Article(id=1241675636712985215, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241675628051755031, articleNumber=null, orderNo=null, doi=10.20043/j.cnki.MPM.202407106, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1720195200000, receivedDateStr=2024-07-06, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773968141009, onlineDateStr=2026-03-20, pubDate=1732464000000, pubDateStr=2024-11-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773968141009, onlineIssueDateStr=2026-03-20, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773968141009, creator=13701087609, updateTime=1773968141009, updator=13701087609, issue=Issue{id=1241675628051755031, tenantId=1146029695717560320, journalId=1227665162245664772, year='2024', volume='51', issue='22', pageStart='4033', pageEnd='4224', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773968138945, creator=13701087609, updateTime=1773968595676, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241677543783322543, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241675628051755031, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241677543783322544, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241675628051755031, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=4191, endPage=4198, ext={EN=ArticleExt(id=1241675637119832734, articleId=1241675636712985215, tenantId=1146029695717560320, journalId=1227665162245664772, language=EN, title=Study on the anti-inflammatory effect of thermal spring water based on a model of Propionibacterium acnes-induced cellular inflammation, columnId=1228016572065837304, journalTitle=Modern Preventive Medicine, columnName=Experimental Technology and Applications, runingTitle=null, highlight=null, articleAbstract=
Objective

To construct a Propionibacterium acnes (P. acnes)-induced cellular inflammation model and to study the anti-inflammatory effect of a thermal water.

Methods

Utilize human immortalized keratinocytes (HaCaT) was used in the experimentation. Co-incubate the cells with thermal spring water and employ the CCK-8 assay to assess cell viability, screening for the optimal intervention time for the thermal spring water. Expose the cells to varying concentrations of P. acnes for different times, measure cell viability, calculate the 50% inhibitory concentration (IC50) of P. acnes, and determine the model induction conditions by testing IL-6 levels. The cells were soaked in thermal spring water for 0.5 hours, followed by co-culturing with P. acnes as the preventive intervention group, or co-culturing with P. acnes first and then soaking the cells in hot spring water for 0.5 hours as the therapeutic intervention group. The levels of cell viability, pro-inflammatory cytokines, and gene expression of inflammation pathways were assessed using the CCK-8 assay, ELISA, and RT-PCR, respectively. Concurrently, control groups with saline solution, tap water, and pure water, along with model and blank controlswere set up.

Results

After treating the cells with thermal spring water and physiological saline for 0.5 hours, cell viabilities were 92.7% and 99.09%, respectively, which led to the selection of 0.5 hours as the intervention time for subsequent experiments.P. acnes at an OD600 of 0.25 was co-incubated with the cells for 5 hours to establish the model. In the preventive intervention, the secretion levels of IL-1β and IL-8, as well as the relative expression levels of IL-6, TNF-α, IL-8, and the genes TLR2 and NF-κB in the cells of the thermal spring water group, were all lower than those in the model group (P<0.05), and significantly lower than those in the saline solution group. In the therapeutic intervention, the contents of IL-6, IL-1β, TNF-α, and IL-8, as well as the expression levels of the genes IL-6, IL-1β, TNF-α, TLR2, and NF-κB in the thermal spring water group, were all lower than those in the model group. The contents of IL-6, IL-1β, TNF-α, and the expression levels of the genes IL-6, IL-1β, IL-8, TLR2, and NF-κB were also significantly reduced compared to those in the saline solution group (P<0.05).

Conclusion

This thermal water may exert its anti-inflammatory effect by down-regulating the expression of TLR2 and NF-κB genes in inflammatory signalling pathway and by reducing the secretion of pro-inflammatory cytokines.

, correspAuthors=null, 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=Yue ZOU, Ying-ying ZHANG, Fen YANG, Hong-xia LI, Bao-chao ZHANG, Yi-hang XU, Rui-xue WANG, Xiao-fang PEI), CN=ArticleExt(id=1241675640563356499, articleId=1241675636712985215, tenantId=1146029695717560320, journalId=1227665162245664772, language=CN, title=基于痤疮丙酸杆菌诱导的细胞炎症模型研究温泉水的抗炎作用, columnId=1228016572288135432, journalTitle=现代预防医学, columnName=实验技术及其应用, runingTitle=null, highlight=null, articleAbstract=
目的

构建痤疮丙酸杆菌(Propionibacterium acnesP.acnes)诱导的细胞炎症模型研究某温泉水的抗炎作用。

方法

使用人永生化角质形成细胞(human immortalized keratinocytes,HaCaT)进行试验。将温泉水与细胞共孵育,用CCK-8法检测细胞存活率,筛选温泉水干预时间;将不同浓度P.acnes与细胞作用不同时间,检测细胞存活率和计算P.acnes的50%抑制浓度(IC50),并检测IL-6含量,确定造模条件;温泉水浸泡细胞0.5 h后,再加入P.acnes共培养作为预防干预组,或先将P.acnes与细胞共培养,再加入温泉水浸泡细胞0.5 h作为治疗干预组,以CCK-8法、ELISA和RT-PCR分别检测细胞存活率、促炎细胞因子及其基因以及炎症通路相关基因表达的水平。同时设置生理盐水、自来水、纯水组以及模型和空白对照。

