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王宗康,谭怡丝,覃一杰,周领航,肖艳波,胡兆勇,谭劲.加味丹玄口康通过Axin1调节口腔上皮损伤细胞增殖活性[J].湖南中医药大学学报英文版,2022,42(12):2008-2015.[Click to copy
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| 加味丹玄口康通过Axin1调节口腔上皮损伤细胞增殖活性 |
| 王宗康,谭怡丝,覃一杰,周领航,肖艳波,胡兆勇,谭劲 |
| (湖南中医药大学第一附属医院, 湖南 长沙 410007;湖南中医药大学, 湖南 长沙 410208) |
| 摘要: |
| 目的 探究加味丹玄口康(DXKK)对口腔上皮损伤细胞增殖活性的影响及轴抑制蛋白1(axis inhibition protein 1, Axin1)的作用。方法 选取不同干扰位点的Axin1干扰小RNA-1(small interfering RNA-1, siRNA-1)、Axin1 si RNA-2和Axin1 si RNA-3干预口腔上皮细胞,qRT-PCR检测Axin1的基因表达水平来筛选Axin1的有效干扰靶点。将细胞随机分为空白对照组、干扰对照组、Axin1干扰组、过表达对照组和Axin1过表达组,采用qRT-PCR检测Axin1 mRNA的表达水平,采用细胞计数试剂盒和平板克隆检测细胞增殖情况,采用流式分析检测细胞周期水平,采用Western blot检测Axin1、β-catenin、增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)、Bax、Bcl-2和cleaved Caspase-3蛋白的表达。采用氢溴酸槟榔碱(arecoline hydrobromide, AH)诱导口腔上皮细胞损伤模型,将口腔上皮细胞随机分为空白对照组、AH刺激组(50μg/m L)、空白血清干预组(10%空白血清+50μg/mL AH)、含DXKK血清干预组(10%含DXKK血清+50μg/mL AH)、含DXKK血清干预+Axin1过表达组(10%含DXKK血清+Axin1过表达质粒+50μg/mL AH),EdU染色检测口腔上皮细胞的增殖活性。结果 与干扰对照组比较,Axin1干扰-1组、Axin1干扰-2组和Axin1干扰-3组Axin1的基因表达水平均显著降低(P<0.01);Axin1干扰组细胞增殖活性和β-catenin、PCNA、Bcl-2蛋白表达均显著升高(P<0.01),Axin1、Bax、cleaved Caspase-3蛋白和Axin1 mRNA的表达均显著降低(P<0.05或P<0.01)。与过表达对照组相比,Axin1过表达组处于G1期的细胞比例、细胞增殖活性和β-catenin、PCNA、Bcl-2蛋白表达均显著降低(P<0.01),处于S期的细胞比例和Axin1、Bax、cleaved Caspase-3蛋白及Axin1 mRNA的表达均显著升高(P<0.01)。与空白对照组比较,AH刺激组Axin1蛋白表达显著增加(P<0.01),细胞增殖活性显著降低(P<0.01)。与空白血清干预组比较,含DXKK血清干预组Axin1蛋白表达需显著降低(P<0.01),细胞增殖活性显著升高(P<0.01)。与含DXKK血清干预组比较,含DXKK血清干预+Axin1过表达组细胞增殖活性显著降低(P<0.05)。结论 Axin1可调控口腔上皮细胞增殖活性。DXKK通过抑制Axin1信号,促进AH诱导的口腔上皮细胞增殖。 |
| 关键词: 口腔黏膜下纤维化 Axin1 加味丹玄口康 口腔上皮细胞 氢溴酸槟榔碱 |
| DOI：10.3969/j.issn.1674-070X.2022.12.009 |
| Received:April 03, 2022 |
| 基金项目:国家自然科学基金项目(81874496) |
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| Jiawei Danxuan Koukang regulates the proliferation activity of oral epithelial injured cells via Axin1 |
| WANG Zongkang,TAN Yisi,TAN Yijie,ZHOU Linghang,XIAO Yanbo,HU Zhaoyong,TAN Jing |
| (The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China;Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective We aimed to explore the effects of Jiawei Danxuan Koukang(JWDXKK) on the proliferation activity of oral epithelial injured cells and the role of axis inhibition protein 1(Axin1).Methods Axin1 small interfering RNA-1(Axin1siRNA-1), Axin1 small interfering RNA-2(Axin1 siRNA-2), and Axin1 small interfering RNA-3(Axin1 siRNA-3) from different interfering positions were selected to interfere with oral epithelial cells, and the expression of Axin1 was detected by quantitative real-time PCR(qRT-PCR) to screen the effective interference targets of Axin1. The cells were divided into the following groups:blank control group, si-NC group, si-Axin1 group, over-expressed control group, and over-expressed Axin1 group. The Axin1 m RNA levels were detected by qRT-PCR. Cell counting kit-8 and plate clone formation assay were applied to examine the cell proliferative activity. Cell cycle levels were detected by flow analysis. The protein expression levels of Axin1, β-catenin, PCNA,Bax, Bcl-2, and cleaved Caspase-3 were identified by Western blot. The oral epithelial cell injury model was induced by arecoline hydrobromide(AH). Oral epithelial cells were then divided into: blank control group, AH group(50 μg/mL AH), blank group(10%blank serum + 50 μg/mL AH), JWDXKK group(10% JWDXKK serum + 50 μg/mL AH), and a JWDXKK + over-expressed Axin1group(10% JWDXKK serum + over-expressed Axin1 + 50 μg/mL AH). EdU incorporation assay was performed to examine the proliferative activity.Results Compared with the si-NC group, Axin1 gene expression in si-Axin1-1, si-Axin1-2, and si-Axin1-3groups significantly decreased(P<0.01). Cell proliferation activity of Axin1 group cells and the protein levels of β-catenin, PCNA,and Bcl-2 increased in the si-Axin1 group(P<0.01), while the protein expression of Axin1, Bax, and cleaved Caspase-3 and Axin1mRNA levels significantly decreased(P<0.05 or P<0.01). Compared with the over-expressed control group, the proportion of cells in G1 phase, cell proliferation activity, and protein expression of β-catenin, PCNA, and Bcl-2 significantly decreased in over-expressed Axin1 group(P<0.01). The proportion of cells in S phase and the Axin1, Bax, cleaved Caspase-3 protein, and Axin1mRNA expression significantly increased(P<0.01). Compared with the blank control group, Axin1 protein expression increased in AH stimulated group(P<0.01), and the proliferation activity was significantly reduced(P<0.01). Compared with the blank group, the Axin1 protein expression decreased in JWDXKK group(P<0.01), and the proliferation activity increased(P<0.01). Compared with the JWDXKK group, the proliferation activity decreased in the JWDXKK + over-expressed Axin1 group(P<0.05).Conclusion Axin1could regulate the proliferative activity of oral epithelial cells. JWDXKK could promote the proliferation of oral epithelial cells with AH induction, possibly by inhibiting Axin1 signaling. |
| Key words: oral submucous fibrosis Axin1 Jiawei Danxuan Koukang oral epithelial cells arecoline hydrobromide |
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