Application of cobra neurotoxin polypeptide molecule in treating nephritis proteinuria

Abstract

After several years of illness, a small amount of albumin in the blood of nephritis patients will begin to leak into the urine. At this time, it is easily overlooked due to the insignificant clinical symptoms or being confused with proteinuria caused by other factors such as hypertension and hyperlipidemia, leading to the progression of the disease. However, when it is found that other indicators reflecting renal function, such as β2-microglobulin, α1-microglobulin, transferrin, and immunoglobulin, are also significantly elevated in the urine, it can be confirmed that pathological changes have occurred in renal function. Currently, there is no other effective treatment method except for hormones, and long-term hormone treatment will bring many side effects to patients. If the disease progresses freely, more proteins will leak into the urine and the kidney damage will further increase. Cobra neurotoxin polypeptide molecules can effectively reduce the leakage of the above various proteins in the urine, control and delay the pathological progression of the kidney, improve renal function, and are of extremely important significance for the treatment of nephritis by inhibiting the autoimmune inflammatory reaction of the kidney.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to the application of cobra neurotoxin polypeptide molecules in the treatment of nephritis proteinuria. Background Art

[0002] Nephritis is a group of diseases mainly characterized by glomerular lesions with multiple etiologies, which can be divided into primary nephritis and secondary nephritis. Primary nephritis may be caused by various infections such as bacteria, viruses or protozoa through immune mechanisms, inflammatory factors and non-immune mechanisms, etc.; secondary nephritis can be caused by diabetes, hypertension, allergic purpura, systemic lupus erythematosus, polycystic kidney disease, gouty nephropathy, etc. Whether it is primary or secondary, since there is currently no effective treatment drug, a considerable number of patients will develop into chronic nephritis, which is difficult to cure. If the development of nephritis cannot be stopped, it is impossible to avoid the worst clinical stage of renal function failure.

[0003] In the traditional concept, conventional urine protein, blood urea nitrogen BUN, serum creatinine Cr, etc. are indicators for judging kidney damage. However, when there is a large amount of proteinuria or an increase in blood urea nitrogen and creatinine, it is very likely that irreversible severe kidney damage has occurred. In fact, in patients with nephritis for many years, a small amount of blood albumin begins to leak into the urine at the beginning, which is the first stage of chronic nephritis, called microalbuminuria. Although microalbuminuria may also be associated with some cardiovascular diseases such as hypertension, hyperlipidemia, and atherosclerosis, when other protein indicators reflecting renal function such as immunoglobulin, β2-microglobulin, α1-microglobulin, transferrin, etc. also increase significantly in urine, it can be confirmed that pathological changes have occurred in renal function. [1-11]

[0004] When glomerular function declines, β2-microglobulin will enter the urine, resulting in a significant increase in its content in the urine. Therefore, urinary β2-microglobulin can directly reflect glomerular function. [12,13] After α1-microglobulin in the blood passes through the glomerular filtration membrane, it will be reabsorbed in the proximal renal tubule, and only a small amount will be excreted in the urine. Therefore, when the content of α1-microglobulin in the urine increases, it can directly reflect the abnormalities of the reabsorption function of the renal tubule and the filtration function of the glomerulus [14,15,16]. Transferrin is an iron-containing protein in plasma, mainly responsible for transporting iron. The transferrin molecule has less negative charge and is easily passed through the charge barrier of the glomerulus. Especially in the early stage of diabetic nephropathy, when the negative charge of the filtration membrane decreases and the filtration pores remain unchanged, urinary transferrin can appear earlier than albumin and can be used as a more sensitive indicator for diagnosing early kidney lesions. When human renal function is disordered, its content will increase significantly [17-21]. Immunoglobulin G is a direct indicator of the body's autoimmune response. Therefore, related renal function indicators such as microalbumin, β2-microglobulin, α1-microglobulin, transferrin, and immunoglobulin G in human urine can directly reflect the renal function and kidney lesion status of the human body. When the kidney is diseased, the contents of urinary microalbumin, β2-microglobulin, α1-microglobulin, transferrin, and immunoglobulin G will increase significantly. There is experimental evidence that the simultaneous detection of multiple microproteins such as urinary microalbumin, β2-microglobulin, α1-microglobulin, transferrin, and immunoglobulin G provides more reliable parameters for clinical practice and overcomes the heterogeneity of various microproteins. [1-3]

