连翘苷调节SphK1/S1P/S1PR1信号通路对流感病毒肺炎大鼠肺损伤的影响

Effects of phillyrin on lung injury in rats with influenza virus pneumonia by regulating the SphK1/S1P/S1PR1 signal pathway

目的 探究连翘苷(KD-1)对流感病毒肺炎大鼠肺损伤的影响以及对鞘氨醇激酶1(SphK1)/1-磷酸鞘氨醇(S1P)/鞘氨醇1磷酸酯受体1(S1PR1)信号通路的调节机制。方法 将Wistar雄性大鼠分为对照组(灌胃等量0.9%NaCl)、模型组(灌胃等量0.9%NaCl)、阳性药物组(灌胃0.02 g·kg^(-1)利巴韦林)、PF-543组(灌胃10 mg·kg^(-1)SphK1抑制药PF-543 Citrate)和低、高剂量实验组(分别灌胃6.5、13 mg·kg^(-1)KD-1)。除对照组外,其余各组大鼠用流感病毒滴鼻建立流感病毒感染肺炎模型。测量大鼠的肺指数,用苏木精-伊红(HE)染色法观察大鼠肺组织的病理损伤情况,用酶联免疫吸附试验(ELISA)法检测支气管肺泡灌洗液(BALF)中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)和IL-6的含量,用蛋白质印迹法检测大鼠肺组织中SphK1、S1P、S1PR1蛋白的表达水平。结果 低、高剂量实验组和PF-543组、阳性药物组、模型组、对照组的肺指数分别为(7.62±0.51)、(5.34±0.46)、(6.53±0.52)、(5.48±0.43)、(12.46±0.87)和(4.41±0.32)mg·g^(-1),IL-1β含量分别为(47.26±2.05)、(25.18±1.58)、(35.75±1.50)、(27.31±1.67)、(62.37±2.51)和(13.28±1.04)ng·L^(-1),TNF-α含量分别为(76.58±4.73)、(51.82±3.90)、(64.81±4.15)、(53.06±3.86)、(98.47±4.92)和(42.71±3.52)ng·L^(-1),IL-6含量分别为(57.62±4.29)、(39.06±3.86)、(48.75±3.83)、(41.23±3.61)、(76.92±5.24)和(28.56±3.17)ng·L^(-1),SphK1蛋白相对表达水平分别为1.07±0.08、0.51±0.04、0.65±0.05、0.53±0.04、1.28±0.09和0.36±0.03,S1P蛋白相对表达水平分别为1.21±0.10、0.57±0.05、0.73±0.06、0.58±0.05、1.39±0.11和0.39±0.03,S1PR1蛋白相对表达水平分别为0.45±0.03、0.83±0.07、0.64±0.05、0.81±0.07、0.28±0.02和1.03±0.07。模型组的上述指标与对照组比较,低、高剂量实验组、PF-543组、阳性药物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 KD-1可能通过抑制SphK1/S1P/S1PR1信号通路减轻流感病毒肺炎大鼠的肺损伤。

Objective To explore the effect of phillyrin(KD-1) on lung injury in rats with influenza virus pneumonia and its regulatory mechanism on the sphingosine kinases 1(SphK1)/sphingosine 1-phosphate(S1P)/S1P receptors 1(S1PR1) signal pathway.Methods Wistar male rats were divided into control group(gavage with equal amount of 0.9% NaCl),model group( gavage with equal amount of 0.9% NaCl),positive drug group(gavage with 0.02 g · kg^(-1) ribaverin),PF-543 group( gavage with 10 mg · kg^(-1) Sph K1 inhibitor PF-543 Citrate) and experimental-L,-H groups( gavage with 6.5,13 mg·kg-1KD-1,respectively).Except the control group,the other rats were treated with influenza virus nasal drip to establish influenza virus infection pneumonia model.The lung index of rats was measured;Hematoxylin-eosin( HE) staining was applied to observe the pathological damage of lung tissue in rats;the contents of interleukin 1β( IL-1β),tumor necrosis factor α( TNF-α) and IL-6 in bronchoalveolar lavage fluid( BALF) were detected by enzyme linked immunosorbent assay( ELISA);Western blot was applied to detect the expression levels of SphK1,S1P and S1PR1 proteins in rat lung tissue.Results The lung indices of experimental-L,-H groups,PF-543 group,positive drug group,model group and control group were( 7.62 ± 0.51),( 5.34 ± 0.46),( 6.53 ± 0.52),( 5.48 ± 0.43),( 12.46 ± 0.87) and( 4.41 ± 0.32) mg·g^(-1);IL-1β content were( 47.26 ± 2.05),( 25.18 ± 1.58),( 35.75 ± 1.50),( 27.31 ± 1.67),( 62.37 ± 2.51) and( 13.28 ± 1.04) ng· L^(-1);the contents of TNF-α were( 76.58 ± 4.73),( 51.82 ± 3.90),( 64.81 ± 4.15),( 53.06 ± 3.86),( 98.47 ± 4.92) and( 42.71 ± 3.52) ng · L^(-1);IL-6 content were( 57.62 ± 4.29),( 39.06 ± 3.86),( 48.75 ± 3.83),( 41.23 ± 3.61),( 76.92 ± 5.24) and( 28.56 ± 3.17) ng·L^(-1);SphK1 protein expression were 1.07 ± 0.08,0.51 ± 0.04,0.65 ± 0.05,0.53 ± 0.04,1.28 ± 0.09 and 0.36 ± 0.03;S1P protein expression were 1.21 ± 0.10,0.57 ± 0.05,0.73 ± 0.06,0.58 ± 0.05,1.39 ± 0.11 and 0.39 ± 0.03;S1PR1protein expression were 0.45 ± 0.03,0.83 ± 0.07,0.64 ± 0.05,0.81 ± 0.07,0.28 ± 0.02 and 1.03 ± 0.07,respectively.Compared with the control group,the above indexes in the model group had statistical significance( all P < 0.05);compared with the model group,the above indexes in experimental-L,-H groups,PF-543 group and positive drug group had statistical significance( all P < 0.05).Conclusion KD-1 may alleviate lung injury in rats with influenza virus pneumonia by inhibiting the SphK1/S1P/S1PR1 signal pathway.