Responses of bladder smooth muscle to the stretch go through extracellular signal‐regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/Nuclear factor‐κB (NF‐κB) Pathway
Aims The present study was designed to study changes and its potential mechanisms in human bladder smooth muscle subjected to stretch. Methods Bioinformatics analyses including differential expression analysis, overrepresentation enrichment analysis, principal component analysis, and weighted gene c...
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| Veröffentlicht in: | Neurourology and urodynamics 2019-08, Vol.38 (6), p.1504-1516 |
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| creator | Li, Yaohui He, Minke Lin, Wenyao Xiang, Zhuoyi Huang, Jiaqi Xu, Peirong Shi, Yi Wang, Hang |
| description | Aims
The present study was designed to study changes and its potential mechanisms in human bladder smooth muscle subjected to stretch.
Methods
Bioinformatics analyses including differential expression analysis, overrepresentation enrichment analysis, principal component analysis, and weighted gene coexpression network analysis were used to analyze a microarray dataset (GSE47080) of partial bladder outlet obstruction (pBOO) in rat to find the potential changes of gene expressions. Bladder from pBOO model and human bladder smooth muscle cells (HBSMCs) subjected to sustained prolonged stretch were collected for Western blot analysis, real‐time polymerase chain reaction, and fluorescence analysis to verify the changes of gene expressions and preliminarily study the potential role of signaling pathway regulation in treatment of pBOO.
Results
The bioinformatics analysis showed that chronic obstruction activated mitogen‐activated protein kinase pathway and changed cytoskeleton structure in bladder smooth muscle. In in vivo experiments in mice, pBOO was verified by cystometry. Partial BOO activated the extracellular signal‐regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/nuclear factor‐κB (NF‐κB) signaling pathway in DM. The messenger RNA (mRNA) expressions of contractile phenotypic proteins increased after pBOO. In in vitro experiments of HBSMCs, mechanical stretch activated ERK/p90RSK/NF‐κB in HBSMCs in a time‐dependent manner. The mRNA expressions of α‐smooth muscle actin and SM22 also increased and filamentous actin (F‐actin) polymerization was enhanced as well. Inhibition of ERK/p90RSK/NF‐κB pathway reversed mechanical stretch‐induced changes of contractile phenotypic expression and F‐action polymerization.
Conclusions
Continuous stretch increases expressions of contractile phenotypic proteins and promotes the polymerization of F‐actin. This process partially goes through ERK/p90RSK/NF‐κB pathway. |
| doi_str_mv | 10.1002/nau.24003 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_2216772808</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>2216772808</sourcerecordid><originalsourceid>FETCH-LOGICAL-c3533-154afc264e69035e7f57ed8dd193e4d319d3e825fcf3eb603c2eacf3f576c6433</originalsourceid><addsrcrecordid>eNp1kU1uEzEYhi0EoiGw4ALIEptkkcY_M56ZZanagqgCSul65NjfZKZ4xsE_Ktn1CJwH7sAhOAkOU7pAYuUfPX5svy9CLyk5poSw5SDjMcsI4Y_QhOaMLERRFI_RhBScL1gmiiP0zPsbQkjJs-opOuKUcE6omKAfa_A7O3jw2DZ4Y6TW4LDvrQ0t7qNXBnCwOLSAfXAQVIu3h6Wzcdti-BqcVGBMNDKd6raDNL_uvjnYpo0AGn_uBukBz87W7-fLXUWw6zbW214afCXwztkA3fBAJWB9lcBVTNcmYSNVsC4Jf35_g2er83E2xx9laG_l_jl60kjj4cX9OEXX52efTt8uLj9cvDs9uVwonqcAaJ7JRjGRgagIz6Fo8gJ0qTWtOGSa00pzKFneqIbDRhCuGMg0T5hQIuN8imajN733SwQf6r7zh1_LAWz0NWM0Bc7KlO4Uvf4HvbHRpVQOlMhIUQouEjUfKeWs9w6aeue6Xrp9TUl9aLROjdZ_Gk3sq3tj3PSgH8i_FSZgOQK3nYH9_0316uR6VP4GCFqvVw</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2264078636</pqid></control><display><type>article</type><title>Responses of bladder smooth muscle to the stretch go through extracellular signal‐regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/Nuclear factor‐κB (NF‐κB) Pathway</title><source>MEDLINE</source><source>Wiley Online Library Journals Frontfile Complete</source><creator>Li, Yaohui ; He, Minke ; Lin, Wenyao ; Xiang, Zhuoyi ; Huang, Jiaqi ; Xu, Peirong ; Shi, Yi ; Wang, Hang</creator><creatorcontrib>Li, Yaohui ; He, Minke ; Lin, Wenyao ; Xiang, Zhuoyi ; Huang, Jiaqi ; Xu, Peirong ; Shi, Yi ; Wang, Hang</creatorcontrib><description>Aims
The present study was designed to study changes and its potential mechanisms in human bladder smooth muscle subjected to stretch.