[0005] In the early stage, the clinical symptoms of nephritis patients are not obvious, and they may have microalbuminuria for many years. During this period, the filtration function of the kidney can basically remain normal. If microalbuminuria can be diagnosed and treated in time at this time, it is possible to achieve a reversible improvement in nephritis; however, if the disease process is allowed to progress freely, more protein will leak into the urine, and this stage can be called the stage of massive proteinuria. As the protein content in the urine increases, the filtration function of the kidney usually begins to decline, and at the same time, the body will retain various wastes, and blood urea nitrogen (BUN) and serum creatinine (Cr) will increase with the progression of kidney damage. At this time, in addition to the common clinical massive proteinuria and edema, patients are also prone to hypertension, anemia, and are accompanied by varying degrees of renal function lesions. At this time, nephrotic patients are about to enter end-stage renal disease. Currently, the treatment of nephritis mainly relies on glucocorticoids, but the side effects of hormones are numerous, including gastrointestinal mucosal bleeding, hypoglycemia, hypotension, various infections, osteoporosis, avascular necrosis of the femoral head, etc., which cause serious harm to the motor system and bones, and may also cause mental excitement.

[0006] The main treatment methods for end-stage renal disease are kidney transplantation or blood purification. However, the long-term treatment process will have a great impact on the physical and mental health of patients. Therefore, strengthening the diagnosis and treatment of early kidney disease in clinical practice is the key to improving the treatment effect and prognosis of kidney disease patients.

[0007] Scientists have tried various methods to develop drugs for the treatment of kidney disease, including using snake venom products to treat kidney disease. For example, Naja atra snake venom has been reported to be used in the treatment of kidney disease (CN Patent 201210228609 Use of Physically Modified Naja atra Snake Venom in the Preparation of Drugs for the Treatment of Acute and Chronic Kidney Diseases). However, there are many types of cobra toxins, and the known components include neurotoxins, cytotoxins, cardiotoxins, nerve growth factors, hemolysin (DLP), CVA protein, membrane-active polypeptides, cobra venom factor, etc., as well as other components such as alkaline phosphomonoesterase, phosphodiesterase, acetylcholinesterase, L-amino acid oxidase, ribonuclease, proteolytic enzyme, etc. For cobra snake venom preparations, the mixed toxins pose a very big hidden danger to clinical safety, especially for the treatment of kidney disease, because acute renal failure after being bitten by a snake is a serious reaction caused by the snake venom mixture. [22, 23, 24] This is because the synergistic effect of the toxin mixture is an important phenomenon existing in snake venom, which may be a strategy to enhance toxicity during the evolution process. There is a synergistic effect between different types of toxins or the same type of toxin complex in various snake venoms. The main toxins such as phospholipase A2 and three-finger toxins (neurotoxins) play important roles in the synergistic process.

[25] And it is this synergistic effect that leads to fatal consequences.

[0008] Studies on the synergistic effect of snake venom mixtures have found that there is a synergistic effect between the toxins of a series of snake species. The interaction of two or more toxin components directly or indirectly enhances the toxicity to a level exceeding the sum of their individual toxicities. From a molecular perspective, the synergistic effect generally may exist in two forms: (1) intermolecular synergistic effect, when two or more toxins interact with one or more (related) targets on a biological pathway, causing a synergistically increased toxicity. (2) supramolecular synergistic effect, when two or more toxins interact with the same target in a synergistic manner or when two or more toxins combine to create a complex with increased toxicity.

[26] An example of the intermolecular synergistic effect is the binding of α-neurotoxin from the Elapidae family to other toxins, resulting in a synergistically effective toxic effect, causing flaccid paralysis and respiratory failure in victims and prey.

[26] In a study on how the venom of the black mamba (cobra) causes high mortality, scientists found that potassium channel blocking activity is only one of them, and there are various pathways. The combined toxicity induced by different toxins at different organ levels is the concern of high mortality.

[27]

[0009] The research by Strydom and Botes (1970) showed that after 48 hours of single use, the venom components isolated from the venom of the relevant Eastern green mamba (cobra) were not lethal, while when used in a similar dose, the whole venom could be fatal within 10 minutes.

[28]

[0010] It is known that co-toxins can enhance the toxicity of certain toxins. Individually, these proteins are less toxic, but when they are co-injected into mice, they will have a strong toxic effect. These toxins are similar to neurotoxins or cytotoxins in terms of amino acid sequence and the number of cysteines. Such toxins with a synergistic effect are called co-toxins.

[29] The venom of Naja atra after physical modification may be able to reduce the toxicity of snake venom, but as a mixture, it has a complex composition and is difficult to quality control, so there is still a possibility of synergistic effects between toxins.

