Pharmaceutical composition and application thereof in preparing medicine for treating swine streptococcal meningitis

Abstract

The invention belongs to the technical field of biological medicine, and discloses a pharmaceutical composition which comprises glucocorticoid and penicillin. Aiming at the problems of poor treatment effect, poor prognosis and the like of clinically treating swine streptococcal meningitis at present, penicillin and glucocorticoid are combined, and meanwhile, the invention discloses a drug combination method. Microscopic examination, change of partial bacteria loading amount, blood brain barrier staining observation and the like are carried out to find that the composition can effectively treat streptococcus suis infection and can quickly reduce brain inflammation, so that the blood brain barrier damaged by streptococcus suis is repaired, and the effect is better than that of a single component; therefore, the prognosis effect of streptococcal meningitis of swine can be improved to the greatest extent. Meanwhile, the composition is a common medicine, and a new feasible thought is provided for clinically treating the streptococcus suis diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a pharmaceutical composition and application thereof in preparing a drug for treating swine streptococcal meningitis. Background Art

[0002] Suicidal meningitis is a common pig disease caused by infection with Streptococcus suis, with high morbidity and mortality. Currently, there is little clinical guidance on the use of drugs to treat suicidal meningitis, and the direction of treatment is very unclear.

[0003] Penicillin is a widely used antibiotic that has a good bactericidal effect on Streptococcus suis. Studies have shown that streptococci frequently occur in pig farms, and that streptococcal meningitis in pigs is very harmful to pigs. Generally, after streptococci infect the brain, the pigs will face death. Moreover, streptococci in pigs are Gram-positive bacteria, and their thick cell walls make many antibiotics ineffective against them. With the frequent use of antibiotics in pig farms, the resistance rate of streptococci in pigs has increased year by year, which brings considerable challenges to the clinical treatment of streptococci in pigs.

[0004] Therefore, it is urgent to seek effective treatment methods for suis streptococcal meningitis according to the characteristics of Streptococcus suis, which is of great significance in reducing the mortality rate of suis streptococcal meningitis and the prognosis after treatment. Summary of the invention

[0005] In view of the problems of poor treatment effect and poor prognosis of current clinical treatment of Streptococcus suis meningitis, a combination of penicillin and glucocorticoid and a combined treatment method are proposed, aiming to effectively treat Streptococcus suis infection and quickly reduce brain inflammation and repair the blood-brain barrier damaged by Streptococcus suis, thereby maximizing the prognosis of Streptococcus suis meningitis.

[0006] The first object of the present invention is to provide a pharmaceutical composition comprising penicillin and glucocorticoid.

[0007] Preferably, the glucocorticoid is hydrocortisone. The glucocorticoid is hydrocortisone, which has a strong anti-inflammatory effect, can effectively reduce brain inflammation and edema, reduce intracranial pressure, thereby relieving the neurological symptoms of sick pigs, reducing brain tissue damage caused by inflammation and intracranial hypertension, and enabling the blood-brain barrier to restore its barrier function through the body's repair effect.

[0008] More preferably, the concentration of penicillin is 20 mg / kg to 50 mg / kg, and the concentration of hydrocortisone is 10 mg / kg to 20 mg / kg.

[0009] Furthermore, the present invention uses highly toxic type 2 Streptococcus suis to establish a mouse meningitis model in the laboratory, and the experimental animals are divided into groups, namely A: blank group (intracerebral injection of normal saline) B: infection group (intracerebral injection of type 2 Streptococcus suis) C: penicillin treatment group (infected mice are injected with 50 mg / kg sodium penicillin through the tail vein) D: glucocorticoid treatment group (infected mice are injected with 20 mg / kg hydrocortisone intramuscularly) E: combined treatment group (infected mice are injected with sodium penicillin and hydrocortisone at the same time); after 3 days of treatment, each experimental mouse begins to measure subsequent indicators. The subsequent indicator measurement includes the following:

[0010] 1) Record the food intake and body weight changes of mice during modeling and treatment;

[0011] 2) The complete brain tissue of mice in each treatment group was obtained and sliced ​​for F480 and HE staining microscopy.