结果

温泉水和生理盐水作用0.5 h时,细胞存活率分别为92.7%和99.09%,将0.5 h作为后续实验的干预时间;OD600值0.25的P.acnes与细胞共孵育5 h作为建模条件。预防式干预,仅温泉水组细胞的IL-1βIL-8分泌量、IL-6、TNF-αIL-8及TLR2、NF-κB基因的相对表达量均低于模型组(P<0.05),且明显低于生理盐水组。治疗式干预,温泉水组细胞的IL-6、IL-1βTNF-αIL-8含量以及IL-6、IL-1βTNF-αTLR2和NF-κB基因表达水平均低于模型组,其中IL-6、IL-1βTNF-α含量以及IL-6、IL-1βIL-8、TLR2、NF-κB基因表达水平也比生理盐水组的明显降低(P<0.05)。

结论

该温泉水可能通过下调TLR2NF-κB炎症信号通路基因,降低促炎细胞因子分泌实现其抗炎作用。

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裴晓方,E-mail:
, copyrightStatement=本刊刊出的所有文章不代表中华预防医学会和本刊编委会的观点,除非特别声明。, copyrightOwner=中华预防医学会和四川大学华西公共卫生学院, extLink=null, articleAbsUrl=null, sourceXml=omDMdlICPPNwUTVbEu9DGw==, magXml=IPSoTTSXVhgDMLEmxn4lsg==, pdfUrl=null, pdf=UuyaTPP/iycLmt0lYsX/6Q==, pdfFileSize=1501787, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=t47jGea3nHwDvGqKqGh8og==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=XfjjtytkmlTi422CxACLyw==, mapNumber=null, authorCompany=null, fund=null, authors=

邹粤(1999—),女,硕士在读,研究方向:微生物,公众健康与检验

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Journal of Inflammation Research, 2019, 12: 181-194., articleTitle=Effect of thermal spring water on human dendritic cell inflammatory response, refAbstract=null), Reference(id=1241826553957520362, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, doi=null, pmid=null, pmcid=null, year=2014, volume=28, issue=4, pageStart=364, pageEnd=371, url=null, language=null, rfNumber=[22], rfOrder=24, authorNames=Prasad AS, journalName=Journal of Trace Elements in Medicine and Biology: Organ of the Society for Minerals and Trace Elements, refType=null, unstructuredReference=Prasad AS. Zinc: an antioxidant and anti-inflammatory agent: role of Zinc in degenerative disorders of aging[J]. Journal of Trace Elements in Medicine and Biology: Organ of the Society for Minerals and Trace Elements, 2014, 28(4): 364-371., articleTitle=Zinc: an antioxidant and anti-inflammatory agent: role of Zinc in degenerative disorders of aging, refAbstract=null), Reference(id=1241826554049795050, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, doi=null, pmid=null, pmcid=null, year=2006, volume=18, issue=1, pageStart=17, pageEnd=25, url=null, language=null, rfNumber=[23], rfOrder=25, authorNames=Chiarini A, Dal Pra I, Pacchiana R, journalName=International Journal of Molecular Medicine, refType=null, unstructuredReference=Chiarini A, Dal Pra I, Pacchiana R, et al. Comano’s (Trentino) thermal water interferes with the expression and secretion of vascular endothelial growth factor-A protein isoforms by cultured human psoriatic keratinocytes: a potential mechanism of its anti-psoriatic action[J]. International Journal of Molecular Medicine, 2006, 18(1): 17-25., articleTitle=Comano’s (Trentino) thermal water interferes with the expression and secretion of vascular endothelial growth factor-A protein isoforms by cultured human psoriatic keratinocytes: a potential mechanism of its anti-psoriatic action, refAbstract=null)], funds=[Fund(id=1241826551373829047, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, awardId=2021ZYSF006, language=CN, fundingSource=宜宾市科技计划资助项目(2021ZYSF006), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1241826540527358579, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, xref=1., ext=[AuthorCompanyExt(id=1241826540535747186, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, companyId=1241826540527358579, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=West China School of Public Health/ West China Fourth Hospital, Sichuan University,Chengdu, Sichuan 610041, China), AuthorCompanyExt(id=1241826540544135795, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, companyId=1241826540527358579, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.四川大学华西公共卫生学院(华西第四医院),四川 成都 610041)]), AuthorCompany(id=1241826540653187706, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, xref=2., ext=[AuthorCompanyExt(id=1241826540665770620, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, companyId=1241826540653187706, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.四川大学华西口腔医学院)]), AuthorCompany(id=1241826540766433925, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, xref=3., ext=[AuthorCompanyExt(id=1241826540774822534, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, companyId=1241826540766433925, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3.重庆市大渡口区疾病预防控制中心)])], figs=[ArticleFig(id=1241826548261655423, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=EN, label=Fig.1, caption=Effect of different types of water on the viability of HaCaT cells, figureFileSmall=GH9f+Xs/byqnyIxc7+wBVg==, figureFileBig=1hULQvWbgm+92u1C4kYNoQ==, tableContent=null), ArticleFig(id=1241826548349735811, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=CN, label=图1, caption=不同种类水对HaCaT细胞存活率的影响, figureFileSmall=GH9f+Xs/byqnyIxc7+wBVg==, figureFileBig=1hULQvWbgm+92u1C4kYNoQ==, tableContent=null), ArticleFig(id=1241826548542673806, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=EN, label=Fig.2, caption=Modeling of P.acnes-induced HaCaT cell inflammation, figureFileSmall=kHVqyiut6pWEKGSGE85HJA==, figureFileBig=rvZLnq1GEOSJn5ayIGBFpA==, tableContent=null), ArticleFig(id=1241826548672697234, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=CN, label=图2, caption=P.acnes诱导HaCaT细胞炎症模型建立

