Article(id=1322923864086426274, tenantId=1146029695717560320, journalId=1255847919539208197, issueId=1322923782029062261, articleNumber=null, orderNo=null, doi=10.13193/j.issn.1673-7717.2025.06.043, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=null, receivedDateStr=null, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1793339227935, onlineDateStr=2026-10-30, pubDate=1749484800000, pubDateStr=2025-06-10, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1793339227935, onlineIssueDateStr=2026-10-30, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1793339227935, creator=13701087609, updateTime=1793339227935, updator=13701087609, issue=Issue{id=1322923782029062261, tenantId=1146029695717560320, journalId=1255847919539208197, year='2025', volume='43', issue='6', pageStart='1', pageEnd='258', issueExtLink='null', onlineDate='null', pubDate='1749484800000', pubDateStr='2025-06-10', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1793339208371, creator='13701087609', updateTime=1793340556755, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1322929437859258468, tenantId=1146029695717560320, journalId=1255847919539208197, issueId=1322923782029062261, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1322929437859258469, tenantId=1146029695717560320, journalId=1255847919539208197, issueId=1322923782029062261, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=209, endPage=212, ext={EN=ArticleExt(id=1322923866179383971, articleId=1322923864086426274, tenantId=1146029695717560320, journalId=1255847919539208197, language=EN, title=Mechanism Research of Icariin Derivative YS-10 in Treatment of Testicular Radiation Injury, columnId=1256183365644968140, journalTitle=Chinese Archives of Traditional Chinese Medicine, columnName=Platform of Provincial Project, runingTitle=null, highlight=null, articleAbstract=
Objective

To observe the therapeutic effect of icariin derivative YS-10 on testicular radiation injury,and to explore its potential mode of action.

Methods

Eighteen 10-week-old male SD rats were selected and randomly divided into three groups,six in each group,which were blank control group(Control group),radiation injury group(Model group)and YS-10 treatment group(YS-10 group).The testicular index and serum testosterone content of each group were detected.HE staining was used to observe the pathological changes of testicular tissue.Histochemical kit method was used to detect the levels of superoxide dismutase(SOD),malondialdehyde(MDA)and reduced glutathione(GSH)in testicular tissue.GC-2 cells were selected and divided into Control group,Model group and YS-10 group.Mouse spermatocyte cell line GC-2 cells were induced by 8 Gy X-ray irradiation(2 Gy/min)for 24 h to construct a radiation injury model,and the treatment group was also intervened with YS-10.CCK-8 was used to detect cell viability in each group.Fluorescent probe was used to detect reactive oxygen species(ROS)and Western blot was used to determine the levels of Kelch-like ECH-associated protein 1(Keap1),GSH,nuclear factor erythroid-derived-2-like 2(Nrf2)and heme oxygenase-1(HO-1)in GC-2 cells were determined by Western blot.

Results

Animal experiments confirmed that compared with those in the Control group,the testicular index and testosterone secretion level of rats in the Model group were significantly reduced(P<0.05),the MDA content in testicular tissues was elevated,and the levels of SOD and GSH were reduced(P<0.05).Compared with that of the Model group,the testicular index of the YS-10 group increased from about 0.81% to 1.25%.The testosterone secretion level increased from about 4.203 U/mg to 5.042 U/mg(P<0.05).The MDA content decreased from about 36.559 nmol/g to 26.882 nmol/g,and the SOD activity of the testicular tissue increased from about 14.727 U/mg increased to 18.417 U/mg,and GSH content approximately increased from 20.21μmol/L to 32.922μmol/L(P<0.05).The cellular experiments confirmed that compared with that of the Control group,the viability of GC-2 cells in the Model group was significantly decreased(P<0.01).The fluorescent probe showed that the average fluorescence intensity of ROSwas increased about 5-fold(P<0.01),the protein expression of Keap1 was elevated about 1.35-fold,and the protein expressions of Nrf2 and HO-1 were decreased about 1.46-fold each(P<0.05).Compared with that in the Model group,the viability of GC-2 cells in the YS-10 group approximately increased from 73.564% to 86.427%(P<0.01),the average fluorescence intensity of ROS approximately decreased from 255 to 78.378(P<0.01),the level of Keap1 protein expression approximately decreased from 0.951 to 0.73,the level of Nrf2 protein expression approximately increased from 0.498 to 0.805,and HO-1 protein expression level approximately increased from 0.492 to 0.599(P<0.05).

Conclusion

YS-10,a derivative of icariin,can effectively alleviate radiation-induced oxidative stress and protect testicular tissue to reduce the effects of radiation.The mechanism of action may be achieved through the Keap1/Nrf2/HO-1 signaling pathway.

