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Article(id=1222469877743472845, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1222469875008790921, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2019-0286, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1555430400000, receivedDateStr=2019-04-17, revisedDate=1559577600000, revisedDateStr=2019-06-04, acceptedDate=null, acceptedDateStr=null, onlineDate=1769389131246, onlineDateStr=2026-01-26, pubDate=1573488000000, pubDateStr=2019-11-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1769389131246, onlineIssueDateStr=2026-01-26, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1769389131246, creator=13701087609, updateTime=1769389131246, updator=13701087609, issue=Issue{id=1222469875008790921, tenantId=1146029695717560320, journalId=1189982191388893191, year='2019', volume='54', issue='11', pageStart='1881', pageEnd='2140', issueExtLink='null', onlineDate='null', pubDate='1573488000000', pubDateStr='2019-11-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1769389130593, creator='13701087609', updateTime=1769389577080, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1222471747778109959, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1222469875008790921, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1222471747778109960, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1222469875008790921, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1903, endPage=1908, ext={EN=ArticleExt(id=1222469878284538088, articleId=1222469877743472845, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Sophoral flavones inhibit proliferation of cardiac fibroblasts via regulating the expression of prohibitin, columnId=1222469877852524754, journalTitle=Acta Pharmaceutica Sinica, columnName=Special Reports: The cardiac safety evaluation and rational use of cardiovascular disease prevention and treatment of Chinese medicine, runingTitle=null, highlight=null, articleAbstract=

The aim of the present study was to investigate the effect of Sophoral flavones on proliferation of cardiac fibroblasts (CFb) induced by high glucose and its underlying mechanism. Cardiac fibroblasts were exposed to different concentration of D-glucose (15, 25 and 35 mmol·L-1) at different time point (24, 48 and 72 h) in order to determine cell proliferation, and the model group was established by culturing CFb with 25 mmol·L-1 D-glucose for 48 h. Sophoral flavones (12.5, 25 and 50 mg·L-1) were employed for intervention. The cell viability was measured by MTT assay, and the levels of transforming growth factor-β1 (TGF-β1), matrix metalloproteinase-2 (MMP-2), collagen Ⅰ and collagen Ⅲ were measured by ELISA. In addition, flow cytometry was employed to detect the cell cycle; while the protein expression of prohibitin (PHB) was observed via immunocytochemistry and Western blot. This animal experiment had been approved by Jilin Medical University Experiment Animal Ethics Review Committee. The results showed that 25 mmol·L-1 glucose could promote the proliferation of CFb; and the contents of TGF-β1, MMP-2, collagen Ⅰ and collagen Ⅲ in the model group were higher than that of control (P < 0.05). The number of cells in S and G2 phase increased under high glucose condition. In the model group, PHB translocation occurred at 6 h and protein expression decreased at 48 h (P < 0.01). Compared with the model group, 12.5-50 mg·L-1 Sophoral flavones reduced the contents of TGF-β1, MMP-2, collagen Ⅰ and collagen Ⅲ, increased the number of G1 phase cells, and increased the expression of PHB protein at 48 h (P < 0.05), with no effect on the nuclear translocation of PHB. These results indicated that Sophoral flavones could prevent the proliferation of CFb induced by high glucose, the mechanism of which may be related to increasing the expression of PHB protein.

