| Objective: Chronic obstructive pulmonary disease(short for COPD) is one of chronic recurring respiratory diseases. Its long-term repeated attacks lead to seriously impaired respiratory functions and reduced quality of life. Meanwhile, COPD has placed a heavier social and financial burden on the society and family because of repeated admission caused by its acute exacerbations. By 2020, COPD will have become the fifth disease placing a financial burden in the world. So it is necessary to choose effective treatment to reduce the double burden of COPD.The predisposing factors of COPD mainly include smoking, air pollution and occupational powders and dusts. Besides, infection is one of main factors making COPD worse. The pathogenesis of COPD is unclear now, but COPD has been proved to be related to the following causes, including repeated increased airway inflammation, proteinase/anti-proteinase, oxidation/antioxidation imbalance, cell apoptosis and airway remodeling. Traditional Chinese Medicine(TCM) has summarized its pathogeneses as follows: people are influenced by smoking and evils of six exogenous pathogens, leading to accumulation of phlegm in the lung, counterflow ascent of lung qi, Qi-movement disturbance, and after a long time, losses of lung function, spleen dysfunction, the failure of kidney to receive qi, blood stasis obstructing the collaterals and finally COPD. During its attack stage, COPD is treated mainly by qi-descending and phlegm-resolving. During its remission stage, COPD is treated mainly by invigorating lung-qi and kidney-qi. Pathogenesis of COPD in TCM is mainly directed against “qi, phlegm and stasis”, so its treatments aim to lower qi, reduce phlegm, invigorate qi and remove blood stasis.HAECs used in the experiments are derived from placental amnion tissues and has been reported to have an anti-inflammatory effect and mitigate the acute lung injury in the literature. The runfeizhisou pill(short for runfei pill) used in the experiment is a classic prescription for COPD, which can regulate qi, invigorate qi, relieve cough, and reduce phlegm. It is clinically used for respiratory tract diseases with chronic cough and asthma.Multiple experiments in the article have been carried out to explore whether human amniotic epithelial cells, runfei pills, and their combination therapy can reverse COPD in rats. They were adopted to observe what effects h AECs and runfeizhisou pills can have on the COPD model rats in terms of pathomorphology, inflammatory factors, cell apoptosis, airway remodeling, and investigate the effect of early interventions on the development of COPD. Besides, the purpose of this study was to describe mechanism of actions that human amniotic cells and runfei pills intervened COPD rats, and therapies for COPD rats.Methods:1 The rat COPD model was established using tobacco smoke as well as intratracheal instillation of lipopolysaccharides(LPS). Human amniotic epithelial cells were extracted and identified from human placentas.2 64 clean Wistar rats(male) were divided by random number table into 8 groups, including normal group, model group, earlier runfei pills treatment group(rats in this group received an earlier intervention with runfei pills), earlier h AECs treatment group(rats in this group received an earlier intervention with h AECs), 1-month runfei pills group(rats in this group received runfei pills for 1 month) and 3-month runfei pills group(rats in this group received runfei pills for 3 month), and h AECs group, and runfei pills+h AECs group(rats in this group were treated with runfei pills as well as h AECs).3 COPD rats were gavaged with runfei pills, and treated with tracheal instillation of amniotic epithelial cells. Afterwards, their serum assays and alveolar lavages were performed to collect alveolar lavage fluid and cut living lung tissues.4 Morphological changes in lung tissues, the degree of emphysema and the thickness of smooth muscles in the airway in COPD rats were observed under light microscope.5 IL-8 and TNF-а levels in the blood and tissues of rats in each group were examined by radioimmunoassay.6 The pulmonary surfactant protein-D(SP-D) level and VEGF levels in BALF in rats’ blood and tissues were detected by ELISA.7 VEGF and VEGFR2 protein expressions in lung tissues were measured by the Western blot.8 the number of apoptotic cells was observed in each group in TUNEL staining.9 TGF-β1 in lung tissues was measured by double antibody sandwich ABC-ELISA.10 MMP2 and MMP8 expressions in rats’ lung tissues were determined by semi-quantitative RT-PCR.Results: 1Changes in rats’ lung tissues and bronchiolar pathomorphologcial observations in different groups were obtained under light microscope. 1.1 In the normal group, the tracheal mucosa cilia did not become shorter and desquamate, and were regularly arranged, with a small amount of lymphocyte infiltration and no mucous plug in the lumen; only a small number of goblet cells were observed in bronchioles, and no alveoli around bronchioles became thin and enlarged, and glandular hyperplasia, edema of bronchiolar walls and thickened airway smooth muscle were not visible. 1.2 In the model group, tracheal mucosa cilia became shorter and desquamated in parts of the airway, and cilia were irregularly arranged, such inflammatory cells infiltration as lymphocytes were observed in the lumen; mucous plugs and inflammatory cells infiltration could be found in the airway. Alveoli became enlarged in surrounding lung tissues, and bubble walls became thin and ruptured. The bronchiolar wall thickened, smooth muscles proliferated and airway lumen became narrowed and more goblet cells were distributed in the bronchiole. 