Construction method and application of streptococcus pneumoniae pneumonia animal model

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a construction method and application of a streptococcus pneumoniae pneumonia animal model. The invention provides a construction method of a streptococcus pneumoniae infected rat pneumonia and kidney injury model. The method comprises the following steps: S1, applying a streptococcus pneumoniae liquid into the respiratory tract of a rat by adopting an atomization bacterium feeding mode; and S2, verifying whether the rat treated in the step S1 is infected with pneumonia or not and whether kidney injury exists or not. According to the method, whether pneumonia is infected or not is judged by detecting inflammatory factors in alveolar lavage and lung HE staining, and whether kidney injury exists or not is judged by detecting the urine protein / creatinine ratio in a rat urine sample and detecting kidney HE staining. The construction method provided by the invention is convenient to operate, accurate in model construction and high in survival rate.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology, and in particular relates to a method for constructing an animal model of Streptococcus pneumoniae and its application. Background Art

[0002] Streptococcus pneumoniae (S.pn) is a common upper respiratory tract pathogen that primarily exists as a commensal bacterium, coexisting with other commensal microorganisms on respiratory epithelial cells. Research data indicate that S.pn, along with Haemophilus influenzae and Moraxella catarrhalis, are three common bacteria responsible for lung infections, with S.pn being the most common. Respiratory colonization by S.pn can be detected in approximately 10% of healthy adults, 20%-40% of healthy children are carriers, and over 60% of infants and children are carriers. S.pn produces numerous well-characterized virulence factors that are crucial in the pathogenesis of infection, including capsular polysaccharides, the pore-forming toxin pneumotoxin, surface proteins (PsaA, PSPA, PSPC, PhtD, PavA, and PsrP), hyaluronidase, and neuraminidase (NA). The pathophysiology of S. pn may involve the production of NA, an enzyme that cleaves N-acetylneuraminic acid residues from glycoproteins on red blood cells, glomerular endothelial cells, and renal tubular epithelial cells, exposing T antigens. The resulting anti-T antibody is a naturally occurring IgM antibody that is present in almost all children over 6 months of age.

[0003] In Traditional Chinese Medicine, the Five Elements correspond to the five internal organs, with metal and water corresponding to the lungs and kidneys, respectively. Metal generates water, so the lungs and kidneys are like mother and child, and the mutual generation of metal and water signifies the common origin of the lungs and kidneys. The "Suwen" chapter on the theory of disease potential mentions that the Shaoyin meridian runs through the kidneys and connects to the lungs. From a meridian perspective, the hand Taiyin Lung Meridian and the foot Shaoyin Kidney Meridian are connected; functionally, the qi of the lungs and kidneys is interconnected, making them closely connected and interdependent. The "Zhengzhi Zhunsheng" states: "From the lungs to the kidneys... the essence and qi are internally deprived, and the fundamentals are not solid. This leads to a loss of qi absorption, with more outflow than inflow." The lung's governing qi is primarily Zong Qi, which is generated by the primordial qi in the kidneys. Zong Qi, in turn, provides the material basis for the production of essence and qi in the kidneys, achieving mutual complementarity and nourishment between the lungs and kidneys. Modern medical research shows that the lungs and kidneys play a synergistic role in maintaining body homeostasis: the lungs regulate O2 / CO2 levels through gas exchange, while the kidneys regulate oxygenation and blood volume through erythropoietin secretion and the renin-angiotensin-aldosterone axis system; the two work together to maintain acid-base balance (the lungs rapidly regulate CO2, and the kidneys metabolically regulate HCO3 - and H +) and affect hemodynamics through hormonal interactions (such as angiotensin conversion mediated by angiotensin-converting enzyme inhibitors). In pathological conditions (such as chronic obstructive pulmonary disease or sepsis), crosstalk between inflammatory mediators in the lung-kidney axis can lead to co-injuries, such as respiratory acidosis or acute kidney injury.

[0004] Pulmonary inflammation caused by Streptococcus pneumoniae infection not only affects the respiratory system but also damages the kidneys. Because kidney damage caused by Streptococcus pneumoniae is mostly a clinical observation, there is currently no animal-level validation or detection of relevant indicators. Therefore, this study aims to construct a model of pneumonia and kidney damage in rats infected with Streptococcus pneumoniae. Summary of the Invention

[0005] The present invention aims to provide a method for constructing a rat model of pneumonia and kidney damage caused by Streptococcus pneumoniae infection and its application. The construction method provided by the present invention is easy to operate, the model is accurately constructed, and the survival rate is high.

[0006] The first object of the present invention is to provide a method for constructing a rat model of pneumonia and kidney damage caused by Streptococcus pneumoniae infection, the method comprising:

[0007] S1: The Streptococcus pneumoniae solution was administered into the respiratory tract of rats by aerosol administration;

[0008] S2: Verify whether the rats treated with S1 are infected with pneumonia and have kidney damage.