[0012] 3) The complete brain tissue of mice was homogenized and then plated to calculate the changes in brain bacterial load.

[0013] 4) The blood-brain barrier was stained and observed by intraperitoneal injection of Evans blue into each group of mice.

[0014] Through the above series of experiments, it was found that the above composition can effectively treat Streptococcus suis infection and can quickly reduce brain inflammation, thereby repairing the blood-brain barrier damaged by Streptococcus suis. The effects are better than those of a single component, and thus can maximize the prognosis of Streptococcus suis meningitis.

[0015] The second object of the present invention is to provide a method for combined treatment of swine streptococcal meningitis, comprising treating a herd of pigs with the pharmaceutical composition.

[0016] Further, the pharmaceutical composition is used to quickly repair the blood-brain barrier. When the above treatment is adopted, the pharmaceutical composition composed of penicillin and hydrocortisone is injected intramuscularly into pigs. More preferably, the concentration of penicillin is 50 mg / kg, and the concentration of hydrocortisone is 20 mg / kg.

[0017] The third object of the present invention is to provide the use of the above-mentioned pharmaceutical composition for treating swine streptococcal meningitis.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention selects drugs with obvious inhibitory effect on Streptococcus suis and low clinical drug resistance rate from commonly used antibiotics for animals, and simultaneously uses potent anti-inflammatory drugs to repair the damage of blood-brain barrier caused by Streptococcus suis, providing a new idea for clinical treatment of Streptococcus suis diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figures show the comparison of body weight and food intake of mice before and after modeling and before and after treatment.

[0021] Figure 2 This is a graph of bacterial load in mouse brain tissue.

[0022] Figure 3 This is a brain tissue section of a mouse whole brain.

[0023] Figure 4 This is a graph showing the experimental results of bacterial load in mouse brain. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0025] Unless otherwise specified, the test methods used in the following embodiments and experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels; the equipment used, unless otherwise specified, are all conventional experimental equipment.

[0026] Example 1 Construction of animal model and drug effects on swine streptococcal meningitis

[0027] 1. Test strain: In this experiment, highly virulent wild Streptococcus 05ZYH33 (deposited in the National Veterinary Microbial Resistance Risk Assessment Laboratory, College of Veterinary Medicine, South China Agricultural University, and also available through commercial channels) was used as the test strain.

[0028] 2. Model grouping: 5-week-old female ICR mice weighing 20 g (purchased from Guangdong Beston Biological Co., Ltd.) were selected as experimental animals, and 50 mice were divided into 5 groups:

[0029] A: blank group (intracerebral injection of normal saline);

[0030] B: infection group (intracerebral injection of Streptococcus suis type 2);

[0031] C: penicillin treatment group (infected mice were injected with sodium penicillin at 50 mg / kg via tail vein);

[0032] D: glucocorticoid treatment group (infected mice were intramuscularly injected with 20 mg / kg of hydrocortisone);

[0033] E: Combined treatment group (infected mice were injected with sodium penicillin and hydrocortisone at the same time).

[0034] 3. Drug treatment: When infected with Streptococcus suis, the mice were first anesthetized with tribromoethanol for a long time, and then the mouse brain epidermis was cut open with a scalpel, the skull was opened with a brain drill, and 10 6 Streptococcus suis. After the mice were raised normally for 1 day, each group was treated with drugs. For example, the combined treatment group was intravenously injected with penicillin at a dose of 2000U / kg and the glucocorticoid was injected intramuscularly at a dose of 20mg / kg. The weight changes of the mice were observed, and the brain tissues were taken for section staining and microscopic examination, and the bacterial load in the brain was calculated by drop plate.

[0035] 4. Results and Analysis

[0036] The comparison results of body weight and food intake of mice before and after modeling and before and after treatment are shown in Figure 1 Among them, the information in the figure is as follows:

[0037] CON: blank group; MOL: modeling group (infection group); PEN: penicillin alone group; GS: glucocorticoid alone group; PEN+GS: penicillin combined with glucocorticoid group. The changes in food intake and body weight reflect the effect of modeling and the prognosis of treatment after single and combined medication. From the above results, it can be seen that the food intake and body weight of mice recovered faster after combined treatment, indicating that the combined medication group has a better effect in vivo than single medication.

