Smad5 mutant and application in preventing and treating diabetes
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- TONGJI UNIV
- Filing Date
- 2024-02-06
- Publication Date
- 2026-06-26
Smart Images
Figure CN118146342B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine, specifically relating to an Smad5 mutant and its application in the prevention and treatment of diabetes. Background Technology
[0002] Diabetes is a group of metabolic diseases characterized by elevated blood glucose levels. In recent years, with the improvement of living standards and changes in dietary structure, the incidence of diabetes has been rising year by year and showing a trend of gradually becoming younger. According to the latest data released by the International Diabetes Federation (IDF), as of 2021, there were approximately 537 million patients worldwide. The research team published the latest epidemiological survey results of diabetes in the internationally renowned medical journal British Medical Journal, showing that the incidence of diabetes in adults in some regions has increased nearly 20 times in the past 30 years [1]. At present, diabetes has become a major public health problem that seriously endangers health and brings a heavy economic burden to society.
[0003] Type 1 diabetes is caused by the destruction of pancreatic β cells due to autoimmunity, resulting in an absolute deficiency of insulin secretion. Type 2 diabetes is caused by a relative deficiency of insulin secretion or insulin resistance due to impaired pancreatic function. Abnormal insulin synthesis and secretion are important characteristics of diabetes. The body needs to sense subtle changes in blood glucose and environment in real time, and then finely regulate the amount of insulin synthesis and secretion [2]. Since pancreatic β cells are in a constantly changing intracellular and extracellular environment, understanding the establishment and maintenance mechanism of β cell homeostasis will help to provide a new understanding of the pathogenesis of diabetes and provide new ideas for the prevention and treatment of diabetes. Pancreatic β cells are very sensitive to changes in the intracellular and extracellular environment. For example, subtle changes in pH inside and outside the β cells will affect the synthesis and secretion of insulin [3-5]. Therefore, pancreatic β cells need to sense complex changes in the intracellular and extracellular environment and precisely regulate insulin synthesis and secretion. Insulin resistance (IR) refers to a pathological state in which the target organs of insulin, including the liver, adipose tissue, skeletal muscle, etc., are less sensitive to the body's secretion of insulin [6-8]. As one of the main target organs of insulin, the liver experiences insulin resistance, which reduces the efficiency of glucose absorption and utilization under the action of insulin. To compensate for this reduced efficiency, the body secretes more insulin, ultimately leading to hyperinsulinemia, decreased hepatic glycogen synthesis, and enhanced gluconeogenesis. Numerous studies have shown that hepatic insulin resistance is involved in the occurrence and development of type 2 diabetes mellitus (T2DM) and is one of the important pathogenic mechanisms of T2DM. The insulin resistance mechanism is complex and is the result of the synergistic effect of multiple factors [9-13]. Therefore, increasing hepatic insulin sensitivity and improving insulin resistance are key to the treatment of T2DM
[14] .
[0004] Currently, treatments for type 1 diabetes include insulin injections, insulin pumps, and islet cell transplantation. Treatments for type 2 diabetes include insulin and oral hypoglycemic agents such as biguanides, sulfonylureas, thiazolidinediones, and alpha-glucosidase inhibitors [15,16]. However, these drugs do not cure diabetes, and all treatments have varying degrees of adverse effects. Therefore, developing more effective treatment strategies to improve insufficient insulin secretion or insulin resistance is an urgent problem to be solved. Summary of the Invention
[0005] The technical problem this invention aims to solve is to overcome the lack of effective treatment strategies in the prior art to improve insufficient insulin secretion or insulin resistance, and to provide an Smad5 mutant and its application in the prevention and treatment of diabetes. Furthermore, this invention also provides methods for protecting pancreatic islet cells from damage, improving glucose tolerance, alleviating insulin resistance, lowering blood glucose, and relieving symptoms of diabetic glucose intolerance. The application of Smad5 protein can protect the islets from damage caused by adverse environmental factors in the body, preventing the occurrence of diabetes; the application of Smad5_K protein can alleviate symptoms of diabetic glucose intolerance and insulin resistance; and the application of baking soda in drinking water can alleviate symptoms of abnormal blood glucose and glucose intolerance in diabetes. These techniques can be used in combination.