[0009] Preferably, the rats in step S1 are male and no less than 3 weeks old.

[0010] Preferably, the rats in step S1 are subjected to swimming stress treatment before being administered bacteria; the swimming stress treatment comprises placing the rats in a warm water pool, forcing them to swim until their mouths and noses are immersed in water for the first time, and then being fished out.

[0011] Preferably, the atomization bacteria feeding method in step S1 adopts an atomizer to feed the bacteria; the atomization time is 5-10 minutes.

[0012] Preferably, the amount of Streptococcus pneumoniae liquid applied in step S1 is 0.5 mL / kg, and the density of Streptococcus pneumoniae is 1x10 8 CFU / mL; the frequency of administering Streptococcus pneumoniae was once every two days until the end of the 7th week.

[0013] Preferably, the method for verifying whether pneumonia is infected in step S2 is to detect inflammatory factors through alveolar lavage fluid;

[0014] The method for detecting inflammatory factors in bronchoalveolar lavage fluid is specifically as follows: after anesthetizing rats with intraperitoneal injection of sodium pentobarbital, the rat trachea is opened, the alveoli are repeatedly flushed with PBS buffer, the bronchoalveolar lavage fluid is collected, and the bronchoalveolar lavage fluid is centrifuged at 4°C and 3000 rpm for 15 minutes, the supernatant is collected, and stored at -80°C for detection of inflammatory factor-related indicators;

[0015] The related indicators of the inflammatory factors are IL-6, IL-10, IL-1β, TNF-α, and IFN-β.

[0016] Preferably, the method for verifying whether pneumonia is infected in step S2 is by detecting lung HE staining;

[0017] The method for detecting lung HE staining is specifically as follows: after rats are anesthetized by intraperitoneal injection of sodium pentobarbital, lung tissue is taken, fixed with 4% paraformaldehyde solution, dehydrated, immersed, and embedded, then stained with hematoxylin and eosin, dehydrated, sealed, and examined under a microscope to observe changes in the lungs and collect images.

[0018] Preferably, the method for verifying whether kidney damage exists in step S2 is to collect urine samples from rats at 3, 5, and 7 weeks after administration of the bacteria, and to detect the urine protein / creatinine ratio and serum renal function biochemical indicators; the method for collecting urine samples from rats is to place the rats in metabolic cages, fasting but not depriving of water, and collecting urine samples from the rats for 24 hours;

[0019] The specific method for detecting serum renal function biochemical indicators is as follows: after rats are anesthetized by intraperitoneal injection of sodium pentobarbital, blood is collected from the abdominal aorta, serum is separated, and the blood sample is allowed to stand for 1 hour and then centrifuged at 3500 rpm for 10 minutes to separate the serum.

[0020] Preferably, the method for verifying whether kidney damage exists in step S2 further comprises detecting kidney HE staining.

[0021] The second object of the present invention is to provide a rat pneumonia and kidney injury model obtained by the above-mentioned method for constructing a rat pneumonia and kidney injury model infected with Streptococcus pneumoniae, and to use the model in screening or treating pneumonia drugs.

[0022] Compared with the existing technology, the beneficial effects of the present invention are: the method for constructing a pneumonia and kidney damage model in rats infected with Streptococcus pneumoniae provided by the present invention provides an animal model for experimental research on the phenomenon of kidney damage caused by clinical pneumonia infection, and provides a methodological reference for basic research on pediatric kidney disease caused by low immunity due to recurrent pneumonia and physical exhaustion. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Schematic diagram of inflammatory factor-related indicators in the bronchoalveolar lavage fluid of model rats in each group.

[0025] Figure 2 Schematic diagram of HE pathological examination of the lungs of model rats in each group. The red arrows represent inflammatory cell infiltration, and the yellow arrows represent alveolar wall thickening.

[0026] Figure 3 Schematic diagram of HE pathological examination of kidneys in model rats of each group. The black arrows represent glomerular atrophy.

[0027] Figure 4 Schematic diagram of urine protein / creatinine of model rats in each group (x±s, n=5-8); compared with the normal group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0028] Figure 5 The graph shows the analysis of serum renal function biochemical indicators in model rats in each group (x±s, n=5-8). Compared with the normal group, *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0029] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Unless otherwise specified, the production processes, experimental methods or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art, and their names and / or abbreviations are conventional names in the field and are very clear and unambiguous in the relevant fields of use. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment to implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0031] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources and are all conventional products that can be purchased through regular commercial channels or prepared according to conventional methods well known to those skilled in the art.

[0032] The experimental materials and main reagents used in the following examples are as follows:

[0033] 1. Experimental Animals

[0034] 3-week-old SD male rats were provided by Hefei Qingyuan Biotechnology Co., Ltd.