, authors=Chenli PAN1, Zhitao WEI2, Juntao SUN2, Xiangxiang ZHANG1, Wanqi WANG1, Hongwei LI1, Yong YANG2, authorsList=Chenli PAN, Zhitao WEI, Juntao SUN, Xiangxiang ZHANG, Wanqi WANG, Hongwei LI, Yong YANG, authorCompany=null, correspAuthors=Yong YANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, 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=1322923867664167601, articleId=1322923864086426274, tenantId=1146029695717560320, journalId=1255847919539208197, language=CN, title=淫羊藿苷衍生物YS-10治疗睾丸放射性损伤的机制研究, columnId=1256183369033965799, journalTitle=中华中医药学刊, columnName=省级项目平台, runingTitle=null, highlight=null, articleAbstract=
目的

观察淫羊藿苷衍生物YS-10对睾丸放射性损伤的治疗效果,并探究其潜在作用机制。

方法

选用10周龄雄性SD大鼠18只,随机分为3组,每组6只,分别为空白对照组(Control组)、放射损伤组(Model组)、YS-10治疗组(YS-10组)。检测各组睾丸指数、血清睾酮含量;HE染色观察睾丸组织病理变化;组化试剂盒检测睾丸组织SOD(超氧化物歧化酶)、MDA(丙二醛)、GSH(还原型谷胱甘肽)水平。选用小鼠精母细胞系GC-2细胞,将细胞分为Control组、Model组、YS-10组。采用8Gy X射线照射(2Gy/Min)诱导GC-2细胞24 h,构建放射损伤模型,治疗组同时使用YS-10进行干预。CCK-8检测各组细胞活力;荧光探针检测ROS;Western blot测定Kelch样ECH关联蛋白(Kelchlike ECH-associated protein 1,Keap1)、核因子E2相关因子(nuclear factor erythroid-derived-2-like 2,Nrf2)、血红素加氧酶-1(heme oxygenase-1,HO-1)蛋白表达水平。

结果

动物实验证实:与Control组相比,Model组大鼠睾丸指数和睾酮分泌水平明显降低(P<0.05),睾丸组织中MDA含量升高,SOD、GSH水平降低(P<0.05)。与Model组相比,YS-10组睾丸指数约由0.81%上升到1.25%,睾酮分泌水平约由4.203 U/mg上升至5.042 U/mg(P<0.05),MDA含量约由36.559 nmol/g下降至26.882 nmol/g,睾丸组织SOD活性约由14.727 U/mg上升至18.417 U/mg,GSH含量约由20.21μmol/L上升至32.922μmol/L(P<0.05)。细胞实验证实:与Control组相比,Model组GC-2细胞活力明显下降(P<0.01),荧光探针显示ROS平均荧光强度约增加5倍(P<0.01),Keap1蛋白表达升高了约1.35倍,Nrf2、HO-1蛋白表达各降低了约1.46倍(P<0.05);与Model组相比,YS-10组GC-2细胞活力约由73.564%增加至86.427%(P<0.01),ROS平均荧光强度约由255降低至78.378(P<0.01),Keap1蛋白表达水平约由0.951降低至0.73,Nrf2蛋白表达水平约由0.498上升至0.805,HO-1蛋白表达水平约由0.492升高至0.599(P<0.05)。

结论

淫羊藿苷衍生物YS-10可有效减轻辐射诱导的氧化应激反应,保护睾丸组织以减少辐射影响。其作用机制可能是通过Keap1/Nrf2/HO-1信号通路来实现的。

, authors=潘陈丽1, 魏志涛2, 孙军涛2, 张香香1, 王婉琦1, 李鸿伟1, 杨勇2, authorsList=潘陈丽, 魏志涛, 孙军涛, 张香香, 王婉琦, 李鸿伟, 杨勇, authorCompany=null, correspAuthors=杨勇, authorNote=

潘陈丽(1997-),女,安徽滁州人,硕士在读,研究方向:中西医结合临床外科疾病。

, correspAuthorsNote=
杨勇(1963-),男,吉林长春人,主任医师、教授,博士研究生导师,博士,研究方向:中西医结合临床外科疾病。E-mail:。
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潘陈丽(1997-),女,安徽滁州人,硕士在读,研究方向:中西医结合临床外科疾病。

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潘陈丽(1997-),女,安徽滁州人,硕士在读,研究方向:中西医结合临床外科疾病。

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注:A:睾丸大体状况;B:睾丸指数和血清睾酮。##与Control组相比,P<0.01;*与Model组比,P<0.05;**与Model组比,P<0.01。

