miR-138的下调通过激活PI3K/AKT信号通路和hTERT减轻糖尿病足溃疡大鼠的炎症反应并促进伤口愈合
Down-Regulation of miR-138 Alleviates Inflammatory Response and Promotes Wound Healing in Diabetic Foot Ulcer Rats via Activating PI3K/AKT Pathway and hTERT.
作者信息
Wang Jian, Zhao Xiaodan, Tian Guichang, Liu Xiaochao, Gui Chengyan, Xu Lin
机构信息
School of Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, People's Republic of China.
Department of Orthopedics, Qufu Hospital of TCM, Qufu, 273100, People's Republic of China.
出版信息
Diabetes Metab Syndr Obes. 2022 Apr 14;15:1153-1163. doi: 10.2147/DMSO.S359759. eCollection 2022.
OBJECTIVE
To study the role of miR-138 on the repair of diabetic foot ulcer (DFU) and further to explore its possible mechanism.
MATERIALS AND METHODS
miR-138 inhibitor, IGF-1, LY294002 were used in DFU rat mode, and the mRNA expression of miR-138 was detected. HE staining was used to observe the histological changes of skin ulcer in rats. The level of inflammation, wound healing, and blood vessel formation-related factors were detected by ELISA and immunohistochemical. The expression of VEGF and PI3K/AKT pathway-related proteins were detected by Western blot. To further determine the underlying mechanism of miR-138 in the repair of DFU, telomerase inhibitor BIBR-1232 was used in HUVECs. Dual-luciferase assay was used to determine the target relationship between miR-138 and hTERT. CCK-8, transwell, and tube formation assays were conducted to observe the biological behavior of HUVECs. Inflammatory cytokines and PI3K/AKT pathway-related proteins were also measured by ELISA and Western blot.
RESULTS
The mRNA expression of miR-138 in DFU rat was increased and ulcer of diabetic foot rats was improved after silencing miR-138. The results of cellular bioactivity in vitro experiment were consistent with that in vivo. Meanwhile, after silencing miR-138, the level of inflammatory cytokines was decreased, while the level of anti-inflammatory and healing factors was increased in vivo and vitro. Moreover, the ratios of p-PI3K/PI3K and p-AKT/AKT were upregulated after treated with miR-138 inhibitor and miR-138 was negatively regulated the expression of hTERT. However, the inhibitory effect on inflammatory response and the promotion effect on wound healing of miR-138 inhibitor were reversed by LY294002 and BIBR-1232.
CONCLUSION
Down-regulation of miR-138 could alleviate inflammatory response and promote wound healing in DFU rats by activating PI3K/AKT pathway and hTERT.
目的
研究miR-138在糖尿病足溃疡（DFU）修复中的作用，并进一步探讨其可能机制。
材料与方法
在DFU大鼠模型中使用miR-138抑制剂、IGF-1、LY294002，并检测miR-138的mRNA表达。采用HE染色观察大鼠皮肤溃疡的组织学变化。通过ELISA和免疫组化检测炎症、伤口愈合及血管生成相关因子水平。采用Western blot检测VEGF和PI3K/AKT通路相关蛋白的表达。为进一步确定miR-138在DFU修复中的潜在机制，在人脐静脉内皮细胞（HUVECs）中使用端粒酶抑制剂BIBR-1232。采用双荧光素酶报告基因检测法确定miR-138与hTERT的靶向关系。进行CCK-8、Transwell和管腔形成实验观察HUVECs的生物学行为。同时通过ELISA和Western blot检测炎症细胞因子及PI3K/AKT通路相关蛋白。
结果
沉默miR-138后，DFU大鼠中miR-138的mRNA表达增加，糖尿病足大鼠的溃疡得到改善。体外细胞生物活性实验结果与体内一致。同时，沉默miR-138后，体内外炎症细胞因子水平降低，抗炎及愈合因子水平升高。此外，用miR-138抑制剂处理后，p-PI3K/PI3K和p-AKT/AKT的比值上调，且miR-138对hTERT的表达具有负调控作用。然而，LY294002和BIBR-1232可逆转miR-138抑制剂对炎症反应的抑制作用及对伤口愈合的促进作用。
结论
下调miR-138可通过激活PI3K/AKT通路和hTERT减轻DFU大鼠的炎症反应并促进伤口愈合。
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