Article(id=1322912259781042475, tenantId=1146029695717560320, journalId=1255847919539208197, issueId=1322912162913596032, articleNumber=null, orderNo=null, doi=10.13193/j.issn.1673-7717.2025.01.021, 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=1793336461253, onlineDateStr=2026-10-30, pubDate=1736438400000, pubDateStr=2025-01-10, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1793336461253, onlineIssueDateStr=2026-10-30, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1793336461252, creator=13701087609, updateTime=1793336461252, updator=13701087609, issue=Issue{id=1322912162913596032, tenantId=1146029695717560320, journalId=1255847919539208197, year='2025', volume='43', issue='1', pageStart='1', pageEnd='258', issueExtLink='null', onlineDate='null', pubDate='1736438400000', pubDateStr='2025-01-10', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1793336438157, creator='13701087609', updateTime=1793340633627, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1322929760048906989, tenantId=1146029695717560320, journalId=1255847919539208197, issueId=1322912162913596032, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1322929760048906990, tenantId=1146029695717560320, journalId=1255847919539208197, issueId=1322912162913596032, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=117, endPage=121, ext={EN=ArticleExt(id=1322912261345517868, articleId=1322912259781042475, tenantId=1146029695717560320, journalId=1255847919539208197, language=EN, title=Effect of Sagittaria Sagittifolia Polysaccharide on Regulating TLR4/MyD88/NF-κB to Inhibit Inflammation and Oxidative Damage in L02 Hepatocyte Apoptosis Injury by Multiple Heavy Metals, columnId=1256183361521967297, journalTitle=Chinese Archives of Traditional Chinese Medicine, columnName=Target on National Project, runingTitle=null, highlight=null, articleAbstract=
Objective

To explore whether Sagittaria Sagittifolia polysaccharide(SSP)plays a protective role in L02 hepatocyte injured by six heavy metals mixture bymediating the Toll-like receptors 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear transcription factor-κB(NF-κB)signaling pathway.

Methods

Firstly,according to the dietary intake of six heavy metals in rice sold in Beijing,a heavy metal ixture was prepared for inducing L02 cell injury. MTT assay was used to detect the effect of SSP(0.25 mg/mL,0.5 mg/mL,1 mg/mL,2 mg/mL)on the L02 cells survival rate with 24 h pre-treatment. Annexinv FITC/propidium iodide(PI)double staining combined with flow cytometry and Hoechst 33342 staining were used for detecting the cell apoptosis. The content of reactive oxygen species(ROS)in cells was detected by using DCFH-DA fluorescence probe. The protein expressions of poly-ADP ribose polymerase1(PARP1),dynamic related protein 1(Drp1),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-17(IL-17),TLR4,MyD88 and NF-κB p65 were detected by Western blot.

Results

MTT assay showed that SSP(1 mg/mL,24 h)had the best protective effect. Compared with those of the model group,the ROS contentand apoptosis rate in SSP treatmentgroup were significantly decreased(P<0.05),and the protein expressions of Drp 1,TNF-α,IL-1β,IL-17,TLR4,MyD88 and NF-κB p65 decreased significantly(P<0.05)and the protein expression of PARP1 increased significantly(P<0.01).

Conclusion

SSP can effectively improve the apoptosis of L02 cells induced by six heavy metals mixture,whichmay be related to the regulation of TLR4/MyD88/NF-κB signaling pathway to inhibit the oxidative damage and inflammatory response.

, authors=Manyu ZHOU, Hongshuang LIU, Bingqing LIU, Xin GAO, Yao LIANG, Yan LIAO, authorsList=Manyu ZHOU, Hongshuang LIU, Bingqing LIU, Xin GAO, Yao LIANG, Yan LIAO, authorCompany=null, correspAuthors=Yan LIAO, 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=1322912266353516877, articleId=1322912259781042475, tenantId=1146029695717560320, journalId=1255847919539208197, language=CN, title=慈姑多糖调控TLR4/MyD88/NF-κB抑制细胞炎症和氧化损伤对多种重金属诱导L02肝细胞凋亡的影响, columnId=1256183362172142382, journalTitle=中华中医药学刊, columnName=国家项目点击, runingTitle=null, highlight=null, articleAbstract=
目的

探索慈姑多糖(Sagittaria Sagittifolia polysaccharide,SSP)是否通过介导Toll样受体4(Toll-like receptors 4,TLR4)/髓样分化因子88(myeloid differentiation factor 88,MyD88)/核转录因子-κB(nuclear transcription factor-kappa B,NF-κB)信号通路对6种重金属联合所致L02肝细胞损伤发挥保护作用。

