Mechanisms of AKR1C3 and PI3K/AKT signaling pathways in human keloid formation
10.3760/cma.j.cn114453-20201227-00647
- VernacularTitle:PI3K/AKT信号通路及AKR1C3在人瘢痕疙瘩形成中的作用机制研究
- Author:
Yiqun MA
1
;
Yang TANG
Author Information
1. 昆明医科大学第一附属医院皮肤科，昆明 650032
- Keywords:
Keloid;
Fibroblasts;
Reactive oxygen species;
Phosphatidylinositol 3-kinases;
Protein serine/threonine kinases;
Aldo-keto reductase family 1 member C3
- From:
Chinese Journal of Plastic Surgery
2022;38(1):83-92
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the possible functional roles and mechanisms of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, reactive oxygen species (ROS), and aldo-keto reductase family 1 member C3 (AKR1C3) in keloid formation.Methods:Nine keloid tissues from patients (six males, three females; aged: 24-40 years old) and six normal skin tissues from healthy controls (four males, two females; aged 24-45 years old) were collected. Tissue samples were embedded or used in fibroblasts isolation and culture. (1) The relative expression of protein kinase B serine phosphorylation site 473[p-AKT (S473)], protein kinase B threonine phosphorylation site 308[p-AKT (T308)], and AKR1C3 in keloid tissues and normal skin tissues was measured using immunohistochemistry. (2) The CCK-8 assay was employed to detect the inhibitory effect of PI3K-specific inhibitor LY294002 (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mmol/L) on the proliferation of keloid fibroblasts and to screen the optimal experimental concentration of LY294002. (3) The ROS assay kit was used to detect the effects of different concentrations (0, 4, 6, 8, 10, 12 mmol/L) of the ROS inhibitor N-acetylcysteine (NAC) and 15 mmol/L LY294002 on ROS levels in keloid fibroblasts. (4) Fibroblasts derived from keloid tissues or normal skin tissues were divided into control group (without any treatment), respectively, dimethyl sulfoxide(DMSO) group (0.002% DMSO treatment), LY294002 group (15 mmol/L LY294002 treatment), and NAC group (6 mmol/L NAC treatment), respectively. Quantitative PCR (qPCR) and Western blotting assays were performed to detect the levels of AKT mRNA, AKR1C3 mRNA and the protein levels of p-AKT(S473), p-AKT(T308), and AKR1C3. Statistical analysis was performed with SPSS 22.0. Data was presented as Mean±SD. Independent sample t-test was used for comparison between two groups, one-way ANOVA was used for comparison between multiple groups, and SNK- q test was used for multiple comparisons between two groups. Results:(1) Immunohistochemistry showed that the expression levels of p-AKT(S473), p-AKT(T308), and AKR1C3 in keloid tissues were 16.75±3.30, 16.20±1.56, 26.69±2.50, which were significantly higher than those in normal skin tissues: 4.02±1.50, 1.82±0.50, 1.47±1.07 ( P<0.01). (2) The proliferation inhibition rate of keloid fibroblasts treated by different concentrations of LY294002 (15-50 mmol/L LY294002) for 24 h was significantly higher than in the control group (0 mmol/L) ( P<0.01). The optimal experimental concentration of LY294002 is 15 mmol/L. (3) After different concentrations of NAC were applied to keloid fibroblasts for 1 h, the differences in ROS level was groups were statistically significant ( P<0.01), and the lowest ROS level was found in the 6 mmol/L NAC group(0.72±0.03). The 15 mmol/L LY294002 group had significantly lower ROS levels than the control group (0 mmol/L) (0.80±0.01 vs. 0.86±0.01, P<0.01). (4) qPCR showed that the mRNA levels of AKT and AKR1C3 were 1.38±0.09 and 1.40±0.05 in keloid fibroblasts in control group, which were significantly higher than those in normal skin fibroblasts: 0.97±0.10, 0.98±0.03( P<0.01). The expression level of AKT mRNA in keloid fibroblasts treated with 15 mmol/L LY294002 was significantly lower than that in normal skin fibroblasts (0.73±0.05 vs. 0.89±0.06, P<0.01). The expression level of AKR1C3 mRNA in keloid fibroblasts treated with 6 mmol/L NAC was significantly lower than that in normal skin fibroblasts (0.43±0.05 vs. 0.86±0.03, P<0.01). Western blotting revealed the expression levels of p-AKT(S473), p-AKT(T308), and AKR1C3 in keloid fibroblasts were 1.19±0.21, 0.92±0.04, 0.73±0.08, significantly higher than those in normal skin fibroblasts (0.24±0.06, 0.33±0.05, 0.31±0.05, P<0.01). After treatment with 15 mmol/L LY294002 or 6 mmol/L NAC, the protein expression of p-AKT (S473) in keloid fibroblasts were 0.92±0.04 and 0.80±0.20; the protein levels of p-AKT (T308) were 0.42±0.04 and 0.81±0.05, which were all significantly higher than the expression levels in normal skin tissue (0.23±0.03, 0.22±0.05, 0.30±0.06, 0.32±0.05). However, The expression of AKR1C3 protein in keloid fibroblasts treated with 15 mmol/L LY294002 (0.23±0.05) was significantly lower than that in normal skin fibroblasts (0.30±0.07), the expression of AKR1C3 protein in keloid fibroblasts treated with 6 mmol/L NAC (0.33±0.07) was significantly higher than that in normal skin fibroblasts (0.28±0.06, P>0.05). Conclusions:Activation of PI3K/AKT signaling pathway and AKR1C3 promoted the proliferation of keloid fibroblasts and keloid formation. Meanwhile, the elevation of AKR1C3 accelerated the increase of ROS level in keloid fibroblasts.