注:图A,不同浓度P.acnes作用不同时间后HaCaT细胞存活率;图B,不同浓度P.acnes作用5 h后HaCaT细胞存活率;图C,不同浓度P.acnes作用HaCaT细胞后IL-6的分泌量;与对照组相比,*P<0.05,****P<0.000 1,ns为差异无统计学意义。

, figureFileSmall=kHVqyiut6pWEKGSGE85HJA==, figureFileBig=rvZLnq1GEOSJn5ayIGBFpA==, tableContent=null), ArticleFig(id=1241826548794332054, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=EN, label=Fig.3, caption=Effect of "prophylactic" interventions on cell survival in models of inflammation, figureFileSmall=1ms+qoLMlEPR/d2VLeNLFg==, figureFileBig=3lmH+eXqCvw4Hw2aOFyliw==, tableContent=null), ArticleFig(id=1241826548899189656, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=CN, label=图3, caption=“预防式”干预对炎症模型细胞存活率的影响

注:*P<0.05,**P<0.01,***P<0.001,ns为差异无统计学意义。

, figureFileSmall=1ms+qoLMlEPR/d2VLeNLFg==, figureFileBig=3lmH+eXqCvw4Hw2aOFyliw==, tableContent=null), ArticleFig(id=1241826549020824473, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=EN, label=Fig.4, caption=Effects of "preventive" interventions on pro-inflammatory cytokine levels and related gene expression, figureFileSmall=/8LwPUCMKLGsWBnCYz/iLg==, figureFileBig=KW0ndQwtl9bogheFcIi20w==, tableContent=null), ArticleFig(id=1241826549146653598, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=CN, label=图4, caption=“预防式”干预对促炎细胞因子水平以及相关基因表达的影响

注:图A,IL-6分泌量;图B,IL-1β分泌量;图C,TNF-α分泌量;图D,IL-8分泌量;图E,IL-6基因相对表达量;图F,IL-1β基因相对表达量;图G,TNF-α基因相对表达量;图H,IL-8基因相对表达量;图I,NF-κB基因相对表达量;图J,TLR-2基因相对表达量。*P<0.05,**P<0.01,***P<0.001,ns为差异无统计学意义。

, figureFileSmall=/8LwPUCMKLGsWBnCYz/iLg==, figureFileBig=KW0ndQwtl9bogheFcIi20w==, tableContent=null), ArticleFig(id=1241826549251511200, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=EN, label=Fig.5, caption=Effect of “therapeutic” interventions on cell survival in models of inflammation, figureFileSmall=2Wsw0DH+gTOpvAly2SWkxg==, figureFileBig=E2lbJJmkLArZ5V6WzOlzzA==, tableContent=null), ArticleFig(id=1241826549360563110, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=CN, label=图5, caption=“治疗式”干预对炎症模型细胞存活率的影响, figureFileSmall=2Wsw0DH+gTOpvAly2SWkxg==, figureFileBig=E2lbJJmkLArZ5V6WzOlzzA==, tableContent=null), ArticleFig(id=1241826549519946668, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=EN, label=Fig.6, caption=Effects of “therapeutic” interventions on pro-inflammatory cytokine levels and related gene expression, figureFileSmall=wRhkcuv5JSy62Q5PMjqeUw==, figureFileBig=8/a/zF6K6GTjEMkzKZ9jmQ==, tableContent=null), ArticleFig(id=1241826549620609966, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=CN, label=图6, caption=“治疗式”干预对促炎细胞因子水平以及相关基因表达的影响

图A,IL-6分泌量;图B,IL-1β分泌量;图C,TNF-α分泌量;图D,IL-8分泌量;图E,IL-6基因相对表达量;图F,IL-1β基因相对表达量;图G,TNF-α基因相对表达量;图H,IL-8基因相对表达量;图I,NF-κB基因相对表达量;图J,TLR-2基因相对表达量。

, figureFileSmall=wRhkcuv5JSy62Q5PMjqeUw==, figureFileBig=8/a/zF6K6GTjEMkzKZ9jmQ==, tableContent=null), ArticleFig(id=1241826549712884657, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=EN, label=Table 1, caption=

