| Objective: Preliminary study indicated earthworm powder has certain positive cure for asthma, such as reduction of airway collagen deposition and inhibition of PAS positive cell, but the effective relief of airway inflammation is unsatisfactory. This study is to investigate the antitussive, expectorant and anti-asthmatic effects of earthworm compound, and so as to provide theoretical basis for its clinical application.Methods:1. Pharmacological study on Earthworm powder① To investigate the effect of earthworm powder on acute asthma model of guinea pig established by using histamine, acetylcholine chloride and ovalbum(OVA).② To determine the effect of earthworm powder on cough of guinea pig and mouse models established by using citric acid and ammonia.③ To observe the expectorant effects of earthworm powder on the model mouse via the method of phenol red shedding in mouse airway.2. Studies on antitussive and expectorant effects of earthworm compound① The ammonia-induced cough model of mouse was set up, and the qualified mice were randomized into 7 groups: low and high earthworm group, low and high ephedra licorice group, low and high earthworm compound group, model group, positive control group and normal control group, and then after intragastric administration for 7 days, to determine the times of cough and cough incubation period of model mice.② Sixty four test mice were randomized into 8 groups as above and intragastric administration for 7 days. The mice were treated via intraperitoneal injection of phenol red solution after 2 h for the last treatment and after 30 min, bleed to death. To flush their bronchial three times with BALF, and determine the BALF OD value and calculate the content of phenol red.3. Studies on anti-asthma effect of earthworm compound① Sixty four chronic allergen-induced asthma Balb/c mice models were randomized into 8 groups. Except normal control group, other groups were sensitized on 1, 8, 15 d, by means of celiac injection of 0.2ml aluminum hydroxide gel(with 20μg OVA), and from 22 d, all model mice were stimulated with 2.5% atomized OVA, with amount of 0.5ml/min and 30 minute per time. The stimulation was taken three times per week and continued six weeks. The normal control group was stimulated with phosphate buffer.Ligation of the left lung, right lung lavage with saline water get alveolar lavage fluid(BALF), precipitation with a total number of white blood cell plate meter and all kinds of inflammatory cells count; Lung tissue after fixed paraffin section, HE staining to observe pathological changes in lung tissue, with preliminary quantitative degree of inflammatory infiltration inflammatory score; Masson staining to observe the degree of fibrosis of lung tissue and skin, and Image- Pro Plus6.0 system quantitative degree of inflammatory cell infiltration; PAS staining observation of goblet cells and mucus gland secretion, with Image quantitative PAS positive cell number- Pro Plus6.0 system; Meanwhie, the ELISA method was applied in determination of IL-4 levels in the serum and BALF..② To observe the survival quality of the model mice and determine the changes of diet and weight. The stimulated mice were bleed to death after last atomization, then collected the blood, centrifuged supermatant, ligated the left lunga and washed the right lung lavage with saline water to extract the brochoalveola lavage(BALF), counted all kinds of inflammatory cells and the total number of cells, respectively. Haematoxylin and Eosin(HE) dyeing to observe lung tissue pathological changes, and inflammatory score to preliminarily quantify the infiltration degree of inflammatory cell; Masson dyeing to observe lung organization subcutaneous fibrosis degree and the Image- Pro Plus 6.0 system to quantitatively analyze the degree of collagen deposition; Periodic acid-schiff stain(PAS) to observe goblet cells and mucus gland secretion and the Image- Pro Plus 6.0 system to quantify PAS positive cells. At the same time, the ELISA method was used for the determination of IL- 4 levels in serum and BALF.Results:1. Low and high content of earthworm powder could notably prolong the incubation period of asthma(p<0.05) and reduce the number of fall of the histamine – induced model guinea pigs within 6 minutes, and also extend the incubation period of asthma guinea pigs by using OVA sensitization and reduce the asthma mortality rate.2. Low and high content of earthworm powder could prolong the cough incubation period of ammonia – induce mice models, decrease cough times and increase airway phenol red in mice, but had no significant difference compared with model group(p>0.05).3. Low and high content of earthworm compound could significantly prolong the cough incubation period of ammonia – induce mice models, decrease cough times and increase airway phenol red in mice, especially for the high content of earthworm compound(p<0.05).4. Compared with the asthmatic model group(p<0.05), Low and high content of earthworm compound could improve the life quality of asthma mice and significantly decrease the number of inflammatory cells in BALF(p<0.05), reduce the airway and perivascular inflammatory infiltration, reduce airway collagen deposition and PAS positive cell number.Conclusion:1. Earthworm powder can significantly prolong the incubation period of asthma in mice, has positive smooth wheezing and antispasmodic effects, but the effects in relieving cough and eliminating phlegm are not significant.2. Earthworm compound is made from earthworm powder, ephedra and liquorice with 6:2:1 ratios, has good effects on relieving cough, expectorant and anti-asthma actions, and can make up the deficiency of earthworm-alone treatment. |
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