, authors=null, authorsList=Xiao-li CUI, Ze SHEN, Chun-yao SONG, Ji-nan JIANG, Jia-xu ZHOU, Zhi-rong LIU, Yan-chun WANG, authorCompany=null, correspAuthors=Yan-chun WANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2019 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1222469879781904709, articleId=1222469877743472845, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=苦参总黄酮通过抗增殖蛋白抑制高糖诱导的心肌成纤维细胞增殖, columnId=1222469878087405790, journalTitle=药学学报, columnName=专题报道：中药心脏安全性评价及合理用于心血管疾病的防治, runingTitle=null, highlight=null, articleAbstract=

探讨苦参总黄酮（sophoral flavones，SF）改善高糖诱导心肌成纤维细胞（cardiac fibroblasts，CFb）增殖的作用及其相关机制。以25 mmol·L-1 D-葡萄糖培养CFb 48 h构建细胞增殖模型，作为模型组；SF（12.5、25和50 mg·L-1）作为干预组。采用MTT法检测细胞活力；ELISA检测转化生长因子-β1（transforming growth factor-β1，TGF-β1）、基质金属蛋白酶-2（matrix metalloproteinase-2，MMP-2）、Ⅰ型胶原蛋白（collagen Ⅰ）和Ⅲ型胶原蛋白（collagen Ⅲ）含量；流式细胞术检测细胞周期变化；Western blot法和免疫荧光法检测抗增殖蛋白（prohibitin，PHB）的表达和定位。动物处置方案经吉林医药学院伦理审查委员会批准。结果表明，25 mmol·L-1葡萄糖可促进CFb增殖；模型组的TGF-β1、MMP-2、collagen Ⅰ和collagen Ⅲ含量高于对照组（P < 0.05）；高糖条件下处于S期和G2期的细胞数量增加。与对照组相比，模型组的PHB在6 h出现核转位，在48 h蛋白表达下降（P < 0.01）；与模型组比较，12.5~50 mg·L-1苦参总黄酮可降低TGF-β1、MMP-2、collagen Ⅰ和collagen Ⅲ的含量，增加了G1期细胞数量，在48 h增加了PHB的蛋白表达（P < 0.05），但未观察到SF对PHB核转位的影响。以上结果表明，SF可以改善高糖诱导的CFb增殖，其作用与促进PHB表达相关。

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*王艳春, Tel / Fax: 86-432-4560023, E-mail:
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Following the incubation of CFb with normal (5 mmol·L<sup>-1</sup>) and high (15, 25, 35 mmol·L<sup>-1</sup>) <i>D</i>-glucose concentrations for 48 h. B: The effect of high glucose (HG) on the proliferation of CFb at different time points. The CFb were incubated with normal (5 mmol·L<sup>-1</sup>) and high (25 mmol·L<sup>-1</sup>) <i>D</i>-glucose concentrations for 24, 48 and 72 h, respectively. C: The effect of SF on the relative proliferation of CFb. Cells were incubated with normal (5 mmol·L<sup>-1</sup>), high (25 mmol·L<sup>-1</sup>) <i>D</i>-glucose concentrations and HG + (12.5, 25, 50 mg·L<sup>-1</sup>) Sophoral flavones (SF) for 48 h. <i>n</i> = 10, <i><span class="mag-xml-overline" style="border-top:1px solid black">x</span></i> ± <i>s</i>. <sup>#</sup><i>P</i> < 0.05, <sup>##</sup><i>P</i> < 0.01 <i>vs</i> Cont (control) group; <sup>*</sup><i>P</i> < 0.05, <sup>**</sup><i>P</i> < 0.01 <i>vs</i> HG (model) group , figureFileSmall=fcXq3vRwvBFXupcy0In7hA==, figureFileBig=kJ/Jx1QoT/1oqtxx2q0tgQ==, tableContent=null), ArticleFig(id=1222469886610232077, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877743472845, language=EN, label=null, caption=null, figureFileSmall=jEaI6WyPFNVvAdHpbs2OIw==, figureFileBig=YNKtSpUHtR22HkIxA5wVxw==, tableContent=null), ArticleFig(id=1222469886731866904, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877743472845, language=CN, label=Figure 