Amplification Primer Sequence List

, figureFileSmall=null, figureFileBig=null, tableContent=
基因序列(5′→3′)
GAPDHForward: CAT GGG TGT GAA CCA TGA GAA GTA T
Reverse: GAC TGT GGT CAT GAG TCC TTC CA
IL-6Forward: CAA TCT GGA TTC AAT GAG GAG AC
Reverse: CTC TGG CTT GTT CCT CAC TAC TC
IL-8Forward: CCA CAC TGC GCC AAC A
Reverse: GCA TCT TCA CTG ATT CTT GGA T
IL-1βForward: CCA TCA GCC AGG ACA GTC AG
Reverse: CCA AAT GTG GCC GTG GTT TC
TNF-αForward: CAA TGG CGT GGA GCT GAG AGA TAA C
Reverse: GAC GGC GAT GCG GCT GAT G
NF-κBForward: ATG GTG GTC GGC TTC GCA AAC
Reverse: CGC CTC TGT CAT TCG TGC TTC C
TLR2Forward: CTA CCA GAT GCC TCC CTC TTA CCC
Reverse: ACC AGC TTC CAA AGT CTT CAG TGT G
), ArticleFig(id=1241826551210251187, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241675636712985215, language=CN, label=表1, caption=

扩增引物序列表

, figureFileSmall=null, figureFileBig=null, tableContent=
基因序列(5′→3′)
GAPDHForward: CAT GGG TGT GAA CCA TGA GAA GTA T
Reverse: GAC TGT GGT CAT GAG TCC TTC CA
IL-6Forward: CAA TCT GGA TTC AAT GAG GAG AC
Reverse: CTC TGG CTT GTT CCT CAC TAC TC
IL-8Forward: CCA CAC TGC GCC AAC A
Reverse: GCA TCT TCA CTG ATT CTT GGA T
IL-1βForward: CCA TCA GCC AGG ACA GTC AG
Reverse: CCA AAT GTG GCC GTG GTT TC
TNF-αForward: CAA TGG CGT GGA GCT GAG AGA TAA C
Reverse: GAC GGC GAT GCG GCT GAT G
NF-κBForward: ATG GTG GTC GGC TTC GCA AAC
Reverse: CGC CTC TGT CAT TCG TGC TTC C
TLR2Forward: CTA CCA GAT GCC TCC CTC TTA CCC
Reverse: ACC AGC TTC CAA AGT CTT CAG TGT G
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基于痤疮丙酸杆菌诱导的细胞炎症模型研究温泉水的抗炎作用
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邹粤 1 , 张莹莹 2 , 杨芬 1 , 李红霞 3 , 张保超 1 , 徐义航 1 , 王瑞雪 1 , 裴晓方 1
现代预防医学 | 实验技术及其应用 2024,51(22): 4191-4198
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现代预防医学 | 实验技术及其应用 2024, 51(22): 4191-4198
基于痤疮丙酸杆菌诱导的细胞炎症模型研究温泉水的抗炎作用
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邹粤1, 张莹莹2, 杨芬1, 李红霞3, 张保超1, 徐义航1, 王瑞雪1, 裴晓方1
作者信息
  • 1.四川大学华西公共卫生学院(华西第四医院),四川 成都 610041
  • 2.四川大学华西口腔医学院
  • 3.重庆市大渡口区疾病预防控制中心
  • 邹粤(1999—),女,硕士在读,研究方向:微生物,公众健康与检验

通讯作者:

裴晓方,E-mail:
Study on the anti-inflammatory effect of thermal spring water based on a model of Propionibacterium acnes-induced cellular inflammation
Yue ZOU1, Ying-ying ZHANG2, Fen YANG1, Hong-xia LI3, Bao-chao ZHANG1, Yi-hang XU1, Rui-xue WANG1, Xiao-fang PEI1
Affiliations
  • West China School of Public Health/ West China Fourth Hospital, Sichuan University,Chengdu, Sichuan 610041, China
出版时间: 2024-11-25 doi: 10.20043/j.cnki.MPM.202407106
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目的

构建痤疮丙酸杆菌(Propionibacterium acnesP.acnes)诱导的细胞炎症模型研究某温泉水的抗炎作用。

方法

使用人永生化角质形成细胞(human immortalized keratinocytes,HaCaT)进行试验。将温泉水与细胞共孵育,用CCK-8法检测细胞存活率,筛选温泉水干预时间;将不同浓度P.acnes与细胞作用不同时间,检测细胞存活率和计算P.acnes的50%抑制浓度(IC50),并检测IL-6含量,确定造模条件;温泉水浸泡细胞0.5 h后,再加入P.acnes共培养作为预防干预组,或先将P.acnes与细胞共培养,再加入温泉水浸泡细胞0.5 h作为治疗干预组,以CCK-8法、ELISA和RT-PCR分别检测细胞存活率、促炎细胞因子及其基因以及炎症通路相关基因表达的水平。同时设置生理盐水、自来水、纯水组以及模型和空白对照。