[0035] 2. Strains

[0036] Streptococcus pneumoniae ATCC49619 was purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0037] 3. Experimental Materials

[0038] Large animal nebulizer (purchased from Zhengzhou Haopai Biotechnology Co., Ltd.), centrifuge, oven and other routine biological instruments.

[0039] BCA quantitative kit (purchased from Lanjieke Technology Co., Ltd.), creatinine kit, urea nitrogen kit, triglyceride kit, albumin kit, total cholesterol kit, total protein kit (purchased from Nanjing Jiancheng Bioengineering Institute), rat IL-6 ELISA kit, rat IL-10 ELISA kit, rat IL-1β ELISA kit, rat TNF-α ELISA kit, rat IFN-β ELISA kit (purchased from Shanghai Jianglai Biotechnology Co., Ltd.).

[0040] Example 1

[0041] Construction of animal model of pneumonia

[0042] 1. The experimental animals were randomly divided into a normal group, a bacteria administration group, and a swimming + bacteria administration group.

[0043] The animals in the bacterial group were given a dose of 0.5 mL / kg to take a certain amount of 1x10 8 Add a quantitative bacterial solution of Streptococcus pneumoniae at a concentration of CFU / mL into the nebulizer cup, select the strong gear of the nebulizer, and select the nebulization time for 5 minutes. After the nebulization is completed, let the experimental animals stay in the nebulizer box for 10-15 minutes to ensure that the experimental animals fully inhale the bacterial solution. Administer bacteria once every two days until the end of the 7th week.

[0044] In the swimming + bacteria administration group, swimming was performed to exhaust the physical strength of the young rats. The rats were placed in a warm water pool (water depth 36 cm, water temperature 30°C) and forced to swim until their mouths and noses were immersed in the water for the first time. The rats were fished out and dried with a towel. This operation was performed once a day, and then the bacteria model was established using the same method as the bacteria administration group.

[0045] The normal group was given normal saline by nebulization, and the operation was the same as that of the bacteria group.

[0046] 2. Detection of inflammatory factors after pneumococcal infection

[0047] At the 7th week of bacterial administration, rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and the trachea of ​​the rats was exposed. 5 ml of PBS buffer was injected into the rat respiratory tract from the upper end of the trachea, and the alveoli were repeatedly flushed downward for 3 times. The alveolar lavage fluid was collected and then centrifuged at 4°C and 3000 rpm (centrifugal radius 14 cm) for 15 minutes. The supernatant was collected and stored at -80°C for the detection of inflammatory factor-related indicators, including IL-6, IL-10, IL-1β, TNF-α, and IFN-β. The results are shown in Figure 2. Figure 1 shown.

[0048] The results showed that compared with the normal group, the concentrations of IL-6, IL-10 and IFN-β in the alveolar lavage fluid of the bacteria-administered group were significantly increased, and the concentrations of IL-6, IL-10, TNF-α and IFN-β in the alveolar lavage fluid of the swimming + bacteria-administered group were also significantly increased, indicating that young rats had lung infection after repeated Streptococcus pneumoniae infection.

[0049] 3. Evaluation of Lung Damage and Detection of Kidney Damage after Streptococcus Pneumoniae Infection

[0050] At the 7th week of bacterial administration, rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and 3 lung and kidney tissues were taken from each group and fixed with 4% paraformaldehyde solution. The tissues were removed from the fixative, trimmed and flattened, and then dehydrated in a dehydrator and immersed in paraffin; embedded in an embedding machine, and then the wax block was cut to a thickness of 3 μm and baked; after the water was dried and the paraffin was baked, it was taken out, dewaxed, and washed with water; then stained with hematoxylin and eosin, and finally dehydrated, sealed, and examined under a microscope. The slides were placed under a microscope to observe changes in the lungs and collect images. The results of lung injury evaluation are as follows: Figure 2 As shown, kidney damage Figure 3 shown.

[0051] Results showed that compared with the normal control group, both the bacteria-administered group and the swimming-plus-bacteria-administered group showed extensive inflammatory cell infiltration and alveolar wall thickening. Kidney HE analysis revealed glomerular atrophy in both the bacteria-administered and swimming-plus-bacteria-administered groups compared with the normal control group. This suggests that lung infection damages both the rats' lungs and kidneys.

[0052] 4. Detection of indicators of proteinuria induced by Streptococcus pneumoniae infection

[0053] Urine samples were collected from rats at the 3rd, 5th, and 7th weeks of bacterial administration. The rats were placed in metabolic cages and fasted but not watered. 24-hour urine samples were collected from the rats for urine protein / creatinine testing to determine whether the urine protein in the rats increased. The results were as follows: Figure 4 shown.

