Article(id=1195664141189951491, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1195664138694341616, articleNumber=1001-2494(2024)02-0151-10, orderNo=null, doi=10.11669/cpj.2024.02.007, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1682438400000, receivedDateStr=2023-04-26, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1762998145625, onlineDateStr=2025-11-13, pubDate=1705852800000, pubDateStr=2024-01-22, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1762998145625, onlineIssueDateStr=2025-11-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1762998145625, creator=13701087609, updateTime=1762998145625, updator=13701087609, issue=Issue{id=1195664138694341616, tenantId=1146029695717560320, journalId=1190317699101192196, year='2024', volume='59', issue='2', pageStart='101', pageEnd='190', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1762998145030, creator=13701087609, updateTime=1762998511460, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1195665675697045692, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1195664138694341616, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1195665675701239997, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1195664138694341616, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=151, endPage=160, ext={EN=ArticleExt(id=1195664141374500870, articleId=1195664141189951491, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Effect of 717 Jiedu Decoction on Local Tissue Damage Repair of Rats After Agkistrodon halys Bite Through NETs-CYR61/CCN1 Signaling Pathway, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

OBJECTIVE To investigate the effect of 717 Jiedu decoction on neutrophil extracellular traps (NETs) and CYR61/CCN1 expression in local tissue of rats after Agkistrodon halys bite. METHODS SD rats were randomly divided into normal control group, Agkistrodon halys bite group and high-dose and low-dose 717 Jiedu decoction groups. The serum and thigh gastrocnemius were collected, and the pathological changes of gastrocnemius in each group were observed by Hematoxylin-Eosin staining(HE) and Masson staining, the ultrastructure of gastrocnemius in each group was observed by transmission electron microscopy, the apoptosis of gastrocnemius cells in each group was detected by TUNEL method, and the serum circulating-free cell (cf-DNA) level was detected by PicoGreen fluorescence. The expression levels of myeloperoxidase (MPO) in serum and gastrocnemius were detected by enzyme-linked immunosorbent assay(ELISA), the change of neutrophil elastase (NE) in gastrocnemius was detected by immunohistochemistry, and the level of citcitinic histone H3 (CitH3) in gastrocnemius was detected by immunofluorescence. The expressions of CYR61/CCN1 protein and its mRNA in gastrocnemius were detected by Western blot and PCR. RESULTS Compared with the normal group, the model group showed significant inflammatory infiltration and bleeding reaction, and the degree of collagen fibrosis increased. Ultrastructure showed that the structure of gastrocnemius cells in the model group was seriously damaged, and the distribution of muscle segments was asymmetrical. The contents of cf-DNA and MPO in serum were significantly increased (P<0.01), the protein expressions of MPO and CitH3 in gastrocnemius were increased (P<0.05 or P<0.01), and the protein and mRNA expressions of CYR61/CCN1 in gastrocnemius were up-regulated. All the administration groups of 717 Jiedu decoction significantly improved the local inflammatory response and bleeding symptoms of rats, reduced the degree of collagen fibrosis, effectively inhibited the apoptosis rate of gastrocnemius cells of pit viper bitten rats, and reduced the contents of cf-DNA and MPO in serum (P<0.05 or P<0.01). The expressions of NE and MPO in gastrocnemius were inhibited in the high-dose 717 Jiedu decoction group, and the expression of CitH3 in gastrocnemius muscle was decreased in all administration groups of 717 Jiedu decoction (P<0.05 or P<0.01), and the high expression of CYR61/CCN1 protein and its mRNA was inhibited. CONCLUSION The venom of Agkistrodon halys can induce the formation of neutrophil NETs in local tissue and the overexpression of CYR61/CCN1. The 717 Jiedu decoction could improve the ultrastructure of gastrocnemius muscle and inhibit cell apoptosis, which may also improve snitch symptoms by regulating the NETs-Cyr61/CCN1 pathway. The signaling molecules of NETs and CYR61/CCN1 are expected to be new targets for the prevention and treatment of local tissue damage caused by Agkistrodon halys venom.

, correspAuthors=YAN Zhangren, WANG Wanchun, 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, authorCompany=null, fund=null, authors=null, authorsList=DONG Degang, YANG yue, ZHENG Xianglong, MAO Wenli, SONG Mei, YAN Zhangren, WANG Wanchun), CN=ArticleExt(id=1195664677398164084, articleId=1195664141189951491, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=717解毒合剂通过NETs-CYR61/CCN1信号通路对蝮蛇咬伤大鼠局部组织损伤修复作用, columnId=1190352405612040510, journalTitle=中国药学杂志, columnName=论著, runingTitle=null, highlight=null, articleAbstract=

目的 探究717解毒合剂对蝮蛇咬伤大鼠局部组织中性粒细胞胞外陷阱(neutrophil extracellular traps, NETs)水平,以及对富含半胱氨酸蛋白61(CYR61/CCN1)表达的影响。方法 将SD大鼠随机分为正常对照组、蝮蛇咬伤组及抗蝮蛇毒血清组及717解毒合剂高、低剂量组。收集血清、大腿腓肠肌,采用苏木素-伊红(Hematoxylin-Eosin staining, HE)和马松(Masson)染色观察各组腓肠肌病理学变化,透射电镜观察腓肠肌超微结构,原位末端凋亡(TUNEL)法检测各组大鼠腓肠肌细胞凋亡情况,PicoGreen荧光检测各组血清游离DNA(cf-DNA)水平,酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测各组血清与腓肠肌中髓过氧化物酶(myeloperoxidase, MPO)的表达水平,免疫组化检测腓肠肌中性粒细胞弹性蛋白酶(neutrophil elastase, NE)的改变,免疫荧光法检测各组大鼠腓肠肌瓜氨酸化组蛋白H3(CitH3)水平,Western blot与聚合酶链式反应(polymerase chain reaction,PCR)技术检测腓肠肌组织CYR61/CCN1蛋白及其mRNA表达情况。结果 与正常组比较,模型组出现显著的炎性浸润与出血反应,且胶原纤维化程度均升高;超微结构显示模型组大鼠腓肠肌细胞结构损伤严重,肌节呈非对称性分布,肌原纤维丝断裂;并分布有大量凋亡细胞,血清cf-DNA、MPO含量显著上升(P<0.01),腓肠肌MPO及CitH3蛋白表达均有升高(P<0.05或P<0.01),腓肠肌CYR61/CCN1蛋白及其mRNA表达均有上调;717解毒合剂各给药组均能显著改善大鼠局部炎性反应及出血症状,降低胶原纤维化程度,有效抑制蝮蛇咬伤大鼠腓肠肌细胞凋亡率,减少血清cf-DNA、MPO含量(P<0.05或P<0.01),717解毒合剂高剂量组抑制了腓肠肌NE、MPO的表达,717解毒合剂各给药组均能降低腓肠肌CitH3的表达(P<0.05或P<0.01),抑制了CYR61/CCN1蛋白及其mRNA高表达。结论 蝮蛇毒诱导局部组织中性粒细胞NETs形成,CYR61/CCN1地过表达,717解毒合剂改善了大鼠腓肠肌超微结构,抑制了细胞凋亡,并可能通过调控NETs-CYR61/CCN1信号通路改善蛇伤症状,NETs信号分子及CYR61/CCN1有望成为蝮蛇毒致局部组织损伤防治的新靶点。

