A method for constructing an animal model of spleen deficiency and dampness obstruction combined with cisplatin-induced renal injury and its application

Abstract

The present invention discloses a method and purpose for constructing an animal model of spleen deficiency and dampness obstruction combined with cisplatin renal injury, and relates to the technical field of animal model construction. The construction method comprises the following steps: (1) placing an experimental animal in a water-containing container for 6-10 hours per day; (2) giving the experimental animal normal saline and fasting for 10-14 hours on odd-numbered days, and giving the experimental animal high-fat feed and lard on even-numbered days, drinking chilled water, and continuously modeling for 50-65 days; (3) on the third to last day before the end of modeling, giving the experimental animal a single injection of 7-10 mg / kg of cisplatin to obtain an animal model of spleen deficiency and dampness obstruction combined with cisplatin renal injury. The present invention combines cisplatin renal injury with an animal model of spleen deficiency and dampness obstruction, and can better replicate the spleen deficiency and dampness obstruction syndrome caused by long-term consumption of fatty and greasy foods, overwork, or improper diet in the clinic, and can provide new ideas and experimental basis for exploring the mechanism of action of cisplatin nephrotoxicity and related clinical symptoms.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction, and in particular to a method for constructing and using an animal model of spleen deficiency and dampness obstruction syndrome combined with cisplatin-induced kidney injury. Background Art

[0002] Cisplatin (DDP) is an inorganic platinum derivative widely used in chemotherapy for various solid tumors. In cancer cells, cisplatin can induce DNA cross-linking, triggering a DNA damage response, which in turn leads to cell cycle arrest and cell death. It has become one of the most promising and widely used chemotherapeutic agents for the treatment of various solid tumors. However, cisplatin is associated with various adverse reactions. Studies have shown that cisplatin is primarily excreted through the kidneys and can accumulate in large quantities in the proximal renal tubules, causing damage to the renal vascular system, ischemic tubular cell death, and decreased glomerular filtration rate, leading to renal abnormalities. Therefore, modern medicine often uses drugs such as diuretics and angiotensin-converting enzyme inhibitors to alleviate cisplatin nephrotoxicity. While these drugs have demonstrated some renal protective effects, they reduce cisplatin accumulation in the renal tubules by increasing urine output and cannot reverse cisplatin metabolism. They also have disadvantages such as poor long-term efficacy and high drug prices. Furthermore, large amounts of fluid replacement can be detrimental to patients and inconvenience clinical practice. How to alleviate cisplatin nephrotoxicity, effectively prevent the occurrence of diseases related to cisplatin nephrotoxicity, and further conduct in-depth research on the mechanism of cisplatin nephrotoxicity has become a serious issue before us.

[0003] In recent years, Traditional Chinese Medicine (TCM) has demonstrated its superiority in preventing and treating the side effects of anti-tumor drugs. Cisplatin-induced nephrotoxicity is categorized by syndrome types, including spleen deficiency and dampness syndrome (SDDS), kidney qi deficiency, liver-kidney yin deficiency, and spleen-kidney yang deficiency. Spleen deficiency and dampness syndrome is particularly common. Clinically, cisplatin-induced nephrotoxicity is categorized as "drug toxicity" in TCM, with cisplatin-induced renal impairment falling under the category of "kidney deficiency." Basic TCM theory posits that the kidneys store vital energy, while the spleen governs transportation and transformation. Kidney deficiency impairs the spleen, while spleen deficiency harms the kidneys, with the two promoting and synergizing with each other. The spleen's role in transportation and transformation is inherently consistent with drug metabolism. The pathogenesis of cisplatin-induced nephrotoxicity is that "drug toxicity" leads to dampness and turbidity in the middle organs, impairing spleen function, and impairing water metabolism, which in turn exacerbates toxic side effects. Clinically, symptoms include low back pain, weight loss or edema, a red tongue with a dry, yellow coating or minimal or no coating, scanty, dark urine or hematuria, and a slippery, rapid, or weak pulse.

[0004] At present, there are many methods for modeling animal models of spleen deficiency and dampness obstruction in traditional Chinese medicine, such as: high-fat feed combined with forced swimming method, high-fat feed combined with standing in water method, bitter and cold purgation combined with hunger and satiety disorder method, climate chamber modeling method, low protein and low choline plus swimming induction method, etc. However, there is no unified and recognized modeling standard, and there are problems such as poor reproducibility, short modeling time, and difficulty in modeling.