[0006] One aspect of the present invention is to provide a Smad5 mutant, the amino acid sequence of which is shown in SEQ NO ID:1.
[0007] Preferably, the nucleotide sequence encoding the Smad5 mutant is shown in SEQ NO ID:2.
[0008] The second aspect of the present invention is to provide an isolated nucleic acid that encodes the Smad5 mutant as described in the first aspect of the invention.
[0009] Preferably, the nucleotide sequence of the isolated nucleic acid is as shown in SEQ. NO As shown in ID:2.
[0010] The third aspect of the present invention is to provide a recombinant expression vector comprising the isolated nucleic acid as described in the second aspect.
[0011] Preferably, the backbone plasmid of the recombinant expression vector is pET-N-His-C-His and pLVX-Tight-Puro.
[0012] The fourth aspect of the present invention provides a transformant comprising the nucleic acid as described in the second aspect of the invention, or the recombinant expression vector as described in the third aspect of the invention.
[0013] Preferably, the host of the transformant is Escherichia coli and mammalian cells.
[0014] The fifth aspect of the present invention provides a method for preparing a Smad5 mutant, comprising culturing a transformant as described in the fourth aspect of the invention to obtain a fermentation product, and obtaining the Smad5 mutant from the fermentation product.
[0015] Preferably, the culture medium used for the culture includes DMEM and 10% fetal bovine serum.
[0016] The sixth aspect of the present invention is to provide a composition comprising Smad5, as described in one aspect of the present invention.
[0017] Preferably, the composition further includes a pharmaceutically acceptable carrier.
[0018] The seventh aspect of the present invention provides the use of Smad5, the Smad5 mutant described in the first aspect, the isolated nucleic acid described in the second aspect, the recombinant expression vector described in the third aspect, the transformant described in the fourth aspect, or the composition described in the sixth aspect in the preparation of a medicament for the prevention and / or treatment of diabetes.
[0019] Preferably, the diabetes is selected from type 1 diabetes and type 2 diabetes.
[0020] The eighth aspect of the present invention is to provide a reagent for regulating acid-base balance, which includes baking soda alkaline drinking water.
[0021] Preferably, the baking soda alkaline drinking water comprises: 0.05-0.2M NaHCO3, pH 7.6-9.5, and 0.5% sugar.
[0022] The ninth aspect of the present invention provides the use of the reagent described in the eighth aspect in the preparation of a medicament for treating diabetes.
[0023] Preferably, the diabetes is selected from type 1 diabetes and type 2 diabetes.
[0024] The tenth aspect of the present invention provides a method for protecting pancreatic islet cells by administering Smad5 or a Smad5 mutant as described in one aspect of the present invention to a subject in need.
[0025] The eleventh aspect of the present invention provides a method for improving glucose intolerance and / or lowering blood glucose by administering Smad5 or a Smad5 mutant as described in one aspect of the invention to a subject in need.
[0026] The twelfth aspect of the present invention provides a method for alleviating insulin resistance, which is to administer Smad5 or a Smad5 mutant as described in one aspect of the invention to a subject in need.
[0027] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.
[0028] The reagents and raw materials used in this invention are all commercially available.
[0029] The positive and progressive effects of this invention are as follows:
[0030] By synthesizing Smad5 and Smad5_K proteins in vitro and injecting them into the body, symptoms of type 1 and type 2 diabetes can be improved. Because these proteins are naturally present in the body or promote metabolism, they have no toxic side effects compared to conventional oral hypoglycemic agents and do not damage the patient's liver or kidneys. Administering alkaline drinking water containing baking soda to lower blood sugar and alleviate diabetes symptoms is more economical and convenient for patients. Attached Figure Description
[0031] Figure 1 This figure shows the experimental results of GFP-Smad5 translocating from the nucleus to the cytoplasm due to high glucose stimulation.