Methods
Bioinformatics analyses including differential expression analysis, overrepresentation enrichment analysis, principal component analysis, and weighted gene coexpression network analysis were used to analyze a microarray dataset (GSE47080) of partial bladder outlet obstruction (pBOO) in rat to find the potential changes of gene expressions. Bladder from pBOO model and human bladder smooth muscle cells (HBSMCs) subjected to sustained prolonged stretch were collected for Western blot analysis, real‐time polymerase chain reaction, and fluorescence analysis to verify the changes of gene expressions and preliminarily study the potential role of signaling pathway regulation in treatment of pBOO.
Results
The bioinformatics analysis showed that chronic obstruction activated mitogen‐activated protein kinase pathway and changed cytoskeleton structure in bladder smooth muscle. In in vivo experiments in mice, pBOO was verified by cystometry. Partial BOO activated the extracellular signal‐regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/nuclear factor‐κB (NF‐κB) signaling pathway in DM. The messenger RNA (mRNA) expressions of contractile phenotypic proteins increased after pBOO. In in vitro experiments of HBSMCs, mechanical stretch activated ERK/p90RSK/NF‐κB in HBSMCs in a time‐dependent manner. The mRNA expressions of α‐smooth muscle actin and SM22 also increased and filamentous actin (F‐actin) polymerization was enhanced as well. Inhibition of ERK/p90RSK/NF‐κB pathway reversed mechanical stretch‐induced changes of contractile phenotypic expression and F‐action polymerization.
Conclusions
Continuous stretch increases expressions of contractile phenotypic proteins and promotes the polymerization of F‐actin. This process partially goes through ERK/p90RSK/NF‐κB pathway.</description><identifier>ISSN: 0733-2467</identifier><identifier>EISSN: 1520-6777</identifier><identifier>DOI: 10.1002/nau.24003</identifier><identifier>PMID: 31033016</identifier><language>eng</language><publisher>United States: Wiley Subscription Services, Inc</publisher><subject>Actin ; Actins - biosynthesis ; Animals ; Bioinformatics ; Bladder ; bladder smooth muscle ; Cats ; Computational Biology ; Cytoskeleton ; DNA microarrays ; Extracellular signal-regulated kinase ; Female ; Gene Expression ; Kinases ; MAP Kinase Signaling System - genetics ; MAPK ; mRNA ; Muscle Contraction ; Muscle, Smooth - physiopathology ; Myocytes, Smooth Muscle - metabolism ; NF-kappa B - genetics ; NF‐κB ; obstruction ; Physical Stimulation ; Polymerase chain reaction ; Polymerization ; Principal components analysis ; Protein kinase ; Proteins ; Rats ; Rats, Sprague-Dawley ; Ribosomal protein S6 ; Ribosomal Protein S6 Kinases, 90-kDa - genetics ; RSK ; Signal transduction ; Signal Transduction - genetics ; signaling pathway ; Smooth muscle ; Urinary Bladder - cytology ; Urinary Bladder - metabolism ; Urinary Bladder - physiopathology ; Urinary Bladder Neck Obstruction - genetics ; Urinary Bladder Neck Obstruction - physiopathology ; Urinary Bladder Neck Obstruction - surgery ; Urinary Catheterization</subject><ispartof>Neurourology and urodynamics, 2019-08, Vol.38 (6), p.1504-1516</ispartof><rights>2019 Wiley Periodicals, Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3533-154afc264e69035e7f57ed8dd193e4d319d3e825fcf3eb603c2eacf3f576c6433</citedby><cites>FETCH-LOGICAL-c3533-154afc264e69035e7f57ed8dd193e4d319d3e825fcf3eb603c2eacf3f576c6433</cites><orcidid>0000-0002-3301-2548</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fnau.24003$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fnau.24003$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,27903,27904,45553,45554</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31033016$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Li, Yaohui</creatorcontrib><creatorcontrib>He, Minke</creatorcontrib><creatorcontrib>Lin, Wenyao</creatorcontrib><creatorcontrib>Xiang, Zhuoyi</creatorcontrib><creatorcontrib>Huang, Jiaqi</creatorcontrib><creatorcontrib>Xu, Peirong</creatorcontrib><creatorcontrib>Shi, Yi</creatorcontrib><creatorcontrib>Wang, Hang</creatorcontrib><title>Responses of bladder smooth muscle to the stretch go through extracellular signal‐regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/Nuclear factor‐κB (NF‐κB) Pathway</title><title>Neurourology and urodynamics</title><addtitle>Neurourol Urodyn</addtitle><description>Aims
The present study was designed to study changes and its potential mechanisms in human bladder smooth muscle subjected to stretch.