方法

首先根据北京市售大米6种重金属膳食摄入比例,构建6种重金属混合染毒液所致L02细胞损伤体外模型。应用MTT法检测不同质量浓度慈姑多糖(0.25、0.5、1、2mg/mL)提前作用24 h对L02细胞存活率的影响;Annexin V-FITC/碘化丙啶(PI)双染流式结合Hoechst 33342荧光检测细胞凋亡;DCFH-DA荧光探针检测细胞中活性氧(reactive oxygen species,ROS)含量变化;Western blot法检测细胞中多聚ADP核糖聚合酶1(poly-ADP ribose polymerase1,PARP1)、动力相关蛋白1(dynamin-related protein 1,Drp1)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素(interleukin,IL)-1β、IL-17、TLR4、MyD88、NFκB p65蛋白表达情况。

结果

MTT结果显示,SSP在1 mg/mL作用24 h保护效果最佳;与模型组相比,SSP治疗组中ROS含量及细胞凋亡率均明显下降(P<0.05),Drp1、TNF-α、IL-1β、IL-17和TLR4、MyD88、NF-κB p65蛋白表达均显著降低(P<0.05),PARP1蛋白表达显著升高(P<0.01)。

结论

SSP可有效改善6种重金属联用诱导的L02细胞凋亡,可能与调控TLR4/MyD88/NF-κB信号通路抑制氧化损伤和炎症反应相关。

, authors=周漫钰, 刘红双, 刘冰清, 高鑫, 梁垚, 廖艳, authorsList=周漫钰, 刘红双, 刘冰清, 高鑫, 梁垚, 廖艳, authorCompany=null, correspAuthors=廖艳, authorNote=

周漫钰(1995-),女,广东揭阳人,博士在读,研究方向:中医养生康复及预防营养。

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廖艳(1975-),女,四川阆中人,教授,博士研究生导师,博士,研究方向:中医养生康复及营养。E-mail:。
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周漫钰(1995-),女,广东揭阳人,博士在读,研究方向:中医养生康复及预防营养。

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周漫钰(1995-),女,广东揭阳人,博士在读,研究方向:中医养生康复及预防营养。

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volume=231, issue=12, pageStart=2570, pageEnd=2581, url=null, language=null, rfNumber=[28], rfOrder=27, authorNames=YANG Y H, KARAKHANOVA S, HARTWIGW, journalName=JCell Physiol, refType=null, unstructuredReference=YANG Y H,KARAKHANOVA S,HARTWIGW,et al.Mitochondria and mitochondrial ROS in cancer:novel targets for anticancer therapy[J].JCell Physiol,2016,231(12):2570-2581., articleTitle=Mitochondria and mitochondrial ROS in cancer:novel targets for anticancer therapy, refAbstract=null), Reference(id=1322912560357441905, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, doi=null, pmid=null, pmcid=null, year=2020, volume=57, issue=10, pageStart=4090, pageEnd=4105, url=null, language=null, rfNumber=[29], rfOrder=28, authorNames=YANG SH, HUANG C Y, HSIEH C Y, journalName=Mol Neurobiol, refType=null, unstructuredReference=YANG SH,HUANG C Y,HSIEH C Y,et al.CDK4 and CDK5 inhibition have comparable mild hypothermia effects in preventing Drp1-dependentmitochondrial fission and neuron death induced by MPP+[J].Mol Neurobiol,2020,57(10):4090-4105., articleTitle=CDK4 and CDK5 inhibition have comparable mild hypothermia effects in preventing Drp1-dependentmitochondrial fission and neuron death induced by MPP+, refAbstract=null), Reference(id=1322912560470688114, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, doi=null, pmid=null, pmcid=null, year=2020, volume=352, issue=null, pageStart=159, pageEnd=187, url=null, language=null, rfNumber=[30], rfOrder=29, authorNames=MILLER D R, CRAMER S D, THORBURN A, journalName=Int Rev Cell Mol Biol, refType=null, unstructuredReference=MILLER D R,CRAMER S D,THORBURN A.The interplay of autophagy and non-apoptotic cell death pathways[J].Int Rev Cell Mol Biol,2020,352:159-187., articleTitle=The interplay of autophagy and non-apoptotic cell death pathways, refAbstract=null), Reference(id=1322912560546185587, tenantId=1146029695717560320, 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label=图1, caption=不同浓度重金属混合液对L02细胞存活率的影响(n=3,±s)

注:*与对照组比较,P<0.05;**与对照组比较,P<0.01。

, figureFileSmall=gMC4vsMe+aMlvQIvdDtB6A==, figureFileBig=z8NfcpPcXueHCg8wwmVQ0A==, tableContent=null), ArticleFig(id=1322912554833543494, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=EN, label=null, caption=null, figureFileSmall=PKnT7h9mkLrR4x7lr/P9rw==, figureFileBig=w2p76qLWwRev7wYgWKc3zw==, tableContent=null), ArticleFig(id=1322912554925818183, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=CN, label=图2, caption=不同浓度慈姑多糖对L02细胞存活率的影响(n=3,±s)

