Feng-Chun Ren, Qing-Wen Xu, Lei-Ling Han, Yan Wu, Hui-Zhi Zhu

1. Anhui University of Chinese Medicine, Hefei 230038, China

2. First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230031, China

Keywords:

ABSTRACT

1. Introduction

Bronchial asthma is one of the most common chronic diseases of respiratory system[1]. It is found that the basic research on asthma has been increasing in recent years. Dexamethasone is one of the main positive control drugs in animal experiments on asthma,but because of its wide range of dosage in clinical use, different modeling animals, modeling cycles and drug administration methods. The doses used in animal experiments were more [2-6]and each dose was not supported by experimental evidence. At present, there is no uniform intragastric dose for the rat model of dexamethasone chronic asthma, but also for the needs of the later experiment of the research group. In order to explore the appropriate intragastric dose of dexamethasone, the effects of different concentrations of dexamethasone on the general condition and pulmonary histology of chronic asthma rats were observed. The aim of this study was to provide experimental evidence for the dosage of dexamethasone in the rat model of chronic asthma.

2.Materials and methods

2.1.Animals

75 male Sprague-Dawley (SD) rats, clean grade, average body mass(180 ±20) g, purchased from Experimental Center of Anhui Medical University (production license No.: SCXK (Anhui) 2017-001), at an average temperature of 20℃-25℃, relative humidity of 40%-70%, The rats could be fed in the environment of free feeding and drinking water, and the rats were fed adaptively for 1 week before the beginning of the experiment. This experiment was approved by the Animal Experimental Ethics Committee of Anhui University of traditional Chinese Medicine.

2.2.Chemicals and instruments

Ovalbumin (OVA) (American Sigma); Dexamethasone acetate tablet 0.75mg (Shanghai Xinyi); Yuyue 402B ultrasonic atomizer(Yuyue Group); Closed atomization box: self-made volume about 600L plexiglass box; JB-P5 embedding machine (Wuhan Junjie);RM2016 pathological slicer (Shanghai Leica); EclipseE100 optical microscope (Nikon, Japan);

2.3.Animal antigen sensitization, challenge and treatment

Seventy-five rats were randomly divided into N group(N group),model group(M group), dexamethasone low dose group(DEXL group), dexamethasone middle dose group(DEXM group) and dexamethasone high dose group(DEXH group), 15 rats in each group. The asthma model was induced by OVA sensitization and challenge. The asthma model was sensitized on the 1st and 8th day, respectively. OVA saline mixture with 10% concentration was prepared by mixing OVA, aluminum hydroxide dry powder and normal saline, respectively. 0.25ml and 0.5ml were injected subcutaneously and intraperitoneally into the left and right groin of each rat. Atomization challenge began on the 15th day, and 1% OVA solution was prepared with OVA and normal saline once a day for 30 minutes for 5 days, from the 20th day to once every other day for another 42 days[7-10].The normal group was treated with the same amount of normal saline at the sensitization and challenge stages. In the process of challenge, rats showed symptoms such as shortness of breath, wheezing, wheezing and hair lodging, which were regarded as successful modeling.

Intervention began on the 20th day of the experiment. The DEXL,DEXM and DEXH groups were given intragastric administration of 0.0625mg/ (kg ·d), 0.125mg/ (kg ·d) and 0.5mg/ (kg ·d)for 42 days, respectively. The other groups were given the same amount of normal saline.

After stopping administration and fasting for 12 hours, the rats were anesthetized with 20% 1.5g/kg intraperitoneally[11]. The left lung of rats was fixed in 10% formaldehyde solution. The overall experimental protocol is shown in figure 1.

Fig.1: Experimental protocol

2.4.Observation and record of general condition of rats

The respiration, mental state, coat color, body mass, food intake,drinking water and death of rats were observed and recorded.

2.5.Lung histology and morphometry

The fixed lung tissue was embedded in paraffin and the thickness of the section was 4 μm. Hematoxylin and eosin staining were used for histological evaluation under light microscope, and Masson trichrome staining was used to show the existence of collagen deposition. The histological analysis was performed by the pathologists who blinded each group. The bronchus in the section was analyzed by Image-Pro Plus image processing software, and the average value was taken as the representative value of the section.The perimeter of bronchial basement membrane (Pbm, μm), the total area of airway wall (Wat, μm2), the area of smooth muscle(Wam, μm2) and the area of airway collagen fibers (Wcol, μm2)were measured. (Wam/Pbm, μm2/μm) and (Wat/Pbm, μm2/μm).μm2/μm) and (Wcol/Pbm, μm2/μm) represent airway wall thickness,airway smooth muscle thickness and airway subepithelial collagen deposition thickness.Histological analyses were performed by a pathologist blinded to the groups.

