Experimental study on promotion of skin radiation damage repair by icarin via HIF-2α/VEGF/Notch pathway to enhance the paracrine function of adipose-derived stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZUO Yuer ¹ , LI Shuangyi ² ^{共同第一作者} , TAN Siyu ² , HU Xiaohao ¹ , LI Zhou ³ ,  LI Haoxi ⁴

1. Department of Plastic and Reconstructive Surgery, Guangxi Academy of Medical Sciences & People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning Guangxi, 530021, P. R. China;
2. Liaoning University of Traditional Chinese Medicine Xinglin College, Shenyang Liaoning, 110000, P. R. China;
3. Department of Radiation Oncology, the First People’s Hospital of Nanning, Nanning Guangxi, 530022, P. R. China;
4. Department of Spine Surgery, Guangxi Academy of Medical Sciences & People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning Guangxi, 530021, P. R. China;

Corresponding author：

LI Haoxi, Email: volvoxc-90@163.com

Keywords：

Icariin; adipose-derived stem cells; repair mechanism; skin radiation damage; rat

DOI：

10.7507/1002-1892.202503089

Video：

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Objective To investigate the effectiveness and preliminary mechanisms of icariin (ICA) in enhancing the reparative effects of adipose-derived stem cells (ADSCs) on skin radiation damagies in rats. Methods Twelve SPF-grade Sprague Dawley rats [body weight (220±10) g] were subjected to a single dose of 10 Gy X-ray irradiation on a 1.5 cm×1.5 cm area of their dorsal skin, with a dose rate of 200 cGy/min to make skin radiation damage model. After successful modeling, the rats were randomly divided into 4 groups (n=3), and on day 2, the corresponding cells were injected subcutaneously into the irradiated wounds: group A received 0.1 mL of rat ADSCs, group B received 0.1 mL of rat ADSCs+1 μmol/L ICA (0.1 mL), group C received 0.1 mL of rat ADSCs pretreated with a hypoxia-inducible factor 2α (HIF-2α) inhibitor+1 μmol/L ICA (0.1 mL), and group D received 0.1 mL of rat ADSCs pretreated with a Notch1 inhibitor+1 μmol/L ICA (0.1 mL). All treatments were administered as single doses. The skin injury in the irradiated areas of the rats was observed continuously from day 1 to day 7 after modeling. On day 28, the rats were sacrificed, and skin tissues from the irradiated areas were harvested for histological examination (HE staining and Masson staining) to assess the repair status and for quantitative collagen content detection. Immunohistochemical staining was performed to detect CD31 expression, while Western blot and real time fluorescence quantitative PCR (qRT-PCR) were used to measure the relative protein and mRNA expression levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), fibroblast growth factor 2 (FGF-2), interleukin 10 (IL-10), transforming growth factor β (TGF-β), HIF-2α, and Notch1, 2, and 3. Results All groups exhibited skin ulcers and redness after irradiation. On day 3, exudation of tissue fluid was observed in all groups. On day 7, group B showed significantly smaller skin injury areas compared to the other 3 groups. On day 28, histological examination revealed that the epidermis was thickened and the dermal fibers were slightly disordered with occasional inflammatory cell aggregation in group A. In group B, the epidermis appeared more normal, the dermal fibers were more orderly, and there was an increase in new blood vessels without significant inflammatory cell aggregation. In contrast, groups C and D showed significantly increased epidermal thickness, disordered and disrupted dermal fibers. Group B had higher collagen fiber content than the other 3 groups, and group D had lower content than group A, with significant differences (P<0.05). Immunohistochemical staining showed that group B had significantly higher CD31 expression than the other 3 groups, while groups C and D had lower expression than group A, with significant differences (P<0.05). Western blot and qRT-PCR results indicated that group B had significantly higher relative expression levels of VEGF, PDGF-BB, FGF-2, IL-10, TGF-β, HIF-2α, and Notch1, 2, and 3 proteins and mRNA compared to the other 3 groups (P<0.05). Conclusion ICA may enhance the reparative effects of rat ADSCs on skin radiation damage by promoting angiogenesis and reducing inflammatory responses through the HIF-2α-VEGF-Notch signaling pathway.

