乙醛脱氢酶2调控巨噬细胞M2型极化促进创面愈合

Aldehyde dehydrogenase 2 promotes wound healing by regulating M2 macrophage polarization

目的验证乙醛脱氢酶2(aldehyde dehydrogenase 2,ALDH2)在创面愈合中的作用并探讨其分子机制。方法构建小鼠全层皮肤切除模型,随机分为对照组和Alda-1(ALDH2激动剂)组,观察创面愈合情况并检测创面组织ALDH2活性。采用Masson染色观察创面胶原纤维含量,免疫组化染色检测ALDH2和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)水平,免疫荧光染色检测Ⅰ型胶原(Col-Ⅰ)、Ⅲ型胶原(Col-Ⅲ)表达和F4/80^(+)、iNOS^(+)、CD206^(+)细胞数。体外实验采用白介素4(interleukin-4,IL-4)诱导RAW264.7细胞M2型极化,分别用Alda-1和ALDH2抑制剂CVT-10216处理细胞。流式细胞仪检测F4/80^(+)CD206^(+)细胞比例,酶联免疫分析测定细胞上清中促炎因子和抗炎因子水平,Western印迹法检测AKT/mTOR信号通路相关蛋白表达。结果与对照组相比,Alda-1组小鼠创面愈合率显著提高,创面的Col-Ⅰ、Col-Ⅲ及α-SMA表达增多;F4/80^(+)细胞差异无统计学意义,i NOS^(+)细胞显著减少,CD206^(+)细胞显著增多。体外实验结果显示,与IL-4组相比,IL-4^(+)Alda-1组F4/80^(+)CD206^(+)细胞比例、抗炎因子和p-AKT、p-m TOR蛋白表达显著升高,促炎因子表达降低;IL-4^(+)CVT-10216组F4/80^(+)CD206^(+)细胞比例、抗炎因子和p-AKT、p-mTOR蛋白表达显著降低,促炎因子表达升高。结论ALDH2通过AKT/mTOR通路诱导巨噬细胞M2型极化,促进小鼠创面愈合。

Objective To verify the role of aldehyde dehydrogenase 2(ALDH2)in wound healing and to explore the underlying mechanisms.Methods A murine excisional wound model was developed and mice were randomly assigned to control group and Alda-1(ALDH2 agonist)group.Wound healing rate and activity of ALDH2 were measured.Masson staining was used to observe the collagen fiber content,and immunohistochemistry was used to detect the expression of ALDH2 andα-smooth muscle actin(α-SMA).The expression of collagen typeⅠandⅢ(Col-Ⅰ,Col-Ⅲ),and the number of F4/80,inducible nitric oxide synthase(iNOS)and CD206 positive(F4/80+,iNOS+,CD206+)cells were analyzed using immunofluorescences.Furthermore,interleukin(IL)-4-stimulated RAW264.7 cells were treated with Alda-1 or CVT-10216(an ALDH2 inhibitor)in vitro.The proportion of F4/80+CD206+cells were analyzed using flow cytometry.Anti-inflammatory and pro-inflammatory cytokines were examined using ELISA.The expression of protein associated with the AKT/mTOR pathway were detected via western blotting.Results Compared with control group,the wound healing rate of mice in the Alda-1 group was significantly improved,and the expression of Col-Ⅰ,Col-Ⅲ,andα-SMA in the wound surface increased.Although the number of F4/80+cells in wounds did not significantly differ,there was a decrease in iNOS+cells and an increase in CD206+cells.In vitro,compared to IL-4 group,IL-4+Alda-1 group exhibited an increased proportion of F4/80+CD206+macrophages and higher levels of anti-inflammatory factors,alongside a reduction in levels of pro-inflammatory factors.Conversely,IL-4+CVT-10216 group demonstrated a decreased proportion of F4/80+CD206+macrophages,lower levels of anti-inflammatory factors with a concomitant increase in proinflammatory factors.Additionally,the protein expressions of p-AKT and p-mTOR were significantly elevated in IL-4+Alda-1 group and diminished in IL-4+CVT-10216 group.Conclusion ALDH2 induces M2 macrophage polarization via AKT/mTOR pathway to promote wound healing in mice.