2.6.Statistical analysis

The measurement data were expressed as mean ±standard deviation.Single factor analysis of variance was used for comparison among groups, and repeated analysis of variance was used for comparison between groups at different time. The data were analyzed by statistical software SPSS23.0, with P ＜ 0. 05 and P ＜ 0. 01 as the test level.

3.Results

3.1.Observation on the general condition of rats

The rats in the N group showed restlessness during atomization, but there were no obvious symptoms such as wheezing and coughing,and the hair color was normal. The rats in each group had different degrees of restlessness, scratching the ear and scratching the nose,gradually less movement, lying wheezing, abdominal muscle convulsions and other symptoms. In the later stage of the M group,wheezing and hair color turned yellow. After intervention, the food intake and body mass of rats in the high dose group decreased significantly, and their mental state was very poor. Some rats showed ocular bloody secretions, hematochezia, mouse claw necrosis and so on. Obvious gastrointestinal flatulence, hyperemia and bleeding were found in dead rats (Fig.2). In the later stage of intervention,the mental state and activity of the rats in the DEXM group were worse than those in the DEXL group. In the DEXH group, 1 died on the 24th and 26th, and 4 died on the 27th and 29th. One rat died on the 48th day, 50th day and 55th day in the DEXM group, and there was no death in the other groups. The samples were killed on the 10th day of intervention because of a large number of deaths in the DEXH group, and did not participate in the histological comparison of each group.

Fig.2: Adverse reactions in the DEXH group

3.2.The increase of body mass and body mass in rats

The increase of body mass and body mass of rats in each group after intervention is shown in Fig.3 and Tab.1. Before intervention,there was no difference in body mass among the groups (P ＞ 0.05).Time had an effect on the change of body mass of rats in each group(P ＜ 0.01), but there was no difference in body mass and increase in body mass between the N group and the M group (P ＞ 0.05).The increase of body mass in the DEXM group and DEXL group was significantly lower than that in the M group (P ＜ 0.01). After 3 weeks of intervention, the increase in body mass in the DEXM group was more obvious than that in the M group (P ＜ 0.05). After one week of intervention, the body mass of rats in the DEXH group was significantly lower than that in the other groups (P ＜ 0.01).

Fig.3: Effects of dexamethasone on body mass and weekly body mass increase of rats in each group

Tab.1 Increase in body mass of rats at each week after intervention (g, n = 9-15, ±s)

Tab.1 Increase in body mass of rats at each week after intervention (g, n = 9-15, ±s)

Note: Compared with group M ▲▲P＜0.01; Compared with DEXM group #P＜0.05.

Group 1w 2w 3w 4w 5w 6w N 17.45±6.47 26.82±5.20 27.27±5.05 28.18±14.32 21.27±17.71 6.64±5.96 M 16.47±16.02 29.40±7.29 30.07±7.29 34.33±9.53 14.40±13.18 10.60±11.00 DEXH -71.07±41.20▲▲DEXM -13.25±9.66▲▲ 32.92±17.68▲▲ -8.92±8.03▲▲ 9.00±28.34▲▲ -20.90±17.51▲▲ -26.00±95.62▲▲DEXL -4.00±13.53▲▲ 33.46±17.09▲▲ 0.23±7.08▲▲ 24.08±36.64▲▲# -13.38±33.80▲▲# 10.92±9.30▲▲#F F time=12.991;F Intergroup=16.503 P P time=0.000;P Intergroup=0.000

Tab.2 Average daily food intake of rats per week after intervention (g, n = 9-15, ±s)

Note: Compared with group N *P＜0.05; Compared with group M ▲▲P＜0.01; Compared with DEXM group ##P＜0.01.

Group 1w 2w 3w 4w 5w 6w N 21.77±0.91 22.25±0.94 23.19±1.11 23.22±1.23 22.65±0.74 22.42±1.54 M 23.49±0.82 24.90±0.49* 25.49±0.60* 25.53±1.76* 24.76±1.47* 24.41±1.09*DEXH 10.25±2.34▲▲ 7.13±4.34 DEXM 21.31±4.15 22.14±1.38 20.75±0.99▲▲ 14.99±1.64▲▲ 12.32±1.86▲▲ 10.52±1.48▲▲DEXL 20.70±3.08 20.93±1.14 20.18±1.15▲▲## 18.07±1.92▲▲## 17.64±1.48▲▲## 16.51±2.35▲▲##F F time=18.498; F intergroup=124.322 P P time=0.000; P intergroup=0.000

Tab.3 Average daily drinking water of rats per week after intervention(ml, n = 9-15, ±s)

Tab.3 Average daily drinking water of rats per week after intervention(ml, n = 9-15, ±s)

Note: Compared with group N **P＜0.01.

