Background
Excess mucus production is an important pathophysiological feature of chronic inflammatory airway diseases. Effective therapies are currently lacking. The
Conclusion
CUR inhibits LPS-induced airway mucus hypersecretion and inflammation through activation of Nrf2 possibly. 姜黄素通过核因子E2相关因子2抑制脂多糖诱导气道黏蛋白5AC表达及气道炎症摘要背景：气道黏液高分泌是慢性气道炎症性疾病重要的病理生理特征，目前仍然缺乏有效的治疗手段。本研究旨在阐述姜黄素(curcumin, CUR)在脂多糖（lipopolysaccharide，LPS）诱导气道黏蛋白Mucin 5AC (MUC5AC) 表达中的调控作用，并从体内外试验探讨其作用机制。 方法：体外试验中，气道上皮细胞(NCI-H292)预先加入姜黄素干预30分钟后，再予脂多糖（LPS）刺激24小时。接着，使用Nrf2 siRNA干扰NCI-H292细胞Nrf2表达，探讨转录因子核因子E2相关因子2(Nuclear factor erythroid 2-related factor 2, Nrf2)的作用。体内试验中，C57BL/6小鼠随机分为3组：空白对照组、LPS干预组和LPS+CUR组(每组7只)。小鼠经气道滴注LPS（100μg/ml）前，预先经腹腔注射生理盐水或姜黄素（50mg/kg）。使用实时荧光定量PCR和Western blotting检测细胞裂解液和肺组织MUC5AC和Nrf2的mRNA和蛋白表达量。收集小鼠肺泡灌洗液分类计数总细胞和中性粒细胞、肺组织HE染色了解小鼠肺组织炎症情况。数据使用ANOVA和t检验分析，以均数±标准差表示。 结果：姜黄素显著抑制LPS诱导NCI-H292细胞黏蛋白基因MUC5AC表达，MUC5AC mRNA和蛋白表达量分别为2.424 ± 0.318、1.060 ± 0.197，均低于LPS干预组的7.169±1.785和2.340±0.209（t=4.534和t=7.716, p均<0.05）；姜黄素可诱导转录因子Nrf2表达，mRNA和蛋白表达量分别为1.952 ± 0.340和2.010 ± 0.209，均高于LPS干预组的1.142±0.176和1.089±0.132（t=-3.661和t=-6.453, p均<0.05）。Nrf2 siRNA抑制Nrf2表达后，姜黄素抑制黏蛋白MUC5AC表达的作用被削弱47.7%，CUR+LPS+Nrf2 siRNA组MUC5AC表达量高于CUR+LPS+阴性对照siRNA组，mRNA表达量分别为6.845 ± 1.478和3.391 ± 0.517（t=-3.821, p<0.05）。姜黄素同样抑制LPS诱导小鼠肺组织黏液表达、上调Nrf2表达，并抑制小鼠肺组织炎症。LPS+CUR组小鼠肺组织MUC5AC mRNA和蛋白表达量为1.672±0.721和0.480 ± 0.191，均低于LPS组的5.961 ± 2.452和2.290 ± 0.834（t=2.906和t=3.665, p均<0.05）；PAS染色见该组小鼠肺组织粉红染颗粒较LPS组少。LPS+CUR组小鼠肺泡灌洗液细胞总数和中性粒细胞数分别为247±30和185±22，低于LPS组的334±24和246±20（t=3.901和t=3.56, p均<0.05）；HE染色镜下见LPS+CUR组小鼠肺泡腔内及周围浸润的炎症细胞明显减少。 结论：姜黄素可能通过激活Nrf2抑制脂多糖诱导气道黏蛋白基因MUC5AC表达和气道炎症。.
Methods
For the in vitro study, human bronchial epithelial (NCI-H292) cells were pretreated with CUR or vehicle for 30 min, and then exposed to LPS for 24 h. Next, nuclear factor erythroid 2-related factor 2 (Nrf2) was knocked down with Nrf2 small interfering RNA (siRNA) to confirm the specific role of Nrf2 in mucin regulation of CUR in NCI-H292 cells. In vivo, C57BL/6 mice were randomly assigned to three groups (n = 7 for each group): control group, LPS group, and LPS + CUR group. Mice in LPS and LPS + CUR group were injected with saline or CUR (50 mg/kg) intraperitoneally 2 h before intratracheal instillation with LPS (100 μg/ml) for 7 days. Cell lysate and lung tissue were obtained to measured Mucin 5AC (MUC5AC) and Nrf2 mRNA and protein expression by a real-time polymerase chain reaction and Western blotting. Bronchoalveolar lavage fluid (BALF) was collected to enumerate total cells and neutrophils. Histopathological changes of the lung were observed. Data were analyzed by one-way analysis of variance. Student's t-test was used when two groups were compared.
Results
CUR significantly decreased the expression of MUC5AC mRNA and protein in NCI-H292 cells exposed to LPS. This effect was dose dependent (2.424 ± 0.318 vs. 7.169 ± 1.785, t = 4.534, and 1.060 ± 0.197 vs. 2.340 ± 0.209, t = 7.716; both P < 0.05, respectively) and accompanied by increased mRNA and protein expression of Nrf2 (1.952 ± 0.340 vs. 1.142 ± 0.176, t = -3.661, and 2.010 ± 0.209 vs. 1.089 ± 0.132, t = -6.453; both P < 0.05, respectively). Furthermore, knockdown of Nrf2 with siRNA increased MUC5AC mRNA expression by 47.7%, compared with levels observed in the siRNA-negative group (6.845 ± 1.478 vs. 3.391 ± 0.517, t = -3.821, P < 0.05). Knockdown of Nrf2 with siRNA also markedly increased MUC5AC protein expression in NCI-H292 cells. CUR also significantly decreased LPS-induced mRNA and protein expression of MUC5AC in mouse lung (1.672 ± 0.721 vs. 5.961 ± 2.452, t = 2.906, and 0.480 ± 0.191 vs. 2.290 ± 0.834, t = 3.665, respectively; both P < 0.05). Alcian blue/periodic acid-Schiff staining also showed that CUR suppressed mucin production. Compared with the LPS group, the numbers of inflammatory cells (247 ± 30 vs. 334 ± 24, t = 3.901, P < 0.05) and neutrophils (185 ± 22 vs. 246 ± 20, t = 3.566, P < 0.05) in BALF decreased in the LPS + CUR group, as well as reduced inflammatory cell infiltration in lung tissue.