[0032] Figure a shows that high glucose (16.7 mM) stimulation causes an increase in cytoplasmic pH in pancreatic islet cells; Figure b shows that high glucose (16.7 mM) stimulation for 10-15 minutes causes GFP-Smad5 to translocate from the nucleus to the cytoplasm; Figure c shows that stimulation of GFP-Smad5 mice with 2 g / kg glucose causes GFP-Smad5 to translocate from the nucleus to the cytoplasm in pancreatic islet cells; Figure d shows the effect of... Figure 1 Statistical analysis of the nucleoplasmic distribution of cGFP-Smad5. **p<0.01,***p<0.001, unpaired two-tailed t-test.
[0033] Figure 2 The study demonstrated that Smad5 knockout specifically in pancreatic β-cells induced elevated blood glucose and impaired glucose tolerance in mice. Figure a shows a schematic diagram of Smad5 knockout in pancreatic β-cells in mice; figure b shows Western blot validation of Smad5 knockout efficiency; figure c shows the elevated fasting blood glucose induced by Smad5 knockout in pancreatic β-cells; and figure d shows the impaired glucose tolerance induced by Smad5 knockout in pancreatic β-cells in mice. Figure 2 e is a pair Figure 2 Statistical graph of glucose tolerance curve; Figure 2 f represents the determination of insulin tolerance in pancreatic β-cell-specific knockout Smad5 mice; Figure 2 g is a pair Figure 2Statistical graph of insulin tolerance curve. **p<0.01,***p<0.001, unpaired two-tailed t-test.
[0034] Figure 3 Smad5 knockout specifically on pancreatic β cells induced abnormal insulin processing and secretion in mice. Specifically, a) Smad5 knockout on pancreatic β cells resulted in decreased C-peptide secretion in mice; b) Smad5 knockout on pancreatic β cells resulted in decreased insulin secretion in mice after fasting and 30 minutes of glucose stimulation; c) Immunofluorescence staining showed that Smad5 knockout increased proinsulin expression in mice; d) [further details needed for accurate translation]. Figure 3 c is a statistical graph of the fluorescence density of proinsulin in mice; e is a Western blot showing that knockout of Smad5 increases proinsulin expression and decreases insulin expression in mouse islets; f is the effect of... Figure 3 Statistical analysis of the proinsulin / insulin ratio. **p<0.01, ***p<0.001, unpaired two-tailed t-test.
[0035] Figure 4 Figure 1 shows the experimental results of GFP-Smad5 overexpressing mice resisting STZ-induced hyperglycemia and weight loss. Figure 2 shows the proportion of normal and hyperglycemic blood glucose in wild-type (WT) and GFP-Smad5 overexpressing (OE) mice one day after streptozotocin (STZ) injection. Figures 3 and 4 show the weight changes and the proportion of weight loss in wild-type (WT) and GFP-Smad5 overexpressing (OE) mice seven days after hyperglycemia. **p<0.01, unpaired two-tailed t-test.
[0036] Figure 5 Figure 1 shows the experimental results of overexpressing the persistently cytoplasmic mutant GFP-Smad5_K in mice to alleviate the glucose tolerance disorder phenotype induced by a high-fat diet. Figure 2 shows that after 2 months of high-fat diet induction, GFP-Smad5 nuclear aggregation was promoted, indicating acidification in pancreatic islet cells in a high-fat-induced type 2 diabetes model. Figure 3 shows the reduction rate of the nuclear-cytoplasmic ratio of GFP-Smad5 induced by glucose in Figure 4. Figure 5 shows the changes in glucose tolerance in wild-type, GFP-Smad5-overexpressing, and GFP-Smad5_K-overexpressing mice after 2 months of high-fat diet induction. Figure 6 shows the statistical results of the glucose tolerance curves. **p<0.01, ***p<0.001, unpaired two-tailed t-test.
[0037] Figure 6Figure 1 shows the experimental results of reducing insulin resistance phenotype in mice by sustained overexpression of the cytoplasmic mutant GFP-Smad5_K. Figure 2 shows the results for the control group, experimental group, and cytoplasmic Smad5-retention rescue group. Mice were 24 weeks old and administered glucose solution intraperitoneally; blood glucose levels were measured at different time points. Figure 3 shows the area under the curve (AUC) of the glucose tolerance test for the three groups. Figure 4 shows the results for the control group, experimental group, and cytoplasmic Smad5-retention rescue group. Mice were 24 weeks old and administered insulin solution intraperitoneally; blood glucose levels were measured at different time points. Figure 5 shows the results for the area under the curve (AUC) of the insulin tolerance test for the three groups. ***p<0.001, unpaired two-tailed t-test.