2, caption= Effect of SF on high glucose-induced morphological changes in CFb. Phase-contrast microscopy images are presented, showing the morphology of CFb following exposure to normal glucose (control, A), HG (model, 25 mmol·L<sup>-1</sup>, B), HG +12.5 mg·L<sup>-1</sup> SF (C), HG + 25 mg·L<sup>-1</sup> SF (D), HG+50 mg·L<sup>-1</sup> SF (E). Magnification, × 200; bar = 100 μm , figureFileSmall=jEaI6WyPFNVvAdHpbs2OIw==, figureFileBig=YNKtSpUHtR22HkIxA5wVxw==, tableContent=null), ArticleFig(id=1222469886845113124, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877743472845, language=EN, label=null, caption=null, figureFileSmall=IEYAojWuq/R7t6HyEBMVfA==, figureFileBig=s9I2aiwhGP9Ck9FNG+5yxw==, tableContent=null), ArticleFig(id=1222469886996108081, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877743472845, language=CN, label=Figure 3, caption= Cell cycle charts at different stages. A: Control group; B: HG model group; C: HG + 12.5 mg·L<sup>-1</sup> SF; D: HG + 25 mg·L<sup>-1</sup> SF; E: HG + 50 mg·L<sup>-1</sup> SF; F: Statistical analysis results. <i>n</i> = 3, <i><span class="mag-xml-overline" style="border-top:1px solid black">x</span></i> ± <i>s</i>. <sup>##</sup><i>P</i> < 0.01 <i>vs</i> control group; <sup>*</sup><i>P</i> < 0.05, <sup>**</sup><i>P</i> < 0.01 <i>vs</i> model group , figureFileSmall=IEYAojWuq/R7t6HyEBMVfA==, figureFileBig=s9I2aiwhGP9Ck9FNG+5yxw==, tableContent=null), ArticleFig(id=1222469887117742911, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877743472845, language=EN, label=null, caption=null, figureFileSmall=1AzRv1Eu9efXUDrmmsEGug==, figureFileBig=ZvMIv74+rhQpVrbUgVy49A==, tableContent=null), ArticleFig(id=1222469887210017610, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877743472845, language=CN, label=Figure 4, caption= Prohibitin (PHB) expression and metastasis under high glucose at different time points (A). Effect of SF on PHB expression and metastasis under HG 48 h (B). PHB proteins are shown in green; Hoechst-labeled nucleus are shown in blue. Bar = 20 μm. Magnification, ×1 200. The effect of HG on the expression of PHB at different time points (C, D). The effect of SF on the expression of PHB under HG at 48 h (E, F). <i>n</i> = 3, <i><span class="mag-xml-overline" style="border-top:1px solid black">x</span></i> ± <i>s</i>. <sup>##</sup><i>P</i> < 0.01 <i>vs</i> control group; <sup>*</sup><i>P</i> < 0.05, <sup>**</sup><i>P</i> < 0.01 <i>vs</i> model group , figureFileSmall=1AzRv1Eu9efXUDrmmsEGug==, figureFileBig=ZvMIv74+rhQpVrbUgVy49A==, tableContent=null), ArticleFig(id=1222469887302292309, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877743472845, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Group TGF-β1/pg·mL-1 MMP-2/ng·mL-1 Collagen Ι/ng·mL-1 Collagen IIΙ/ng·mL-1