Group 1w 2w 3w 4w 5w 6w N 53.77±4.19 52.86±9.28 48.57±6.87 52.99±7.71 51.69±5.44 49.74±6.20 M 39.20±1.97** 40.54±7.53 41.70±3.29 43.75±3.24 45.89±1.94 43.75±3.24 DEXH 40.80±6.86** 26.44±18.94 DEXM 38.88±3.68** 39.71±3.45 44.88±16.2 41.90±12.13 40.36±18.00 38.41±24.74 DEXL 36.00±2.42** 40.14±2.43 46.81±9.37 42.97±8.63 47.91±9.34 43.52±10.99 F F time=0.716; F intergroup=12.938 P P time=0.545; P intergroup=0.000

3.3.Average daily food intake and drinking water of rats in each Intervention week

The average daily food intake and drinking water of rats intervened in each week are shown in Fig.4 and tables 2 and 3. The analysis of variance of repeated measurement showed that the intervention time could affect the food intake of rats (P ＜ 0.01). After one week of intervention, it was found that the intervention time could affect the food intake of rats (P ＜ 0.01). There was no significant difference in the average daily food intake between the M group and the N group (P ＞ 0.05), but the average daily food intake in the M group was higher than that in the N group (P ＜ 0.05). After one week of intervention, the average daily food intake of rats in the DEXH group was significantly less than that in the M group (P ＜ 0.01). There was no significant difference between the DEXM and DEXL groups and the M group (P ＞ 0.05), but the average daily food intake in the DEXM and DEXL groups was significantly lower than that in the M group (P ＜ 0.01). After the third week of intervention, the average daily food intake of rats in the DEXM group was significantly lower than that in the DEXL group (P ＜ 0.01). The analysis of variance of repeated measurement showed that the intervention time had no effect on the drinking water of rats (P ＞ 0.05). The average daily drinking water of rats in each group was significantly lower than that in the N group (P ＜ 0.01), but there was no difference between the two groups (P ＞ 0.05). There was no significant difference in the average daily drinking water among the groups in the following weeks (P ＞ 0.05).

Fig.4: Effects of dexamethasone on average daily food intake and drinking water of rats in each group after intervention

3.4.Effects of dexamethasone on inflammatory cell infiltration, airway wall and smooth muscle thickness in lung tissue

As shown in fig.5 and tab.4, compared with the N group, there was obvious inflammatory infiltration in and around the airway in the model group, and the thickness of airway wall and smooth muscle increased significantly (P ＜ 0.01). However, the airway inflammatory cell infiltration in the DEXM and DEXL groups was significantly lower than that in the M group, and the airway wall thickness and smooth muscle thickness in the DEXL group were significantly lighter than those in the M group (P ＜ 0.01). The mean value of rats in the DEXL group was lower than that in the M group.However, there was no significant difference between the DEXM group and the DEXM group (P ＞ 0.05).

Fig.5: Effects of dexamethasone on inflammatory infiltration of lung tissue,thickness of airway wall and airway smooth muscle in rats (HE staining ×200)

3.5.Effect of dexamethasone on collagen fiber deposition in airway epithelium

As shown in fig.6 and Tab.4, there is a small deposition of collagen fibers in the airway wall and surrounding vessels in normal rats. In the M group, the airway wall and surrounding blood vessels were widely distributed, and the deposition of collagen fibers in the airway epithelium was significantly increased (P ＜ 0.01). Compared with the M group, the deposition of subepithelial collagen fiber in the DEXM group and DEXL group was inhibited (P ＜ 0.05), but there was no significant difference between the DEXM group and the DEXL group (P ＞ 0.05).