, correspAuthors=严张仁, 王万春, authorNote=null, correspAuthorsNote=
*王万春,男,教授 研究方向:毒蛇咬伤的临床与基础 Tel:(0791)86362691;
严张仁,男,教授 研究方向:中医外科学 Tel:(0791)86362691
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董德刚,男,副教授,硕士生导师 研究方向:中药药理与毒理

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董德刚,男,副教授,硕士生导师 研究方向:中药药理与毒理

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董德刚,男,副教授,硕士生导师 研究方向:中药药理与毒理

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Green arrows-inflammatory cell infiltration; Black arrows-bleeding.

, figureFileSmall=uJlltIb14AIYMV/6D9bJNQ==, figureFileBig=dhXvNjzbgoHN19r5HMrDBA==, tableContent=null), ArticleFig(id=1197098411250991189, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=CN, label=图1, caption=各组大鼠腓肠肌组织苏木素-伊红(HE)染色结果(×400)

绿色箭头-炎症细胞浸润;黑色箭头-出血。

, figureFileSmall=uJlltIb14AIYMV/6D9bJNQ==, figureFileBig=dhXvNjzbgoHN19r5HMrDBA==, tableContent=null), ArticleFig(id=1197098411334877271, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=EN, label=Fig.2, caption=Masson staining results of gastrocnemius tissue in each group. n=8, x -±s

Green arrows-collagen fibers; Red arrows-head muscle fibers, cytoplasm, cellulose, keratin, and red blood cells; 1)P<0.01, compared with control group;2)P<0.01, compared with model group.

, figureFileSmall=nWj9qy8dxI4JNrO1OWaZJw==, figureFileBig=MB8iDdatgcGUHz5oQNWXcQ==, tableContent=null), ArticleFig(id=1197098411439734873, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=CN, label=图2, caption=各组腓肠肌组织马松(Masson)染色结果(×400). n=8, x -±s

绿色箭头-胶原纤维;红色箭头-肌纤维、胞质、纤维素、角蛋白及红细胞;与正常对照组比较,1)P<0.01;与模型组比较,2)P<0.01。

, figureFileSmall=nWj9qy8dxI4JNrO1OWaZJw==, figureFileBig=MB8iDdatgcGUHz5oQNWXcQ==, tableContent=null), ArticleFig(id=1197098411506843739, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=EN, label=Fig.3, caption=Ultrastructural changes of gastrocnemius of rats by TEM(×7 000), figureFileSmall=Nu+szCsoeg0hDRiOQWh8bg==, figureFileBig=iubebibB10uRavm3V97+iw==, tableContent=null), ArticleFig(id=1197098411565563997, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=CN, label=图3, caption=大鼠腓肠肌透射电镜观察超微结构变化(×7 000), figureFileSmall=Nu+szCsoeg0hDRiOQWh8bg==, figureFileBig=iubebibB10uRavm3V97+iw==, tableContent=null), ArticleFig(id=1197098411641061471, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=EN, label=Fig4, caption=Apoptosis of rat gastrocnemius by TUNEL assay(×200), figureFileSmall=/ZpjICe3PDCjVFCau1jCKw==, figureFileBig=dUjjmWldUrPHyMpK/vuf8w==, tableContent=null), ArticleFig(id=1197098411724947553, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=CN, label=图4, caption=大鼠腓肠肌TUNEL荧光细胞凋亡(×200), figureFileSmall=/ZpjICe3PDCjVFCau1jCKw==, figureFileBig=dUjjmWldUrPHyMpK/vuf8w==, tableContent=null), ArticleFig(id=1197098411783667811, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=EN, label=Fig.5, caption=cf-DNA and MPO levels in serum and MPO levels in gastrocnemius of rats. n=8, x -±s

A-cf-DNA in serum; B-MPO in serum; C-MPO in gastrocnemius;1) P<0.01, compared with control group;2)P<0.05,3)P<0.01, compared with model group.

, figureFileSmall=qhe+d8NiwyFHQPr1EjeTPg==, figureFileBig=GbbdylPOWPrrwyeE59ss9Q==, tableContent=null), ArticleFig(id=1197098411854970980, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=CN, label=图5, caption=大鼠血清游离DNA(cf-DNA)水平、髓过氧化物酶(MPO)水平及腓肠肌MPO水平. n=8, x -±s

A-血清cf-DNA;B-血清MPO;C-腓肠肌MPO;与正常对照组比较,1)P <0.01;与模型组比较,2)P<0.05,3)P<0.01。

, figureFileSmall=qhe+d8NiwyFHQPr1EjeTPg==, figureFileBig=GbbdylPOWPrrwyeE59ss9Q==, tableContent=null), ArticleFig(id=1197098411934662758, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=EN, label=Fig.6, caption=Results of NE IHC staining of gastrocnemius tissue of rats(×400). n=8, x -±s

1)P<0.01, compared with control group;2)P<0.01, compared with model group.

, figureFileSmall=ydmJJlrms0969/YNtA9gXw==, figureFileBig=pbiyJMFH5LnAe6hmPczl7Q==, tableContent=null), ArticleFig(id=1197098411993383016, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=CN, label=图6, caption=大鼠腓肠肌组织中性粒细胞弹性蛋白酶(NE)免疫组化染色结果(×400). n=8, x -±s

与正常对照组比较,1)P<0.01;与模型组比较,2)P<0.01。

, figureFileSmall=ydmJJlrms0969/YNtA9gXw==, figureFileBig=pbiyJMFH5LnAe6hmPczl7Q==, tableContent=null), ArticleFig(id=1197098412064686185, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=EN, label=Fig.7, caption=Results of NE IF staining of gastrocnemius tissue of rats(×400). n=8, x -±s

1)P<0.05,2)P<0.01, compared with control group;3)P<0.05,4)P<0.01, compared with model group.