[0038] Figure 7 The figure shows the experimental results demonstrating that sustained overexpression of the cytoplasmic mutant GFP-Smad5_K better alleviates the insulin resistance phenotype in mice compared to wild-type GFP-Smad5 mice. Figure a shows the changes in insulin tolerance in GFP-Smad5-overexpressing and GFP-Smad5_K-overexpressing mice after 2 months of a high-fat diet. Figure b shows the statistical analysis of the insulin tolerance curves. *p<0.05, unpaired two-tailed t-test.
[0039] Figure 8 Figure 1 shows the experimental results of how sodium bicarbonate alkaline drinking water improved the glucose tolerance phenotype induced by a high-fat diet. Figure 2 shows the feeding patterns of normal drinking water (Neutral water, NW) and sodium bicarbonate alkaline drinking water; Figures 3 and 4 show that sodium bicarbonate alkaline drinking water promotes GFP-Smad5 cell cytoplasmic aggregation, indicating that it can improve the acidification phenotype of pancreatic islet cells induced by a high-fat diet; Figure 5 shows the fasting and postprandial blood glucose levels after 50 days of feeding with normal drinking water or sodium bicarbonate alkaline drinking water (0.2M NaHCO3, pH 9.5, 0.5% sugar); Figure 6 shows the changes in glucose tolerance in mice fed with normal drinking water or sodium bicarbonate alkaline drinking water; Figure 7 shows the effects of sodium bicarbonate alkaline drinking water on glucose tolerance in mice fed with normal drinking water or sodium bicarbonate alkaline drinking water; Figure 8 shows the effects of sodium bicarbonate alkaline drinking water on glucose tolerance in mice fed with normal drinking water or sodium bicarbonate alkaline drinking water. Figure 8 Statistical analysis of e-glucose tolerance curves. *p<0.05,**p<0.01,***p<0.001, unpaired two-tailed t-test.
[0040] Figure 9 Figure a shows the experimental results of the concentration range of sodium bicarbonate alkaline drinking water in improving the glucose tolerance phenotype induced by a high-fat diet. Figure a shows the changes in glucose tolerance in mice after 50 consecutive days of administration of commercially available sodium bicarbonate alkaline drinking water (2.5 mM NaHCO3, pH 7.6) or different concentrations of sodium bicarbonate alkaline drinking water (25 mM, 50 mM, 100 mM, 150 mM, 200 mM NaHCO3, pH 7.6). Figure b shows the statistical analysis of the glucose tolerance curves in figure a. *p<0.05, unpaired two-tailed t-test.
[0041] Figure 10Figure 1 shows the experimental results of sodium bicarbonate alkaline drinking water concentration and pH range for improving the glucose tolerance disorder phenotype induced by a high-fat diet. Figure 2 shows the changes in glucose tolerance in mice after 50 days of feeding with normal drinking water or sodium bicarbonate alkaline drinking water (0.2M NaHCO3, pH 8.5, 0.5% sugar). Figure 3 shows the changes in glucose tolerance in mice after 50 days of feeding with normal drinking water or sodium bicarbonate alkaline drinking water (0.2M NaHCO3, pH 8.5, 0.5% sugar). Figure 4 shows the changes in glucose tolerance in mice after 50 days of feeding with normal drinking water or sodium bicarbonate alkaline drinking water (0.2M NaHCO3, pH 8.5, 0.5% sugar). Figure 10 Figure a shows the statistical analysis of glucose tolerance curves; c represents the changes in glucose tolerance in mice after 50 days of feeding with normal drinking water or alkaline drinking water (0.15M NaHCO3, pH 8.5, 0.5% sugar); d represents the changes in glucose tolerance in mice after 50 days of feeding with normal drinking water or alkaline drinking water (0.15M NaHCO3, pH 8.5, 0.5% sugar); and d represents the changes in glucose tolerance in mice after 5 Figure 10 Figure c shows the statistical analysis of glucose tolerance curves, while e represents the changes in glucose tolerance in mice after 50 days of feeding with normal drinking water or alkaline drinking water containing baking soda (0.1M NaHCO3, pH 8.5, 0.5% sugar). Figure 10 f is a pair Figure 10 Statistical analysis of the e-plot glucose tolerance curve. *p<0.05, unpaired two-tailed t-test. Detailed Implementation
[0042] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.