[0038] Therefore, the above results indicate that the combined drug group has a more significant effect on the feeding status and weight recovery of mice than penicillin alone or glucocorticoids alone in the treatment of swine streptococcal meningitis.

[0039] The bacterial load in mouse brain tissue is shown in Figure 2 , where: ns represents P value>0.5: no significant difference;

[0040] The symbol “*” represents (P<0.05); “**” (P<0.01); “***” (P<0.001); “****” (P<0.0001).

[0041] We took brain tissues from mice in different treatment groups and put them into 2ml epoxies. We added 1ml PBS (phosphate buffer) into each epoxies and then used a freezing homogenizer to homogenize the brain tissues and then used a plate to calculate the changes in the bacterial load in the brain. By analyzing the changes in the bacterial load in the brain, we can obtain the inhibitory effects of penicillin alone and combined with glucocorticoids on Streptococcus suis. The results showed that the bacterial load in the brain of the combined drug group was more significantly reduced than that of the single drug group. This indicates that the relief of inflammation may have enabled the blood-brain barrier to resume its barrier function, effectively preventing some Streptococcus suis from entering the brain tissue.

[0042] Mouse whole brain tissue sections Figure 3First, the macrophages were stained orange by F480 staining. Macrophages are a type of inflammatory cell. When inflammation occurs in the body's tissues, they will gather at the site of inflammation. Therefore, the number of macrophages reflects the inflammation in the brain. Comparison of the F480 staining results of several groups showed that the combined medication group had a more obvious inhibitory effect on inflammation.

[0043] Example 2 Combination therapy group - penicillin combined with glucocorticoids to repair the integrity of the blood-brain barrier

[0044] Based on the experimental results of brain bacterial load in Example 1, we speculate that the use of glucocorticoids combined with penicillin can quickly repair the blood-brain barrier, so that the blood-brain barrier destroyed by Streptococcus suis can play a strong barrier role again, so that a part of Streptococcus suis cannot reach the brain tissue, thereby reducing the bacterial load of the brain tissue. Therefore, we use 1ml of Evans blue to judge the integrity of the blood-brain barrier by intraperitoneal injection of mice. Half an hour after intraperitoneal injection of 1ml of 2% Evans blue dye solution into the mouse, anesthetize the mouse and then perfuse it through the mouse heart, slowly inject 0.9 sodium chloride solution (containing heparin) through the apex of the heart, cut the right atrium until the fluid flowing out of the right atrium is clear, and stop perfusion. Separate the brain tissue. If the blood-brain barrier is destroyed, there will be obvious coloring in the brain tissue. On the contrary, if the integrity of the blood-brain barrier is good, the brain tissue will not be stained with Evans blue. After removing the brain tissue, use dimethylformamide to extract the Evans blue in the brain tissue and then measure OD 620 The integrity of the blood-brain barrier was further demonstrated by quantifying Evans blue at 100 nm. Figure 4 . It also showed that the combined drug group had the best effect in protecting the integrity of the blood-brain barrier of mice.

[0045] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that It consists of penicillin and hydrocortisone.

2. The pharmaceutical composition according to claim 1, characterized in that The concentration of the penicillin is 20 mg / kg to 50 mg / kg, and the concentration of the hydrocortisone is 10 mg / kg to 20 mg / kg.

3. Use of the pharmaceutical composition according to claim 1 in the preparation of drugs for preventing and treating swine streptococcal meningitis.

4. The use according to claim 3, characterized in that: The control object of the drug is pigs.

5. The use according to claim 3 or 4, characterized in that: The pharmaceutical composition is used for rapidly repairing the blood-brain barrier.

6. A method for combined treatment of swine streptococcal meningitis, characterized in that: The pharmaceutical composition consisting of penicillin and hydrocortisone is injected intramuscularly into pigs.

7. The method for combined treatment of suis streptococcal meningitis according to claim 6, characterized in that: The concentration of the penicillin is 50 mg / kg, and the concentration of the hydrocortisone is 20 mg / kg.

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