Tab.4 Histological changes of lungs in rats of each group (n = 6, ±s)

Tab.4 Histological changes of lungs in rats of each group (n = 6, ±s)

Note: Compared with group N **P＜0.01; Compared with group M ▲P＜0.05, ▲▲P＜0.01;

Group Wam/Pbm(μm2/μm) Wat/Pbm(μm2/μm) Wcol/Pbm(μm2/μm)N 12.5575±1.2058 5.0999±0.6487 3.6726±0.4141 M 25.9620±1.1457** 7.0909±0.9565** 5.9197±0.8099**DEXM 16.8484±2.7220▲▲ 5.7311±0.6068▲▲ 4.6791±0.6746▲DEXL 15.8397±1.2585▲▲ 5.0979±0.6123▲▲ 4.4371±0.7891▲F 67.247 10.184 10.963 P 0.000 0.000 0.000

Fig.6: Effect of dexamethasone on subcutaneous collagen deposition in lung tissue of rats in each group (Masson staining × 200)

4.Discussion

Bronchial asthma is a serious and common chronic respiratory disease, and the global incidence of asthma is still on the rise [1].Current studies have shown that the theory of airway inflammation is the most basic pathogenesis of asthma. Airway remodeling is not only the inevitable result of repairing airway injury caused by airway inflammation, but also the basis of relatively irreversible lung function damage. The proliferation of airway epithelium and airway smooth muscle and airway subcutaneous fibrosis play an important role in the formation mechanism of airway remodeling[1].Glucocorticoid is the main drug in the treatment of asthma, mainly inhaled glucocorticoid, but it still needs short-term or even long-term oral dosage form therapy in some patients with acute exacerbation or refractory asthma [1,12]. The basic research on bronchial asthma focuses on these pathological changes, especially in the study of new drugs and new methods, and dexamethasone is a common positive control drug in animal experiments[13-15]. Studies have shown that dexamethasone can not only reduce airway inflammation in asthma model rats, but also reduce airway epithelial injury, reduce airway smooth muscle proliferation and inhibit airway subepithelial collagen fiber deposition[16-19]. The results are consistent with the experimental results of this experiment. However, in many studies,the dosage of dexamethasone is not uniform, and there is a lack of detailed description of the side effects and even death caused by dexamethasone, so the retrieved intervention dose may not be suitable for the rat model of chronic asthma. In this study, 0.5mg/(kg ·d)[20,21](equivalent to 8 tablets per day of 70kg adults),which is common in literature retrieval, was selected as high dose intragastric perfusion. The equivalent dose of 0.125mg/ (kg ·d)(equivalent to 2 tablets per day of 70kg adults) was intragastrically administered to rats with the lowest initial dose of clinical routine.

The equivalent dose of 0.0625mg/ (kg ·d) (equivalent to 1 tablet per day of 70kg adults) was intragastrically administered to rats with a routine clinical maintenance dose. The results showed that 0.5mg/(kg ·d) caused a large number of deaths in a short period of time,and the adverse reactions were obvious, so this dose was not suitable as a positive control dose in the animal experiment of chronic asthma. Therefore, this dose is not suitable for positive control in animal experiments with chronic asthma. 0.125mg/ (kg ·d) and 0.0625mg/ (kg ·d) could significantly reduce airway inflammation and airway remodeling in rats, and there was no difference between the two groups, so both of them could be used as positive control in the rat model of chronic asthma. However, in the DEXM and late stage of the intervention, the spirit of the former rats gradually deteriorated, the food intake gradually decreased, the body mass decreased continuously, and a small amount of death began to occur,and the intervention time in this experiment was only 42 days. It is not the longest time for the establishment and intervention of chronic asthma rat model, so it is suggested that dexamethasone 0.0625mg/(kg ·d) may be more suitable for the positive control of chronic asthma rat model.

In this experiment, no appropriate samples were taken from the other groups at the same time in the DEXH group to compare the therapeutic effects of the same period, and no more tests were made, including blood and alveolar lavage fluid. Therefore, it is impossible to compare the effects of different modeling time on airway pathological changes in asthmatic rats, and it is impossible to fully compare the intervention effects of three concentrations of dexamethasone on chronic asthma rat model, which are the shortcomings and shortcomings of this experiment. In this experiment, it was also found that modeling could increase the food intake of rats, but the increase of body mass of rats was not parallel to it. At the beginning of modeling, the amount of drinking water of rats was significantly affected, but the effect disappeared in the later stage, and the mechanism was not clear. In order to further verify the study in the later stage of the experiment.

In conclusion, the intragastric doses of dexamethasone 0.125mg/(kg ·d) and 0.0625mg/ (kg ·d) could significantly reduce airway inflammation and airway epithelial and airway smooth muscle proliferation in a six-week chronic asthma rat model. It can effectively inhibit airway collagen deposition and can be used as an appropriate dose of positive control drugs in chronic asthma rat model for 6 weeks, but there are fewer adverse reactions in the later stage of 0.0625mg/ (kg ·d). It may be more suitable to intervene for 6 weeks or even longer intervention time of chronic asthma rat model.