[0011] In addition, technicians have also tried to use a mixture of cobra neurotoxins to treat diabetic nephropathy. The mixture of neurotoxins has higher safety than snake venom because a series of toxins such as cardiotoxin, cytotoxin, phospholipase A2, etc. have been removed, reducing the high toxicity caused by the synergistic effect of the mixed snake venom. However, they are still a mixture of several neurotoxins and still have the possibility of causing a synergistic effect. For example, a side effect of the cobra neurotoxin mixture "Kebotai" is that it may cause respiratory depression. Although it is not yet possible to exactly know the role played by each toxin in such synergy, the most effective way to avoid such serious side effects is to replace the mixture with monomeric molecules.

[0012] From the publicly published literature known, it is the first time to study the therapeutic effect on nephritis at the level of cobra neurotoxin monomeric molecules in this invention; at the same time, the comprehensive treatment of various urinary microproteins in the early stage of nephritis in this invention, including β2-microglobulin, α1-microglobulin, transferrin, and immunoglobulin G, is also reported for the first time. The cobra neurotoxin monomeric molecule can control the inflammatory reaction process caused by autoimmunity through the regulatory effect on the nicotinic acetylcholine receptor, [30, 31] and can block and delay the progression of kidney disease in the initial stage of nephritis, which has extremely important clinical significance for the early diagnosis and early treatment of nephritis. Summary of the Invention

[0013] The present invention discloses the application of a group of cobra neurotoxin polypeptide molecules in the treatment of nephrotic proteinuria. This group of cobra neurotoxin polypeptide molecules can reduce the urinary levels of albumin and other immunoglobulins, β2-microglobulin, α1-microglobulin, and transferrin that reflect renal function. The decrease in the levels of these urinary proteins represents the alleviation of the renal autoimmune response and the alleviation of renal tissue pathological changes. Another advantage of the present invention is that this group of cobra neurotoxin polypeptide monomer molecules can avoid the serious side effects caused by the synergistic effect of snake venom mixtures or neurotoxin mixtures. The last advantage of the present invention is that the cobra neurotoxin polypeptide molecules are a kind of biological products that can be strictly quality-controlled. The clear amino acid sequence of the product ensures its purity and quality control standards, improving the safety and efficacy of the product. Detailed implementation mode

[0014] Implementation method:

[0015] The adriamycin-induced rat nephritis model can cause rats to present typical manifestations of nephrotic syndrome, and the pathological changes of its animal model are similar to those of human minimal change nephropathy and focal segmental glomerulosclerosis nephropathy; the streptozotocin (STZ)-induced rat diabetic nephropathy can cause rats to present typical manifestations of diabetic nephropathy, and the pathological changes of its animal model are similar to those of human diabetic nephropathy with minimal change.

[0016] The present invention observes the therapeutic efficacy of cobra neurotoxin polypeptide molecules on various urinary microproteins in nephritis by establishing an adriamycin-induced rat nephritis model and a streptozotocin (STZ)-induced diabetic nephritis model recognized by professionals in the field. The following further illustrates the above scheme with specific embodiments. It should be understood that these embodiments are used to illustrate the present invention rather than to limit the scope of the present invention.

[0017] Implementation steps:

[0018] I. Establishment of the adriamycin-induced rat nephritis model

[0019] A. Experimental animals and grouping

[0020] 80 male SD rats, weighing 160 - 180 g, were randomly divided into a control group of 20 rats and a model group of 60 rats. Among the rats that survived after successful modeling, 40 were randomly selected and randomly divided into a cobra neurotoxin polypeptide treatment group of 20 rats and a model group of 20 rats. The above 3 groups of rats in the control group, treatment group, and model group were each randomly divided into 2 groups, with 10 rats in each group, for the experiments of cobra neurotoxin polypeptide SEQ ID No.1 and cobra neurotoxin polypeptide SEQ ID No.2, and the remaining rats were removed from the group.

[0021] B. Modeling method

[0022] A glomerulosclerosis rat model was established by unilateral nephrectomy combined with two intravenous injections of adriamycin into the tail vein at an interval of two weeks.

[32] After unilateral nephrectomy, the rats were allowed to rest for 3 days, and then on the 4th and 18th days, 3 mg / kg and 2 mg / kg of adriamycin were injected into the tail vein respectively. Seven days after the second injection of adriamycin in the surviving rats, urinary protein was detected. A urinary protein quantification > 100 mg / 24 h indicated successful model establishment. The rats were randomly divided into the model group or the treatment group and continuously observed until the end of the 8th week. In the control group, 0.5 mL of normal saline was injected into the tail vein on the 4th and 18th days respectively. During the 8-week observation period, the rats in the treatment group were intragastrically administered with cobra neurotoxin polypeptide at 20 μg / kg once a day for 8 consecutive weeks. The rats in the model group and the control group were intragastrically administered with normal saline once a day for 8 consecutive weeks.

