Smad5 mutant and uses thereof in prevention and treatment of diabetes

Abstract

A Smad5 mutant and uses thereof in the prevention and treatment of diabetes. The amino acid sequence of the Smad5 mutant is as shown in SEQ NO ID:1. The invention further relates to a nucleic acid encoding the Smad5 mutant, a recombinant expression vector containing the nucleic acid, and a transformant. The uses also comprise uses of the nucleic acid, the recombinant expression vector containing the nucleic acid, and the transformant in the preparation of a medicament for preventing and / or treating diabetes. The Smad5 mutant can improve symptoms of type 1 and type 2 diabetes in vivo, and, compared with conventional oral hypoglycemic drugs, has no toxic or side effects and does not cause damage to the liver or kidneys of patients. In addition, the invention relates to a reagent for regulating acid-base balance, comprising alkaline drinking water containing baking soda; the reagent can lower blood glucose, alleviate diabetic symptoms, and is more economical and convenient for patients to use.

Description

A Smad5 mutant and its application in preventing and treating diabetes This application claims priority to Chinese patent application No. 2024101713283, filed on February 6, 2024. This application incorporates the entire text of the aforementioned Chinese patent application. Technical Field The present invention belongs to the field of biomedicine, and particularly relates to a Smad5 mutant and its application in preventing and treating diabetes. Background Art Diabetes is a group of metabolic diseases characterized by elevated blood sugar levels. In recent years, with the improvement of living standards and changes in dietary structure, the incidence of diabetes has increased year by year and is showing a trend of gradually becoming younger. According to the latest data released by the International Diabetes Federation (IDF), as of 2021, there are approximately 537 million patients worldwide. The research team published the latest diabetes epidemiological survey results 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] . Type 1 diabetes is caused by autoimmune destruction of pancreatic beta cells, resulting in an absolute lack of insulin secretion, while type 2 diabetes is caused by impaired pancreatic function, resulting in a relative lack of insulin secretion or insulin resistance. Abnormal insulin synthesis and secretion are important characteristics of diabetes. The body needs to sense subtle changes in blood sugar and the environment in real time, and then finely regulate 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 us gain a new understanding of the pathogenesis of diabetes and provide new ideas for diabetes prevention and treatment. Pancreatic β cells are very sensitive to changes in the internal and external environment. For example, slight changes in the pH inside and outside the β cells will affect the synthesis and secretion of insulin. [3-5] Therefore, pancreatic β cells need to sense changes in the complex intracellular and extracellular environment and precisely regulate insulin synthesis and secretion. Insulin resistance (IR) refers to the target organs of insulin, including the liver, adipose tissue, skeletal muscle, etc., which are insensitive to the body's secretion of insulin. Reduced insulin sensitivity, a pathological condition [6-8]The liver is one of the most important target organs of insulin. When the liver becomes insulin resistant, the absorption and utilization efficiency of glucose under the action of insulin decreases. To compensate for this decrease in efficiency, the body secretes more insulin, which eventually leads to hyperinsulinemia, decreased liver glycogen synthesis, and increased gluconeogenesis. A large number of research results have shown that hepatic insulin resistance runs through the occurrence and development of type 2 diabetes mellitus (T2DM) and is one of the important pathogenesis of T2DM. The IR mechanism is complex and is the result of the synergistic action of multiple factors. [9-13] Therefore, increasing liver insulin sensitivity and improving insulin resistance are the key to treating T2DM.

