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Objective:To investigate the role of the TLR4-NFκB-TNFαinflammation 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(1μg/mL) was added to 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 factorα(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 realtime reverse transcription polymerase chain reaction(RT-PCR).Results:(1) The concentrations of LDH and TNFαin 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 TNFαwere 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±410.12μm~2 and 2378.15±175.29μm~2,versus 418.62±24.42μm~2 and 1721.74±202.87μm~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±133.82μm~2 and 1996.37±256.67μm~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 pretreated group(1.92±0.28 and 5.17±0.77,respectively,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 pretreated group(1.34±0.22 and 1.74±0.26,respectively,P<0.05).The concentration of HSP70 did not show any statistical differences in all groups(P>0.05).Conclusions:The TLR4-NFκB-TNFαpathway 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-TNFαpathway and are dose-dependent.
Objective: To investigate the role of the TLR4-NFκB-TNFαinflammation 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 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. These concentrations of tumor necrosis factor α (TNFα) and heat shock protein 70 (HSP70) in the supernatant were detected by enzyme linked immunosorbent assay. These 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 realtime reverse transcription polymerase chain reaction (RT-PCR) .Results: (1) The c oncentrations of LDH and TNFαin 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, Compared with the LPS control group, the concentrations of LDH and TNFαwere 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 ± 410.12μm ~ 2 and 2378.15 ± 175.29μm ~ 2, versus 418.62 ± 24.42μm ~ 2 and 1721.74 ± 202.87μm ~ 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 proteasome in expression areas were signiThe expressions of TLR4 and NFκB protein expression IOD values were significantly decreased in Sal B 10 ~ (-5) mol / L pre-treated group (1251.54 ± 133.82μm ~ 2 and 1996.37 ± 256.67μm ~ 2, respectively, P < (3) The TLR4 and NFκB mRNA expressions (2 ~ (-ΔΔ)) were also significantly decreased in the Sal B 10 -5 mol / L pretreated group (1.92 ± 0.28 and 5.17 ± 0.77, respectively, P < 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 (2 ~ (-ΔΔ) CT value) were significantly decreased in the Sal B 10 ~ (-5) mol / L pretreated group (1.34 ± 0.22 and 1.74 ± 0.26, respectively, P <0.05) of HSP70 did not show any statistical differences in all groups (P> 0.05) .Conclusions: The TLR4-NFκB-TNFαpathway was rapidly activated and was independent of HSP70 in the early phase of neonatal cardiomyocyte injury induced by LPS. ffects of Sal B may be through inhibiting the TLR4-NFκB-TNFαpathway and are dose-dependent.