, figureFileSmall=MHPUUSBcj/Xaz084FErtYQ==, figureFileBig=HrRPKMWe266+F6KtsOOK1w==, tableContent=null), ArticleFig(id=1322923872735081190, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923864086426274, language=EN, label=null, caption=null, figureFileSmall=83r4oGtwyy1D73A6r/QpaQ==, figureFileBig=aoBjIBa/GM5dpQH9Pm3YTQ==, tableContent=null), ArticleFig(id=1322923872814772967, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923864086426274, language=CN, label=图2, caption=各组大鼠睾丸组织HE染色结果分析

注:A:HE染色(×20);B:生精小管直径和面积计算。##与Control组相比,P<0.01;*与Model组比,P<0.05。

, figureFileSmall=83r4oGtwyy1D73A6r/QpaQ==, figureFileBig=aoBjIBa/GM5dpQH9Pm3YTQ==, tableContent=null), ArticleFig(id=1322923874538631912, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923864086426274, language=EN, label=null, caption=null, figureFileSmall=ZmYQSi+FX4A9s8zx9Dgw8A==, figureFileBig=9sLpbM/45TKgY58EN05ovw==, tableContent=null), ArticleFig(id=1322923874635100905, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923864086426274, language=CN, label=图3, caption=各组大鼠睾丸组织中SOD、MDA、GSH水平

注:A:GSH含量;B:MDA含量;C:SOD活性。#与Control组相比,P<0.05;##与Control组相比,P<0.01。*与Model组比,P<0.05;**与Model组比,P<0.01。

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注:A:造模剂量;B:YS-10给药剂量;C:给药剂量的确定。#与Control组相比,P<0.05;##与Control组相比,P<0.01;**与Model组比,P<0.01。

, figureFileSmall=j8FXA+D7cItCdwgm1qwUNg==, figureFileBig=buR84DAJ+sREgFVVoO+u+w==, tableContent=null), ArticleFig(id=1322923874861593324, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923864086426274, language=EN, label=null, caption=null, figureFileSmall=x+3In1CzJ+wiJ1M3D/FJJw==, figureFileBig=qDw36b0TJ3dOdOhp4ycAHQ==, tableContent=null), ArticleFig(id=1322923874949673709, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923864086426274, language=CN, label=图5, caption=ROS在各组GC-2细胞中的表达水平

注:A:荧光探针;B:定量分析ROS平均荧光强度。##与Control组比,P<0.01;**与Model组比,P<0.01。

, figureFileSmall=x+3In1CzJ+wiJ1M3D/FJJw==, figureFileBig=qDw36b0TJ3dOdOhp4ycAHQ==, tableContent=null), ArticleFig(id=1322923875046142702, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923864086426274, language=EN, label=null, caption=null, figureFileSmall=3cHFzQ4pe7i12rFuP9EqJg==, figureFileBig=9IB2VHe1GDZsKgD7IxU6AA==, tableContent=null), ArticleFig(id=1322923875113251567, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923864086426274, language=CN, label=图6, caption=Keap1/Nrf2/HO-1通路在各组GC-2细胞中的蛋白表达水平

注:A:免疫印迹;B:定量分析蛋白相对表达水平。#与Control组比,P<0.05;##与Control组比,P<0.05;*与Model组比,P<0.05;**与Model组比,P<0.01。

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淫羊藿苷衍生物YS-10治疗睾丸放射性损伤的机制研究
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潘陈丽 1 , 魏志涛 2 , 孙军涛 2 , 张香香 1 , 王婉琦 1 , 李鸿伟 1 , 杨勇 2
中华中医药学刊 | 省级项目平台 2025,43(6): 209-212
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中华中医药学刊 |省级项目平台 2025 , 43 (6) : 209 -212
淫羊藿苷衍生物YS-10治疗睾丸放射性损伤的机制研究
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潘陈丽1, 魏志涛2, 孙军涛2, 张香香1, 王婉琦1, 李鸿伟1, 杨勇2
作者信息
  • 1.长春中医药大学,吉林 长春 130117
  • 2.长春中医药大学附属医院,吉林 长春 130021
通讯作者:
杨勇(1963-),男,吉林长春人,主任医师、教授,博士研究生导师,博士,研究方向:中西医结合临床外科疾病。E-mail:。
作者简介:

