Objective To explore the inhibitory effect of Xinhui tangerine peel polysaccharides on the mouse skin fibroblasts so as to understand the underlying molecular mechanism.Methods We separated and purified the components of tangerine peel polysaccharides from Xinhui tangerine peel.C57BL/6 mouse skin fibroblasts were isolated and cultured for the cellular experimental study.We set up a blank control group(normal culture)and low-,medium-and high-dose experimental groups(50,100 and 200 μg/mL tangerine peel polysaccharides).After 24 hours of treatment,cell survival rate was assessed by CCK-8 assay.The mRNA and protein expression levels of collagen type Ⅰ(Col1a1),collagen type Ⅲ(Col1a3),TGF-β1 and ACTA2 in fibroblasts were examined by RT-qPCR and Western blotting.Smad3 was examined by Western blotting.Results The cell survival rate in the blank control group,the medium-and high-dose experimental groups was 100％,(90.54±6.74)％,and(78.90±4.24)％,respectively.The relative expression level of Col1a1 protein was 1.13±0.15,0.57±0.16,and 0.48±0.05,respectively;the relative expression level of Col3a1 protein was 0.81±0.13,0.49±0.11 and 0.50±0.03;the relative expression level of TGF-β1 protein was 1.11±0.15,0.60±0.13,and 0.33±0.11;the relative expression level of p-Smad3/Smad3 proteins was 0.96±0.05,0.75±0.06 and 0.71±0.03.The mRNA expression level of Col1a1 was 1.01±0.17,0.58±0.11,and 0.52±0.12;the mRNA expression level of Col3a1 was 1.01±0.12,0.56±0.19,and 0.65±0.14;the expression level of ACTA2 mRNA was 1.01±0.13,0.24±0.04,and 0.22±0.07;the mRNA expression level of TGF-β1 was 1.00±0.09,0.50±0.10,and 0.49±0.15.The relative expression levels of p-Smad3/Smad3 proteins were 0.86±0.06,0.66±0.06,0.55±0.13,0.43±0.09,0.35±0.06,and 0.27±0.12,respectively,after time-related treatment with high-dose tangerine peel polysaccharides.The above indicators in medium-and high-dose tangerine peel polysaccharide groups showed statistically significant differences compared to those in the blank control group(P＜0.05).Conclusion Tangerine peel polysaccharides can inhibit the cell proliferation and the synthesis of keloid-related genes on fibroblasts by inhibiting TGF-β1/Smad3 signaling pathway.

Objective To explore the inhibitory effect of Xinhui tangerine peel polysaccharides on the mouse skin fibroblasts so as to understand the underlying molecular mechanism.Methods We separated and purified the components of tangerine peel polysaccharides from Xinhui tangerine peel.C57BL/6 mouse skin fibroblasts were isolated and cultured for the cellular experimental study.We set up a blank control group(normal culture)and low-,medium-and high-dose experimental groups(50,100 and 200 μg/mL tangerine peel polysaccharides).After 24 hours of treatment,cell survival rate was assessed by CCK-8 assay.The mRNA and protein expression levels of collagen type Ⅰ(Col1a1),collagen type Ⅲ(Col1a3),TGF-β1 and ACTA2 in fibroblasts were examined by RT-qPCR and Western blotting.Smad3 was examined by Western blotting.Results The cell survival rate in the blank control group,the medium-and high-dose experimental groups was 100％,(90.54±6.74)％,and(78.90±4.24)％,respectively.The relative expression level of Col1a1 protein was 1.13±0.15,0.57±0.16,and 0.48±0.05,respectively;the relative expression level of Col3a1 protein was 0.81±0.13,0.49±0.11 and 0.50±0.03;the relative expression level of TGF-β1 protein was 1.11±0.15,0.60±0.13,and 0.33±0.11;the relative expression level of p-Smad3/Smad3 proteins was 0.96±0.05,0.75±0.06 and 0.71±0.03.The mRNA expression level of Col1a1 was 1.01±0.17,0.58±0.11,and 0.52±0.12;the mRNA expression level of Col3a1 was 1.01±0.12,0.56±0.19,and 0.65±0.14;the expression level of ACTA2 mRNA was 1.01±0.13,0.24±0.04,and 0.22±0.07;the mRNA expression level of TGF-β1 was 1.00±0.09,0.50±0.10,and 0.49±0.15.The relative expression levels of p-Smad3/Smad3 proteins were 0.86±0.06,0.66±0.06,0.55±0.13,0.43±0.09,0.35±0.06,and 0.27±0.12,respectively,after time-related treatment with high-dose tangerine peel polysaccharides.The above indicators in medium-and high-dose tangerine peel polysaccharide groups showed statistically significant differences compared to those in the blank control group(P＜0.05).Conclusion Tangerine peel polysaccharides can inhibit the cell proliferation and the synthesis of keloid-related genes on fibroblasts by inhibiting TGF-β1/Smad3 signaling pathway.

[Objective] To investigate the role of miR-199a-3p on mouse skin scar fibroblasts and the potential target of miR-199a-3p. [Methods] A mouse skin keloid model was established. The mRNA levels of miR-199a-3p, Smad1 and keloid related genes in keloid tissues and normal skin tissues were detected by Real-time quantitative PCR. C57BL/6 mouse skin fibroblasts were isolated and cultured for the cellular experimental study. miR-199a-3p mimic and Smad1 siRNA matter were transiently transfected into mouse skin fibroblasts by liposome reagent. the interaction between miR-199a-3p and the 3′-UTR of Smad1 was confirmed by the dual luciferase reporter assay. The expressions of Smad1 and keloid-related genes at mRNA and protein levels after transfection of miR-199a-3p mimic were determined. The expressions of Smad1 and keloid-related genes at protein level after transfection of miR-199a-3p mimic and Smad1 siRNA were determined by Western blot assay. [Results] Compared with normal skin tissues, the expressions of Smad1 (t=-4.403, P=0.010) and keloid related genes, Col1a1(t=-3.334, P=0.016), Col3a1(t=-5.927, P=0.001) and ACTA2(t=-3.673, P=0.010), were significantly increased in keloid tissues, while miR-199a-3p (t=7.059, P<0.001) expression was significantly decreased. Over-expression of miR-199a-3p could significantly decrease the expressions of keloid-related genes, Col1a1 (t=5.514, P=0.005), Col3a1 (t=5.132, P=0.014) and ACTA2 (t=4.136, P=0.026), in mouse skin fibroblasts. Moreover, the dual luciferase reporter assay revealed that miR-199a-3p could interact with the 3′-UTR of Smad1. miR-199a-3p was observed to inhibit Smad1 at mRNA expression level (t=3.556, P=0.024), and at the post-transcriptional level (t=3.781, P=0.019). Meanwhile, miR-199a-3p mimic, in parallel to Smad1 siRNA, decreased the expressions of keloid-related genes, Col1a1 (F=18.804; P=0.003, 0.022), Col3a1 (F=33.212; P=0.001, 0.001) and α-SMA (F=10.181; P=0.020, 0.028), and decreased the proliferation of skin fibroblasts (F=18.622; P=<0.001, <0.001). [Conclusion] miR-199a-3p inhibits the formation of keloid by targeting Smad1.