大黄素调控TLR4/NF-κB信号通路对大鼠感染性创面愈合的研究

Research of emodin regulating TLR4/NF-κB signaling pathway on heal infected wound in rats

目的研究大黄素对大鼠感染性创面愈合的作用,及其对Toll样受体4(TLR4)/核因子κB(NF-κB)信号通路的调节作用。方法用全层皮肤缺损法构建感染性创面大鼠模型,将造模成功的大鼠随机分为模型组、对照组和低、中、高剂量实验组,每组12只;另取12只正常大鼠作为正常组。低、中、高剂量实验组分别外敷大黄素150、300、600μg·d^(-1),每次1次,同时均灌胃给予200 mg·kg^(-1)大黄素,每天1次;对照组外敷莫匹罗星软膏0.1 g·d^(-1),每天1次;正常组和模型组均灌胃给予等体积0.9%NaCl,每天1次。6组大鼠均连续给药21 d。比较各组大鼠的创面愈合率及动态血流值,用酶联免疫吸附试验法分别测定血清中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β水平及创面组织中羟脯氨酸(HYP)、过氧化氢酶(CAT)水平,用蛋白质印迹法测定创面组织中TLR4和NF-κB蛋白的表达水平。结果中、高剂量实验组和对照组、模型组的创面愈合率分别为(50.70±1.26)%、(75.51±0.50)%、(69.44±1.65)%和(28.58±5.09)%;中、高剂量实验组和对照组、模型组、正常组的动态血流值分别为(123.71±3.71)、(155.61±2.47)、(146.29±2.04)、(79.47±4.76)和(158.00±1.93)mL·min^(-1),TNF-α分别为(81.05±2.32)、(56.69±1.59)、(69.53±2.39)、(139.46±2.46)和(46.49±5.71)ng·L^(-1),IL-1β分别为(59.87±0.83)、(42.66±1.30)、(48.37±3.34)、(88.79±1.84)和(33.93±2.19)ng·L^(-1),HYP分别为(24.79±0.82)、(31.12±1.16)、(25.83±1.12)、(16.88±2.20)和(32.95±1.18)μg·mg^(-1),CAT分别为(24.99±1.47)、(34.52±1.26)、(31.00±1.15)、(13.70±2.09)和(34.23±0.70)U·mg^(-1),TLR4蛋白相对水平分别为0.74±0.05、0.53±0.05、0.55±0.04、1.09±0.07和0.23±0.05,NF-κB蛋白相对水平分别为0.69±0.06、0.44±0.04、0.52±0.03、1.10±0.04和0.22±0.10。中、高剂量实验组的上述指标与模型组比较,在统计学上差异均有统计学意义(均P<0.05)。结论大黄素能够改善大鼠感染性创面愈合及动态血流量,抑制创面组织的炎症水平和氧化应激反应,其机制可能与调节TLR4/NF-κB通路有关。

Objective To investigate the effects of emodin on the healing of infected wound and the regulation of Toll-likereceptor 4(TLR4)/nuclear factor kappa-B(NF-κB)signaling pathway in rats.Methods The model of infectious wounds rats were constructed by the full-thickness skin defect method.The successful model rats were randomly divided into model group,control group and experimental-L,-M,-H groups with 12 rats per group;another 12 normal rats were taken as the normal group.Experimental-L,-M,-H groups was applied with emodin 150,300 and 600μg·d^(-1)and 200 mg·kg^(-1)emodin by gavage,respectively.Control group was applied with mopirolacin cream 0.1 g·d^(-1)on rat wounds,once a day.Normal and model groups were treated with 0.9%NaCl.Six groups were treated for 21 days.The wound healing rates and dynamic blood flow were measured.The levels of tumor necrosis factor(TNF)-αand interleukin(IL)-1βin serum and the hydroxyproline(HYP)and catalase(CAT)in wound tssue were measured by enzyme linked immunosorbent assay.The protein levels of TLR4 and NF-κB in wound were determined by Western blot.Results The wound healing rates of experimental-M,-H groups,control group and model group were(50.70±1.26)%,(75.51±0.50)%,(69.44±1.65)%and(28.58±5.09)%,respectively.The dynamic blood flow values of experimental-M,-H groups,control group,model group and blank group were(123.71±3.71),(155.61±2.47),(146.29±2.04),(79.47±4.76)and(158.00±1.93)mL·min^(-1);the levels of TNF-αwere(81.05±2.32),(56.69±1.59),(69.53±2.39),(139.46±2.46)and(46.49±5.71)ng·L^(-1);the levels of IL-1βwere(59.87±0.83),(42.66±1.30),(48.37±3.34),(88.79±1.84)and(33.93±2.19)ng L^(-l);the levels of HYP were(24.79±0.82),(31.12±1.16),(25.83±1.12),(16.88±2.20)and(32.95±1.18)μg·mg^(-1);the levels of CAT were(24.99±1.47),(34.52±1.26),(31.00±1.15),(13.70±2.09)and(34.23±0.70)U·mg^(-1);the relative expression levels of TLR4 protein were 0.74±0.05,0.53±0.05,0.55±0.04,1.09±0.07 and 0.23±0.05;the relative expression levels of NF-κB protein were 0.69±0.06,0.44±0.04,0.52±0.03,1.10±0.04 and 0.22±0.10,respectively.Compared with the model group,the above indexes in the experimental-M,-H groups were statistically significant(all P<0.05).Conclusion Emodin can improve the infectious wound healing and dynamic blood flow in rats,and inhibit the inflammatory levels and oxidative stress response in wound tissue,and the mechanism may be related to the regulation of TLR4/NF-κB pathway.