[0005] Chinese patent CN110558286A discloses a method for establishing an animal model of simple obesity due to spleen deficiency and dampness obstruction, comprising: S1: purchasing healthy, clean-grade male SD rats weighing 180-220g; S2: adaptive feeding: the purchased healthy rats are first fed a mixture of regular feed and high-fat feed for one week, and the food intake per gram of body weight is calculated; then, based on a food intake per gram of body weight of at least 100%, adaptive feeding is performed on a high-fat feed for another week; S3: establishing a spleen deficiency and dampness obstruction obesity model: the selected rats are placed in an environment of 22-28°C and 40-50% humidity for 8-11 weeks according to a specific feeding schedule, and then the weight of each rat is measured. Rats weighing ≥500g are selected; S4: the selected rats are scored according to specific indicators, and successful animal models of simple obesity due to spleen deficiency and dampness obstruction are selected. This invention provides an animal model of simple obesity due to spleen deficiency and dampness obstruction that resembles the human condition, providing a reference for the treatment of simple obesity due to spleen deficiency and dampness obstruction.

[0006] No animal models for spleen deficiency and dampness-stagnation syndrome combined with cisplatin-induced nephropathy have been reported in the prior art. To conduct targeted research on the clinical treatment, development, and screening of new drugs for spleen deficiency and dampness-stagnation nephropathy, and to further explore the mechanism of cisplatin-induced nephrotoxicity in this syndrome, it is imperative to construct an animal model of spleen deficiency and dampness-stagnation nephropathy similar to the human syndrome. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for constructing an animal model of spleen deficiency and dampness obstruction syndrome combined with cisplatin-induced renal injury and its application.

[0008] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides a method for constructing an animal model of spleen deficiency and dampness accumulation combined with cisplatin-induced renal injury, comprising the following steps:

[0010] (1) Place the experimental animals in a container containing water for 6-10 hours per day;

[0011] (2) On odd-numbered days, the experimental animals were given normal saline and fasted for 10-14 hours. On even-numbered days, the experimental animals were given high-fat feed and lard and drank chilled water. The model was established for 50-65 consecutive days.

[0012] (3) On the third day before the end of modeling, the experimental animals were given a single injection of 7-10 mg / kg of cisplatin to obtain an animal model of spleen deficiency and dampness obstruction combined with cisplatin renal injury.

[0013] Specifically, step (1) includes any mammals other than humans, such as sheep, horses, pigs, goats, camels, antelopes, dogs, mice, rats, guinea pigs, and hamsters.

[0014] Furthermore, the experimental animals described in step (1) include rats and mice.

[0015] Furthermore, the rats include but are not limited to SD rats, Wistar rats, Fisher 344 rats, SHR rats, ACI rats, and LEW rats.

[0016] Furthermore, the rats are SD rats.

[0017] Furthermore, the mice include but are not limited to BALB / c mice, C57BL mice, C3H / He mice, KM mice, ICR mice, NIH mice, CFW mice, LACA mice, nude mice, and Scid mice.

[0018] Specifically, the height of the water in the container in step (1) is 2-6 cm; further, the height of the water in the container in step (1) is 4 cm.

[0019] Specifically, in step (1), the experimental animals are placed in the water container for 8 hours.

[0020] According to some embodiments of the present invention, the experimental animals are placed in a 4 cm deep water pool from 8:00 am to 16:00 pm every day.

[0021] Specifically, during the experiment, the sleeping time of the experimental animals was controlled to be 8 h.

[0022] Specifically, the recording method of odd and even days is: the first day of the experiment is recorded as an odd day, the second day is recorded as an even day, the third day is recorded as an odd day, the fourth day is recorded as an even day, and so on.

[0023] Specifically, the fasting time in step (2) is 12 hours.

[0024] Specifically, the amount of physiological saline administered in step (2) is 6-9 mL / kg, and the temperature of the physiological saline is 4°C.

[0025] Specifically, the amount of lard administered in step (2) is 6-9 mL / kg.

[0026] Specifically, the lard is composed of 100% pig fat, has an energy of 3700 kJ, and is heated and melted before oral administration.

