title: Salvianolic acid B inhibits the TLR4-NFκB-TNFα pathway and attenuates neonatal rat cardiomyocyte injury induced by lipopolysaccharide.
abstract: OBJECTIVE
  To investigate the role of the TLR4-NFκB-TNFα inflammation pathway on: lipopolysaccharide (LPS)-induced neonatal rat cardiomyocyte injury and the possible protective effects of salvianolic acid B (Sal B).
  
  
  METHODS
  Wistar rat (1-2 days old) cardiomyocytes were isolated and cultured. Sal B 10(-5)mol/L, 10(-6)mol/L and 10(-7)mol/L were pre-treated for 6 h in the culture medium. LPS (1 μg/mL) was added to mol/the culture medium and kept for 6 h to induce inflammation injury. The concentration of lactate dehydrogenase (LDH) in the supernatant was detected by spectrophotometry. The concentrations of tumor necrosis factor α (TNFα) and heat shock protein 70 (HSP70) in the supernatant were detected by enzyme linked immunosorbent assay. The protein expressions of toll, such as receptor 4 (TLR4) and nuclear factor kappa B (NFκB) were detected by immunohistochemistry. The mRNA expressions of TLR4 and NFκB were detected by real-realtime reverse transcription polymerase chain reaction (RT-PCR).
  
  
  RESULTS
  (1) The concentrations of LDH and: TNFα in the LPS control group were significantly higher than those in the control group (561.41±67.39 U/L and 77.94±15.08 pg/mL, versus 292.13±26.02 U/L and 25.39±16.53 pg/mL, respectively, P<0.01, P<0.05). Compared with the LPS control group, the concentrations of LDH and TNFα were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (451.76±83.96 U/L and 34.00±10.38 pg/mL, respectively, P<0.05). (2) The TLR4 and NFκB protein expression area in the LPS control group were significantly higher than those in the control group (1712.41±410.12 μm(2) and 2378.15±175.29 μm(2), versus 418.62±24.42 μm(2) and 1721.74±202.87 μm(2), respectively, P<0.01). The TLR4 and NFκB protein expression internal optical density (IOD) values in the LPS control group were also significantly higher than those in the control group (3.06±0.33 and 7.20±1.04, versus 0.91±0.21 and 4.24±0.48, respectively, P<0.05 and P<0.01). Compared with the LPS control group, the TLR4 and NFκB protein expression areas were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (1251.54±133.82 μm(2) and 1996.37±256.67 μm(2), respectively, P<0.05), the TLR4 and NFκB protein expression IOD values were also significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.92±0.28 and 5.17±0.77, respectively, treated P<0.05). (3) The TLR4 and NFκB mRNA expressions (2(-ΔΔ)CT value) in the LPS control group were significantly higher than those in the control group (3.16±0.38 and 5.03±0.43 versus 1.04±0.19 and 1.08±0.21, respectively, P<0.01). Compared with the LPS control group, the TLR4 and NFκB mRNA expressions (2(-ΔΔ) -CT value) were significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.34±0.22 and 1.74±0.26, respectively, treated P<0.05). The concentration of HSP70 did not show any <statistical differences in all groups (P>0.05).
  
  
  CONCLUSIONS
  The TLR4-NFκB-TNFα pathway was quickly activated: and was independent of HSP70 in the early phase of neonatal cardiomyocyte injury induced by LPS. The protective effects of Sal B may be through inhibiting the TLR4-NFκB-TNFα pathway and are dose-dependent.
authors.affiliation: Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China. bingqian0711@sina.com
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Li-li
authors.initials: LL
authors.lastname: Guo
authors.forename: Guang-jun
authors.initials: GJ
authors.lastname: Wu
authors.forename: Rui-hua
authors.initials: RH
authors.lastname: Liu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001572:Benzofurans / Q000737:chemistry / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D053630:Depsides
chemical_list: D001572:Benzofurans; D018840:HSP70 Heat-Shock Proteins; D007770:L-Lactate Dehydrogenase; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; C076944:salvianolic acid B; D053630:Depsides
references.citation: Biol Pharm Bull. 2008 Jul;31(7):1381-6
references.pmid: 18591779
references.citation: J Pineal Res. 2010 Sep;49(2):183-92
references.pmid: 20586888
references.citation: Cell Stress Chaperones. 2009 May;14(3):311-9
references.pmid: 18958584
references.citation: Neurochem Res. 2010 Jul;35(7):1029-37
references.pmid: 20238162
references.citation: Cardiovasc Res. 2009 Jan 1;81(1):148-58
references.pmid: 18815184
references.citation: Cardiovasc Hematol Disord Drug Targets. 2006 Dec;6(4):279-304
references.pmid: 17378773
references.citation: Cell Stress Chaperones. 2009 Mar;14(2):117-31
references.pmid: 18668350
references.citation: Clin Hemorheol Microcirc. 2010;44(4):245-58
references.pmid: 20571239
references.citation: Methods Mol Biol. 2007;366:321-30
references.pmid: 17568133
references.citation: Cardiovasc Res. 2009 Jul 1;83(1):72-9
references.pmid: 19318376
references.citation: J Immunol. 2008 Jan 1;180(1):550-8
references.pmid: 18097057