[0014] . Currently, the treatment for type 1 diabetes includes insulin injections, insulin pumps, and islet cell transplantation. The treatment for type 2 diabetes includes insulin and oral hypoglycemic drugs such as biguanides, sulfonylureas, thiazolidinediones, and α-glucosidase inhibitors. [15,16] However, these drugs cannot fundamentally cure diabetes and various therapeutic drugs have adverse reactions to varying degrees. Therefore, developing more treatment strategies that can effectively improve insufficient insulin secretion or insulin resistance is an urgent problem to be solved. Summary of the Invention The technical problem to be solved by the present invention is to overcome the lack of therapeutic strategies that can effectively improve insufficient insulin secretion or insulin resistance in the prior art. The present invention provides a Smad5 mutant and its use in preventing and treating diabetes. Furthermore, the present invention provides methods for protecting pancreatic islet cells from damage, improving impaired glucose tolerance, alleviating insulin resistance, lowering blood sugar, and alleviating the symptoms of impaired glucose tolerance in diabetes. The application of Smad5 protein can protect pancreatic islets from damage caused by adverse environmental conditions and prevent the onset of diabetes; the application of Smad5_K protein can alleviate the symptoms of impaired glucose tolerance and insulin resistance in diabetes; and the application of baking soda in drinking water can alleviate the symptoms of impaired glucose tolerance and glucose tolerance in diabetes. These techniques can be used in combination. One of the technical aspects of the present invention is to provide a Smad5 mutant, the amino acid sequence of which is shown in SEQ NO ID: 1. Preferably, the nucleotide sequence encoding the Smad5 mutant is shown in SEQ NO ID: 2. A second technical aspect of the present invention is to provide an isolated nucleic acid encoding the Smad5 mutant as described in the first technical aspect. Preferably, the nucleotide sequence of the isolated nucleic acid is shown in SEQ NO ID: 2. The third technical aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid as described in the second technical aspect. Preferably, the backbone plasmids of the recombinant expression vector are pET-N-His-C-His and pLVX-Tight-Puro. A fourth technical aspect of the present invention provides a transformant comprising the nucleic acid as described in the second technical aspect, or the recombinant expression vector as described in the third technical aspect. Preferably, the host of the transformant is Escherichia coli or mammalian cells. A fifth technical aspect of the present invention provides a method for preparing a Smad5 mutant, comprising culturing the transformant as described in the fourth technical aspect to obtain a fermentation product, and obtaining the Smad5 mutant from the fermentation product. Preferably, the culture medium used in the culture comprises DMEM and 10% fetal bovine serum. A sixth technical aspect of the present invention provides a composition comprising Smad5 and the Smad5 mutant as described in one of the technical aspects. Preferably, the composition further comprises a pharmaceutically acceptable carrier. The seventh technical aspect of the present invention provides the use of Smad5, the Smad5 mutant as described in the first technical aspect, the isolated nucleic acid as described in the second technical aspect, the recombinant expression vector as described in the third technical aspect, the transformant as described in the fourth technical aspect, or the composition as described in the sixth technical aspect in the preparation of a drug for preventing and / or treating diabetes. Preferably, the diabetes is selected from type 1 diabetes and type 2 diabetes. An eighth technical aspect of the present invention is to provide a reagent for regulating acid-base balance, which includes baking soda alkaline drinking water. Preferably, the baking soda alkaline drinking water comprises: 0.05-0.2M NaHCO3, pH 7.6-9.5; and 0.5% sugar. A ninth technical aspect of the present invention provides use of the reagent described in the eighth technical aspect in the preparation of a drug for treating diabetes. Preferably, the diabetes is selected from type 1 diabetes and type 2 diabetes. A tenth technical aspect of the present invention provides a method for protecting pancreatic islet cells, comprising administering Smad5 or the Smad5 mutant according to one of the technical aspects to a subject in need thereof. An eleventh technical aspect of the present invention provides a method for improving impaired glucose tolerance and / or lowering blood sugar, which comprises administering Smad5 or the Smad5 mutant as described in one of the technical aspects to a subject in need thereof. A twelfth technical aspect of the present invention provides a method for alleviating insulin resistance, comprising administering Smad5 or the Smad5 mutant as described in one of the technical aspects to a subject in need thereof. On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention. The reagents and raw materials used in the present invention are commercially available. The positive progress effect of the present invention is: By synthesizing Smad5 and Smad5_K proteins in vitro and injecting them into the body, the drug improves symptoms of type 1 and type 2 diabetes. Because these proteins are already present in the body or promote metabolism, compared to conventional oral hypoglycemic drugs, it has no toxic side effects and does not damage the patient's liver or kidneys. Giving alkaline water containing baking soda to lower blood sugar and alleviate diabetic symptoms is more economical and convenient for patients. