Experimental study of bilobalide against rat Pneumocystis carinii pneumonia - Master's thesis - Dissertation

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Pneumoeystis carinii pneumonia (PCP) is an opportunistic pathogenic infectious disease that occurs in people with impaired or defective immune function. The pathological damage is mainly caused by lung tissue. With the advancement of society, the improvement of people's living standards and the prolongation of life expectancy, in recent years, high-risk groups such as patients with advanced diabetes, cancer chemotherapy and AIDS patients have further expanded, making this disease an important cause of death in this population. The main drugs against PCP at home and abroad are sulfonamides, pentamidines, etc. Although these drugs can receive good curative effect, there are obvious toxic side effects, and the incidence of drug resistance is also significantly increased. In view of the side effects of western medicine, some Chinese scholars _2I5j used traditional Chinese medicine Astragalus, Ginkgo, Garlic, Artemisia annua L. to treat Pneumocystis carinii pneumonia, which has a certain therapeutic effect. This study was conducted through animal experiments.

The efficacy of combined Astragalus injection against Pneumocystis carinii pneumonia was compared with that of compound sulfamethoxazole, and the efficacy of the two treatment regimens was compared to provide a theoretical basis for clinical treatment.

1. Materials and Method

1.1 Establishment of PCP model: 4o clean grade sD rats weighing 180-220g (purchased by Experimental Animal Center of Hunan University of Traditional Chinese Medicine) were used, and open squirrel cages were fed with ordinary pellets. Two rats were randomly selected and subcutaneously injected with 0.5 mL of physiological saline under sterile conditions as a normal control group. The remaining rats were injected subcutaneously with dexamethasone sodium phosphate (1IT-g/only, 2 times/week) under sterile conditions in the groin. In the experimental group and the control group, 1 g/L of tetracycline hydrochloride was added to the drinking water. Prevent secondary bacterial infections. Rat changes were observed at any time, including changes in food intake, water intake and body weight, hair color changes, and activity. After 9 weeks of continuous induction, 2 rats in the experimental group and 2 rats in the normal control group were randomly aseptically killed. The lung slices were printed on the clean slides to make lung tissue prints. After fixed drying, staining was performed by hexamethylenetetramine silver (GMS) rapid staining. Under the oil microscope, there was no abnormality in the control group. A large number of Pneumocystis carinii cysts confirmed the establishment of the PCP rat model.

1.2 Drugs and reagents:

(Sigma), Astragalus Injection (Chengdu Diao Jiuyi Pharmaceutical Factory), dissolve 40rag with 0.8ml absolute ethanol

Then add 9.2ml of Astragalus injection to make aqueous solution of Astragalus membranaceus; dexamethasone sodium phosphate (Tianjin Pharmaceutical Factory); tetracycline powder (Huabei Pharmaceutical Factory Tianxing Co., Ltd.); compound sulfamethoxazole tablets (Kunming Pharmaceutical Group Co., Ltd.) Limited). 1.3 Experimental rats grouping and treatment: After the successful establishment of the model, the injection of dexamethasone sodium phosphate was stopped, while the experimental rats naturally died during the induction process, and 33 PCP rats were randomly divided into 3 groups, each group 11 only. Group A was infected with the control group and no medication was given. Group B was treated with aqueous solution of Astragalus membranaceus, 0.5 mL/only, intraperitoneal injection, 2 times/day for 10 consecutive days; Group C was compound sulfamethoxine In the azole treatment group, the compound sulfamethoxazole was made into a suspension, and the compound sulfamethoxazole was dissolved in 20 mL of NS, 1 ml/time/day gavage, treatment for 10 days, and all the sections were observed after 1 week of withdrawal. Kill, aseptically take the lungs.