1.3 In the earlier intervention groups with runfei pills and human amniotic cells, compared with COPD model group, part of the bronchial lumen thickened, and there were a small amount of lymphocytes and infiltration, fewer mucous plugs and fewer fibrous tissue hyperplasia in the lumen. Besides, inflammatory cell infiltration was lessened in bronchial walls at all levels, under mucosa of blood vessels and in surrounding lung tissues, and there were a smaller amount of alveolar damage, with the bronchiolar smooth muscle thickening. 1.4 In the h AECs group, tracheal mucosa cilia were regularly arranged, with few cilia lodging. There were less inflammatory cells infiltration in the lumen, thickening of wall, and pulmonary bullae compared to the COPD model group. 1.5 In the runfei group, thickening of airway lumen, formation of mucous plugs, and fibrous tissue hyperplasia were lessened. Besides, inflammatory cell infiltration was reduced in bronchiolar walls, under mucosa of blood vessels and in the surrounding lung tissues. There were a smaller number of surrounding alveolar damage and partial hyperemia and edema in surrounding blood vessels. 1.6 In the h AECs+runfei pills group, tracheal mucosa cilia were regularly arranged, and a small amount of inflammatory cells infiltration was in the airway; part of the alveolar walls became thin or ruptured, with less mucous plugs in airway, and less alveolar damage around the trachea, and bronchiolar smooth muscle partly thickened. 1.7 In the 3-month runfei pills group: less tracheal mucosa epithelium desquamated, cilia were arranged regularly, and a small amount of inflammatory cell infiltration was visible in the walls; a small amount of mucus was observed in the bronchiolar lumen, and partial walls thickened, and smooth muscle hyperplasia was observed. Compared to the COPD model group, fewer alveolar enlarged and bubble walls became thin in this group. 2 Changes of pulmonary emphysema in each group were found. 2.1 There was a significant difference in MLI and MAN between normal group and model group(P<0.01), which indicated the COPD model group was established. 2.2 MLI and MAN in the model group were compared with those in the earlier intervention groups with runfei pills and human amniotic cells(P<0.05),which indicated the earlier intervention could mitigate pulmonary emphysema. 2.3 MLI and MAN in the model group were compared with those in each treatment group(P<0.05). The pulmonary emphysema in each treatment group was less severe than that in model group and there was statistically significant(model group vs all treatment groups). 2.4 There was a significant difference in MLI and MAN between h AECs group and runfei group(P<0.05). The pulmonary emphysema in h AECs group was less severe than that in runfei group and there was statistically significant(h AECs group vs runfei group). 2.5 MLI in h AECs group was compared to that in h AECs+runfei group(P <0.05),MAN in h AECs group was compared to that in h AECs+runfei group(P>0.05),which indicated pulmonary emphysema did not improve when runfei pills were added to the h AECs group. However, MLI and MAN in runfei group was compared to that in h AECs+runfei group(P<0.01), which demonstrated pulmonary emphysema improved significantly when h AECs treatment was added to the runfei group. 2.6 Runfei group vs 3-month runfei group(P<0.05) demonstrated a prolonged treatment improved pulmonary emphysema greater than a short-term treatment. 3 Inflammatory factors such as IL-8,TNF-α and SP-D in the blood and tissues of rats in each group were compared. 3.1 SP-D, TNF-α and IL-8 levels in the blood and tissues of rats in the normal group were compared with those in the model group(P<0.01), which suggested the contents of SP-D,TNF-α and IL-8 in the blood and tissues of rats in the normal group were lower. 3.2 SP-D, TNF-α and IL-8 levels in the blood and tissues of rats in the model group, the earlier intervention groups with runfei pills and human amniotic cells were compared. The earlier intervention groups could reduce SP-D, TNF-α and IL-8 levels in the blood and tissues of rats. SP-D, TNF-α and IL-8 levels in the blood and tissues of rats in the earlier intervention groups with runfei pills and human amniotic cells were compared with those in the model grup(P<0.05, P<0.01), which suggested the earlier intervention can inhibitory inflammatory factor.But SP-D in the blood and tissues of rats in the earlier intervention groups were compared with those in the model group(P>0.05),which indicated the inflammatory response is more obvious in the early formation of COPD 3.3 SP-D, TNF-α and IL-8 levels in the blood and tissues of rats in the model group, h AECs group, runfei group, h AECs +runfei group and 3-month runfei group were compared.SP-D level in the blood and tissues of rats were compared(P<0.05, model group vs each treatment group), which indicated treatments in the h AECS group, runfei group and h AECs+runfei group can slow the progression of COPD. SP-D, TNF-α and IL-8 in the blood and tissues of rats in the h AECs group was compared that those in the runfei group(P<0.05), showing SP-D level in the h AECs group was bettrer than that in the runfei group. However, SP-D, TNF-α and IL-8 in the blood and tissues of rats were compared(P>0.05, h AECs group vs h AECs+runfei group), which suggested h AECS+runfei pills could not significantly lower the SP-D level.The h AECs treatment added to the runfei pills treatment could lower SP-D level significantly(P<0.05, runfei group vs h AECs+runfei group) and h AECs+runfei treatment could mitigate the condification of COPD. The prolonged treatment was more effective than the short-term one(P<0.01, runfei group vs 3-month runfei group).IL-8 and TNF-α levels in the blood and tissues were compared(P<0.05, all treatment groups vs model group), which suggested IL-8 and TNF-α levels in the blood and tissues were higher in model group, and each treatment in treatment groups could lower their levels. Compared to runfei group, the h AECs could lower their levels more(P<0.01). However, runfei pills added to h AECs treatment could not lower their levels significantly(P>0.05). The h AECs treatment added to the runfei treatment could lower their levels significantly compared to the single runfei treatment. Besides, prolonged treatments could inhibit inflammatory factors effectively(P<0.01, runfei group vs 3-month runfei group). 