[0043] Example 1: Smad5 regulates insulin synthesis and secretion
[0044] Previous research by the inventors revealed that Smad5 can act as a cytoplasmic pH sensor to detect environmental stress and regulate metabolic responses through nucleoplasmic shuttle to maintain intracellular metabolic homeostasis [17,18]. The inventors have now further discovered that under normal conditions, GFP-Smad5 is located in the nucleus of pancreatic β-cells, and glucose stimulation causes GFP-Smad5 to accumulate in the cytoplasm, indicating that glucose stimulation leads to an increase in intracellular pH in pancreatic β-cells. Figure 1 To investigate the effect of conditional Smad5 knockout in pancreatic β-cells on pancreatic islet function in mice, changes in blood glucose and glucose tolerance were examined in knockout mice. After Smad5 knockout, mice exhibited elevated fasting blood glucose and significantly abnormal glucose tolerance, indicating that pancreatic β-cell-specific Smad5 knockout reduces the ability of mice to process glucose. Figure 2 Serum C-peptide and insulin levels were detected by ELISA, and the results showed a decrease in serum C-peptide and insulin levels. Subsequently, immunofluorescence staining and Western blot analysis of proinsulin expression in the pancreas and serum of Smad5 knockout mice showed that Smad5 knockout increased proinsulin expression, indicating that Smad5 knockout caused abnormal proinsulin processing and impaired insulin synthesis. Figure 3 The above indicates that Smad5 plays an important role in regulating insulin synthesis and secretion.
[0045] Example 2: Smad5 protects pancreatic islet cells from damage
[0046] Experimental methods:
[0047] 1.1 Mouse preparation: 6-week-old c57 mice weighing about 20g were purchased from Slack, Inc. GFP-Smad5 overexpression mice were obtained from laboratory-constructed models. All mice were housed in the SPF-grade breeding room of Tongji University Animal Center.
[0048] 1.2 Intraperitoneal injection modeling: Mice were fasted for 24 hours before the experiment, followed by a single intraperitoneal injection of streptozotocin (STZ) (120 mg / kg body weight) to destroy pancreatic β cells, thus obtaining a type 1 diabetic mouse model. The normal control group received an equal volume of citric acid solution intraperitoneally. Blood glucose was measured via tail vein 24 hours later; a blood glucose level above 11.2 mmol / L was considered hyperglycemia.
[0049] Experimental results:
[0050] Intraperitoneal injection of streptozotocin (STZ) induced pancreatic β-cell toxicity in wild-type mice and mice overexpressing GFP-Smad5. Results showed that 7 days after STZ injection, the rates of hyperglycemia and weight loss were significantly lower in GFP-Smad5 mice compared to wild-type mice. Figure 4 The results (ac) indicate that overexpression of GFP-Smad5 has a protective effect on pancreatic β cells.
[0051] Example 3: Smad5_K mutant improves glucose intolerance
[0052] Experimental methods:
[0053] 1.1 Mouse preparation: GFP-Smad5_K mutant (SEQ ID NO:1) overexpression mice were obtained from laboratory-constructed models and were housed in the SPF-grade breeding room of Tongji University Animal Center.
[0054] 1.2 Establishment of a high-fat diet-induced type 2 diabetes model: Wild-type mice, GFP-Smad5 overexpressing mice, and GFP-Smad5_K mutant overexpressing mice were fed a high-fat diet (Research Diet, D12492) for 2 months to establish a high-fat diet-induced diabetes model.
[0055] 1.3 Glucose Tolerance Test (GTT): Mice were fasted for 15-18 hours beforehand, weighed, and their tail veins were punctured with a needle to collect blood. Fasting blood glucose levels were measured and recorded using a glucometer. Based on the mice's weight, 2 g / kg of glucose solution was injected intraperitoneally. Blood glucose levels were collected and recorded at 15 min, 30 min, 60 min, 90 min, and 120 min after the injection.