注:*与对照组比较,P<0.05。

, figureFileSmall=PKnT7h9mkLrR4x7lr/P9rw==, figureFileBig=w2p76qLWwRev7wYgWKc3zw==, tableContent=null), ArticleFig(id=1322912555013898568, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=EN, label=null, caption=null, figureFileSmall=kOM+T5xAfRafLf3vkABLOQ==, figureFileBig=BAUXv/a6gC8VKFsucy9FbQ==, tableContent=null), ArticleFig(id=1322912555081007433, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=CN, label=图3, caption=不同浓度慈姑多糖对重金属混合液诱导的L02细胞存活率的影响(n=3,±s), figureFileSmall=kOM+T5xAfRafLf3vkABLOQ==, figureFileBig=BAUXv/a6gC8VKFsucy9FbQ==, tableContent=null), ArticleFig(id=1322912555169087818, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=EN, label=null, caption=null, figureFileSmall=RvEVDnp0y7omIRckyqvuHA==, figureFileBig=GGOuSsojes3ab0o9rkEu8A==, tableContent=null), ArticleFig(id=1322912555244585291, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=CN, label=图4, caption=慈姑多糖对重金属混合液诱导的L02细胞凋亡的影响(n=3,±s)

注:**与对照组比较,P<0.01;ΔΔ与模型组比较,P<0.01。

, figureFileSmall=RvEVDnp0y7omIRckyqvuHA==, figureFileBig=GGOuSsojes3ab0o9rkEu8A==, tableContent=null), ArticleFig(id=1322912555328471372, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=EN, label=null, caption=null, figureFileSmall=1qqVSo/rEzw2gXUNYWkQZg==, figureFileBig=JKW7lKBEoEajQrgxnE/7aw==, tableContent=null), ArticleFig(id=1322912555429134669, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=CN, label=图5, caption=慈姑多糖对重金属混合液诱导的L02细胞ROS释放及Drp1蛋白表达的影响(n=3,±s)

注:**与对照组比较,P<0.01;*与对照组比较,P<0.05;Δ与模型组比较,P<0.05。

, figureFileSmall=1qqVSo/rEzw2gXUNYWkQZg==, figureFileBig=JKW7lKBEoEajQrgxnE/7aw==, tableContent=null), ArticleFig(id=1322912555504632142, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=EN, label=null, caption=null, figureFileSmall=HNuePHIiVsWHeeUek0OszQ==, figureFileBig=N+l13HDOYnYPk2NOpo87LQ==, tableContent=null), ArticleFig(id=1322912555605295439, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=CN, label=图6, caption=慈姑多糖对重金属混合液诱导的L02细胞TNF-α、IL-1β、IL-17蛋白表达的影响(n=3,±s)

注:**与对照组比较,P<0.01;ΔΔ与模型组比较,P<0.01。

, figureFileSmall=HNuePHIiVsWHeeUek0OszQ==, figureFileBig=N+l13HDOYnYPk2NOpo87LQ==, tableContent=null), ArticleFig(id=1322912555697570128, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=EN, label=null, caption=null, figureFileSmall=BSaLnGebIJ/3ukcc15FyKg==, figureFileBig=AZqRcrU3D6cr0PweX4ZJZA==, tableContent=null), ArticleFig(id=1322912555777261905, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=CN, label=图7, caption=慈姑多糖对重金属混合液诱导的L02细胞TLR4、MyD88、NF-κB p65蛋白表达的影响(n=3,±s)

注:**与对照组比较,P<0.01;Δ与模型组比较,P<0.05。

, figureFileSmall=BSaLnGebIJ/3ukcc15FyKg==, figureFileBig=AZqRcrU3D6cr0PweX4ZJZA==, tableContent=null), ArticleFig(id=1322912555861147986, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
重金属化合物大米中重金属含量/(mg·人-1·d-1)元素质量分数化合物质量/(mg)
K2Cr2O70.167 40.353 741 47823.946 10
CdCl2·2.5H2O0.001 50.491 227 9730.154 52
(CH3COO)2Pb·3H2O0.001 90.546 174 8060.176 03
CuSO4·5H2O0.194 40.255 999 94238.425 70
MnCl2·4H2O0.010 90.277 778 0921.985 61
ZnSO4·7H2O1.053 20.226 480 824235.312 00
合计300
), ArticleFig(id=1322912555953422675, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322912259781042475, language=CN, label=表1, caption=

