Use of L-cysteine as feline AIM protein activator and use for improving renal function in felines

Abstract

The invention belongs to the technical field of animals, and particularly relates to application of L-cysteine as a feline AIM protein activator and application of L-cysteine in improvement of renal functions of felines. The invention provides an application of L-cysteine or physiologically acceptable salt thereof as a feline AIM protein activator, an application of L-cysteine or physiologically acceptable salt thereof in improving renal functions of felines, and an application of L-cysteine or physiologically acceptable salt thereof in a composition for improving renal functions and preventing or treating nephropathy of felines. The present application also provides a composition for improving renal function, preventing or treating nephropathy for felines, comprising L-cysteine or a physiologically acceptable salt thereof. According to the application, research finds that L-cysteine can promote dissociation of an IgM-AIM complex in cat serum and activate AIM protein; therefore, the L-cysteine can be used as an AIM protein activator for improving renal functions of felines and preventing or treating nephropathy, and has a wide application prospect.

Description

Technical Field

[0001] The present application belongs to the field of animal technology, and specifically, relates to the use of L-cysteine ​​as an activator of AIM protein of felines and the use of L-cysteine ​​for improving renal function of felines. Background Art

[0002] AIM protein (apoptosis inhibitor of macrophage, encoded by the cd5l gene) is a circulating protein in the blood. AIM circulates in an inactive state by binding to immunoglobulin M (IgM) pentamers in the blood. During acute renal failure, AIM separates from IgM and enters the kidneys, marking dead cells in the kidneys, triggering macrophages to recognize and phagocytize dead cells and other debris, so as to relieve and recover from renal inflammation.

[0003] There are two types of cat AIM proteins, 3SRCR and 4SRCR, with different molecular weights of approximately 37 and 49 kDa respectively (Reference: Scientific Reports, volume 6, Article number: 35251 (2016)). Its amino acid sequence is different from that of humans by a key amino acid cluster. This charged amino acid cluster causes AIM to bind tightly to IgM, 1,000 times more tightly than mice, and is not easy to dissociate. AIM cannot be taken into the urine and cannot enter the kidneys to complete the task of marking garbage cells, resulting in the inability to destroy and clean up dead cells and other debris in the body. This means that from the moment a cat is born, all dead cells and debris produced in the body will accumulate in the kidneys, which will cause renal overdraft over time, resulting in a high chance of acute kidney injury (AKI) or other kidney diseases in cats. Therefore, the difficulty in activating AIM protein is the reason for the high incidence of renal failure in cats. If the cat's AIM protein can be separated from IgM and the AIM protein can be activated, macrophages can effectively clear dead cells and debris in the body, thereby reducing the occurrence of kidney inflammation and kidney disease. It is expected that the life span of cats can be extended (literature: Scientific Reports, volume 6, Article number: 35251 (2016); Seminars in Immunopathology (2018) 40: 567-575).

[0004] In 2016, Professor Miyazaki's team reported the following research results: The researchers used "AIM cat mice" as research subjects. The key cluster of the SRCR3 binding domain of the AIM protein in these mice was expressed as a cat structure, and the performance was similar to that of AIM in cats, and it was difficult to dissociate from IgM. Subsequently, these mice showed symptoms of acute kidney injury, and then they were treated with recombinant AIM to observe the effect. The experimental results showed that when ischemia / reperfusion (IR)-induced acute renal failure (AKI) occurred, AIM-deficient (AIM-) mice were unable to clear debris, showed persistent renal inflammation, and had a higher mortality rate due to progressive renal dysfunction than wild-type mice. Treating IR-induced AKI mice with recombinant AIM can clear debris, improve renal pathology, and increase survival rate. Therefore, injecting recombinant AIM into mice with acute kidney injury has a certain therapeutic effect and improves the survival rate of mice (literature: Scientific Reports, volume 6, Article number: 35251 (2016)).

[0005] A drug for cats that is directly injected with AIM recombinant protein has entered clinical research in Japan. A 23-year-old cat with kidney disease was given a continuous injection of AIM for 4 months. It was able to help the cat with abnormal renal function to clean up blood waste and prevent the kidney disease from worsening, but it could not restore the cat's kidney function.

