Application of morchella polysaccharide nano-selenium in preparation of preparation for improving insulin resistance

Abstract

The invention discloses an application of morchella polysaccharide nano-selenium in preparation of a preparation for improving insulin resistance. The morchella polysaccharide nano-selenium is MSP4-SeNPs, the particle size of the morchella polysaccharide nano-selenium is 67.45 + / -0.52 nm, and the selenium content of the morchella polysaccharide nano-selenium is 52.6 + / -3.21 mg / kg; cell experiments prove that the improvement effect of the MSP4-SeNPs with the concentration of 6.25 mu g / mL on cell insulin resistance is equivalent to the improvement effect of a metformin solution with the concentration of 100 mu g / mL, and the MSP4-SeNPs can be applied to preparation of a preparation for improving insulin resistance. Animal experiments prove that the MSP4-SeNPs with the concentration of 4 mg / kg has the treatment effect on glucose metabolism disorder syndrome or metabolic syndrome caused by insulin resistance equivalent to that of a metformin solution with the concentration of 100 mg / kg, can be particularly used for treating type II diabetes mellitus, and has the advantages of low dosage and no side effect compared with the existing medicine metformin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, more specifically, relates to the application of a Morchella polysaccharide nano-selenium in the preparation of a preparation for improving insulin resistance. BACKGROUND

[0002] Insulin resistance is the core pathological link of type II diabetes, hypertension, metabolic syndrome and other diseases, and improving insulin resistance is expected to improve or treat some diseases related to insulin resistance. The preparation for improving insulin resistance aims to improve insulin resistance by directly or indirectly enhancing the sensitivity of the body to insulin, and the insulin sensitizer can directly enhance the sensitivity of the insulin signaling pathway or target tissue to insulin.

[0003] As a class of drugs for improving metabolic disorders by enhancing insulin sensitivity, the mechanism of action and clinical application of insulin sensitizer has been verified in many studies. For example, insulin sensitizer can reduce hepatic gluconeogenesis and glucose output, thereby reducing fasting and postprandial blood glucose levels; by reducing free fatty acid and triglyceride levels, increasing high-density lipoprotein, and improving dyslipidemia; by inhibiting atherosclerosis, improving vascular endothelial function, reducing inflammation, and reducing the risk of cardiovascular events. It is clinically suitable for type II diabetes, especially for insulin-resistant type II diabetes patients, and can also be used for polycystic ovary syndrome and non-alcoholic fatty liver disease and other metabolic syndrome-related diseases.

[0004] The main types of existing insulin sensitizer include biguanides, thiazolidinediones, GLP-1 receptor agonists and SGLT2 inhibitors. Among them, biguanides (such as metformin) can improve insulin resistance by inhibiting hepatic glucose output, increasing peripheral tissue glucose uptake and utilization, and are suitable for obese type II diabetes patients, especially those with hypertension and dyslipidemia; thiazolidinediones (such as rosiglitazone, pioglitazone) can enhance the sensitivity of adipocytes to insulin by activating PPAR-γ receptors and inhibiting hepatic glucose production; GLP-1 receptor agonists (such as liraglutide, semaglutide) can improve glycemic control by promoting insulin secretion, inhibiting glucagon release and delaying gastric emptying; SGLT2 inhibitors (such as empagliflozin, dapagliflozin) inhibit renal glucose reabsorption, promote urinary glucose excretion, and indirectly improve insulin sensitivity.

[0005] However, these insulin sensitizers also have certain use contraindications and side effects. For example, biguanides need to start from a small dose, monitor kidney function, and avoid the risk of lactic acidosis; thiazolidinediones are contraindicated in patients with severe heart dysfunction and liver dysfunction; GLP-1 receptor agonists can cause gastrointestinal reactions such as nausea and vomiting; and SGLT2 inhibitors need to pay attention to the risk of urogenital system infection. Therefore, it is more beneficial to improve metabolic disorders and achieve the treatment of type II diabetes and metabolic syndrome to study a preparation for improving insulin resistance with a wide range of applications and no side effects. SUMMARY

[0006] In view of the above defects or improvement needs of the prior art, the present application provides an application of a morel polysaccharide nano selenium in the preparation of a preparation for improving insulin resistance, which aims to find that the morel polysaccharide nano selenium can significantly improve the insulin resistance state of cells and can be applied to the preparation of a preparation for improving insulin resistance, thereby solving the technical problem that the existing preparations for improving insulin resistance have certain side effects.

[0007] To achieve the above-mentioned purpose, according to one aspect of the present application, an application of morel polysaccharide nano selenium is provided, which is MSP4-SeNPs and is applied to the preparation of a preparation for improving insulin resistance. In some embodiments, the morel polysaccharide nano selenium is applied to the preparation of a preparation for improving the insulin resistance of cells, and the concentration of the morel polysaccharide nano selenium is 1.5-25 μg / mL or 1.5-25 μg / mg; preferably, the concentration of the morel polysaccharide nano selenium is 1.5-6.25 μg / mL or 1.5-6.25 μg / mg, and more preferably, the concentration of the morel polysaccharide nano selenium is 6.25 μg / mL or 6.25 μg / mg.

[0008] Preferably, the application is that the morel polysaccharide nano selenium is applied to the preparation of an insulin sensitizer.

[0009] Preferably, the application is that the morel polysaccharide nano selenium is applied to the preparation of a drug for improving or treating metabolic disorder-related diseases caused by insulin resistance.

[0010] Preferably, the application is that the morel polysaccharide nano selenium is applied to the preparation of a drug for treating glucose metabolism disorder syndrome caused by insulin resistance.

[0011] Preferably, the application is that the morel polysaccharide nano selenium is applied to the preparation of a drug for treating type II diabetes caused by insulin resistance. In some embodiments for treating type II diabetes caused by insulin resistance, the mass concentration of the morel polysaccharide nano selenium is 0.08-0.4 mg / kg or 0.08-0.4 mg / L.

[0012] Preferably, the application, the Morchella polysaccharide nano selenium is applied to prepare a medicine for improving insulin resistance of metabolic syndrome. In some embodiments for improving insulin resistance of metabolic syndrome, the mass concentration of the Morchella polysaccharide nano selenium is 0.08-0.4 mg / kg or 0.08-0.4 mg / L.

[0013] Preferably, the application, the Morchella polysaccharide nano selenium is a spherical MSP4-SeNPs complex, the average particle size of which is 67-68 nm, and the selenium content is 49-56 mg / L, and in some embodiments, the average particle size is 67.45±0.52 nm, and the selenium content is 52.6±3.21 mg / kg.

[0014] Preferably, the application, the Morchella polysaccharide MSP4 is composed of mannose, glucose and galactose, and the molecular weight is 2.98×10 5 The α-type polysaccharide is an α-type polysaccharide with 1,4-glycosidic bond as the main chain and branches at O-6, O-3 and O-2.

[0015] Preferably, the application, the MSP4-SeNPs complex is prepared by the following method:

[0016] Sodium selenite is used as a selenium source, ascorbic acid is used as a reducing agent, and Morchella polysaccharide MSP4 is used as a stabilizing agent. The Morchella polysaccharide MSP4 solution and the mixed solution of ascorbic acid-sodium selenite are mixed in a mass ratio of 1:1, and the reaction is stirred at 38°C for 2h. SeO3 2- is reduced to nano selenium SeNPs in the reaction solution, and the reduced nano selenium SeNPs interact with O-H and N-H of the Morchella polysaccharide MSP4 to form a stable spherical structure, thereby obtaining the MSP4-SeNPs complex. The concentration of MSP4 in the Morchella polysaccharide solution is 7 mg / mL, and the molar ratio of ascorbic acid to sodium selenite in the mixed solution of ascorbic acid-sodium selenite is 2:1.

