丹玄口康对ANE诱导的口腔黏膜下纤维化TGFβ1/Smad信号通路的影响

Effect of Danxuan Koukang on ANE induced TGF beta 1/Smad signaling pathway in oral submucosal fibrosis

目的探讨丹玄口康对ANE诱导的口腔黏膜下纤维化TGFβ_1/Smad信号通路的影响。方法体外培养大鼠口腔黏膜FB,以ANE为诱导剂,丹玄口康药物血清为干预物,以TGFβ_1/Smad信号通路阻断剂LY2109761为阳性对照物,采用免疫细胞化学法检测细胞内Ⅰ型胶原蛋白、磷酸化Smad2/3蛋白的表达变化,用酶联免疫吸附剂测定法检测培养上清Ⅰ型胶原的含量,采用荧光定量PCR检测Ⅰ型胶原m RNA的表达变化。结果 ANE刺激FB后,细胞和上清液中Ⅰ型胶原的表达、Smad2/3的磷酸化胞核的转位水平和Ⅰ型胶原m RNA的表达均高于正常组(P<0.05);ANE刺激FB的同时给予LY2109761干预后,细胞和上清液中Ⅰ型胶原的表达、磷酸化Smad2/3的胞核转位水平和Ⅰ型胶原m RNA的表达均有所降低(P<0.05);ANE刺激FB的同时给予丹玄口康含药血清干预后,细胞和上清液中I型胶原的表达、磷酸化Smad2/3的胞核转位水平和I型胶原m RNA的表达均有所降低(P<0.05)。结论丹玄口康可通过调控TGFβ_1/Smads信号转导通路,抑制ANE刺激的FB胶原蛋白的合成,从而发挥抗纤维化作用。

Objective To explore the effect of Danxuan Koukang on ANE induced TGF beta 1/Smad signaling pathway in Oral Submucosal Fibrosis.Methods Oral submucosal FB of rats was cultured in vitro,and ANE was used as an inducer.The serum of Danxuan Koukang was taken as intervention,and TGF beta 1/Smad signal pathway blocker LY2109761 was used as a positive control.The expression of typeⅠcollagen and phosphorylated Smad2/3 protein in cells was detected by immunocytochemistry.The content of typeⅠcollagen in culture supernatant was detected by enzyme-linked immunosorbent assay,and the expression of collagen type I mRNA was detected by real-time quantitative PCR.Results After ANE stimulated FB,the expression of type I collagen,the translocation of Smad2/3 phosphorylated nucleus and the expression of type I collagen mRNA in the cells and supernatants were higher than those in the normal group(P<0.05).When ANE stimulated FB and was intervened by LY2109761,the expression of typeⅠcollagen,the nuclear translocation level of phosphorylated Smad2/3 and the expression of collagen mRNA of typeⅠin the cells and supernatants were decreased(P<0.05).ANE stimulated FB and intervened by the serum of Danxuan Koukang.After the intervention,the expression ofⅠcollagen,the nuclear translocation level of phosphorylated Smad2/3 and the expression of I type collagen mRNA in the cells and supernatants were decreased(P<0.05).Conclusion Danxuan Koukang can inhibit the synthesis of FB collagen stimulated by ANE by regulating the TGF beta 1/Smads signal transduction pathway,thus exerting anti fibrosis effect.