[0006] In terms of cat epidemiology research, a research paper on the cause of death in cats reported: 3,108 cats that underwent autopsy at the William R. Pritchard Veterinary Teaching Hospital at the University of California, Davis from January 1, 1989 to October 31, 2019 were used as research subjects to determine life expectancy and factors affecting mortality; this cat epidemiology study evaluated gender factors, environmental factors, age, and causes of death. The gender included 5.66% females, 39.86% sterilized females, 6.95% males, 47.49% sterilized males, and 84.2% were mixed-race cats. The age of 2,974 cats was determined, with a median of 9.07 years. The results of the study found that cancer was the most common cause of death (35.81%), and 62.84% of cats had abnormal kidney tissue (but only 13.06% of cats had abnormal kidney tissue as the main cause of death). The above authoritative research shows that 62.84% of cats have kidney problems, that is, more than 60% of the autopsy cats suffer from chronic kidney disease. This confirms the defect of AIM protein in cats discovered by Professor Miyazaki.

[0007] Therefore, cat food series, health products, and kidney disease prevention or treatment drugs based on the AIM mechanism, such as kidney prescription food, have a solid scientific research foundation and broad market demand.

[0008] Currently, only in Japan are there any cat AIM protein products available, including the following:

[0009] AIM30 series cat food: Developed by Markan, a comprehensive pet food manufacturer, it contains the "A-30" amino acid ingredient that can activate the cat's AIM protein. These products are designed to prevent kidney disease, especially for kittens who have not yet had kidney damage.

[0010] AIM nutritional supplement: The product name is "Japanese-made AIM 30 cat kidney health nutritional supplement", which is a powdered supplement that allows cats to take the amino acid "A-30", which effectively activates AIM in the cat's body and promotes the removal of waste in the body.

[0011] Cat Treats: In addition to cat food and nutritional supplements, there are also cat treats with added AIM protein-related ingredients. These products are also designed to support cats' kidney health.

[0012] The "A-30" amino acids contained in the existing AIM30 series of foods include a combination of cystine, methionine, taurine, etc. through food formula analysis. Methionine and taurine are both essential amino acids added to cat food. There is no report on whether these amino acids can affect the activation function of cat AIM protein. Finding more effective AIM protein activators that can be used in cat food or health products has positive significance for improving the high incidence and dysfunction of cat kidney disease. Summary of the invention

[0013] In response to the technical problems of high incidence and abnormal function of cat kidney disease, the present applicant has found through research that L-cysteine ​​can promote the dissociation of IgM-AIM complex in cat serum and activate AIM protein; therefore, L-cysteine ​​can be used as an AIM protein activator for improving renal function of cats or preparing compositions for improving renal function, preventing or treating kidney disease, such as prescription food (such as kidney prescription food), food (staple food, snacks, etc.), food additives, health products, nutritional supplements, etc., to improve renal function, repair kidney damage, and prevent or treat kidney disease.

[0014] In a first aspect, the present application provides the use of L-cysteine ​​or a physiologically acceptable salt thereof as an activator of feline AIM protein or in the preparation of an activator of feline AIM protein.

[0015] In some embodiments, the AIM protein activator can promote the dissociation of the IgM-AIM complex.

[0016] In a second aspect, the present application provides L-cysteine ​​or a physiologically acceptable salt thereof for use in improving renal function, preventing or treating nephropathy in a feline. In some embodiments, the L-cysteine ​​or a physiologically acceptable salt thereof can be administered to a feline via the gastrointestinal tract. The administration to a feline via the gastrointestinal tract can be administered to a feline orally.

[0017] In a third aspect, the present application provides the use of L-cysteine ​​or a physiologically acceptable salt thereof in a composition for improving renal function, preventing or treating kidney disease in a feline; or the use of L-cysteine ​​or a physiologically acceptable salt thereof in the preparation of a composition for improving renal function, preventing or treating kidney disease in a feline.

[0018] In some embodiments, the composition can be administered to a feline enterally or parenterally. The enteral administration to a feline can be administered to a feline orally, and the parenteral administration can be administered to a feline intravenously, subcutaneously, intraarterially, intraperitoneally, and the like.

[0019] In some embodiments, the composition is a prescription food, food, food additive, health product, nutritional supplement. The food includes staple food, snacks (such as canned food, cat strips, freeze-dried food, etc.), etc.