重金属混合染毒液的配制

, figureFileSmall=null, figureFileBig=null, tableContent=
重金属化合物大米中重金属含量/(mg·人-1·d-1)元素质量分数化合物质量/(mg)
K2Cr2O70.167 40.353 741 47823.946 10
CdCl2·2.5H2O0.001 50.491 227 9730.154 52
(CH3COO)2Pb·3H2O0.001 90.546 174 8060.176 03
CuSO4·5H2O0.194 40.255 999 94238.425 70
MnCl2·4H2O0.010 90.277 778 0921.985 61
ZnSO4·7H2O1.053 20.226 480 824235.312 00
合计300
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慈姑多糖调控TLR4/MyD88/NF-κB抑制细胞炎症和氧化损伤对多种重金属诱导L02肝细胞凋亡的影响
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周漫钰 , 刘红双 , 刘冰清 , 高鑫 , 梁垚 , 廖艳
中华中医药学刊 | 国家项目点击 2025,43(1): 117-121
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中华中医药学刊 |国家项目点击 2025 , 43 (1) : 117 -121
慈姑多糖调控TLR4/MyD88/NF-κB抑制细胞炎症和氧化损伤对多种重金属诱导L02肝细胞凋亡的影响
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周漫钰, 刘红双, 刘冰清, 高鑫, 梁垚, 廖艳
作者信息
  • 北京中医药大学中医学院,北京 102446
通讯作者:
廖艳(1975-),女,四川阆中人,教授,博士研究生导师,博士,研究方向:中医养生康复及营养。E-mail:。
作者简介:

周漫钰(1995-),女,广东揭阳人,博士在读,研究方向:中医养生康复及预防营养。

Effect of Sagittaria Sagittifolia Polysaccharide on Regulating TLR4/MyD88/NF-κB to Inhibit Inflammation and Oxidative Damage in L02 Hepatocyte Apoptosis Injury by Multiple Heavy Metals
Manyu ZHOU, Hongshuang LIU, Bingqing LIU, Xin GAO, Yao LIANG, Yan LIAO
Affiliations
  • School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 102446, China
出版时间: 2025-01-10 doi: 10.13193/j.issn.1673-7717.2025.01.021
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目的

探索慈姑多糖(Sagittaria Sagittifolia polysaccharide,SSP)是否通过介导Toll样受体4(Toll-like receptors 4,TLR4)/髓样分化因子88(myeloid differentiation factor 88,MyD88)/核转录因子-κB(nuclear transcription factor-kappa B,NF-κB)信号通路对6种重金属联合所致L02肝细胞损伤发挥保护作用。

方法

首先根据北京市售大米6种重金属膳食摄入比例,构建6种重金属混合染毒液所致L02细胞损伤体外模型。应用MTT法检测不同质量浓度慈姑多糖(0.25、0.5、1、2mg/mL)提前作用24 h对L02细胞存活率的影响;Annexin V-FITC/碘化丙啶(PI)双染流式结合Hoechst 33342荧光检测细胞凋亡;DCFH-DA荧光探针检测细胞中活性氧(reactive oxygen species,ROS)含量变化;Western blot法检测细胞中多聚ADP核糖聚合酶1(poly-ADP ribose polymerase1,PARP1)、动力相关蛋白1(dynamin-related protein 1,Drp1)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素(interleukin,IL)-1β、IL-17、TLR4、MyD88、NFκB p65蛋白表达情况。

结果

MTT结果显示,SSP在1 mg/mL作用24 h保护效果最佳;与模型组相比,SSP治疗组中ROS含量及细胞凋亡率均明显下降(P<0.05),Drp1、TNF-α、IL-1β、IL-17和TLR4、MyD88、NF-κB p65蛋白表达均显著降低(P<0.05),PARP1蛋白表达显著升高(P<0.01)。

结论

SSP可有效改善6种重金属联用诱导的L02细胞凋亡,可能与调控TLR4/MyD88/NF-κB信号通路抑制氧化损伤和炎症反应相关。

慈姑多糖  /  多种重金属  /  TLR4/MyD88/NF-κB通路  /  氧化应激  /  炎症反应  /  L02细胞
Objective

To explore whether Sagittaria Sagittifolia polysaccharide(SSP)plays a protective role in L02 hepatocyte injured by six heavy metals mixture bymediating the Toll-like receptors 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear transcription factor-κB(NF-κB)signaling pathway.

Methods

Firstly,according to the dietary intake of six heavy metals in rice sold in Beijing,a heavy metal ixture was prepared for inducing L02 cell injury. MTT assay was used to detect the effect of SSP(0.25 mg/mL,0.5 mg/mL,1 mg/mL,2 mg/mL)on the L02 cells survival rate with 24 h pre-treatment. Annexinv FITC/propidium iodide(PI)double staining combined with flow cytometry and Hoechst 33342 staining were used for detecting the cell apoptosis. The content of reactive oxygen species(ROS)in cells was detected by using DCFH-DA fluorescence probe. The protein expressions of poly-ADP ribose polymerase1(PARP1),dynamic related protein 1(Drp1),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-17(IL-17),TLR4,MyD88 and NF-κB p65 were detected by Western blot.

Results

MTT assay showed that SSP(1 mg/mL,24 h)had the best protective effect. Compared with those of the model group,the ROS contentand apoptosis rate in SSP treatmentgroup were significantly decreased(P<0.05),and the protein expressions of Drp 1,TNF-α,IL-1β,IL-17,TLR4,MyD88 and NF-κB p65 decreased significantly(P<0.05)and the protein expression of PARP1 increased significantly(P<0.01).

