Modeling method and application of simple anaphylactoid purpura animal model

Abstract

The invention provides a modeling method and application of a simple anaphylactoid purpura animal model. The modeling method comprises the following steps: adaptively feeding SPF rats for 3 days; the hot traditional Chinese medicine is continuously injected into the stomach for 7 days, and 1mL / 100g body mass is injected every day; on the seventh day and the fourteenth day, an abdominal cavity sensitizer is injected to the abdominal cavity of the rat twice; shaving a local area on the back of the rat on the 25th day, performing inhalation anesthesia on the rat, performing warm water local wet compress, and then removing local stubbles by using a scraper; injecting a caudal vein sensitizer into the caudal vein of the rat; and after 30 minutes, intradermal injection of an intradermal sensitizer is carried out, 4-5 points are divided, waiting is carried out for 24 hours, so that the antigen is completely excited, and molding is completed. The hot traditional Chinese medicine comprises the following raw materials: cinnamon, fructus piperis longi and rhizoma zingiberis. The modeling time is shorter, the modeling rate is high, the cost is lower, and blood routine, renal function, immunity and inflammation index detection results and skin and kidney pathological results of model rats are all highly similar to those of simple / blood-heat syndrome anaphylactoid purpura patients.

Description

Technical Field

[0001] This invention belongs to the field of animal model technology, and in particular relates to a method for creating a simple allergic purpura animal model and its application. Background Technology

[0002] Henoch-Schönlein purpura (HSP) is a systemic syndrome characterized by capillary and small vessel inflammation, and is the most common vasculitis in children. From a modern medical perspective, its etiology is primarily due to various allergens acting on individuals with a genetic predisposition, leading to B cell activation, the production of immune complexes dominated by IgA, and the mediation of systemic immune disorders. Main manifestations include non-thrombocytopenic purpura of the skin, joint swelling and pain, abdominal pain and gastrointestinal bleeding, and kidney damage. The disease has a high recurrence rate, a prolonged course, and numerous complications. Based on the main sites of HSP involvement, HSP is classified into several types: simple (cutaneous), articular, abdominal, renal, and mixed.

[0003] The simple type, primarily characterized by skin symptoms, is the most common type of allergic purpura. Typical manifestations include symmetrically distributed purplish-red rashes on the extremities (especially the lower extremities), which do not blanch on pressure and may be accompanied by mild itching or pain. The rash initially appears as pinpoint- to soybean-sized petechiae or ecchymoses, either isolated or clustered, with swelling and blisters being rare. Some patients may experience nonspecific symptoms such as low-grade fever and fatigue. Simple allergic purpura does not involve the gastrointestinal tract.

[0004] From the perspective of traditional Chinese medicine, simple allergic purpura falls under the category of blood-heat syndrome. It refers to the invasion of external heat pathogens (such as wind-heat or heat toxin) into the interior, or emotional stagnation transforming into fire, or spicy food that aggravates heat, leading to excessive heat in the blood (heat pathogens penetrate deep into the blood layer). The heat forces the blood to flow erratically, without obvious dampness. Therefore, the rash is mostly bright red or dark red, with a bright color (because of the excessive heat in the blood, the heat forces the blood to overflow, making the blood color bright).

[0005] Meanwhile, blood heat syndrome may also manifest in systemic symptoms such as thirst with a preference for cold drinks (excessive heat injures body fluids), irritability and anger (heat disturbs the mind), mild joint pain, or only a slight burning sensation (heat obstructs the flow of qi, without dampness stickiness, and the pain is not heavy or turbid), and rarely gastrointestinal symptoms.

[0006] To better understand the pathogenesis of Henoch-Schönlein purpura (HSP) and aid in more effective clinical diagnosis and treatment, constructing animal models is a common approach. However, the symptoms of different HSP subtypes vary significantly, making universal models less practical and unable to provide adequate research for different subtypes. Therefore, it is necessary to establish different models for different symptom subtypes.

