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 10−5mol/L, 10−6mol/L and 10−7mol/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 10−5mol/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 μm2 and 2378.15±175.29 μm2, versus 418.62±24.42 μm2 and 1721.74±202.87 μm2, 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 10−5mol/L pre-treated group (1251.54±133.82 μm2 and 1996.37±256.67 μm2, respectively, P<0.05), the TLR4 and NFκB protein expression IOD values were also significantly decreased in the Sal B 10−5mol/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−ΔΔCT 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 10−5mol/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.
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Supported by the Foundation of Guang’anmen Hospital, China Academy of Chinese Medical Sciences (No. 81359)
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Wang, J., Zhang, Y., Guo, Ll. et al. Salvianolic acid B inhibits the TLR4-NFκB-TNFα pathway and attenuates neonatal rat cardiomyocyte injury induced by lipopolysaccharide. Chin. J. Integr. Med. 17, 775–779 (2011). https://doi.org/10.1007/s11655-011-0877-x
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DOI: https://doi.org/10.1007/s11655-011-0877-x