结果

温泉水和生理盐水作用0.5 h时,细胞存活率分别为92.7%和99.09%,将0.5 h作为后续实验的干预时间;OD600值0.25的P.acnes与细胞共孵育5 h作为建模条件。预防式干预,仅温泉水组细胞的IL-1βIL-8分泌量、IL-6、TNF-αIL-8及TLR2、NF-κB基因的相对表达量均低于模型组(P<0.05),且明显低于生理盐水组。治疗式干预,温泉水组细胞的IL-6、IL-1βTNF-αIL-8含量以及IL-6、IL-1βTNF-αTLR2和NF-κB基因表达水平均低于模型组,其中IL-6、IL-1βTNF-α含量以及IL-6、IL-1βIL-8、TLR2、NF-κB基因表达水平也比生理盐水组的明显降低(P<0.05)。

结论

该温泉水可能通过下调TLR2NF-κB炎症信号通路基因,降低促炎细胞因子分泌实现其抗炎作用。

温泉水  /  痤疮丙酸杆菌  /  HaCaT  /  抗炎作用
Objective

To construct a Propionibacterium acnes (P. acnes)-induced cellular inflammation model and to study the anti-inflammatory effect of a thermal water.

Methods

Utilize human immortalized keratinocytes (HaCaT) was used in the experimentation. Co-incubate the cells with thermal spring water and employ the CCK-8 assay to assess cell viability, screening for the optimal intervention time for the thermal spring water. Expose the cells to varying concentrations of P. acnes for different times, measure cell viability, calculate the 50% inhibitory concentration (IC50) of P. acnes, and determine the model induction conditions by testing IL-6 levels. The cells were soaked in thermal spring water for 0.5 hours, followed by co-culturing with P. acnes as the preventive intervention group, or co-culturing with P. acnes first and then soaking the cells in hot spring water for 0.5 hours as the therapeutic intervention group. The levels of cell viability, pro-inflammatory cytokines, and gene expression of inflammation pathways were assessed using the CCK-8 assay, ELISA, and RT-PCR, respectively. Concurrently, control groups with saline solution, tap water, and pure water, along with model and blank controlswere set up.

Results

After treating the cells with thermal spring water and physiological saline for 0.5 hours, cell viabilities were 92.7% and 99.09%, respectively, which led to the selection of 0.5 hours as the intervention time for subsequent experiments.P. acnes at an OD600 of 0.25 was co-incubated with the cells for 5 hours to establish the model. In the preventive intervention, the secretion levels of IL-1β and IL-8, as well as the relative expression levels of IL-6, TNF-α, IL-8, and the genes TLR2 and NF-κB in the cells of the thermal spring water group, were all lower than those in the model group (P<0.05), and significantly lower than those in the saline solution group. In the therapeutic intervention, the contents of IL-6, IL-1β, TNF-α, and IL-8, as well as the expression levels of the genes IL-6, IL-1β, TNF-α, TLR2, and NF-κB in the thermal spring water group, were all lower than those in the model group. The contents of IL-6, IL-1β, TNF-α, and the expression levels of the genes IL-6, IL-1β, IL-8, TLR2, and NF-κB were also significantly reduced compared to those in the saline solution group (P<0.05).

Conclusion

This thermal water may exert its anti-inflammatory effect by down-regulating the expression of TLR2 and NF-κB genes in inflammatory signalling pathway and by reducing the secretion of pro-inflammatory cytokines.