潘陈丽(1997-),女,安徽滁州人,硕士在读,研究方向:中西医结合临床外科疾病。

Mechanism Research of Icariin Derivative YS-10 in Treatment of Testicular Radiation Injury
Chenli PAN1, Zhitao WEI2, Juntao SUN2, Xiangxiang ZHANG1, Wanqi WANG1, Hongwei LI1, Yong YANG2
Affiliations
  • 1.Changchun University of Chinese Medicine,Changchun 130117,Jilin,China
  • 2.The Affiliated Hospital of Changchun University of Chinese Medicine,Changchun 130021,Jilin,China
出版时间: 2025-06-10 doi: 10.13193/j.issn.1673-7717.2025.06.043
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目的

观察淫羊藿苷衍生物YS-10对睾丸放射性损伤的治疗效果,并探究其潜在作用机制。

方法

选用10周龄雄性SD大鼠18只,随机分为3组,每组6只,分别为空白对照组(Control组)、放射损伤组(Model组)、YS-10治疗组(YS-10组)。检测各组睾丸指数、血清睾酮含量;HE染色观察睾丸组织病理变化;组化试剂盒检测睾丸组织SOD(超氧化物歧化酶)、MDA(丙二醛)、GSH(还原型谷胱甘肽)水平。选用小鼠精母细胞系GC-2细胞,将细胞分为Control组、Model组、YS-10组。采用8Gy X射线照射(2Gy/Min)诱导GC-2细胞24 h,构建放射损伤模型,治疗组同时使用YS-10进行干预。CCK-8检测各组细胞活力;荧光探针检测ROS;Western blot测定Kelch样ECH关联蛋白(Kelchlike ECH-associated protein 1,Keap1)、核因子E2相关因子(nuclear factor erythroid-derived-2-like 2,Nrf2)、血红素加氧酶-1(heme oxygenase-1,HO-1)蛋白表达水平。

结果

动物实验证实:与Control组相比,Model组大鼠睾丸指数和睾酮分泌水平明显降低(P<0.05),睾丸组织中MDA含量升高,SOD、GSH水平降低(P<0.05)。与Model组相比,YS-10组睾丸指数约由0.81%上升到1.25%,睾酮分泌水平约由4.203 U/mg上升至5.042 U/mg(P<0.05),MDA含量约由36.559 nmol/g下降至26.882 nmol/g,睾丸组织SOD活性约由14.727 U/mg上升至18.417 U/mg,GSH含量约由20.21μmol/L上升至32.922μmol/L(P<0.05)。细胞实验证实:与Control组相比,Model组GC-2细胞活力明显下降(P<0.01),荧光探针显示ROS平均荧光强度约增加5倍(P<0.01),Keap1蛋白表达升高了约1.35倍,Nrf2、HO-1蛋白表达各降低了约1.46倍(P<0.05);与Model组相比,YS-10组GC-2细胞活力约由73.564%增加至86.427%(P<0.01),ROS平均荧光强度约由255降低至78.378(P<0.01),Keap1蛋白表达水平约由0.951降低至0.73,Nrf2蛋白表达水平约由0.498上升至0.805,HO-1蛋白表达水平约由0.492升高至0.599(P<0.05)。

结论

淫羊藿苷衍生物YS-10可有效减轻辐射诱导的氧化应激反应,保护睾丸组织以减少辐射影响。其作用机制可能是通过Keap1/Nrf2/HO-1信号通路来实现的。

睾丸  /  放射损伤  /  YS-10  /  氧化应激
Objective

To observe the therapeutic effect of icariin derivative YS-10 on testicular radiation injury,and to explore its potential mode of action.

Methods

Eighteen 10-week-old male SD rats were selected and randomly divided into three groups,six in each group,which were blank control group(Control group),radiation injury group(Model group)and YS-10 treatment group(YS-10 group).The testicular index and serum testosterone content of each group were detected.HE staining was used to observe the pathological changes of testicular tissue.Histochemical kit method was used to detect the levels of superoxide dismutase(SOD),malondialdehyde(MDA)and reduced glutathione(GSH)in testicular tissue.GC-2 cells were selected and divided into Control group,Model group and YS-10 group.Mouse spermatocyte cell line GC-2 cells were induced by 8 Gy X-ray irradiation(2 Gy/min)for 24 h to construct a radiation injury model,and the treatment group was also intervened with YS-10.CCK-8 was used to detect cell viability in each group.Fluorescent probe was used to detect reactive oxygen species(ROS)and Western blot was used to determine the levels of Kelch-like ECH-associated protein 1(Keap1),GSH,nuclear factor erythroid-derived-2-like 2(Nrf2)and heme oxygenase-1(HO-1)in GC-2 cells were determined by Western blot.