[0007] The existing Chinese patent CN 115413627 B provides a method for constructing and applying an animal model of abdominal allergic purpura. However, this model is mainly used for abdominal allergic purpura, with gastrointestinal symptoms such as abdominal pain, vomiting, and gastrointestinal bleeding as the main symptoms, and skin symptoms are not obvious. From the perspective of traditional Chinese medicine, abdominal allergic purpura falls under the category of damp-heat syndrome. Damp-heat syndrome often presents with joint swelling and pain (mostly lower limb joints, such as knees and ankles), accompanied by a feeling of heaviness (dampness obstructing the joints, hindering the flow of qi and blood), abdominal distension, poor appetite (dampness trapping the spleen and stomach, causing abnormal digestion), nausea and vomiting, sticky and unsatisfactory stools (damp-heat descending to the intestines), and dark yellow and scanty urine (damp-heat descending to the bladder). Even if a rash is present, it is mostly dark red or purplish-red, and the rash may merge into patches, or be accompanied by mild swelling and blisters (dampness easily leads to water retention, causing skin swelling). Therefore, this model is not applicable to the symptoms of simple / blood-heat syndrome allergic purpura.

[0008] In their 2014 Chinese Journal of Comparative Medicine article, "Comparison of Hematologic Immunological Indicators in Rats, Rabbits, and Children with Allergic Purpura," Li Yanhong et al. established rabbit and rat models of allergic purpura. While most hematologic changes in these models were similar to those in humans, they did not differentiate between types of allergic purpura, making them general models. Furthermore, the methodology concluded that "the proportion of rats exhibiting symptoms is lower than that of rabbits… In contrast, the rabbit model is easier to operate, has more stable disease progression, and its larger size makes it easier to observe."

[0009] The above-mentioned model takes a long time to establish, with the total steps taking more than 6 weeks. Moreover, the rabbit model, which is more similar to the changes in human blood indicators, is also expensive. Therefore, it is necessary to continue to improve it and provide a modeling method that has a shorter modeling time, a higher rate of symptom appearance, an immune response that is closer to human blood, and is more suitable for animal models of simple / blood-heat syndrome allergic purpura. Summary of the Invention

[0010] This invention addresses the problems of existing animal models being unable to better suit simple / blood-heat syndrome allergic purpura, such as long modeling time, low symptom incidence, or high cost, by providing a method and application for establishing a simple allergic purpura animal model.

[0011] To achieve the above objectives, the technical solution provided by the present invention is as follows: A method for establishing a simple allergic purpura animal model includes the following steps: S1. Adaptively feed SPF rats for 3 days; S2. Administer hot-natured Chinese medicine via gavage for 7 consecutive days, 1 mL / 100g body weight per day; On days S3, 7, and 14, rats were injected intraperitoneally twice with an intraperitoneal sensitizer. S4. On day 25, a local area was shaved off on the back of the rats. The rats were given inhalation anesthesia. After applying warm water to the local area, the stubble was removed with a scraper. S5. Inject a tail vein sensitizer into the tail vein of rats; S6. After injecting the sensitizer into the tail vein for 30 minutes, inject the sensitizer intradermally at 4-5 points, and wait 24 hours for the antigen to be fully stimulated to complete the modeling.

[0012] The warming Chinese herbs in step S1 are: cinnamon, long pepper, and dried ginger.

[0013] Furthermore, the heat-inducing Chinese medicine consists of cinnamon, long pepper, and dried ginger in a mass ratio of 1:1:1. These are decocted twice with 6 times their volume of water, filtered, and the decoctions are combined and concentrated to 3 g of raw herbs per mL.

[0014] Furthermore, in step S3, the intraperitoneal sensitizer is an emulsion solution of 20 mg / ml ovalbumin and 1 mg / ml Freund's complete adjuvant in a 1:1 ratio.

[0015] Furthermore, in step S3, the intraperitoneal sensitizer is injected in doses of 0.5 mL each time.

[0016] Furthermore, in step S5, the tail vein sensitizer is prepared with physiological saline containing 10 mg / ml ovalbumin.

[0017] Furthermore, in step S5, 0.25 ml of the tail vein sensitizer was injected into each rat.

[0018] Furthermore, in step S6, the intradermal sensitizer is ovalbumin saline at 3 mg / ml.

[0019] Furthermore, in step S6, 100 μl of intradermal sensitizer is injected intradermally at each point.

[0020] The present invention also provides the application of the rat model of simple allergic purpura constructed by the above modeling method in screening and testing drugs for the treatment of simple allergic purpura.