[0020] In a fourth aspect, the present application provides a composition for improving renal function, preventing or treating renal disease in felines, comprising L-cysteine ​​or a physiologically acceptable salt thereof.

[0021] In some embodiments, a composition for improving renal function, preventing or treating renal disease in felines further comprises vitamin B1 or a physiologically acceptable salt thereof, and / or L-carnitine or a physiologically acceptable salt thereof.

[0022] In some embodiments, a composition for improving renal function, preventing or treating renal disease in felines comprises L-cysteine ​​or a physiologically acceptable salt thereof, and vitamin B1 or a physiologically acceptable salt thereof; or it comprises L-cysteine ​​or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof; or it comprises L-cysteine ​​or a physiologically acceptable salt thereof, vitamin B1 or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof.

[0023] In some embodiments, a composition for improving renal function, preventing or treating kidney disease in felines comprises L-cysteine ​​or a physiologically acceptable salt thereof, and vitamin B1 or a physiologically acceptable salt thereof; wherein the weight ratio of L-cysteine ​​or a physiologically acceptable salt thereof, and vitamin B1 or a physiologically acceptable salt thereof is 0.0001-10000:1, 0.01-1000:1, 0.1-1000:1, 1-1000:1, 10-1000:1, preferably 20-500:1, more preferably 50-200:1, such as 100:1, 150:1, 200:1.

[0024] In some embodiments, a composition for improving renal function, preventing or treating kidney disease in felines comprises L-cysteine ​​or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof; wherein the weight ratio of L-cysteine ​​or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof is 1:0.0001-10000, 1:0.001-1000, 1:0.01-100, preferably 1:0.1-10, more preferably 1:0.2-5, such as 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5.

[0025] In some embodiments, a composition for improving renal function, preventing or treating renal disease in felines comprises L-cysteine ​​or a physiologically acceptable salt thereof, vitamin B1 or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof; wherein the weight ratio of L-cysteine ​​or a physiologically acceptable salt thereof, vitamin B1 or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof is (0.0001-10000): 1: (0.0001-1 0000), (0.01-1000):1:(0.01-1000), (0.1-1000):1:(0.1-1000), (1-1000):1:(1-1000), (10-1000):1:(10-1000), preferably (20-500):1:(20-500), more preferably (50-200):1:(50-200), such as 100:1:100, 150:1:150, 200:1:200.

[0026] In some embodiments, the composition can be administered to a feline enterally or parenterally. The enteral administration to a feline can be administered to a feline orally, and the parenteral administration can be administered to a feline intravenously, subcutaneously, intraarterially, intraperitoneally, and the like.

[0027] In some embodiments, the composition is a prescription food, food, food additive, health product, nutritional supplement. The food includes staple food, snacks (such as canned food, cat strips, freeze-dried food, etc.). In some embodiments, the composition for improving renal function, preventing or treating kidney disease in felines may also contain other ingredients used in prescription food, food, food additive, health product, nutritional supplement.

[0028] In a fifth aspect, the present application provides a method for improving renal function, preventing or treating kidney disease in a feline, which comprises administering L-cysteine ​​or a pharmaceutically acceptable salt thereof, or the composition described in the fourth aspect to the feline.

[0029] In some embodiments, the L-cysteine ​​or a pharmaceutically acceptable salt thereof, or the composition of the fourth aspect can be administered to the feline enterally (such as orally) or parenterally (such as intravenously, subcutaneously, intraarterially, intraperitoneally, etc.).

[0030] In some embodiments, the method for improving renal function, preventing or treating nephropathy in a feline, the L-cysteine ​​or a pharmaceutically acceptable salt thereof, or the composition described in the fourth aspect can be administered once or multiple times according to the situation. The dosage can be adjusted according to the conditions of the feline subject and the renal function.

[0031] In some embodiments, the method for improving renal function, preventing or treating kidney disease in a feline comprises administering L-cysteine ​​or a pharmaceutically acceptable salt thereof, or the composition described in the fourth aspect, wherein the composition is a prescription food, food, food additive, health product, or nutritional supplement.