[0023] C. Observation indicators and detection methods

[0024] After the last treatment, the rats were placed in a metabolic cage to collect 10 mL of urine, centrifuged at 3500 r / min for 10 min, the supernatant was extracted and placed in a cryopreservation tube, and stored in a -80°C refrigerator for later use. Enzyme-linked immunosorbent assay was used to detect urinary α1-microglobulin, β2-microglobulin, microalbumin, transferrin, and immunoglobulin G (IgG), and the operation was carried out according to the instructions of the ELISA kit.

[0025] The rats were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital (50 mg / kg). After the corneal reflex disappeared, the abdominal cavity was opened to expose the abdominal aorta, and 10 mL of whole blood was collected from the abdominal aorta and placed in a centrifuge tube. After standing for 2 h, it was centrifuged at 3500 r / min for 10 min, the supernatant was extracted and placed in a cryopreservation tube, and stored in a -80°C refrigerator for later use. A fully automatic biochemical analyzer was used to detect serum creatinine, urea nitrogen, cholesterol, and triglyceride.

[0026] D. Table-1 shows the experimental results of the effect of cobra neurotoxin polypeptide of SEQ ID No.1 on the renal function indexes of adriamycin-induced nephropathy in rats

[0027] (Table-1)

[0028]

[0029] 1) The effect of cobra neurotoxin polypeptide on microalbuminuria (UALB) in adriamycin-induced nephropathy in rats. Compared with the control group, microalbuminuria (UALB) in the two groups of rats after model establishment was significantly increased; compared with the model group, microalbuminuria in the cobra neurotoxin polypeptide group was significantly decreased.

[0030] ** indicates that P < 0.01 when the cobra neurotoxin polypeptide group is compared with the model group.

[0031] 2) Effects of cobra neurotoxin polypeptide on urinary α1-microglobulin (α1-MG) in adriamycin-induced nephropathy in rats. Compared with the control group, urinary α1-MG in the two groups of rats after modeling was significantly increased; compared with the model group, urinary α1-MG in the cobra neurotoxin polypeptide group was significantly decreased. ** indicates P < 0.01 when the cobra neurotoxin polypeptide group is compared with the model group.

[0032] 3) Effects of cobra neurotoxin polypeptide on urinary β2-microglobulin (β2-MG) in adriamycin-induced nephropathy in rats. Compared with the control group, microalbuminuria in the two groups of rats after modeling was significantly increased; compared with the model group, microalbuminuria in the cobra neurotoxin polypeptide group was significantly decreased.

[0033] # indicates P < 0.05 when the cobra neurotoxin polypeptide group is compared with the model group.

[0034] 4) Effects of cobra neurotoxin polypeptide on urinary transferrin (TRF) in adriamycin-induced nephropathy in rats. Compared with the control group, urinary transferrin in the two groups of rats after modeling was significantly increased; compared with the model group, urinary transferrin in the cobra neurotoxin polypeptide group was significantly decreased.

[0035] # indicates P < 0.05 when the cobra neurotoxin polypeptide group is compared with the model group.

[0036] 5) Effects of cobra neurotoxin polypeptide on urinary immunoglobulin (IgG) in adriamycin-induced nephropathy in rats. Compared with the control group, immunoglobulins in the two groups of rats after modeling were significantly increased; compared with the model group, immunoglobulins in the cobra neurotoxin polypeptide group were significantly decreased.

[0037] # indicates P < 0.05 when the cobra neurotoxin polypeptide group is compared with the model group.

[0038] E. Table - 2 shows the experimental results of the effects of SEQ ID No.2 cobra neurotoxin polypeptide on renal function indexes in adriamycin-induced nephropathy in rats

[0039] (Table - 2)

[0040]

[0041] 1. Effects of cobra neurotoxin polypeptide on urinary microalbuminuria (UALB) in adriamycin-induced nephropathy in rats. Compared with the control group, urinary microalbuminuria (UALB) in the two groups of rats after modeling was significantly increased; compared with the model group, urinary microalbuminuria in the cobra neurotoxin polypeptide group was significantly decreased.

[0042] # indicates P < 0.05 when the cobra neurotoxin polypeptide group is compared with the model group.