[0056] Experimental results:
[0057] High-fat diet (HFD) treatment induces glucose intolerance and insulin resistance in mice. This study aimed to investigate whether cytoplasmic Smad5 improves the glucose intolerance phenotype induced by HFD. Mice overexpressing the GFP-Smad5_K mutant mimicked cytoplasmic alkalization, with Smad5 persistently expressed in the cytoplasm. After two months of HFD-feeding, mice fed a high-fat diet to GFP-Smad5_K showed significantly improved glucose tolerance compared to control mice and GFP-Smad5-overexpressing mice. Figure 5 (ab), Smad5 in the cytoplasm can improve the glucose intolerance phenotype induced by a high-fat diet.
[0058] Smad5_K protein sequence (SEQ ID NO:1)
[0059] MTSMASLFSFTSPAVKRLLGWKQGDEEEKWAEKAVDALVAALAAAAGAMEELEKALSSPGQPSKCVTIPRSLDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPLDICEFPFGS KQKEVCINPYHYKRVESPVLPPVLVPRHNEFNPQHSLLVQFRNLSHNEPHMPQNATFPDSFHQPNNTPFPLSPNSPYPPSPASSTYPNSPASSGPGSPFQLPADTPPPAYMPPDDQ MGQDNSQPMDTSNNMIPQIMPSISSRDVQPVAYEEPKHWCSIVYYELNNRVGEAFHASSTSVLVDGFTDPSNNKSRFCLGLLSNVNRNSTIENTRRHIGKGVHLYYVGGEVYAECL SDSSIFVQSRNCNFHHGFHPTTVCKIPSSCSLKIFNNQEFAQLLAQSVNHGFEAVYELTKMCTIRMSFVKGWGAEYHRQDVTSTPCWIEIHLHGPLQWLDKVLTQMGSPLNPISSVS
[0060] Smad5_K nucleotide sequence (SEQ ID NO:2)
[0061]
[0062] pET-N-His-C-His vector sequence (SEQ ID NO:3)
[0063]
[0064] pLVX-Tight-Puro vector sequence (SEQ ID NO:4)
[0065]
[0066] Example 4: Smad5_K mutant alleviates insulin resistance
[0067] Experimental methods:
[0068] 1.1 Mouse preparation: GFP-Smad5_K mutant overexpression mice were obtained from laboratory-constructed models and were housed in the SPF-grade breeding room of Tongji University Animal Center.
[0069] 1.2 Glucose Tolerance Test (GTT): Mice were fasted for 15-18 hours beforehand, weighed, and their tail veins were punctured with a needle to collect blood. Fasting blood glucose levels were measured and recorded using a glucometer. Based on the mice's weight, 2 g / kg of glucose solution was injected intraperitoneally. Blood glucose levels were collected and recorded at 15 min, 30 min, 60 min, 90 min, and 120 min after the injection.
[0070] 1.3 Insulin Tolerance Test (ITT): Mice were fasted for 15-18 hours beforehand, weighed, and their tail veins were punctured with a needle to collect blood. Fasting blood glucose levels were measured and recorded using a glucometer. Based on the mice's weight, 0.75 U / kg of insulin solution was injected intraperitoneally. Blood glucose levels were collected and recorded at 15 min, 30 min, 60 min, 90 min, and 120 min after insulin injection.
[0071] Experimental results:
[0072] The effects of Smad5KO-GFP-Smad5_K on insulin sensitivity and glucose tolerance in mice were assessed. Smad5KO-GFP-Smad5_K rescue mice showed improved glucose tolerance, reversing the glucose tolerance impairment caused by Smad5 knockout. Figure 6 The presence of Smad5 in the cytoplasm suggests that Smad5 may play an important role in metabolic regulation. Further investigation was conducted to examine the effect of Smad5KO-GFP-Smad5_K on insulin sensitivity rescue in mice under normal dietary conditions. The results showed that the AUC of mice rescued by Smad5KO-GFP-Smad5_K recovered to the control group level (ab), indicating that Smad5KO-GFP-Smad5_K restored the AUC of mice to the level of the control group. Figure 6 (cd). The above results indicate that, under normal dietary conditions, cytoplasmic overexpression of Smad5 in mice can rescue insulin resistance impairment caused by Smad5 knockout, suggesting that cytoplasmic Smad5 can reduce insulin resistance in mice.