[0032] In some embodiments, the method for improving renal function, preventing or treating nephropathy in felines, the single administration amount of L-cysteine ​​or its pharmaceutically acceptable salt can be 0.01-10000, 0.1-1000, 1-1000, 10-1000 mg / kg, preferably 20-500, 50-200 mg / kg, such as 20, 30, 40, 50, 60, 80, 100, 150, 200 mg / kg. The single administration dose of vitamin B1 or its physiologically acceptable salt can be 0.01-1000, 0.1-100 mg / kg, preferably 0.2-50, 0.5-30 mg / kg, such as 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0 mg / kg. The single dose of L-carnitine or a physiologically acceptable salt thereof can be 0.01-1000, 0.1-1000, 1-1000, 10-1000 mg / kg, preferably 20-500, 50-200 mg / kg, such as 20, 30, 40, 50, 60, 80, 100, 150, 200 mg / kg.

[0033] The single administration amount described in the present application refers to a single administration dose based on the body weight of a feline, such as "0.01-1000 mg / kg", which means a single administration of 0.01-1000 mg per kg based on the body weight of the feline.

[0034] The feline animals mentioned in the present application refer to animals classified in the family Felidae, such as cats, lions, tigers, leopards, etc., preferably cats, such as pet cats.

[0035] The improvement of renal function mentioned in the present application preferably refers to the improvement of renal abnormality, renal dysfunction, renal hypofunction, and renal damage caused by the inability to dissociate the IgM-AIM complex and activate the AIM protein.

[0036] The kidney disease described in the present application includes acute renal failure, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, IgM nephropathy or kidney disease associated with collagen disease. In some embodiments, the kidney disease is acute renal failure or chronic renal failure.

[0037] Compared with the prior art, the beneficial effects of this application are:

[0038] The application, by in vitro and in vivo experiments, clearly verifies that L-cysteine ​​can promote the dissociation of IgM-AIM complex in cat serum, activates AIM protein. Free AIM protein can mark dead cells and other fragments, so that macrophages can effectively remove dead cells and fragments in vivo, thereby improving renal function, renal injury, reducing the generation of renal inflammation and nephropathy. Therefore, L-cysteine ​​can be used for improving renal function, preventing or treating nephropathy of feline animals as AIM protein activator, for preparing the composition of feline animals improving renal function, preventing or treating nephropathy, such as prescription food (such as kidney prescription food), food, food additives, health products, nutritional supplements, etc., can improve renal function, prevent or treat nephropathy, with wide application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which:

[0040] Figure 1 This is a diagram showing the in vitro effect of L-cysteine ​​and methionine in promoting the dissociation of the IgM-AIM complex.

[0041] Figure 2 This is a graph showing the in vitro effect of L-cysteine ​​in promoting the dissociation of the IgM-AIM complex.

[0042] Figure 3A The figure shows the in vitro effect of different concentrations of L-cysteine ​​and cystine on promoting the dissociation of IgM-AIM complex.

[0043] Figure 3B This is a graph showing the in vitro effects of different concentrations of L-carnitine and cystine in promoting the dissociation of the IgM-AIM complex.

[0044] Figure 3C This is a graph showing the in vitro effects of different concentrations of vitamin B1 and cystine in promoting the dissociation of the IgM-AIM complex.

[0045] Figure 4A The figure shows the in vivo effect of L-cysteine ​​in promoting the dissociation of IgM-AIM complex.

[0046] Figure 4B This is a diagram showing the in vivo effect of L-carnitine in promoting the dissociation of the IgM-AIM complex.

[0047] Figure 4C This is a diagram showing the in vivo effect of vitamin B1 in promoting the dissociation of the IgM-AIM complex.

[0048] Figure 5 The figure shows the in vivo effect of the combined use of L-cysteine, vitamin B1 and L-carnitine on promoting the dissociation of the IgM-AIM complex. DETAILED DESCRIPTION

[0049] The specific implementation of the present application is described in detail below. It should be understood that the specific implementation described here is only used to illustrate and explain the present application, and is not used to limit the present application.

[0050] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0051] Features illustrated or described as part of one embodiment or certain embodiments can be used on another embodiment or certain other embodiments to yield a still further embodiment.

[0052] Before describing the present application in detail, it should be understood that the terms used herein are only intended to describe specific embodiments and are not intended to limit the scope of the present application, which is limited only by the appended claims. In order to more fully understand the present application described herein, the following terms are used, and their definitions are as follows. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by a person of ordinary skill in the art to which the present application belongs.