, figureFileSmall=RgxZvH8JDf9woXPajN/NOA==, figureFileBig=yBIme434OAeEaPasPmVv4w==, tableContent=null), ArticleFig(id=1197098412123406443, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=CN, label=图7, caption=大鼠腓肠肌组织NE免疫荧光染色结果(×400). n=8, x -±s

与正常对照组比较,1)P<0.05,2)P<0.01;与模型组比较,3)P<0.05,4)P<0.01。

, figureFileSmall=RgxZvH8JDf9woXPajN/NOA==, figureFileBig=yBIme434OAeEaPasPmVv4w==, tableContent=null), ArticleFig(id=1197098412186321005, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=EN, label=Fig.8, caption=Expression of CYR61 protein and its mRNA in gastrocnemius of rats. n=3, x -±s

A-Western blot; B-the relative expression of CYR61 protein; C-the relative expression of CYR61 mRNA;1)P<0.05,2)P<0.01, compared with control group;3)P<0.05,4)P<0.01, compared with model group.

, figureFileSmall=84/3a5yPi+a7MnnZemJ/ZA==, figureFileBig=t4Xzg35vW04djfQXemdhtw==, tableContent=null), ArticleFig(id=1197098412249235567, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195664141189951491, language=CN, label=图8, caption=大鼠腓肠肌CYR61蛋白及其mRNA表达. n=3, x -±s

A-免疫印迹图;B-CYR61蛋白相对表达量;C-CYR61 mRNA相对表达量; 与正常对照组比较,1)P<0.05,2)P<0.01;与模型组比较,3)P<0.05,4)P<0.01。

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717解毒合剂通过NETs-CYR61/CCN1信号通路对蝮蛇咬伤大鼠局部组织损伤修复作用
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董德刚 1 , 杨悦 2 , 郑向龙 2 , 毛文丽 3 , 宋梅 4 , 严张仁 3, * , 王万春 3, *
中国药学杂志 | 论著 2024,59(2): 151-160
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中国药学杂志 | 论著 2024, 59(2): 151-160
717解毒合剂通过NETs-CYR61/CCN1信号通路对蝮蛇咬伤大鼠局部组织损伤修复作用
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董德刚1, 杨悦2, 郑向龙2, 毛文丽3, 宋梅4, 严张仁3, *, 王万春3, *
作者信息
  • 1 江西中医药大学生命科学学院, 南昌 330004
  • 2 江西中医药大学附属医院中医外科, 南昌 330006
  • 3 南昌医学院公共学科教学部, 南昌 330004
  • 董德刚,男,副教授,硕士生导师 研究方向:中药药理与毒理

通讯作者:

*王万春,男,教授 研究方向:毒蛇咬伤的临床与基础 Tel:(0791)86362691;
严张仁,男,教授 研究方向:中医外科学 Tel:(0791)86362691
Effect of 717 Jiedu Decoction on Local Tissue Damage Repair of Rats After Agkistrodon halys Bite Through NETs-CYR61/CCN1 Signaling Pathway
DONG Degang1, YANG yue2, ZHENG Xianglong2, MAO Wenli3, SONG Mei4, YAN Zhangren3, *, WANG Wanchun3, *
Affiliations
  • 1 School of Life Sciences, Jiangxi University of Chinese Medicine, Nanchang 330004, China
  • 2 Department of TCM Surgery, Affiliated Hospital of Jiangxi University of Chinese Medicine, Nanchang 330006, China
  • 3 Department of Public Science, Nanchang Medical College, Nanchang 330004, China
出版时间: 2024-01-22 doi: 10.11669/cpj.2024.02.007
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目的 探究717解毒合剂对蝮蛇咬伤大鼠局部组织中性粒细胞胞外陷阱(neutrophil extracellular traps, NETs)水平,以及对富含半胱氨酸蛋白61(CYR61/CCN1)表达的影响。方法 将SD大鼠随机分为正常对照组、蝮蛇咬伤组及抗蝮蛇毒血清组及717解毒合剂高、低剂量组。收集血清、大腿腓肠肌,采用苏木素-伊红(Hematoxylin-Eosin staining, HE)和马松(Masson)染色观察各组腓肠肌病理学变化,透射电镜观察腓肠肌超微结构,原位末端凋亡(TUNEL)法检测各组大鼠腓肠肌细胞凋亡情况,PicoGreen荧光检测各组血清游离DNA(cf-DNA)水平,酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测各组血清与腓肠肌中髓过氧化物酶(myeloperoxidase, MPO)的表达水平,免疫组化检测腓肠肌中性粒细胞弹性蛋白酶(neutrophil elastase, NE)的改变,免疫荧光法检测各组大鼠腓肠肌瓜氨酸化组蛋白H3(CitH3)水平,Western blot与聚合酶链式反应(polymerase chain reaction,PCR)技术检测腓肠肌组织CYR61/CCN1蛋白及其mRNA表达情况。结果 与正常组比较,模型组出现显著的炎性浸润与出血反应,且胶原纤维化程度均升高;超微结构显示模型组大鼠腓肠肌细胞结构损伤严重,肌节呈非对称性分布,肌原纤维丝断裂;并分布有大量凋亡细胞,血清cf-DNA、MPO含量显著上升(P<0.01),腓肠肌MPO及CitH3蛋白表达均有升高(P<0.05或P<0.01),腓肠肌CYR61/CCN1蛋白及其mRNA表达均有上调;717解毒合剂各给药组均能显著改善大鼠局部炎性反应及出血症状,降低胶原纤维化程度,有效抑制蝮蛇咬伤大鼠腓肠肌细胞凋亡率,减少血清cf-DNA、MPO含量(P<0.05或P<0.01),717解毒合剂高剂量组抑制了腓肠肌NE、MPO的表达,717解毒合剂各给药组均能降低腓肠肌CitH3的表达(P<0.05或P<0.01),抑制了CYR61/CCN1蛋白及其mRNA高表达。结论 蝮蛇毒诱导局部组织中性粒细胞NETs形成,CYR61/CCN1地过表达,717解毒合剂改善了大鼠腓肠肌超微结构,抑制了细胞凋亡,并可能通过调控NETs-CYR61/CCN1信号通路改善蛇伤症状,NETs信号分子及CYR61/CCN1有望成为蝮蛇毒致局部组织损伤防治的新靶点。