[0017] Overall, compared with the prior art, the above technical solutions conceived by the present application can achieve the following beneficial effects:

[0018] The present application discloses a method for improving insulin resistance by using Morel polysaccharide nano selenium (MSP4-SeNPs). The method comprises the following steps: preparing a Morel polysaccharide nano selenium (MSP4-SeNPs) solution; and adding the Morel polysaccharide nano selenium (MSP4-SeNPs) solution into a cell culture medium to improve insulin resistance. The present application finds that the Morel polysaccharide nano selenium (MSP4-SeNPs) can significantly improve the insulin resistance of cells, and can be applied to the preparation of a preparation for improving insulin resistance. The cell experiment proves that the improvement effect of the Morel polysaccharide nano selenium (MSP4-SeNPs) with a concentration of 6.25 μg / mL on the insulin resistance of cells is equivalent to that of metformin with a concentration of 100 μg / mL. In addition, the animal experiment proves that the treatment effect of the Morel polysaccharide nano selenium (MSP4-SeNPs) with a concentration of 4 mg / kg on the glucose metabolism disorder syndrome or metabolic syndrome caused by insulin resistance is equivalent to that of the metformin solution with a concentration of 100 mg / kg, and the Morel polysaccharide nano selenium (MSP4-SeNPs) can be used for treating type II diabetes, and has a low dosage and no side effects compared with the existing drug metformin. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a purification elution curve of Morel polysaccharide (MSP);

[0020] Figure 2 is a transmission electron microscope image of Morel polysaccharide nano selenium (MSP4-SeNPs);

[0021] Figure 3 is a Fourier infrared spectrum and ultraviolet-visible spectrum diagram of MSP, SeNOs and MSP4-SeNPs, Figure 3 wherein A is a Fourier infrared spectrum diagram, and B is an ultraviolet-visible spectrum diagram;

[0022] Figure 4 is an effect of MSP4-SeNPs on cell activity of HepG2 and IR-HepG2 cells and an effect of insulin concentration and action time on an insulin resistance model;

[0023] Figure 5 is an effect of MSP-4-SeNPs on glucose metabolism of IR-HepG2 cells, wherein A is an effect of MSP-4-SeNPs on glucose uptake of IR-HepG2 cells, B is an effect of MSP-4-SeNPs on glycogen content of IR-HepG2 cells, C is an effect of MSP-4-SeNPs on hexokinase (HK) content of IR-HepG2 cells, and D is an effect of MSP-4-SeNPs on pyruvate kinase (PK) content of IR-HepG2 cells;

[0024] Figure 6Figure 7 is the effect of MSP-4-SeNPs on the antioxidant indexes of IR-HepG2 cells, wherein A is the effect of MSP-4-SeNPs on the content of malondialdehyde (MDA) in IR-HepG2 cells, B is the effect of MSP-4-SeNPs on the content of triglyceride (TG) in IR-HepG2 cells, C is the effect of MSP-4-SeNPs on the content of superoxide dismutase (SOD) in IR-HepG2 cells, and D is the effect of MSP-4-SeNPs on the content of glutathione peroxidase (GSH-Px) in IR-HepG2 cells;

[0025] Figure 7 Figure 8 is the effect of MSP4-SeNPs on the body weight of healthy mice;

[0026] Figure 8 Figure 9 is the effect of MSP4-SeNPs on the food intake of healthy mice;

[0027] Figure 9 Figure 10 is the pathological tissue sections of the heart, liver and kidney of mice after intragastrical administration of MSP4-SeNPs;

[0028] Figure 10 Figure 11 is the biochemical indexes of the liver and kidney of mice after intragastrical administration of MSP4-SeNPs, wherein A is the effect of MSP4-SeNPs on the HK content of the liver of mice, B is the effect of MSP4-SeNPs on the PK content of the liver of mice, C is the effect of MSP4-SeNPs on the glycogen content of the liver of mice, D is the effect of MSP4-SeNPs on the HK content of the kidney of mice, and E is the effect of MSP4-SeNPs on the PK content of the kidney of mice;

[0029] Figure 11 Figure 12 is the effect of MSP4-SeNPs on the body weight of model mice;

[0030] Figure 12 Figure 13 is the effect of MSP4-SeNPs on the food intake and water intake of model mice (after intragastrical administration for 4 weeks);

[0031] Figure 13 Figure 14 is the effect of MSP4-SeNPs on the fasting blood glucose (FBG) of model mice;

[0032] Figure 14 Figure 15 is the effect of MSP4-SeNPs on the glucose tolerance (OGTT) and the area under the blood glucose curve (AUS) of model mice;

[0033] Figure 15Fig. 4 is the influence of MSP4-SeNPs on insulin and HOMA-IR index of model mice, wherein Fig. 4A is the influence of MSP4-SeNPs on insulin content of model mice, and Fig. 4B is the influence of MSP4-SeNPs on HOMA-IR index of model mice;

[0034] Figure 16 Fig. 5 is the influence of MSP4-SeNPs on serum biochemical indexes of model mice, wherein Figs. 5A to 5D are the influence of MSP4-SeNPs on serum GLU, GSP, TG and TP contents of model mice, Figs. 5E to 5G are the influence of MSP4-SeNPs on serum ALP, AST and ALT activities of model mice, and Fig. 5H is the influence of MSP4-SeNPs on serum UA content of model mice;

[0035] Figure 17 Fig. 6 is the influence of MSP4-SeNPs on liver biochemical indexes of model mice, wherein Fig. 6A is the liver glycogen content of mice in different groups, Fig. 6B is the total protein (TP) content of livers of mice in different groups, Fig. 6C is the MDA content in livers of mice in different groups, Fig. 6D is the GSH-Px activity in livers of mice in different groups, Fig. 6E is the SOD activity in livers of mice in different groups, Fig. 6F is the HK activity in livers of mice in different groups, and Fig. 6G is the PK activity in livers of mice in different groups. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application in combination with the accompanying drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as there is no conflict.

[0037] The present application is based on the preparation of Morel polysaccharide nano selenium (MSP4-SeNPs) from extracted Morel polysaccharide, and the construction of an insulin resistance cell model (IR-HepG2) to study the influence of MSP4-SeNPs on cell insulin resistance. The results show that Morel polysaccharide nano selenium can significantly improve the insulin resistance state of cells, and the cell experiment results unexpectedly found that 6.25 μg / mL of MSP4-SeNPs has the same effect on improving cell insulin resistance as 100 μg / mL of metformin. Therefore, the present Morel polysaccharide nano selenium can be used to prepare a preparation for improving insulin resistance.

[0038] Insulin resistance is the core pathological link of diseases such as type II diabetes, hypertension and metabolic syndrome. We further studied the therapeutic effect of Morel polysaccharide nano selenium on metabolic disorder-related diseases caused by insulin resistance, and the in vivo animal model experiment proved that Morel polysaccharide nano selenium can be used to treat type II diabetes caused by insulin resistance and improve metabolic syndrome caused by insulin resistance.