[0053] definition

[0054] Unless otherwise specified, the following terms used in this application have the following definitions.

[0055] Unless otherwise stated, the terms "comprising," "including," and "containing" and similar expressions should be interpreted in this specification and claims in an open and inclusive sense as "including but not limited to."

[0056] The term "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0057] The term "AIM protein" refers to AIM protein (apoptosis inhibitor of macrophage, encoded by the cd5l gene), which is a circulating protein in the blood. AIM circulates in an inactive state by binding to immunoglobulin M (IgM) pentamers in the blood. During acute renal failure, AIM separates from IgM and enters the kidneys, marking dead cells in the kidneys, triggering macrophages to recognize and phagocytize dead cells and other debris, so as to relieve and recover from renal inflammation.

[0058] The cat AIM protein used in the examples of the present application is a recombinant protein, about 37 kDa, and the amino acid sequence is as follows.

[0059] Seq ID No.1:

[0060] MGWSCIILFLVATATGVHSSFSRVRLVGGDHRCEGRVELQQDDEWVTVCDDYWNMDSVAVLCRELGCGAARKTMSGTVYGPVTPKDQKVFIHLFRCNGIEESLSQCEREDAIGCSHVEDAGAVCEPIYTGPGILGPESVRLADGPGRCQGRVEVKFRGEWSSVCQAGWSFAAAKVVCRQLG CGRATLTRRGCNKATQGQGAIWQRKASCSGQEVSLQDCLSEVWEHNCTHNEDVWVECEDPFALKLVGGRSHCEGRLEVLHKGEWGSVCDDGWGQDADRVVCRQLGCGQPLSPPVKVRRRFGPGVGRIWLDDVKCSGKEPSLEQCLHRSWGYHNCNHREDVAVVCEEQQSGLPDAHHHHHH*.

[0061] The term "physiologically acceptable salt" in the context of the present invention refers to any salt that is physiologically tolerated (most often meaning not toxic - in particular lacking toxicity caused by counterions). When used in medicine, it usually refers to pharmaceutically acceptable salts. These physiologically acceptable salts can be formed with cations or bases, and can also be formed with anions or acids.

[0062] The feline animals mentioned in the present application refer to animals classified in the family Felidae, such as cats, lions, tigers, leopards, etc. For example: lion (Panthera leo), leopard (Panthera pardus), tiger (Panthera tigris), snow leopard (Panthera uncia), jaguar (Panthera onca), clouded leopard (Neofelis nebulosa), Bornean clouded leopard (Neofelis diardi), puma (Puma concolor), cheetah (Acinonyx jubatus), slender-waisted cat (Herpailurus yagouaroundi), African golden cat (Caracal aurata), Persian wildcat (Caracalcaracal), serval (Leptailurus serval), Bornean golden cat (Catopuma badia), golden cat (Catopumatemminckii), clouded cat (Pardofelis marmorata), South American steppe cat (Leopardus colocola), Joe’s cat (Leopardus geoffroyi), South American forest cat (Leopardus guigna), tabby cat (Leopardus guttulus), mountain cat (Leopardus jacobita), Leopardus pardalis, Leopardus tigrinus, Leopardus wiedii, Canada lynx, Lynx canadensis, Lynx lynx, Iberian lynx, Lynx pardinus, Lynx rufus, Prionailurus bengalensis, Bornean leopard cat, Prionailurus javanensis, Prionailurus planiceps, Rusty-spotted cat, Prionailurus viverrinus, Manul, Felis bieti, Domestic cat, Felis catus, Jungle cat, Felischaus, Felis lybica, Desert cat, Felis margarita, Black-footed cat, Felis nigripes, Spotted cat, Felis silvestris) etc.

[0063] Freund's adjuvant is the most commonly used adjuvant in animal experiments. It is divided into incomplete Freund's adjuvant and complete Freund's adjuvant. Incomplete Freund's adjuvant is a mixture of oil (paraffin oil or vegetable oil) and emulsifier (lanolin or leaf Tween 80). The ratio of the components is 1 to 5:1, which can be determined according to needs, usually 2:1. Adding BCG (final concentration of 2 to 20 mg / ml) or dead Mycobacterium tuberculosis to incomplete Freund's adjuvant is complete Freund's adjuvant (FCA).