717解毒合剂  /  蝮蛇咬伤  /  中性粒细胞胞外陷阱  /  细胞外基质  /  富含半胱氨酸蛋白61  /  局部组织

OBJECTIVE To investigate the effect of 717 Jiedu decoction on neutrophil extracellular traps (NETs) and CYR61/CCN1 expression in local tissue of rats after Agkistrodon halys bite. METHODS SD rats were randomly divided into normal control group, Agkistrodon halys bite group and high-dose and low-dose 717 Jiedu decoction groups. The serum and thigh gastrocnemius were collected, and the pathological changes of gastrocnemius in each group were observed by Hematoxylin-Eosin staining(HE) and Masson staining, the ultrastructure of gastrocnemius in each group was observed by transmission electron microscopy, the apoptosis of gastrocnemius cells in each group was detected by TUNEL method, and the serum circulating-free cell (cf-DNA) level was detected by PicoGreen fluorescence. The expression levels of myeloperoxidase (MPO) in serum and gastrocnemius were detected by enzyme-linked immunosorbent assay(ELISA), the change of neutrophil elastase (NE) in gastrocnemius was detected by immunohistochemistry, and the level of citcitinic histone H3 (CitH3) in gastrocnemius was detected by immunofluorescence. The expressions of CYR61/CCN1 protein and its mRNA in gastrocnemius were detected by Western blot and PCR. RESULTS Compared with the normal group, the model group showed significant inflammatory infiltration and bleeding reaction, and the degree of collagen fibrosis increased. Ultrastructure showed that the structure of gastrocnemius cells in the model group was seriously damaged, and the distribution of muscle segments was asymmetrical. The contents of cf-DNA and MPO in serum were significantly increased (P<0.01), the protein expressions of MPO and CitH3 in gastrocnemius were increased (P<0.05 or P<0.01), and the protein and mRNA expressions of CYR61/CCN1 in gastrocnemius were up-regulated. All the administration groups of 717 Jiedu decoction significantly improved the local inflammatory response and bleeding symptoms of rats, reduced the degree of collagen fibrosis, effectively inhibited the apoptosis rate of gastrocnemius cells of pit viper bitten rats, and reduced the contents of cf-DNA and MPO in serum (P<0.05 or P<0.01). The expressions of NE and MPO in gastrocnemius were inhibited in the high-dose 717 Jiedu decoction group, and the expression of CitH3 in gastrocnemius muscle was decreased in all administration groups of 717 Jiedu decoction (P<0.05 or P<0.01), and the high expression of CYR61/CCN1 protein and its mRNA was inhibited. CONCLUSION The venom of Agkistrodon halys can induce the formation of neutrophil NETs in local tissue and the overexpression of CYR61/CCN1. The 717 Jiedu decoction could improve the ultrastructure of gastrocnemius muscle and inhibit cell apoptosis, which may also improve snitch symptoms by regulating the NETs-Cyr61/CCN1 pathway. The signaling molecules of NETs and CYR61/CCN1 are expected to be new targets for the prevention and treatment of local tissue damage caused by Agkistrodon halys venom.

