Abstract：
BACKGROUND: The reactive oxygen species (ROS) surge in the chronic wound tissue of diabetic ulcers (DUs) aggravates the inflammatory response. The oxidative stress state during inflammation will exacerbate inflammation and cause tissue damage, resulting in prolonged wound healing. Shengjihuayu Formula (SJHYF) is a renowned Chinese medicine prescription for treating chronic wounds in diabetic ulcers. Growing clinical evidence has demonstrated that SJHYF exhibits superior therapeutic efficacy and has a favorable safety profile. However, the underlying mechanisms by which SJHYF ameliorates oxidative damage under pathological conditions of DUs remain unclear.
OBJECTIVE: To investigate the cytoprotective properties of SJHYF on hydrogen peroxide (H2O2)-induced cell damage in human HaCaT keratinocytes and to explore its potential targets and molecular pathways in treating DUs using RNA-seq.
METHODS: HaCaT cells were incubated with H2O2 for 24 h to construct an oxidative stress cell model. Cell viability and proliferation were measured using the MTT and EdU assays, respectively. Cell migration was assessed using the scratch assay, and the fluorescence intensity of ROS was measured using the DCFH-DA probe. The chemical components of SJHYF were analyzed by UPLC-Q-TOF/MS, while the therapeutic effects of SJHYF on H2O2-induced HaCaT cells were analyzed using RNA-Seq. The potential target genes were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). At the same time, the pathway phenotype expression of SJHYF on the protection of H2O2-induced HaCaT cells was explored using Western Blot.
RESULTS: The application of SJHY at a concentration of 0.25 mg/mL promoted cell proliferation, cell migration, and reduced ROS production. In addition, SJHYF was detected to have a total of 93 active compounds, including key components such as Galloyl-beta-D-glucose, Danshensu, Procyanidin B2, Catechin, and Alkannin. The RNA-seq analysis identified several core targets namely KRT17, TGM1, JUNB, PRDX5, TXNIP, PRDX1, HSP90AA1, HSP90AB1, HSPA8, and TNF-α. Western blot revealed the presence of the JNK/c-Jun/MMPs pathway and its related transcription factors.
CONCLUSIONS: SJHYF displays significant protective effects on H2O2-induced oxidative cell damage in HaCaT cells via blocking the JNK/c-Jun/MMPs pathway.
OBJECTIVE: To investigate the cytoprotective properties of SJHYF on hydrogen peroxide (H2O2)-induced cell damage in human HaCaT keratinocytes and to explore its potential targets and molecular pathways in treating DUs using RNA-seq.
METHODS: HaCaT cells were incubated with H2O2 for 24 h to construct an oxidative stress cell model. Cell viability and proliferation were measured using the MTT and EdU assays, respectively. Cell migration was assessed using the scratch assay, and the fluorescence intensity of ROS was measured using the DCFH-DA probe. The chemical components of SJHYF were analyzed by UPLC-Q-TOF/MS, while the therapeutic effects of SJHYF on H2O2-induced HaCaT cells were analyzed using RNA-Seq. The potential target genes were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). At the same time, the pathway phenotype expression of SJHYF on the protection of H2O2-induced HaCaT cells was explored using Western Blot.
RESULTS: The application of SJHY at a concentration of 0.25 mg/mL promoted cell proliferation, cell migration, and reduced ROS production. In addition, SJHYF was detected to have a total of 93 active compounds, including key components such as Galloyl-beta-D-glucose, Danshensu, Procyanidin B2, Catechin, and Alkannin. The RNA-seq analysis identified several core targets namely KRT17, TGM1, JUNB, PRDX5, TXNIP, PRDX1, HSP90AA1, HSP90AB1, HSPA8, and TNF-α. Western blot revealed the presence of the JNK/c-Jun/MMPs pathway and its related transcription factors.