Thermal spring water  /  Propionibacterium acnes  /  HaCaT  /  Anti-inflammatory effect
邹粤, 张莹莹, 杨芬, 李红霞, 张保超, 徐义航, 王瑞雪, 裴晓方. 基于痤疮丙酸杆菌诱导的细胞炎症模型研究温泉水的抗炎作用. 现代预防医学, 2024 , 51 (22) : 4191 -4198 . DOI: 10.20043/j.cnki.MPM.202407106
Yue ZOU, Ying-ying ZHANG, Fen YANG, Hong-xia LI, Bao-chao ZHANG, Yi-hang XU, Rui-xue WANG, Xiao-fang PEI. Study on the anti-inflammatory effect of thermal spring water based on a model of Propionibacterium acnes-induced cellular inflammation[J]. Modern Preventive Medicine, 2024 , 51 (22) : 4191 -4198 . DOI: 10.20043/j.cnki.MPM.202407106
温泉水是一种富含矿物质和微量元素的天然矿物水,泡温泉被认为具有促进血液循环和加速新陈代谢,帮助缓解肌肉紧张和关节疼痛,提高睡眠质量、改善皮肤状况,以及对某些疾病具有预防、辅助治疗及康复作用等健康效应。据此,有些学者进行研究,提供了泡温泉在缓解和辅助治疗呼吸道疾病、风湿病、肌肉骨骼疾病、皮肤病等方面的证据[1],如Beylot-Barry等采用随机对照试验,评估温泉疗法对银屑病患者的影响,发现温泉疗法组患者的生活质量指数、疼痛和瘙痒症状均明显改善[2]
值得注意的是,上述疾病多与炎症密切相关,因此,有学者采用细胞模型对温泉水的抗炎作用进行了初探。如Zöller等[3]采用含72%温泉水的溶液配制细胞培养液,培养HaCaT细胞,发现其能显著抑制1% Triton X-100溶液引起的细胞损伤等。Karagülle等人[4]将高糖Dulbecco ’s Modified Eagle液体培养液分别用温泉水稀释(温泉水浓度分别为10%、25%及50%)后培养HaCaT细胞,发现在加入10%温泉水的培养组中,TNF-αIL-1αVEGF的基因表达降低,提示温泉水具有抗炎潜力,并且可能与该温泉水富含二氧化硅和硫化氢相关。
尽管上述研究基于细胞实验证明了温泉水具有潜在的抗炎作用,然而,在这些实验中,是将温泉水作为溶剂,配制细胞培养液,或者将温泉水用培养液稀释后与细胞共孵育,作用时间至少24 h[35-6]、甚至72 h[4],而通常温泉浴的时长仅为0.5 h,且泡温泉时皮肤接触的是温泉水,而非含培养液的温泉水。因此,本研究拟探讨直接使用温泉水处理HaCaT细胞后对细胞的影响,并设置纯水、自来水和生理盐水作为对照组,以比较不同水溶液对细胞影响的差异。
此外,本项目组前期在四川宜宾筠连县巡司温泉村进行人群调查,该地温泉富含溴、碘、锶、锂等微量元素[7],不少村民自报泡温泉后,痤疮有所改善。痤疮是一种常见的慢性炎症性皮肤病,P.acnes是引发痤疮皮损炎症的重要因素[8],已有学者将P.acnes与HaCaT细胞共培养,建立痤疮的炎症反应模型,研究植物提取物的抗炎作用[9-10],因此,本研究将采用该模型探讨温泉水的抗炎作用,而且采取“预防”和“治疗”两种干预模式,即先用水浸泡细胞再建模和建模后再用水浸泡细胞,检测细胞存活率、促炎细胞因子的含量以及炎症信号通路相关基因表达的水平,评估温泉水对痤疮丙酸杆菌诱导炎症反应的“预防”和“治疗”效果。
综上,本研究拟建立P.acnes诱导的HaCaT细胞炎症模型,评估温泉水的抗炎效果,特别是通过设立生理盐水、自来水和纯水对照实验,评估温泉水的抗炎优势,研究结果将为该温泉水健康效应研究积累宝贵资料。
项目组成员于2023年3月在该温泉出水口采用无菌技术采集温泉水样,过滤后常温保存。经测定温泉水pH值为7.69,属弱碱性,富含大量Na+、Ca2+、Mg2+、K+、SO42-和HCO3-等离子,以及人体必需的Fe、Cu、Zn、Se和Si等微量元素。
P.acnes(ATCC6919)和HaCaT细胞均为本实验室保存;DMEM培养基(美国Gibco公司);CCK-8试剂盒(日本同仁化学研究所);ELISA试剂盒(武汉菲恩生物科技有限公司);BCA蛋白浓度测定试剂盒(上海碧云天生物技术有限公司);FastPure Complex Cell/Tissue Total RNA Isolation Kit试剂盒(南京诺维赞生物科技股份有限公司);MightyScript第一链cDNA合成Master Mix以及2 × SG Fast qPCR Master Mix(生工生物工程(上海)股份有限公司)。
温泉水、纯水、自来水经0.22 μm滤膜过滤除菌后分装,常温储存备用;生理盐水经高压灭菌后分装备用。
HaCaT于DMEM完全培养基(10%胎牛血清、0.5 ml 100×青链霉素)在37℃、5% CO2下培养,当细胞融合率到达80%~90%时进行相应实验,或使用0.25%胰酶消化、传代。
1×105个/ml的HaCaT细胞100 μL分别加于96孔板培养,24 h后,将细胞分别暴露于温泉水、生理盐水、自来水、纯水各0.5 h、1 h、1.5 h、2 h、3 h后,每孔分别加入110 μl CCK-8工作液(CCK-8试剂:DMEM培养基=1∶10),继续孵育1 h,测定各孔450 nm处吸光度,计算细胞存活率(细胞存活率=(OD实验- OD空白)/(OD对照- OD空白)× 100%),选择对细胞存活率无明显影响的最长时间作为后续的干预时间。