Control
    24 h 5.58 ± 0.41 0.074 ± 0.01 0.159 ± 0.017 7.42 ± 0.520
    48 h 6.11 ± 0.21 0.095 ± 0.009 0.150 ± 0.006 8.06 ± 0.630
    72 h 6.52 ± 0.10 0.088 ± 0.013 0.145 ± 0.013 9.90 ± 1.26
HG (25 mmol·L-1)
    24 h 6.55 ± 0.18## 0.103 ± 0.004## 0.208 ± 0.019## 9.26 ± 0.700##
    48 h 7.23 ± 0.18# 0.091 ± 0.004 0.209 ± 0.014## 11.80 ± 0.730##
    72 h 8.05 ± 0.48## 0.108 ± 0.005# 0.194 ± 0.016## 11.70 ± 1.82
), ArticleFig(id=1222469887419732831, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877743472845, language=CN, label=Table 1, caption=

Effect of HG on transforming growth factor-β1 (TGF-β1), matrix metalloproteinase-2 (MMP-2), collagen Ⅰ and collagen Ⅲ content in CFb at different time points. n = 10, x ± s. # P < 0.05, ## P < 0.01 vs control group at same time

, figureFileSmall=null, figureFileBig=null, tableContent=
Group TGF-β1/pg·mL-1 MMP-2/ng·mL-1 Collagen Ι/ng·mL-1 Collagen IIΙ/ng·mL-1
Control
    24 h 5.58 ± 0.41 0.074 ± 0.01 0.159 ± 0.017 7.42 ± 0.520
    48 h 6.11 ± 0.21 0.095 ± 0.009 0.150 ± 0.006 8.06 ± 0.630
    72 h 6.52 ± 0.10 0.088 ± 0.013 0.145 ± 0.013 9.90 ± 1.26
HG (25 mmol·L-1)
    24 h 6.55 ± 0.18## 0.103 ± 0.004## 0.208 ± 0.019## 9.26 ± 0.700##
    48 h 7.23 ± 0.18# 0.091 ± 0.004 0.209 ± 0.014## 11.80 ± 0.730##
    72 h 8.05 ± 0.48## 0.108 ± 0.005# 0.194 ± 0.016## 11.70 ± 1.82
), ArticleFig(id=1222469887524590444, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877743472845, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Group TGF-β1/pg·mL-1 MMP-2/ng·mL-1 Collagen Ⅰ/ng·mL-1 Collagen Ⅲ/ng·mL-1
Control 6.59 ± 0.335 0.081 ± 0.005 0.169 ± 0.006 9.261 ± 0.485
HG (25 mmol·L-1) 7.56 ± 0.060## 0.101 ± 0.004## 0.222 ± 0.007## 13.56 ± 1.028##
HG+SF (12.5 mg·L-1) 7.46 ± 0.079 0.091 ± 0.005* 0.220 ± 0.005 12.66 ± 0.534*
HG+SF (25 mg·L-1) 7.19 ± 0.240* 0.088 ± 0.009* 0.198 ± 0.006** 9.453 ± 0.478**
HG+SF (50 mg·L-1) 6.77 ± 0.197** 0.085 ± 0.006** 0.177 ± 0.005** 8.684 ± 0.221**
), ArticleFig(id=1222469887663002488, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877743472845, language=CN, label=Table 2, caption=

Effect of SF on TGF-β1, MMP-2, collagen I and collagen III content in CFb induced by high glucose at 48 h. n = 4, x ± s. ##P < 0.01 vs control group; *P < 0.05, **P < 0.01 vs model group

, figureFileSmall=null, figureFileBig=null, tableContent=
Group TGF-β1/pg·mL-1 MMP-2/ng·mL-1 Collagen Ⅰ/ng·mL-1 Collagen Ⅲ/ng·mL-1
Control 6.59 ± 0.335 0.081 ± 0.005 0.169 ± 0.006 9.261 ± 0.485
HG (25 mmol·L-1) 7.56 ± 0.060## 0.101 ± 0.004## 0.222 ± 0.007## 13.56 ± 1.028##
