摘要
目的:探究皮肤损伤愈合中基质刚度对巨噬细胞功能的调控作用及机制,阐明机械敏感离子通道Piezo1介导的力学-免疫-代谢调控通路。方法:制备刚度分别为(6.77±1.35)kPa(Soft组)与(16.68±1.82)kPa(Stiff组)的甲基丙烯酰化明胶(GelMA)水凝胶,并完成体外表征;建立小鼠全层皮肤缺损模型(分为Sham、Soft、Stiff组),通过标准化影像结合苏木精-伊红染色、Masson染色,检测皮损愈合相关指标;分离培养小鼠骨髓来源巨噬细胞(BMDMs)并接种于两种水凝胶表面,另设Piezo1激动剂(Yoda1)/抑制剂(GsMTx4)药理学干预组,采用免疫荧光、实时荧光定量PCR、Western blot检测Piezo1表达、细胞骨架相关指标以及促炎因子诱导型一氧化氮合酶(iNOS)、白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)、IL-1β与促修复因子IL-10、转化生长因子-β(TGF-β)、精氨酸酶-1(Arg1)的基因或蛋白表达水平;通过转录组测序(RNA-seq)进行京都基因与基因组百科全书(KEGG)富集分析,并检测糖酵解/磷酸戊糖途径相关代谢指标(葡萄糖剩余量、乳酸生成量、NADPH/NADP^(+)比值)及关键酶基因(G6PD及PKM2)表达。结果:成功构建低刚度、高刚度水凝胶,两者均具有良好生物相容性。体内实验显示,Stiff组小鼠创面愈合率、肉芽组织厚度及胶原沉积均显著高于其他两组(P<0.05)。体外实验表明,Stiff组可上调BMDMs中Piezo1表达、促进细胞骨架重组,同时下调促炎因子、上调促修复因子表达(P<0.05);此外,Stiff组巨噬细胞糖酵解与磷酸戊糖途径激活(P<0.001),葡萄糖剩余量降低(P<0.05),乳酸生成量、NADPH/NADP^(+)比值及G6PD、PKM2基因表达均显著升高(P<0.05),上述效应可被Piezo1激动剂/抑制剂逆转。结论:高刚度GelMA水凝胶可通过激活巨噬细胞Piezo1,调控其糖酵解与磷酸戊糖代谢通路,推动巨噬细胞向修复表型转化,进而促进皮肤损伤愈合。
Objective:To investigate the regulatory effect and underlying mechanism of matrix stiffness on macrophage function during skin wound healing,and to clarify the mechano-immune-metabolic regulatory pathway mediated by the mechanosensitive ion channel Piezo1.Methods:Gelatin-methacryloyl(GelMA)hydrogels with stiffness of(6.77±1.35)kPa(Soft group)and(16.68±1.82)kPa(Stiff group)were fabricated and characterized.A mouse model of full-thickness skin defect was established.Mice were randomly divided into Sham,Soft,and Stiff groups.Wound healing was assessed via standardized imaging and histological evaluation(HE and Masson staining).Mouse bone-marrow-derived macrophages(BMDMs)were isolated,cultured,and seeded on the hydrogels.Additional groups treated with a Piezo1 agonist(Yoda1)or inhibitor(GsMTx4)were established for pharmacological intervention.The expression of Piezo1,cytoskeletal-related indicators,and the gene or protein expression levels of pro-inflammatory factors including inducible nitric oxide synthase(iNOS),interleukin(IL)-6,tumor necrosis factor-α(TNF-α),IL-1βand pro-repair factors including IL-10,transforming growth factor-β(TGF-β),and arginase-1(Arg1)were detected using immunofluorescence,real-time quantitative PCR and Western blot.RNA sequencing was performed,followed by Kyoto Encyclopedia of Genes and Genomes enrichment analysis.Moreover,the metabolic indicators related to the glycolysis/pentose phosphate pathways(residual glucose lactic acid production,and NADPH/NADP^(+) ratio)and the expression of key enzyme genes(G6PD and PKM2)were determined.Results:Soft and stiff hydrogels were successfully constructed and exhibited good biocompatibility.In vivo,the wound healing rate,granulation tissue thickness,and collagen deposition in the Stiff group were significantly increased(P<0.05).In vitro,the Stiff group upregulated Piezo1 expression in BMDMs,promoted cytoskeletal reorganization,downregul-ated pro-inflammatory factors,and upregulated repair-related factors(P<0.05).Furthermore,the glycolysis and pentose phosphate pathways were significantly activated in macrophages of the Stiff group(P<0.001),residual glucose was reduced,and lactic acid production,NADPH/NADP^(+) ratio,as well as the expression of G6PD and PKM2 genes,were all significantly elevated(P<0.05).These effects could be reversed by the intervention of Piezo1 agonist or inhibitor.Conclusions:Stiff hydrogels promote macrophage polarization toward a reparative phenotype by activating Piezo1 and subsequently reprogramming glycolytic and pentose phosphate metabolic pathways,ultimately accelerating skin wound healing.
作者
梁玙璠
刘文豪
董佳欢
程佳宇
何小宁
李蓓
LIANG Yufan;LIU Wenhao;DONG Jiahuan;CHENG Jiayu;HE Xiaoning;LI Bei(State Key Laboratory of Oral&Maxillofacial Reconstruction and Regeneration,National Clinical Research Center for Oral Diseases,Shaanxi International Joint Research Center for Oral Diseases,Xi'an 710032,China;Department of Histopathology,School of Stomatology,The Air Force Military Medical University,Xi'an 710032,China)
出处
《口腔生物医学》
2026年第2期79-88,共10页
Oral Biomedicine
基金
陕西省重点研发计划项目(2024SF-GJHX-29)。
