Method for constructing rabbit ear acne model

Abstract

The invention belongs to the technical field of medical experimental animal models, and particularly relates to a construction method of a rabbit ear acne model for studying acne pathogenesis and drug curative effect. A healthy New Zealand large-ear white rabbit is selected, a testosterone solution is subcutaneously injected to induce endocrine disorder, oleic acid is smeared at an opening of an external auditory canal on the inner side of an ear, dermatobacterium acnes suspension is subcutaneously injected, and whether acne-like lesions such as erythema, papule and comedo appear on the skin of the ear or not is observed. The method is easy and convenient to operate, low in cost, good in repeatability, capable of simulating the pathological process of human acne and suitable for acne pathogenesis research and drug screening.

Description

Technical Field

[0001] This invention belongs to the field of medical experimental animal model technology, specifically relating to a method for constructing a rabbit ear acne model. Background Technology

[0002] Acne is a common chronic inflammatory skin disease that primarily affects adolescents, significantly impacting their appearance, mental health, and quality of life. Its pathogenesis is complex, involving multiple factors, including excessive sebum secretion in the pilosebaceous unit, abnormal follicular keratinization, microbial infection such as Propionibacterium acnes, and pathological processes that trigger immune and inflammatory responses. This multifactorial nature makes acne prevention and treatment research highly challenging.

[0003] Establishing suitable animal models is a crucial research tool for exploring the pathogenesis and drug intervention strategies of acne. Commonly used animal models for acne include mouse ear models and hamster models. These models offer valuable insights into the biological mechanisms of excessive sebaceous gland secretion and abnormal keratinization. However, due to differences in the structure, physiological function, and inflammatory response patterns of sebaceous glands among different species, these models have significant limitations in simulating the pathological characteristics of human acne. For example, mouse sebaceous glands are relatively sparsely distributed, and their skin microecological environment and immune response differ significantly from those of humans; while hamster models have more developed sebaceous glands, their hair follicle distribution differs considerably from that of humans, affecting the accuracy of the models and the extrapolation of research results.

[0004] Rabbit ear models are considered ideal for studying acne due to their abundant sebaceous glands and skin structure similar to humans. In particular, the thinner epidermis and well-developed hair follicles and sebaceous glands in rabbit ears are more closely related to the development and distribution of human sebaceous glands, providing an excellent platform for studying the pathophysiological mechanisms of acne.

[0005] Existing methods for constructing rabbit ear acne models suffer from the following technical deficiencies: First, single-inducer modeling has limitations. CN111206016A, which only uses intradermal injection of Propionibacterium acnes, can only trigger a local inflammatory response and cannot simulate the pathological process of abnormal sebaceous gland secretion. CN106492438A, which only uses topical application of chemicals such as coal tar and oleic acid, is unlikely to induce typical inflammatory papules, pustules, and other core acne lesions, which is inconsistent with the multifactorial pathogenesis of clinical acne. Second, existing combined modeling methods (such as coal tar + Propionibacterium acnes) often use simultaneous drug administration, which can easily lead to the inhibition of bacterial activity by chemicals, reducing the success rate of modeling. Furthermore, the timing and dosage of drug administration are not precisely optimized, resulting in large phenotypic fluctuations and poor reproducibility. There is currently no composite modeling scheme that simultaneously integrates the three core mechanisms of "endocrine disorder-lipid abnormality-bacterial infection." Third, the bacterial infection process lacks optimization. Existing models mostly use common Propionibacterium acnes strains directly without screening for virulence factors, resulting in significant differences in bacterial colonization ability and inflammation induction ability, poor model consistency, and an inability to simulate the pathological characteristics of severe acne caused by infection with highly virulent strains in clinical practice.

[0006] Therefore, there is an urgent need to develop a rabbit ear acne model that can comprehensively consider multiple pathological aspects such as sebaceous gland function, follicular keratosis, microbial infection, and inflammatory immune response. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for constructing a rabbit ear acne model based on "compound induction by androgens + oleic acid + highly virulent Propionibacterium acnes". This method has the advantages of fully replicating the multifactorial pathogenesis of clinical acne, improving the pathological consistency between the model and clinical acne, and increasing the success rate, stability and consistency of modeling.