[0021] The advantages of this invention lie in its ability to shorten the modeling time (25 days) by adjusting the use of heat-inducing drugs, feeding methods, and the timing of intraperitoneal sensitization injections. The model achieves a high success rate with no model deaths. The rats' blood routine, renal function, immune, and inflammatory marker results, as well as skin and kidney pathological findings, are highly similar to those of patients with simple / blood-heat allergic purpura. This model exhibits both significant skin purpura and mild renal impairment, making it more suitable as an animal model for simple / blood-heat allergic purpura, for screening or testing treatments and drugs for this condition. Compared to existing rabbit models, it has lower modeling costs and better accuracy in studies of simple / blood-heat allergic purpura. Attached Figure Description

[0022] Figure 1 The dorsal skin lesions of mice in the model group and the control group. Figure 2 Skin pathological features of rats in the model group (HE staining) Figure 3 The pathological features of the kidneys in the model group rats (HE staining) Figure 4 The pathological features of the kidneys in the model group rats (PAS staining) Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The modeling method includes the following steps: S1. Adaptively feed SPF rats for 3 days; S2. Administer hot-natured Chinese medicine via gavage for 7 consecutive days, 1ml / 100g body weight per day; On days S3, 7, and 14, rats were injected intraperitoneally twice with an intraperitoneal sensitizer. S4. On day 25, use an electric shaver to shave a local area on the back of the rat, administer inhalation anesthesia to the rat, apply warm water compresses to the local area, and then use a scraper to remove the stubble. S5. Inject a tail vein sensitizer into the tail vein of rats; S6. After injecting the sensitizer into the tail vein for 30 minutes, inject the sensitizer intradermally at 4-5 points, and wait 24 hours for complete antigen activation.

[0025] The heat-inducing Chinese medicine in step S1 is as follows: weigh out cinnamon, long pepper, and dried ginger in a mass ratio of 1:1:1, add 6 times the volume of water and decoct twice, filter, combine the decoctions, and concentrate to 3 g of raw medicine / mL.

[0026] In step S3, the intraperitoneal sensitizer is an emulsion solution of 20 mg / ml ovalbumin and 1 mg / ml Freund's complete adjuvant in a 1:1 ratio.

[0027] In step S5, the tail vein sensitizer is prepared with physiological saline containing 10 mg / ml ovalbumin.

[0028] In step S6, the intradermal sensitizer is ovalbumin saline at 3 mg / ml.

[0029] Example 1. Modeling object: Twelve male SPF rats, aged 6 weeks, of the SD strain, were used as the model group (6 rats) and the control group (6 rats).

[0030] 2. Modeling drugs 2.1 Hot-natured Chinese herbs: 10g cinnamon, 10g long pepper, and 10g dried ginger are decocted twice with 6 times their volume of purified water. The decoctions are filtered, combined, and concentrated to 3g crude herb / mL. Store at 4℃ for later use.

[0031] 2.2 Intraperitoneal sensitizer: 1 ml of emulsion solution containing 20 mg / ml ovalbumin and 1 mg / ml Freund's complete adjuvant in a 1:1 ratio.

[0032] 2.3 Tail vein sensitizer: Prepare ovalbumin at 10 mg / ml with physiological saline.

[0033] 2.4 Intradermal sensitizer: 3 mg / ml ovalbumin saline.

[0034] 3. The modeling method includes the following steps: 3.1 Adaptive feeding of SPF rats (6 weeks old, male, SD strain) for 3 days (D1-D3); 3.2 Administer warming Chinese herbal medicine via gavage for 7 consecutive days (D4-D10), 1 ml / 100g body weight per day; 3.3 At 7-day intervals (D11 and D18), rats were injected intraperitoneally twice with an intraperitoneal sensitizer, with 0.5 ml injected into each rat; 3.4 On day 25, a 5*5cm area was shaved off the back of the rats using an electric shaver. After inhalation anesthesia and local application of warm water, the stubble was removed using a scraper. 3.5 Inject the tail vein sensitizer into the tail vein of rats, 0.25 ml per rat; 3.6 Thirty minutes after the tail vein injection of the sensitizer, the intradermal sensitizer was injected at four points, 100 μl at each point; the antigen was fully challenged after 24 hours.

[0035] The control group and the model group had the same diet, but the medications used in procedures such as gavage and injection were replaced with an equal volume of physiological saline.

[0036] 4. Modeling Results The study used a control group and a model group, with 6 rats in each group. No rats died during the experiment, but all rats in the model group developed significant hemorrhages on their skin.

[0037] 4.1 Symptoms of Blood-Heat Syndrome in Rats Compared with the control group, all rats in the model group showed signs of restlessness, reduced food intake, increased water intake, and slower weight gain after 7 days of oral administration of hot Chinese medicine.