Conclusion

SSP can effectively improve the apoptosis of L02 cells induced by six heavy metals mixture,whichmay be related to the regulation of TLR4/MyD88/NF-κB signaling pathway to inhibit the oxidative damage and inflammatory response.

Sagittaria Sagittifolia polysaccharides(SSP)  /  multiple heavy metals  /  TLR4/MyD88/NF-κB signaling pathway  /  oxidative stress  /  inflammation  /  L02 hepatocyte
周漫钰, 刘红双, 刘冰清, 高鑫, 梁垚, 廖艳. 慈姑多糖调控TLR4/MyD88/NF-κB抑制细胞炎症和氧化损伤对多种重金属诱导L02肝细胞凋亡的影响. 中华中医药学刊, 2025 , 43 (1) : 117 -121 . DOI: 10.13193/j.issn.1673-7717.2025.01.021
Manyu ZHOU, Hongshuang LIU, Bingqing LIU, Xin GAO, Yao LIANG, Yan LIAO. Effect of Sagittaria Sagittifolia Polysaccharide on Regulating TLR4/MyD88/NF-κB to Inhibit Inflammation and Oxidative Damage in L02 Hepatocyte Apoptosis Injury by Multiple Heavy Metals[J]. Chinese Archives of Traditional Chinese Medicine, 2025 , 43 (1) : 117 -121 . DOI: 10.13193/j.issn.1673-7717.2025.01.021
随着农业生产现代化和工业化的快速发展,土壤重金属污染问题日益凸显,重金属可通过土壤蓄积污染农作物,经膳食摄入进而危害人体健康[1]。在实际暴露中,多种重金属通常以一定比例同时进入人体,产生复杂的毒性作用[2]。目前单一重金属的毒性研究已较为全面[3-5],但多种重金属膳食摄入的研究并不多见。近年来的研究发现,重金属的损伤机制与氧化损伤和炎症反应密切相关。其中,Toll样受体4(Toll-like receptors 4,TLR4)作为一种模式识别受体,是多种组织损伤的感受器,髓样分化因子88(myeloid differentiation factor 88,MyD88)是TLR4的关键衔接蛋白,可激活下游的核转录因子-κB(nuclear transcription factor-kappa B,NF-κB)亚基向细胞核移位,激活诱导型一氧化氮合酶,同时也可促使炎症因子[如肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)]释放,加重氧化应激和炎症反应[6-8]。因此,TLR4/MyD88/NF-κB信号通路成为评价重金属损伤和药物筛选的重点考察路径之一[9]。
慈姑多糖(Sagittaria Sagittifolia polysaccharide,SSP)作为慈姑的主要活性成分,具有抗凋亡[10]、抗氧化和免疫调节[11]等作用,本课题组前期研究发现提前给予慈姑多糖可明显改善6种重金属所致损伤[12-14],可能与调节核转录因子E2相关因子2(nuclear factor erythroid-2-related factor 2,Nrf2)信号通路减轻氧化应激所致细胞凋亡相关,但作用机制研究尚不深入。故本研究模拟北京市售大米6种重金属的膳食摄入比例[15],构建多种重金属混合染毒L02肝细胞损伤模型,探讨慈姑多糖是否通过调节TLR4/MyD88/NF-κB信号通路,对多种重金属肝损伤发挥预防性保护作用,为SSP解毒保肝作用的深入研究提供依据。
人正常肝细胞系L02细胞,由北京中医药大学生命科学学院惠赠。
