Protective Effects of Xiyanping Injection on Lipopolysaccharide-induced Acute Lung Injury in Mice
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摘要:
目的 探讨喜炎平注射液对脂多糖（lipopolysaccharide，LPS）诱导的急性肺损伤（acute lung injury，ALI）小鼠的保护作用，初步探讨其作用机制。 方法 将120只小鼠随机分为正常对照组（NS）、ALI模型组（LPS）、喜炎平注射液低剂量组（XYP-L，18.96 mg/kg）、中剂量组（XYP-M，37.92 mg/kg）、高剂量组（XYP-H，75.83 mg/kg）和地塞米松阳性药组（DXM，3.03 mg/kg），共6组，每组20只。每天固定时间点给予各药物组腹腔注射对应药物，NS组和LPS组给予腹腔注射等体积的0.9%氯化钠注射液，每天腹腔注射给药1次，连续3 d。末次给药1 h后，NS组气管滴注等量生理盐水，其余各组气管滴注LPS。造模24 h后，收集小鼠肺组织标本和支气管肺泡灌洗液，计算肺系数。肉眼观察肺组织大体病理表现及显微镜下观察组织切片病理形态学改变，在显微镜下计数肺泡灌洗液（broncho alveolar lavage fluid，BALF）中总细胞数，二喹啉甲酸（bicin-choninic acid，BCA）法检测BALF中总蛋白含量，酶联免疫吸附分析（enzyme linked immunosorbent assay，ELISA）法检测BALF中炎症细胞因子肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-8（IL-8）的含量，丙二醛（malondialdehyde ，MDA）和超氧化物歧化酶（superoxide dismutase，SOD）试剂盒检测小鼠肺组织中MDA含量和SOD的活力。 结果 与NS组比较，LPS组小鼠肺组织病理损伤严重，肺系数、BALF总细胞数、总蛋白浓度、TNF-α、IL-1β、IL-8含量明显升高（P < 0.001），肺组织MDA含量明显升高（P < 0.001）、SOD活力明显降低（P < 0.001）。与LPS组比较，给药组小鼠肺组织损伤均有所改善，肺系数、BALF总细胞数、总蛋白浓度、TNF-α、IL-1β、IL-8含量明显降低（P < 0.05），肺组织中MDA含量明显降低（P < 0.05）、SOD活力明显升高（P < 0.05）。 结论 喜炎平注射液对LPS诱导的小鼠ALI有一定的防治作用，其作用机制与抗炎和抗氧化有关，可通过下游炎症因子的释放来缓解肺部炎症，同时通过抗氧化作用抑制LPS引起的肺组织氧化应激。 Abstract:Objective To explore the protective effects of Xiyanping injection against lipopolysaccharide （lipopolysaccharide, LPS）-induced acute lung injury （acute lung injury, ALI）in mice, and preliminarily investigate its mechanism. Methods The mice were randomly divided into the normal control group （NS）, ALI model group （LPS）, Xiyanping injection low dosage group （XYP-L, 18.96 mg/kg）, medium dosage group （XYP-M, 37.92 mg/kg）, and high dosage group （XYP-H, 75.83 mg/kg） and dexamethasone positive drug group （DXM, 3.03 mg/kg）, with 20 in each group. Each group was injected intraperitoneally with the corresponding drug, once a day at the same time for 3 consecutive days. the NS and LPS groups were given the same 0.9% sodium chloride injection volume. Except for NS group, LPS was instilled into the trachea to induce the acute lung injury in mice 1 hour after the last administration of the above drugs. The lung tissue samples and broncho alveolar lavage fluid （BALF） samples were collected 24 hours after the intratracheal instillation of LPS. The lung coefficient was calculated. The pathological and morphological changes of lung tissue were observed macroscopically and microscopially. The total cell count in BALF was calculated under the microscope. The concentration of total protein in BALF was measured using the BCA （bicin-choninic acid） method. The content of inflammatory cytokines tumor necrosis factor-α（TNF-α）, interleukin-1β（IL-1β）, and interleukin-8 （IL-8）in BALF was detected using the ELISA （enzyme linked immunosorbent assay） method. The levels of malondialdehyde （MDA） and superoxide dismutase （SOD） in mouse lung tissue were measured using MDA and SOD assay kits. Results Compared with the NS group, the LPS group showed severe pathological damage in the lung tissue, increased lung coefficient, total cell count, total protein concentration, and elevated levels of TNF-α, IL-1β, and IL-8 in BALF （P < 0.01）. The LPS group also exhibited the higher levels of MDA in the lung tissue （P < 0.001）, decreased SOD activity （P < 0.001）. The treatment groups showed the improvements in the lung tissue damage compared to the LPS group. The treatment groups exhibited the decreased lung coefficient, total cell count, total protein concentration, TNF-α, IL-1β, and IL-8 levels in BALF （P < 0.05）, reduced MDA content in lung tissue （P < 0.001）, and increased SOD activity （P < 0.05）. Conclusions Xiyanping injection has a certain protective effect on LPS-induced ALI in mice, and its mechanism is related to anti-inflammatory and antioxidant effects. It can alleviate the lung inflammation through inhibiting the release of downstream inflammatory factors and oxidative stress in the lung tissue caused by LPS through antioxidant effects. -