[0008] To address the problems in the existing technology, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for constructing a rabbit ear acne model, comprising the following steps:

[0010] (1) The expression levels of virulence factor genes (lipA, kdpA, hly, cpa) were detected by qPCR to screen for highly virulent Propionibacterium acnes CC-01.

[0011] (2) Prepare a compound induction model by combining oleic acid solution, androgen solution and CC-01 bacterial solution.

[0012] According to an embodiment of the present invention, the relative expression levels of virulence factors of the highly virulent Propionibacterium acnes CC-01 are all >5 (using ATCC 6919 as a control).

[0013] According to an embodiment of the present invention, the oleic acid solution is sterile filtered through a 0.22 μm filter membrane using anhydrous ethanol as a solvent, and has a mass concentration of 5-8%.

[0014] According to an embodiment of the present invention, the androgen is testosterone, which is sterile filtered through a 0.22 μm filter membrane using olive oil as a solvent, and has a concentration of 2-4 mg / mL.

[0015] According to an embodiment of the present invention, the concentration of the CC-01 bacterial solution is 1×10⁻⁶. 7 -5×10 7 CFU / mL.

[0016] According to an embodiment of the present invention, the model was established for 10 days, with 10-20 mg / kg of testosterone solution injected subcutaneously daily. Except for the 4th day, oleic acid was applied to the skin at the opening of the external auditory canal on the inner side of the ear (0.15 mL / ear) daily. On the 4th day, 0.05 mL of bacterial solution was injected subcutaneously into the opening of the external auditory canal on the inner side of the ear, with 3 injection points per ear.

[0017] Secondly, this invention provides the application of the constructed rabbit ear acne model in screening anti-acne drugs. Thirdly, this invention provides a model evaluation method:

[0018] (1) Macroscopic evaluation: Referring to the grading standard for acne lesions, observe and record the area of ​​erythema, number of papules, number of pustules and nodule formation in the rabbit ear experimental area. The success rate of modeling is calculated by the proportion of rabbit ears with typical inflammatory papules (diameter ≥1mm) and pustules.

[0019] (2) Microscopic evaluation: Rabbit ear skin lesions were routinely embedded in paraffin, sectioned, stained with HE, and observed under a microscope to detect the degree of follicular hyperkeratosis, sebaceous gland hyperplasia, and inflammatory cell infiltration. The expression levels of inflammatory factors (TNF-α, IL-6) in the skin lesions were detected by ELISA.

[0020] The beneficial effects of this invention are:

[0021] This invention innovatively employs a three-factor composite induction scheme of "androgens + oleic acid + highly virulent Propionibacterium acnes". By specifically screening highly virulent Propionibacterium acnes strains and exploring and optimizing experimental conditions such as oleic acid concentration, bacterial concentration and injection frequency, it achieves a synergistic simulation of the inflammatory response induced by endocrine disorders, abnormal sebaceous gland secretion and infection by highly virulent strains, while avoiding toxic reactions. It overcomes the limitations of existing single-factor / two-factor modeling and the lack of optimization of the bacterial infection link, and fully reproduces the pathogenesis of clinical acne (especially severe acne). Attached Figure Description

[0022] Figure 1 This is a culture diagram of acne-causing bacteria provided in Embodiment 1 of the present invention.

[0023] Figure 2 This is an appearance diagram of the rabbit ear acne model constructed according to Embodiment 2 of the present invention.

[0024] Figure 3 This is a histopathological section (HE staining) of rabbit ear skin tissue from Example 2 of the present invention.

[0025] Figure 4 This is a histopathological section (HE staining) of rabbit ear skin tissue from Example 2 of the present invention.

[0026] Figure 5 This is a histopathological section (HE staining) of rabbit ear skin tissue from Example 2 of the present invention.

[0027] Figure 6 This is a graph showing the bacterial count results of Propionibacterium acnes in rabbit ear skin tissue in Example 3 of the present invention. Detailed Implementation

[0028] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. After reading this invention, any modifications of the present invention in various equivalent forms, or direct or indirect applications in other related technical fields, made by those skilled in the art, shall fall within the scope defined by the appended claims.

[0029] Experimental Example 1: Isolation, Identification, and Virulence Gene Screening of Clinical Propionibacterium acnes Strains

[0030] Animal preparation: Select healthy SPF-grade New Zealand white rabbits, weighing 1.8-2.2kg, regardless of sex, and acclimatize them for 5-7 days. The environmental conditions are a temperature of 20-24℃, humidity of 55-65%, a circadian rhythm of 12h / 12h, and free access to food and water.