[0027] Specifically, the high-fat feed in step (2) comprises, by weight, 50-55 parts of ordinary feed, 8-15 parts of casein, 15-25 parts of sucrose, 10-20 parts of lard, 1-3 parts of cholesterol, 0.1-1 parts of sodium cholate, 0.1-1 parts of calcium hydrogen phosphate, and 0.1-2 parts of calcium carbonate.

[0028] Furthermore, the high-fat feed described in step (2) comprises, by weight, 52.6 parts of ordinary feed, 10 parts of casein, 20 parts of sucrose, 15 parts of lard, 1.2 parts of cholesterol, 0.2 parts of sodium cholate, 0.6 parts of calcium hydrogen phosphate, and 0.4 parts of calcium carbonate.

[0029] According to some embodiments of the present invention, the high-fat feed is composed of: 52.6% ordinary feed, 10% casein, 20% sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, 0.6% calcium hydrogen phosphate (feed grade type I), and 0.4% calcium carbonate, and the above percentages are mass percentages.

[0030] Specifically, the common feed comprises, by weight, 15-20 parts of protein, 4-6 parts of fat, 50-65 parts of carbohydrates, 1-5 parts of crude fiber, 5-10 parts of ash, 1-5 parts of calcium, 0.1-2 parts of phosphorus, 1-5 parts of minerals, 4-6 parts of amino acids and 0.1-1 parts of vitamins.

[0031] Furthermore, the common feed comprises, by weight, 18.5 parts of protein, 4.6 parts of fat, 58.9 parts of carbohydrates, 3.2 parts of crude fiber, 6.8 parts of ash, 1.28 parts of calcium, 0.92 parts of phosphorus, 1.48 parts of minerals, 4.12 parts of amino acids and 0.2 parts of vitamins.

[0032] According to some embodiments of the present invention, the common feed includes 18.5% protein, 4.6% fat, 58.9% carbohydrates, 3.2% crude fiber, 6.8% ash, 1.28% calcium, 0.92% phosphorus, 1.48% minerals, 4.12% amino acids and 0.2% vitamins, and the percentages are by mass.

[0033] Specifically, the chilled water in step (2) is drinking water frozen at -20°C, the drinking bottle is wrapped with insulation cotton during feeding, and the chilled water is replaced every 12 hours.

[0034] According to some embodiments of the present invention, the continuous modeling time in step (2) is 63 days.

[0035] Specifically, the methods of administering cisplatin in step (3) include but are not limited to intraperitoneal injection, intramuscular injection, tail vein injection, intradermal injection, and subcutaneous injection.

[0036] Furthermore, in step (3), the cisplatin is administered by intraperitoneal injection.

[0037] Specifically, the injection amount of cisplatin in step (3) is 8 mg / kg.

[0038] In another aspect, the present invention provides an animal model of spleen deficiency and dampness accumulation syndrome combined with cisplatin-induced renal injury constructed by the above-mentioned construction method.

[0039] In another aspect, the present invention provides the use of the spleen deficiency and dampness stagnation syndrome combined with cisplatin-induced renal injury animal model constructed by the above-mentioned construction method in screening or preparing drugs for spleen deficiency and dampness stagnation type cisplatin-induced renal injury.

[0040] The beneficial effects of the present invention are:

[0041] 1. The present invention is based on the etiology and pathogenesis of traditional Chinese medicine, and adopts the combination of external dampness, irregular diet and forced standing to construct an animal model of spleen deficiency and dampness obstruction. At the same time, based on clinical practice, the inducement of the disease (cisplatin renal injury) is imposed on the basis of the traditional Chinese medicine syndrome model (spleen deficiency and dampness obstruction), giving full play to the advantages of combining traditional Chinese medicine theory with modern medicine.

[0042] 2. This invention combines cisplatin-induced renal injury with an animal model of spleen deficiency and dampness retention for the first time. The modeling methods are able to effectively replicate the clinical characteristics of spleen deficiency and dampness retention caused by long-term consumption of fatty and greasy foods, excessive fatigue, or dietary irregularities. The optimal cisplatin injection concentration and frequency can avoid animal mortality. Multiple experimental evaluations have demonstrated that the model construction method is highly reproducible and can be used to screen traditional Chinese medicines that can alleviate cisplatin-induced renal injury by strengthening the spleen and removing dampness. This approach provides new insights and experimental evidence for exploring the mechanisms of cisplatin nephrotoxicity and clinical research on related syndromes. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The experimental flow chart is shown in Figure 2.