[0043] 2. Effect of cobratoxin polypeptide on urinary α1-microglobulin (α1-MG) in adriamycin-induced nephropathy in rats. Compared with the control group, urinary α1-microglobulin in the two groups of rats after modeling was significantly increased; compared with the model group, urinary α1-microglobulin in the cobratoxin polypeptide group was significantly decreased.

[0044] ** indicates P < 0.01 when the cobratoxin polypeptide group is compared with the model group.

[0045] 3. Effect of cobratoxin polypeptide on urinary β2-microglobulin (β2-MG) in adriamycin-induced nephropathy in rats. Compared with the control group, microalbuminuria in the two groups of rats after modeling was significantly increased; compared with the model group, microalbuminuria in the cobratoxin polypeptide group was significantly decreased.

[0046] # indicates P < 0.05 when the cobratoxin polypeptide group is compared with the model group.

[0047] 4. Effect of cobratoxin polypeptide on urinary transferrin (TRF) in adriamycin-induced nephropathy in rats. Compared with the control group, urinary transferrin in the two groups of rats after modeling was significantly increased; compared with the model group, urinary transferrin in the cobratoxin polypeptide group was significantly decreased.

[0048] # indicates P < 0.05 when the cobratoxin polypeptide group is compared with the model group.

[0049] 5. Effect of cobratoxin polypeptide on urinary immunoglobulin (IgG) in adriamycin-induced nephropathy in rats. Compared with the control group, urinary immunoglobulin in the two groups of rats after modeling was significantly increased; compared with the model group, urinary immunoglobulin in the cobratoxin polypeptide group was significantly decreased.

[0050] # indicates P < 0.05 when the cobratoxin polypeptide group is compared with the model group.

[0051] II. Establishment of a streptozotocin (STZ)-induced rat nephritis model

[0052] A. Experimental animals and grouping

[0053] Eighty Wistar rats, weighing 180±10 g, were randomly divided into a control group of 20 rats and a model group of 60 rats. Among the rats that survived after successful modeling, 40 were randomly selected and randomly divided into a cobra neurotoxin polypeptide treatment group of 20 rats and a model group of 20 rats. Finally, the above-mentioned 20 rats in the control group, 20 rats in the cobra neurotoxin polypeptide treatment group, and 20 rats in the model group were each randomly divided into 2 groups of 10 rats each for experiments on cobra neurotoxin polypeptide SEQ ID No.1 and cobra neurotoxin polypeptide SEQ ID No.2, and the remaining rats were removed from the group. The cobra neurotoxin polypeptide treatment group was gavaged with cobra neurotoxin polypeptide at 20 μg / kg once a day for 8 consecutive weeks. The model group and the control group were gavaged with normal saline once a day for 8 consecutive weeks.

[0054] B. Modeling method

[0055] The rats in the model group were fed and watered normally. Each rat was first intraperitoneally injected with 0.5 ml of Freund's complete adjuvant (CFA), and the next day, streptozotocin (STZ) solution was intraperitoneally injected. Before use, it was prepared into a 1% concentration with 0.1 mmol / L citric acid buffer at pH 4.5 and intraperitoneally injected at 55 mg / kg. One week after the model group was established, tail vein blood was collected to detect blood glucose. Random blood glucose maintained above 16.7 mmol / L, and urine sugar 3+ to 4+ was regarded as successful establishment of the diabetes model.

[0056] C. Observation indicators and detection methods

[0057] After the last treatment, the rats were placed in a metabolic cage to collect 10 mL of urine, centrifuged at 3500 r / min for 10 min, the supernatant was extracted and placed in a cryopreservation tube, and stored in a -80°C refrigerator for later use. Enzyme-linked immunosorbent assay was used to detect urinary α1-microglobulin, β2-microglobulin, microalbumin, transferrin, and immunoglobulin G (IgG), and the operation was carried out according to the instructions of the ELISA kit.

[0058] The rats were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital (50 mg / kg). After the corneal reflex disappeared, the abdomen was opened to expose the abdominal aorta, and 10 mL of whole blood was collected from the abdominal aorta and placed in a centrifuge tube. After standing for 2 h, it was centrifuged at 3500 r / min for 10 min, the supernatant was extracted and placed in a cryopreservation tube, and stored in a -80°C refrigerator for later use. A fully automatic biochemical analyzer was used to detect serum creatinine, urea nitrogen, cholesterol, and triglyceride.

