Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China.
Chin J Integr Med. 2011 Oct;17(10):775-9. doi: 10.1007/s11655-011-0877-x. Epub 2011 Nov 19.
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).
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).
(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).
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.
探讨 TLR4-NFκB-TNFα 炎症通路在脂多糖（LPS）诱导的新生大鼠心肌细胞损伤中的作用及丹酚酸 B（Sal B）的可能保护作用。
分离培养 Wistar 大鼠（1-2 天龄）心肌细胞。Sal B 10（-5）mol/L、10（-6）mol/L 和 10（-7）mol/L 预孵育 6 h，加入 LPS（1μg/mL）孵育 6 h 诱导炎症损伤。用分光光度法检测上清液中乳酸脱氢酶（LDH）的浓度。酶联免疫吸附试验（ELISA）检测上清液中肿瘤坏死因子α（TNFα）和热休克蛋白 70（HSP70）的浓度。用免疫组化法检测 toll 样受体 4（TLR4）和核因子 kappa B（NFκB）等蛋白的表达。实时荧光定量聚合酶链反应（RT-PCR）检测 TLR4 和 NFκB 的 mRNA 表达。
（1）LPS 对照组上清液中 LDH 和 TNFα 浓度明显高于对照组（561.41±67.39 U/L 和 77.94±15.08 pg/mL 与 292.13±26.02 U/L 和 25.39±16.53 pg/mL，均 P<0.01，P<0.05）。与 LPS 对照组相比，Sal B 10（-5）mol/L 预处理组上清液中 LDH 和 TNFα 浓度明显降低（451.76±83.96 U/L 和 34.00±10.38 pg/mL，均 P<0.05）。（2）LPS 对照组 TLR4 和 NFκB 蛋白表达面积明显高于对照组（1712.41±410.12 μm（2）和 2378.15±175.29 μm（2）与 418.62±24.42 μm（2）和 1721.74±202.87 μm（2），均 P<0.01）。LPS 对照组 TLR4 和 NFκB 蛋白表达内部光密度（IOD）值也明显高于对照组（3.06±0.33 和 7.20±1.04 与 0.91±0.21 和 4.24±0.48，均 P<0.05，P<0.01）。与 LPS 对照组相比，Sal B 10（-5）mol/L 预处理组 TLR4 和 NFκB 蛋白表达面积明显减小（1251.54±133.82 μm（2）和 1996.37±256.67 μm（2），均 P<0.05），TLR4 和 NFκB 蛋白表达 IOD 值也明显减小（1.92±0.28 和 5.17±0.77，均 P<0.05）。（3）LPS 对照组 TLR4 和 NFκB mRNA 表达（2（-ΔΔ）CT 值）明显高于对照组（3.16±0.38 和 5.03±0.43 与 1.04±0.19 和 1.08±0.21，均 P<0.01）。与 LPS 对照组相比，Sal B 10（-5）mol/L 预处理组 TLR4 和 NFκB mRNA 表达（2（-ΔΔ）CT 值）明显降低（1.34±0.22 和 1.74±0.26，均 P<0.05）。各组 HSP70 浓度均无统计学差异（P>0.05）。
LPS 诱导新生大鼠心肌细胞损伤早期 TLR4-NFκB-TNFα 通路迅速激活，与 HSP70 无关。Sal B 的保护作用可能是通过抑制 TLR4-NFκB-TNFα 通路，且呈剂量依赖性。