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the experimental results of high glucose stimulation causing GFP-Smad5 to translocate from the cell nucleus to the cytoplasm. a represents that high glucose (16.7 mM) stimulation causes an increase in the cytoplasmic pH of pancreatic islet cells, and starvation treatment is performed with a glucose-free buffer (135 mmol NaCl, 3.6 mmol KCl, 0.5 mmol NaH2PO4, 0.5 mmol MgSO4, 1.5 mmol CaCl2, 2.0 mmol NaHCO3, 10 mmol Cells were treated with HEPES (0.1% BSA) for 30 minutes; b shows high glucose (16.7 mM) stimulation within 10-15 minutes, causing GFP-Smad5 to translocate from the nucleus to the cytoplasm. pHe refers to extracellular pH. c shows 2 g / kg glucose stimulation of GFP-Smad5 mice, which causes GFP-Smad5 to translocate from the nucleus to the cytoplasm of pancreatic islet cells. Fasting treatment involves depriving mice of food for 15-18 hours. d shows the statistical analysis of the nuclear and cytoplasmic distribution of GFP-Smad5 in Figure 1c. **p<0.01, ***p<0.001, unpaired two-tailed t-test. Figure 2 shows that pancreatic β-cell-specific knockout of Smad5 causes elevated blood glucose and impaired glucose tolerance in mice. (a) Schematic diagram of pancreatic β-cell-specific knockout of Smad5 in mice, obtained by crossing Smad5 flox mice with INS2-cre mice (The Jackson Laboratory, No. 003573); (b) Western blot verification of Smad5 knockout efficiency; (c) Pancreatic β-cell-specific knockout of Smad5 causes elevated fasting blood glucose in mice; (d) Pancreatic β-cell-specific knockout of Smad5 causes impaired glucose tolerance in mice, where 5 months refers to the age of the mice; (e) Statistical graph of the glucose tolerance curve in Figure 2d; (f) Statistical graph of insulin tolerance in pancreatic β-cell-specific knockout of Smad5 mice; (g) Statistical graph of the insulin tolerance curve in Figure 2f. **p<0.01, ***p<0.001, unpaired two-tailed t-test. Figure 3 shows abnormal insulin processing and secretion in mice caused by pancreatic β-cell-specific knockout of Smad5. (a) Shows decreased C-peptide secretion in mice caused by pancreatic β-cell-specific knockout of Smad5; (b) Shows decreased insulin secretion in mice following fasting and glucose stimulation for 30 minutes caused by pancreatic β-cell-specific knockout of Smad5; (c) Immunofluorescence staining shows increased proinsulin expression in mice caused by Smad5 knockout; (d) Statistical analysis of proinsulin fluorescence density in mice shown in Figure 3c; (e) Western blot analysis shows increased proinsulin expression and decreased insulin expression in mouse pancreatic islets caused by Smad5 knockout; (f) Statistical analysis of the proinsulin / insulin ratio in Figure 3e. **p<0.01, ***p<0.001, unpaired two-tailed t-test. Figure 4 shows the experimental results of GFP-Smad5 overexpression mice resisting STZ-induced hyperglycemia and weight loss. Among them, a is the ratio of normal blood sugar to hyperglycemia in wild-type mice (WT) and GFP-Smad5 overexpression mice (OE) 1 day after streptozotocin (STZ) injection, and bc is the weight change and weight loss of wild-type mice (WT) and GFP-Smad5 overexpression mice (OE) 7 days after hyperglycemia. Ratio. **p<0.01, unpaired two-tailed t-test. Figure 5 shows experimental results demonstrating that mice overexpressing the persistently cytoplasmic mutant GFP-Smad5_K alleviate the high-fat diet-induced impaired glucose tolerance phenotype. (a) High-fat diet induction for two months promotes nuclear accumulation of GFP-Smad5, indicating acidification in pancreatic islet cells in this high-fat-induced model of type 2 diabetes. (b) shows the glucose-induced decrease in the nuclear-cytoplasmic ratio of GFP-Smad5 relative to (a). (c) Changes in glucose tolerance in wild-type, GFP-Smad5-overexpressing, and GFP-Smad5_K-overexpressing mice after two months of high-fat diet induction. (d) Glucose tolerance curves. **p<0.01, ***p<0.001, unpaired two-tailed t-test. Figure 6 shows the experimental results showing that sustained overexpression of the cytoplasmic mutant GFP-Smad5_K can alleviate the insulin resistance phenotype in mice. (a) 24-week-old mice were intraperitoneally administered with glucose solution, and blood glucose levels were measured at different time points (n=5). (b) The area under the curve (AUC) of the three groups of mice during the glucose tolerance test. (c) 24-week-old mice were intraperitoneally administered with insulin solution, and blood glucose levels were measured at different time points (n=9). (d) The area under the curve (AUC) of the three groups of mice during the insulin tolerance test. ***p<0.001, unpaired two-tailed t-test. Figure 7 shows experimental results demonstrating that sustained overexpression of the cytoplasmic mutant GFP-Smad5_K significantly alleviates the insulin resistance phenotype in mice compared to wild-type GFP-Smad5 mice. (a) Changes in insulin tolerance in GFP-Smad5-overexpressing and GFP-Smad5_K-overexpressing mice after two months of high-fat diet. (b) Statistics of insulin tolerance curves. *p<0.05, unpaired two-tailed t-test. Figure 8 shows experimental results demonstrating that baking soda-alkaline drinking water improves the impaired glucose tolerance phenotype induced by a high-fat diet. (a) Schematic diagram of the feeding pattern of neutral water (NW) and baking soda-alkaline drinking water. (b) and (c) Schematic diagram of baking soda-alkaline drinking water promoting GFP-Smad5 intracytoplasmic accumulation, demonstrating that it can improve the high-fat-induced acidification phenotype in pancreatic islets. (d) Schematic diagram of fasting and fed blood glucose levels after 50 days of feeding with either neutral water or baking soda-alkaline drinking water (0.2M NaHCO3, pH 9.5, 0.5% sugar). (e) Schematic diagram of the glucose tolerance curves in Figure 8e. *p<0.05, **p<0.01, ***p<0.001, unpaired two-tailed