HG+SF (12.5 mg·L-1) 7.46 ± 0.079 0.091 ± 0.005* 0.220 ± 0.005 12.66 ± 0.534*
HG+SF (25 mg·L-1) 7.19 ± 0.240* 0.088 ± 0.009* 0.198 ± 0.006** 9.453 ± 0.478**
HG+SF (50 mg·L-1) 6.77 ± 0.197** 0.085 ± 0.006** 0.177 ± 0.005** 8.684 ± 0.221**
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苦参总黄酮通过抗增殖蛋白抑制高糖诱导的心肌成纤维细胞增殖
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崔晓丽 1 , 沈泽 2 , 宋春垚 2 , 蒋济南 3 , 周佳旭 3 , 刘志荣 3 , 王艳春 1, *
药学学报 | 专题报道：中药心脏安全性评价及合理用于心血管疾病的防治 2019,54(11): 1903-1908
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药学学报 |专题报道：中药心脏安全性评价及合理用于心血管疾病的防治 2019 , 54 (11) : 1903 -1908
苦参总黄酮通过抗增殖蛋白抑制高糖诱导的心肌成纤维细胞增殖
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崔晓丽1, 沈泽2, 宋春垚2, 蒋济南3, 周佳旭3, 刘志荣3, 王艳春1, *
作者信息
  • 1.吉林医药学院 基础医学院, 吉林 吉林 132013
  • 2.吉林医药学院 临床医学院, 吉林 吉林 132013
  • 3.吉林医药学院 药学院, 吉林 吉林 132013
通讯作者:
*王艳春, Tel / Fax: 86-432-4560023, E-mail:
Sophoral flavones inhibit proliferation of cardiac fibroblasts via regulating the expression of prohibitin
Xiao-li CUI1, Ze SHEN2, Chun-yao SONG2, Ji-nan JIANG3, Jia-xu ZHOU3, Zhi-rong LIU3, Yan-chun WANG1, *
Affiliations
  • 1. Basic Medicine of Jilin Medical University, Jilin 132013, China
  • 2. The Clinical Medicine of Jilin Medical University, Jilin 132013, China
  • 3. The Pharmacy College of Jilin Medical University, Jilin 132013, China
出版时间: 2019-11-12 doi: 10.16438/j.0513-4870.2019-0286
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摘要
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探讨苦参总黄酮（sophoral flavones，SF）改善高糖诱导心肌成纤维细胞（cardiac fibroblasts，CFb）增殖的作用及其相关机制。以25 mmol·L-1 D-葡萄糖培养CFb 48 h构建细胞增殖模型，作为模型组；SF（12.5、25和50 mg·L-1）作为干预组。采用MTT法检测细胞活力；ELISA检测转化生长因子-β1（transforming growth factor-β1，TGF-β1）、基质金属蛋白酶-2（matrix metalloproteinase-2，MMP-2）、Ⅰ型胶原蛋白（collagen Ⅰ）和Ⅲ型胶原蛋白（collagen Ⅲ）含量；流式细胞术检测细胞周期变化；Western blot法和免疫荧光法检测抗增殖蛋白（prohibitin，PHB）的表达和定位。动物处置方案经吉林医药学院伦理审查委员会批准。结果表明，25 mmol·L-1葡萄糖可促进CFb增殖；模型组的TGF-β1、MMP-2、collagen Ⅰ和collagen Ⅲ含量高于对照组（P < 0.05）；高糖条件下处于S期和G2期的细胞数量增加。与对照组相比，模型组的PHB在6 h出现核转位，在48 h蛋白表达下降（P < 0.01）；与模型组比较，12.5~50 mg·L-1苦参总黄酮可降低TGF-β1、MMP-2、collagen Ⅰ和collagen Ⅲ的含量，增加了G1期细胞数量，在48 h增加了PHB的蛋白表达（P < 0.05），但未观察到SF对PHB核转位的影响。以上结果表明，SF可以改善高糖诱导的CFb增殖，其作用与促进PHB表达相关。

糖尿病性心肌病  /  苦参总黄酮  /  高糖  /  心肌成纤维细胞  /  增殖  /  抗增殖蛋白

The aim of the present study was to investigate the effect of Sophoral flavones on proliferation of cardiac fibroblasts (CFb) induced by high glucose and its underlying mechanism. Cardiac fibroblasts were exposed to different concentration of D-glucose (15, 25 and 35 mmol·L-1) at different time point (24, 48 and 72 h) in order to determine cell proliferation, and the model group was established by culturing CFb with 25 mmol·L-1 D-glucose for 48 h. Sophoral flavones (12.5, 25 and 50 