Methods
Bioinformatics analyses including differential expression analysis, overrepresentation enrichment analysis, principal component analysis, and weighted gene coexpression network analysis were used to analyze a microarray dataset (GSE47080) of partial bladder outlet obstruction (pBOO) in rat to find the potential changes of gene expressions. Bladder from pBOO model and human bladder smooth muscle cells (HBSMCs) subjected to sustained prolonged stretch were collected for Western blot analysis, real‐time polymerase chain reaction, and fluorescence analysis to verify the changes of gene expressions and preliminarily study the potential role of signaling pathway regulation in treatment of pBOO.
Results
The bioinformatics analysis showed that chronic obstruction activated mitogen‐activated protein kinase pathway and changed cytoskeleton structure in bladder smooth muscle. In in vivo experiments in mice, pBOO was verified by cystometry. Partial BOO activated the extracellular signal‐regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/nuclear factor‐κB (NF‐κB) signaling pathway in DM. The messenger RNA (mRNA) expressions of contractile phenotypic proteins increased after pBOO. In in vitro experiments of HBSMCs, mechanical stretch activated ERK/p90RSK/NF‐κB in HBSMCs in a time‐dependent manner. The mRNA expressions of α‐smooth muscle actin and SM22 also increased and filamentous actin (F‐actin) polymerization was enhanced as well. Inhibition of ERK/p90RSK/NF‐κB pathway reversed mechanical stretch‐induced changes of contractile phenotypic expression and F‐action polymerization.
Conclusions
Continuous stretch increases expressions of contractile phenotypic proteins and promotes the polymerization of F‐actin. This process partially goes through ERK/p90RSK/NF‐κB pathway.</description><subject>Actin</subject><subject>Actins - biosynthesis</subject><subject>Animals</subject><subject>Bioinformatics</subject><subject>Bladder</subject><subject>bladder smooth muscle</subject><subject>Cats</subject><subject>Computational Biology</subject><subject>Cytoskeleton</subject><subject>DNA microarrays</subject><subject>Extracellular signal-regulated kinase</subject><subject>Female</subject><subject>Gene Expression</subject><subject>Kinases</subject><subject>MAP Kinase Signaling System - genetics</subject><subject>MAPK</subject><subject>mRNA</subject><subject>Muscle Contraction</subject><subject>Muscle, Smooth - physiopathology</subject><subject>Myocytes, Smooth Muscle - metabolism</subject><subject>NF-kappa B - genetics</subject><subject>NF‐κB</subject><subject>obstruction</subject><subject>Physical Stimulation</subject><subject>Polymerase chain reaction</subject><subject>Polymerization</subject><subject>Principal components analysis</subject><subject>Protein kinase</subject><subject>Proteins</subject><subject>Rats</subject><subject>Rats, Sprague-Dawley</subject><subject>Ribosomal protein S6</subject><subject>Ribosomal Protein S6 Kinases, 90-kDa - genetics</subject><subject>RSK</subject><subject>Signal transduction</subject><subject>Signal Transduction - genetics</subject><subject>signaling pathway</subject><subject>Smooth muscle</subject><subject>Urinary Bladder - cytology</subject><subject>Urinary Bladder - metabolism</subject><subject>Urinary Bladder - physiopathology</subject><subject>Urinary Bladder Neck Obstruction - genetics</subject><subject>Urinary Bladder Neck Obstruction - physiopathology</subject><subject>Urinary Bladder Neck Obstruction - surgery</subject><subject>Urinary Catheterization</subject><issn>0733-2467</issn><issn>1520-6777</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kU1uEzEYhi0EoiGw4ALIEptkkcY_M56ZZanagqgCSul65NjfZKZ4xsE_Ktn1CJwH7sAhOAkOU7pAYuUfPX5svy9CLyk5poSw5SDjMcsI4Y_QhOaMLERRFI_RhBScL1gmiiP0zPsbQkjJs-opOuKUcE6omKAfa_A7O3jw2DZ4Y6TW4LDvrQ0t7qNXBnCwOLSAfXAQVIu3h6Wzcdti-BqcVGBMNDKd6raDNL_uvjnYpo0AGn_uBukBz87W7-fLXUWw6zbW214afCXwztkA3fBAJWB9lcBVTNcmYSNVsC4Jf35_g2er83E2xx9laG_l_jl60kjj4cX9OEXX52efTt8uLj9cvDs9uVwonqcAaJ7JRjGRgagIz6Fo8gJ0qTWtOGSa00pzKFneqIbDRhCuGMg0T5hQIuN8imajN733SwQf6r7zh1_LAWz0NWM0Bc7KlO4Uvf4HvbHRpVQOlMhIUQouEjUfKeWs9w6aeue6Xrp9TUl9aLROjdZ_Gk3sq3tj3PSgH8i_FSZgOQK3nYH9_0316uR6VP4GCFqvVw</recordid><startdate>201908</startdate><enddate>201908</enddate><creator>Li, Yaohui</creator><creator>He, Minke</creator><creator>Lin, Wenyao</creator><creator>Xiang, Zhuoyi</creator><creator>Huang, Jiaqi</creator><creator>Xu, Peirong</creator><creator>Shi, Yi</creator><creator>Wang, Hang</creator><general>Wiley Subscription Services, Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7T5</scope><scope>7TK</scope><scope>H94</scope><scope>K9.