以3×105个/孔的密度接种HaCaT细胞于24孔培养板,24 h后,弃原培养液,每孔分别加入500 μl OD600为0.05、0.10、0.25、0.50、1.00的P.acnes菌悬液,每个浓度设置4个复孔,并设置细胞对照组。分别培养3、4、5、6、7 h后,采用CCK-8法评估P.acnes对HaCaT细胞存活率的影响,计算P.acnes的50%抑制浓度(Half Maximal Inhibitory Concentration,IC50),选出细菌的作用时间和浓度,并基于选出时间,采用不同浓度菌悬液作用细胞,收集细胞培养上清液,采用ELISA法检测IL-6含量,验证建模是否成功。
本研究分别采用“预防”和“治疗”两种干预方式进行实验。均将HaCaT细胞以3×105个/孔的密度接种于24孔板培养24 h后弃培养液。“预防式”干预每组分别加入1 ml相应水溶液,空白对照组和模型组每孔加入1 ml DMEM培养基,每组设置4个复孔;培养0.5 h后,空白对照组每孔加入500 μl DMEM培养基,其余各组每孔加入500 μl OD600为0.25的P.acnes菌悬液,继续培养5 h。“治疗式”干预中,除空白对照组外各组每孔均加入500 μl OD600为0.25的痤疮丙酸杆菌菌悬液,培养5 h后,各组分别加入1 ml相应水溶液,但空白对照组和模型组每孔分别加入1 ml DMEM培养基后继续培养。
两种方式干预后,采用CCK-8法测定并计算细胞存活率。
两种方式干预后,收集各孔培养液于2 500 r/min离心5 min,取上清液,采用ELISA法检测IL-6、IL-8、IL-1β、TNF-α含量,并用BCA蛋白定量法对各细胞因子测定结果进行校正。
两种方式干预后,收集细胞,提取其总RNA并逆转录合成cDNA。按照95℃ 3 min,95℃ 3 s,60℃ 30 s,95℃ 15 s,60℃ 1 min,95℃ 15 s的条件进行RT-PCR,共40个循环。以GAPDH作为内参基因,待测基因包括IL-6、IL-8、IL-1βTNF-αNF-κBTLR2。以2-△△Ct法计算目的基因mRNA的相对表达量:△Ct=Ct待测基因 - Ct内参基因,△△Ct=△Ct处理组 - △Ct对照组,各基因引物见表1
实验结果以均数±标准差()表示,采用SPSS 25.0和GraphPad Prism 9.5.0软件进行统计分析。采用单因素方差分析或Kruskal-Wallis H检验进行多组间比较,Tukey’s、SNK或Dunnett-t检验进行组间两两比较,检验水准α=0.05。
图1所示,在0.5~3 h的作用时间内,自来水和纯水组HaCaT细胞的存活率均低于10%,而温泉水和生理盐水作用0.5 h时,细胞存活率分别为92.7%和99.09%,因此,将选择0.5 h作为后续实验中水溶液的干预时间。
图2-A所示,不同浓度P.acnes与HaCaT细胞共培养5 h后,细胞存活率随菌液浓度增加而降低,且呈剂量-反应关系。根据细胞存活率,计算获得P.acnes对HaCaT细胞的IC50值为OD600=0.2649。不同浓度P.acnes作用细胞5 h后,促炎细胞因子IL-6分泌量显著增加(P<0.05)。结合IC50值及IL-6分泌量,选取对细胞产生一定损伤,但尚未造成细胞完全死亡,且可使IL-6分泌量显著增加的菌悬液浓度和作用时间,即OD600为0.25的P.acnes菌悬液与细胞共孵育5 h作为建立炎症细胞模型的条件。
(1)对炎症模型细胞存活率的影响
图3所示,模型组、温泉水组以及生理盐水组的细胞存活率分别为50.17%、46.40%和45.45%,与模型组相比,温泉水及生理盐水组细胞的存活率均有所下降,且差异具有统计学意义(P<0.05),但两组间差异无统计学意义(P>0.05),而自来水和纯水组作用0.5 h后,细胞存活率均降低至10%以下,因此后续实验不再设置该两组。
(2)“预防式”干预对促炎细胞因子水平以及相关基因表达的影响
图4所示,与模型组相比,温泉水组的IL-1β、IL-8分泌量降低,IL-6、TNF-αIL-8及TLR2、NF-κB基因的相对表达量也明显降低,且差异具有统计学意义(P<0.05),IL-6、TNF-α的分泌量以及IL-1β基因的相对表达量也有下降趋势,但差异无统计学意义(P>0.05),而生理盐水组的这些因子及其相关基因表达均无明显降低。进一步与生理盐水组相比,温泉水组IL-6、IL-1β含量,IL-6、IL-1βTNF-αNF-κBTLR2相对表达量均降低,差异具有统计学意义(P<0.05)。结果提示,进行预防式干预,温泉水处理0.5 h的抗炎作用优于生理盐水。
(1)“治疗式”干预对炎症模型细胞存活率的影响
图5所示,模型组、温泉水组以及生理盐水组的细胞存活率分别为48.72%、43.12%和43.48%,与模型组相比,温泉水组和生理盐水组细胞存活率均有所下降,且差异具有统计学意义(P<0.05),但温泉水组和生理盐水组之间差异无统计学意义(P>0.05),自来水和纯水组细胞存活率均低于10%,故后续实验也不再设置该两组。
(2)“治疗式”干预对促炎细胞因子水平以及相关基因表达的影响
图6所示,与模型组相比,温泉水组的IL-6、IL-1β、TNF-α、IL-8分泌量降低,IL-6、IL-1βTNF-α基因及TLR2NF-κB基因的相对表达量也明显降低,且差异具有统计学意义(P<0.05),IL-8基因的相对表达量也有下降趋势,但差异无统计学意义(P>0.05),而生理盐水组的这些因子及其相关基因表达均无明显降低。进一步与生理盐水组相比,温泉水组IL-6、IL-1βTNF-α含量,IL-6、IL-1βIL-8及TLR2、NF-κB的相对表达量均明显降低,差异具有统计学意义(P<0.05)。说明温泉水抗炎效果亦优于生理盐水。