717 Jiedu decoction  /  Agkistrodon halys bite  /  neutrophil extracellular trap  /  extracellular matrix  /  CYR61/CCN1  /  local tissue
董德刚, 杨悦, 郑向龙, 毛文丽, 宋梅, 严张仁, 王万春. 717解毒合剂通过NETs-CYR61/CCN1信号通路对蝮蛇咬伤大鼠局部组织损伤修复作用. 中国药学杂志, 2024 , 59 (2) : 151 -160 . DOI: 10.11669/cpj.2024.02.007
DONG Degang, YANG yue, ZHENG Xianglong, MAO Wenli, SONG Mei, YAN Zhangren, WANG Wanchun. Effect of 717 Jiedu Decoction on Local Tissue Damage Repair of Rats After Agkistrodon halys Bite Through NETs-CYR61/CCN1 Signaling Pathway[J]. Chinese Pharmaceutical Journal, 2024 , 59 (2) : 151 -160 . DOI: 10.11669/cpj.2024.02.007
毒蛇咬伤是一类严重危害人类健康的危重症。据统计,毒蛇咬伤影响了世界热带和亚热带地区的数百万人,全世界每年约有500万人被蛇咬伤,其中超过10万人死亡,多达40万人截肢和永久性缺陷[1]。蝮蛇是我国主要蛇伤蛇种,蝮蛇咬伤占江西省毒蛇咬伤的88%以上[2]。其毒性程度高,如未获得及时或有效医治,轻者致残,重者危及生命。随着气温的逐年升高,蝮蛇咬伤事件发生率已呈上升趋势,而我国基层地区以老弱幼为主,不少蛇伤患者预后仍不理想。蝮蛇咬伤能引起出血、水肿、疼痛及肌坏死等显著局部损伤效应。抗蛇毒血清能显著降低了毒蛇咬伤的死亡率,但在治疗蛇咬伤引起的局部病理效应方面是有限的[3-4]。寻找蝮蛇咬伤局部组织损伤有效治疗方法已成为临床急需。
近年来,在毒液诱导的组织损伤研究方面取得了重大成果,包括所涉及的毒素筛选与鉴定,以及局部组织对毒液应激反应等方面。但蛇毒诱导局部组织损伤的确切机制尚不明确[5]。中性粒细胞是机体最重要的炎性细胞,通过释放嵌入瓜氨酸化组蛋白 H3(citrullinated histone H3, CitH3)、髓过氧化物酶(myeloperoxidase, MPO)和细胞外染色质等产生类似于渔网的纤维结构,称为中性粒细胞胞外陷阱 (neutrophil extracellular traps, NETs)。现有研究证实,中性粒细胞是第1个到达蛇伤组织的炎症细胞,迅速募集并激活,产生包括趋化性、炎症介质合成、活性氧以及NETs形成等效应,NETs在蛇毒诱导的局部组织损伤发生与修复过程中起关键作用[6]
本研究前期实验证实蝮蛇毒能引起炎症反应与氧化应激显著的局部组织损伤,经验方717解毒合剂能显著改善局部损伤症状,降低血清中透明质酸(HA)、Ⅳ型胶原(Col-Ⅳ),层粘连蛋白(LN)等细胞外基质(extracellular matrix,ECM)成分含量,加快创面愈合[7-8]。ECM是一种高度动态和复杂的网络结构,几乎存在于所有组织中,是细胞赖以生存的微环境,ECM与细胞相互作用以调节多种功能,包括分化、增殖和迁移。中性粒细胞是重要的免疫细胞,在炎症期间协调一系列复杂事件中发挥关键作用。中性粒细胞还可以通过提供特定的基质重塑酶(如中性粒细胞弹性蛋白酶和金属蛋白酶)、产生NETs来介导 ECM 重塑,ECM 也可以通过调节中性粒细胞的功能来重塑炎症微环境,从而驱动疾病进展[9]
富含半胱氨酸61(cysteine rich 61, Cyr61/CCN1)是一种ECM相关蛋白,可发挥多种生理功能,包括介导ECM的形成、细胞增殖、分化和黏附,以及创面愈合、组织修复和血管形成等[10-11]。Cyr61/CCN1是一种新型促炎因子,异常表达的CCN1与多种病理有关,包括各种急慢性炎症、组织纤维化及肿瘤等,尤其涉及伤口愈合的稳态、炎症、增殖和重塑各个阶段[10,12]。但其在毒蛇咬伤进展中作用机制尚不清楚。本实验将以蝮蛇咬伤为研究对象,围绕NETs-CYR61/CCN1信号通路,探讨717解毒合剂抗蝮蛇咬伤局部组织损伤作用及其可能机制,以期探寻治疗蛇伤局部损伤的新靶点,为中医药治疗蛇伤提供理论依据。
SPF级SD大鼠40只(体质量180~220 g),雌雄各半(湖南斯莱克景达实验动物有限公司),许可证号:SCXK(湘)2019-0004。饲养条件:温度20~26 ℃,湿度:40%~70%。各12 h明暗周期的饲养室,普通饲料喂养,自由进食和饮水。
蝮蛇毒(福建龙海市春林农场,批号:20150927);717解毒合剂(院内制剂,批号: 040843,组方:金银花-野菊花-紫花地丁-蒲公英-黄连-黄柏-半边莲-七叶一枝花-蝉蜕-防风-白芷-生大黄-车前草=3∶3∶3∶3∶2∶2∶3∶3∶2∶2∶2∶2∶3,广东一方制药公司),按比例配制成1 g·mL-1悬液进行灌胃,超声振荡30 min,生药定容浓度为1 g·mL-1。所制颗粒各项指标均等符合2020年版《中国药典》一部附录ⅠC颗粒剂项下要求。
Masson染色液(货号:G1006,武汉Servicebio公司);丽春红酸性品红染色液(货号:R23039,上海源叶生物公司);苏木精-伊红染色液(货号:G1100)、PicoGreen dsDNA(货号:P9740)、TUNEL试剂盒(货号:G1501)(北京索莱宝公司);髓过氧化物酶(MPO,货号:MM-0337R1)、中性粒细胞弹性蛋白酶(Neutrophil Elastase, NE)(货号:MM-0306R1)(上海酶联生物科技有限公司);瓜氨酸化组蛋白 H3(CitH3)(货号:ab5103)、β-tubulin一抗(货号:ab21057)(英国Abcam公司);Neutrophil Elastase一抗(货号:AF0010,美国Affinity公司);CYR61 (货号:DF6250,武汉Proteintech公司);山羊抗兔 IgG cy3(货号:AS007,武汉ABdonal公避开);PVDF膜(IPVH00010,美国Millipore公司);抗蝮蛇毒血清(批号:20210404,上海赛伦生物,每支6000 u);其他均为国产分析纯。
