黄芩苷通过p38 MAPK/NLRP3通路对脂多糖诱导大鼠急性肺损伤的影响

Baicalin Alleviates LPS-Induced Acute Lung Injury in Rats Through p38 MAPK/NLRP3 Pathway

目的:观察黄芩苷对脂多糖(LPS)诱导急性肺损伤模型大鼠的疗效及相关作用机制。方法:采用将80只健康雄性SD大鼠随机分为正常组,模型组,黄芩苷低、中、高剂量组,地塞米松组(DEX),SB203580组和黄芩苷+SB203580组(BA+SB203580),每组10只。黄芩苷低、中、高剂量组分别腹腔注射不同剂量(10,50,100 mg·kg^(-1))的BA溶液;DEX组腹腔注射5 mg·kg^(-1)的DEX溶液;SB203580组腹腔注射0.5 mg·kg^(-1)的SB203580溶液;黄芩苷+SB203580组腹腔注射100 mg·kg^(-1)的BA溶液和0.5 mg·kg^(-1)的SB203580溶液;正常组和模型组均注射等体积的生理盐水,每天给药1次,连续7 d,末次给药1 h后,除正常组,其余大鼠均给予气管滴注5 mg·kg^(-1)LPS构建急性肺损伤模型,正常组大鼠气管滴注等体积的生理盐水溶液,12 h后结束实验,取材。苏木素-伊红(HE)染色观察肺组织病理学改变,进行支气管肺泡灌洗液(BALF)中细胞总数和中性粒细胞计数;测定肺组织湿/干重比、血清中超氧化物歧化酶(SOD)和丙二醛(MDA)的水平;免疫荧光法检测肺组织中活性氧(ROS)的含量,酶联免疫吸附测定法(ELISA)测定BALF中细胞因子白细胞介素-1β(IL-1β),白细胞介素-18(IL-18),白细胞介素-6(IL-6),肿瘤坏死因子-α(TNF-α)的含量;免疫组化(IHC)检测p-p38丝裂原活化蛋白激酶(MAPK)的表达的定位表达及蛋白免疫印迹法(Western blot)检测肺组织中p-p38 MAPK,硫氧还蛋白互作蛋白(TXNIP),NOD样受体蛋白3(NLRP3),半胱氨酸天冬氨酸蛋白水解酶-1(Caspase-1)蛋白表达情况。结果:与正常组比较,模型组大鼠肺组织出现炎性病理变化,肺湿/干质量,BALF中细胞总数及中性粒细胞数目增高(P<0.01),SOD活性下降(P<0.01),ROS和MDA含量升高(P<0.01),细胞因子IL-1β,IL-18,IL-6,TNF-α和p-p38 MAPK,NLRP3,Caspase-1的表达量均上调(P<0.01)。与模型组比较,黄芩苷组,SB203580组和黄芩苷+SB203580组可有效减轻LPS所致肺组织病理变化,降低肺湿/干质量,BALF中细胞总数及中性粒细胞数目(P<0.05,P<0.01),升高SOD活性(P<0.05,P<0.01),并降低ROS和MDA水平(P<0.05,P<0.01),细胞因子IL-1β,IL-18,IL-6,TNF-α和p-p38 MAPK,NLRP3,Caspase-1的表达量均明显下降(P<0.05,P<0.01)。结论:黄芩苷可有效保护LPS所致急性肺损伤,其作用机制可能与抑制p38 MAPK/NLRP3信号通路减轻炎症反应有关。

Objective:To investigate the effects and mechanism of baicalin(BA)on lipopolysaccharide(LPS)-induced acute lung injury in rats.Method:Eighty healthy male SD rats were randomly divided into the control group,model group,low-dose BA(BA-L)group,medium-dose BA(BA-M)group,high-dose BA(BA-H)group,dexamethasone(DEX)group,SB203580 group,and BA+SB203580 group,with 10 rats in each group.The rats in the BA-L,BA-M,and BA-H groups were injected intraperitoneally with different doses(10,50,100 mg·kg^(-1))of BA solution,the ones in the DEX group with 5 mg·kg^(-1)DEX solution,the ones in the SB203580 group with 0.5 mg·kg^(-1)SB203580 solution,the ones in the BA+SB203580 group with 100 mg·kg^(-1)BA solution and 0.5 mg·kg^(-1)SB203580,and those in both the control group and model group with the same volume of normal saline,once per day,for seven successive days.One hour after the last administration,rats in all groups except for the control group were given 5 mg·kg^(-1)LPS via intratracheal instillation for inducing the acute lung injury,whereas those in the control group received the same volume of normal saline solution.Twelve hours later,the lung tissues were sampled and stained with htoxylineosin(HE)for observing the pathological changes,followed by the counting of the total number of cells and neutrophils in bronchoalveolar lavage fluid(BALF).The wet/dry weight ratio of the lung tissue and the contents of serum superoxide dismutase(SOD)and malondialdehyde(MDA)were measured.The activity of reactive oxygen species(ROS)in the lung tissue was detected by immunofluorescence and the levels of interleukin-1β(IL-1β),interleukin-18(IL-18),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in BALF by enzyme-linked immunosorbent assay(ELISA).Immunohistochemistry(IHC)was conducted to determine the relative expression of p-p38 mitogen-activated protein kinase(MAPK)and Western blotting was carried out to detect the protein expression levels of p-p38 MAPK,thioredoxin interacting protein(TXNIP),NOD-like receptor protein 3(NLRP3),and cysteinyl aspartate specific protease-1(Caspase-1)in the lung tissue.Result:Compared with the control group,the model group displayed inflammatory pathological changes in lung tissue,elevated wet/dry weight ratio,total number of cells and neutrophils in BALF,and ROS and MDA levels(P<0.01),decreased SOD activity(P<0.01),and up-regulated IL-1,IL-18,IL-6,TNF-α,p-p38 MAPK,NLRP3,and Caspase-1 expression(P<0.01).Compared with the model group,BA at different doses,SB203580,and BA+SB203580 all effectively alleviated the pathological changes in lung tissue induced by LPS,reduce the lung wet/dry weight ratio,the total number of cells and neutrophils in BALF,and ROS and MDA levels(P<0.05,P<0.01),enhanced the activity of SOD(P<0.05,P<0.01),and down-regulated the expression of IL-1β,IL-18,IL-6,TNF-α,p-p38 MAPK,NLRP3,and Caspase-1 in lung tissue(P<0.05,P<0.01).Conclusion:BA has a protective effect against LPS-induced acute lung injury,which may be related to its inhibition of p38MAPK/NLRP3 signaling pathway and the improvement of inflammatory response.