尽管已有学者采用细胞模型对温泉水的抗炎作用进行了初探,但在实验中他们所采用的干预方式不能体现泡温泉的实际情况,一是作用时间长,二是作用过程中均含有细胞培养液中的营养成分,因此,本研究创新性建立了基于P.acnes诱导的细胞炎症模型研究温泉水抗炎作用的方法,通过与生理盐水、自来水、纯水处理组比较,发现四川省筠连县巡司的温泉水能够明显降低P.acnes诱导产生的促炎细胞因子的含量以及炎症信号通路相关基因表达的水平,从体外实验证明了该温泉水具有潜在的抗炎作用。需要说明的是,本研究在建模前首先采用纸片法和牛津杯法评估了温泉水对P.acnes的抑制作用,发现温泉水本身对该菌并无直接的抑菌效果(结果未在正文中呈现),故采用P.acnes诱导的细胞炎症模型是可行的。
本研究选用的HaCaT细胞保留了分化能力[11]、表达参与病原体识别的Toll样受体(Toll-Like Receptor,TLR)[12]以及在细菌刺激下产生多种细胞因子的能力[13-14],是皮肤生物学研究中常用的细胞类型之一。该细胞与细菌共培养,会表现出对细菌的识别和反应。有研究发现,P.acnes能产生降解皮肤成分的酶,并刺激角质细胞释放促炎细胞因子,如IL-6、IL-8、IL-1βTNF-α,进而引发剧烈的炎症反应[15-17],而且通过被皮肤表面的TLR受体识别后,激活细胞内TLRs和下游信号细胞核因子NF-κB,促进炎性细胞因子等靶基因的显著表达[18]。因此,基于P.acnes诱导的细胞炎症模型已被用于研究植物提取物的抗炎作用[919],本研究成功建立了该模型并用于温泉水的抗炎作用研究。
已有温泉水抗炎作用研究中,尽管采用了HaCaT细胞,但是温泉水的作用条件是在有细胞培养液存在的情况下,而且作用时间至少24 h,与人们泡温泉的情形相距甚远,且未采用P.acnes共培养建立炎症模型。本研究首先通过测定水溶液作用后HaCaT细胞的存活率,发现用温泉水和生理盐水浸泡细胞0.5 h,细胞存活率分别为92.7%和99.09%,因此,选择0.5 h作为干预时间,这与人们在实际生活中浸泡温泉的持续时间相一致。进一步通过测定不同浓度P.acnes与HaCaT细胞共培养5 h后的细胞存活率及IL-6的含量,确定了P.acnes诱导的细胞炎症模型的建模条件。在此基础上,采用两种干预模式作用于细胞炎症模型,检测发现用纯水和自来水浸泡细胞0.5 h后,细胞存活率低于10%,而温泉水组和生理盐水组的细胞存活率与模型对照组接近,分析由于纯水和自来水浸泡组的低渗透压作用,导致细胞死亡。非常有趣的是,两种干预模式的结果均显示,温泉水组促炎细胞因子的含量以及相关基因表达的水平均低于生理盐水组,说明该温泉水具有潜在的抗炎作用,而且可能具有预防和辅助治疗痤疮的效果,这与本研究人群调查中发现的泡该温泉似乎对痤疮有改善作用的结果一致。
筠连巡司温泉,位于四川省筠连县巡司镇,富含多种矿物质和微量元素,水温51.2℃[7]。据报道,长期浸泡该温泉的居民,其风心病、风湿性关节炎等慢性疾病及感染性疾病的患病率均较低[20]。温泉水为何能起到抗炎效果,可能与其化学成分、热和免疫调节作用有关[21]。通过水质分析发现,该温泉属于典型的NaCL型温泉(Na+为1 566.67 mg/L、Cl-为2 238.00 mg/L),但在本研究中,生理盐水组并未表现出有降低炎性细胞因子的作用,推测其抗炎作用可能与其富含溴、碘、锶、锂[7]以及硫酸盐、碳酸氢盐、硅、硼等成分相关。Karagulle等人[4]的研究发现,硅、锌[22]、碳酸氢钠和硼成分也可能是温泉水抑制促炎细胞因子的原因。另有学者将白细胞介素的抑制归因于硒和锶[23]。而上述成分在该温泉水中的含量不低。
综上,本研究基于P.acnes诱导的细胞炎症模型成功建立了体外研究温泉水抗炎作用的方法,发现四川省筠连县巡司的温泉水具有潜在的抗炎作用,研究结果对进一步探索该温泉的健康效应、促进该温泉资源的开发具有重要的意义,同时也创新了该模型用于研究水溶液的体外抗炎作用的干预方式,具有进一步的应用价值。
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2024年第51卷第22期
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doi: 10.20043/j.cnki.MPM.202407106
  • 接收时间:2024-07-06
  • 首发时间:2026-03-20
  • 出版时间:2024-11-25
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  • 收稿日期:2024-07-06
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    1.四川大学华西公共卫生学院(华西第四医院),四川 成都 610041
    2.四川大学华西口腔医学院
    3.重庆市大渡口区疾病预防控制中心

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2种不同金属材料的力学参数

Family
属数
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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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