCK8 method.The experiment was divided into four groups:（1）Control group:2ml medium;（2）Melatonin group:2ml medium containing 1*10-3m M melatonin;（3）HIF-1αinhibitors（YC-1）group:2ml medium containing 2.5*10-4m M YC-1;（4）Melatonin+YC-1group:2ml medium containing melatonin and YC-1.The expression of Smad2,Smad3 and CTGF m RNA was detected by QPCR,and the expression of CTGF protein was detected by Western blot.SPSS statistics 24 was used to analyze the results.The results were expressed as mean±standard deviation（x±s）.Two sample t-test and one way ANOVA were used to compare the mean differences among groups.SNK method was used to analyze the differences among groups.The difference was statistically significant（P<0.05）.Results The relative expression of Smad2 m RNA in fibroblasts composed of Melatonin（0.74±0.37）was lower than that in control group（1.01±0.16）（P<0.05）.The relative expression of Smad2 m RNA in fibroblasts composed of YC-1（0.56±0.36）was lower than that in control group（1.01±0.16）（P<0.05）.The relative expression of Smad2 m RNA in fibroblasts composed of Melatonin+YC-1（0.56±0.36）was lower than that in control group（1.01±0.16）（P<0.05）.The relative expression of Smad3m RNA in melatonin group was 0.61±0.29,which was lower than 1.01±0.16 in the control group（P<0.05）.The relative expression of Smad3 m RNA in YC-1 group was0.83±0.57,which was lower than that in control group（1.02±0.19）（P>0.05）;.The relative expression of Smad3 m RNA in Melatonin+YC-1 group was 0.49±0.15,which was lower than 1.02±0.19 in the control group（P<0.05）.The relative expression of CTGF m RNA in 1*10-3m M melatonin group was 0.60±0.24,which was lower than 1.01±0.16 in control group（P<0.05）.The relative expression of CTGF protein in melatonin group was 0.26±0.09,which was lower than 0.68±0.03 in control group（P<0.05）.The relative expression of CTGF m RNA in 2.5*10-4m M YC-1 group was 0.65±0.35,which was lower than 1.01±0.16 in the control group（P<0.05）.The relative expression of CTGF protein in YC-1 group was 0.36±0.23,which was lower than 0.68±0.03 in the control group（P<0.05）.The relative expression of CTGF m RNA in melatonin+YC-1 group was 0.54±0.42,which was lower than 1.01±0.16in the control group（P<0.05）.The relative expression of CTGF protein in melatonin+YC-1 group was 0.28±0.06,which was lower than 0.68±0.03 in the control group（P<0.05）.Conclusion The results showed that inhibition of HIF-1αcould down regulate the expression of Smad2 and CTGF in hypertrophic scar fibroblasts.The results suggested that melatonin could affect the expression of Smad2 and CTGF through HIF-1α. |
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