1.4 Observation indicators and methods: After the three groups of rats were killed, the lung tissue was taken, and the lung lobe was cut into a 1 cm diameter print. The stain was stained by GMS. Under the oil microscope, 100 oils were counted per lung print. The total number of cysts in the field of view was calculated, and the average number of Pneumocystis cysts in the lung prints of each group was calculated. The lung tissue was fixed in 10% formaldehyde solution, and pathological sections were stained with HE for observation.

I. 5 Statistical analysis: The average number of Pneumocystis cysts in each group of mouse lung prints was processed by SPSS15.0 software for statistical analysis.

2. result

2.1 Observation of the basic situation of the disease. Before treatment, the rats in the three groups generally had less anorexia, body hair fluffiness, dark coat color, and difficulty in breathing. After treatment with Astragalus membranaceus aqueous solution or compound sulfamethoxazole, the rats in Groups B and C increased their food intake, hair color and dyspnea.

2.2 Changes in lung weight and body weight in rats. From (Table 1), the average body weight of the rats in each group decreased at the end of the experiment, and the A model control group decreased most significantly, which was greater than the B group and the C group, but the difference was not statistically analyzed. Significance (P>0.05). The average lung weight and lung weight/body weight ratio of the three groups of rats were found to be the highest, and the lung weight and lung weight/body weight ratio were the highest in group A, and the difference was statistically significant (P<0.05). ).

2.3 The degree of infection of Pneumocystis and the number of cysts in lung prints. After the establishment of the rat model of Pneumocystis carinii pneumonia, the lungs were treated by the corresponding Chinese and Western medicine, and the lungs were observed under aseptic operation. The number of cysts of Pneumocystis carinii infected in the lung tissue of each group was observed. We found that the Pc infection rate in lung tissue prints of group A rats was very high, and after drug treatment, the Pc infection rate of lung tissue prints in group B and group C was significantly reduced (see Figure 1).

The lung prints of each group of lungs showed the average number of cysts of Pneumocystis per 100 oil fields (see Table 2). After analysis, the number of cysts of Pneumocystis per 100 oil fields was significantly lower in group B and group C than in group A (P<0.01).

2.4 Pathological examination of lung tissue. PCP model control group (group A): moderate to severe interstitial pneumonia, alveolar septum widening, alveolar cavity became smaller, alveolar epithelial hyperplasia, interval widened, cells with lymphocytes and monocytes Infiltration, part of the alveolar cavity is filled with light red foamy exudate (Fig. 1); Astragalus membranaceus aqueous solution treatment group (group B): mild interstitial pneumonia, occasionally a small amount of eosinophilic foam in the alveoli, visible foam cell aggregation There are fewer inflammatory cells and narrower alveolar septum (Fig. 2); compound sulfamethoxazole tablets treatment group (group C): mild interstitial pneumonia, alveolar cavity size, wide alveolar septum, alveolar space A small amount of eosinophilic foam cells can be seen (Figure 3)

3. discuss

At present, the most commonly used methods for establishing PCP animal models at home and abroad are the use of glucocorticoids (cortisone acetate or dexamethasone) to inhibit the PCP model induced by the rat immune system. This experiment used this method to successfully model, further indicating the use of ground plugs. It is a stable and reliable method to establish a rat PCP model. Modern pharmacological studies have shown that Astragalus has obvious anti-hypoxia, anti-inflammatory, analgesic and immune-modulating functions.

Has a certain anti-Pneumocystis carinii pneumonia effect L 2-3]. In this experiment, bilobalide combined with astragalus injection was used to treat Pneumocystis carinii pneumonia in rats. The lung mass/body mass value and the mean number of Pneumocystis per 100 oil fields were used as evaluation indicators. And combined with pathological comprehensive observation to judge the anti-Pneumocystis effect of the drug. The above research results show

Combined Huangqi injection has obvious therapeutic effect on rat PCP, and it is found to be toxic and side effects. therefore,

Combined Astragalus injection is expected to be applied to the clinical treatment of Pneumocystis carinii, but its mechanism of action needs further research.