t-test. Figure 9 shows the experimental results of a range of concentrations of baking soda in alkaline drinking water to improve the impaired glucose tolerance phenotype induced by a high-fat diet. (a) shows the changes in glucose tolerance in mice after 50 consecutive days of drinking water with commercially available baking soda (2.5mM NaHCO3, pH 7.6) or with varying concentrations of baking soda (25mM, 50mM, 100mM, 150mM, and 200mM NaHCO3, pH 7.6). (b) shows the statistical analysis of the glucose tolerance curve in (a). *p < 0.05, unpaired two-tailed t-test. Figure 10 shows experimental results showing the concentration and pH ranges of sodium bicarbonate (BN)-alkaline drinking water that improves high-fat diet-induced impaired glucose tolerance. (a) shows changes in glucose tolerance in mice fed with either normal water or sodium bicarbonate (0.2M NaHCO3, pH 8.5, 0.5% sugar) for 50 days; (b) shows the statistical analysis of the glucose tolerance curve in Figure 10a; (c) shows changes in glucose tolerance in mice fed with either normal water or sodium bicarbonate (0.15M NaHCO3, pH 8.5, 0.5% sugar) for 50 days; (d) shows the statistical analysis of the glucose tolerance curve in Figure 10c; (e) shows changes in glucose tolerance in mice fed with either normal water or sodium bicarbonate (0.1M NaHCO3, pH 8.5, 0.5% sugar) for 50 days; and (f) shows the statistical analysis of the glucose tolerance curve in Figure 10e. *p < 0.05, unpaired two-tailed t-test. DETAILED DESCRIPTION The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications. Example 1 Smad5 regulates insulin synthesis and secretion 1. Experimental methods: 1.1 Isolation of mouse pancreatic islets Mice were killed by cervical dislocation, and the abdomen was disinfected with 75% alcohol. The abdominal cavity was quickly opened under sterile conditions. The common bile duct was found under a stereomicroscope and clamped with hemostats at the entrance of the common bile duct into the duodenum. A section of the common bile duct was carefully separated. Under a stereomicroscope, collagenase P (Roche, Catalog No. 11213857001) was injected into the common bile duct using a 2ml syringe. The pancreas was observed to rapidly expand, and there was no cyst in the abdominal cavity. After the enzyme solution leaks out and the pancreas fully expands, the needle is removed and the entire pancreas is quickly removed with ophthalmic scissors. The pancreas is placed in a 50ml centrifuge tube and digested in a 37°C water bath for 17 minutes. Digestion is terminated with 4°C pre-cooled 1640 culture medium (containing 10% fetal bovine serum) and the pancreatic tissue is thoroughly vibrated. The digestion solution is filtered through a 50-mesh filter to remove the islet tissue mixture. Density gradient centrifugation is performed to obtain a resuspension of islets. Islets are manually picked under a stereomicroscope. Under the microscope, the islets appear as white, round, and uniformly dense granules. This process is repeated 3-6 times until no or only a small amount of impurities are observed under the microscope. 1.2 Determination of intracellular pH of glucose-stimulated pancreatic islet cells The intracellular pH-sensitive probe BCECF-AM (Biyuntian, Catalog No. S1006) was used to measure glucose-stimulated intracellular pH in pancreatic islet cells. Islet cells were treated with 1 μM BCECF-AM for 60 minutes. BCECF-AM was dissolved in Kreb's buffer (135 mmol NaCl, 3.6 mmol KCl, 0.5 mmol NaH2PO4, 0.5 mmol MgSO4, 1.5 mmol CaCl2, 2.0 mmol NaHCO3, 10 mmol HEPES, 0.1% BSA). Excitation at 440 nm and 490 nm was used, and emission at 535 nm was read using a multifunctional microplate reader. The 490 / 440 nm ratio reflects changes in intracellular pH. Intracellular pH standard curves were prepared using Kreb's buffer at pH 6.5, 6.8, 7.0, 7.4, or 7.8 containing 10 μM nigericin (Selleck, Cat. No. 28643-80-3). Referring to Figure 1a, intracellular pH was measured after treatment with 4.5 mM glucose (dissolved in Kreb's buffer). This was followed by a 30-minute pretreatment with Kreb's buffer without glucose. The intracellular pH was then measured over a 5-minute period under starvation conditions. The intracellular pH was then measured after treatment with 2.8 mM or 16.7 mM glucose for 20 minutes (Figure 1a). Emission light at 535 nm was then read using a multi-functional microplate reader, and the 490 / 440 nm ratio, which reflects the intracellular pH change, was measured (Figure 1b). 1.3 High glucose stimulation causes an increase in the pH of pancreatic β cells in vivo In vivo acid-base reporter mice (GFP-Smad5 transgenic mice) were used to indicate changes in the pH of pancreatic β cells. Mice were starved for 15-18 hours and intraperitoneally injected with glucose (2g glucose / kg body weight). 20 minutes later, the pancreas was removed and fixed with paraformaldehyde for 2 hours. Immunofluorescence staining was then used to observe changes in the nuclear and cytoplasmic distribution of GFP-Smad5 in pancreatic β cells (GFP antibody: Aves, GFP-1020). Insulin indicated pancreatic islet cells (insulin antibody: Cell Signaling, #8138). The results are shown in Figure 1. c and d. 1.4 Smad5 flox mouse preparation: Smad5 flox mice were generated in the laboratory. Seven- to eight-week-old C57BL / 6N female mice were used as embryo donors. Superovulation was induced by injection of serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The C57BL / 6N females were then crossed with C57BL / 6N males. Fertilized eggs were collected from the oviducts. 1 μg of Cas9 mRNA (Addgene #41815), 1 μg of sgRNA (sequence CAGCCGTGAAGCGATTGTT, SEQ ID NO: 7), and 5 μg of the Donor-mSmad5 donor plasmid (Donor-mSmad5 vector sequence (SEQ ID NO: 6)) were mixed and microinjected into the cytoplasm of the fertilized eggs. The fertilized eggs were cultured in vitro for 24 hours and then transplanted into the oviducts of pseudopregnant ICR mice. All mice were housed in the SPF-grade animal center of Tongji University. 