</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-3301-2548</orcidid></search><sort><creationdate>201908</creationdate><title>Responses of bladder smooth muscle to the stretch go through extracellular signal‐regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/Nuclear factor‐κB (NF‐κB) Pathway</title><author>Li, Yaohui ; He, Minke ; Lin, Wenyao ; Xiang, Zhuoyi ; Huang, Jiaqi ; Xu, Peirong ; Shi, Yi ; Wang, Hang</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3533-154afc264e69035e7f57ed8dd193e4d319d3e825fcf3eb603c2eacf3f576c6433</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Actin</topic><topic>Actins - biosynthesis</topic><topic>Animals</topic><topic>Bioinformatics</topic><topic>Bladder</topic><topic>bladder smooth muscle</topic><topic>Cats</topic><topic>Computational Biology</topic><topic>Cytoskeleton</topic><topic>DNA microarrays</topic><topic>Extracellular signal-regulated kinase</topic><topic>Female</topic><topic>Gene Expression</topic><topic>Kinases</topic><topic>MAP Kinase Signaling System - genetics</topic><topic>MAPK</topic><topic>mRNA</topic><topic>Muscle Contraction</topic><topic>Muscle, Smooth - physiopathology</topic><topic>Myocytes, Smooth Muscle - metabolism</topic><topic>NF-kappa B - genetics</topic><topic>NF‐κB</topic><topic>obstruction</topic><topic>Physical Stimulation</topic><topic>Polymerase chain reaction</topic><topic>Polymerization</topic><topic>Principal components analysis</topic><topic>Protein kinase</topic><topic>Proteins</topic><topic>Rats</topic><topic>Rats, Sprague-Dawley</topic><topic>Ribosomal protein S6</topic><topic>Ribosomal Protein S6 Kinases, 90-kDa - genetics</topic><topic>RSK</topic><topic>Signal transduction</topic><topic>Signal Transduction - genetics</topic><topic>signaling pathway</topic><topic>Smooth muscle</topic><topic>Urinary Bladder - cytology</topic><topic>Urinary Bladder - metabolism</topic><topic>Urinary Bladder - physiopathology</topic><topic>Urinary Bladder Neck Obstruction - genetics</topic><topic>Urinary Bladder Neck Obstruction - physiopathology</topic><topic>Urinary Bladder Neck Obstruction - surgery</topic><topic>Urinary Catheterization</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Li, Yaohui</creatorcontrib><creatorcontrib>He, Minke</creatorcontrib><creatorcontrib>Lin, Wenyao</creatorcontrib><creatorcontrib>Xiang, Zhuoyi</creatorcontrib><creatorcontrib>Huang, Jiaqi</creatorcontrib><creatorcontrib>Xu, Peirong</creatorcontrib><creatorcontrib>Shi, Yi</creatorcontrib><creatorcontrib>Wang, Hang</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Immunology Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>MEDLINE - Academic</collection><jtitle>Neurourology and urodynamics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Li, Yaohui</au><au>He, Minke</au><au>Lin, Wenyao</au><au>Xiang, Zhuoyi</au><au>Huang, Jiaqi</au><au>Xu, Peirong</au><au>Shi, Yi</au><au>Wang, Hang</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Responses of bladder smooth muscle to the stretch go through extracellular signal‐regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/Nuclear factor‐κB (NF‐κB) Pathway</atitle><jtitle>Neurourology and urodynamics</jtitle><addtitle>Neurourol Urodyn</addtitle><date>2019-08</date><risdate>2019</risdate><volume>38</volume><issue>6</issue><spage>1504</spage><epage>1516</epage><pages>1504-1516</pages><issn>0733-2467</issn><eissn>1520-6777</eissn><abstract>Aims
The present study was designed to study changes and its potential mechanisms in human bladder smooth muscle subjected to stretch.
