摘要
目的探讨大气混合污染物(PM10、SO2、NO2及CO)对大鼠肺组织Clara细胞分泌蛋白(CCSP)的影响,为大气污染致肺损伤机制的研究提供科学依据。
方法32只Wistar大鼠随机分为3个实验组(低、中、高剂量组)和1个对照组。一次性气管注入染尘,低、中、高剂量组大鼠分别气管注入1ml含7.5、15、22.5mg PM10的生理盐水混悬液,对照组大鼠注入1ml生理盐水。低、中、高剂量组大鼠分别静态吸入SO2、NO2、CO空气混合气,SO2、NO2、CO浓度分别为7.5、6、200㎎/m3,15、12、400㎎/m3,22.5、18、600㎎/m3。每天吸入2h,连续30d。对照组大鼠吸入正常空气。肺组织HE染色观察形态学变化。ELISA法测定血清、支气管肺泡灌洗液(BALF)中CCSP水平;RT-PCR法检测肺组织CCSP mRNA表达;肺组织免疫组化染色观察Clara细胞变化。
结果本实验染毒剂量可致肺组织损伤。高剂量组血清中CCSP浓度升高、肺组织中CCSP阳性细胞积分值下降。实验组BALF中CCSP水平下降。肺组织CCSP mRNA表达各组间无差异。
结论本实验各染毒剂量对大鼠肺组织形态有损害作用。血清中CCSP浓度增高,提示大气混合污染物致肺上皮细胞通透性增强,肺泡毛细血管膜的完整性受到破坏。BALF中CCSP水平下降、肺组织免疫组化染色可见部分Clara细胞脱落和大量Clara细胞浆内CCSP褐色变浅,表明大气混合污染物损害Clara细胞,并且抑制Clara细胞的分泌功能。
Objectve Respiratory system is target organ for air pollutants. Meta analysis of dose-response relationships between particulates exposure and death rates indicated that for every increase of 10mg respirable particulate (PM10) there will be a 0.38% increase in acute death rate. Epidemiological studies showed concentration of respirable particulate, and SO2 in air were correlated with the death rates of COPD, cardiopulmonary diseases as well as lung cancer. Exposure to acid gas, NO, and PM25 was correlated with significant reduction in lung function. It is known that SO2 and total suspended particulate (TSP) present the combined effect on cardiovascular diseases. SO2 and smoke combined together exert far more hazard than SO2 alone. In fact, the adverse health effect of air pollution is generated by the combination of various air pollutants, in which PM10 exerts significant direct effect on human health, resulting in wide range of damages on respiratory, cardiovascular and endocrinal system. In order to provide theoretic base for exploration the mechanism of lung tissue injury caused by air pollution, rats were exposed to air mixed pollutants (PM10, SO2, NO2, CO) to observe their effect on Clara cell secretory protein (CCSP) as well as the change of expression of mRNA in lung.
Method 32 Wistar rats were purchased from laboratory center of China Medical University. half male half female, weight 200～240mg each. They were randomly divided into three test groups (low, middle and high dose) and a control group, 8 rats in each group. Rats were anaesthetized then intratracheally injected with 0, 7.5, 15, 22.5mg PM10 suspended in 1ml saline for four groups respectively. Low, middle and high dose group statically inhaled 12 times diluted SO2,, NO2, CO mixed air. The concentration of SO2, NO2, CO for low dose groups were 7.5, 6, 200mg/m3, for middle dose group15, 12, 400mg/m3, for high dose group 22.5, 18, 600 mg/m3 respectively, which equal 50, 100, 150 times of secondary level of daily air standard set by national environmental quality standard (GB3095 -1996). The exposure lasted for 30 days, 2 hours daily. Control group inhaled room air. Examination of the level of CCSP in serum and in BALF by ELISA, and CCSP mRNA expression in lung tissue by RT-PCR, as well as expression of CCSP protein in lung tissue by immunohistochemistry.