Results

Animal experiments confirmed that compared with those in the Control group,the testicular index and testosterone secretion level of rats in the Model group were significantly reduced(P<0.05),the MDA content in testicular tissues was elevated,and the levels of SOD and GSH were reduced(P<0.05).Compared with that of the Model group,the testicular index of the YS-10 group increased from about 0.81% to 1.25%.The testosterone secretion level increased from about 4.203 U/mg to 5.042 U/mg(P<0.05).The MDA content decreased from about 36.559 nmol/g to 26.882 nmol/g,and the SOD activity of the testicular tissue increased from about 14.727 U/mg increased to 18.417 U/mg,and GSH content approximately increased from 20.21μmol/L to 32.922μmol/L(P<0.05).The cellular experiments confirmed that compared with that of the Control group,the viability of GC-2 cells in the Model group was significantly decreased(P<0.01).The fluorescent probe showed that the average fluorescence intensity of ROSwas increased about 5-fold(P<0.01),the protein expression of Keap1 was elevated about 1.35-fold,and the protein expressions of Nrf2 and HO-1 were decreased about 1.46-fold each(P<0.05).Compared with that in the Model group,the viability of GC-2 cells in the YS-10 group approximately increased from 73.564% to 86.427%(P<0.01),the average fluorescence intensity of ROS approximately decreased from 255 to 78.378(P<0.01),the level of Keap1 protein expression approximately decreased from 0.951 to 0.73,the level of Nrf2 protein expression approximately increased from 0.498 to 0.805,and HO-1 protein expression level approximately increased from 0.492 to 0.599(P<0.05).

Conclusion

YS-10,a derivative of icariin,can effectively alleviate radiation-induced oxidative stress and protect testicular tissue to reduce the effects of radiation.The mechanism of action may be achieved through the Keap1/Nrf2/HO-1 signaling pathway.