mg·L-1) were employed for intervention. The cell viability was measured by MTT assay, and the levels of transforming growth factor-β1 (TGF-β1), matrix metalloproteinase-2 (MMP-2), collagen Ⅰ and collagen Ⅲ were measured by ELISA. In addition, flow cytometry was employed to detect the cell cycle; while the protein expression of prohibitin (PHB) was observed via immunocytochemistry and Western blot. This animal experiment had been approved by Jilin Medical University Experiment Animal Ethics Review Committee. The results showed that 25 mmol·L-1 glucose could promote the proliferation of CFb; and the contents of TGF-β1, MMP-2, collagen Ⅰ and collagen Ⅲ in the model group were higher than that of control (P < 0.05). The number of cells in S and G2 phase increased under high glucose condition. In the model group, PHB translocation occurred at 6 h and protein expression decreased at 48 h (P < 0.01). Compared with the model group, 12.5-50 mg·L-1 Sophoral flavones reduced the contents of TGF-β1, MMP-2, collagen Ⅰ and collagen Ⅲ, increased the number of G1 phase cells, and increased the expression of PHB protein at 48 h (P < 0.05), with no effect on the nuclear translocation of PHB. These results indicated that Sophoral flavones could prevent the proliferation of CFb induced by high glucose, the mechanism of which may be related to increasing the expression of PHB protein.

diabetic cardiomopathy  /  Sophoral flavones  /  high glucose  /  myocardial fibrosis  /  proliferation  /  prohibitin
崔晓丽, 沈泽, 宋春垚, 蒋济南, 周佳旭, 刘志荣, 王艳春. 苦参总黄酮通过抗增殖蛋白抑制高糖诱导的心肌成纤维细胞增殖. 药学学报, 2019 , 54 (11) : 1903 -1908 . DOI: 10.16438/j.0513-4870.2019-0286
Xiao-li CUI, Ze SHEN, Chun-yao SONG, Ji-nan JIANG, Jia-xu ZHOU, Zhi-rong LIU, Yan-chun WANG. Sophoral flavones inhibit proliferation of cardiac fibroblasts via regulating the expression of prohibitin[J]. Acta Pharmaceutica Sinica, 2019 , 54 (11) : 1903 -1908 . DOI: 10.16438/j.0513-4870.2019-0286
正文
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在糖尿病引起的多系统并发症中, 糖尿病性心肌病最为严重。其中, 心肌成纤维细胞(cardiac fibroblasts, CFb)过度增殖所致的心肌纤维化是糖尿病心肌病(diabetic cardiomopathy, DCM)的早期病理变化[1], 可影响心脏的收缩和舒张功能[2, 3]。因此, 在细胞水平研究高糖(high glucose, HG)对CFb的影响, 以确定有效靶点, 对改善DCM具有重要意义。苦参总黄酮(Sophoral flavones, SF)是苦参中最具生物活性的化合物之一, 具有广泛的生理活性, 包括抗菌作用、抗氧化作用及抗炎作用等[4-7]。本研究团队前期研究发现, 在糖尿病大鼠模型上, 观察到SF有降低血糖的倾向, 与卡托普利联合用药, 降血糖的作用更加明显; SF单用或与卡托普利联合应用均可以改善糖尿病所致的心肌纤维化及心肌损伤[8]。在前期工作的基础上, 本研究进一步观察SF对高糖诱导CFb增殖的影响及其机制。
材料与方法
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动物    采用1~3日龄SD大鼠乳鼠, SPF级雌雄不限, 购自辽宁长生生物技术股份有限公司, 动物合格证号: SCXK (辽) 2015-0001。实验过程中对动物处置方案经吉林医药学院伦理审查委员会批准。