1.5 Mice with pancreatic β-cell-specific Smad5 knockout (Smad5 cKO) were generated by crossing Smad5flox mice with INS2-cre mice (The Jackson Laboratory, No. 003573) (Figure 2a). Mice without Smad5 knockout served as a control group (Ctrl). Western blot was used to verify the efficiency of Smad5 knockout, as shown in Figure 2b. 1.6 Blood glucose determination in mice: Mice were fasted for 15-18 hours and blood glucose was measured using a Roche blood glucose meter and blood glucose test strips. Glucose tolerance in mice: Mice were fasted for 15-18 hours and intraperitoneally injected with glucose (2g glucose / kg body weight). Blood glucose levels were measured at 0, 15, 30, 60, and 120 minutes. Results show changes in blood glucose and glucose tolerance in knockout mice after 5 months, as shown in Figures 2c-g. 1.7 Determination of insulin and proinsulin secretion in mice: Serum was collected from mice after fasting and glucose stimulation (2 g glucose / kg body weight) for 30 minutes, and detected using an insulin ELISA kit (company: Millipore, product number: EZRMI-13K) and a proinsulin ELISA kit (company: Mercodia, product number: 10-1232-01). 2. Results and Discussion The inventors' previous studies have found that Smad5 can act as a cytoplasmic pH sensor to detect environmental stress and regulate metabolic reactions through nucleoplasmic shuttling to maintain intracellular metabolic homeostasis. [17,18] The inventors have further discovered that under normal circumstances, GFP-Smad5 is located in the nucleus of pancreatic β cells and glucose stimulates Glucose stimulation caused GFP-Smad5 to accumulate in the cytoplasm, indicating that glucose stimulation increases the intracellular pH of pancreatic β cells (Figure 1). To investigate the effects of conditional Smad5 knockout in pancreatic β cells on pancreatic islet function in mice, changes in blood glucose and glucose tolerance were assessed. Smad5 knockout mice exhibited elevated fasting blood glucose and significantly impaired glucose tolerance, indicating that pancreatic β-cell-specific Smad5 knockout reduced the body's ability to process glucose (Figure 2c-g). ELISA assays for serum C-peptide and insulin levels revealed decreased levels. Immunofluorescence staining and Western blot analysis of proinsulin expression in the islets and serum of Smad5 knockout mice revealed increased proinsulin expression, indicating that Smad5 knockout leads to abnormal proinsulin processing and impaired insulin synthesis (Figure 3). These findings suggest that Smad5 plays a crucial role in regulating insulin synthesis and secretion. Example 2 Smad5 protects pancreatic islet cells from damage Experimental methods: 1.1 Mouse preparation: 6-week-old C57 mice weighing approximately 20 g were purchased from Slack, and GFP-Smad5 overexpressing mice were obtained from a laboratory-established model. All mice were housed in an SPF-grade breeding room at the Tongji University Animal Center. 1.2 Intraperitoneal Injection Modeling: Mice were fasted for 24 hours prior to the experiment. Then, a single intraperitoneal injection of streptozotocin (STZ) (120 mg / kg body weight) was administered to destroy pancreatic β cells, creating a type 1 diabetic mouse model. A normal control group received an equal volume of citric acid solution via intraperitoneal injection. Twenty-four hours later, blood glucose was collected via the tail vein for measurement. A blood glucose level above 11.2 mmol / L was considered hyperglycemic. Experimental results: Wild-type mice and mice overexpressing GFP-Smad5 were intraperitoneally injected with streptozotocin (STZ), which can induce pancreatic β-cell toxicity. Seven days after STZ injection, the GFP-Smad5 mice experienced significantly less hyperglycemia and weight loss than the wild-type mice (Figures 4a-c), indicating that overexpression of GFP-Smad5 protects pancreatic β-cells. Example 3 Smad5_K mutant improves impaired glucose tolerance Experimental methods: 1.1 Mouse Preparation: GFP-Smad5_K mutant (SEQ ID NO: 1) overexpressing mice were obtained from the laboratory. Seven- to eight-week-old C57BL / 6N female mice were used as embryo donors. Superovulation was induced by injection of serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) and then crossed with C57BL / 6N male mice. Fertilized eggs were collected from the oviducts. 1 μg of Cas9 mRNA (Addgene #41815), 1 μg of sgRNA (sequence CTGGAGTTGCAGATCACGA), and 5 μg of the GFP-Smad5_K donor plasmid (backbone plasmid: Rosa26-puro-CAG vector (SEQ ID NO: 5)) were mixed and microinjected into the cytoplasm of the zygotes. The zygotes were cultured in vitro for 24 hours and then transplanted into the oviducts of pseudopregnant ICR mice. All mice were housed in an SPF-grade breeding room at the Animal Center of Tongji University. 1.2 Construction of high-fat diet-induced type 2 diabetes model: Wild-type, GFP-Smad5-overexpressing, and GFP-Smad5_K mutant-overexpressing mice aged approximately 8 weeks were fed a high-fat diet (Research Diet, D12492) for 2 months to establish a high-fat diet-induced diabetes model. 