testis  /  radiation damage  /  YS-10  /  oxidative stress
潘陈丽, 魏志涛, 孙军涛, 张香香, 王婉琦, 李鸿伟, 杨勇. 淫羊藿苷衍生物YS-10治疗睾丸放射性损伤的机制研究. 中华中医药学刊, 2025 , 43 (6) : 209 -212 . DOI: 10.13193/j.issn.1673-7717.2025.06.043
Chenli PAN, Zhitao WEI, Juntao SUN, Xiangxiang ZHANG, Wanqi WANG, Hongwei LI, Yong YANG. Mechanism Research of Icariin Derivative YS-10 in Treatment of Testicular Radiation Injury[J]. Chinese Archives of Traditional Chinese Medicine, 2025 , 43 (6) : 209 -212 . DOI: 10.13193/j.issn.1673-7717.2025.06.043
随着核能核技术的飞速发展,人们受辐射影响的几率越来越高[1]。其中,医源性辐射损伤最常见的原因是放射治疗,这是一种常见的恶性肿瘤治疗方法。在放疗过程中,射线会不可避免地对靶细胞以外的邻近正常组织和细胞造成损伤。睾丸作为维持男性生育能力的重要器官,对电离辐射(IR)极为敏感[2]。局部照射或全身照射后精子数量和质量降低是放射性睾丸损伤常见不良反应[3]。此外,放射性事故(如核事故或放射性污染事件)也会导致大量人群暴露于放射性物质中[4-5],睾丸受到的损伤可能对未来的生育能力产生长期影响。目前,尚缺乏有效治疗手段用于预防或治疗睾丸放射性损伤。因此,寻求一种高效、低毒的睾丸放射性损伤治疗手段和药物已成为当前研究的热点与难点。
目前,针对睾丸放射性损伤发病机制方向的早期研究相对较少。现有研究表明,放射治疗后组织中活性氧(ROS)水平升高,而氧化应激又是导致睾丸生殖细胞DNA损伤的主要原因[6]。提示氧化应激损伤可能在睾丸放射性损伤的发生发展中发挥重要作用[7]。放射治疗后,可通过抗氧化剂修复组织病理损伤[8]。
中医应用传统中药淫羊藿治疗男性生殖功能障碍已有数千年的历史[9],淫羊藿苷(ICA)及其代谢产物(ICAⅡ)作为淫羊藿的有效活性成分,多年来一直被用于治疗勃起功能障碍、生精功能障碍、睾丸功能损伤等[10-12]。然而考虑到淫羊藿的提取成本过高,ICA和ICAⅡ的生物利用度低,在临床环境中使用范围有限[13],而我们采用人工合成的淫羊藿苷衍生物(YS-10),这一药物不仅具有8-烯基山柰酚类黄酮的共同结构,且比从植物分离物中提取的ICAⅡ成本更低[14]。最近,YS-10被报道用于治疗放射性损伤[15]。然而,YS-10能否用于治疗睾丸放射性损伤,及其潜在的作用机制尚不明确。
在本研究中,我们拟探讨淫羊藿苷衍生物YS-10对睾丸放射性损伤的调控作用,初步明确YS-10的作用通路及机制。本研究也将为YS-10这一潜在治疗药物的机制研究与临床转化提供参考依据与研究基础。
10周龄雄性SD大鼠18只,体质量260~310 g,购买地:北京科宇动物养殖中心。[实验动物生产许可号:SCXK(京)2018-0010]。动物饲养于长春中医药大学附属医院动物繁育中心。实验根据长春中医药大学附属医院动物伦理委员会颁发的项目许可(第2024390号)进行,本实验遵守长春中医药大学动物护理和使用指南。
小鼠精母细胞系GC-2spd(ts)购于上海中乔新舟生物科技。使用DMEM高糖完全培养基培养GC-2细胞。高糖完全培养基中含有DMEM基础培养基、10%胎牛血清和1%青霉素-链霉素混合溶液。并将细胞置于37℃、5%CO2恒温恒湿培养箱中培养。
YS-10(成都格力浦生物技术);苏木素-伊红染色试剂盒(武汉索莱宝,G1120);总SOD活性检测试剂盒(上海碧云天,S0101S);脂质氧化(MDA)检测试剂盒(上海碧云天,S0131S);还原型谷胱甘肽(GSH)测定试剂盒(南京建成,A006-1-1);活性氧检测试剂盒(上海碧云天,S0033S);Keap1、Nrf2、HO-1抗 体(江 苏 亲 科,AF5266、AF0639、AF5393);医用直线加速器(日本西门子,MD-7745)。
随机选取6只大鼠为空白组(Control组)。其余12只大鼠给予单次20 Gy的前列腺X射线照射。通过腹腔注射3%戊巴比妥钠(30 mg/kg)麻醉大鼠,将麻醉后大鼠置于医用直线加速器下,进行单次剂量20 Gy照射(2 Gy/min)。辐照后将大鼠随机分为两组:实验组(Model组)和YS-10组。辐照完成后24 h,治疗组给予YS-10灌胃治疗(2.5 mg·kg-1·day-1),YS-10给药4周的同时,剩下两组给予等量的生理盐水对大鼠进行灌胃。治疗结束2周后,经戊巴比妥钠麻醉后,收集全血,颈椎脱臼法处死大鼠,摘取睾丸组织,进行后续检测。
将大鼠称量体质量后,作腹部中线切口,腹主动脉取血。充分暴露睾丸后,摘除睾丸并称重,计算各组大鼠睾丸指数:睾丸指数=[(睾丸质量/g)/(身体质量/g)]×100%。按照Elisa试剂盒说明书,检测各组大鼠睾丸组织中血清睾酮的含量。
严格按照生化试剂盒说明书检测各项氧化应激指标。将睾丸组织制成10%组织匀浆液后,利用化学比色法检测SOD和GSH,硫代巴比妥酸(TBA)法检测MDA。