图 1 肺组织大体病理和显微病理改变（HE，200×）
NS：正常对照组；LPS：ALI模型组；XYP-L：喜炎平注射液低剂量组；XYP-M：喜炎平注射液中剂量组；XYP-H：喜炎平注射液高剂量组；DXM：地塞米松阳性药组。
Figure 1. Gross and microscopic pathological changes of lung tissues （HE，200×）
图 2 各组小鼠平均体重和肺系数（$\bar x \pm s $，n = 8）
A：各组小鼠平均体重；B：各组小鼠肺系数；NS：地塞米松阳性药组。与NS组比较，*P < 0.05，**P < 0.01，***P < 0.001；与LPS组比较，#P < 0.05，##P < 0.01。
Figure 2. The average weight and lung coefficient of mice in each group（$\bar x \pm s $，n = 8）
表 1 各组小鼠病理组织切片肺泡炎评分[（$\bar x \pm s $），分]
Table 1. Alveolitis score of mouse lung tissue [（$\bar x \pm s $），points]
组别 肺泡炎评分 n P 与NS比较 与LPS比较 NS 1.67±0.18 10 LPS 2.71±0.28*** 10 0.0010 XYP-L 2.38±0.38** 10 0.0010 0.3279 XYP-M 2.33±0.44** 10 0.0023 0.2104 XYP-H 2.17±0.25*# 10 0.0388 0.0201 DXM 2.13±0.35# 10 0.0719 0.0100 NS：正常对照组；LPS：ALI模型组；XYP-L：喜炎平注射液低剂量组；XYP-M：喜炎平注射液中剂量组；XYP-H：喜炎平注射液高剂量组；DXM：地塞米松阳性药组；与NS组比较，*P < 0.05，**P < 0.01，*** P < 0.0001 ；与LPS组比较，#P < 0.05。
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表 2 BALF总细胞数和总蛋白浓度（$\bar x \pm s $，n = 10）
Table 2. Total cell counts and protein concentration in BALF（$\bar x \pm s $，n = 10）
组别 总细胞数
（×105）P 总蛋白浓度
（mg/mL）P 与NS比较 与LPS比较 与NS比较 与LPS
比较NS 3.31±1.12 2.27±0.58 LPS 10.13±4.17*** 0.000 3.81±0.47*** 0.000 XYP-L 4.45±1.95### 0.396 0.000 2.74±0.57## 0.115 0.001 XYP-M 3.21±1.07### 0.939 0.000 2.43±0.64### 0.576 0.000 XYP-H 5.49±2.28## 0.108 0.001 2.26±0.46### 0.988 0.000 DXM 3.25±1.54### 0.962 0.000 2.29±0.2### 0.943 0.000 与NS组比较，***P < 0.001；与LPS组比较， ##P < 0.01，###P < 0.001。
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表 3 BALF中TNF-α含量（$\bar x \pm s $，n = 6）
Table 3. The contents of TNF-α in BALF（$\bar x \pm s $，n = 6）
组别 TNF-α P 与NS比较 与LPS比较 NS 6.05±2.12 LPS 16.62±6.76*** 0.000 XYP-L 9.54±1.97## 0.094 0.001 XYP-M 9.55±3.09## 0.094 0.001 XYP-H 6.11±2.24### 0.976 0.000 DXM 6.7±2.18### 0.750 0.000 与NS组比较， ***P < 0.001；与LPS组比较， ##P < 0.01，###P < 0.001。
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表 5 BALF中IL-8含量（$\bar x \pm s $，n = 6）
Table 5. The contents of IL-8 in BALF（$\bar x \pm s $，n = 6）
组别 IL-8 P 与NS比较 与LPS比较 NS 73.49±19.33 LPS 306.45±70.12*** 0.000 XYP-L 234.05±145.15** 0.002 0.133 XYP-M 161.48±52.28## 0.070 0.004 XYP-H 109.06±60.18### 0.454 0.000 DXM 132.44±82.57## 0.218 0.001 与NS组比较，*P < 0.05，**P < 0.01，***P < 0.001；与LPS组比较，#P < 0.05，##P < 0.01，### P <0.001。
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表 4 BALF中IL-1β含量（$\bar x \pm s $，n = 6）
Table 4. The contents of IL-1β in BALF（$\bar x \pm s $，n = 6）
组别 IL-1β P 与NS比较 与LPS比较 NS 2.38±0.27 LPS 5.22±2.32*** 0.000 XYP-L 3.39±0.97# 0.110 0.005 XYP-M 3.17±0.47*## 0.206 0.002 XYP-H 2.26±0.36### 0.842 0.000 DXM 2.54±1.06### 0.804 0.000 与NS组比较，*P < 0.05，***P < 0.001；与LPS组比较， #P < 0.05，##P < 0.01，###P < 0.001。
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表 6 肺组织MDA含量和SOD活性（$\bar x \pm s $，n = 6）
Table 6. The contents of MDA and SOD activity in lung tissue（$\bar x \pm s $，n = 6）
组别 MDA
（nmoL/g）P SOD（U/g） P 与NS比较 与LPS比较 与NS比较 与LPS比较 NS 41.50±3.09 22968.25 ±1616.73 LPS 65.05±10.68*** 0.000 13884.96 ±999.61***0.000 XYP-L 54.98±1.77**# 0.003 0.019 17737.02 ±706.5**#0.005 0.029 XYP-M 43.86±6.09### 0.553 0.000 19573.48 ±673.39##0.052 0.003 XYP-H 42.99±3.57### 0.707 0.000 21019.01 ±2879.11 ###0.247 0.000 DXM 41.52±2.41### 0.996 0.000 22831.06 ±4354.56 ###0.934 0.000 与NS组比较，*P < 0.05，**P < 0.01，***P < 0.001；与LPS组比较，#P < 0.05，##P < 0.01，###P < 0.001。
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