[0044] Figure 2 The body weight changes of rats in each group.

[0045] Figure 3 (A) is the average food intake of rats in each group. Figure 3 (B) in the figure shows the average water intake of rats in each group. In the figure, *p<0.05, **p<0.01.

[0046] Figure 4 (A) is the fecal water content of rats in each group. Figure 4 (B) is the tail-pulling resistance time of rats in each group. Figure 4(C) in each group represents the serum D-xylose content of rats. In the figure, *p < 0.05, **p < 0.01.

[0047] Figure 5 (A) in each group represents the thymus index of rats. Figure 5 (B) in each group represents the spleen index of rats. In the figure, *p < 0.05, **p < 0.01.

[0048] Figure 6 (A) in each group represents the urea nitrogen content of rats. Figure 6 (B) in each group represents the creatinine content of rats. Figure 6 (C) in each group represents the cisplatin accumulation in the renal tissue of rats. Figure 6 (D) in each group represents the pathological structure of rats. In the figure, *p < 0.05, **p < 0.01.

[0049] Figure 7 (A) in each group represents the expression level of KIM-1 in rats. Figure 7 (B) in each group represents the expression level of NGAL in rats. In the figure, *p < 0.05, **p < 0.01. Detailed implementation methods

[0050] In order to make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the following combines specific embodiments to further clarify the present invention. However, the following embodiments are only the preferred embodiments of the present invention, not all. Based on the embodiments in the implementation manner, other embodiments obtained by those skilled in the art without creative efforts all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used is all conventional equipment, and the equipment materials used in each embodiment are the same.

[0051] Example 1 Construction method of a rat model of spleen deficiency and dampness obstruction combined with cisplatin-induced kidney injury

[0052] 1. Experimental materials

[0053] 1.1 Experimental animals

[0054] 24 SPF-grade male SD rats weighing 250 - 300 g, license number: SCXK(Guangdong)2022 - 0063, were housed in the Experimental Animal Center of Guangdong Provincial Hospital of Traditional Chinese Medicine, license number: SYXK(Guangdong)2018 - 0094, and the housing conditions were SPF-level environment. The experiment passed the experimental animal welfare ethics review of Guangdong Provincial Hospital of Traditional Chinese Medicine, and the ethics review approval number was 2022083.

[0055] 1.2 Experimental drugs

[0056] Refined lard was purchased from Linyi Xincheng Jinluo Meat Products Group Co., Ltd.; cisplatin injection was purchased from Jiangsu Hausen Pharmaceutical Group Co., Ltd., specification: 30 mg / 5 mL.

[0057] 1.3 Experimental Reagents

[0058] High-grade nitric acid was purchased from Tianjin Komeiou Chemical Reagent Co., Ltd.; inductively coupled plasma-mass spectrometry (ICP-MS) tuning fluid, platinum (Pt), and bismuth (Bi) ICP-MS standard solutions were purchased from Agilent Technologies; D-xylose was purchased from Aladdin Biotechnology Co., Ltd., catalog number X101012; urea nitrogen (Ure) test kit (urease method) and creatinine (Cre) determination kit (sarcosine oxidase method) were purchased from Nanjing Jiancheng Bioengineering Institute, catalog number C013-2-1 for the urea nitrogen test kit and catalog number C011-2-1 for the creatinine determination kit; anti-kidney injury molecule 1 (KIM-1) antibody was purchased from Novus Biologicals; and anti-neutrophil gelatinase-associated lipid carrier protein (NGAL) antibody was purchased from Santa Clara Biotechnology Co., Ltd. Cruz Biotechnology Company; Hematoxylin-eosin staining (HE) staining kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. with the catalog number C0105M; Immunohistochemistry (IHC) reaction kit was purchased from Wuhan Boster Biotechnology Co., Ltd. with the catalog number SA1020.

[0059] 1.4 Experimental Instruments

[0060] Table 1 Experimental instruments

[0061]

[0062]

[0063] 2. Experimental methods

[0064] 2.1 Animal grouping

[0065] After 3 days of adaptive feeding, SPF SD rats were randomly divided into 4 groups, 6 rats in each group: Control group, SDDS group, Control+DDP group, and SDDS+DDP group.

