Regulatory Role of PI3K/Akt Signaling Pathway in Hypertrophic Scar
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摘要:
目的 探讨PI3K/AKT信号通路在增生性瘢痕中的调控作用。 方法 构建兔耳增生性瘢痕模型，并随机分为4组：正常兔耳皮肤组（A）、瘢痕模型组（B）、DMSO刺激模型组（C）、PI3K特异性抑制剂（LY294002）刺激模型组（D）。通过组织病理学观察兔耳瘢痕的形态学变化；免疫组化和Western blot检测p-Akt[S473]和p-Akt[T308]的表达量；免疫荧光双染色评估p-Akt[S473]和p-Akt[T308]的共定位情况；以及TUNEL评估皮肤组织中成纤维细胞的凋亡水平。 结果 D组中瘢痕厚度及成纤维细胞数明显高于B和C组。免疫组化和Western blot检测结果表明，p-Akt[T308]和p-Akt[S473]在B组中的表达量明显高于A组，但在D组中显著低于C组。免疫荧光表明，p-Akt[T308]和p-Akt[S473]主要定位于瘢痕组织中的细胞浆内，且在D组中的共表达量低于B组。TUNEL检测结果证实，与A组相比，B组皮肤组织中细胞凋亡水平明显下调，且D组中细胞凋亡水平高于C组（P < 0.01）。 结论 阻断PI3K/Akt信号通路可通过抑制Akt磷酸化，导致成纤维细胞凋亡水平上调，进而缓解瘢痕增生进程。 Abstract:Objective To explore the regulatory role of PI3K/Akt signaling pathway in the hypertrophic scar. Methods The rabbit ear hypertrophic scar model was established and randomly divided into four groups, including normal rabbit ear skin group （A）, hypertrophic scar model group （B）, DMSO-stimulated hypertrophic scar model group （C）, and PI3K-specific inhibitor （LY294002） stimulated model group （D）. Morphological changes of rabbit ear scar were observed by histopathology. The expression of phosphorylation of Akt[S473] and Akt[T308] was examined by immunohistochemistry and western blot. Immunofluorescence double staining was performed to assess the co-localization of p-Akt[S473] and p-Akt[T308]. The TUNEL assay was used to detect the fibroblasts’ apoptosis in skin tissues. Results Scar thickness and fibroblast count were significantly higher in the D group than that in the B and C groups, but the difference was not statistically significant between the B group and the C group. The results of immunohistochemistry and western blot assay confirmed that the expression of p-Akt[T308] and p-Akt[S473] were significantly higher in the B group than in the A group, but significantly lower in the D group than in the C group. The expression of p-Akt[T308] and p-Akt[S473] was significantly enhanced in the B group compared with the C group. Immunofluorescence double staining showed that p-Akt[T308] and p-Akt[S473] were mainly co-localized in the cytoplasm in scar tissue, and their co-expression was lower in the D group than in the B group. TUNEL assay results confirmed that the apoptosis level of fibroblast was significantly downregulated in skin tissue in the B group compared with the A group, and the apoptosis level of fibroblast was higher in the D group than in the C （P < 0.01）. Conclusion Blocking the PI3K/Akt signaling pathway can accelerate fibroblast apoptosis by inhibiting Akt phosphorylation, and then alleviate hypertrophic scar formation. -
Key words:
- Hypertrophic scar /
- LY294002 /
- Phosphatidylinositol 3-kinase /
- Apoptosis
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图 1 各组组织形态学变化
A：增生瘢痕模型组；B：DMSO处理增生瘢痕组；C：LY294002处理增生瘢痕组。
Figure 1. Histomorphology changes in different groups
图 2 免疫组化染色检测兔耳创面中p-Akt[T308]和p-Akt[S473]的表达量（200×）
A：正常兔耳皮肤组；B：增生瘢痕模型组；C：DMSO处理增生瘢痕组；D：LY294002处理增生瘢痕组。
Figure 2. The expression of p-Akt[T308] and p-Akt[S473] in rabbit ear trauma was determined by immunohistochemical staining （200×）
图 3 Western blot检测总Akt、p-Akt[T308]、p-Akt[S473]蛋白在兔耳创面中的表达水平
A：Western blot检测总Akt、p-Akt[T308]、p-Akt[S473]蛋白；B：总Akt、p-Akt[T308]、p-Akt[S473]蛋白统计值； 与A组比较，*P < 0.05；与C组比较，#P < 0.05 。
Figure 3. The protein expression of total Akt，p-Akt[T308]，p-Akt[S473] in the tissues of rabbit ear trauma was examined by western blot
图 4 免疫荧光双染分析p-Akt[T308]和p-Akt[S473]在瘢痕组织中表达情况（40×）
A：增生瘢痕模型组；B：LY294002处理增生瘢痕组；注：p-Akt[T308]为绿色，p-Akt[S473]为红色。
Figure 4. The expression of p-Akt[T308] and p-Akt[S473] in the scar tissue was determined by immunofluorescence double staining （40×）
图 5 TUNEL染色分析兔耳创面组织中成纤维细胞凋亡情况（200×）
A ：正常兔耳皮肤组；B：增生瘢痕模型组；C：DMSO处理增生瘢痕组；D：LY294002处理增生瘢痕组。
Figure 5. TUNEL assay was used to evaluate fibroblast apoptosis in the tissues of rabbit ear trauma （200×）
