摘要
Objective： To investigate the role of the TLR4-NF K B-TNFa 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 pro-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. The concentrations of tumor necrosis factor （TNF a） 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 （NF K B） were detected by immunohistochemistry. The mRNA expressions of TLR4 and NF K B were detected by realtime reverse transcription polymerase chain reaction （RT-PCR）. Results： （1） The concentrations of LDH and TNF a 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 TNF α were significantly decreased in the Sal B 10-5mol/L pro-treated group （451.76 ± 83.96 U/L and 34.00± 10.38 pg/mL, respectively, P〈0.05）. （2） The TLR4 and NF K B 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 μ m2 and 1721.74 ± 202.87μ m2, respectively, P〈0.01）. The TLR4 and NF K B 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 NF K B protein expression areas were significantly decreased in the Sal B 10Smol/L pre-treated group （1251.54± 133.82 μ m2 and 1996.37 ± 256.67 μ m2, respectively, P〈0.05）, the TLR4 and NF K B protein expression IOD values were also significantly decreased in the Sal B 10-5mol/L pretreated group （1.92 ±0.28 and 5.17 ±0.77, respectively, P〈0.05）. （3） The TLR4 and NF K B mRNA expressions （2△△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 NF KB mRNA expressions （2△△CT value） were significantly decreased in the Sal B 10-5mol/L pre- treated 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-NF K B-TNF α 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-NF K B-TNF a pathway and are dose-dependent.
Objective： To investigate the role of the TLR4-NF K B-TNFa 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 pro-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. The concentrations of tumor necrosis factor （TNF a） 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 （NF K B） were detected by immunohistochemistry. The mRNA expressions of TLR4 and NF K B were detected by realtime reverse transcription polymerase chain reaction （RT-PCR）. Results： （1） The concentrations of LDH and TNF a 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 TNF α were significantly decreased in the Sal B 10-5mol/L pro-treated group （451.76 ± 83.96 U/L and 34.00± 10.38 pg/mL, respectively, P〈0.05）. （2） The TLR4 and NF K B 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 μ m2 and 1721.74 ± 202.87μ m2, respectively, P〈0.01）. The TLR4 and NF K B 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 NF K B protein expression areas were significantly decreased in the Sal B 10Smol/L pre-treated group （1251.54± 133.82 μ m2 and 1996.37 ± 256.67 μ m2, respectively, P〈0.05）, the TLR4 and NF K B protein expression IOD values were also significantly decreased in the Sal B 10-5mol/L pretreated group （1.92 ±0.28 and 5.17 ±0.77, respectively, P〈0.05）. （3） The TLR4 and NF K B mRNA expressions （2△△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 NF KB mRNA expressions （2△△CT value） were significantly decreased in the Sal B 10-5mol/L pre- treated 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-NF K B-TNF α 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-NF K B-TNF a pathway and are dose-dependent.
基金
Supported by the Foundation of Guang'anmen Hospital,China Academy of Chinese Medical Sciences(No.81359)