CONCLUSIONS: SJHYF displays significant protective effects on H2O2-induced oxidative cell damage in HaCaT cells via blocking the JNK/c-Jun/MMPs pathway.
摘要：
背景：糖尿病溃疡（DU）的慢性伤口组织中的活性氧（ROS）激增加剧了炎症反应。炎症过程中的氧化应激状态会加剧炎症，引起组织损伤，导致伤口愈合时间延长。生积化瘀方（SJHYF）是治疗糖尿病溃疡慢性伤口的著名中药方剂。越来越多的临床证据表明，SJHYF表现出优异的治疗效果，并具有良好的安全性。然而,SJHYF在DU病理条件下改善氧化损伤的潜在机制尚不清楚.
目的：研究SJHYF对过氧化氢（H2O2）诱导的人HaCaT角质形成细胞损伤的细胞保护特性，并探讨其使用RNA-seq治疗DU的潜在靶标和分子途径。
方法：HaCaT细胞与H2O2孵育24小时，构建氧化应激细胞模型。使用MTT和EdU测定法测量细胞活力和增殖,分别。使用划痕试验评估细胞迁移，使用DCFH-DA探针测量ROS的荧光强度。通过UPLC-Q-TOF/MS分析SJHYF的化学成分，同时使用RNA-Seq分析SJHYF对H2O2诱导的HaCaT细胞的治疗作用。使用逆转录-定量聚合酶链反应(RT-qPCR)验证潜在的靶基因。同时,采用WesternBlot法探讨SJHYF对H2O2诱导的HaCaT细胞保护作用的通路表型表达。
结果：以0.25mg/mL的浓度应用SJHY促进细胞增殖，细胞迁移，并减少了ROS的产生。此外,SJHYF检测到共有93种活性化合物，包括关键成分，如没食子酰-β-D-葡萄糖，丹参素,原花青素B2，儿茶素，和碱宁。RNA-seq分析确定了几个核心靶标，即KRT17，TGM1，JUNB，PRDX5、TXNIP、PRDX1、HSP90AA1、HSP90AB1、HSPA8和TNF-α。Westernblot显示存在JNK/c-Jun/MMPs通路及其相关转录因子。
结论：SJHYF通过阻断JNK/c-Jun/MMPs通路对H2O2诱导的HaCaT细胞氧化损伤具有明显的保护作用。
目的：研究SJHYF对过氧化氢（H2O2）诱导的人HaCaT角质形成细胞损伤的细胞保护特性，并探讨其使用RNA-seq治疗DU的潜在靶标和分子途径。
方法：HaCaT细胞与H2O2孵育24小时，构建氧化应激细胞模型。使用MTT和EdU测定法测量细胞活力和增殖,分别。使用划痕试验评估细胞迁移，使用DCFH-DA探针测量ROS的荧光强度。通过UPLC-Q-TOF/MS分析SJHYF的化学成分，同时使用RNA-Seq分析SJHYF对H2O2诱导的HaCaT细胞的治疗作用。使用逆转录-定量聚合酶链反应(RT-qPCR)验证潜在的靶基因。同时,采用WesternBlot法探讨SJHYF对H2O2诱导的HaCaT细胞保护作用的通路表型表达。
结果：以0.25mg/mL的浓度应用SJHY促进细胞增殖，细胞迁移，并减少了ROS的产生。此外,SJHYF检测到共有93种活性化合物，包括关键成分，如没食子酰-β-D-葡萄糖，丹参素,原花青素B2，儿茶素，和碱宁。RNA-seq分析确定了几个核心靶标，即KRT17，TGM1，JUNB，PRDX5、TXNIP、PRDX1、HSP90AA1、HSP90AB1、HSPA8和TNF-α。Westernblot显示存在JNK/c-Jun/MMPs通路及其相关转录因子。
结论：SJHYF通过阻断JNK/c-Jun/MMPs通路对H2O2诱导的HaCaT细胞氧化损伤具有明显的保护作用。