将睾丸组织冠状切开,用4%多聚甲醛固定,脱水包埋,切片厚度4μm。每个样本中随机选取5个视野。在光学显微镜下观察睾丸的病理变化,并计算睾丸组织切片中生精小管的直径和面积。
将对数生长期的GC-2细胞(5×103个/孔)接种于96孔板,每组设6个复孔,分别给予0、2、4、6、8、10、12 Gy X射线照射(2 Gy/Min),照射24 h后,加入CCK-8后37℃孵育1 h。多功能酶标仪450 nm处检测吸光度。细胞活力% =(A实验组-A空白组)/(A对照组-A空白组)×100%。
取适量YS-10称重,使用DMSO溶液溶解后,通过细胞培养基将药物溶液配制成浓度最高的母液,置于-20℃储存备用。使用前,按照实验所需浓度要求对母液进行稀释。收取细胞后,分别给予0、2.5、5、10、20、40、80、100μmol/L的YS-10药物处理细胞,每个药物浓度设置6个复孔。药物培养24 h后,加入CCK-8后37℃孵育1 h。多功能酶标仪450 nm处检测吸光度,并根据吸光度计算细胞的存活率。计算公式同1.8。
将细胞接种于6孔板中培养过夜,建立细胞放射损伤模型,使用YS-10处理24 h,在用无血清细胞培养基代替原培养基培养细胞的同时,在严格的避光条件下,每孔加入适量的10μmol DCFH-DA荧光探针37℃孵育30 min。孵育完成后,使用PBS洗涤细胞2次,利用荧光显微镜观察细胞成像结果。根据绿色荧光强度变化分析GC-2细胞内ROS的水平。
在YS-10给药处理完成后,使用23 cm细胞刮收集细胞,将裂解液加入细胞后吹打混匀,超声裂解细胞,高速离心后,提取蛋白,采用BCA法定量并计算蛋白浓度,10%的SDS-PAG凝胶电泳,转膜后,使用5%脱脂奶粉室温封闭1h。按照抗体说明书分别加入Keap1、Nrf2、HO-1、GAPDH抗体(抗体稀释浓度分别为1∶1000、1∶1000、1∶1000、1∶2000)4℃孵育过夜,使用TBST溶液快速洗膜,5 min×3次,室温下二抗孵育1 h,二抗抗体稀释度均为1∶10 000,TBST溶液快速洗膜,5 min×3次,洗膜完成后,利用高灵敏ECL化学发光法显色。根据成像分析系统分析不同蛋白条带的表达水平。
利用ImageJ软件对所有组织学和western blot图像进行校正和定量,并计算阳性区域的密度和比例。利用SPSS 26.0软件和GraphPad Prism 8.0软件对数据结果进行统计分析并绘制统计图像。以均数±标准差表示所有数据结果。多组间数据进行两两比较时,使用单因素方差分析。认为P<0.05,差异具有统计学意义。
从插页ⅩⅩⅩⅢ图1可知,Control组睾丸大小适中,形态无异常,与Control组相比,Model组大鼠睾丸可见明显的组织血肿,睾丸体积减小。经过YS-10治疗后睾丸组织的充血现象和水肿程度减轻,睾丸体积增加。与Control组相比,Model组睾丸指数和睾酮分泌水平明显降低(P<0.05);与Model组相比,YS-10组睾丸指数和睾酮分泌水平明显增加(P<0.05)。
HE染色后,镜下观察发现,Control组大鼠睾丸结构完整,生精细胞排列紧密,各类细胞形态正常。间质组织结构完好无损,生精小管内可见大量精子,精子形态无异常。而Model组大鼠睾丸体积减小,睾丸萎缩,组织结构模糊,间质细胞和生精小管排列紊乱,生精细胞和精子细胞减少,间质组织出现大量空泡变性。而YS-10组与Model组相比,生精小管结构变清晰,生精上皮厚度明显增加,精子数量增多。
与Control组相比,Model组睾丸生精小管直径和面积明显降低(P<0.01);与Model组相比,YS-10组睾丸生精小管直径和面积明显增加(P<0.05)。结果见插页ⅩⅩⅩⅢ图2。
与Control组比较,Model组大鼠睾丸组织中MDA含量明显增加,SOD活性与GSH含量显著降低(P<0.05)。与Model组比较,YS-10组大鼠MDA含量减少,SOD活性与GSH含量增加(P<0.05)。结果如插页ⅩⅩⅩⅢ图3所示。
如插页ⅩⅩⅩⅣ图4所示,X射线诱导的小鼠精母细胞系GC-2细胞的放射性损伤的最佳诱导剂量为8 Gy(P<0.01),最佳诱导时间为24 h。如图所示,与Control组相比,YS-10在2.5~10μmol/L浓度范围内,GC-2细胞活力均未受到抑制,表明YS-10在该浓度范围内对GC-2细胞无毒性作用,安全性较高。进一步实验表明,与Model组相比,2.5μmol/L组GC-2细胞活力增加(P<0.01)。与Model组相比,5μmol/L组和10μmol/L组的GC-2细胞活力差异无统计学意义(P>0.05)。依据此实验结果,后续我们选定8 Gy作为造模剂量,2.5μmol/L作为YS-10的给药剂量。
荧光探针检测可见ROS呈现绿色荧光,与Control组相比,Model组细胞ROS平均荧光强度明显升高(P<0.01);YS-10组的细胞ROS平均荧光强度较Model组降低(P<0.01)。见插页ⅩⅩⅩⅣ图5。
如插页ⅩⅩⅩⅣ图6所示,与Control组比较,Model组GC-2细胞中Keap1蛋白表达升高了约1.35倍,Nrf2、HO-1蛋白表达各降低了约1.46倍(P<0.05)。与Model组比较,YS-10组GC-2细胞Keap1蛋白表达降低约1.3倍,Nrf2蛋白表达增加约1.61倍,HO-1蛋白表达均升高了约1.22倍(P<0.05)。