[0038] 4.2 Blood routine examination indicators related to allergic purpura Table 1. Blood routine examination indicators of model group and control group Inspection indicators Control group (n=6) Model group (n=6) leukocyte 6 (4, 7) 18 (15, 23)* neutrophil percentage 23 (17, 27) 47 (35, 50)* absolute neutrophil count 1.07 (0.83, 1.62) 7.05 (5.64, 9.25)* lymphocyte percentage 71 (66, 80) 38 (35, 42)* absolute lymphocyte count 7.16 (6.46, 8.02) 4.12 (3.06, 4.68)* Percentage of eosinophils 1.10 (0.93, 1.20) 1.45 (1.40, 1.80)* Absolute value of eosinophils 0.06 (0.05, 0.09) 0.15 (0.13, 0.35)* platelets 518 (453, 566) 546 (523, 611) Mean hemoglobin content 19.20 (19.03, 19.30) 20.00 (19.75, 20.78)* Mean hemoglobin concentration 335 (329, 341) 376 (373, 381)* Note: * indicates the model group compared to the control group. p <0.05 The complete blood count (CBC) indicators of the model group rats are shown in Table 1. The white blood cell count, neutrophil percentage, absolute neutrophil count, eosinophil percentage, absolute eosinophil count, mean eosinophil content, and mean eosinophil concentration were significantly higher in the model group than in the control group (P < 0.05). The lymphocyte percentage and lymphocyte percentage were significantly lower in the model group than in the control group (P < 0.05). There was no difference in platelet count between the model group and the control group. The CBC results of the model group are consistent with the characteristics of patients with simple allergic purpura.

[0039] 4.3 Examination indicators for kidney damage related to allergic purpura Table 2. Renal damage indicators in the model group and the control group detection indicators Control group (n=6) Model group (n=6) serum creatinine 34.2 (34.0, 34.9) 47.8 (46.7, 48.8)* urea nitrogen 12.5 (12.3, 12.5) 29.4 (28.8, 30.3)* Note: * indicates the model group compared to the control group. p <0.05 As shown in Table 2, the serum creatinine and blood urea nitrogen levels in the model group rats were significantly higher than those in the control group (P < 0.05), which were highly similar to the renal function test results of patients with simple / blood-heat syndrome allergic purpura.

[0040] 4.4 Immunological and inflammatory indicators related to allergic purpura Table 3. Immunological and inflammatory indicators of the model group and the control group detection indicators Control group (n=6) Model group (n=6) IgA 34.2 (34.0, 34.9) 47.8 (46.7, 48.8)* IgG 12.5 (12.3, 12.5) 29.4 (28.8, 30.3)* C3 0.58 (0.55, 0.61) 0.29 (0.28, 0.30)* C4 0.20 (0.19, 0.24) 0.12 (0.10, 0.14)* CIC 0.66 (0.62, 0.68) 1.57 (1.47, 1.67)* SAA 112 (81, 155) 335 (305, 379)* Note: * indicates the model group compared to the control group. p <0.05 As shown in Table 3, the levels of IgA, IgG, CIC, and SAA in the model group rats were significantly higher than those in the control group (P < 0.05), while C3 and C4 were significantly lower than those in the control group (P < 0.05), which is similar to the immune inflammation manifestations of patients with allergic purpura.

[0041] 4.5 Skin lesions in rats with allergic purpura like Figure 1 As shown, within 24 hours after modeling, the antigen was fully stimulated. The skin lesions on the back of the model group rats were observed within 24 hours. Starting from 6 hours, obvious bright red to dark red rashes gradually appeared on the epidermis at the injection sites of the sensitizing agent. The rashes were bright in color, without blisters or swelling, which were highly similar to the skin symptoms of patients with simple / blood-heat syndrome allergic purpura. All rats in the model group showed obvious skin lesions, with a symptom incidence rate of 100%.

[0042] 4.6 Skin pathological manifestations in rats with allergic purpura (HE staining) like Figure 2 As shown, after HE staining of the skin of rats in the model group, a large number of necrotic cell fragments and extensive epidermal loss were visible on the surface of the damaged skin tissue; and a large number of inflammatory cells, mainly lymphocytes and granulocytes, were scattered and infiltrated.