[0059] D. Table-3 shows the experimental results of the effect of cobra neurotoxin polypeptide SEQ ID No.1 on renal function indexes in streptozotocin (STZ)-induced rat nephropathy

[0060] (Table-3)

[0061]

[0062] 1. Effects of cobra neurotoxin polypeptide on microalbuminuria (UALB) in streptozotocin (STZ)-induced diabetic nephropathy in rats. Compared with the control group, microalbuminuria (UALB) in the two groups of rats after modeling was significantly increased; compared with the model group, microalbuminuria in the cobra neurotoxin polypeptide group was significantly decreased. **Indicates P < 0.01 when the cobra neurotoxin polypeptide group is compared with the model group.

[0063] 2. Effects of cobra neurotoxin polypeptide on urinary α1-microglobulin (α1-MG) in streptozotocin (STZ)-induced diabetic nephropathy in rats. Compared with the control group, urinary α1-microglobulin in the two groups of rats after modeling was significantly increased; compared with the model group, urinary α1-microglobulin in the cobra neurotoxin polypeptide group was significantly decreased. **Indicates P < 0.01 when the cobra neurotoxin polypeptide group is compared with the model group.

[0064] 3. Effects of cobra neurotoxin polypeptide on urinary β2-microglobulin (β2-MG) in streptozotocin (STZ)-induced diabetic nephropathy in rats. Compared with the control group, urinary β2-microglobulin in the two groups of rats after modeling was significantly increased; compared with the model group, urinary β2-microglobulin in the cobra neurotoxin polypeptide group was significantly decreased. **Indicates P < 0.01 when the cobra neurotoxin polypeptide group is compared with the model group.

[0065] 4. Effects of cobra neurotoxin polypeptide on urinary transferrin (TRF) in streptozotocin (STZ)-induced diabetic nephropathy in rats. Compared with the control group, urinary transferrin in the two groups of rats after modeling was significantly increased; compared with the model group, urinary transferrin in the cobra neurotoxin polypeptide group was significantly decreased. #Indicates P < 0.05 when the cobra neurotoxin polypeptide group is compared with the model group.

[0066] 5. Effects of cobra neurotoxin polypeptide on urinary immunoglobulin (IgG) in streptozotocin (STZ)-induced diabetic nephropathy in rats. Compared with the control group, urinary immunoglobulin in the two groups of rats after modeling was significantly increased; compared with the model group, urinary immunoglobulin in the cobra neurotoxin polypeptide group was significantly decreased. #Indicates P < 0.05 when the cobra neurotoxin polypeptide group is compared with the model group.

[0067] E. Table - 4 shows the experimental results of the effects of SEQ ID No.2 cobra neurotoxin polypeptide on renal function indices in streptozotocin (STZ)-induced diabetic nephropathy in rats

[0068] (Table - 4)

[0069]

[0070] 1. Effects of cobra neurotoxin polypeptide on microalbuminuria (UALB) in streptozotocin (STZ)-induced diabetic nephropathy in rats. Compared with the control group, the microalbuminuria (UALB) of the two groups of rats after modeling increased significantly; compared with the model group, the microalbuminuria of the cobra neurotoxin polypeptide group decreased significantly. ** indicates P < 0.01 when the cobra neurotoxin polypeptide group is compared with the model group.

[0071] 2. Effects of cobra neurotoxin polypeptide on urinary α1-microglobulin (α1-MG) in streptozotocin (STZ)-induced diabetic nephropathy in rats. Compared with the control group, the urinary α1-microglobulin of the two groups of rats after modeling increased significantly; compared with the model group, the urinary α1-microglobulin of the cobra neurotoxin polypeptide group decreased significantly. ** indicates P < 0.01 when the cobra neurotoxin polypeptide group is compared with the model group.

[0072] 3. Effects of cobra neurotoxin polypeptide on urinary β2-microglobulin (β2-MG) in streptozotocin (STZ)-induced diabetic nephropathy in rats. Compared with the control group, the urinary β2-microglobulin of the two groups of rats after modeling increased significantly; compared with the model group, the urinary β2-microglobulin of the cobra neurotoxin polypeptide group decreased significantly. ** indicates P < 0.01 when the cobra neurotoxin polypeptide group is compared with the model group.

[0073] 4. Effects of cobra neurotoxin polypeptide on urinary transferrin (TRF) in streptozotocin (STZ)-induced diabetic nephropathy in rats. Compared with the control group, the urinary transferrin of the two groups of rats after modeling increased significantly; compared with the model group, the urinary transferrin of the cobra neurotoxin polypeptide group decreased significantly. # indicates P < 0.05 when the cobra neurotoxin polypeptide group is compared with the model group.