1.3 Glucose tolerance test (GTT) detection: Mice were fasted for 15-18 hours in advance, weighed, and the tail vein of the mice was punctured with a needle to collect blood. The fasting blood glucose of the mice was measured and recorded using a blood glucose meter. According to the weight of the mice, 2g / kg glucose solution was injected intraperitoneally, and the blood glucose values were collected and recorded 15min, 30min, 60min, 90min and 120min after the injection of glucose solution. Experimental results: High-fat diet (HFD) treatment can cause phenotypes such as impaired glucose tolerance and insulin resistance in mice. This experiment wanted to know whether Smad5 in the cytoplasm can improve the impaired glucose tolerance phenotype induced by a high-fat diet. High glucose (Glucose, G) stimulation promotes the transfer of GFP-Smad5 to the cytoplasm of pancreatic islet cells of GFP-Smad5 mice, indicating that high glucose stimulation causes intracellular alkalinization of pancreatic islet cells (Figure 5a). Compared with mice fed with standard chow, high glucose stimulation inhibits the transfer of GFP-Smad5 to the cytoplasm of pancreatic islet cells of mice fed with a high-fat diet, indicating that the intracellular acidification of pancreatic islet cells of mice fed with a high-fat diet is caused (Figure 5b). Overexpression of GFP-Smad5_K mice simulates cytoplasmic alkalinization, and it is continuously expressed in the cytoplasm. Feeding overexpression GFP-Smad5_K mice were fed a high-fat diet for 2 months. Compared with control mice and GFP-Smad5 overexpressing mice, the glucose tolerance level of GFP-Smad5_K overexpressing mice was significantly improved ( Figure 5 , cd). Cytoplasmic Smad5_K can improve the abnormal glucose tolerance phenotype induced by a high-fat diet. Smad5_K protein sequence (SEQ ID NO: 1) Smad5_K nucleotide sequence (SEQ ID NO: 2) pET-N-His-C-His vector sequence (SEQ ID NO: 3) pLVX-Tight-Puro vector sequence (SEQ ID NO: 4) Rosa26-puro-CAG vector sequence (SEQ ID NO: 5) Donor-mSmad5 vector sequence (SEQ ID NO: 6) Example 4 Smad5_K mutant alleviates insulin resistance Experimental methods: 1.1 Mouse preparation: GFP-Smad5_K mutant overexpressing mice were obtained from the laboratory model and were housed in the SPF-grade breeding room of the Tongji University Animal Center. 1.2 Glucose tolerance test (GTT) detection: Mice were fasted for 15-18 hours in advance, weighed, and the tail vein of the mice was punctured with a needle to collect blood. The fasting blood glucose of the mice was measured and recorded using a blood glucose meter. According to the weight of the mice, 2g / kg glucose solution was injected intraperitoneally, and the blood glucose values were collected and recorded 15min, 30min, 60min, 90min and 120min after the injection of glucose solution. 1.3 Insulin tolerance test (ITT) detection: Mice were fasted for 15h-18h in advance, weighed, and the tail vein of the mice was punctured with a needle to collect blood. The fasting blood glucose of the mice was measured and recorded with a blood glucose meter. According to the weight of the mice, 0.75U / kg of insulin solution was injected intraperitoneally, and the blood glucose values were collected and recorded 15min, 30min, 60min, 90min and 120min after the injection of the insulin solution. Experimental results: The insulin sensitivity and glucose tolerance of Smad5 KO-GFP-Smad5_K rescued mice were detected. Smad5 KO-GFP-Smad5_K rescued mice can improve glucose tolerance and rescue the insulin sensitivity and glucose tolerance of mice. Smad5 knockout resulted in impaired glucose tolerance (Figure 6, ab), suggesting that cytoplasmic Smad5 may play an important role in metabolic regulation. Further testing examined the ability of Smad5 KO-GFP-Smad5_K to rescue insulin sensitivity in mice under normal dietary conditions. The results showed that the AUC of Smad5 KO-GFP-Smad5_K rescued mice returned to the level of the control group (Figure 6, cd). These results indicate that, under normal dietary conditions, cytoplasmic overexpression of Smad5 can rescue the impaired insulin tolerance caused by Smad5 knockout in mice, indicating that cytoplasmic Smad5 can reduce insulin resistance in mice. GFP-Smad5 and GFP-Smad5_K overexpressing mice were fed a high-fat diet for 2 months. Compared with GFP-Smad5 overexpressing mice, the insulin tolerance level of GFP-Smad5_K overexpressing mice was significantly improved (Figure 7 ab). Cytoplasmic Smad5 can improve the insulin tolerance phenotype induced by a high-fat diet. Example 5: Acid-base balance regulating agent can lower blood sugar and alleviate symptoms of impaired diabetic tolerance Experimental methods: 1.1 Mouse preparation: 4-6 week old C57 mice were purchased from Slack and housed in the SPF-grade breeding room of the Tongji University Animal Center. 1.2 Baking Soda Alkaline Drinking Water Model: Control group: normal sterilized tap water + high-fat diet were fed for 7 weeks. Experimental group: Baking soda alkaline drinking water (0.2M NaHCO3, pH 9.5, 0.5% sugar) + high-fat diet for 7 weeks; 1.3 Baking Soda Alkaline Drinking Water Model - Baking Soda Concentration and pH Range Determination: Control group (commercially available baking soda alkaline drinking water concentration): baking soda alkaline drinking water (2.5 mM NaHCO3, pH 7.6, 0.5% sugar) + high-fat diet for 7 weeks. Experimental group: a. Baking soda alkaline drinking water (0.025M NaHCO3, pH 7.6, 0.5% sugar) plus a high-fat diet for 7 weeks; b. Baking soda alkaline drinking water (0.05M NaHCO3, pH 7.6, 0.5% sugar) + high-fat diet for 7 weeks; c. Baking soda alkaline drinking water (0.1M NaHCO3, pH 7.6, 0.5% sugar) + high-fat diet for 7 weeks; d. Baking soda alkaline drinking water (0.15M NaHCO3, pH 7.6, 0.5% sugar) + high-fat diet for 7 weeks; e. Baking soda alkaline drinking water (0.2M NaHCO3, pH 7.6, 0.5% sugar) + high-fat diet for 7 weeks; For the pH range determination of baking soda, three pH values, pH 7.6, pH 8.5, and pH 9.5 (see method 1.2 and Figure 8 for pH 9.5, method 1.3 and Figure 9 for pH 7.6, and Figure 10 for pH 8.5) were selected to detect the pH range of baking soda alkaline drinking water. 