[0039] Based on this discovery, the present application proposes an application of Morel polysaccharide nano selenium in preparing a preparation for improving insulin resistance. In some embodiments, for improving cell insulin resistance, the concentration of the Morel polysaccharide nano selenium is 1.5-25 μg / mL or 1.5-25 μg / mg, preferably the concentration of the Morel polysaccharide nano selenium is 1.5-6.25 μg / mL or 1.5-6.25 μg / mg, more preferably the concentration of the Morel polysaccharide nano selenium is 6.25 μg / mL or 6.25 μg / mg, and the improvement effect of the Morel polysaccharide nano selenium on cell insulin resistance is equivalent to that of a metformin solution with a concentration of 100 μg / mL.

[0040] In some embodiments, the Morel polysaccharide nano selenium is applied in preparing an insulin sensitizer; preferably, the Morel polysaccharide nano selenium is applied in preparing a drug for improving or treating a metabolic disorder related disease caused by insulin resistance.

[0041] In some embodiments, the Morel polysaccharide nano selenium is applied in preparing a drug for treating a glucose metabolism disorder syndrome caused by insulin resistance, especially a drug for treating type II diabetes caused by insulin resistance or a drug for improving metabolic syndrome caused by insulin resistance. In some embodiments, for treating type II diabetes caused by insulin resistance or improving metabolic syndrome caused by insulin resistance, the mass concentration of the Morel polysaccharide nano selenium is 0.08-0.4 mg / kg or 0.08-0.4 mg / L, preferably the concentration of the Morel polysaccharide nano selenium is 0.324 mg / kg, and the hypoglycemic effect of the Morel polysaccharide nano selenium on type II diabetes caused by insulin resistance is equivalent to that of a metformin solution with a concentration of 100 mg / kg.

[0042] The Morel polysaccharide nano selenium is a spherical MSP4-SeNPs complex, the average particle size of which is 67-68 nm, and the selenium content is 49-56 mg / L. The smaller the particle size of the Morel polysaccharide nano selenium, the greater the activity thereof. It is surprisingly found that in animal experiments, the treatment effect of a MSP4-SeNPs solution with a concentration of 4 mg / kg on type II diabetes is equivalent to that of a metformin solution with a concentration of 100 mg / kg.

[0043] The Morel polysaccharide MSP4 is an α-type polysaccharide with a molecular weight of 2.98 x 10 5 The α-type polysaccharide is an α-type polysaccharide with a 1,4-glycosidic bond as a main chain and branches at O-6, O-3 and O-2.

[0044] In some embodiments, the MSP4-SeNPs complex is prepared by the following method:

[0045] The sodium selenite is used as the selenium source, the ascorbic acid is used as the reducing agent, the Morchella polysaccharide MSP4 is used as the stabilizer, the Morchella polysaccharide MSP4 solution is mixed with the mixed solution of ascorbic acid-sodium selenite according to the mass ratio of 1:1, and the mixed solution is stirred at 38 DEG C for 2h, so that SeO3 2- is reduced to nano selenium SeNPs in the reaction liquid, the reduced nano selenium SeNPs interact with O-H and N-H of the Morchella polysaccharide MSP4 to form a stable spherical structure, and the MSP4-SeNPs complex is obtained; the concentration of MSP4 in the Morchella polysaccharide solution is 7 mg / mL, and the molar ratio of ascorbic acid to sodium selenite in the mixed solution of ascorbic acid-sodium selenite is 2:1.

[0046] The following is an example

[0047] The Morchella polysaccharide nano selenium is prepared by a chemical reduction method, ascorbic acid (VC) is used as a reducing agent, sodium selenite (Na2SeO3) is used as a selenium donor, and Morchella polysaccharide is used as a stabilizer. The Morchella polysaccharide is extracted from Morchella by a water extraction and alcohol precipitation method.

[0048] Example 1 Preparation of Morchella polysaccharide nano selenium

[0049] (1) Extraction and purification of Morchella polysaccharide (MSP-4)

[0050] The Morchella polysaccharide (MSP) is extracted by a water extraction and alcohol precipitation method, the DEAE-52 cellulose filler is activated, and the filler is soaked in 0.1 mol / L hydrochloric acid and 0.1 mol / L sodium hydroxide respectively, then filtered with deionized water to neutral, and then packed into a column. 0.5 g of Morchella polysaccharide (MSP) sample is dissolved in an appropriate amount of water and filled into a purification column, and the flow rate is adjusted to 1.0 mL / min. Distilled water, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L and 0.4 mol / L NaCl solutions are used for gradient elution respectively. The eluate is collected in test tubes and the tube number is recorded. The absorbance of each tube solution is measured by the sulfuric acid phenol method, and the elution curve is drawn according to the tube number and absorbance as the horizontal and vertical coordinates, as shown in Figure 1 The eluate under the main elution peak is combined, concentrated and freeze-dried to obtain the purified Morchella polysaccharide.

[0051] The Smartarose CL-4B gel filler is packed into a column, and the operation is the same as above except that the filler does not need to be activated. After the eluate is combined, concentrated and freeze-dried, the purified Morchella polysaccharide MSP-4 is obtained. The elution curve of the Morchella polysaccharide is drawn with the tube number as the horizontal coordinate and the absorbance as the vertical coordinate. The main peak appears at 0.2 mol / L NaCl eluent, the eluate is combined and freeze-dried to obtain the Morchella polysaccharide, which is named MSP-4.

[0052] MSP-4 is an alpha-type polysaccharide consisting of mannose, glucose and galactose, with 1,4-glycosidic bond as the main chain and multiple branches, and the molecular weight of MSP-4 is 2.98 x 10 5 Da, with 1,4-glycosidic bond as the main chain, branching at O-6, O-3 and O-2, and the structure is as follows:

[0053]

[0054] (2) Preparation of Morel polysaccharide nano selenium

[0055] We optimized the preparation process by single factor experiment response surface analysis (reaction time, temperature, Vc / Na2SeO3 molar ratio, MSP-4 concentration), as follows:

[0056] A certain amount of Morel polysaccharide MSP-4 powder was taken in a beaker, ultrapure water was added, and a glass rod was used to stir and dissolve uniformly to prepare a Morel polysaccharide solution (MSP-4 concentration of 1 mg / mL, 3 mg / mL, 5 mg / mL, 7 mg / mL, 9 mg / mL). A Na2SeO3 solution with a concentration of 20 mmol / L was prepared, and Vc was added to the Na2SeO3 solution to make the molar ratio of Vc:Na2SeO3 in the solution gradient at 2:1, 3:1, 4:1, 5:1, 6:1, and a sodium selenite-ascorbic acid mixed solution was prepared. The sodium selenite-ascorbic acid mixed solution was mixed with the Morel polysaccharide (MSP-4) solution at a mass ratio of 1:1, and was stirred uniformly and then transferred to a magnetic stirring water bath. By precisely controlling the reaction temperature and time, four times the volume of anhydrous ethanol was added after the reaction, and the precipitate was collected and dissolved in deionized water, and was dialyzed with a dialysis bag and then freeze-dried to obtain the MSP4-SeNPs complex.

[0057] Generally, the smaller the size of polysaccharide nano selenium particles, the more stable they are. The optimal conditions were determined as follows: MSP-4 concentration of 7 mg / mL, Vc / Na2SeO3 molar ratio of 2:1, and reaction at 38°C for 2 hours. Under these conditions, the MSP4-SeNPs obtained had the smallest particle size, were uniform spherical, and the particle size was 67.20 ± 0.20 nm, and the selenium content was 52.6 ± 3.21 mg / L. The transmission electron micrograph of Morel polysaccharide nano selenium (MSP4-SeNPs) is shown in Figure 2 The Fourier infrared spectrum and ultraviolet-visible spectrum of MSP, SeNOs and MSP4-SeNPs are shown in Figure 3 .

