[Effect and mechnism study of Shengjiyuhong cream on transdifferentiation and collagen production of normal human dermal fibroblasts].

OBJECTIVE

To explore the effects of Shengjiyuhong cream(SJYHC) on proliferation, transdifferentiation, collagen production and TGF-β1/Smads signaling of normal human dermal fibroblasts (NHDFs).

METHODS

Primary cultured NHDFs between 3-6 passages derived from 6 hypertrophic scar samples were all treated with TGF-β1 (0,2,5,10 ng/ml)stimulation added 5 μg/ml SJYHC or not. After culturing 72 h,CCK-8 solution was added to record absorbance at 450 nm to test proliferation of NHDFs. Fluorescence quantitative PCR was used for testing mRNA expression of α-SMA and type Ⅰ and Ⅲ collagen. Digestion method was to test the hydroxyproline content in the supernatant liquor. Western Blot was used for testing protein expression of α-SMA, type Ⅰ and Ⅲ collagen and Smad2,Smad3,P-Smad2 and P-Smad3.One-way analysis of variance were used to analyze differences among more than two groups, while LSD-t test as post hoc test were used to make paired-comparisons among the groups. P ＜ 0.05 indicated significant difference.

RESULTS

With the stimulation of 2,5,10 ng/ml TGF-β1,the absorbance values(A values),mRNA and protein expression of α-SMA and type Ⅰ and Ⅲ collagen, hydroxyproline content, and protein expression of Smad2,Smad3,P-Smad2 and P-Smad3 were all elevated contrasted with the control group (P ＜ 0.05,P ＜ 0.01).Without TGF-β1 stimulation, SJYHC only increased the absorbance values(A values) from 1.645 ±0.052 to 1.796 ±0.060(P ＜0.05),while mRNA and protein expression of α-SMA and type Ⅰ and Ⅲ collagen, hydroxyproline content, and protein expression of Smad2,Smad3,P-Smad2 and P-Smad3 were infinitely variable(P ＞ 0.05).With stimulation of 2,5 ng/ml TGF-β1,SJYHC elevated the the absorbance values (A values) from 1.814 ± 0.052,1.970 ± 0.045 to 1.981 ± 0.061,2.133 ± 0.059 (P ＜ 0.05).While stimulated with 10 ng/ml TGF-β1,SJYHC declined the absorbance values(A values) from 2.130 ± 0.050 to 1.958 ± 0.045 (P ＜ 0.05). With stimulation of 2,5,10 ng/ml TGF-β1,mRNA expression of α-SMA were declined by SJYHC from 1.04 ±0.06,2.42 ±0.07,7.17±0.11 to 0.28 ±0.06,0.36 ±0.06,1.89 ±0.08 respectively, protein expression from 0.48± 0.05,1.17 ±0.09,2.04 ±0.09 to 0.18 ±0.03,0.21 ±0.08,0.91 ±0.11 respectively (P＜0.01),mRNA expression of Col Ⅰ from 0.73 ± 0.08,1.52 ± 0.08,3.05 ± 0.11 to 0.45 ± 0.07 0.46 ± 0.05,1.28±0.09 respectively, protein expression from 0.36 ±0.11,0.94 ±0.10,2.13 ±0.13 to 0.21 ± 0.13,0.24 ±0.08,0.87 ±0.09 respectively (P ＜0.01),mRNA expression of Col Ⅲ from 1.51 ±0.09,3.28 ±0.09,6.96 ±0.14 to 0.66 ±0.08,0.69 ±0.08,2.23 ±0.10 respectively, protein expression from 0.26 ± 0.08,0.96 ±0.09,1.96 ±0.15 to 0.08 ±0.02,0.12 ±0.02,0.43 ±0.06 respectively (P ＜0.01),hydroxyproline content from (7.219 ±0.590) μg/ml,(8.745 ±0.514) μg/ml,(10.969 ± 0.489) μg/ml to (6.242 ±0.225) μg/ml,(6.603±0.336) μg/ml,(7.516±0.511) μg/ml (P＜ 0.05).Under stimulation of 5 ng/ml TGF-β1,SJYHC had no significant effect on protein expression of Smad2 and Smad3 (P ＞ 0.05),while protein expression of P-Smad2,P-Smad3 were all declined from 0.56±0.08,0.87 ±0.13 to 0.31 ±0.07,0.46 ± 0.05 (P ＜0.01).

CONCLUSIONS

SJYHC may accelerate wound healing and prevent HS by promoting proliferation, inhibiting transdifferation and collagen production and secretion of NHDFs.