慈姑多糖,由云南产新鲜慈姑制备,制备工艺及方法同前期文献[16-17]。重铬酸钾(K2Cr2O7)、四水氯化锰(MnCl2·4H2O)、水合氯化镉(CdCl2·2.5H2O)、七水硫酸锌(ZnSO4·7H2O)、五水硫酸铜(CuSO4·5H2O),均购买于上海源叶生物科技有限公司(批号分别为P299156、s27140、s50703、s24313、s24260);三水乙酸铅[(CH3COO)2Pb·3H2O],购于上海阿拉丁生化科技股份有限公司(批号s48936);所有化学试剂均为分析纯。胰蛋白酶(货号25200056),购于Invitrogen公司;青霉素、链霉素(货号PSL01),购于Caisson公司;DMEM高糖培养基、胎牛血清、活性氧(reactive oxygen species,ROS)检测试剂盒、Annexin VFITC细胞凋亡检测试剂盒、Hoechst33342染色液,购于北京兰博利德生物技术有限公司(货号分别为01-100-1ACS-1、04-001-1ACS、S0033S、C1062S、C1025);MTT噻唑兰购于Amresco公司(货号0793-1);磷酸缓冲盐溶液(phosphate buffer saline,PBS),购于北京雷根生物技术有限公司(货号cc0004);TLR4、MyD88、NF-κB p65抗体购于Abcam公司(货号分别为ab13556、ab219413、ab32536);动力相关蛋白1(dynamin-related protein 1,Drp1)购于Abways公司(货号AY2001);IL-1β(11E5)、IL-17(G-4)抗体购于Santa cruz公司(货号为sc-374218、sc-52012);多聚ADP核糖聚合酶1(poly-ADP ribose polymerase1,PARP1)、TNF-α、β-actin抗体及山羊抗兔、山羊抗鼠二抗均购于proteintech公司(货号分别为13371-1-AP、17590-1-AP、60008-1-Ig、SA00001-2、SA00001-1)。
HERA·Cell 150i型CO2培养箱(赛默飞世尔科技有限公司);离心机(Eppendorf 5418R);酶标仪(赛默飞世尔科技有限公司);A008流式细胞仪(Beckman Coulter公司);A010共聚焦显微镜(日本奥林巴斯公司);电泳仪及电转仪、凝胶成像系统(美国Bio-Rad公司)。
参考北京市居民通过大米途径摄入铬、锰、镉、锌、铜、铅6种常见重金属的含量比例[15]配制重金属染毒液(见表1),DMEM培养液配制16 mg/mL慈姑多糖母液,放于4℃冰箱保存,不同药物浓度用DMEM培养液稀释,现配现用。
将对数生长期的L02细胞,以每孔5×103个的密度接种于96孔培养板上,待细胞生长至80%进行重金属混合染毒液毒性实验及慈姑多糖无毒浓度实验。慈姑多糖处理组终浓度分别为0.25、0.5、1、2、4 mg/mL,重金属处理组终浓度分别为25、50、100、200、400、800、1600 ng/mL,每组设置4个复孔,继续放置5% CO2、37℃的培养箱中培养24 h,采用MTT法用全自动酶标仪在波长570 nm处测定其吸光度(A),并计算细胞存活率。细胞存活率=(A实验组-A调零组)/(A对照组-A调零组)×100%。
取对数生长期L02细胞,接种于96孔板,每孔5×103个细胞,待细胞生长至80%进行实验。将细胞分为:对照组、模型组(重金属染毒液900 ng/mL)、慈姑多糖给药组(重金属染毒液900 ng/mL+慈姑多糖)。慈姑多糖给药组提前加入不同质量浓度慈姑多糖(0.25、0.5、1、2 mg/mL)培养24 h后,模型组与慈姑多糖给药组均加入重金属染毒液。每组4个复孔,继续培养24 h后,应用MTT法检测细胞存活率,应用全自动酶标仪在570 nm波长处测其吸光度,并计算出细胞活力。
取对数生长期L02细胞,以每孔5×104个细胞密度接种于6孔板中,待细胞长至80%进行实验。去除给药完成的细胞培养液,应用无血清细胞培养液洗涤细胞2次,加入2 mL稀释好的DCFH-DA。将6孔板置于37℃含5% CO2培养箱内孵育30 min后,应用无血清培养液洗涤孔内细胞3次,置于荧光显微镜下观察各组ROS含量变化,并在30 min内完成检测,拍照。应用Image J图像分析软件记录每张图片的平均荧光强度,实验重复3次。
将培养好的细胞吸弃培养液,应用PBS洗涤1次,加入1 mL Hoechst 33342工作液,置于37℃培养箱内孵育30 min后,吸弃工作液,用无血清的培养液洗涤3次,每次5 min,随后置于荧光显微镜下观察。此外,分组处理结束后收集细胞,计数后每组取0.5 mL单细胞悬液,加入5 μL Annexin V-FITC和10 μL碘化丙锭溶液(PI),混匀后室温避光反应15min,置于冰盒内,随即采用流式细胞仪检测细胞凋亡率。
取对照组、模型组、1 mg/mL慈姑多糖给药组处理结束后的L02细胞,离心收集细胞,用含有PMSF、蛋白酶和磷酸酶抑制剂的RIPA液充分裂解,提取细胞总蛋白,BCA法测定蛋白浓度,制备蛋白样品。以β-actin为内参,SDS-PAGE凝胶电泳蛋白,电转至PVDF膜后应用5%的脱脂牛奶封闭1 h,孵育一抗4℃过夜,分别为PARP1(1∶1000)、Drp1(1∶1000)、TNF-α(1∶1000)、IL-1β(1∶200)、IL-17(1∶200)、TLR4(1∶1000)、MyD88(1∶1000)、NF-κB p65(1∶1000)、β-actin(1∶5000)。第2天室温复温1 h,TBST洗膜3次,每次5 min。随后室温孵育二抗1 h(1∶5000),TBST洗涤3次,使用ECL试剂进行显色,通过Image J软件对条带灰度进行定量分析。