1.3 Glucose tolerance test (GTT) detection: Mice were fasted for 15-18 hours in advance, weighed, and the tail vein of the mice was punctured with a needle to collect blood. The fasting blood glucose of the mice was measured and recorded using a blood glucose meter. According to the weight of the mice, 2g / kg glucose solution was injected intraperitoneally, and the blood glucose values were collected and recorded 15min, 30min, 60min, 90min and 120min after the injection of glucose solution. Experimental results: 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 and simultaneously given normal drinking water (Neutral water, NW) or baking soda alkaline drinking water (0.2M NaHCO3, pH 9.5, alkaline water, AW) (Figure 8a). Giving mice baking soda alkaline drinking water for 50 consecutive days promoted the translocation of GFP-Smad5 to the cytoplasm. In addition, giving mice baking soda alkaline drinking water also promoted the high-glucose-induced translocation of GFP-Smad5 to the cytoplasm (Figure 8bc). These data indicate that baking soda alkaline drinking water can improve the symptoms of pancreatic islet cell acidification induced by a high-fat diet. Giving mice baking soda alkaline drinking water for 50 consecutive days reduced blood glucose levels under high-fat diet-induced feeding and improved the high-fat-induced impaired glucose tolerance phenotype (Figure 8df), indicating that baking soda alkaline drinking water can improve blood glucose clearance defects. In order to further study the concentration of baking soda in alkaline drinking water and to clarify the baking soda sold in the market Does alkaline drinking water improve the impaired glucose tolerance phenotype induced by high-fat diet? Mice were given alkaline drinking water containing a commercially available concentration of baking soda (2.5mM NaHCO3, pH 7.6) or different concentrations of baking soda (25mM, 50mM, 100mM, 150mM, and 200mM NaHCO3, pH 7.6) for 50 consecutive days. The results showed that drinking water containing 50mM-200mM NaHCO3 improved the impaired glucose tolerance phenotype induced by high-fat diet. Therefore, the concentration range of baking soda in alkaline drinking water was: 50mM to 200mM NaHCO3. Next, the pH range of baking soda alkaline drinking water was studied. Mice were given normal drinking water (Neutral water, NW) or baking soda alkaline drinking water (0.1M, 0.15M, 0.2M NaHCO3, pH 8.5, alkaline water, AW) (Figure 10). Giving mice different concentrations of baking soda alkaline drinking water with a pH of 8.5 for 50 consecutive days improved the high-fat-induced impaired glucose tolerance phenotype (Figure 10); combined with the effective baking soda alkaline drinking water pH 7.6 used in Figure 9 and the effective baking soda alkaline drinking water pH 9.5 used in Figure 8, the pH range of baking soda alkaline drinking water is: pH 7.6 to pH 9.5. To sum up, the concentration and pH range in which baking soda alkaline drinking water works are: The concentration range of baking soda alkaline drinking water is: 50mM NaHCO3 to 200mM NaHCO3 The pH range of baking soda alkaline drinking water is: pH 7.6 to pH 9.5 References: 1.Li,Y.,et al.,Prevalence of diabetes recorded in mainland China using 2018diagnostic criteria from the American Diabetes Association:national cross sectional study.BMJ,2020.369:p.m997. 2.Tokarz,V.L.,P.E.MacDonald,and A.Klip,The cell biology of systemic insulin function.J Cell Biol,2018.217(7):p.2273-2289. 3.Rocheleau,J.V.,et al.,Pancreatic islet beta-cells transiently metabolize pyruvate.J Biol Chem,2002.277(34):p.30914-20. 4.Haythorne,E.,et al.,Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic beta-cells.Nat Commun,2019.10(1):p.2474. 5.Tsalamandris,S.,et al.,The Role of Inflammation in Diabetes:Current Concepts and Future Perspectives.Eur Cardiol,2019.14(1):p.50-59. 6.DeFronzo,R.A.and D.Tripathy,Skeletal Muscle Insulin Resistance Is the Primary Defect in Type 2Diabetes.Diabetes Care,2009.32:p.S157-S163. 7.Muoio,D.M.and C.B.Newgard,Molecular and metabolic mechanisms of insulin resistance andβ-cell failure in type 2 diabetes.Nature Reviews Molecular Cell Biology,2008.9(3):p.193-205. 8.Muoio,D.M.,et al.,Metabolic Mechanisms of Muscle Insulin Resistance.Type 2 Diabetes Mellitus:An Evidence-Based Approach to Practical Management,2008:p.35-47. 9.Basu,R.,et al.,Obesity and type 2 diabetes impair insulin-induced suppression of glycogenolysis as well as gluconeogenesis.Diabetes,2005.54(7):p.1942-1948. 10.Krssak,M.,et al.,Alterations in postprandial hepatic glycogen metabolismin type 2diabetes.Diabetes,2004.53(12):p.3048-3056. 11.Lewis,G.F.,et al.,Direct and indirect control of hepatic glucose production by insulin.Cell Metabolism,2021.33(4):p.709-720. 12.Michael,M.D.,et al.,Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction.Molecular Cell,2000.6(1):p.87-97. 13.Dong,X.C.,et al.,Irs1 and Irs2 signaling is essential for hepatic glucose homeostasis and systemic growth.Journal of Clinical Investigation,2006.116(1):p.101-114. 14.Zimmet,P.,K.G.M.M.Alberti,and J.Shaw,Global and societal implications of the diabetes epidemic.Nature,2001.414(6865):p.782-787. 15.Kupfer,S.,et al.,Glycemic efficacy of concomitant administration of pioglitazone with the extended-release metformin fortamet.Diabetes,2007.56:p.A565-A565. 16.Meng,G.,et al.,Synthetic optimization of rosiglitazone and related intermediates for industrial purposes.Research on Chemical Intermediates,2016.42(3):p.2023-2033. 17.Fang,Y.,et al.,Smad5 acts as an intracellular pH messenger and maintains bioenergetic homeostasis.Cell Res,2017.27(9):p.1083-1099. 18.Orlowski,J.,SMAD5 signaling:more than meets the nuclei.Cell Res,2017.27(9):p.1075-1076. The above examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. It should be understood that although some embodiments of the present invention are illustrated herein, according to this disclosure, those skilled in the art will recognize that many modifications can be made without departing from the spirit and intended scope of the present invention. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and their equivalents.