Result At the dose level in our experiment, varying degree of lung injuries were found on tissue slides stained by HE, especially exposed to high dose. It was observed that serum CCSP in high dose group increased obviously than that in control. The difference was significant in statistics. The level of serum CCSP in low and middle groups were lower than in control, but not statistically significant. CCSP in BALF in low, middle, high dose groups decreased statistically significantly than in control. On the other hand no statistical significance of expression of CCSP mRNA was observed among different dose groups in our experiment. Integral optical density of CCSP positive cell was obviously lower than in control. The difference was statistically significant.
Discussion After exposure to air mixed pollutants, varying degree of pathological changes in slides of lung stained by HE was observed. Especially in high dose group, indicating the dose level in our experiment was able to cause lung impairment in rats. The changes during the process of impairment are recognized as follows.
CCSP is the main antiinflamatory protein secreted by nonciliated Clara cell in bronchiole and terminal bronchiole. It can penetrate to intralayer fluid and occur high level in BALF as well as leak out passively from epithelial cell in lung to blood. Therefore Serum CCSP is a biomarker for identify permeability of lung-blood barrier in clinical and experimental test, especially in animal experimental study exposed to ozone, silicon, asbestos. In our study serum level of CCSP was measured by ELISA. It was found 30 days after instillation of air mixed pollutants serum CCSP increased obviously, suggesting permeability of epithelial cell increases, integrity of alveolar epitheliar cell membranes was affected. In combination the results of immunohistochemical image analysis with pathological changes in our study, it was clear that injury of lung structure in high dose group was obvious and that serum increased level of CCSP was due to passive leak caused by injury of lung structure after prolonged exposure to mixed air pollutants. In low and middle dose groups the effect was not obvious, and no dose-effect relationship between air mixed pollutants and serum CCSP was found.
The level of CCSP in BALF reflect secretory function of Clara cell, therefore it is often used as an index for lung injury in animal test caused by environmental contamination. Our study indicated after exposure for 30 days levels of CCSP in BALF in test groups significantly decreased than control group, suggesting that air mixed pollutants acted on lung tissue, damaged to secretary function of Clara cell. Test air (SO2, NO2, CO) was a mixture of irritant and asphyxiating gas. It destroyed normal lung structure and physical function, damaged to Clara cell in bronchiole and terminal bronchiole then lead to lower secretary functioning. It is considered that mechanical irritation of dust itself in lung may irritate and impair Clara cell, then damages secretory function of Clara cell, reduces CCSP level in BALF. The CCSP level in Clara cell obviously decreased in high dose group observed on pathological slide, suggesting air mixed pollutants may inhibit secretory function of Clara cell.
Expression of mRNA examined by RT-PCR is the highest in lung and in bronchiole in various organs, its gene control is carried out at the time of transcription or after transcription. IFN-γthrough enhances expression of HNF3β, accelerates transcription of CCSP mRNA, and delays its decomposition, then promotes CCSP protein synthesis. TNF-αenhances the stability of mRNA, raises the level of CCSP mRNA in cell, accelerates translation of its protein, then increases the level of CCSP. Expression of mRNA in lung 30 days after exposure in our experiment did not show significant differences among different groups, suggesting that action of air mixed pollutants on CCSP might occur after transcription.
Analysis the result of immnohistochistry assay showed that value of integral optical density for CCSP positive cells in high dose group was obviously reduced than that in control group, suggesting the reduction the number of CCSP positive cells. Observation of tissue slides found that individual Clara cells dropped off around bronchiole in high dose group. On the contrary large amount of dark brown Clara cell plasma was observed in control group, however in high dose group it obviously lessened, indicating that CCSP reduction was related both to fall off of Clara cell and the reduction secretion of CCSP.
Conclusions Exposure to the dose levels in our experiment, pathological changes in lung tissue have generated and there are the relationship between dose and effects. The effects of air mixed pollutants include:
1. At various dose levels in our experiment, lung injuries were found on tissue slides stained by HE.
2. It was showed that after instillation of air mixed pollutants, serum CCSP increased obviously, suggesting permeability of epithelial cell increases, integrity of alveolar epitheliar cell membranes was affected.
3. It was observed that CCSF level in BALF reduced in the experiment and the result of immnohistochistry assay showed that some Clara cells dropped off and large amount of dark brown Clara cell plasma lessened. The results indicate that air mixed pollutants impaired Clara cell and inhibit secretory function of Clara cell.
引文
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