基于代谢组学研究中药定喘汤对中性粒细胞型哮喘小鼠的疗效及作用机制

Study the mechanism of Dingchuan decoction in neutrophilic asthma mice on serum metabolomics

目的基于代谢组学的方法, 从内源性代谢物的变化分析定喘汤对中性粒细胞型哮喘的干预效果及其可能机制。方法 40只C57BL/6小鼠随机分为5组, 分别为正常组(A组)、中性粒细胞型哮喘模型组(B组)、定喘汤低剂量治疗组(C组)、定喘汤中剂量治疗组(D组)、定喘汤高剂量治疗组(E组), 每组8只。B、C、D、E组采用卵清白蛋白(OVA)和完全弗氏佐剂(CFA)联合诱导致敏小鼠建立中性粒细胞型哮喘模型, C、D、E组采用不同生药浓度的定喘汤进行干预治疗。采用Buxco小动物肺功能检测仪评估小鼠的气道反应性;提取肺泡灌洗液(BALF)采用计数板计数其白细胞总数, 细胞涂片染色进行分类计数;HE染色观察肺组织的病理改变。采用高效液相色谱联用四级杆飞行时间质谱(UPLC-Q-TOF-MS/MS)检测各组小鼠的血清代谢物, 采用常用的主成分分析(PCA), 偏最小二乘判别分析(PLS-DA)、正交偏最小二乘判别分析(OPLS-DA)模型对血清中代谢物图谱进行统计分析, 从内源性代谢物的变化来反映定喘汤的干预效果及其可能机制。结果 (1)一般行为观察:除A组小鼠外, 其他各组小鼠在激发期间均出现不同程度的哮喘症状。定喘汤各治疗组(C、D、E组)小鼠症状较B组程度减轻。(2)气道反应性:B组小鼠对乙酰甲胆碱(MCh)的气道反应性随着吸入浓度的升高而增加, 各浓度MCh水平下的气道阻力均明显高于A组(均P<0.01);C、D、E组气道反应性低于B组(均P<0.01);D、E组小鼠气道反应性低于C组(均P<0.05);D、E组气道反应性比较差异无统计学意义(P>0.05)。(3)气道炎症细胞浸润:B组小鼠BALF中白细胞总数、中性粒细胞百分率明显高于A组(均P<0.01);C、D、E组白细胞总数、中性粒细胞百分率低于B组(均P<0.01);D、E组小鼠白细胞总数、中性粒细胞百分率低于C组(均P<0.01);D、E组之间比较差异无统计学意义(均P>0.05)。(4)肺组织病理改变:A组小鼠肺组织未见病理改变。B组可见支气管管腔变狭窄, 管腔内黏膜皱褶增生、上皮细胞脱落、肿胀及可见黏液栓、肺泡及肺组织结构破坏, 支气管及血管周围大量炎症细胞浸润等典型病理学改变, 其中以中性粒细胞及淋巴细胞浸润最明显;定喘汤各治疗组肺组织结构破坏、支气管黏膜水肿及炎症细胞浸润较B组均明显改善, 其中D组小鼠肺组织病理改变相对较轻。(5)代谢组学分析:各组小鼠血清代谢物经PCA、PLS-DA以及OPLS-DA分析显示, A组和B组小鼠血清代谢物存在明显差异。通过代谢通路分析发现, 不同生药浓度的定喘汤干预治疗可以在不同程度上改善哮喘疾病所致的代谢紊乱。结论定喘汤能有效减轻中性粒细胞型哮喘小鼠的气道炎症、气道高反应, 有效调节中性粒细胞型哮喘所致的代谢异常。

Objective Based on the method of metabonomics,the intervention effect of Dingchuan decoction on neutrophilic asthma and its possible mechanism were analyzed from the changes of endogenous metabolites.Methods Forty C57BL/6 mice were randomly divided into five groups:normal group(group A),neutrophilic asthma model group(group B),Dingchuan decoction low dose treatment group(group C),Dingchuan decoction medium dose treatment group(group D),and Dingchuan decoction high dose treatment group(group E),with 8 mice in each group.B/C/D/E group used ovalbumin(OVA)and complete Freund′s adjuvant(CFA)to induce sensitized mice to establish neutrophilic asthma model,and C/D/E group used Dingchuan decoction with different concentrations of crude drugs for intervention treatment.Buxco small animal lung function tester was used to evaluate the airway reactivity of mice;The total number of white blood cells in bronchoalveolar lavage fluid(BALF)was counted by counting plate,and the number was classified by cell smear staining;The pathological changes of lung tissue were observed by hematoxylin and eosin(HE)staining.The serum metabolites of mice in each group were detected by high performance liquid chromatography coupled with four pole time of flight mass spectrometry(UPLC-Q-TOF-MS/MS).The commonly used principal component analysis(PCA),partial least squares discriminant analysis(PLS-DA),orthogonal partial least squares discriminant analysis(OPLS-DA)models were used for statistical analysis of the metabolite profiles in serum.The intervention effect of Dingchuan decoction and its possible mechanism were reflected from the changes of endogenous metabolites.Results(1)General behavior observation:except mice in group A,mice in other groups showed asthma symptoms of different degrees during the challenge period.The symptoms of mice in each treatment group(group C,D,E)of Dingchuan decoction were less than those in group B.(2)Airway reactivity:the airway reactivity of mice in group B to methacholine(MCh)increased with the inhalation concentration,and the airway resistance at each concentration of MCh was significantly higher than that in group A(all P<0.01);the airway reactivity in group C,D and E was lower than that in group B(all P<0.01);the airway reactivity in group D and E was lower than that in group C(all P<0.05).There was no significant difference in airway reactivity between group D and E(P>0.05).(3)Airway inflammatory cell infiltration:the total number of white blood cells(WBC)and percentage of neutrophil in BALF of group B were significantly higher than those of group A(all P<0.01).The total WBC and percentage of neutrophil in group C,D and E were lower than those in group B(all P<0.01).The total number of WBC and percentage of neutrophil in group D and E were lower than those in group C(all P<0.01).There was no significant difference between group D and group E(all P>0.05).(4)Pathological changes of lung tissues:no pathological changes were observed in the lung tissues of group A mice.In group B,typical pathological changes such as bronchial lumen stenosis,intraluminal mucosal folds hyperplasia,epithelial cell exfoliation,swelling,mucous embolus,alveolar and lung tissue structure destruction,massive inflammatory cell infiltration around bronchus and blood vessels were observed,among which neutrophil and lymphocyte infiltration were the most obvious.The damage of lung tissue structure,bronchial mucosa edema and inflammatory cell infiltration in Dingchuan decoction treatment groups were significantly improved compared with group B,and the pathological changes of lung tissue in group D were relatively light.(5)Metabolomics analysis:PCA,PLS-DA and OPLS-DA analysis of serum metabolites in each group showed that serum metabolites in group A and group B were significantly different.The metabolic pathway analysis showed that Dingchuan decoction with different crude drug concentrations could improve the metabolic disorders caused by asthma in different degrees.Conclusions Dingchuan decoction can effectively reduce airway inflammation and airway hyperresponsiveness in mice with neutrophil asthma,and effectively regulate metabolic abnormalities caused by neutrophil asthma.