1.	Borrelli MR, Shen AH, Lee GK, et al. Radiation-induced skin fibrosis: Pathogenesis, current treatment options, and emerging therapeutics. Ann Plast Surg, 2019, 83(4S Suppl 1): S59-S64.
2.	宁艳, 甘慧敏, 黄东琳, 等. 脂肪干细胞及细胞因子在皮肤放射性损伤修复中的研究进展. 辐射研究与辐射工艺学报, 2023, 41(2): 13-24.
3.	Hamel KM, Liimatta KQ, Belgodere JA, et al. Adipose-derived stromal/stem cell response to tumors and wounds: Evaluation of patient age. Stem Cells Dev, 2022, 31(19-20): 579-592.
4.	Szabó R, Rácz CP, Dulf FV. Bioavailability improvement strategies for icariin and its derivates: A review. Int J Mol Sci, 2022, 23(14): 7519. doi: 10.3390/ijms23147519.
5.	Gao J, Xiang S, Wei X, et al. Icariin promotes the osteogenesis of bone marrow mesenchymal stem cells through regulating sclerostin and activating the Wnt/β-catenin signaling pathway. Biomed Res Int, 2021, 2021: 6666836. doi: 10.1155/2021/6666836.
6.	Yang A, Yu C, Lu Q, et al. Mechanism of action of icariin in bone marrow mesenchymal stem cells. Stem Cells Int, 2019, 2019: 5747298. doi: 10.1155/2019/5747298.
7.	Liao J, Wei Q, Zou Y, et al. Notch signaling augments bmp9-induced bone formation by promoting the osteogenesis-angiogenesis coupling process in mesenchymal stem cells (MSCs). Cell Physiol Biochem, 2017, 41(5): 1905-1923.
8.	Whyte JL, Ball SG, Shuttleworth CA, et al. Density of human bone marrow stromal cells regulates commitment to vascular lineages. Stem Cell Res, 2011, 6(3): 238-250.
9.	Terlizzi V, Kolibabka M, Burgess JK, et al. The pericytic phenotype of adipose tissue-derived stromal cells is promoted by NOTCH2. Stem Cells, 2018, 36(2): 240-251.
10.	Fan JJ, Cao LG, Wu T, et al. The dose-effect of icariin on the proliferation and osteogenic differentiation of human bone mesenchymal stem cells. Molecules, 2011, 16(12): 10123-10133.
11.	梁至洁, 黄东琳, 朱丹丹, 等. 淫羊藿苷优化大鼠脂肪干细胞促进超长随意皮瓣存活的实验研究. 中国临床新医学, 2022, 15(12): 1140-1146.
12.	唐茂涵, 张艳杰, 刘银, 等. 三甲氧苄嗪对脂肪来源间充质干细胞氧化应激损伤的影响及机制研究. 四川大学学报 (医学版), 2017, 48(2): 173-178.
13.	Malekzadeh H, Surucu Y, Chinnapaka S, et al. Metformin and adipose-derived stem cell combination therapy alleviates radiation-induced skin fibrosis in mice. Stem Cell Res Ther, 2024, 15(1): 13. doi: 10.1186/s13287-023-03627-7.
14.	Li Z, Gan H, Liang A, et al. Promoting repair of highly purified stromal vascular fraction gel combined with advanced platelet-rich fibrin extract for irradiated skin and soft tissue injury. Ann Transl Med, 2022, 10(17): 933. doi: 10.21037/atm-22-3956.
15.	张炜强, 傅丽琴, 黎秋生. MEBT/MEBO联合ADSC治疗慢性难愈合创面的疗效分析. 中国烧伤创疡杂志, 2021, 33(5): 317-320.
16.	刘志燕, 饶振, 盛小伍, 等. 脂肪干细胞对大鼠急性放射性皮肤损伤的干预作用. 中南大学学报 (医学版), 2019, 44(2): 150-157.
17.	Chinnapaka S, Yang KS, Samadi Y, et al. Allogeneic adipose-derived stem cells mitigate acute radiation syndrome by the rescue of damaged bone marrow cells from apoptosis. Stem Cells Transl Med, 2021, 10(7): 1095-1114.
18.	尹小芳, 秦岭, 杨超, 等. ADSCs、VEGF和GM-CSF修复成纤维细胞放射性损伤的机制研究. 中国美容医学, 2023, 32(8): 29-34.
19.	Lin Z, Shibuya Y, Imai Y, et al. Therapeutic potential of adipose-derived stem cell-conditioned medium and extracellular vesicles in an in vitro radiation-induced skin injury model. Int J Mol Sci, 2023, 24(24): 17214. doi: 10.3390/ijms242417214.
20.	蔡卫超, 曹卫红, 胡大利, 等. mRNA水平探究人脂肪干细胞对SD大鼠放射性皮肤损伤的炎症修复作用. 中国现代医生, 2023, 61(23): 25-30.
21.	Berry CE, Abbas DB, Lintel HA, et al. Adipose-derived stromal cell-based therapies for radiation-induced fibrosis. Adv Wound Care (New Rochelle), 2024, 13(5): 235-252.
22.	Felix RG, Bovolato ALC, Cotrim OS, et al. Adipose-derived stem cells and adipose-derived stem cell-conditioned medium modulate in situ imbalance between collagen Ⅰ- and collagen Ⅴ-mediated IL-17 immune response recovering bleomycin pulmonary fibrosis. Histol Histopathol, 2020, 35(3): 289-301.
23.	Rautiainen S, Laaksonen T, Koivuniemi R. Angiogenic effects and crosstalk of adipose-derived mesenchymal stem/stromal cells and their extracellular vesicles with endothelial cells. Int J Mol Sci, 2021, 22(19): 10890. doi: 10.3390/ijms221910890.
24.	Horton JA, Hudak KE, Chung EJ, et al. Mesenchymal stem cells inhibit cutaneous radiation-induced fibrosis by suppressing chronic inflammation. Stem Cells, 2013, 31(10): 2231-2241.
25.	Chiossone L, Conte R, Spaggiari GM, et al. Mesenchymal stromal cells induce peculiar alternatively activated macrophages capable of dampening both innate and adaptive immune responses. Stem Cells, 2016, 34(7): 1909-1921.
26.	Zhao J, Du F, Shen G, et al. The role of hypoxia-inducible factor-2 in digestive system cancers. Cell Death Dis, 2015, 6(1): e1600. doi: 10.1038/cddis.2014.565.
27.	Hamidian A, von Stedingk K, Munksgaard Thorén M, et al. Differential regulation of HIF-1α and HIF-2α in neuroblastoma: Estrogen-related receptor alpha (ERRα) regulates HIF2A transcription and correlates to poor outcome. Biochem Biophys Res Commun, 2015, 461(3): 560-567.
28.	De Bock K, Georgiadou M, Carmeliet P. Role of endothelial cell metabolism in vessel sprouting. Cell Metab, 2013, 18(5): 634-647.
29.	Pietras A, von Stedingk K, Lindgren D, et al. JAG2 induction in hypoxic tumor cells alters Notch signaling and enhances endothelial cell tube formation. Mol Cancer Res, 2011, 9(5): 626-636.