[0074] 5. Effects of cobra neurotoxin polypeptide on urinary immunoglobulin (IgG) in streptozotocin (STZ)-induced diabetic nephropathy in rats. Compared with the control group, the urinary immunoglobulin of the two groups of rats after modeling increased significantly; compared with the model group, the urinary immunoglobulin of the cobra neurotoxin polypeptide group decreased significantly. # indicates P < 0.05 when the cobra neurotoxin group is compared with the model group.

[0075] Cobra neurotoxin polypeptide can also reduce the levels of serum creatinine, blood urea nitrogen, cholesterol, and triglyceride in the rats after modeling, but there is no significant difference compared with the model group (P > 0.05). This may be because in the early stage of nephritis, the changes of these indicators such as serum creatinine, blood urea nitrogen, cholesterol, and triglyceride are not significant, and small sample analysis cannot make statistical significance. Therefore, the decrease of these indicators needs to be confirmed after further expanding the sample size.

[0076] References:

[0077] 1. Zhang Guiyun. Application of Urinary Microprotein Determination in the Early Diagnosis of Nephropathy. Contemporary Medicine 2013, 000(027) - 146~146

[0078] 2. Gao Xuehui. Clinical Application of Urinary Microprotein in Early Renal Injury. Shaanxi Journal of Medical Laboratory Sciences (ShaanxiJLab Sci), Vol. 16, No. 2, May 2001

[0079] 3. Song Xue et al. Clinical Observation and Diagnostic Value of Different Urinary Protein Tests in Kidney Diseases. International Journal of Urology and Nephrology, Vol. 32, No. 2, March 2012

[0080] 4. Zhu Xiaobin. Discussion on the Application Value of Urinary Renal Function Detection in the Clinical Diagnosis of Early Diabetic Nephropathy. Guide of China Medicine 2017, 015(002) -

[0081] 5. Wang Xiao. Discussion on the Clinical Significance of Urinary Microprotein Detection in Judging Diabetic Nephropathy. Journal of Zhejiang Chinese Medical University, 2008, 32(6): 751~752

[0082] 6. Su Cainv et al. Discussion on the Early Diagnostic Value of Serum and Urinary Microprotein Determination in Diabetic Nephropathy. Journal of Radioimmunology, 2001, 14(4): 502

[0083] 7. Duan Liping et al. Analysis of the Types and Contents of Urinary Microproteins in Primary Nephrotic Syndrome and Lupus Nephritis. China Tropical Medicine 2003, 003(006) - 771~772

[0084] 8. Yang Fang et al. Relationship between Changes in Urinary Microprotein and Renal Damage in Reflux Nephropathy. Journal of Jinan University (Natural Science and Medicine Edition), 2000, 021(004) - 104~106

[0085] 9. Shen Shunbiao et al. Clinical Significance of Urinary Microprotein Content in IgA Nephropathy. Chinese Journal of Nephrology 1995, 000(001) - 34~34

[0086] 10. Teng Shoufeng et al. Value of Three - item Urinary Microprotein in the Early Diagnosis of Diabetic Renal Injury. Chinese Journal of Misdiagnostics 2009, 009(034)

[0087] 11. Shao Aihua et al. Observation on the Results of Urinary Microprotein Determination in Patients with Various Nephropathies. Journal of Zhejiang College of Traditional Chinese Medicine 2003, 027(005) - 31~32

[0088] 12. Huo Meifeng. Value of β2 - Microglobulin in the Early Diagnosis of Diabetic Nephropathy. Chinese General Practice, 2012(12): 119 - 120

[0089] 13. Kang Ping, et al. Significance of β2-microglobulin in early renal damage of diabetes mellitus. Heilongjiang Medical Journal, 2008

[0090] 14. Lu Qingyun, et al. Clinical significance of α1 and β2-microglobulin in early renal damage of essential hypertension [J]. Western Journal of Traditional Chinese Medicine, 2014: 131-133

[0091] 15. Wang Chunwei. Clinical value of urinary α1-microglobulin in diabetic nephropathy. Journal of Practical Medical Techniques, 2007, 014(025)

[0092] 16. Yan Jingchun. Clinical significance of urinary α1-microglobulin detection in secondary nephropathy. Chinese Journal of Coal Industry Medicine, 2014, 017(006)

[0093] 17. Wei Wenfeng. Significance of urinary microtransferrin in early diagnosis of diabetic nephropathy. Labeled Immunoassays and Clinical Medicine, 2002