实验结果采用SPSS 25.0统计软件进行统计学分析,实验数据用±s表示,多组间比较采用单因素方差分析,组间两两比较采用LSD检验。以P<0.05为差异具有统计学意义。
见图1。不同浓度重金属混合液作用L02细胞24 h后,L02细胞存活率随着重金属混合液浓度升高而逐渐下降,计算得到LC50浓度为912.67 ng/mL,故后续取900 ng/m L作为L02细胞的重金属混合液造模浓度。
见图2。不同浓度慈姑多糖处理L02细胞24 h后,在0.25~2mg/mL浓度范围内L02细胞生存率在95%以上,提示此范围内慈姑多糖无明显细胞毒性;在4 mg/mL时细胞生存率降至90%以下,说明在此浓度产生细胞毒性,差异具有统计学意义(P<0.05)。故后续选择0.25~2 mg/mL浓度范围进行实验。
见图3。MTT实验结果显示,提前给予慈姑多糖24 h后,不同浓度慈姑多糖(0.25、0.5、1、2 mg/mL)均能提高重金属混合液损伤24 h后的细胞存活率,且1 mg/mL浓度的慈姑多糖保护作用最强。后续选择慈姑多糖1 mg/mL作用24 h开展研究。
见插页ⅩⅣ图4。与对照组相比,模型组中细胞早期凋亡率显著升高(P<0.01);与模型组相比,慈姑多糖给药组能显著降低细胞凋亡率(P<0.01)。相较于对照组,模型组中细胞的细胞核呈致密浓染,平均荧光强度高于对照组,差异具有显著统计学意义(P<0.01);相对于模型组,慈姑多糖给药组中凋亡细胞显著减少(P<0.01)。与对照组相比,模型组中PARP1蛋白表达显著降低(P<0.01);与模型组相比,慈姑多糖给药组PARP1蛋白表达显著升高(P<0.01)。
见插页ⅩⅤ图5。与对照组相比,模型组细胞中平均荧光强度明显升高,即ROS含量升高(P<0.01),且Drp1蛋白表达升高(P<0.05);相对于模型组,慈姑多糖给药组能显著降低细胞中ROS含量及Drp1蛋白表达水平(P<0.05)。
见图6。与对照组比较,模型组中TNF-α、IL-1β、IL-17蛋白表达升高,差异具有显著统计学意义(P<0.01);与模型组相比,慈姑多糖给药组中TNF-α、IL-1β、IL-17蛋白表达均降低(P<0.01)。
见图7。与对照组比较,模型组中TLR4、MyD88、NF-κB p65蛋白表达升高,差异具有显著统计学意义(P<0.01);与模型组比较,慈姑多糖给药组中TLR4、MyD88、NF-κB p65蛋白表达均降低,差异有统计学意义(P<0.05)。
重金属污染目前仍是一个全球性的挑战问题,肝脏作为重要的代谢器官,是重金属积累和毒性损伤的主要靶器官。镉、铜和铬等重金属可产生ROS并导致氧化应激,被认为是重金属诱导肝损伤的主要机制之一[18-19]。相较于肝癌细胞系(HepG2),人正常肝细胞L02与人体内组织的生理生化条件更加吻合[20]。故本研究利用L02肝细胞损伤模型进行深入研究。
天然提取物如枸杞多糖[21]、葛根素[22]等可通过抗氧化、抗炎、调节自噬等途径拮抗单一重金属损伤,成为重金属解毒的研究热点。慈姑多糖作为慈姑的主要活性成分,具有显著抗脂质过氧化[23]、抗凋亡[10]、抗氧化和免疫调节[11]等作用,且前期实验已证实其可通过调节Nrf2信号通路减轻氧化应激和细胞凋亡、改善6种重金属所致小鼠和HepG2肝细胞损伤[10-12],但慈姑多糖对细胞的保护作用是否还涉及炎症反应,炎症反应、氧化应激和细胞凋亡之间是否存在着信号串扰,相互串扰的信号通路又可能是什么尚不清楚。
细胞凋亡是一种严格控制的程序性细胞死亡过程,对生物体维持内环境稳态及系统的生长发育有重要作用,但异常凋亡是一种病理过程。Caspase家族是细胞凋亡的重要执行者,其中Caspase-3的活化可裂解PARP,阻止PARP介导的DNA损伤修复,破坏细胞稳态,导致细胞凋亡[24]。研究表明,在重金属诱导的肝损伤中,细胞凋亡是造成肝毒性损伤的重要机制,氧化应激、线粒体损伤、炎症反应、内质网应激等均对细胞凋亡的发生发展具有调节作用[25-26]。在本研究中,重金属混合物增加了L02细胞凋亡率并降低PARP1蛋白表达,而慈姑多糖干预能显著降低细胞凋亡率并上调PARP1,提示6种重金属混合物可能通过诱导细胞异常凋亡产生肝细胞毒性,而慈姑多糖可通过改善异常凋亡发挥肝保护作用。