精子发生是一个高度复杂、保守的细胞分裂和分化的过程。它包括精原细胞的增殖和分化、精母细胞的减数分裂和精子细胞的生成[16]。在精子发生过程中,一些睾丸结构和细胞发挥着重要作用[17]。其中,生精小管是精子发生的主要场所,各级细胞的正常发育和分化,是精子不断生成的重要保障[18]。本实验结果显示,Model组大鼠睾丸生精功能受损,伴随生精小管异常。YS-10组可恢复睾丸组织生精功能及生精小管形态。
氧化应激在辐射诱导的睾丸损伤中的致病作用已被证实[19]。辐射诱导ROS的积累,过量的ROS可能对生物分子产生有害影响,如DNA损伤、蛋白质氧化和脂质过氧化,导致细胞和组织损伤[20-21]。MDA是ROS引起的脂质过氧化级联反应的代谢物,是一种高毒性分子,提示氧化应激。它还引起细胞膜内不饱和脂肪酸的降解,是组织损伤的标志[22]。GSH和GPx(谷胱甘肽过氧化物 酶)在氧化应激中对自由基的解毒都起着重要作用[23]。除此之外,SOD、GPx等抗氧化酶可以保护男性生殖器官免受ROS的有害影响[24]。本研究结果显示:与对照组相比,睾丸组织中MDA含量升高,SOD、GSH水平降低;YS-10处理后MDA含量降低,SOD、GSH表达升高。说明放射后对睾丸组织产生了有害影响,通过过量产生ROS自由基导致氧化应激。而YS-10作为一种抗氧化剂,可以显著改善组织过氧化状态,抑制氧化应激损伤,从而恢复睾丸功能。在另一项研究中,淫羊藿苷可以通过调节内源性抗氧化水平来减轻辐射诱导的氧化应激反应[25]。该项实验结果与本研究结论一致。
ROS作为一种含氧分子,具有高度的化学活性。在生物学领域,活性氧是氧气正常代谢的天然副产物,并在细胞信号传导和体内平衡过程中发挥着重要作用[26]。ROS对精子细胞的功能和结构有双重影响。虽然它对精子细胞中一些正常的顶体反应过程是必要的,但它高浓度(称为氧化应激)时,可能会抑制精子的运动并改变其形态,导致精子不育[27]。本实验表明,与Control组相比,Model组GC-2细胞活力下降,ROS平均荧光强度增加。YS-10组GC-2细胞活力增加,ROS平均荧光强度减少。说明YS-10通过减少ROS诱导的氧化应激反应增强了细胞活力。
Nrf2是一种转录因子,属于碱性亮氨酸拉链转录因子的帽领家族成员。Nrf2在生物体中具有重要的生理功能,主要参与调节细胞内氧化应激反应和相关基因的表达。在正常情况下,Nrf2与细胞质中的Keap1结合。Keap1促进Nrf2的泛素化并阻止其进入细胞核[28]。在氧化应激条件下,Keap1将Nrf2解离,并将其易位至细胞核中。Nrf2结合细胞核中的抗氧化反应元件,从而上调下游抗氧化基因的表达,如HO-1和NAD(P)H脱氢酶醌1(NQO1)[29]。激活Nrf2及其下游抗氧化基因,可减轻电离辐射引起的精子发生功能障碍[30]。本实验表明,与Control组相比,放射后GC-2细胞中Keap1蛋白表达增加,Nrf2及其下游的HO-1蛋白表达减少,说明经放射后,细胞内的氧化应激反应被激活,下游抗氧化基因被上调。YS-10处理可以逆转Keap1和Nrf2、HO-1的蛋白表达水平,提示YS-10可以显著改善组织过氧化状态,维持氧化还原平衡,从而保护睾丸生精功能。
综上所述,淫羊藿苷衍生物YS-10可有效减轻辐射诱导的睾丸氧化应激反应,减轻睾丸组织损伤。其防治睾丸放射性损伤的作用机制可能是通过Keap1/Nrf2/HO-1信号通路来实现的。
  • 吉林省自然科学基金项目(YDZJ202301ZYTS138)
  • 吉林省卫生健康委员会科技能力提升项目(2022JC048)
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2025年第43卷第6期
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doi: 10.13193/j.issn.1673-7717.2025.06.043
  • 首发时间:2026-10-30
  • 出版时间:2025-06-10
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吉林省自然科学基金项目(YDZJ202301ZYTS138)
吉林省卫生健康委员会科技能力提升项目(2022JC048)
作者信息
    1.长春中医药大学,吉林 长春 130117
    2.长春中医药大学附属医院,吉林 长春 130021

通讯作者:

杨勇(1963-),男,吉林长春人,主任医师、教授,博士研究生导师,博士,研究方向:中西医结合临床外科疾病。E-mail:。
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2种不同金属材料的力学参数
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genus
种数
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species
占总种数比例
Percentage of
total species (%)
属
Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
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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