[0058] The range of hydroxyl (O-H) characteristic peak is 3650 cm -1 -3000 cm -1 , Figure 3 The middle hydroxyl is from 3403 cm-1 Shift to 3381 cm -1 , indicating the existence of stretching vibration phenomenon, which proves the hydrogen bond interaction between MSP and MSP-SeNPs. The imine group (N-H) absorption peak ranges from 1650 cm -1 -1500 cm -1 , Figure 3 The imine group (N-H) in the middle of the range from 1639 cm -1 to 1647 cm -1 , indicating that selenium interacts with the imine group. The peak spectrum of SeNPs is quite different, especially the appearance of new absorption peaks at 1731 cm -1 and 1406 cm -1 . Therefore, the structural characterization shows that SeNPs may interact with O-H and N-H of MSP to form a stable structure. In the preparation of Morel polysaccharide nano selenium, sodium selenite is reduced to elemental selenium, and with the progress of the reaction, selenium particles gradually aggregate and are strongly adsorbed by Morel polysaccharide molecules to form a stable spherical structure.

[0059] Example 2 Safety evaluation of Morel polysaccharide nano selenium

[0060] MSP4-SeNPs was prepared according to the preparation method of Example 1, and CCK-8 method was used to evaluate the effect of MSP4-SeNPs on the cell viability of IR-HepG2. The specific experimental method is as follows:

[0061] 2.1 Culture of HepG2 cells

[0062] (1) Resuscitation of HepG2 cells: Take the frozen HepG2 cell tube out of the liquid nitrogen and immediately put it into a 37℃ water bath, gently shake the frozen tube for about 1 minute to make it evenly heated until the ice crystals completely melt. Transfer the melted cell suspension to a 15mL centrifuge tube, slowly add 3mL of preheated complete culture medium, gently blow and mix evenly, then seal. Use the centrifuge to centrifuge at 1000rpm for 3 minutes, carefully discard the supernatant and reserve the cell pellet. Add 5mL of fresh culture medium to the centrifuge tube, gently blow to form a uniform cell suspension, and transfer it to a T25 culture bottle. Shake the culture bottle by cross method to make the cells evenly distributed. Place the culture bottle in a 37℃, 5% CO2 incubator and incubate. When the culture medium turns yellow, replace it in time. Monitor the cell morphology and adhesion by inverted microscope every day.

[0063] (2) HepG2 cell passage: remove the cell bottle full of about 80-90% cells from the incubator, observe under the inverted microscope: whether the cell density reaches the passage standard, whether the cell morphology is normal and whether there are signs of pollution (turbidity, black spots and other abnormalities). When the cell density reaches 80%-90% and above, perform 1:3 ratio cell passage operation. Remove the culture solution, wash the culture bottle with 2 mL PBS buffer for 2-3 times to completely remove the residual cell debris and dead cells. Then, remove the PBS solution, add 2 mL trypsin-EDTA digestion solution, and quickly place it in the CO2 cell incubator for reaction for 2-3 minutes. Add 2-3 mL complete medium to terminate digestion, and transfer the cell suspension to a 10 mL centrifuge tube and seal. Use the centrifuge to centrifuge at 1000 rpm for 3 minutes, and discard the supernatant. Add 3 mL of fresh culture medium, mix gently, and make a uniform cell suspension. Then take 1 mL of cell suspension and inoculate into a T25 culture bottle pre-added with an appropriate amount of complete culture medium. Use cross oscillation method to make the cells evenly distributed, and then place it in a 37°C, 5% CO2 incubator for culture.

[0064] 2.2 Effect of MSP4-SeNPs on HepG2 cell viability

[0065] DMEM high glucose medium as the solvent, MSP4-SeNPs powder was sequentially gradient diluted, prepared into MSP4-SeNPs solution with concentration of 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL. The experimental groups are as follows:

[0066] (1) Experimental group: HepG2 cells + different concentrations of MSP4-SeNPs solution (1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL);

[0067] (2) Control group: HepG2 cells + complete medium (DMEM medium containing 10% bovine serum and 1% penicillin-streptomycin);

[0068] (3) Blank group: only contains DMEM high glucose medium (no cells).

[0069] Each group sets 6 replicate wells to reduce experimental error. Adjust the log phase of HepG2 cells to 1 x 10 6The cells were seeded in 96-well plates at a density of 5000 cells / mL in 100 μL / well. After 24 h incubation at 37 °C in 5% CO2, the supernatant was removed after the cells were adhered. In the experimental group, 100 μL of different concentrations of MSP4-SeNPs solution (1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL) was added to each well. In the control group, 100 μL of complete medium was added to each well. After 24 h of continuous culture, the supernatant was removed and washed twice with PBS buffer. 100 μL of DMEM high glucose medium and 10 μL of CCK-8 solution were added to the experimental group, control group and blank group, respectively. Then incubate at 37 °C, 5% CO2, avoid light for 2 h. Then use the microplate reader to measure the absorbance (OD value) of each well at 450 nm wavelength. Calculate the cell viability according to the following formula.

[0070]

[0071] Note: A 实验 , A 空白 , A 对照 The absorbance values at 450 nm of the experimental group, blank group and control group after 2 h of incubation, respectively.

[0072] 2.3 Construction of IR-HepG2 cell model

[0073] (1) Insulin stock solution preparation method: accurately weigh 4 mg of recombinant human insulin, dissolve in 2 mL of hydrochloric acid solution with pH of 1-2, mix thoroughly, filter sterilize through 0.22 μm microporous filter membrane, and store at -20 °C after aliquoting. When used, mix 300 μL of stock solution with 10.2 mL of DMEM high glucose medium to prepare a 10 -5 mol / L insulin working solution, and then obtain serial concentration solutions of 10 -6 to 10 -11 mol / L by gradient dilution.

[0074] (2) Effect of insulin concentration on HepG2 cell viability

[0075] Experimental grouping: ① Model group: HepG2 cells + different concentrations of insulin solution (10 -5 mol / L, 10 -6 mol / L, 10 -7 mol / L, 10 -8 mol / L, 10 -9 mol / L); ② Control group: HepG2 cells + complete medium; ③ Blank group: only DMEM high glucose medium (without cells).

[0076] Each group of 6 holes, will be adjusted to 1 x 10 6 -5 -9 mol / L), the control group was added with complete medium. Continue to cultivate 24h, 2 times of PBS washing, each well was added with 100 μL DMEM high glucose medium and 10 μL CCK-8 reagent, incubated for 2h, then measured the absorbance at 450nm, calculated the relative cell viability according to the formula, the effect of insulin concentration on HepG2 cell viability was shown in the upper left graph in Figure 4 each value represents the mean ± SD (n = 5), different lowercase letters represent significant difference (p < 0.05) between groups by multiple comparison analysis.

[0077] (3) IR-HepG2 cell model construction

[0078] Grouping as above "cell viability experiment", each group of 6 holes. After cell adhesion, the model group was treated with 100 μL of different concentrations of insulin solution (10 -5 -11 mol / L) for 24h, 36h, 48h, 60h. Glucose consumption determination: after discarding the culture solution and PBS washing, replaced with 100 μL of phenol red-free DMEM high glucose medium, continued to cultivate for 24h. Collected the supernatant, GOD-POD method was used to detect the glucose content, calculated the difference of glucose consumption between the model group and the control group, to determine the best modeling concentration and action time, the results were shown in the lower graph in Figure 4 each value represents the mean ± SD (n = 5), different lowercase letters represent significant difference (p < 0.05) between groups by multiple comparison analysis. The best modeling condition selected by the experiment was using 10 -7 -8 mol / L recombinant human insulin culture solution to act on cells for 36h to construct insulin-resistant HepG2 cells (IR-HepG2 cells).