[0094] 18. Li Xiaoxia, et al. Observation and comparison of urinary microtransferrin and albumin detection in early glomerular injury. Journal of Medical Forum, 2010

[0095] 19. Zhu Guangbo, et al. Clinical significance of determination of urinary microtransferrin in diabetic patients. Shaanxi Medical Journal, 1999

[0096] 20. Dong Hui, et al. Clinical significance of combined detection of microglobulin and transferrin in early diagnosis of diabetic nephropathy. Journal of Tianjin Medical University, 2002, 8(2): 253

[0097] 21. Martin P, et al. Urinary transferring and early predictor of glomerular dysfunction [J]. Ann Clin Biochem, 1988, 5(Suppl): 158.

[0098] 22. Liu Qian. Efficacy of hemoperfusion combined with continuous blood purification in the rescue of acute renal failure caused by snakebite. Chinese and Foreign Medical Research, 2013, 000(036)

[0099] 23. Zhang Qionglin. Nursing experience of continuous renal replacement therapy combined with traditional Chinese medicine hoop therapy in the treatment of severe snakebite. Family Psychologist, 2014, 010(009)

[0100] 24. Zhang Youqi, et al. Research progress on snake venom toxin-induced kidney injury. Medical Recapitulate, 2010, 016(002)

[0101] 25. Shengwei Xiong, Chunhong Huang. Synergistic strategies of predominant toxins in snake venoms. Toxicology Letters 287(2018)142 - 154

[0102] 26. Laustsen, A.H. Toxin synergism in snake venoms. Toxin Reviews, 35(3 - 4), 165 - 170. DOI:10.1080 / 15569543.2016

[0103] 27. Anil Kumar, Varun Gupta. Neurological Implications of Dendrotoxin: A Review. EC PHARMACOLOGY AND TOXICOLOGY. May 25, 2018

[0104] 28. Strydom DJ, Botes DP. Snake venom toxins - I. Preliminary studies on the separation of toxins of elapidae venoms. Toxicon 8:203 - 9. 1970

[0105] 29. Joubert, F.J., Taljaard, N, Snake venoms. The amino - acid sequence of Protein S2C4 from Dendroaspis jamesoni kaimosae(Jameson’s mamba) venom. Hoppe Seylers. Z. Physiol. Chem. 360, 571 - 580. 1979

[0106] 30. Zhou Guowu et al. α7 nicotinic acetylcholine receptor and inflammation regulation. Progress in Modern Biomedicine, 2009, Issue 14

[0107] 31. Liu Zhigang. Regulatory role of α7 nicotinic acetylcholine receptor in inflammation. Guide of China Medicine, 2014, Issue 03

[0108] 32. Wei Na et al. Establishment of a modified adriamycin - induced nephropathy rat model. Journal of Xi'an Jiaotong University(Medical Sciences), 2009, (4):445 - 452.

Claims

1. Use of a composition comprising a therapeutically effective amount of cobra neurotoxin polypeptide and a pharmaceutically acceptable carrier thereof in the preparation of a medicament for treating diabetic nephropathy, characterized in that, The effect of the drug in diabetic nephropathy is to reduce proteinuria; the polypeptide sequence of cobra neurotoxin is as shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The application according to claim 1, wherein The proteinuria mentioned above refers to proteinuria caused by the leakage of one or more of albumin, immunoglobulin, β2-microglobulin, α1-microglobulin or transferrin into urine, resulting in an increase in the content of these proteins in urine, exceeding the normal range defined medically.

3. The application according to claim 1, wherein The proteinuria includes proteinuria caused by an increase in one or more of the protein indicators of albumin, immunoglobulin, β2-microglobulin, α1-microglobulin, and transferrin.

4. The application according to claim 1, characterized in that, The cobra neurotoxin polypeptide is derived from isolation and extraction from natural snake venom, or chemical polypeptide synthesis, or production from prokaryotic or eukaryotic hosts using recombinant technology.

5. The application according to claim 4, characterized in that, For the cobra neurotoxin polypeptide produced by recombination, depending on the host used in the recombinant production scheme, the polypeptide is glycosylated or non-glycosylated, contains or does not contain disulfide bonds, and includes or does not include the starting methionine residue.

6. The application according to claim 1, wherein The usage methods of the drug include intravenous injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, rectal administration, intradermal administration or transdermal administration.

7. The application according to claim 1, characterized in that, The dosage of the cobra neurotoxin polypeptide is from 1 μg / Kg to 350 μg / kg each time, with a frequency of once a day or multiple times a day.

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