试剂    SF (纯度 > 80%, 西安草翠芯生物科技有限公司)溶于改良型DMEM高糖培养基(上海源培生物科技股份有限公司), 并用高糖培养基进一步稀释至所需质量浓度(12.5、25和50 mg·L-1); 转化生长因子-β1 (TGF-β1)、基质金属蛋白酶-2 (MMP-2)、Ⅰ和Ⅲ型胶原蛋白(collagen Ⅰ和Ⅲ) ELISA试剂盒(语纯生物科技股份有限公司); β-Actin、抗增殖蛋白(PHB)等多克隆抗体(美国Abcam公司); Triton X-100、BCA蛋白浓度测定试剂盒(BCA Protein Assay Kit)、细胞周期试剂盒和BeyoECL Moon (极超敏ECL化学发光试剂盒) (碧云天公司)。
CFb的原代培养    取1~3日龄SD大鼠乳鼠, 无菌消毒后开胸取心脏, 制备成1 mm3左右的组织于预冷磷酸盐缓冲液(PBS)中; 0.125%胰酶反复消化。收集消化液, 离心(1 000 r·min-1, 5 min), 弃上清, 加入含10%血清的DMEM培养液重悬细胞, 差速贴壁120 min后获得CFb。本实验采用2~4代CFb。
细胞活性检测    将CFb分为对照组(含葡萄糖5 mmol·L-1)、高糖模型组(25 mmol·L-1葡萄糖)和3个用药组(高糖25 mmol·L-1 + SF 12.5、25或50 mg·L-1)。各条件孵育48 h, 保留上清液, 用含10% MTT培养基作用4 h。二甲基亚砜(DMSO) 150 μL溶解甲瓒结晶, 450 nm测定吸光度值。
TGF-β1、MMP-2、collagen Ⅰ和collagen Ⅲ含量检测    根据ELISA试剂盒说明, 加入标准品50 μL, 样品10 μL。然后将辣根过氧化物酶(horseradish peroxidase, HRP)偶联剂100 μL加入各孔中, 胶条覆盖, 37 ℃孵育60 min, 冲洗后在每孔中加入溶液A和溶液B各50 μL。轻轻混合并在37 ℃恒温孵育箱中孵育15 min。最后, 在每孔中加入终止溶液50 μL。在15 min内于450 nm处读取吸光度值, 检测各组TGF-β1、MMP-2、collagen Ⅰ和collagen Ⅲ的含量。
细胞周期检测    收集各组细胞, 置于预冷的PBS中洗涤两次, 70%冰乙醇固定, 4 ℃过夜。用含50 μL RNase和25 μL碘化丙啶的染料缓冲液(535 μL)重悬样品。37 ℃避光孵育30 min, 过滤, 在流式细胞仪上检测。
免疫荧光染色检测PHB核转移    将细胞置于24孔板中, 每孔1×104个细胞, 按实验分组处理后, 用4%多聚甲醛固定30 min, 用0.1% Triton X-100渗透10 min, 5%山羊血清阻断非特异性结合。随后, FITC结合的山羊抗兔PHB多克隆抗体检测PHB, 并用BX51荧光显微镜对染色细胞进行可视化。
PHB蛋白表达分析    预冷PBS洗涤细胞样品两次, 裂解。BCA蛋白检测试剂盒测定蛋白质浓度。蛋白(每孔30 μg)在100 V电压下分离20 min, 200 V电压下分离50 min, 转膜120 min, 5%脱脂奶封闭1 h, 抗体孵育, 用化学发光法对膜进行扫描处理, 测定总蛋白/β-actin的吸光度值。
统计学分析    所有数据均以均值±标准差表示, 统计分析使用SPSS 16.0软件和GraphPad 5.0软件分析处理, 各组比较采用方差分析和t检验, P < 0.05被认为具有统计学差异。
结果
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1 高浓度葡萄糖对CFb活性的影响
1.1 不同浓度葡萄糖对CFb活性的影响
选取15、25和35 mmol·L-1葡萄糖分别与CFb孵育48 h。结果显示, 与对照组相比, 25和35 mmol·L-1葡萄糖可提高CFb活力(P < 0.01)。后续实验采用25 mmol·L-1葡萄糖作用CFb制备增殖模型(图 1A)。
1.2 葡萄糖作用不同时间对CFb活性的影响
采用25 mmol·L-1葡萄糖分别处理CFb 24、48和72 h。结果显示, 与对照组24 h比较, 对照组细胞在48 h条件下有增殖(P < 0.05), 72 h增殖无统计学差异(P > 0.05), 高糖组在24和48 h均有增殖(P < 0.01) (图 1B)。因此, 采用25 mmol·L-1葡萄糖作用CFb 48 h制备CFb增殖模型, 作为高糖(HG)模型组。
2 SF对CFb活性的影响
与HG模型组相比, SF (12.5~50 mg·L-1)处理48 h, CFb的活力显著降低(P < 0.05或P < 0.01), 且随SF剂量的增加, 改善CFb增殖作用增强(图 1C)。结果表明, SF (12.5~50 mg·L-1)可改善CFb增殖。因此, 采用12.5~50 mg·L-1 SF用于后续机制研究。
3 SF对CFb形态变化的影响
将细胞分别培养48 h后, 用倒置相差显微镜观察各组细胞形态。结果可见, 对照组细胞较大, 胞浆丰富(图 2A); HG模型组细胞生长密集, 细胞体积较小, 增生活跃, 呈火焰状(图 2B); 与HG模型组比较, SF (12.5~50 mg·L-1)组细胞数量减少, 且随浓度增加, 药物抑制细胞增殖的作用增强(图 2C~E)。
4 SF对TGF-β1、MMP-2、collagen Ⅰ和collagen Ⅲ含量的影响
与对照组比较, HG模型组的TGF-β1含量增加(P < 0.01); MMP-2含量在24和48 h增加(P < 0.01、P < 0.05); collagen Ⅰ分泌增加(P < 0.01); collagen Ⅲ在24和48 h分泌增多(P < 0.01) (表 1)。此结果验证了HG作用48 h可成功制备增殖模型。与HG模型组比较, 12.5~50 mg·L-1 SF可抑制MMP-2和collagen Ⅲ分泌(P < 0.01、P < 0.05); 25和50 mg·L-1 SF组可抑制TGF-β1和collagen Ⅰ分泌(P < 0.01、P < 0.05) (表 2)。结果表明, 12.5~50 mg·L-1 SF可通过改善细胞外基质含量而改善CFb增殖。
5 SF对细胞周期的影响
25 mmol·L-1葡萄糖可显著增加S期和G2期细胞数量, 减少G1期细胞数量。与HG模型组相比, SF组中G1期细胞数量明显增加, S期降低, 提示SF可使细胞周期阻滞在G1期(图 3A~F)。
6 SF对PHB表达的影响