[0043] 4.7 Pathological manifestations of the kidneys in rats with allergic purpura (HE staining, PAS staining) like Figure 3 As shown, in the model group rats, HE staining at 40.0x revealed a small number of necrotic cell fragments around the glomeruli in the kidney cortex, a small number of atrophied renal tubules with narrowed lumens, decreased eosinophilicity of the renal tubular epithelial cells, a small number of dilated renal tubules with irregular lumens, flattened renal tubular epithelial cells, and a small amount of connective tissue hyperplasia without necrosis. Fibroblasts and collagen fibers were interspersed, accompanied by a small amount of inflammatory cell infiltration, mainly lymphocytes, and a few eosinophilic flocculent masses within the renal tubules. The connective tissue between the urinary tubules was the renal interstitium, with no significant interstitial hyperplasia; a small amount of interstitial vascular congestion was observed.

[0044] like Figure 4 As shown, in the model group rats (PAS staining_40.0x), the cell nuclei appeared light blue under PAS staining, while the glomerular mesangial matrix and the brush border of the renal tubules appeared red. Glomeruli were evenly distributed in the renal cortex, with no significant thickening of Bowman's capsule. Mesangial matrix proliferation was observed in many glomeruli, but the number of mesangial cells did not increase significantly, and no obvious crescents were observed. A small number of proximal convoluted tubules showed broken or incomplete brush borders. This indicates that the model rats exhibited milder kidney damage symptoms, more consistent with the symptoms of simple / blood-heat syndrome allergic purpura primarily characterized by skin lesions.

[0045] In summary, the results of routine blood tests, renal function tests, immune function tests, inflammatory marker tests, and skin and kidney pathological findings in the model group rats were highly similar to those in patients with simple / blood-heat syndrome allergic purpura. Having both significant skin purpura and mild renal impairment, these rats can serve as an animal model of blood-heat syndrome allergic purpura for screening or testing drugs to treat simple / blood-heat syndrome allergic purpura.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for establishing a simple allergic purpura animal model, characterized in that, Includes the following steps: S1. Adaptively feed SPF rats for 3 days; S2. Administer hot-natured Chinese medicine via gavage for 7 consecutive days, 1 mL / 100g body weight per day; On days S3, 7, and 14, rats were injected intraperitoneally twice with an intraperitoneal sensitizer. S4. On day 25, a local area was shaved off on the back of the rats. The rats were given inhalation anesthesia. After applying warm water to the local area, the stubble was removed with a scraper. S5. Inject a tail vein sensitizer into the tail vein of rats; S6. After injecting the sensitizer into the tail vein for 30 minutes, inject the sensitizer intradermally at 4-5 points, and wait 24 hours for the antigen to be fully stimulated to complete the modeling process. The ingredients of the warming Chinese medicine in step S1 are: cinnamon, long pepper, and dried ginger.

2. The method for establishing a simple allergic purpura animal model as described in claim 1, characterized in that: The heat-inducing Chinese medicine consists of cinnamon, long pepper, and dried ginger in a 1:1:1 ratio by weight. The herbs are decocted twice with 6 times their volume of water, filtered, and the decoctions are combined and concentrated to 3 g of raw herbs per mL.

3. The method for establishing a simple allergic purpura animal model as described in claim 1, characterized in that: In step S3, the intraperitoneal sensitizer is an emulsion solution of 20 mg / ml ovalbumin and 1 mg / ml Freund's complete adjuvant in a 1:1 ratio.

4. The method for establishing a simple allergic purpura animal model as described in claim 1 or 3, characterized in that: In step S3, 0.5 mL of the intraperitoneal sensitizer is injected each time.

5. The method for establishing a simple allergic purpura animal model as described in claim 1, characterized in that: In step S5, the tail vein sensitizer is prepared with physiological saline containing 10 mg / ml ovalbumin.

6. The method for establishing a simple allergic purpura animal model as described in claim 1 or 5, characterized in that: In step S5, each rat was injected with 0.25 ml of the tail vein sensitizer.

7. The method for establishing a simple allergic purpura animal model as described in claim 1, characterized in that: In step S6, the intradermal sensitizer is ovalbumin saline at 3 mg / ml.

8. The method for establishing a simple allergic purpura animal model as described in claim 1 or 7, characterized in that: In step S6, inject 100 μl of intradermal sensitizer into each spot.

9. The application of a rat model of simple allergic purpura constructed by the modeling method according to any one of claims 1 to 8 in screening and testing drugs for treating simple allergic purpura.

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