虫草素对脂多糖诱导的急性肺损伤模型大鼠的保护作用及TLR4/NF-κB通路的影响

Protective effect of cordycepin on lipopolysaccharide-induced acute lung injury in a rat model by regulation of the TLR4/NF-κB pathway activity

目的探讨虫草素对脂多糖诱导的急性肺损伤(ALI)大鼠的保护作用及Toll样受体4(TLR4)/核因子-κB(NF-κB)通路的影响。方法SPF级成年SD大鼠60只,12只大鼠作为对照组;48只通过脂多糖诱导建立ALI模型大鼠,随机分成模型组、虫草素低(5 mg/kg)、中(10 mg/kg)、高(20 mg/kg)剂量组,每组12只。虫草素各剂量组大鼠腹腔注射相应剂量的虫草素,模型组和对照组大鼠腹腔注射等量生理盐水,连续给药1周(1次/d)。检测大鼠动脉血氧分压(PaO_(2))、二氧化碳分压(PaCO_(2))、肺组织肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、TLR4、NF-κB信使核糖核酸(mRNA)和蛋白水平,观察肺组织形态学并进行肺损伤评分。结果对照组大鼠肺泡组织结构正常;模型组肺泡组织受损,毛细血管扩张,红细胞渗漏和肺组织液分泌,可见炎症细胞浸润;虫草素干预后,肺泡组织趋于正常,肺泡壁变薄,红细胞、分泌液减少,炎症细胞浸润减少。与对照组比较,模型组大鼠PaO_(2)水平[(52.97±4.13)mmHg比(90.05±7.09)mmHg,P<0.05]降低,PaCO_(2)[(48.03±7.56)mmHg比(29.13±4.01)mmHg,P<0.05]、肺损伤评分[(9.58±1.03)分比(0.00±0.00)分,P<0.05]、肺组织TNF-α[(30.16±8.12)pg/mg比(19.02±4.03)pg/mg,P<0.05]、IL-6[(25.47±7.16)pg/mg比(9.03±1.95)pg/mg,P<0.05]、IL-1β[(77.51±16.34)pg/mg比(40.22±9.83)pg/mg,P<0.05]、TLR4、NF-κB mRNA[TLR4 mRNA:(3.26±0.52)比(1.00±0.02),NF-κB mRNA:(3.38±0.47)比(1.00±0.03),P均<0.05]和蛋白[TLR4蛋白:(1.09±0.15)比(0.31±0.02),NF-κB蛋白:(1.43±0.22)比(0.21±0.03),P均<0.05]水平升高。与模型组比较,虫草素低、中、高剂量组大鼠PaO_(2)水平[(63.11±5.02)mmHg、(72.28±5.63)mmHg、(80.16±6.50)mmHg比(52.97±4.13)mmHg,P均<0.05]依次升高,PaCO_(2)[(42.24±6.90)mmHg、(38.55±5.28)mmHg、(33.92±4.77)mmHg比(48.03±7.56)mmHg,P均<0.05],肺损伤评分[(6.92±0.87)分、(4.03±0.61)分、(2.19±0.33)分比(9.58±1.03)分,P均<0.05],肺组织TNF-α[(27.09±7.05)pg/mg、(25.01±6.52)pg/mg、(22.93±5.14)pg/mg比(30.16±8.12)pg/mg,P均<0.05],IL-6[(20.59±5.08)pg/mg、(17.02±4.07)pg/mg、(13.28±3.13)pg/mg比(25.47±7.16)pg/mg,P均<0.05],IL-1β[(60.44±13.05)pg/mg、(57.17±11.02)pg/mg、(50.23±10.27)pg/mg比(77.51±16.34)pg/mg,P均<0.05],TLR4、NF-κB mRNA[TLR4 mRNA:(2.57±0.31)、(2.03±0.18)、(1.61±0.10)比(3.26±0.52),NF-κB mRNA:(2.74±0.40)、(2.05±0.26)、(1.43±0.12)比(3.38±0.47),P均<0.05],TLR4和NF-κB蛋白[TLR4蛋白:(0.92±0.10)、(0.73±0.08)、(0.56±0.05)比(1.09±0.15),NF-κB蛋白:(1.05±0.16)、(0.86±0.11)、(0.50±0.07)比(1.43±0.22),P均<0.05]水平依次降低。结论虫草素可改善ALI大鼠肺功能和肺损伤,降低肺部炎症反应,其机制可能与虫草素抑制ALI大鼠肺组织TLR4、NF-κB mRNA和蛋白表达进而抑制TLR4/NF-κB通路的激活有关。