为了探究慈姑多糖改善重金属混合物诱导肝细胞凋亡的相关分子机制,本研究进一步测定了ROS含量、Drp1蛋白表达。在生理条件下,ROS参与能量转换、氧平衡等生理环节,是一种重要的信号分子,其生成和清除的动态平衡对维持细胞氧化还原稳态尤为关键。但当机体受到重金属等外源性刺激时,氧化与抗氧化平衡失调,细胞中产生过量的ROS,导致机体发生氧化损伤,进而可导致线粒体功能损伤、炎症反应、DNA损伤、细胞凋亡及恶性转变等[27]。而线粒体作为产生ROS的主要场所,在ROS异常升高时成为ROS的直接损伤靶点。氧化损伤的发生会导致线粒体形态及正常生理功能遭到破坏,引起线粒体功能障碍[28],表现为线粒体嵴的肿胀和消失、线粒体裂变异常增加、氧化磷酸化过程和能量产生受损、线粒体膜电位下降、代谢途径改变等。线粒体动力相关蛋白Drp1是介导线粒体分裂的主要蛋白,具有促进线粒体裂变及ROS蓄积的作用,其过度表达可使线粒体出现碎片化[29]。线粒体受氧化应激损伤后,线粒体外膜通透性改变,线粒体内的细胞色素C被释放到细胞质中,可同凋亡蛋白酶激活因子相结合,构成细胞色素C/凋亡蛋白酶激活因子凋亡体,导致细胞的凋亡。本研究结果发现,模型组中ROS含量及Drp1蛋白表达显著升高;经慈姑多糖提前干预后,细胞中ROS的产生及Drp1蛋白表达显著减少,提示慈姑多糖可能通过减少ROS产生、缓解线粒体功能损伤,进而减轻细胞凋亡来发挥保护作用。
前期研究发现,重金属混合物可导致小鼠肝脏组织炎性细胞浸润,提示多种重金属可诱导炎症反应[12]。TNF-α作为炎症反应中心,不仅可刺激中性粒细胞、单核细胞等进一步释放IL-6、IL-1β、IL-17等炎症因子,导致炎症反应的级联放大和过度激活,从而导致肝脏组织细胞的炎性坏死,还可以通过Caspase-8的大量活化来激活凋亡通路[30]。TNF-α的生物效应主要由TNFR1和TNFR2两种受体介导,TNF-α结合受体TNFR1后,与肿瘤坏死因子受体相关死亡结构域形成三联复合物,募集RIP1和TRAF2,进而结合FADD、RAIDD等衔接蛋白,募集并激活Caspase-8,继而激活外源性凋亡途径,是导致肝细胞凋亡的主要机制。本研究结果发现,模型组TNFα、IL-1β、IL-17蛋白表达显著升高,经慈姑多糖提前干预后,上述蛋白表达显著下降,提示慈姑多糖通过减轻炎症反应诱导的细胞凋亡,发挥细胞保护作用。
为了进一步探究慈姑多糖改善氧化应激和炎症反应的调控机制,检测了L02细胞中TLR4、MyD88、NF-κB p65蛋白的表达量。Toll样受体家族是重要的炎症识别受体,该类跨膜蛋白能识别受体及复合物的胞外区域,从而激发下游分子通路[31]。TLR4作为机体中参与免疫调节的重要蛋白,是最早被发现的I型跨膜蛋白受体,其胞内区的TIR结构域可与接头蛋白MyD88死亡结构域和中间域相结合,激活下游的NF-κB易转位为核磷酸化,调节TNF-α、IL-6等免疫炎症因子表达。此外,上调TLR4/MyD88/NF-κB还可激活一氧化氮合酶,造成大量NO、MDA、ROS生成,引起严重的氧化应激,导致组织氧化损伤[7]。研究表明,下调TLR4/MyD88/NF-κB信号通路可减轻金属Cd诱导的肝损伤[9]。本研究结果发现,经慈姑多糖干预后,TLR4、MyD88、NF-κB p65蛋白表达均较模型组显著下降,提示慈姑多糖可能通过下调TLR4/MyD88/NFκB信号通路来发挥保护作用。
综上,慈姑多糖可能通过下调TLR4/MyD88/NF-κB信号通路关键蛋白表达来减少ROS、Drp1、TNF-α、IL-1β和IL-17的生成,进而减轻氧化应激损伤和炎症反应导致的细胞凋亡,以此拮抗6种重金属的联合毒性,发挥L02肝细胞保护作用。本研究丰富了慈姑多糖发挥解毒保肝作用的实验依据,也提示慈姑多糖可能成为潜在的解毒保肝健康产品的重点研发对象,值得进一步深入研究。
  • 国家重点研发计划项目(2018YFC1706800)
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2025年第43卷第1期
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doi: 10.13193/j.issn.1673-7717.2025.01.021
  • 首发时间:2026-10-30
  • 出版时间:2025-01-10
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国家重点研发计划项目(2018YFC1706800)
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    北京中医药大学中医学院,北京 102446

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廖艳(1975-),女,四川阆中人,教授,博士研究生导师,博士,研究方向:中医养生康复及营养。E-mail:。
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2种不同金属材料的力学参数
科
Family
属数
Number of
genus
种数
Number of
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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