Claims

1. A Smad5 mutant, characterized in that: The amino acid sequence of the Smad5 mutant is shown in SEQ NO ID:

1.

2. The Smad5 mutant according to claim 1, wherein The nucleotide sequence encoding the Smad5 mutant is shown in SEQ NO ID:

2.

3. An isolated nucleic acid, characterized in that It encodes the Smad5 mutant according to claim 1 or 2; preferably, the nucleotide sequence of the isolated nucleic acid is shown in SEQ NO ID:

2.

4. A recombinant expression vector, characterized in that: It comprises the isolated nucleic acid according to claim 3; preferably, the backbone plasmids of the recombinant expression vector are pET-N-His-C-His and pLVX-Tight-Puro.

5. A transformant, characterized in that It comprises the nucleic acid according to claim 3, or the recombinant expression vector according to claim 4; preferably, the host of the transformant is Escherichia coli or mammalian cells.

6. A method for preparing a Smad5 mutant, characterized in that: The method comprises culturing the transformant according to claim 5 to obtain a fermentation product, and obtaining the Smad5 mutant from the fermentation product; preferably, the culture medium used in the culture comprises DMEM and 10% fetal bovine serum.

7. A composition comprising Smad5 and the Smad5 mutant according to claim 1 or 2; preferably, the composition further comprises a pharmaceutically acceptable carrier.

8. Use of Smad5, the Smad5 mutant according to claim 1 or 2, the isolated nucleic acid according to claim 3, the recombinant expression vector according to claim 4, or the transformant according to claim 5 in the preparation of a medicament for preventing and / or treating diabetes; preferably, the diabetes is selected from type 1 diabetes and type 2 diabetes.

9. A reagent for regulating acid-base balance, characterized in that It includes baking soda alkaline drinking water.

10. The reagent according to claim 9, characterized in that The baking soda alkaline drinking water comprises: 0.05-0.2M NaHCO3, pH 7.6-9.5; and 0.5% sugar.

11. Use of the reagent according to claim 9 or 10 in the preparation of a medicament for treating diabetes; preferably, the diabetes is selected from type 1 diabetes and type 2 diabetes.

12. A method for protecting pancreatic islet cells, characterized in that: Smad5 or the Smad5 mutant of claim 1 or 2 is administered to a subject in need thereof.

13. A method for improving impaired glucose tolerance and / or lowering blood sugar, characterized in that: Smad5 or the Smad5 mutant of claim 1 or 2 is administered to a subject in need thereof.

14. A method for alleviating insulin resistance, characterized in that: Smad5 or the Smad5 mutant of claim 1 or 2 is administered to a subject in need thereof.

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