4 The situation of cell apoptosis and apoptotic index(AI) were observed. 4.1 the situation of cell apoptosis in TUNEL stainingThere were a small number of scattered apoptotic cells in the normal group; there were more apoptotic positive cells found in TUNEL staining in lung tissues in the model group. The number of apoptotic cells was alleviated in different degrees in each treatment group compared with the model group. 4.2 Apoptotic index(AI) in each group was compared.AI in the model group was significantly reduced than that in each treatment group(P<0.05). AI in the h AECs group was lower than that in the runfei group(P<0.05).But AI in the h AECs+runfei group was lower than that in the h AECs group and in the runfei group(P<0.05), which indicated additive treatment was better than a single treatment. 5 VEGF and VEGFR2 protein expressions in lung tissues were measured by the Western blot.VEGF and VEGFR2 protein expressions in rats’ lung tissues were significantly lower in the model group than in h AECs group, in the runfei group and in h AECs+runfei group and in 3-month runfei group. VEGF and VEGFR2 protein expressions in rats’ lung tissues were significantly higher in h AECs group than in runfei group. The two protein expressions were lower in h AECs+runfei group and in 3-month runfei group than in a single treatment group. 6 VEGF levels in BALF were measured.VEGF levels in BALF were significantly lower in the model group than in the normal group(P<0.05), which showed that VEGF levels in BALF were low and became higher after each treatment in all treatment groups(P<0.05).Compared to that in runfei group, VEGF level was increased in h AECs group(P<0.01), and there was no significant difference in VEGF level between h AECs group and the h AECs+runfei group(P>0.05). Combined treatment could increase VEGF level in BALF(P<0.01, runfei group vs h AECs+runfei group). The runfei group compared to the 3-month runfei group has significanfly difference(P<0.05). 7 Thickness of smooth muscle in each group were compared.Smooth muscles in the model group were significantly thicker than those in the normal group(P<0.01). Smooth muscles after treatments in each group became thinner than those in the model group(P<0.05). Smooth muscles in the h AECs group became thinner than those in runfei group(P<0.05). There was no difference in the thickness of smooth muscle between h AECs group and h AECs+runfei group. The smooth muscle in the runfei group was thicker than those in h AECs+runfei group(P<0.05) and in 3-month runfei group(P<0.05), which suggested collaborative treatment and prolonged treatment could decrease the thickness of the smooth muscle. 8 MMP2 and MMP8 expressions in rats’ lung tissues were compared in each group.MMP2 and MMP8 protein expressions were significantly higher in the model group than in the normal group. The two expressions were lower in the model group than in each treatment group. However, the two expressions were higher in 3-month runfei group than in h AECs+runfei group, lower in h AECs group than in runfei group, and higher in h AECs group and in runfei group than in h AECs +runfei group. 9 TGF-β1 contents in the lung homogenate were compared.TGF-β1 contents in the lung homogenate were higher in model group than in each treatment group(P<0.05).TGF-β1 contents were lower in h AECs group than in runfei group. Runfei pills added to the h AECS treatment did not reduce TGF-β1 contents(P>0.05).However, runfei pills+h AECs treatments could decrease the TGF-β1 level significantly. The TGF-β1 level was lower in 3-month runfei group than in 1-month runfei group(P<0.01).Conclusion:1 A COPD model can be replicated by tobacco smoke as well as intratracheal instillation of lipopolysaccharides(LPS). There are cell apoptosis and airway remodeling in COPD rats. The airway remodeling becomes reversible by interventions.2 Early interventions by h AECs and runfeizhisou pills could reduce IL-8 and TNF-α level, formation of pulmonary bullae and slow the progression of COPD,but had no obvious effect on SP-D,we speculated inflammation in the early formation of COPD is more obvious.3 Compared to those in model group, more inflammation has been inhibited and less cell apoptosis has occurred and airway remodeling has improved better in h AECs group, runfei group, h AECs+runfei group and 3-month runfei group. Besides, the outcome is better in h AECs group than in runfei group.4 There are synergistic therapeutic effects in treating COPD rats in h AECs+runfei group.5 The h AECs treatment has an advantage over inhibiting inflammation, and its effect on the cell apoptosis may be related to properties of its stem cell.6 Therapeutic effects of a prolonged runfei pill treatment are more ideal than that of a short-term treatment. |
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