Objective To investigate the effects of cordycepin on lipopolysaccharide-induced acute lung injury(ALI)in an animal model and the underlying molecular events.Methods A total of 60 adult Sprague-Dawley rats in specific pathogen-free(SPF)condition were divided into control,ALI model,ALI+cordycepin low(5 mg/kg),ALI+medium(10 mg/kg)and ALI+high(20 mg/kg)dose groups(n=12 per group).The model rats were established by intraperitoneal injection of lipopolysaccharide and randomly divided into model and treatment groups.After that,the rats were treated with daily intraperitoneal injection of the corresponding doses of cordycepin for one week,the control rats were injected with same amount of saline.The arterial partial pressure of oxygen(PaO_(2)),partial pressure of carbon dioxide(PaCO_(2)),and mRNA and protein levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β),Toll like receptor 4(TLR4)/nuclear factor-κB(NF-κB)in lung tissue were measured.Lung histology was assessed for lung injury score.Results The control group of rats had normal alveolar structure,whereas the model rats showed damaged alveolar sac,diluted capillaries with leakage of the red blood cells,and lung tissue fluid secretion,as well as the inflammatory cell infiltration.However,after treatment with cordycepin,the alveolar sac became normal,the alveolar wall was thinner with reduced red blood cells and fluid secretion as well as a few of inflammatory cell infiltration.Compared with the control rats,level of PaO_(2)(52.97±4.13 vs.90.05±7.09 mmHg;P<0.05)was decreased in the model rats,whereas levels of PaCO_(2)(48.03±7.56 vs.29.13±4.01 mmHg;P<0.05),lung injury score(9.58±1.03 vs.0.00±0.00;P<0.05),TNF-α(30.16±8.12 vs.19.02±4.03 pg/mg;P<0.05),IL-6(25.47±7.16 vs.9.03±1.95 pg/mg;P<0.05),IL-1β(77.51±16.34 vs.40.22±9.83 pg/mg;P<0.05),TLR4(3.26±0.52 vs.1.00±0.02;P<0.05),and NF-κB mRNA(3.38±0.47 vs.1.00±0.03;P<0.05)and TLR4(1.09±0.15 vs.0.31±0.02;P<0.05)and NF-κB proteins(1.43±0.22 vs.0.21±0.03;P<0.05)were all increased in lung tissues of the model rats.In contrast,compared with the model rats,the PaO_(2) levels(63.11±5.02,72.28±5.63and 80.16±6.50 vs.52.97±4.13 mmHg;all P<0.05)were increased in low,medium and high doses of cordycepin treated rats,respectively,whereas levels of PaCO_(2)(42.24±6.90,38.55±5.28,and 33.92±4.77 vs.48.03±7.56 mmHg;all P<0.05),lung injury score(6.92±0.87,4.03±0.61,and 2.19±0.33 vs.9.58±1.03;all P<0.05),TNF-α(27.09±7.05,25.01±6.52,and 22.93±5.14 vs.30.16±8.12 pg/mg;All P<0.05),IL-6(20.59±5.08,17.02±4.07,and 13.28±3.13 vs.25.47±7.16 pg/mg;All P<0.05),IL-1β(60.44±13.05,57.17±11.02,and 50.23±10.27 vs.77.51±16.34 pg/mg;All P<0.05),TLR4(2.57±0.31,2.03±0.18,and 1.61±0.10 vs.3.26±0.52;P<0.05),and NF-κB mRNA(2.74±0.40,2.05±0.26,and 1.43±0.12 vs.3.38±0.47;P<0.05)and TLR4(0.92±0.10,0.73±0.08,and 0.56±0.05 vs.1.09±0.15)and NF-κB proteins(1.05±0.16,0.86±0.11,and 0.50±0.07 vs.1.43±0.22;P<0.05)were all decreased in lung tissues of low,medium and high doses of cordycepin treated rats,respectively.Conclusion Cordycepin was able to protect lung functions and injuries by reduction of lung inflammatory response in ALI rats and the underlying mechanism could be related to cordycepin inhibition of TLR4 and NF-κB expression in lung tissues of ALI rats.