[0079] 2.4. MSP4-SeNPs on IR-HepG2 cell viability

[0080] According to the best modeling insulin concentration (10 -7 ​​​​mol / L) and exposure time (36 h) were used to establish an IR-HepG2 cell model. A blank group, a model group (IR-HepG2), a positive control group (metformin 100 μg / mL), and drug groups (MSP4-SeNPs: 1.5625 μg / mL, 6.2500 μg / mL, 25.000 μg / mL, 100.00 μg / mL) were set up, with six replicate wells in each group. The blank group consisted of a culture medium group without cells. All groups except the blank group were induced to establish an IR-HepG2 cell model. After the model was successful, the blank group and the model group were supplemented with serum-free culture medium, the positive control group was added with serum-free culture medium containing 100 μg / mL metformin, and the drug-treated group was added with serum-free culture medium containing the corresponding MSP4-SeNPs concentrations of 1.5625 μg / mL, 6.2500 μg / mL, 25.000 μg / mL, and 100.00 μg / mL, respectively, and incubated at 37°C and 5% CO2 for 24 hours. Subsequently, the cell proliferation was evaluated according to the previous CCK-8 method to explore the effect of MSP4-SeNPs on the viability of IR-HepG2 cells. The CCK-8 method was used to evaluate the effect of MSP4-SeNPs on the cell viability of IR-HepG2, and the results are as follows. Figure 4 As shown in the upper middle right figure, each value in the figure represents the mean ± SD (n = 5), and different lowercase letters indicate significant differences between the groups in multiple range analysis (p < 0.05).

[0081] Depend on Figure 4 The results showed that when IR-HepG2 cells were treated with MSP4-SeNPs at concentrations ≤100 μg / mL, the cell viability of IR-HepG2 cells remained above 85%, which is within the safe concentration range. When IR-HepG2 cells were treated with MSP4-SeNPs at concentrations between 6.25 and 25 μg / mL, there was no statistically significant difference in the effect on cell viability with increasing MSP4-SeNPs concentrations.

[0082] Based on the above experimental results, it can be determined that MSP4-SeNPs have good biosafety in the concentration range of 0-100 μg / mL, which can be used as a safe concentration range for subsequent experiments.

[0083] Example 3: Morchella polysaccharide nano-selenium improves cellular insulin resistance

[0084] This example studies the new application of Morchella polysaccharide nano-selenium. The results show that Morchella polysaccharide nano-selenium can improve insulin resistance, as follows:

[0085] Experimental Methods: Effects of MSP4-SeNPs on Glucose Metabolism in IR-HepG2 Cells

[0086] According to the best modeling insulin concentration (10 -7 mol / L) and action time (36h) to construct IR-HepG2 cell model. Set up blank group, model group (IR-HepG2), positive control group (metformin 100 μg / mL), drug group (MSP4-SeNPs: 1.5625 μg / mL, 6.2500 μg / mL, 25.000 μg / mL, 100.00 μg / mL), each group set 6 replicates, the blank group is the culture medium group without cells. Except the blank group, the rest groups are induced to construct IR-HepG2 cell model. After the model is successful, the blank group and the model group are supplemented with serum-free medium, the positive control group is added with serum-free medium containing 100 μg / mL metformin, and the drug treatment group is added with serum-free medium containing MSP4-SeNPs with corresponding concentrations of 1.5625 μg / mL, 6.2500 μg / mL, 25.000 μg / mL, 100.00 μg / mL, respectively, and incubated at 37℃, 5% CO2 for 24 hours.

[0087] Subsequently, according to the glucose determination kit instructions, the supernatant of each well was collected, and the glucose content was determined using the glucose detection kit (GOD-POD method) to evaluate the effect of MSP4-SeNPs on the glucose consumption of IR-HepG2 cells, and the results are shown in Figure 2A; the glycogen content detection kit was used for detection to evaluate the effect of MSP4-SeNPs on the glycogen content of IR-HepG2 cells, and the results are shown in Figure 2B; the BCA protein concentration determination kit was used to determine the protein content in the cell supernatant, and the HK content in the IR-HepG2 cells was determined according to the HK test kit instructions, and the results are shown in Figure 2C; the BCA protein concentration determination kit was used to determine the protein content in the cell supernatant, and the PK content in the IR-HepG2 cells was detected according to the PK test kit instructions, and the results are shown in Figure 2D. Among them, the glucose content, glycogen content, HK content and PK content detection kit are shown in the following table. Figure 5 Figure 5 Figure 5 Figure 5

[0088] Table 1 Glucose content detection kit

[0089]

[0090]

[0091] Table 2 Glycogen content detection kit

[0092] Reagent Blank well Calibration well Sample well Working solution (μL) 300 300 300 ddH2O (μL) 3 0 0 Calibration (μL) 0 3 0 Sample (μL) 0 0 3

[0093] Table 3 HK content detection kit ​​​​

[0094] Reagent name Reagent one Reagent two Reagent three Reagent four Reagent five Reagent six Sample Assay tube (uL) 400 400 80 80 40 8 30

[0095] Table 4 PK content detection kit

[0096]

[0097] The BCA protein concentration assay kit was used to detect the contents of malondialdehyde (MDA), triglyceride (TG), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the cells. Figure 6 The effect of MSP-4-SeNPs on the content of malondialdehyde (MDA) in IR-HepG2 cells is shown in Figure 6 As shown in Figure A, the effect of MSP-4-SeNPs on triglyceride (TG) content in IR-HepG2 cells is shown in Figure 4. Figure 6 As shown in Figure B, the effect of MSP-4-SeNPs on the superoxide dismutase (SOD) content in IR-HepG2 cells is shown in Figure 4. Figure 6 As shown in C, and the effect of MSP-4-SeNPs on the content of glutathione peroxidase (GSH-Px) in IR-HepG2 cells is shown in Figure 6 As shown in D.

[0098] Hexokinase (HK), the first rate-limiting enzyme in glycolysis, catalyzes the phosphorylation of glucose to produce glucose-6-phosphate (G6P); decreased HK activity may be associated with metabolic disorders. Pyruvate kinase (PK), the third rate-limiting enzyme in glycolysis, catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate and generates ATP; decreased PK activity may inhibit ATP production, leading to insufficient energy supply. Regulating the activities of HK and PK is a key mechanism for improving glucose metabolism and also indirectly reflects its association with insulin resistance.

[0099] Depend on Figure 5 The results showed that compared with the normal HepG2 cells in the control group, the glucose consumption and glycogen content of the model group cells were significantly reduced, as well as the HK and PK enzyme activities were significantly reduced, indicating that the insulin resistance model was successfully constructed.

[0100] Depend on Figure 5It can be seen that compared with the model group, the administration of MSP-4-SeNPs at a concentration of 1.5-25 μg / mL can significantly promote the uptake and utilization of glucose by IR-HepG2 cells, enhance the activity of HK and PK, and significantly improve the cellular insulin resistance state (P<0.05), and its effect is better than that of 100 μg / mL MSP-4-SeNPs; it shows that Morel polysaccharide nanoselenium can be used as an insulin sensitizer to improve cellular insulin resistance, and Morel polysaccharide nanoselenium has a better effect on improving cellular insulin resistance in the low concentration range (1.5-25 μg / mL) and higher concentration (25-100 μg / mL). Among them, the improvement effect of the 6.25 μg / mL MSP4-SeNPs group on cellular insulin resistance is comparable to that of the positive control drug metformin (100 μg / mL).