[0073] Feeding GFP-Smad5 and GFP-Smad5_K mice with a high-fat diet for 2 months resulted in significantly improved insulin tolerance compared to GFP-Smad5 overexpression mice. Figure 7(ab), Smad5 in the cytoplasm can improve the insulin intolerance phenotype induced by a high-fat diet.
[0074] Example 5: The acid-base balance regulating agent can lower blood glucose and alleviate symptoms of diabetic intolerance.
[0075] Experimental methods:
[0076] 1.1 Mouse preparation: 4-6 week old c57 mice were purchased from Slack Company and housed in the SPF-grade breeding room of Tongji University Animal Center.
[0077] 1.2 Baking soda alkaline drinking water model:
[0078] Control group: fed with normal sterilized tap water and a high-fat diet for 7 weeks.
[0079] Experimental group:
[0080] Feed the baby with a high-fat diet and alkaline drinking water solution (0.2M NaHCO3, pH 9.5, 0.5% sugar) for 7 weeks.
[0081] 1.3 Baking soda alkaline drinking water model - determination of baking soda concentration and pH range:
[0082] Control group (commercially available baking soda alkaline drinking water concentration): baking soda alkaline drinking water (2.5mM NaHCO3, pH 7.6, 0.5% sugar) + high-fat diet for 7 weeks.
[0083] Experimental group:
[0084] a. Feed the animal with a baking soda alkaline drinking water (0.025M NaHCO3, pH 7.6, 0.5% sugar) and a high-fat diet for 7 weeks;
[0085] b. Feed the animal with a high-fat diet and alkaline drinking water (0.05M NaHCO3, pH 7.6, 0.5% sugar) for 7 weeks;
[0086] c. Feed the animal with a high-fat diet and alkaline drinking water solution containing baking soda (0.1M NaHCO3, pH 7.6, 0.5% sugar) for 7 weeks.
[0087] d. Feed the animals with a high-fat diet for 7 weeks, using alkaline drinking water containing baking soda (0.15M NaHCO3, pH 7.6, 0.5% sugar);
[0088] e. Feed the child with a high-fat diet and alkaline drinking water solution containing baking soda (0.2M NaHCO3, pH 7.6, 0.5% sugar) for 7 weeks.
[0089] The pH range of baking soda was determined using pH 7.6, pH 8.5, and pH 9.5 (pH 9.5 is defined in Method 1.2). Figure 8 pH 7.6, see Method 1.3 and... Figure 9 pH 8.5 Figure 10 Three pH values were used to test the pH range of baking soda-alkaline drinking water.
[0090] 1.3 Glucose Tolerance Test (GTT): Mice were fasted for 15-18 hours beforehand, weighed, and their tail veins were punctured with a needle to collect blood. Fasting blood glucose levels were measured and recorded using a glucometer. Based on the mice's weight, 2 g / kg of glucose solution was injected intraperitoneally. Blood glucose levels were collected and recorded at 15 min, 30 min, 60 min, 90 min, and 120 min after the injection.
[0091] Experimental results:
[0092] Baking soda buffers acidic substances in the body and can be used to treat kidney disease. C57 wild-type mice were fed a high-fat diet, along with either normal drinking water (Neutral water, NW) or alkaline drinking water (0.2M NaHCO3, pH 9.5, AW). Figure 8 (a) Administering sodium bicarbonate-alkaline drinking water to mice for 50 consecutive days promoted the translocation of GFP-Smad5 into the cytoplasm. Furthermore, administering sodium bicarbonate-alkaline drinking water to mice also promoted high-glucose-induced translocation of GFP-Smad5 into the cytoplasm. Figure 8 These data (b-7c) indicate that sodium bicarbonate alkaline drinking water can improve symptoms of pancreatic islet cell acidification induced by a high-fat diet. Administering sodium bicarbonate alkaline drinking water to mice for 50 consecutive days reduced blood glucose levels following a high-fat diet and improved the high-fat-induced glucose intolerance phenotype. Figure 8 The df) indicates that alkaline drinking water with baking soda can improve blood sugar clearance defects.