病理切片机 (型号:RM2016,上海徕卡公司);分析天平(BS110S,北京赛多利斯公司);脱水机(Donatello,意大利DIAPATH公司);包埋机(JB-P5,武汉俊杰电子公司);透射电子显微镜(HT7700)、光学显微镜(CX43)、荧光显微镜(CKX53)(日本OLYMPUS公司);多功能酶标仪(S/N502000011,瑞士TECAN公司);高速冷冻离心机(Biofuge stratos,德国Hereaus公司);凝胶成像分析仪(WD-9413)、蛋白垂直电泳仪(DYY-6C)(北京六一生物科技有限公司);超高灵敏度化学发光成像系统(Chemi DocTM XRS+,上海伯乐生命医学),荧光定量PCR仪(PRISM 7700,美国ABI)。
SD大鼠适应性喂养1周后,随机分成5组:正常对照组(control);蝮蛇咬伤模型组(model);抗蝮蛇毒血清组(serum)及717解毒合剂低(717JD-L)、高剂量组(717JD-H),每组8只。除正常对照组以外,其余各组均用蝮蛇毒制成蝮蛇咬伤动物模型。
717解毒合剂各组在造模前6 d给药生药量1.65、3.3 g·kg-1(分别为临床给药量的6与12倍),每天灌胃给药1次,共6 d。control组给予正常饮食及灌胃等体积生理盐水。
serum组自注射蛇毒后2 h开始给药,将血清(离心管中为1 mL)用0.9%氯化钠生理盐水稀释8倍,获得8 mL蛇毒血清稀释液,按每只0.6 mL尾静脉注射。
蝮蛇毒在临用前以生理盐水作溶媒配制为0.05 g·mL-1的蝮蛇毒溶液,造模时大鼠按0.002 5 mL·g-1体质量进行肌肉注射。将大鼠左后肢剪毛,再将蝮蛇毒溶液注射至大鼠左后肢腓肠肌,注毒后棉签压迫进针口约10 s,伤口匀涂碘伏消毒。control组注射等量生理盐水。
造模成功:大鼠未死亡,只限中毒症状,于注射后5 min左右局部变为青色,行走不利,个别大鼠有躁狂症状,注射后1 h左右局部变为暗紫色,肿硬,甚则水肿水疱,行动迟缓。约4 h后大鼠毛发竖起,无光泽,弓背,精神萎靡,活动明显减少。
各组在造模成功24 h后取样,各组取样前均禁食过夜,按照40 mg·kg-1体重腹腔内注射质量分数3%戊巴比妥钠溶液麻醉后,经腹主动脉抽取空腹血4 mL,静置1 h后3 000 r·min-1离心10 min,获得血清,处死后摘取各组腓肠肌,取少量置4%多聚甲醛进行固定,其余腓肠肌经液氮速冻后-80 ℃保存,备用。
取大鼠腓肠肌组织,置于4%多聚甲醛溶液中固定48 h后,常规石蜡包埋、切片,二甲苯脱蜡,乙醇梯度水洗,常规苏木精-伊红(HE)染色,树胶封片。HE染色时,石蜡切片于二甲苯脱蜡,酒精梯度水洗,苏木精染色,盐酸乙醇分化,伊红染色,用乙醇脱水,二甲苯透明、树胶封片;马松Masson染色时,常规脱水包埋,切片脱蜡,Weigert铁苏木素,体积分数1%盐酸乙醇分化,丽春红酸性品红染液染,磷钼酸溶液5 min, 苯胺蓝复染,体积分数1%冰乙酸处理,乙醇多次脱水,二甲苯透明,树胶封固。镜检。
取上述部分腓肠肌置于预冷的生理盐水中洗净血污后,切取1 mm×1 mm×1 mm,迅速投入电镜固定液4 ℃固定2~4 h。磷酸盐缓冲液(PBS)漂洗,体积分数1%的锇酸0.1 mol·L-1 pH 7.4 PBS室温固定2 h。PBS漂洗,常规组织脱水、渗透、包埋、切片,铀铅双染色,透射电镜观测。
选取视腓肠肌部位蜡块切片,烤片后脱蜡至水,蛋白酶K修复,PBS冲洗,TUNEL反应液37 ℃孵育22 min,PBS冲洗后,切片稍甩干后在圈内滴加破膜工作液覆盖组织,常温下孵育20 min,PBS冲洗后室温平衡,按片子数量和组织大小取TUNEL试剂盒内适量末端脱氧核糖核酸转移酶(terminal deoxynucleotidyl transferase, TDTase),dUTP,buffer按1∶5∶50比例混合,加到圈内覆盖组织,切片平放于湿盒内,37 ℃恒温箱孵育2 h,湿盒内加少量水保持湿度,去除PBS后在圈内滴加4',6-联脒-2-苯基吲哚(4',6-diamidino-2-phenylindole, DAPI)染液,避光室温孵育10 min,用抗荧光淬灭封片剂封片。荧光显微镜下观察,波长330~380 nm。
采用Picogreen 试剂盒测定,用已知浓度的小牛胸腺嘧啶DNA(TE缓冲液配置)作为标准品工作液。取稀释10倍后的血清50 μL加入黑色96孔微孔板,加入50 μL经200倍稀释的Picogreen染料工作溶液,室温孵育10 min后,以TE缓冲液为空白,激发波长488 nm,发射波长520 nm,测定样品和control组的荧光值。依据标准品曲线计算血清样品cf-DNA浓度。
将各组腓肠肌组织按照质量(g)∶体积(mL)=1∶9加入生理盐水进行组织匀浆,10 000 r·min-1离心8 min,获得上清,ELISA法检测血清以及腓肠肌MPO含量,严格按照说明书步骤进行。
将大鼠腓肠肌组织石蜡切片予以常规脱蜡脱水后,将切片放入切片架中,加入柠檬酸抗原修复缓冲液(pH 6.0),加热煮沸进行高压抗原修复后自然冷却,弃去抗原修复液,将切片用PBS(pH 7.4)淋洗;加入体积分数3%双氧水去除内源性过氧化物酶,室温孵育10 min,PBS充分淋洗。PBS浸洗玻片3次,每次5 min,吸水纸吸干组织周围的PBS,在玻片上滴加5%BSA,37 ℃封闭30 min。用吸水纸吸掉组织周围的封闭液,每张玻片滴加足够量的稀释好的一抗:Neutrophil Elastase(1∶100);4 ℃孵育过夜,结合山羊抗兔(1∶100),经DAB显色、封片,镜下观察阳性表达情况。
将大鼠腓肠肌组织石蜡切片予以常规脱蜡脱水后,将切片放入切片架中,加入柠檬酸抗原修复缓冲液(pH 6.0),加热煮沸进行高压抗原修复后自然冷却,弃去抗原修复液,将切片用PBS淋洗,体积分数0.5%TritonX-100室温通透20 min。PBS浸洗玻片3次,每次5 min,吸水纸吸干组织周围的PBS,在玻片上滴加质量分数5%BSA,37 ℃封闭30 min。用吸水纸吸掉组织周围的封闭液,每张玻片滴加足够量的稀释好的一抗:CitH3(1∶200);4 ℃孵育过夜荧光二抗Cy3(1∶200),湿盒中在较暗处37 ℃孵育45 min。PBS充分淋洗,滴加DAPI避光孵育3 min,对标本进行染核,用PBS冲洗多余的DAPI,水冲洗,吹干、封片、镜检。
剪取少量腓肠肌加入用裂解液裂解,离心15 min,收集上清。用BCA试剂盒测定蛋白浓度,取50 μg蛋白进行SDS-PAGE电泳分离,将分离的蛋白电转移至PVDF膜上。封闭液室温封闭1 h,加入一抗孵育(1∶1 000),4 ℃过夜。TIBS充分洗膜后,加入二抗(1∶2 000)室温孵育1 h,洗膜后加入ECL发光液,显影、洗片、晾干。凝胶成像仪扫描,并采用Quantity one软件对其进行光密度分析,以β-tubulin作为内参,进行灰度分析,蛋白表达量用目的蛋白/β-tubulin×100%表示。
