β2肾上腺素受体通过TGF-β1/Smad3信号通路调控创面愈合中的纤维增生

The role of β2 adrenergic receptor in fibrogenesis during wound healing via TGF-β1/Smad3 signaling pathway

目的探讨β2肾上腺素受体(β2 adrenergic receptor,ADRB2)在创面愈合过程中对纤维增生的调控机制。方法将12只小鼠背部皮肤随机注射非特异性敲减ADRB2基因的腺相关病毒(AAV-ADRB2组,n=6)和对照病毒(AAV-NC组,n=6)21 d后,在背部建立全层皮肤缺损创面愈合模型。记录术后第1、3、5、7天创面愈合率;采用H-E染色、Masson染色和免疫组化染色观察创面组织结构、纤维化程度及α-平滑肌肌动蛋白表达;定量PCR检测ADRB2、基质金属蛋白酶(matrix metalloproteinase,MMP)mRNA水平;Western印迹法检测胶原纤维1A1、胶原纤维3A1、TGF-β1、Smad3蛋白表达水平。结果术后第5、7天,AAV-ADRB2组创面愈合率显著下降(P<0.05),伴随表皮增厚、炎症细胞增多、纤维细胞减少、胶原沉积降低;α-SMA水平显著下降(P<0.05);COL1A1/COL3A1比例减少(P<0.05);ADRB2 mRNA水平显著降低(P<0.01),MMP-1和MMP-8 mRNA水平升高(P<0.01);TGF-β1/Smad3蛋白水平显著下降(P<0.05)。结论ADRB2敲减通过抑制TGF-β1/Smad3信号通路,减少创面纤维化反应和结缔组织含量,提高MMP mRNA水平,降低Ⅰ型/Ⅲ型胶原比例。

Objective To explore the underlying mechanism of β2 adrenergic receptor(ADRB2)in fibrogenesis during wound healing.Methods Non-specific ADRB2 gene knockdown adeno-associated virus(AAV-ADRB2 group,6 mice)and control virus(AAV-NC group,6 mice)was injected randomly into the back skin of 12 mice for 21 days,a full-thickness skin defected wound healing murine model was established.Wound healing rates were recorded at the 1st,3rd,5th,and 7th day after operation.Histological examinations by H-E staining,Masson staining,and immunohistochemistry were conducted to observe wounded skin tissue structure,fibrosis,and α-SMA protein expression;quantitative PCR was employed to analyze ADRB2 and matrix metalloproteinase(MMP)mRNA levels;Western blotting was utilized to assess the protein expression levels of COL1A1,COL3A1,TGF-β1,and Smad3.Results On postoperative day 5 and 7,the wound healing rate of the AAV-ADRB2 group significantly decreased(P<0.05),accompanied by a series pathological changes,including thickened epidermis,exaggerated inflammation,reduced fibroblast count,and inhibited collagen deposition;theα-SMA expression showed a significant decrease(P<0.05),and the ratio of COL1A1 to COL3A1 decreased(P<0.05);ADRB2 mRNA levels significantly decreased(P<0.01),while MMP-1 and MMP-8 mRNA levels increased(P<0.01);the protein levels of TGF-β1 and Smad3 exhibited a significant decrease(P<0.05).Conclusions ADRB2 knockdown reduced fibrosis during wound healing and degenerated connective tissue content around the wound bed by inhibiting the TGF-β1/Smad3 signaling pathway,which leads to an increase in MMP mRNA levels and a decrease in the ratio of type Ⅰ to type Ⅲ collagen.