免疫荧光结果显示, 与对照组相比, HG模型组PHB在6、12和24 h表达增多, 48 h时PHB表达量减少。在25 mmol·L-1葡萄糖作用6 h后PHB从胞浆向胞核内转移, 胞核中PHB表达量增多(图 4A)。与HG模型组比较, 25 mg·L-1 SF作用细胞48 h可以促进PHB的表达, 但没有阻断PHB向核内转移(图 4B)。Western blot结果显示:与对照组比较, HG模型组PHB在12和24 h表达有升高趋势, 但无统计学差异, 48 h蛋白表达量降低(P < 0.01) (图 4C、D), 故在HG作用48 h条件下观察SF的作用; 与HG模型组比较, SF可促进PHB的表达, 随SF浓度增加, PHB表达量增多(P < 0.01, 图 4E、F)。
讨论
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糖尿病性心肌病发生于糖尿病患者, 排除高血压性心脏病、冠状动脉粥样硬化性心脏病及其他心脏病变的心肌疾病, 最终可发展为心力衰竭。心肌纤维化是糖尿病心肌病发展到一定阶段出现的病理现象, 主要表现为CFb过度增殖及细胞外基质(ECM)的过度沉积。本研究团队前期工作也证实, 在糖尿病大鼠模型上, 随着时间延长, 伴有心肌胶原含量增加, 心肌组织结构紊乱, 心脏功能受到抑制[8]。
本研究以5 mmol·L-1葡萄糖(即常规培养基中所含葡萄糖浓度)作为对照组, 观察不同浓度葡萄糖(15、25和35 mmol·L-1)在不同时间对CFb的影响。MTT结果表明, 15、25和35 mmol·L-1葡萄糖在48 h呈现出促进CFb增殖的趋势, 其中25和35 mmol·L-1的作用具有统计学意义。在作用持续时间方面, 高糖作用24、48和72 h都有促进CFb增殖作用。因此, 选取25 mmol·L-1葡萄糖作用于CFb 48 h为模型组。
机体内的TGF-β主要有3种类型, 其中TGF-β1不仅参与人体内多种器官的创伤与修复, 还通过激活成纤维细胞促使其分泌大量的ECM[9-11], 是主要的促纤维化因子[12]。研究表明, 高糖是诱导TGF-β1表达和活化的主要因素[9]。胶原是ECM的最重要成分, 在心脏内主要是Ⅰ型和Ⅲ型胶原蛋白。胶原蛋白的降解需要MMP-2的存在。在正常心脏结构中, MMP的表达和功能都受到严格的调控, 但在病理状态下, MMP的表达和活性增加会导致ECM失衡, 从而影响心脏的收缩、舒张功能[13]。研究中观察到25 mmol·L-1葡萄糖可促进TGF-β1、MMP-2的增多和细胞外基质Ⅰ型胶原蛋白、Ⅲ型胶原蛋白的过度分泌, 表明增殖模型建立成功。SF在12.5~50 mg·L-1内可抑制CFb增殖, 同时对以上指标均有改善作用, 细胞增殖周期也证实SF能够通过G0/G1阻滞改善高糖诱导的CFb增殖。
PHB家族是一种高度保守的多功能蛋白, 在酵母、植物、蠕虫、果蝇和哺乳动物中均有表达[14, 15], 其参与了细胞增殖、细胞凋亡、转录调控、发育及线粒体生物合成等过程[16]。PHB主要定位在线粒体中, 在细胞核及细胞质中也有表达, 其在不同位置的表达及其含量与其功能密切相关[17]。以往的研究表明, PHB表达的变化与ECM的沉积有关, PHB表达上调可有效缓解肺纤维化, 成为治疗组织纤维化的新靶点[18, 19]。本研究结果显示, PHB在CFb的胞浆及胞核中均有表达, 在高糖作用于CFb 6 h后PHB从胞浆向胞核内转移, 48 h整体表达量降低, 此结果与Western blot结果一致, 表明在高糖诱导CFb增殖过程中, PHB参与了调控。25 mg·L-1 SF可以促进PHB的表达, 提示在SF改善心肌纤维化过程中PHB参与其中, 但尚未观察到其对PHB核转移的影响。
综上所述, 本研究以高糖建立CFb增殖模型, SF具有改善细胞增殖的作用。在CFb增殖的过程中, 各靶点的变化时间是不一致的, 它们之间的关系是本研究下一步关注的内容。
基金
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  • 吉林省卫生厅资助项目(2012Z060)
  • 国家级大学生创新创业训练计划资助项目(201613706021)
文献
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参考文献 引证文献
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2019年第54卷第11期
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doi: 10.16438/j.0513-4870.2019-0286
  • 接收时间：2019-04-17
  • 首发时间：2026-01-26
  • 出版时间：2019-11-12
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  • 收稿日期：2019-04-17
  • 修回日期：2019-06-04
基金
吉林省卫生厅资助项目(2012Z060)
国家级大学生创新创业训练计划资助项目(201613706021)
作者信息
    1.吉林医药学院 基础医学院, 吉林 吉林 132013
    2.吉林医药学院 临床医学院, 吉林 吉林 132013
    3.吉林医药学院 药学院, 吉林 吉林 132013

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*王艳春, Tel / Fax: 86-432-4560023, E-mail:
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Number of
genus		 种数
Number of
species		 占总种数比例
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total species (%)		 属
Genus		 种数
Number of
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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