[0054] The results showed that the urine protein / creatinine ratio of the bacteria-administered group was significantly higher than that of the normal group at the 3rd and 5th weeks, and there were also significant differences in the urine protein / creatinine ratio of the swimming + bacteria-administered group at the 3rd, 5th and 7th weeks, indicating that rats infected with Streptococcus pneumoniae showed kidney damage with increased urine protein.

[0055] 5. Detection of renal function indicators after Streptococcus pneumoniae infection

[0056] At week 7 of the bacterial administration group, rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and blood was collected from the abdominal aorta to separate the serum. The blood samples were allowed to stand for 1 hour and then centrifuged at 3500 rpm for 10 minutes to separate the serum for the detection of renal function biochemical indicators. The renal function biochemical indicators included creatinine, urea nitrogen, total cholesterol, albumin, total protein, and triglycerides. The results are shown in Table 1. Figure 5 shown.

[0057] The results showed that serum creatinine, urea nitrogen, and total cholesterol levels in the bacteria-administered group were significantly higher than those in the normal group, while albumin and total protein levels were significantly lower. In the swimming + bacteria-administered group, serum creatinine, urea nitrogen, total cholesterol, and triglyceride levels were significantly higher, while albumin and total protein levels were significantly lower. These results indicate that serum renal function indicators in rats infected with Streptococcus pneumoniae change.

[0058] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for constructing a rat model of pneumonia and kidney damage caused by Streptococcus pneumoniae infection, characterized in that: The construction method comprises: S1: The Streptococcus pneumoniae solution was administered into the respiratory tract of rats by aerosol administration; S2: Verify whether the rats treated with S1 are infected with pneumonia and have kidney damage.

2. The construction method according to claim 1, characterized in that The rats in step S1 are male and no less than 3 weeks old.

3. The construction method according to claim 1, characterized in that In step S1, the rats are subjected to swimming stress treatment before being administered bacteria; the swimming stress treatment comprises placing the rats in a warm water pool, forcing them to swim until their mouths and noses are immersed in water for the first time, and then being fished out.

4. The construction method according to claim 1, characterized in that In step S1, the atomization method of feeding bacteria is to use an atomizer to feed bacteria; the atomization time is 5-10 minutes.

5. The construction method according to claim 1, characterized in that The amount of Streptococcus pneumoniae liquid applied in step S1 is 0.5 mL / kg, and the density of Streptococcus pneumoniae is 1x10 8 CFU / mL; the frequency of administering Streptococcus pneumoniae was once every two days until the end of the 7th week.

6. The construction method according to claim 1, characterized in that The method for verifying whether pneumonia is infected in step S2 is to detect inflammatory factors through alveolar lavage fluid; The method for detecting inflammatory factors in bronchoalveolar lavage fluid is specifically as follows: after anesthetizing rats with intraperitoneal injection of sodium pentobarbital, the rat trachea is opened, the alveoli are repeatedly flushed with PBS buffer, the bronchoalveolar lavage fluid is collected, and the bronchoalveolar lavage fluid is centrifuged at 4°C and 3000 rpm for 15 minutes, the supernatant is collected, and stored at -80°C for detection of inflammatory factor-related indicators; The related indicators of the inflammatory factors are IL-6, IL-10, IL-1β, TNF-α, and IFN-β.

7. The construction method according to claim 1, characterized in that The method for verifying whether pneumonia is infected in step S2 is by detecting lung HE staining; The method for detecting lung HE staining is specifically as follows: after rats are anesthetized by intraperitoneal injection of sodium pentobarbital, lung tissue is taken, fixed with 4% paraformaldehyde solution, dehydrated, immersed, and embedded, then stained with hematoxylin and eosin, dehydrated, sealed, and examined under a microscope to observe changes in the lungs and collect images.

8. The construction method according to claim 1, wherein: The method for verifying whether kidney damage exists in step S2 is to collect urine samples from rats 3, 5, and 7 weeks after administration of the bacteria, and to detect the urine protein / creatinine ratio and serum renal function biochemical indicators; The method for collecting rat urine samples is to place the rats in a metabolic cage, fasting but not depriving of water, and collect the rat urine samples for 24 hours; The specific method for detecting serum renal function biochemical indicators is as follows: after rats are anesthetized by intraperitoneal injection of sodium pentobarbital, blood is collected from the abdominal aorta, serum is separated, and the blood sample is allowed to stand for 1 hour and then centrifuged at 3500 rpm for 10 minutes to separate the serum.

9. The construction method according to claim 1, characterized in that: The method for verifying whether kidney damage exists in step S2 further includes detecting kidney HE staining.

10. Use of the rat pneumonia and kidney injury model obtained by the method for constructing a rat pneumonia and kidney injury model infected with Streptococcus pneumoniae according to any one of claims 1 to 9 in screening or treating pneumonia drugs.

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