[0031] Strain culture: Clinical acne strains were collected, inoculated onto reinforced Brucella blood agar medium, and anaerobic cultured at 37°C for 48-72 h. Single grayish-white, round, smooth, β-hemolytic colonies were picked.

[0032] Virulence gene screening: PCR technology was used to detect key virulence genes of the strain, including cAMP factor gene (cfb), hemolysin gene (hly), capsular polysaccharide gene (cap), and lipase gene (lip). Strains with expression levels of three or more virulence factor genes significantly higher than those of the standard strain ATCC 6919 (expression level ≥2-fold) were selected as candidate high-virulence strains (to avoid strains with excessively strong or weak virulence).

[0033] Virulence verification: Candidate strains were prepared into 1×10⁻⁶ samples. 8CFU / mL bacterial suspension was injected intradermally into rabbit ears at a rate of 0.05 mL / point. Skin inflammatory reactions (size and time of appearance of erythema and papules) were observed within 72 hours. The strain with the most significant inflammatory reaction and the earliest appearance time (≤24 hours) was selected as the strain for subsequent model construction.

[0034] The specific groupings and results are shown in Tables 1 and 2 below:

[0035] Table 1. Results of model strain screening

[0036]

[0037] Table 2. Expression levels of virulence genes

[0038]

[0039] As shown in Table 1, the clinical isolate 3 (named CC-01) showed the earliest onset of inflammation (20h) and the most significant inflammatory response in the skin lesions. As shown in Table 2, all four virulence factor genes of strain CC-01 were highly expressed, and the results are shown in Table 2. It has the strongest virulence, with a relative expression level of virulence factors >5 (using ATCC 6919 as a control). It is suitable as a highly virulent strain for model construction.

[0040] Experimental Example 2: Exploring the Modeling Conditions for Rabbit Ear Acne Model

[0041] Thirty healthy New Zealand white rabbits were randomly divided into 10 groups (n=3 per group). Using "oleic acid concentration × bacterial concentration × injection frequency" as variables, and in addition to androgen injection (3 mg / mL, 15 mg / kg body weight, once daily for 10 consecutive days) and oleic acid application to the skin at the opening of the external auditory canal on the inner side of the ear (0.15 mL / ear once daily, 3 days before and 7 days after bacterial infection), bacterial infection (0.05 mL / point subcutaneously injected at the opening of the external auditory canal on the inner side of the ear, 3 points per ear) was used to explore suitable experimental conditions. Observed indicators included model establishment success rate, skin lesion phenotype, and toxic reactions (skin necrosis, rabbit weight loss ≥10%, systemic inflammation, etc.).

[0042] The specific groupings and results are shown in Table 3 below:

[0043] Table 3 Comparison of modeling effects and toxic reactions under different experimental conditions

[0044]

[0045] Table 3 shows that the suitable experimental conditions are: oleic acid concentration of 5-8% and highly virulent Propionibacterium acnes concentration of 1×10⁻⁶. 7 -5×10 7With a CFU / mL concentration and an injection frequency of once on day 4, the modeling success rate is 100%, the skin lesion phenotype is typical, and there are no toxic reactions. If the concentration is exceeded, toxic reactions such as skin necrosis and weight loss may occur, or the modeling effect may be poor.

[0046] Example 1: Culture and identification of *Propionibacterium acnes*

[0047] Standard strains ATCC 6919 and ATCC 11827, and clinical isolate CC-01 were inoculated onto reinforced Brucella blood agar plates and anaerobically cultured at 37°C for 48-72 hours. Figure 1 As shown, the clinical isolate CC-01 forms grayish-white, round, smooth colonies with a diameter of 1-2 mm, and a narrow β-hemolytic ring appears around the colonies.

[0048] Morphological identification: Single colonies on blood agar plates were smeared, Gram stained, and observed under a microscope (×1000). Gram-positive bacilli were visible, which were slender rods or slightly curved, arranged singly, in pairs or short chains, without spores or flagella, consistent with the typical morphological characteristics of Propionibacterium acnes.