采用Trizol法提取腓肠肌组织的RNA,利用Primer 5.0软件合成引物,Cyr61上游引物5'-ACTTCATGGTCCCAGTGCGC-3',下游引物5'-AAATCCGGGTTTCTTTCACA-3';内参GAPDH基因上游引物5'-CCTGCACCACCAACTGCTTA-3',下游引物5'-ATGACCTTGCCCACAGCCT-3'。加入目的基因引物后,用试剂盒扩增PCR产物,质量分数2%琼脂糖凝胶进行电泳,采用Quantity One软件进行灰度值分析。用Sequence Detection Soft Wareversion 软件检测样本循环阈值(cycle threshold,Ct)值,经2-△△Ct计算基因相对表达量。
应用SPSS 25.0软件进行统计学分析,定量结果采用均数±标准差($ \bar{x} \pm s$)表示,两组之间定量数值比较采用独立样本t检验,比较采用单因素方差分析。检验水准α=0.05,P<0.05表示差异显著。
HE染色结果显示,control组腓肠肌组织结构完整,肌细胞排列整齐,细胞间隙小,无炎症细胞浸润、出血现象;model组创面肌肉组织结构紊乱,肌细胞变性坏死严重、细胞间隙变宽,炎症细胞浸润明显,出血严重;serum组肌纤维损伤有所减轻,偶见正常细胞,但胞浆质化,炎症细胞浸润明显;717JD-L组细胞核聚集与炎性浸润减轻,正常细胞数增多;717JD-H组腓肠肌结构较为完整,细胞间隙变小,炎症细胞减少(图1)。
Masson染色显示,胶原纤维为蓝色表达,肌纤维、胞质、纤维素、角蛋白及红细胞为红色表达;经胶原容积分数(collagen volume fraction,CVF)计算胶原组织所占比例,与control组比较,其余各组胶原纤维化程度均升高(均有P<0.01);各给药组与model组比较纤维化有显著下降(均有P<0.01),且717JD-H组改善作用明显优于serum组,见图2
超微结构显示,control组大鼠腓肠肌细胞结构正常,细胞质均匀,细胞器数量正常,肌纤维排列整齐,粗细均一,肌节呈对称性分布,肌原纤维丝大多结构致密。model组腓肠肌细胞结构损伤相对严重,细胞膜大面积破损、崩解,整体呈坏死征象,细胞质大面积溶解,细胞器数量减少,大多肿胀、游离,纤维溶解、松散,局部断裂,少量粗细不均,肌节呈非对称性分布,肌原纤维丝断裂。serum组腓肠肌细胞呈明显水肿,细胞器明显肿胀、变大,肌纤维局部断裂,大面积肌纤维松散,细胞质稀疏,细胞器数量明显减少,肌原纤维丝稀疏、溶解。717JD-L组肠肌细胞呈明显水肿,细胞器明显肿胀、变大,肌纤维局部断裂,大面积融合变粗,肌节呈对称性分布,肌原纤维丝大多结构致密,小区域断裂。717JD-L组腓肠肌细胞呈轻度水肿,细胞器轻度肿胀,少量变大,肌纤维结构致密,局部明显变粗,细胞质稀疏,细胞器数量较多,局部基质变淡,部分严重者呈灶性改变,膜内可见髓样结构(图3)。
TUNEL检测结果显示,model组分布有大量凋亡细胞(棕黄色),其凋亡细胞数目明显高于control组;各给药组均可见少量凋亡细胞,但717JD-H组凋亡细胞少于serum组与717JD-L组。提示717解毒合剂有效抑制蝮蛇咬伤大鼠腓肠肌细胞凋亡率,且呈剂量依赖关系,见图4
与control组比较,除717JD-H组外(P>0.05),其余各组cf-DNA水平均显著升高(均有P<0.01);与model组比较,各给药组cf-DNA均有显著降低(P<0.01),且717JD-H组cf-DNA水平稍低于serum组(图5A)。与control组比较,蛇伤模型组血清与腓肠肌MPO含量均显著升高(均有P<0.01);与model组比较,各给药组大鼠血清中MPO均有所降低(P<0.05或P<0.01)(图5B),但serum组腓肠肌MPO无显著差异(P>0.05),而717JD-H组腓肠肌MPO显著降低(P<0.05)(图5C)。
免疫组化结果显示,与control组比较,腓肠肌NE显著升高,serum组与717JD-L组有统计学差异(均有P<0.01),而717JD-H组无显著差异(P>0.05);与model组比较,serum组与717JD-L组差异无统计学意义(均有P>0.05),而717JD-H组腓肠肌组织NE表达呈显著下降(P<0.01),见图6
免疫荧光结果显示,与control组比较,model组CitH3蛋白表达显著升高(P<0.01),与Model组比较,serum组无显著差异(P>0.05);而717JD-L、717JD-H组CitH3蛋白表达均显著降低(均有P<0.01),见图7
与control组比较,其余各组腓肠肌中CYR61蛋白及其mRNA过表达,差异有统计学意义(P<0.05或P<0.01);与model组比较,各给药组均抑制了腓肠肌CYR61蛋白及其mRNA表达水平,差异有统计学意义(P<0.05或P<0.01)。结果表明蝮蛇伤大鼠腓肠肌CYR61蛋白及其mRNA上调,717解毒合剂能抑制蝮蛇伤大鼠腓肠肌CYR61蛋白及其mRNA高表达。见图8
毒蛇咬伤是一个全球性的公共卫生问题。毒蛇咬伤局部病理效应包括出血、水肿、疼痛、肌坏死、皮肤坏死以及继发性感染等,是致残或导致永久性后遗症的主要原因,带来严重的病理生理、社会和心理后果。局部损伤效应产生的主要机制有炎性浸润,氧化应激,细胞外基质成分的降解及血运失衡等。蛇毒使局部组织产生显著的炎症反应和白细胞浸润,浸润细胞主要是多形核白细胞,多为中性粒细胞和富含巨噬细胞的浸润,使咬伤组织释放包括金属蛋白酶、细胞因子、趋化因子及生长因子等多种介质[13-14]。在受伤的局部组织中,一方面,高度活化的中性粒细胞将非致密染色质、颗粒蛋白释放到胞外;另一方面,过多募集的中性粒细胞必然产生一种区别于细胞凋亡和细胞坏死的新型细胞死亡方式,被称为NETosis[15]。NETosis能介导非感染性组织损伤的关键分子,如蛋白激酶C(PKC)、Ca2+载体、活性氧(ROS)、MPO、NE、肽酰基精氨酸脱亚氨酶4(PAD-4)和CitH3等产生[16-17]。这两方面的共同作用形成了NETs。可见,蛇伤后局部组织产生的NETs是探讨其毒性发生、发展及其转归的重要内容,具有深入研究的价值。
