异鼠李素对长波紫外线照射人皮肤成纤维细胞ER/TGF-β1/Smad3信号通路的影响

Effects of isorhamnetin on ER/TGF-β1/Smads3 signaling pathways of UVA-induced human dermal fibroblasts

目的:研究异鼠李素对长波紫外线(ultraviolet A, UVA)损伤的人皮肤成纤维细胞雌激素受体(estrogen receptor, ER)/TGF-β1/Smads3信号通路的影响。方法:将人皮肤成纤维细胞按随机数字表法分为空白组、模型组、雌二醇组及异鼠李素100、10、1、0.1、0.01、0.001 μmol/L组,除空白组外,其余各组建立细胞光老化模型。相应药物干预后,采用MTT法检测细胞增殖率,筛选出异鼠李素有效浓度。再将细胞按随机数字表法分为空白组、模型组、雌二醇组、异鼠李素组,以及TGF-β1阻断剂组、Samd3阻断剂组、COL1A1阻断剂组。除空白组外,其余各组建立细胞光老化模型。相应药物干预后,采用实时定量PCR和Western blot方法检测各组人皮肤成纤维细胞TGF-β1、Smad3、Ⅰ型胶原α1(collagen, type Ⅰ, alpha 1, COL1A1)mRNA和蛋白表达。结果:与模型组比较,0.001 μmol/L组异鼠李素组人皮肤成纤维细胞增殖率升高( P<0.01);异鼠李素组TGF-β1 mRNA[(0.956±0.020)比(0.718±0.036)]、Smad3 mRNA[(0.981±0.044)比(0.753±0.047)]、COL1A1 mRNA[(0.998±0.032)比(0.786±0.031)]、TGF-β1蛋白[(0.761±0.026)比(0.542±0.023)]、Smad3蛋白[(0.776±0.016)比(0.551±0.025)]、COL1A1蛋白[(0.792±0.025)比(0.584±0.012)]表达水平升高( P<0.01)。与异鼠李素组比较,TGF-β1阻断剂组、Smad3阻断剂组、COL1A1阻断剂组TGF-β1 mRNA[(0.762±0.051)、(0.802±0.012)、(0.828±0.030)比(0.967±0.026)]、Smad3 mRNA[(0.784±0.027)、(0.816±0.015)、(0.830±0.032)比(0.998±0.021)]、COL1A1 mRNA[(1.082±0.025)、(1.101±0.012)、(1.138±0.011)比(1.263±0.022)]、TGF-β1蛋白[(0.675±0.028)、(0.682±0.026)、(0.722±0.015)比(0.862±0.014)]、Smad3蛋白[(0.712±0.013)、(0.764±0.012)、(0.778±0.011)比(0.901±0.015)]、COL1A1蛋白[(0.738±0.016)、(0.770±0.038)、(0.792±0.026)比(0.964±0.017)]表达降低( P<0.05)。 结论:异鼠李素可促进UVA照射后的人皮肤成纤维细胞胶原蛋白合成,其作用机制与调控成纤维细胞ER/TGF-β1/Smad3信号通路有关。

Objective To observe the effect of isorhmnetin on the ER/TGF-β1/Smad3 signaling pathways in human dermal fibroblasts(HSF)damaged by UVA.Methods HSF were divided into control group,model group,estradiol group,isorhmnetin groups with 100,10,1,0.1,0.01,0.001μmol/L by random number table method,and cell photoaging models were established in all groups excepting the control group.After the intervention with corresponding drugs,cell proliferation rates were detected with MTT method,and the effective concentration of isorhmnetin was screened.Then the cells were divided into control group,model group,estradiol group,isorhmnetin group,TGF-β1 blocker group,Samd3 blocker group,and COL1A1 blocker group.Cell photoaging models were established in all groups excepting the control group.After intervened with corresponding drugs,the TGF-β1,Smad3,Ⅰcollagenα1(collagen,typeⅠ,alpha 1,COL1A1)mRNA and protein expression in all groups were detected by the real-time quantitative PCR and the Wester blot method.Results The proliferation rate of isor administration group were increased than those in the control group(P<0.01).Compared to the UVA irradiation group,the expression of TGF-β1 mRNA(0.956±0.020 vs.0.718±0.036),Smad3 mRNA(0.981±0.044 vs.0.753±0.047),COL1A1 mRNA(0.998±0.032 vs.0.786±0.031),TGF-β1 protion(0.761±0.026 vs.0.542±0.023),Smad3 protion(0.776±0.016 vs.0.551±0.025),COL1A1 protion(0.792±0.025 vs.0.584±0.012)in isor administration group significantly increased(P<0.01).Compared to the isor administration group,the TGF-β1 mRNA(0.762±0.051,0.802±0.012,0.828±0.030 vs.0.967±0.026),Smad3 mRNA(0.784±0.027,0.816±0.015,0.830±0.032 vs.0.998±0.021),COL1A1 mRNA(1.082±0.025,1.101±0.012,1.138±0.011 vs.1.263±0.022),TGF-β1 protion(0.675±0.028,0.682±0.026,0.722±0.015 vs.0.862±0.014),Smad3 protion(0.712±0.013,0.764±0.012,0.778±0.011 vs.0.901±0.015),COL1A1 protion(0.738±0.016,0.770±0.038,0.792±0.026 vs.0.964±0.017)in the TGF-β1 blocker group,Smad3 blocker group and COL1A1 blocker group significantly decreased(P<0.01).Conclusions Isorhmnetin can promote the collagen synthesis of photo aging HSF cells,and its mechanism is related to the regulation of ERβ/TGF-β1 signaling pathway.