黄芩素通过SIRT1通路调控OSA模型气道免疫炎症的机制研究

Mechanism of baicalein regulating airway immune inflammation in OSA model through SIRT1 pathway

摘要:

目的:探讨黄芩素通过沉默调节蛋白1(SIRT1)通路调控阻塞性睡眠呼吸暂停(OSA)模型气道免疫炎症的机制。方法:30只C57BL/6雌性小鼠随机分为5组:对照组、慢性间歇性缺氧(CIH)组、黄芩素-L组、黄芩素-H组、黄芩素+EX527组,每组6只。除对照组外,其他组模拟60次/h呼吸暂停,持续5天。黄芩素-L组、黄芩素-H组每天灌胃给药5 mg/kg黄芩素、20mg/kg黄芩素,连续治疗5 d。收集小鼠支气管肺泡灌洗液(BALF)。将RAW264.7巨噬细胞分为对照(Con)组、CIH组、黄芩素-L组、黄芩素-H组、黄芩素+EX527组。将各组细胞培养插入物与支气管上皮细胞BEAS-2B共培养,以模拟体内炎症对气道损伤的影响。采用酶联免疫吸附试验检测培养基和BALF中IL-1β、IL-5和IL-6水平。MTT法测定BEAS-2B细胞活力。蛋白质印迹法测定SIRT1、NF-κB p65(P65)、p-NF-κB p65(P-p65)蛋白表达。结果:与CIH组相比,黄芩素-L组和黄芩素-H组小鼠BALF中的总细胞、嗜酸性粒细胞、巨噬细胞、嗜中性粒细胞数目和IL-5、IL-1β、IL-6蛋白水平均显著降低(P＜0.05)。与黄芩素-H组相比,黄芩素+EX527组小鼠BALF中的总细胞、嗜酸性粒细胞、巨噬细胞、嗜中性粒细胞数目和IL-5、IL-1β、IL-6蛋白水平均显著增加(P＜0.05)。与CIH组相比,黄芩素-L组和黄芩素-H组RAW 264.7细胞培养上清液中IL-6和IL-1β蛋白浓度显著降低(P＜0.05)。然而,当施用EX-527抑制SIRT1时,黄芩素对RAW 264.7细胞释放IL-6和IL-1β的抑制作用减弱。共培养结果表明,CIH诱导的巨噬细胞炎症导致BEAS-2B细胞活力显著降低(P＜0.05),TUNEL阳性细胞数、P-p65/P65表达显著增加(P＜0.05)。与CIH组相比,黄芩素-L组和黄芩素-H组BEAS-2B细胞活力显著增加(P＜0.05),TUNEL阳性细胞数、P-p65/P65表达显著降低(P＜0.05)。与黄芩素-H组相比,黄芩素+EX527组BEAS-2B细胞活力显著降低(P＜0.05),TUNEL阳性细胞数、P-p65/P65表达显著增加(P＜0.05)。结论:黄芩素通过激活SIRT1来调节NF-κB通路,从而减少巨噬细胞的炎症反应并减轻BEAS-2B细胞损伤,改善OSA模型的肺损伤和气道炎症。

Abstract:

Objective To explore the mechanism of baicalein regulating airway immune inflammation in obstructive sleep apnea (OSA) model through 1(SIRT1 pathway. Methods Thirty C57BL/6 female mice were randomly divided into five groups: control group, chronic intermittent hypoxia (CIH) group, baicalein -L group, baicalein -H group and baicalein +EX527 group, with 6 mice in each group. Except for the control group, the other groups simulated 60 times /h apnea for 5 days. Baicalein -L group and baicalein -H group were given 5 mg/kg baicalein and 20mg/kg baicalein by gavage every day for 5 days. Bronchoalveolar lavage fluid (BALF) of mice was collected. RAW264.7 macrophages were divided into control group (Con), CIH group, baicalein -L group, baicalein -H group and baicalein +EX527 group. Cell culture inserts of each group were co-cultured with bronchial epithelial cells BEAS-2B to simulate the effect of inflammation on airway injury in vivo. The levels of IL-1β, IL-5 and IL-6 in culture medium and BALF were detected by enzyme-linked immunosorbent assay. The viability of BEAS-2B cells was determined by MTT assay. The expressions of SIRT1, NF-κB p65(P65) and p-NF-κB p65(P-p65) were determined by western blot. Results Compared with CIH group, the number of total cells, eosinophils, macrophages and neutrophils in BALF of baicalein -L group and baicalein -H group and the protein levels of IL-5, IL-1β and IL-6 decreased significantly (P < 0.05). Compared with baicalein -H group, the number of total cells, eosinophils, macrophages and neutrophils and the protein levels of IL-5, IL-1β and IL-6 in BALF of mice in baicalein +EX527 group increased significantly (P < 0.05). Compared with CIH group, the concentrations of IL-6 and IL-1β protein in the supernatant of RAW 264.7 cells in baicalein -L group and baicalein -H group decreased significantly (P < 0.05). However, when EX-527 was administered to inhibit SIRT1, baicalein inhibited the release of IL-6 and IL-1β from RAW 264.7 cells. The results of co-culture showed that the macrophage inflammation induced by CIH significantly decreased the activity of BEAS-2B cells (P < 0.05), and the number of TUNEL positive cells and the expression of P-p65/P65 increased significantly (P < 0.05). Compared with CIH group, the activity of BEAS-2B cells in baicalein -L group and baicalein -H group increased significantly (P < 0.05), and the number of TUNEL positive cells and the expression of P-p65/P65 decreased significantly (P < 0.05). Compared with baicalein -H group, BEAS-2B cells in baicalein +EX527 group decreased significantly (P < 0.05), while TUNEL positive cells and P-p65/P65 expression increased significantly (P < 0.05). Conclusions Baicalein regulates NF-κB pathway by activating SIRT1, thus reducing the inflammatory reaction of macrophages, alleviating the damage of BEAS-2B cells, and improving lung injury and airway inflammation in OSA model.