[0101] Insulin resistance occurs when insulin's efficiency in promoting glucose uptake and utilization decreases due to various factors, leading to the body's compensatory overproduction of insulin. People with elevated plasma insulin levels become insensitive to insulin. Clinically, insulin resistance can lead to metabolic syndrome and glucose metabolism disorders such as type 2 diabetes. This cell-based experiment found that MSP4-SeNPs significantly increased hexokinase (HK) and pyruvate kinase (PK) activity in IR-HepG2 cells, boosting glycogen content and effectively improving glucose metabolism disorders.

[0102] Antioxidant index itself can be used as one of the physiological indicators to evaluate insulin resistance cells. Figure 6 It can be seen that compared with the control group, the MDA and TG levels in the IR-HepG2 cells of the model group were significantly increased, while the SOD and GPx levels were significantly decreased; compared with the model group, intervention with a concentration of 1.5-6.25 μg / mL MSP4-SeNPs or 100 μg / mL metformin can significantly reduce the MDA and TG levels in IR-HepG2 cells, and significantly increase the SOD and GPx levels, indicating that the antioxidant level of insulin-resistant cells was restored after administration, indicating that both MSP4-SeNPs and metformin can improve the symptoms of insulin resistance.

[0103] Metformin (Met) is a drug widely used in the treatment of metabolic-related diseases, its mechanism of action is mainly through the inhibition of hepatic glucose production and secretion, thereby improving cellular insulin resistance, suitable for type II diabetes (as a first-line treatment drug), metabolic syndrome. The improvement effect of the Morel polysaccharide nano selenium on insulin resistance can be comparable to that of metformin, therefore, it is speculated that the Morel polysaccharide nano selenium can be used for preparing a drug for treating metabolic disorder-related diseases caused by insulin resistance, such as a drug for treating glucose metabolism disorder or metabolic syndrome caused by insulin resistance, and especially a drug for treating type II diabetes caused by insulin resistance.

[0104] Example 4 Safety evaluation of Morel polysaccharide nano selenium in vivo

[0105] (1) Animal grouping and administration method

[0106] According to the stratified random method, the mice after one week of adaptive feeding were randomly divided into a blank control group and an MSP4-SeNPs treatment group (low-dose group: 0.5 mg / kg, 1 mg / kg, medium-dose group: 2 mg / kg, high-dose group: 4 mg / kg, 6 mg / kg), 6 in each group, and the initial body weight was recorded. Subsequently, the mice in the MSP4-SeNPs treatment group were fasted for 8 hours within 24 hours, and then were respectively gavaged with 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg and 6 mg / kg of MSP4-SeNPs solution, and the gavage volume was accurately controlled to be 0.2 mL, while the mice in the blank control group were gavaged with the same volume of deionized water. During the continuous 14-day gavage treatment, the appearance characteristics and behavior of all experimental mice were continuously monitored, and the first 24 hours were observed in detail. After the end of the experiment, the final body weight of the mice was measured.

[0107] (2) Determination of organ coefficients

[0108] On the 15th day of gavage treatment, the mice fasted overnight were sacrificed by cervical dislocation after ether anesthesia, and the internal organ morphological changes were observed by systematic dissection. The fresh organs such as heart, liver, spleen, lung and kidney were weighed, and the organ coefficients were calculated according to the following formula:

[0109]

[0110] (3) Histopathological detection

[0111] The mouse heart, liver and kidney tissues were taken and fixed in 4% paraformaldehyde fixing solution for more than 24 hours, and after paraffin embedding and sectioning, HE staining was performed. The prepared tissue sections were observed under an optical microscope at 200 times magnification, and the changes in tissue structure were evaluated.

[0112] (4) Serum biochemical index determination

[0113] After blood collection through the orbital venous plexus, the serum was separated by centrifugation at 3000 rpm / min for 15 minutes. The enzyme activity and metabolites in the serum were determined using the detection kit produced by Kewei Biological Technology Co., Ltd.:

[0114] ① Enzyme activity index: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) activity;

[0115] ② Metabolic index: total cholesterol (TC), triglyceride (TG), urea nitrogen (BUN), glucose (GLU) content.

[0116] (5) Liver and kidney biochemical index determination

[0117] The mouse liver and kidney tissues were taken, and the biochemical kit (glycogen content test box, HK content test box, PK content test box) was used to measure the glycogen, HK, and PK contents of the mouse liver; and the HK and PK contents of the kidney.

[0118] (6) Data processing and analysis

[0119] All experimental data were expressed as mean ± standard error (Mean ± SEM), and statistical analysis was performed using SPSS 27.0, while data visualization and chart drawing were performed using GraphPad Prism 10. Single factor analysis of variance (ANOVA) was used for statistical analysis of differences with p<0.05, and the results were as follows.

[0120] ① Effect of MSP4-SeNPs on body weight and food intake of mice

[0121] For normal and healthy male mice, different doses of MSP4-SeNPs were given for 14 days, and the body weight and food intake of the mice were recorded, and the results are shown in Figure 7 and Figure 8 .

[0122] As can be seen from Figure 7 , during the last 7 days of observation, the body weight of male mice in each group showed a stable growth trend, indicating that the growth state of experimental animals was good. Statistical analysis showed that there was no significant difference in body weight change at different time points between each dosing group and the blank control group (P>0.05).

[0123] And as can be seen from Figure 8 , after being given different doses of MSP4-SeNPs, the daily food intake of mice in each group also showed no statistical difference compared with the blank control group (P>0.05), indicating that the tested substance had no significant effect on the normal feeding behavior of experimental animals.

[0124] ②Effects of MSP4-SeNPs on mouse organ indices

[0125] The organ index is an important indicator for evaluating the degree of organ development and drug toxicity. After the experiment, the main organs of the mice were anatomically observed. The results of the mouse organ index are shown in the table below.

[0126] Table 5 Analysis results of mouse organ index

[0127]

[0128] As shown in Table 5, the organs of mice in all experimental groups (including heart, liver, spleen, lungs, kidneys, etc.) developed normally, with no pathological changes. Specifically, the size of each organ was normal, the surface color was uniform, and no abnormalities such as edema, atrophy, hyperplasia or exudative lesions were observed. This shows that under the experimental conditions, the intervention of MSP4-SeNPs did not cause obvious anatomical damage to the main organs of mice, and all organs maintained a good physiological state. After treatment with different doses of MSP4-SeNPs (0.5-6 mg / kg.bw), there was no significant difference in the organ indexes of mice in each treatment group compared with the blank control group (P>0.05), indicating that MSP4-SeNPs are highly safe in vivo.

[0129] ③ Histopathological analysis of the heart, liver and kidney of mice by MSP4-SeNPs

[0130] The results of pathological analysis of mouse heart, liver and kidney tissues were as follows: Figure 9 shown.