[0066] 2.2 Modeling method

[0067] Rats in the SDDS and SDDS+DDP groups were placed in a 4-cm-deep pool of water from 8:00 AM to 4:00 PM daily to simulate a humid environment and were given an 8-hour sleep schedule. On odd-numbered days, rats were fed 2 mL of 4°C normal saline and fasted for 12 hours. On even-numbered days, rats were fed a high-fat diet (52.6% standard diet, 10% casein, 20% sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, 0.6% calcium hydrogen phosphate (feed grade type I), and 0.4% calcium carbonate. All percentages are by mass) and 2 mL of lard (100% porcine fat, 3700 kJ energy, melted and gavage administered to rats). Rats were given chilled water for 63 consecutive days. The control and control+DDP groups were housed as usual. On day 60, rats in the Control+DDP group and the SDDS+DDP group were intraperitoneally injected with 8 mg / kg DDP injection, while the other groups were injected with normal saline. On day 63, an animal model of spleen deficiency and dampness obstruction combined with cisplatin-induced renal injury was established. Figure 1 .

[0068] Conventional feeding refers to feeding rats a basic maintenance diet and adequate room-temperature drinking water without any other intervention. The composition of each serving of basic maintenance diet is 18.5% protein, 4.6% fat, 58.9% carbohydrates, 3.2% crude fiber, 6.8% ash, 1.28% calcium, 0.92% phosphorus, 1.48% minerals, 4.12% amino acids, and 0.2% vitamins. The above percentages are by mass.

[0069] Chilled water was 600 mL of drinking water frozen in a -20°C refrigerator. When feeding rats, the drinking bottle was wrapped with insulation cotton and the chilled water was replaced every 12 hours.

[0070] 3. Model Evaluation

[0071] 3.1 Macroscopic manifestations of TCM syndromes during modeling

[0072] Starting from the first day of modeling, the rats' hair growth, food intake, defecation, urination, and activity were recorded daily, and body weight was recorded every six days. Based on the clinical manifestations of spleen deficiency and dampness obstruction syndrome and referring to works such as "Standards of Traditional Chinese Medicine Syndromes," "Chinese Medicine Dampness Diseases," and "Chinese Medicine Syndrome Pathology," the following macroscopic diagnostic criteria for the rat spleen deficiency and dampness obstruction model were established: ① Fatigue, laziness, and huddling; ② Dejected demeanor, with withered, sparse, and erect fur, and easy hair loss; ③ Decreased urine volume, soft or loose stools, increased rate of floating stools, a positive tail-pull defecation test, and anal redness and swelling during defecation, or even rectal prolapse; ④ Slow movement, or even gait crookedness; ⑤ No or slow weight gain, significantly less than or even less than that of the normal group; ⑥ Decreased food intake; and ⑦ Significant gastrointestinal bloating. Items ①-④ are primary symptoms, and items ⑤-⑦ are secondary symptoms. The presence of three of these primary symptoms, or two of these primary symptoms plus two of these secondary symptoms, constitutes a preliminary conclusion that the "Spleen Deficiency and Dampness Obstruction" syndrome model has been successfully established.

[0073] 3.2 Analysis of feces moisture content

[0074] Fresh rat feces were collected within 3 hours and weighed, dried at 80°C for 8 hours, and the moisture content of the feces was determined according to the formula [(wet weight of feces - dry weight of feces) / wet weight of feces] × 100%.

[0075] 3.3 Tail pull resistance test

[0076] The rat's tail was lifted up in the air, and the time it took for it to stop spinning and give up struggling was recorded.

[0077] 3.4D-Xylose Uptake Experiment

[0078] Rats were fasted for 2 hours and orally administered 3% D-xylose (1 mL / 100 g). Blood samples were collected 1 hour after intervention, and serum was separated by centrifugation. D-xylose was measured using a D-xylose assay kit according to the manufacturer's instructions.

[0079] 3.5 Determination of spleen index and thymus index

[0080] After euthanasia, the rats were immediately excised and weighed, and the thymus index (thymus weight relative to body weight) and spleen index (spleen weight relative to body weight) were measured.