1. Borrelli MR, Shen AH, Lee GK, et al. Radiation-induced skin fibrosis: Pathogenesis, current treatment options, and emerging therapeutics. Ann Plast Surg, 2019, 83(4S Suppl 1): S59-S64.
2. 宁艳, 甘慧敏, 黄东琳, 等. 脂肪干细胞及细胞因子在皮肤放射性损伤修复中的研究进展. 辐射研究与辐射工艺学报, 2023, 41(2): 13-24.
3. Hamel KM, Liimatta KQ, Belgodere JA, et al. Adipose-derived stromal/stem cell response to tumors and wounds: Evaluation of patient age. Stem Cells Dev, 2022, 31(19-20): 579-592.
4. Szabó R, Rácz CP, Dulf FV. Bioavailability improvement strategies for icariin and its derivates: A review. Int J Mol Sci, 2022, 23(14): 7519. doi: 10.3390/ijms23147519.
5. Gao J, Xiang S, Wei X, et al. Icariin promotes the osteogenesis of bone marrow mesenchymal stem cells through regulating sclerostin and activating the Wnt/β-catenin signaling pathway. Biomed Res Int, 2021, 2021: 6666836. doi: 10.1155/2021/6666836.
6. Yang A, Yu C, Lu Q, et al. Mechanism of action of icariin in bone marrow mesenchymal stem cells. Stem Cells Int, 2019, 2019: 5747298. doi: 10.1155/2019/5747298.
7. Liao J, Wei Q, Zou Y, et al. Notch signaling augments bmp9-induced bone formation by promoting the osteogenesis-angiogenesis coupling process in mesenchymal stem cells (MSCs). Cell Physiol Biochem, 2017, 41(5): 1905-1923.
8. Whyte JL, Ball SG, Shuttleworth CA, et al. Density of human bone marrow stromal cells regulates commitment to vascular lineages. Stem Cell Res, 2011, 6(3): 238-250.
9. Terlizzi V, Kolibabka M, Burgess JK, et al. The pericytic phenotype of adipose tissue-derived stromal cells is promoted by NOTCH2. Stem Cells, 2018, 36(2): 240-251.
10. Fan JJ, Cao LG, Wu T, et al. The dose-effect of icariin on the proliferation and osteogenic differentiation of human bone mesenchymal stem cells. Molecules, 2011, 16(12): 10123-10133.
11. 梁至洁, 黄东琳, 朱丹丹, 等. 淫羊藿苷优化大鼠脂肪干细胞促进超长随意皮瓣存活的实验研究. 中国临床新医学, 2022, 15(12): 1140-1146.
12. 唐茂涵, 张艳杰, 刘银, 等. 三甲氧苄嗪对脂肪来源间充质干细胞氧化应激损伤的影响及机制研究. 四川大学学报 (医学版), 2017, 48(2): 173-178.
13. Malekzadeh H, Surucu Y, Chinnapaka S, et al. Metformin and adipose-derived stem cell combination therapy alleviates radiation-induced skin fibrosis in mice. Stem Cell Res Ther, 2024, 15(1): 13. doi: 10.1186/s13287-023-03627-7.
14. Li Z, Gan H, Liang A, et al. Promoting repair of highly purified stromal vascular fraction gel combined with advanced platelet-rich fibrin extract for irradiated skin and soft tissue injury. Ann Transl Med, 2022, 10(17): 933. doi: 10.21037/atm-22-3956.
15. 张炜强, 傅丽琴, 黎秋生. MEBT/MEBO联合ADSC治疗慢性难愈合创面的疗效分析. 中国烧伤创疡杂志, 2021, 33(5): 317-320.
16. 刘志燕, 饶振, 盛小伍, 等. 脂肪干细胞对大鼠急性放射性皮肤损伤的干预作用. 中南大学学报 (医学版), 2019, 44(2): 150-157.