[0093] To further investigate the concentration of baking soda in alkaline drinking water and to clarify whether commercially available alkaline drinking water can improve the phenotype of high-fat induced glucose intolerance, mice were given commercially available alkaline drinking water at concentrations of 2.5 mM NaHCO3, pH 7.6, or different concentrations of alkaline drinking water (25 mM, 50 mM, 100 mM, 150 mM, 200 mM NaHCO3, pH 7.6) for 50 consecutive days. The results showed that administering alkaline drinking water containing 50 mM-200 mM NaHCO3 to mice improved the phenotype of high-fat induced glucose intolerance. Therefore, the optimal concentration range for alkaline drinking water is 50 mM to 200 mM NaHCO3.
[0094] Next, the pH range of sodium bicarbonate alkaline drinking water was investigated. Mice were given either normal drinking water (Neutral water, NW) or sodium bicarbonate alkaline drinking water (0.1M, 0.15M, 0.2M NaHCO3, pH 8.5, alkaline water, AW). Figure 10 ), administering mice with alkaline drinking water at pH 8.5 for 50 consecutive days can improve the high-fat induced glucose intolerance phenotype. Figure 10 ); combined Figure 9 The effective baking soda used resulted in an alkaline drinking water pH of 7.6. Figure 8 The effective baking soda used in the alkaline drinking water has a pH of 9.5. Therefore, the pH range of baking soda alkaline drinking water is from pH 7.6 to pH 9.5.
[0095] In summary, the concentration and pH range at which baking soda is effective in making drinking water alkaline are as follows:
[0096] The concentration range of baking soda in alkaline drinking water is: 50mM NaHCO3 to 200mM NaHCO3.
[0097] The pH range for alkaline drinking water made with baking soda is from pH 7.6 to pH 9.5.
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[0117] The above embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. It should be understood that although some embodiments of the invention have been exemplified herein, those skilled in the art will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention. It should also be understood that the terminology used herein is used only to describe particular embodiments and is not intended to be limiting, as the scope of the invention will be defined only by the appended claims and their equivalents.
Claims
1. An Smad5 mutant, characterized in that, The amino acid sequence of the Smad5 mutant is shown in SEQ NO ID:
1.
2. The Smad5 mutant as described in claim 1, characterized in that, The nucleotide sequence encoding the Smad5 mutant is shown in SEQ NO ID:
2.
3. An isolated nucleic acid, characterized in that, Its encoding is the Smad5 mutant as described in claim 1 or 2.
4. The isolated nucleic acid as described in claim 3, characterized in that, The nucleotide sequence of the isolated nucleic acid is shown in SEQ NO ID:
2.
5. A recombinant expression vector, characterized in that, It contains the isolated nucleic acid as described in claim 3 or 4.
6. The recombinant expression vector as described in claim 5, characterized in that, The backbone plasmids of the recombinant expression vector are pET-N-His-C-His and pLVX-Tight-Puro.
7. A transformant, characterized in that, It contains the nucleic acid as described in claim 3 or 4, or the recombinant expression vector as described in claim 5 or 6.
8. The transformant as described in claim 7, characterized in that, The host of the transformant is Escherichia coli or mammalian cells.
9. A method for preparing Smad5 mutants, characterized in that, It includes culturing the transformant as described in claim 7 or 8 to obtain a fermentation product, and obtaining the Smad5 mutant from the fermentation product.
10. The method as described in claim 9, characterized in that, The culture medium used for the culture includes DMEM and 10% fetal bovine serum.
11. A composition comprising the Smad5 mutant as described in claim 1 or 2.
12. The composition according to claim 11, characterized in that, The composition also includes a pharmaceutically acceptable carrier.
13. The use of the Smad5 mutant as described in claim 1 or 2, the isolated nucleic acid as described in claim 3 or 4, the recombinant expression vector as described in claim 5 or 6, or the transformant as described in claim 7 or 8 in the preparation of a medicament for the prevention and / or treatment of diabetes.
14. The application as described in claim 13, characterized in that, The diabetes mellitus mentioned is selected from type 1 diabetes and type 2 diabetes.