Methods
Bioinformatics analyses including differential expression analysis, overrepresentation enrichment analysis, principal component analysis, and weighted gene coexpression network analysis were used to analyze a microarray dataset (GSE47080) of partial bladder outlet obstruction (pBOO) in rat to find the potential changes of gene expressions. Bladder from pBOO model and human bladder smooth muscle cells (HBSMCs) subjected to sustained prolonged stretch were collected for Western blot analysis, real‐time polymerase chain reaction, and fluorescence analysis to verify the changes of gene expressions and preliminarily study the potential role of signaling pathway regulation in treatment of pBOO.
Results
The bioinformatics analysis showed that chronic obstruction activated mitogen‐activated protein kinase pathway and changed cytoskeleton structure in bladder smooth muscle. In in vivo experiments in mice, pBOO was verified by cystometry. Partial BOO activated the extracellular signal‐regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/nuclear factor‐κB (NF‐κB) signaling pathway in DM. The messenger RNA (mRNA) expressions of contractile phenotypic proteins increased after pBOO. In in vitro experiments of HBSMCs, mechanical stretch activated ERK/p90RSK/NF‐κB in HBSMCs in a time‐dependent manner. The mRNA expressions of α‐smooth muscle actin and SM22 also increased and filamentous actin (F‐actin) polymerization was enhanced as well. Inhibition of ERK/p90RSK/NF‐κB pathway reversed mechanical stretch‐induced changes of contractile phenotypic expression and F‐action polymerization.
Conclusions
Continuous stretch increases expressions of contractile phenotypic proteins and promotes the polymerization of F‐actin. This process partially goes through ERK/p90RSK/NF‐κB pathway.</abstract><cop>United States</cop><pub>Wiley Subscription Services, Inc</pub><pmid>31033016</pmid><doi>10.1002/nau.24003</doi><tpages>13</tpages><orcidid>https://orcid.org/0000-0002-3301-2548</orcidid></addata></record> |
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| issn | 0733-2467 1520-6777 |
| language | eng |
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| source | MEDLINE; Wiley Online Library Journals Frontfile Complete |
| subjects | Actin Actins - biosynthesis Animals Bioinformatics Bladder bladder smooth muscle Cats Computational Biology Cytoskeleton DNA microarrays Extracellular signal-regulated kinase Female Gene Expression Kinases MAP Kinase Signaling System - genetics MAPK mRNA Muscle Contraction Muscle, Smooth - physiopathology Myocytes, Smooth Muscle - metabolism NF-kappa B - genetics NF‐κB obstruction Physical Stimulation Polymerase chain reaction Polymerization Principal components analysis Protein kinase Proteins Rats Rats, Sprague-Dawley Ribosomal protein S6 Ribosomal Protein S6 Kinases, 90-kDa - genetics RSK Signal transduction Signal Transduction - genetics signaling pathway Smooth muscle Urinary Bladder - cytology Urinary Bladder - metabolism Urinary Bladder - physiopathology Urinary Bladder Neck Obstruction - genetics Urinary Bladder Neck Obstruction - physiopathology Urinary Bladder Neck Obstruction - surgery Urinary Catheterization |
| title | Responses of bladder smooth muscle to the stretch go through extracellular signal‐regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/Nuclear factor‐κB (NF‐κB) Pathway |
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