[0131] Depend on Figure 9 It can be seen that the myocardial fiber bundles of each group of mice were arranged neatly and orderly, and no pathological changes such as fiber breakage or degeneration were observed; the hepatic cord structure was clear and arranged regularly; no abnormal phenomena such as fatty degeneration, congestion or congestion were observed, and no obvious histopathological changes were observed; the renal corpuscles and renal tubules in the renal tissue sections were clear, without abnormal lesions, and no abnormalities were found in the renal capsules and glomeruli within the renal corpuscles. Densa was also observed, but no obvious pathological changes were observed.

[0132] Compared with the blank control group, the organ tissue structure in each treatment group maintained normal morphology, with no obvious pathological changes. These results indicate that under the experimental conditions, MSP4-SeNPs intervention did not cause significant histopathological damage to important organs such as the heart and liver in mice, indicating its high safety.

[0133] ④Analysis of MSP4-SeNPs on mouse serum biochemical indicators

[0134] The collected mouse serum was also analyzed for biochemical indicators, and the results are shown in the following table.

[0135] Table 6 Analysis results of biochemical indicators of mouse serum

[0136]

[0137] Note: Each value represents the mean ± SD (n = 6), and different lowercase letters indicate significant differences between groups by multiple range analysis (p < 0.05).

[0138] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are sensitive biomarkers of hepatocyte injury, and elevated serum levels may be closely related to the following pathological changes: disruption of hepatocyte membrane integrity; inflammatory lesions of the liver; necrotic injury to the liver; drug / toxicant-induced liver toxicity.

[0139] As can be seen from Table 6, compared with the control group, the MSP4-SeNPs treatment group of mice had no statistically significant difference in serum ALT and AST, and the metabolic indicators total cholesterol (TC) and blood glucose (GLU) also had no significant difference between groups, indicating that MSP4-SeNPs intervention for 14 days did not cause obvious liver and kidney damage.

[0140] ⑤Effect of MSP4-SeNPs on biochemical indicators of liver and kidney tissues

[0141] The detection results of the biochemical indicators of the liver and kidney of the mice in each group in this experiment are shown in Table 7. Figure 10 From Table 7, Figure 10 From Table 7, Figure 10 From Table 7,

[0142] Example 5 Hypoglycemic effect of Morel polysaccharide nano-selenium on insulin-resistant type II diabetic mice

[0143] In this example, male C57BL / 6J mice were selected as experimental subjects, and a method of feeding high-sugar and high-fat feed combined with intraperitoneal injection of streptozotocin (STZ) was used to construct an insulin-resistant type II diabetic mouse model, and the specific experiments are as follows:

[0144] (1) After one week of adaptive feeding, a batch of mice were selected and randomly and equally divided into two groups according to the established experimental design. The mice in the blank control group continued to be fed with standard maintenance feed throughout the experiment to maintain their normal physiological state and nutritional needs. The model mice were fed with high-sugar and high-fat special feed.

[0145] (2) After eight weeks of feeding, the model mice fed with high-sugar and high-fat feed were subjected to fasting treatment, i.e. fasting for eight hours but maintaining water supply. The model mice were injected intraperitoneally with STZ (50 mg / kg) citrate buffer solution once a day for three consecutive days. At the same time, in order to establish an effective comparison, the blank control mice were injected intraperitoneally with an equal volume of citrate buffer solution, and the injection frequency and method were consistent with the model group to ensure the fairness and scientificity of the experimental conditions.

[0146] (3) During the fasting recovery phase, all mice except the blank control group continued to be fed with high-sugar and high-fat feed. This feeding method continued until the predetermined experimental time node was reached. One week after the intraperitoneal injection was completed, all mice were fasted for 6-8 hours but not watered to ensure that their metabolic states were relatively consistent before blood sampling. Subsequently, blood sampling was performed through the tail vein of the mice, and the collected blood samples were used to determine the fasting blood glucose (FBG) value. This index is crucial for evaluating the glucose metabolism status of mice. When the FBG measurement is greater than 11.0 mmol / L, it indicates that the diabetic mouse model has been successfully established, and these mice are subsequently used for further experimental research to explore the pathogenesis of diabetes and related treatment strategies.

[0147] (4) During the experiment, all mice except the normal control group continued to be fed with high-fat and high-sugar feed. Based on the results of the previous acute toxicity test, the MSP4-SeNPs in this experiment were selected at two doses of 1.0 mg / kg and 4.0 mg / kg for anti-diabetic efficacy evaluation.

[0148] (5) During the experiment, the mice were grouped and administered as shown in Table 7:

[0149] Table 7 Mouse grouping and administration

[0150]

[0151]

[0152] According to Huang Jihan et al. published in "Animal and animal and human equivalent dose conversion in pharmacological tests", according to the standard weight animal from animal a to animal b mg / kg dose conversion table (the value in the table is the conversion coefficient Rab) in the mouse a row, adult b column R ab = 0.081, the concentration of Morel polysaccharide nano selenium is converted to adult low dose of 1.0 mg / kg x 0.081 = 0.081 mg / kg; adult high dose is 4 mg / kg x 0.081 = 0.324 mg / kg. According to the body surface area conversion coefficient of human and mouse (12 times), these two concentrations are equivalent to 50-200 μg selenium per day for a 70 kg adult, which is lower than the maximum safe intake of selenium (400 μg / d) for Chinese residents, so the selected dose meets the safety requirements.

[0153] The normal mice of the blank group were used as controls, and the successfully modeled mice were randomly divided into four different groups, namely the T2DM model group, the T2DM model combined with metformin treatment group, the T2DM model combined with MSP4-SeNPs low dose treatment group (MSP4-SeNPs concentration of 1.0 mg / kg) and the T2DM model combined with MSP4-SeNPs high dose treatment group (MSP4-SeNPs concentration of 4.0 mg / kg), and at least 5 mice were ensured in each group to ensure the reliability and statistical significance of the experimental data. For the mice in the blank control group, we gave them normal physiological feed to raise them; while for the mice in the T2DM model group, the T2DM model combined with metformin treatment group, the T2DM model combined with MSP4-SeNPs low dose treatment group and the T2DM model combined with MSP4-SeNPs high dose treatment group, we continued to feed them high-fat feed to maintain their T2DM state.

[0154] (6) After the completion of the grouping operation, a gavage administration phase with a duration of 4 weeks was carried out immediately. The mice were subjected to daily gavage treatment according to the sample dose, and the blank group and the model group were subjected to daily gavage with double distilled water, once a day, with a fixed gavage volume of 0.2 mL, and the gavage duration was 28 days. During this phase, the activity state, hair condition, food intake, water intake, and bedding moisture of the mice were recorded daily, and the body weight was measured at regular intervals every week to ensure accurate understanding of the body weight change. During the administration period, the mice were subjected to fasting blood glucose (FBG) detection under fasting (without water restriction), and the food intake and water intake changes were recorded synchronously. After the last administration, the mice were fasted for 12 hours (with free water), weighed, and then the blood samples were collected through the orbit, and the mice were anesthetized using ether. Subsequently, the mice were sacrificed by cervical dislocation, and immediately subjected to gross anatomy. During the dissection, the fresh organs such as heart, liver, kidney, and pancreas were quickly removed and weighed, and the organ index was calculated. Part of the removed organs were fixed in 4% paraformaldehyde solution for subsequent pathological observation and sectioning; and the other part of the organs were stored in liquid nitrogen for subsequent possible experimental needs.

[0155] Experimental results:

[0156] (1) Effect of Morchella polysaccharide nano-selenium on food intake and water intake of type II diabetic mice

[0157] The body weight, food intake, and water intake of the mice in each group were recorded during the experiment, and the results are shown in Figure 11 and Figure 12 .