17. Chinnapaka S, Yang KS, Samadi Y, et al. Allogeneic adipose-derived stem cells mitigate acute radiation syndrome by the rescue of damaged bone marrow cells from apoptosis. Stem Cells Transl Med, 2021, 10(7): 1095-1114.
18. 尹小芳, 秦岭, 杨超, 等. ADSCs、VEGF和GM-CSF修复成纤维细胞放射性损伤的机制研究. 中国美容医学, 2023, 32(8): 29-34.
19. Lin Z, Shibuya Y, Imai Y, et al. Therapeutic potential of adipose-derived stem cell-conditioned medium and extracellular vesicles in an in vitro radiation-induced skin injury model. Int J Mol Sci, 2023, 24(24): 17214. doi: 10.3390/ijms242417214.
20. 蔡卫超, 曹卫红, 胡大利, 等. mRNA水平探究人脂肪干细胞对SD大鼠放射性皮肤损伤的炎症修复作用. 中国现代医生, 2023, 61(23): 25-30.
21. Berry CE, Abbas DB, Lintel HA, et al. Adipose-derived stromal cell-based therapies for radiation-induced fibrosis. Adv Wound Care (New Rochelle), 2024, 13(5): 235-252.
22. Felix RG, Bovolato ALC, Cotrim OS, et al. Adipose-derived stem cells and adipose-derived stem cell-conditioned medium modulate in situ imbalance between collagen Ⅰ- and collagen Ⅴ-mediated IL-17 immune response recovering bleomycin pulmonary fibrosis. Histol Histopathol, 2020, 35(3): 289-301.
23. Rautiainen S, Laaksonen T, Koivuniemi R. Angiogenic effects and crosstalk of adipose-derived mesenchymal stem/stromal cells and their extracellular vesicles with endothelial cells. Int J Mol Sci, 2021, 22(19): 10890. doi: 10.3390/ijms221910890.
24. Horton JA, Hudak KE, Chung EJ, et al. Mesenchymal stem cells inhibit cutaneous radiation-induced fibrosis by suppressing chronic inflammation. Stem Cells, 2013, 31(10): 2231-2241.
25. Chiossone L, Conte R, Spaggiari GM, et al. Mesenchymal stromal cells induce peculiar alternatively activated macrophages capable of dampening both innate and adaptive immune responses. Stem Cells, 2016, 34(7): 1909-1921.
26. Zhao J, Du F, Shen G, et al. The role of hypoxia-inducible factor-2 in digestive system cancers. Cell Death Dis, 2015, 6(1): e1600. doi: 10.1038/cddis.2014.565.
27. Hamidian A, von Stedingk K, Munksgaard Thorén M, et al. Differential regulation of HIF-1α and HIF-2α in neuroblastoma: Estrogen-related receptor alpha (ERRα) regulates HIF2A transcription and correlates to poor outcome. Biochem Biophys Res Commun, 2015, 461(3): 560-567.
28. De Bock K, Georgiadou M, Carmeliet P. Role of endothelial cell metabolism in vessel sprouting. Cell Metab, 2013, 18(5): 634-647.
29. Pietras A, von Stedingk K, Lindgren D, et al. JAG2 induction in hypoxic tumor cells alters Notch signaling and enhances endothelial cell tube formation. Mol Cancer Res, 2011, 9(5): 626-636.

Chinese Journal of Reparative and Reconstructive Surgery

Latest ArticlesExperimental study on promotion of skin radiation damage repair by icarin via HIF-2α/VEGF/Notch pathway to enhance the paracrine function of adipose-derived stem cells

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