积雪草酸影响HaCaT细胞增殖和对咪喹莫特诱导银屑病小鼠模型的作用研究

发布时间：2018-04-18 12:09

本文选题：积雪草酸 + 银屑病　；参考：《江苏大学》2017年硕士论文

【摘要】：背景:积雪草酸是一种主要存在于伞形科植物积雪草中的乌苏烷型五环三萜酸,其资源丰富,目前主要分布于我国长江流域以南地区。现有研究表明,积雪草酸具有抗肿瘤、抗炎症、抗氧化、抗抑郁等多种药理活性。银屑病是一种病理表现为角质形成细胞(keratinocyte,KC)的过度增殖和炎症细胞浸润的慢性皮肤病。HaCaT细胞是自发永生化的人角化细胞系,已广泛用于许多皮肤疾病的研究。本实验拟探讨积雪草酸对咪喹莫特诱导的小鼠银屑病模型及其对角质形成细胞的作用和机制。方法:建立咪喹莫特诱导小鼠银屑病模型,考察积雪草酸对小鼠皮损组织的影响;测定血清中IL-17、IL-23、TNF-α的含量来检测炎症因子的释放;检测小鼠皮损组织中PCNA、Bax、Bcl-2蛋白的表达。建立TNF-α诱导HaCaT细胞增殖的细胞模型,考察积雪草酸抑制TNF-α诱导HaCaT细胞增殖的分子机制。以不同浓度积雪草酸作用于TNF-α诱导HaCaT细胞24 h,MTT法检测细胞活性;流式细胞术检测细胞周期;免疫荧光染色法检测NF-κB的核转位;Western blot法检测细胞蛋白NF-κB、p-NF-κB的表达。研究积雪草酸对HaCaT细胞凋亡的影响:MTT法检测细胞活性;DCFH-DA染色检测细胞内及线粒体ROS生成;JC-1染色检测线粒体膜电位,荧光素酶法检测ATP含量,分析线粒体功能。结果:通过对皮肤的外观观察以及组织HE染色发现,积雪草酸对咪喹莫特诱导小鼠银屑病模型皮肤的银屑样症状具有较好的改善效果。检测小鼠血清中IL-17、IL-23、TNF-α的含量发现,咪喹莫特造成小鼠银屑病模型时,IL-17、IL-23、TNF-α含量显著升高(P0.01),积雪草酸给药处理后能够明显降低TNF-α的升高,但是对IL-17、IL-23无明显影响。检测小鼠皮损组织蛋白的表达水平发现,积雪草酸处理后,细胞核抗原PCNA表达水平降低、细胞凋亡蛋白Bax表达水平升高、Bcl-2表达水平降低。细胞水平研究结果显示,积雪草酸能够显著抑制TNF-α诱导的HaCaT细胞增殖,阻滞细胞周期于G2期。同时我们发现积雪草酸能够抑制TNF-α诱导的NF-κB磷酸化,并阻止其转位入核。此外,积雪草酸处理能够诱导HaCaT细胞内ROS生成增多,促使线粒体膜电位倒塌,抑制细胞内ATP的生成,最终诱导角质形成细胞凋亡。结论:积雪草酸具有良好改善小鼠银屑病的作用,表现为抑制角质形成细胞的增殖。在细胞水平,积雪草酸能够抑制TNF-α诱导HaCaT细胞增殖并显著能够损伤HaCaT细胞的线粒体,诱导细胞发生凋亡。其机制的可能与积雪草酸能够抑制TNF-α诱导的NF-κB通路活化相关。
[Abstract]:Background: Asiaticoic acid is a kind of ursolane-type triterpenoid acid which mainly exists in the umbellidae plants. It is rich in resources and is mainly distributed in the south of the Yangtze River basin in China.Current studies have shown that oxalic acid has many pharmacological activities, such as anti-tumor, anti-inflammatory, anti-oxidation, anti-depressant and so on.Psoriasis is a chronic dermatosis, characterized by excessive proliferation of keratinocytes and infiltration of inflammatory cells in keratinocytes. HaCaT cells are spontaneous immortalized human keratinized cell lines, which have been widely used in the study of many skin diseases.The aim of this study was to investigate the effect and mechanism of oxalic acid on the psoriasis model induced by Imiquimote in mice and on keratinocytes.Methods: the model of psoriasis induced by imiquimod was established, the effect of oxalic acid on the skin lesions of mice was investigated, the levels of IL-17, IL-23 and TNF- 伪 in serum were determined to detect the release of inflammatory factors, and the expression of PCNA Baxax-Bcl 2 protein in the lesions of mice was detected.To establish a cell model of HaCaT cell proliferation induced by TNF- 伪, and to investigate the molecular mechanism of cytosolic oxalic acid inhibiting TNF- 伪 -induced proliferation of HaCaT cells.TNF- 伪 -induced HaCaT cells were treated with different concentrations of oxalic acid for 24 h. Flow cytometry was used to detect the cell cycle, and immunofluorescence staining was used to detect the nuclear translocation of NF- 魏 B and the expression of NF- 魏 B p-NF- 魏 B protein.The effect of oxalic acid on apoptosis of HaCaT cells; DCFH-DA staining was used to detect cell viability and mitochondrial ROS production; JC-1 staining was used to detect mitochondrial membrane potential; luciferase method was used to detect ATP content and mitochondrial function.Results: through the observation of skin appearance and tissue HE staining, it was found that snowy oxalic acid had a better effect on the silver-like symptoms of psoriasis model mice induced by Imiquimod.The content of IL-17, IL-23, TNF- 伪 in serum of mice was detected. It was found that the content of IL-17, IL-23, TNF- 伪 in mice psoriasis model was significantly increased when imiquimod was induced, but the increase of TNF- 伪 was significantly decreased after the administration of arachalic acid, but there was no significant effect on IL-17 IL-23.It was found that the expression of nuclear antigen (PCNA) was decreased and the expression of apoptotic protein (Bax) was increased and Bcl-2 was decreased after treated with Asiatidic acid.The results of cell-level study showed that cytosolic oxalic acid could significantly inhibit the proliferation of HaCaT cells induced by TNF- 伪 and arrest the cell cycle in G2 phase.At the same time, we found that oxalic acid can inhibit the phosphorylation of NF- 魏 B induced by TNF- 伪 and prevent its translocation into the nucleus.In addition, Asiaticoic acid could induce the increase of ROS production in HaCaT cells, induce the collapse of mitochondrial membrane potential, inhibit the formation of intracellular ATP, and eventually induce keratinocyte apoptosis.Conclusion: oxalic acid can improve psoriasis in mice and inhibit the proliferation of keratinocytes.At the cellular level, oxalic acid could inhibit the proliferation of HaCaT cells induced by TNF- 伪, significantly damage the mitochondria of HaCaT cells and induce apoptosis.The mechanism may be related to the inhibition of the activation of NF- 魏 B pathway induced by TNF- 伪 by oxalic acid.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

【参考文献】