库尔勒香梨中性多糖通过调节Nrf2/NF-κB/NLRP3改善小鼠过敏性哮喘研究

Study on the effect of neutral polysaccharide of Pyrus sinkiangensis Yu on allergic asthma in mice by regulating Nrf2/NF-κB/NLRP3

关键词:  库尔勒香梨中性多糖  过敏性哮喘  氧化应激  气道炎症

Key words:  Pyrus sinkiangensis Yu. neutral polysaccharide  allergic asthma  oxidative stress  airway inflammatio

摘要:

摘 要：目的：研究库尔勒香梨中性多糖（Pyrus sinkiangensis Yu. neutral polysaccharide, PSNP-1、PSNP-2）对卵清蛋白（Ovalbumin，OVA）诱导的急性过敏性哮喘小鼠的影响及其机制。方法：108 只 BALB/c 小鼠随机分成正常组，模型组，地塞米松阳性药组，PSNP-1低（10 mg/kg）、中（20 mg/kg）、高（40 mg/kg）剂量组，PSNP-2低、中、高剂量组，每组12只。腹腔注射含卵清蛋白（OVA, 98%）和氢氧化铝混合溶液进行致敏，5％ OVA溶液连续雾化激发7 d，药物干预7 d。取材支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF），ELISA检测、生化检测、HE、PAS及Masson染色，qRT-PCR检测肺组织中 TLR4、NLRP3、IL-18、IL-1β、Caspase-1、GSDMD基因mRNA表达水平；免疫组织化学染色法检测小鼠肺组织中NLRP3、Caspase-1、GSDMD、ASC、Nrf2、HO-1、NF-κBp65、IκBα蛋白表达水平；Western blot法检测肺组织中NLRP3、Caspase-1、ASC、GSDMD-N蛋白表达水平。结果：与模型组相比，PSNP-1、PSNP-2对哮喘小鼠肺脏支气管的炎细胞浸润，粘液分泌、纤维化以及支气管上皮变性、增生等病理学改变有所缓解，并显著调节炎症细胞因子、氧化因子释放水平，qRT-PCR结果显示，TLR4、NLRP3、IL-18、IL-1β、Caspase-1、GSDMDmRNA表达水平显著下降（P<0.05）。免疫组化结果显示小鼠肺组织中NF-κBp65、NLRP3、Caspase-1、GSDMD、ASC、下降（P<0.05），IκBα、Nrf2、HO-1蛋白表达水平显著升高（P<0.05）。Western blot结果显示肺组织中NLRP3、Caspase-1、ASC、GSDMD-N蛋白表达水平显著下降（P<0.05），IκBα蛋白表达水平显著升高（P<0.05）。结论：PSNP-1、PSNP-2可有效改善哮喘小鼠气道炎症、氧化应激，其机制可能与Nrf2通路所介导的氧化应激，NF-κB/NLRP3通路调节的炎症免疫损伤有关。

Abstract:

ABASTACT: ObJECTIVE: To study the effects of Pyrus sinkiangensis Yu. neutral polysaccharide (PSNP-1, PSNP-2) on Ovalbumin (OVA) -induced acute allergic asthma in mice and its mechanism.METHODS: 108 BALB/c mice were randomly divided into normal group, model group, dexamethasone positive group, PSNP-1 low (10 mg/kg), medium (20 mg/kg) and high (40 mg/kg) dose groups, and PSNP-2 low, medium and high dose groups, with 12 mice in each group. A mixture of ovalbumin (OVA, 98%) and aluminum hydroxide was injected into the abdomen for sensitization. The 5% OVA solution was continuously atomized for 7 days, and the drug intervention lasted for 7 days.bronchoalveolar lavage fluid (BALF) was collected for ELISA, biochemical detection, HE, PAS and Masson staining. The mRNA expression levels of TLR4, NLRP3, IL-18, IL-1β, Caspase-1 and GSDMD in lung tissues were detected by qRT-PCR. The expression levels of NLRP3, Caspase-1, GSDMD, ASC, Nrf2, HO-1, NF-κBp65, IκBα in lung tissues were detected by immunohistochemical staining. The expression levels of NLRP3, Caspase-1, ASC and GSDMD-N in lung tissues were detected by Western blot.RESULTS:Compared with the model group, PSNP-1 and PSNP-2 alleviated pathological changes such as inflammatory cell infiltration, mucus secretion, fibrosis and bronchial epithelial degeneration and hyperplasia in asthmatic mice, and significantly regulated the release levels of inflammatory cytokines and oxidizing factors. qRT-PCR results showed that, The mRNA expression levels of TLR4, NLRP3, IL-18, IL-1β, Caspase-1 and GSDMD were significantly decreased (P<0.05). Immunohistochemical results showed that NF-κB p65, NLRP3, Caspase-1, GSDMD, ASC, were decreased (P<0.05), and the expression levels of IκBα, Nrf2, HO-1 were significantly increased (P<0.05). Western blot results showed that the protein expression levels of NLRP3, Caspase-1, ASC and GSDMD-N were significantly decreased (P<0.05), and the protein expression levels of IκBα were significantly increased (P<0.05).CONCLUSION:PSNP-1 and PSNP-2 can effectively improve airway inflammation and oxidative stress in asthmatic mice, and the mechanism may be related to oxidative stress mediated by Nrf2 pathway and inflammatory immune damage regulated by NF-κB/NLRP3 pathway.