[0064] "TTBS": It is a buffer commonly used in biological experiments in the field, mainly used in the washing steps of experiments such as Western Blot and immunohistochemistry (IHC), which can effectively reduce nonspecific binding. It can be prepared using conventional methods in the field, such as weighing 8.8g NaCl and dissolving it in an appropriate amount of ddH2O, and adding 10mL Tris-HCl with pH=8.0 and 500μL Tween 20 to make the volume 1L, and store it at room temperature for use.

[0065] Example 1. Synthesis of AIM protein and preparation of mouse anti-cat AIM protein polyclonal antibody

[0066] The rAIM protein was synthesized by GenScript according to the sequence of the AIM protein.

[0067] Take 50μg rAIM protein (0.29mg / mL) and mix it with an equal volume of complete Freund's adjuvant, and immunize mice by intraperitoneal injection. Turn the mouse over, tilt the head down, insert the needle at 45 degrees, close to the midline of the abdomen, slowly inject and quickly withdraw the needle. On the 14th day after the initial immunization, perform a booster immunization. Collect 100μl of blood after the initial immunization. Mix 50μg rAIM protein with an equal volume of incomplete Freund's adjuvant, and immunize mice by intraperitoneal injection. Perform the third immunization 14 days later, and the method is the same as the second immunization. After 14 days, remove the mouse's eyeballs to collect blood, collect 1mL of blood, place the blood at 37℃ for 2h, centrifuge at 12000rpm for 10min, collect serum, and store at -20℃. The antibody titer of the serum was detected by ELISA using pre-immune serum as negative control. The titers of the prepared anti-AIM protein polyclonal antibodies of four mice, A, B, C and D, were 1:128000, 1:128000, 1:128000 and 1:256000, respectively, and all of them could be used for immunoblotting assay.

[0068] Example 2: In vitro screening of the effect of additives on the dissociation of IgM-AIM complex

[0069] Add 20 μL of cat serum to a 0.5 mL EP tube, and add 5 mM of the test sample, and use ddH2O to make up the difference in the control group. Mix well and incubate at 39°C. After incubation for different times, extract the total protein of cat serum (incubation time is 0-24 h), and analyze the AIM level by immunoblotting.

[0070] The total protein of cat serum was extracted by the following method: cat serum incubated for different times was taken out, the protein concentration in the serum was measured by the BCA method, 200 ng of cat serum was taken after calculation, PBS was added to make up to a total volume of 40 μL, and then 10 μL of 5× loading buffer (non-reduced) was added, and 10 μL was taken for sample loading and immunoblotting detection after mixing.

[0071] Immunoblotting analysis of AIM levels: Immunoblotting (WB) was performed using a 4%-20% gradient SDS-PAGE gel for electrophoresis, 30 μg of total protein sample was added to each well, and 30 ng of rAIM was taken as a positive control. The membrane was transferred at 4°C for 3.5 hours using a 300 mA current. After the transfer was completed, it was blocked with 5% skim milk powder for 1 hour. Subsequently, the mouse anti-cat AIM polyclonal antibody prepared in Example 1 with a dilution of 1:1500 was used as the primary antibody and incubated overnight at 4°C. Then, a secondary antibody (HRP (horseradish peroxidase) labeled goat anti-mouse, diluted 1:5000) was used for incubation at room temperature for 2 hours, and then the NC membrane was washed 6 times / 5 minutes with TTBS. Finally, it was exposed in a chemiluminescence instrument, and the experimental results were marked and analyzed according to the size of the protein Marker.

[0072] like Figure 1-2 As shown, through immunoblot analysis, we found that with the increase of co-incubation time with cat serum, L-cysteine ​​and the combination of L-cysteine ​​+ methionine showed a promoting effect on the dissociation of IgM-AIM complex at different time points, among which the effect of co-incubation for 24h was the most significant, but no obvious change was observed when methionine was used alone.

[0073] Concentration gradient experiments were performed on L-cysteine, L-carnitine, vitamin B1, and cystine to verify their dissociation effects on the IgM-AIM complex in cat serum. 20 μL of cat serum was added to a 0.5 mL EP tube, and different concentrations of additives were added. ddH2O was used to make up the difference in the control group. After mixing, the mixture was incubated at 39°C for 24 hours. After incubation, the total protein of cat serum was extracted according to the above method, and the AIM level was analyzed by immunoblotting.

