黄连解毒汤调控lncRNA NEAT1减轻脓肿创面炎症反应的机制研究

Effect of Huanglian Jiedu Decoction on reducing inflammation of abscess from the perspective of lncRNA NEAT1

关键词:  黄连解毒汤  lncRNA NEAT1  炎症反应  皮肤创面

Key words:  huang lian jiedu decoction  lncRNA NEAT1  inflammation  skin wound

摘要:

目的 从lncRNA NEAT1调控角度探讨黄连解毒汤减轻脓肿创面炎症反应的作用机制。方法 将C57BL/6小鼠分为空白组、模型组、黄连解毒汤组、对照组。经干预后麻醉下取材。动物实验：形态学观察创面组织情况；Real-time PCR和Western blotting分别检测脓肿创面组织中lncRNA NEAT1、miRNA-146a-5p变化及RELA、TNF-ɑ、TGF-β1、α-SMA、Collagen Ⅲ蛋白表达情况。细胞实验：Real-time PCR检测lncRNA NEAT1、miR-146a-5p变化；流式细胞术检测细胞凋亡情况；FISH检测荧光强度变化；Western blotting检测相关蛋白表达情况。结果 与模型组、对照组比较，黄连解毒汤组创面修复较佳，创面中炎性细胞浸润程度明显减少；与模型组、对照组比较，黄连解毒汤组lncRNA NEAT1及RELA、TNF-ɑ、TGF-β1蛋白表达降低，miR-146a-5p及α-SMA、Collagen Ⅲ蛋白表达升高(P<0.05)；与LPS+Lenti-control组比较，LPS+Lenti-sh-NEAT1组lncRNA NEAT1水平降低，miR-146a-5p水平升高(P<0.05)；黄连解毒汤可降低LPS诱导小鼠成纤维细胞凋亡率及lncRNA NEAT1荧光强度；与空白组比较，LPS组RELA、TNF-ɑ、TGF-β1蛋白表达增高，α-SMA、Collagen Ⅲ蛋白表达降低(P<0.05)，经sh-NEAT1后，黄连解毒汤可减弱以上蛋白表达(P<0.05)。结论 黄连解毒汤可通过lncRNA NEAT1减轻脓肿创面炎症反应，与调控miR-146a、RELA、TNF-ɑ、TGF-β1、α-SMA、Collagen Ⅲ表达关系密切。

Abstract:

OBJECTIVE To explore the effect and mechanism of Huanglian Jiedu Decoction in reducing inflammation of abscess from the perspective of lncRNA NEAT1. METHODS C57BL/6 mice were divided into blank group, model group, Huanglian Jiedu Decoction group and control group. After the intervention, the mice were killed under anesthesia, and the skin tissue of the abscess wound was separated and collected. Animal experiment: the wound tissue of each group was observed by morphology. Real-time PCR and Western blotting were used to detect lncRNA NEAT1, miR-146a-5p gene level changes and RELA, TNF-ɑ, TGF-β1, α-SMA, Collagen Ⅲ protein expression respectively. Cell experiments: Real-time PCR was used to detect the changes of lncRNA NEAT1 and miR-146a-5p gene levels. The effect of flow cytometry on apoptosis rate was detected. Fluorescence intensity was measured by FISH.The expression of related proteins was detected by Western blotting. RESULTS Compared with model and control group, The wound was repaired well and the inflammatory cell infiltration in the wound of the Huanglian Jiedu decoction group was reduced. Compared with the model and control group, the level of lncRNA NEAT1 and protein expressions of RELA, TNF-ɑ,TGF-β1 in the Huanglian Jiedu Decoction group were significantly reduced, while the gene level of miR-146a-5p and the protein expressions of α-SMA and Collagen III were significantly increased(P<0.05). Compared with LPS+Lenti-control group, lncRNA NEAT1 gene level in LPS+Lenti-sh-NEAT1 group was significantly decreased, while miR-146a-5p gene level was increased(P<0.05). Flow cytometry showed that Huanglian Jiedu decoction could reduce the apoptosis rate and fluorescence intensity of lncRNA NEAT1(P<0.05). Compared with the blank group, the protein expressions of RELA, TNF-ɑ,TGF-β1 in LPS group were significantly increased, while the protein expressions of α-SMA and Collagen III were significantly decreased (P<0.05). After sh-NEAT1 treatment, The regulatory effects of Huanglian Jiedu Decoction on the expression of above molecule were reduced(P<0.05). CONCLUSION Huanglian Jiedu Decoction can reduce the inflammatory state of mouse abscess wound through lncRNA NEAT1, which is closely related to the regulation of miR-146a, RELA, TNF-ɑ, TGF-β1, α-SMA and Collagen III expression.