[0081] 3.6 Determination of cisplatin content in renal tissue

[0082] Kidney tissue was pre-cooled at -80°C for 24 hours and then vacuum-dried. The tissue powder was weighed and placed in a digestion vessel. 5 mL of nitric acid was then added and pre-digested at room temperature for 30 minutes. The sample was digested and acid removed using a microwave digester, and the volume was diluted to 10 mL with 1% nitric acid. Pt content was determined by ICP-MS using 500 ng / mL Bi as the internal standard. DDP content (ng / mg) = [Pt weight × 300.05 / 195.08] / kidney tissue weight (the relative molecular masses of DDP injection and Pt are 300.05 and 195.08, respectively).

[0083] 3.7 HE staining and IHC reaction analysis

[0084] The renal tissue was dehydrated, embedded, cut into 3.5 μm samples, stained according to the instructions of HE and IHC kits, and photographed and analyzed using a pathological microscope after sealing.

[0085] 4. Results

[0086] 4.1 Macroscopic manifestations of TCM syndromes during modeling

[0087] During the modeling process, the rats in the Control group and the Control+DDP group had clean and smooth fur, were agile, and had a uniform weight gain. The rats in the SDDS group and the SDDS+DDP group showed signs of grouping, listlessness, arched backs, slow movements, with withered fur, easy hair loss, and mushy stools. Their weight decreased from the 48th day of modeling ( Figure 2 ). The average daily food intake and water intake of rats in each group were analyzed. It was found that compared with the Control group, the SDDS group and the SDDS+DDP group showed a poorer appetite (p<0.01). At the same time, the water intake of rats in the SDDS+DDP group showed a significant difference (p<0.05) ( Figure 3 A in Figure 3 (B) The manifestations of rats in the SDDS+DDP group were consistent with the classic clinical syndrome of spleen deficiency and dampness obstruction.

[0088] 4.2 Analysis of fecal moisture content, tail resistance time, and serum D-xylose content

[0089] Compared with the Control group, the fecal water content of rats in the SDDS group and the SDDS+DDP group increased (p<0.01). Compared with the Control+DDP group, the fecal water content of rats in the SDDS+DDP group increased (p<0.01), while there was no significant difference between the SDDS group and the SDDS+DDP group. This result is consistent with the observed symptomology. Figure 4 A in.

[0090] The tail-pulling resistance time can reflect the degree of physical fatigue of rats. Compared with the Control group, the tail-pulling resistance time of rats in the SDDS group and the SDDS+DDP group was shortened (p<0.01). Compared with the Control+DDP group, the rats in the SDDS+DDP group gave up struggling earlier (p<0.01), and the tail-pulling resistance time of the SDDS+DDP group was the shortest. The results showed that the SDDS+DDP group had symptoms of "spleen deficiency". Results are shown in Figure 4 B in.

[0091] The D-xylose content in the serum can reflect the ability of rats to absorb nutrients. Compared with the control group, the serum D-xylose content of rats in the SDDS group and SDDS+DDP group was reduced (SDDS group p<0.05, SDDS+DDP group p<0.01), indicating that the gastrointestinal absorption function of rats was weakened after modeling. Figure 4 C in.

[0092] 4.3 Thymus Index and Spleen Index Analysis

[0093] Compared with the Control group, the thymus and spleen indexes of the SDDS group and the SDDS+DDP group were both reduced (p<0.01). The thymus index of the Control+DDP group was significantly reduced (p<0.01), while the spleen index had no statistical difference. Compared with the Control+DDP group, the thymus and spleen indexes of the SDDS+DDP group were significantly reduced (p<0.01). This indicates that the immune organ function of the rats may be impaired after modeling. Figure 5 A in Figure 5 B in.

[0094] 4.4 Analysis of Renal Function, Cisplatin Accumulation in Renal Tissue, and Pathological Structure

[0095] Compared with the Control group, the Ure and Cre levels of rats in the Control+DDP group and the SDDS+DDP group were increased (p<0.01). Compared with the SDDS group, the Ure and Cre levels of rats in the SDDS+DDP group were significantly increased (p<0.01). Similarly, compared with the Control+DDP group, the Ure (p<0.05) and Cre (p<0.01) levels of rats in the SDDS+DDP group were increased. This indicates that the renal function of rats was severely damaged after modeling. Results are shown in Figure 6 A in Figure 6 B in.