一般地,NETs被广泛认为具有抗感染作用并介导炎症消退,但新近的研究发现过量或未及时清除的NETs会导致炎症加重和组织损伤[18-19]。因此,NETs具有抗炎与促炎双重作用,是一把双刃剑。近年来,蛇毒引起的组织损伤产生NETs或NETosis已被探索。Swethakumar等[20]研究发现锯鳞蝰(Echis carinatus)毒液通过PKC/细胞外信号调节蛋白激酶(ERK)/c-Jun氨基末端激酶(JNK)信号轴以ROS依赖性方式激活烟酰胺腺嘌呤二核苷酸(NADH)氧化酶(NOX)和PAD-4来诱导NETosis,眼镜蛇(Naja naja)毒液通过激活PAD-4酶来诱导NETosis,并提出NETosis是蛇毒药效学中一个新探索的领域,研究其对蛇毒诱导的各种病理生理特性的影响具有重要意义。研究表明,蛇毒中如蛇毒丝氨酸蛋白酶(SVSPs)、蛇毒金属蛋白酶(SVMPs)、磷脂酶A2(PLA2s)和蛇毒透明质酸酶(SVHYs),及L-氨基酸氧化酶等酶类能直接或间接诱导NETs产生,从而引起局部炎症与组织坏死[21-22]。可见,蛇伤后,咬伤组织中性粒细胞过度激活,诱导NETosis使过量的NETs形成是蛇毒引起局部组织损伤的主要原因。
Katkar等[23]研究发现锯鳞蝰毒液诱导中性粒细胞在咬伤部位积聚,并介导NETs形成,阻塞血管,从而促进组织破坏,而当小鼠同时注射毒液和降解NETs的脱氧核糖核酸酶Ⅰ(DNaseⅠ)时,注射毒液部位中性粒细胞减少,且不会出现NETs、毒液积聚和组织破坏,提出通过DNaseⅠ降解NETs是防止毒液诱导组织损伤。DNaseⅠ通过清除由中性粒细胞聚集引起的NETs来促进伤口愈合[24]。Rudresha等[22]发现四乙基秋兰姆二硫化物(TTD)能抑制锯鳞蝰中SVMP诱导的NETosis,并降低PAD-4、CitH3和MPO表达水平,认为TTD具有治疗蛇毒介导的组织坏死的潜力。植物来源的DNase可以通过调控NETs有效治疗毒液诱导的小鼠组织损伤[25]。鉴于NETs的双重效应,重塑NETs的形成与降解的平衡是有效治疗蛇伤局部效应的重要方向。
本研究结果显示,蝮蛇咬伤能引起显著的炎性浸润与出血反应,胶原纤维化程度升高;血清cf-DNA,及MPO含量显著上升,腓肠肌NE、MPO及CitH3含量均有升高。其中,MPO是中性粒细胞功能与活化的重要标志物[26]。即蝮蛇毒能显著引发多种NETs关键标志物的高表达,提示蝮蛇咬伤后,大鼠局部损伤组织NETs迅速形成,过度激活的NETs是蝮蛇毒介导组织进一步病理损伤的重要原因。中药复方717解毒合剂各给药组均能显著改善大鼠局部炎性反应及出血症状,降低胶原纤维化程度,有效抑制蝮蛇咬伤大鼠腓肠肌细胞凋亡率,降低血清cf-DNA、MPO含量,717JD-H组抑制了腓肠肌NE、MPO的表达,717解毒合剂各给药组均能降低腓肠肌CitH3的表达。这些结果提示,717解毒合剂能显著抑制蝮蛇咬伤大鼠血清及腓肠肌NETs标志物高表达,因此,推测调控NETs表达水平是该复方治疗蝮蛇咬伤局部组织损伤效应的重要机制。
此外,本研究从CYR61/CCN1信号通路角度,初步探讨了蝮蛇咬伤引发局部组织NETs产生,以及717解毒合剂调控NETs水平的生物学机制。现有研究结果显示,NETs的过度形成能诱导NF-κB、IL-6、NLRP3炎症小体等炎症信号激活[27-29]。而这些炎症信号能显著促进了新型炎症分子CYR61/CCN1表达[30-32]。本研究结果显示,蝮蛇咬伤诱导NETs过度形成,而过量的NETs激活上述炎症信号从而介导CYR61/CCN1高表达。另一方面,CCN1作为桥接分子,结合磷脂酰丝氨酸,促进了细胞凋亡与风化,并导致旺盛的中性粒细胞积累和延迟愈合[33]。两方面的相互作用共同导致蛇伤局部组织损伤加重。而717解毒合剂各给药组能显著降低NETs水平,并抑制CYR61/CCN1高表达,结合现有研究,提示717解毒合剂调控NETs水平,进而抑制CYR61/CCN1信号可能是其改善蝮蛇咬伤症状的重要机制,在今后工作中课题组将以NETs-CYR61/CCN1为新靶点,进一步探讨蝮蛇咬伤局部组织损伤进展及其中医药防治详细机制。
  • 国家自然科学基金地区项目资助(81360288)
  • 国家自然科学基金地区项目资助(81960874)
  • 国家自然科学基金地区项目资助(82260937)
  • 江西省自然科学基金重点项目资助(20202ACB206010)
  • 江西中医药大学首批科技创新团队资助项目资助(CXTD22009)
  • 江西省教育厅科技一般项目资助(GJJ201236)
  • 江西中医药大学中西医结合一级学科资助(江西省双一流学科)
  • 江西中医药大学中西医结合一级学科资助(zxyylxk20220103)
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2024年第59卷第2期
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doi: 10.11669/cpj.2024.02.007
  • 接收时间:2023-04-26
  • 首发时间:2025-11-13
  • 出版时间:2024-01-22
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  • 收稿日期:2023-04-26
基金
国家自然科学基金地区项目资助(81360288)
国家自然科学基金地区项目资助(81960874)
国家自然科学基金地区项目资助(82260937)
江西省自然科学基金重点项目资助(20202ACB206010)
江西中医药大学首批科技创新团队资助项目资助(CXTD22009)
江西省教育厅科技一般项目资助(GJJ201236)
江西中医药大学中西医结合一级学科资助(江西省双一流学科)
江西中医药大学中西医结合一级学科资助(zxyylxk20220103)
作者信息
    1 江西中医药大学生命科学学院, 南昌 330004
    2 江西中医药大学附属医院中医外科, 南昌 330006
    3 南昌医学院公共学科教学部, 南昌 330004

通讯作者:

*王万春,男,教授 研究方向:毒蛇咬伤的临床与基础 Tel:(0791)86362691;
严张仁,男,教授 研究方向:中医外科学 Tel:(0791)86362691
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

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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