[0049] Biochemical identification: The biochemical characteristics of the strains were tested, including their fermentation of sugars such as glucose, lactose, maltose, sucrose, and mannitol, as well as the activities of enzymes such as arginine dihydrolase, urease, and esterase. The results are shown in Table 4. All the strains screened could ferment glucose and maltose to produce acid, but did not ferment lactose, sucrose, or mannitol; they were positive for arginine dihydrolase, negative for urease, and positive for esterase, consistent with the biochemical characteristics of *Propionibacterium acnes*.

[0050] Table 4. Biochemical identification results of Propionibacterium acnes

[0051] Identification indicators 1(ATCC6919) 2(ATCC11827) CC-01 Glucose fermentation + + + Maltose fermentation + + + lactose fermentation - - - Arginine dihydrolase + + + Urease - - - esterase + + +

[0052] Note: "+" indicates a positive result; "-" indicates a negative result.

[0053] Example 2: Construction of a rabbit ear acne model

[0054] Ten healthy New Zealand white rabbits were randomly divided into two groups: a control group and an experimental group (n=5 per group). The experimental group received subcutaneous injections of testosterone (3 mg / mL, 15 mg / kg body weight, once daily for 10 days) and 6% oleic acid was applied to the skin at the opening of the external auditory canal on the inner side of the ear (0.15 mL / ear once daily, 3 days before and 7 days after bacterial infection). On day 4, the rabbits were infected with clinical strain CC-01 (bacterial concentration 5 × 10⁻⁶). 7CFU / mL, 0.05 mL / point subcutaneously injected at the opening of the external auditory canal on the inner side of the ear (3 points per ear). The control group received no treatment. The ear skin was observed daily for obvious erythema, acne, and follicular dilation. On day 15, samples were collected for HE staining to detect follicular keratosis, sebaceous gland hyperplasia, and the degree of inflammatory cell infiltration.

[0055] The results are as follows Figure 2 Obvious erythema, acne, and follicular dilation were visible on the skin of the ear. HE staining was performed after sampling on day 15; hyperkeratosis of the hair follicles was observed in the experimental group. Figure 3 ), sebaceous gland hyperplasia and hypertrophy ( Figure 4 ) and inflammatory cell infiltration ( Figure 5 The expression of inflammatory factors (TNF-α, IL-6) was significantly increased compared to the control group (Table 5).

[0056] Table 5

[0057] Group IL-6 (pg / mL) TNF-α (pg / mL) Blank group 132.1±1.8 7.9±1.6 experimental group 315.5±10.3 24.2±5.1

[0058] Example 3: Evaluation of the efficacy of anti-acne drugs

[0059] Experimental grouping and model construction: Twenty healthy New Zealand white rabbits were selected and rabbit ear acne model was constructed according to the method in Example 2 (100% success rate). After modeling, the rabbits were randomly divided into two groups of 10 each: model group (physiological saline) and positive control group (clindamycin gel, commercially available, concentration 1%). Ten healthy New Zealand white rabbits were also selected as the blank group (no modeling, only physiological saline was applied).

[0060] Dosage regimen: All groups were administered the drug once daily for 7 consecutive days, starting on the first day after modeling. During administration, the drug was evenly applied to the lesion area (2cm × 2cm) of the rabbit ear using a sterile cotton swab, with a dosage of 0.1mL per ear. The model group and the control group received an equal volume of physiological saline, while the positive control group received the drug. Rabbits were fasted for 1 hour after administration. The condition of the rabbit ear skin was observed daily during this period, and any adverse reactions such as allergies or irritation were recorded.

[0061] Efficacy evaluation:

[0062] (1) Macroscopic efficacy evaluation: On the 7th day of administration, the area of ​​erythema, number of papules, number of pustules and number of nodules in the rabbit ear lesion area were recorded according to the acne lesion improvement scoring criteria. The lesion improvement rate was calculated (improvement rate = (score before administration - score after administration) / score before administration × 100%). Among them, the erythema area was scored according to the proportion of the experimental area (0 points: no erythema; 1 point: <25%; 2 points: 25%-50%; 3 points: 51%-75%; 4 points: >75%), and the papules / pustules / nodules were scored according to the number (0 points: none; 1 point: 1-3; 2 points: 4-6; 3 points: 7-10; 4 points: >10). The total score was the sum of the scores of each indicator.

