甘露消毒丹及其拆方对肺炎支原体干预的NR8383细胞炎症因子表达的影响

Effect of Ganlu Xiaodu Micropills and its decoction parts on the expression of inflammatory factors in NR8383 cells treated with Mycoplasma pneumoniae

目的研究甘露消毒丹及其拆方对肺炎支原体(MP)感染的大鼠肺泡巨噬细胞(NR8383)炎症因子表达的影响。方法选取35只8周龄SPF级雌性Wistar大鼠,体重(200±10)g,按随机数字表法将其分为空白血清组(生理盐水2 ml/d)、甘露消毒丹血清组[33 g/(kg·d)]、阿奇霉素血清组[0.063 g/(kg·d)]及拆方1血清组[12.3g/(kg·d)]、拆方2血清组[20.8 g/(kg·d)],每组7只。每组按剂量给药,1次/d,连续给药1周制备含药血清。将NR8383细胞分别使用空白血清和含药血清进行处理,使用CCK-8法检测并比较不同血清处理的NR8383细胞与未处理细胞活力的差异。将NR8383细胞分为对照组、模型组、甘露消毒丹组、阿奇霉素组及拆方1、2组,除对照组外,各组均用10^(6)~10^(7) CCU/ml浓度的MP菌液干预4 h以建立MP感染模型,随后加入相应含药血清孵育24 h。收集各组细胞及上清液,采用qPCR和蛋白质印迹法检测各组Toll样受体4(TLR4)、MyD88、核因子κB(NF-κB)mRNA和相应蛋白的表达情况,酶联免疫吸附试验检测各组肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-10的表达水平。结果各血清处理的细胞存活率均低于无处理的细胞(P<0.05)。各血清处理细胞存活率比较,差异无统计学意义(P>0.05)。模型组TLR4、MyD88、NF-κB-p50 mRNA表达及蛋白水平均高于对照组,甘露消毒丹组、拆方2组TLR4、MyD88、NF-κB-p50 mRNA表达及蛋白水平均低于模型组,拆方1组MyD88 mRNA表达及TLR4蛋白水平低于模型组(P<0.05)。甘露消毒丹组TLR4、MyD88 mRNA表达及TLR4、MyD88、NF-κB-p50蛋白水平低于拆方1组,NF-κB-p50 mRNA表达及TLR4蛋白水平高于拆方2组(P<0.05)。甘露消毒丹组、拆方2组TNF-α、IL-1β水平低于模型组,拆方1组IL-1β水平低于模型组,拆方2组IL-10低于模型组(P<0.05)。甘露消毒丹组TNF-α、IL-1β、IL-10水平均低于拆方1组,TNF-α、IL-1β水平高于拆方2组(P<0.05)。结论MP感染可促进NR8383细胞的TNF-α、IL-1β、IL-10表达,甘露消毒丹及其拆方含药血清可通过抑制TNF-α、IL-1β的表达水平减轻炎症反应,其机制可能与TLR4/MyD88/NF-κB通路有关。

Objective To study the effect of Ganlu Xiaodu Micropills and its decoction on the expression of inflammatory factors in rat alveolar macrophages(NR8383)infected with Mycoplasma pneumoniae(MP).Methods Thirty-five female Wistar rats of SPF grade aged eight weeks,weighing(200±10)g,were selected.According to the random number table method,they were divided into blank serum group(normal saline 2 ml/d),Ganlu Xiaodu Micropills serum group(33 g/[kg·d]),Azithromycin serum group(0.063 mg/[kg·d]),decoction part 1 serum group(12.3 g/[kg·d]),decoction part 2 serum group(20.8 g/[kg·d]),with seven rats in each group.Each group of doses was given once a day for one week to prepare drug-containing serum.NR8383 cells were treated with blank serum and drug-containing serum,respectively,CCK-8 assay was used to detect and compare the difference of viability between NR8383 cells treated with different serum and untreated cells.NR8383 cells were divided into control group,model group,Ganlu Xiaodu Micropills group,Azithromycin group,and decoction part 1,2 groups.Except for the control group,the other groups were treated with 10^(6)-10^(7) CCU/ml MP bacterial solution for four hours to establish MP infection model,and then the corresponding drug-containing serum was added and incubated for 24 hours.The cells and supernatant were collected,and qPCR and Western blot were used to detect the mRNA and protein expression of Toll-like receptor 4(TLR4),MyD88,and nuclear factorκB(NF-κB)in each group.The expression levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-10 in each group were detected by enzyme-linked immunosorbent assay.Results The survival rate of cells treated with each serum were lower than those of cells without treatment(P<0.05).There was no significant difference in the survival rate of cells treated with different serum(P>0.05).The expression levels of TLR4,MyD88,and NF-κB-p50 mRNA and protein in the model group were higher than those in the control group,and the expression levels of TLR4,MyD88,and NF-κB-p50 mRNA and protein in the Ganlu Xiaodu Micropills group and the decoction part 2 group were lower than those in the model group,the expression of MyD88 mRNA and TLR4 protein in the decoction part were lower than those in the model group(P<0.05).The mRNA expression of TLR4 and MyD88,and the protein levels of TLR4,MyD88,NF-κB-p50 in Ganlu Xiaodu Micropills group were lower than those in decoction part 1 group,and the mRNA expression of NF-κB-p50 and the protein level of TLR4 were higher than those in decoction part 2 group(P<0.05).The levels of TNF-αand IL-1βin Ganlu Xiaodu Micropills group and decoction part 2 group were lower than those in model group,the level of IL-1βin decoction part 1 group was lower than that in model group,and the level of IL-10 in decoction part 2 group was lower than that in model group(P<0.05).The levels of TNF-α,IL-1β,and IL-10 in Ganlu Xiaodu Micropills group were lower than those in decoction part 1 group,and the levels of TNF-αand IL-1βwere higher than those in decoction part 2 group(P<0.05).Conclusion MP infection can promote the expression of TNF-α,IL-1β,and IL-10 in NR8383 cells.Ganlu Xiaodu Micropills and its decoction containing serum can reduce the inflammatory response by inhibiting the expression of TNF-αand IL-1β,and the mechanism may be related to the TLR4/MyD88/NF-κB pathway.