[0096] The content of DDP in the kidney of rats in each group was determined by ICP-MS. Figure 6As shown in Figure C, the amount of DDP accumulated in the renal tissue of SDDS rats after DDP administration was significantly higher than that in the Control+DDP group (p<0.01). HE analysis showed that the renal tissue structure of SDDS rats was more severely damaged after DDP treatment: specifically, there was tubular degeneration and necrosis (cytoplasmic vacuolation), a large number of damaged tubules and sloughed epithelial cells in the tubular lumen, disordered cell arrangement, and infiltration of inflammatory cells in the renal interstitium. This preliminarily indicates that the spleen deficiency and dampness obstruction type cisplatin renal injury model was successfully established. Results are shown in Figure 6 D in.

[0097] 4.5 Expression determination of renal injury markers KIM-1 and NGAL

[0098] KIM-1 and NGAL have been reported to be the most effective biomarkers for diagnosing renal injury. After injection of DDP, the expression of KIM-1 and NGAL in the Control+DDP group and the SDDS+DDP group was significantly increased compared with the Control group (p<0.01). Compared with the Control+DDP group, the expression of KIM-1 and NGAL in the SDDS+DDP group was significantly increased (p<0.01). This indicates that the spleen deficiency and dampness obstruction type cisplatin renal injury model was successfully established. The results are shown in Figure 7 A in Figure 7 B. Combined with results 4.4-4.5, it suggests that the syndrome of spleen deficiency and dampness obstruction will aggravate cisplatin-induced renal injury.

[0099] Comparative Example 1

[0100] The difference between Comparative Example 1 and Example 1 is that the modeling method is different. In the method for constructing the spleen deficiency and dampness resistance type cisplatin renal injury model in Comparative Example 1, the DDP injection dose is 4 mg / kg, and the number of injections is 1 injection on the 60th day and 61st day respectively. The other model construction steps are the same as in Example 1.

[0101] Comparative Example 2

[0102] The difference between Comparative Example 1 and Example 1 is that the modeling time is different. The modeling time of Comparative Example 2 is 73 days, cisplatin is injected on the 70th day, and the rest of the modeling method is the same as Example 1.

[0103] Comparative Example 3

[0104] The difference between Comparative Example 3 and Example 1 is that in Comparative Example 3, room temperature water is drunk on even-numbered days, and the rest of the modeling method is the same as that of Example 1.

[0105] Experimental results:

[0106] In Comparative Example 1, the model animals showed wiry hair, weight loss, and slowed movements, but no obvious renal tissue pathological damage was observed. In Comparative Example 2, the model animals showed wiry hair, weight loss, slowed movements, and renal tissue pathological damage. The renal tissue damage was not significantly different from that in Example 1, but the modeling period was prolonged compared with Example 1. In Comparative Example 3, some model animals showed weight loss and slowed movements, and the serum D-xylose content was not significantly different from that of normal rats. The modeling success rate was lower than that in Example 1.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing an animal model of spleen deficiency and dampness accumulation combined with cisplatin-induced renal injury, characterized in that: The following steps are involved: (1) Place SD rats in a container containing water for 8 hours per day; (2) On odd-numbered days, SD rats were given normal saline and fasted for 12 hours. On even-numbered days, SD rats were given high-fat diet and lard and drank chilled water. The model was established for 63 consecutive days. (3) On the third day before the end of modeling, SD rats were given a single injection of cisplatin 8 mg / kg to obtain an animal model of spleen deficiency and dampness obstruction combined with cisplatin-induced renal injury.

2. The construction method according to claim 1, characterized in that The amount of lard administered in step (2) was 6-9 mL / kg.

3. The construction method according to claim 1, characterized in that The high-fat feed in step (2) comprises, by weight, 50-55 parts of ordinary feed, 8-15 parts of casein, 15-25 parts of sucrose, 10-20 parts of lard, 1-3 parts of cholesterol, 0.1-1 parts of sodium cholate, 0.1-1 parts of calcium hydrogen phosphate, and 0.1-2 parts of calcium carbonate.

4. The construction method according to claim 1, characterized in that The administration of cisplatin in step (3) includes intraperitoneal injection, intramuscular injection, tail vein injection, intradermal injection, and subcutaneous injection.

5. Use of the spleen deficiency and dampness retention syndrome combined with cisplatin-induced renal injury animal model constructed by the construction method according to any one of claims 1 to 4 in screening or preparing drugs for cisplatin-induced renal injury of the spleen deficiency and dampness retention type.

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