[0074] like Figures 3A-3C As shown, the results demonstrated that L-cysteine, L-carnitine, and vitamin B1 could dissociate the IgM-AIM complex in cat serum, but cystine had no significant effect.

[0075] Example 3: In vivo evaluation of the effect of additives on the dissociation of IgM-AIM complex

[0076] Choose healthy experimental cat, oral administration (2mL / group), respectively water, L-cysteine ​​(200mg), L-carnitine (200mg), vitamin B1 (1mg) and additive combination group (L-cysteine ​​200mg, L-carnitine 200mg, vitamin B11mg), every group 2, L-cysteine ​​group, the body weight of two cats is respectively 2.75,3.0kg, L-carnitine group, the body weight of two cats is respectively 2.3,4.3kg, vitamin B1 group, the body weight of two cats is respectively 3.9,3.5kg, additive combination group, the body weight of two cats is respectively 2.2,3.8kg.And at 0d, 3d, 6d and 9d, respectively collect blood sample, extract the total protein of cat serum according to the same method as in embodiment 2, by immunoblotting detection analysis AIM level, to assess AIM protein activation situation.

[0077] like Figures 4A-4C , Figure 5 As shown, L-cysteine, L-carnitine, vitamin B1 and the additive combination group can promote the dissociation of IgM-AIM complex in vivo.

[0078] The present application verifies through in vitro and in vivo experiments that L-cysteine ​​can promote the dissociation of the IgM-AIM complex in cat serum and activate AIM protein, and can be used for improving renal function, preventing or treating kidney disease in cats, and for preparing compositions for improving renal function, preventing or treating kidney disease in cats, such as prescription food, food, food additives, health products, nutritional supplements, etc., to improve renal function, prevent or treat kidney disease, and has broad application prospects.

[0079] The protection scope of the present application is not limited to the above-mentioned embodiments. Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the scope and spirit of the present invention. If these changes and modifications fall within the scope of the claims of the present application and their equivalents, the intention of the present application also includes these changes and modifications.

[0080] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

[0081] In addition, the various implementation modes of the present application may be arbitrarily combined, and as long as they do not violate the concept of the present application, they should also be regarded as the contents disclosed in the present application.

Claims

1. Use of L-cysteine ​​or a physiologically acceptable salt thereof as an activator of AIM protein in felines.

2. The use according to claim 1, characterized in that The AIM protein activator can promote the dissociation of the IgM-AIM complex.

3. Use of L-cysteine ​​or a physiologically acceptable salt thereof in improving renal function in felines.

4. The use according to claim 3, wherein the L-cysteine ​​or a physiologically acceptable salt thereof is administered to a feline enterally or parenterally.

5. The use according to claim 4, wherein the L-cysteine ​​or a physiologically acceptable salt thereof is orally administered to a feline.

6. Use of L-cysteine ​​or a physiologically acceptable salt thereof in the preparation of a composition for improving renal function, preventing or treating renal disease in felines.

7. The use according to claim 6, characterized in that: The composition is administered to the feline enterally or parenterally.

8. The use according to claim 7, characterized in that: The composition is administered orally to a feline.

9. The use according to any one of claims 6 to 8, characterized in that: The composition is a prescription food, food, food additive, health product, or nutritional supplement.

10. A composition for improving renal function, preventing or treating renal disease in felines, characterized in that: Contains L-cysteine ​​or a physiologically acceptable salt thereof.

11. The composition according to claim 10, characterized in that The composition further comprises vitamin B1 or a physiologically acceptable salt thereof, and / or L-carnitine or a physiologically acceptable salt thereof.

12. The composition according to claim 11, characterized in that It contains L-cysteine ​​or a physiologically acceptable salt thereof, and vitamin B1 or a physiologically acceptable salt thereof; or contains L-cysteine ​​or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof; or contains L-cysteine ​​or a physiologically acceptable salt thereof, vitamin B1 or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof.

13. The composition according to any one of claims 10 to 12, characterized in that The composition is administered to the feline enterally or parenterally.

14. The composition according to claim 13, characterized in that The composition is administered orally to a feline.

15. The composition according to claim 13, characterized in that The composition is a prescription food, food, food additive, health product, or nutritional supplement.

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