[0063] (2) Microscopic pathological evaluation: On the 7th day of drug administration, 3 rabbits were randomly selected from each group, and rabbit ear skin lesions were taken, routinely embedded in paraffin, sectioned, stained with HE and observed under a microscope to evaluate the degree of follicular hyperkeratosis, sebaceous gland hyperplasia and inflammatory cell infiltration (scoring criteria: 0 points: no abnormality; 1 point: mild abnormality; 2 points: moderate abnormality; 3 points: severe abnormality).

[0064] (3) Molecular index evaluation: The expression levels of inflammatory factors (IL-6, TNF-α) in the skin lesion tissue were detected by ELISA.

[0065] (4) Evaluation of bacterial load: Take rabbit ear tissue homogenate and count the bacterial load.

[0066] Experimental results:

[0067] (1) Macroscopic efficacy results: 14 days after administration, the total score of skin lesions in the model control group did not decrease significantly (improvement rate <5%), and a large number of papules and pustules still existed; the skin lesion improvement rate in the positive control drug group (clindamycin gel) was 62.3%, and the number of erythema, papules and pustules was significantly reduced; the skin lesion improvement rates in the low, medium and high dose groups of the test drug were 35.1%, 68.7% and 82.5%, respectively. Among them, the skin lesions in the high dose group almost completely disappeared, with only mild erythema remaining, and no papules, pustules and nodules.

[0068] (2) Microscopic pathological results: The model group still showed severe follicular hyperkeratosis, sebaceous gland hyperplasia and inflammatory cell infiltration (total score 2.8 points); the pathological abnormalities in the positive control group were significantly reduced (total score 1.2 points).

[0069] (3) Molecular marker results: Compared with the model group, the expression levels of inflammatory factors (IL-6, TNF-α) in the positive control drug group were significantly downregulated (P<0.01). The specific data are shown in Table 6 below:

[0070] Table 6

[0071] Group IL-6 (pg / mL) TNF-α (pg / mL) normal control group 151.3±5.9 5.3±2.3 Model control group 260.5±12.6 13.9±6.2 Positive control group (clindamycin gel) 173.4±13.6 7.1±5.6

[0072] (4) Evaluation of bacterial load: Compared with the model group, the bacterial load in the positive control group was significantly reduced ( Figure 6 ).

[0073] The rabbit ear acne model successfully constructed in this invention can stably simulate the core pathological features of acne development, such as excessive sebaceous gland secretion, abnormal keratinization of the pilosebaceous duct, inflammatory cell infiltration, and colonization of Propionibacterium acnes, providing a reliable in vivo experimental platform for the pharmacodynamic evaluation of acne treatment drugs.

Claims

1. A method for constructing a rabbit ear acne model, characterized in that, Includes the following steps: (1) The expression levels of virulence factor genes lipA, kdpA, hly, and cpa were detected by qPCR to screen for highly virulent Propionibacterium acnes CC-01. (2) Prepare a compound induction model by combining oleic acid solution, androgen solution and CC-01 bacterial solution.

2. The construction method according to claim 1, characterized in that, The virulence factor of the highly virulent Propionibacterium acnes CC-01 was compared with ATCC 6919 as a control, and the relative expression level of both was >5.

3. The construction method according to claim 1, characterized in that, The oleic acid solution was sterile filtered through a 0.22 μm filter membrane using anhydrous ethanol as the solvent.

4. The construction method according to claim 3, characterized in that, The oleic acid solution has a mass concentration of 5-8%.

5. The construction method according to claim 1, characterized in that, The androgen is testosterone, which is sterile filtered through a 0.22μm filter membrane using olive oil as a solvent.

6. The construction method according to claim 5, characterized in that, The testosterone concentration is 2-4 mg / mL.

7. The construction method according to claim 1, characterized in that, The concentration of the CC-01 bacterial culture was 1×10⁻⁶. 7 -5×10 7 CFU / mL.

8. The construction method according to claim 1, characterized in that, Step (2) The model was created for a total of 10 days. 10-20 mg / kg of testosterone solution was injected subcutaneously every day. Except for the 4th day, 0.15 mL of oleic acid was applied to the skin at the opening of the external auditory canal on the inner side of the ear every day. On the 4th day, 0.05 mL of bacterial solution was injected subcutaneously at the opening of the external auditory canal on the inner side of the ear. There were 3 injection points in each ear.

9. The application of the rabbit ear acne model constructed by the method for constructing a rabbit ear acne model according to any one of claims 1 to 8 in screening anti-acne drugs.

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