表 1 各组增生块相对增生厚度测量结果[n = 5，（
$ \bar x \pm s $ ）]Table 1. The thickness of scar tissue in different groups [n = 5，（
$ \bar x \pm s $ ）]组别 n 厚度（mm） F P B组 5 2.23 ± 0.38 1.177 0.37 C组 5 2.09 ± 0.10 D组 5 1.94 ± 0.08* 与C组相比，*P < 0.05。 
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表 2 各组凋亡指数的变化（n = 5，
$ \bar x \pm s $ ）Table 2. Changes of fibroblast apoptosis index （n = 5，
$ \bar x \pm s $ ）组别 n 凋亡率（%） F P A组 5 2.38 ± 0.41 16.357 < 0.001* B组 5 2.02 ± 0.16△ C组 5 2.05 ± 0.26 D组 5 3.66 ± 0.42##△△ *P < 0.05；与A组相比，△P < 0.05；与B组相比，##P < 0.01；与C组相比，△△P < 0.01。
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[1] Ogawa R,Dohi T,Tosa M,et al. The latest strategy for keloid and hypertrophic scar prevention and treatment: The nippon medical school (NMS) protocol[J]. J Nippon Med Sch,2021,88(1):2-9. doi: 10.1272/jnms.JNMS.2021_88-106 [2] Raktoe R S,van Haasterecht L,Antonovaite N,et al. The effect of TGFβRI inhibition on extracellular matrix structure and stiffness in hypertrophic scar-specific fibroblast-derived matrix models[J]. Biochem Biophys Res Commun,2021,559(6):245-251. [3] Mercurio L,Albanesi C,Madonna S. Recent updates on the involvement of PI3K/AKT/mTOR molecular cascade in the pathogenesis of hyperproliferative skin disorders[J]. Front Med (Lausanne),2021,8(4):665647. [4] Liu C,Chen K,Wang H,et al. Gastrin attenuates renal ischemia/reperfusion injury by a PI3K/Akt/bad-mediated anti-apoptosis signaling[J]. Front Pharmacol,2020,11(11):540479. [5] He T,Zhang Y,Liu Y,et al. MicroRNA-494 targets PTEN and suppresses PI3K/AKT pathway to alleviate hypertrophic scar formation[J]. J Mol Histol,2019,50(4):315-323. doi: 10.1007/s10735-019-09828-w [6] Tu T,Huang J,Lin M,et al. CUDC-907 reverses pathological phenotype of keloid fibroblasts in vitro and in vivo via dual inhibition of PI3K/Akt/mTOR signaling and HDAC2[J]. Int J Mol Med,2019,44(5):1789-1800. [7] Md Mokhtar A H,Malik I A,Abd Aziz N A A,et al. LY294002,a PI3K pathway inhibitor,prevents leptin-induced adverse effects on spermatozoa in sprague-dawley rats[J]. Andrologia,2019,51(3):e13196. doi: 10.1111/and.13196 [8] Long H Y,Wang C Y,Yang Y E. LY294002,a PI3K inhibitor,attenuates Tourette syndrome in rats[J]. Metab Brain Dis,2017,32(5):1619-1625. doi: 10.1007/s11011-017-0051-z [9] Luo L,Li J,Wu Y,et al. Adiponectin,but Not TGF-β1,CTGF,IL-6 or TNF-α,may be a potential anti-inflammation and anti-fibrosis factor in keloid[J]. J Inflamm Res,2021,14(3):907-916. [10] Wu X,Pu L,Chen W,et al. LY294002 attenuates inflammatory response in endotoxin-induced uveitis by downregulating JAK3 and inactivating the PI3K/Akt signaling[J]. Immunopharmacol Immunotoxicol,2022,44(4):510-518. doi: 10.1080/08923973.2022.2055565 [11] Chen H,Xu K,Sun C,et al. Inhibition of ANGPT2 activates autophagy during hypertrophic scar formation via PI3K/AKT/mTOR pathway[J]. An Bras Dermatol,2022,98(1):26-35. [12] Ma F, Shen J, Zhang H, et al. A novel lncRNA FPASL regulates fibroblast proliferation via the PI3K/AKT and MAPK signaling pathways in hypertrophic scar[J]. Acta Biochim Biophys Sin （Shanghai）, 2022. http://doi.org/10.3724/abbs.2022122. [Online ahead of print]. [13] Zhi Y, Wang H, Huang B, et al. Panax Notoginseng Saponins suppresses TRPM7 via the PI3K/AKT pathway to inhibit hypertrophic scar formation in vitro. Burns, 2021, 47（4）: 894-905. [14] Shi W,Wu Y,Bian D. p75NTR silencing inhibits proliferation,migration,and extracellular matrix deposition of hypertrophic scar fibroblasts by activating autophagy through inhibiting the PI3K/Akt/mTOR pathway[J]. Can J Physiol Pharmacol,2021,99(4):349-359. doi: 10.1139/cjpp-2020-0219 [15] Wu X,Wang Z,Wu G,et al. Tetramethylpyrazine induces apoptosis and inhibits proliferation of hypertrophic scar-derived fibroblasts via inhibiting the phosphorylation of AKT[J]. Front Pharmacol,2020,11(5):602. -
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