[0158] As shown in Figure 11 , during the entire duration of the drug intervention, the body weight of the control group mice showed a stable and gradually increasing trend, and the difference in body weight gain between the drug intervention groups and the control group did not reach statistical significance, mainly because the body weight and dietary changes of this male C57BL / 6J mouse in the II diabetes model were not large.

[0159] As shown in Figure 12 , compared with the control group mice, the model group mice showed increased food intake and water intake, which was similar to the symptoms of human type II diabetes. In addition, the model group mice generally showed increased food intake, which may be related to insulin resistance, leading to abnormal function of hypothalamic neurons regulating appetite; the water intake of the model group mice was significantly increased, mainly due to osmotic diuresis caused by hyperglycemia.

[0160] Compared with the model group, the food intake and water intake of the type II diabetes model mice were improved after administration of low-dose Morel polysaccharide nano selenium (L-MSP4-SeNPs) or high-dose Morel polysaccharide nano selenium (H-MSP4-SeNPs).

[0161] (2) Blood glucose regulation of Morel polysaccharide nano selenium on type II diabetes mice

[0162] In order to accurately measure the fasting blood glucose level of mice in each group, the fasting blood glucose level of mice in each group was measured by tail vein blood, and the fasting blood glucose level changes of mice in different periods between groups were as shown in Figure 13

[0163] As can be seen from Figure 13 , the blood glucose level of the control group mice was maintained within the normal range, while the blood glucose level of the model group mice exceeded 15 mmol / L, which was significantly higher than that of the control group mice. This result more strongly proves that the type II diabetes model is successfully constructed.

[0164] Compared with the model group mice, the fasting blood glucose (FBG) of the model mice in the low-dose Morel polysaccharide nano selenium (L-MSP4-SeNPs) group and the high-dose Morel polysaccharide nano selenium (H-MSP4-SeNPs) group were significantly reduced, and the fasting blood glucose (FBG) of the model mice showed a gradual downward trend with the extension of the treatment time. It can be seen that Morel polysaccharide nano selenium can effectively reduce the fasting blood glucose level of diabetic mice, and the hypoglycemic effect of the high-dose group (Morel polysaccharide nano selenium concentration 4.0 mg / kg) is equivalent to that of the positive control group (metformin concentration 100 mg / kg).

[0165] (3) Effect of Morel polysaccharide nano selenium on glucose tolerance (OGTT) of type II diabetes mice

[0166] After administration for 4 weeks, the mice were subjected to oral glucose tolerance test, and the blood glucose changes within 0-120 min were determined, and the results are shown in the left of Figure 14

[0167] The blood glucose of all experimental group mice reached the peak level 30 minutes after sugar loading, and then showed a downward trend, in which the blood glucose peak of each drug group (metformin, L / H-MSP4-SeNPs) was significantly lower than that of the model group (P<0.05). Compared with the model group, the hypoglycemic effect of each administration group was in the order of Met group>H-MSP4-SeNPs group>L-MSP4-SeNPs group.

[0168] In order to further evaluate the blood glucose changes, the area under the blood glucose curve (AUS) was analyzed, and the results are shown in the right of Figure 14

[0169] ​​​The results show that the AUS value of the model group mice is significantly higher than that of the control group, and the AUS value of each group is significantly reduced after intervention of different doses of MSP4-SeNPs (P<0.05), which indicates that different doses of MSP4-SeNPs can effectively improve the impaired glucose tolerance of diabetic mice, delay the progression of the disease, and the intervention effect of the high-dose group (H-MSP4-SeNPs) is better than that of the low-dose group (L-MSP4-SeNPs), and the glucose-lowering effect of the H-MSP4-SeNPs group is comparable to that of the positive control group.

[0170] From the experimental results, it can be seen that Morel polysaccharide nano selenium can effectively reduce the fasting blood glucose level of diabetic mice. Among them, the glucose-lowering effect of high-dose Morel polysaccharide nano selenium (H-MSP4-SeNPs) and the positive control group is the most significant, and high-dose MSP4-SeNPs shows glucose-lowering efficiency comparable to that of the positive drug metformin, which can be used as a substitute for metformin for the preparation of drugs for treating type II diabetes.

[0171] (4) Effect of MSP4-SeNPs on the organ index of type II diabetic mice

[0172] After the experiment, the organ index of each group of mice was analyzed, and the results are shown in Table 8.

[0173] Table 8 Analysis of organ index of mice

[0174]

[0175] Note: Different lowercase letters represent significant differences (p<0.05) between groups

[0176] As shown in Table 8, compared with the control group, the liver and pancreas organ index of the model group mice was significantly increased, and liver swelling and pancreatic hypertrophy were observed in the model group mice, among which the liver swelling may be due to increased metabolic load, and the pancreatic hypertrophy reflects endocrine compensatory changes; and the abnormal organ index is positively correlated with the course of diabetes.

[0177] Compared with the model group, the liver and pancreas organ index of the mice treated with L-MSP4-SeNPs and H-MSP4-SeNPs were reduced to different degrees, and the liver swelling and pancreatic hypertrophy of the mice were relieved after intragastric administration of metformin, L-MSP4-SeNPs and H-MSP4-SeNPs. At the same time, there was no statistical difference (P>0.05) in the organ index of the heart and kidney of the mice between each group. It is proved that Morel polysaccharide nano selenium can relieve the damage of liver and pancreas in diabetic patients after intervention, and the effect of H-MSP4-SeNPs in relieving pancreatic damage in diabetic patients is comparable to that of metformin.

[0178] (5) Effect of MSP4-SeNPs on serum insulin content of type II diabetic mice

[0179] After the experiment, the serum insulin content (as shown in FIG. 5A) and HOMA-IR index (as shown in FIG. 5B) of each group of mice were analyzed. Figure 15 Figure 15

[0180] As can be seen from FIG. 5A and FIG. 5B, compared with the control group, the serum insulin and HOMA-IR index (an index for evaluating insulin resistance) of the type II diabetic model group of mice were significantly increased, and the higher the HOMA-IR index, the more serious the insulin resistance. Insulin resistance refers to the decrease in the sensitivity of cells (such as muscle, fat and liver cells) to insulin, which causes insulin to be unable to effectively promote glucose into cells, thereby causing blood glucose levels to rise; again proving that the type II diabetic mouse model was successfully constructed by high-sugar high-fat / STZ combination induction. Figure 15 After 28 days of treatment with Morel polysaccharide nano-selenium, compared with the model group, the HOMA-IR value of mice treated with low-dose and high-dose MSP4-SeNPs was significantly lower than that of the model group, indicating that Morel polysaccharide nano-selenium can significantly improve insulin resistance; at the same time, the insulin level of the mice was significantly reduced, and the effect of high-dose MSP4-SeNPs was better than that of low-dose MSP4-SeNPs, indicating that MSP4-SeNPs can alleviate the increase of insulin and improve insulin resistance in a dose-dependent manner.

[0181] (6) Effect of MSP4-SeNPs on serum biochemical indicators of type II diabetic mice

[0182] After the experiment, the serum biochemical indicators of mice were detected, and the effect of MSP4-SeNPs on serum biochemical indicators of type II diabetic mice is shown in FIG. 6. These biochemical indicators can comprehensively evaluate liver function, kidney function, lipid metabolism and blood glucose status.

[0183] Figure 16 As can be seen from the results, compared with the control group, the serum glucose (GLU), glycosylated serum protein (GSP), triglyceride (TG), total protein (TP) and uric acid (UA) contents of the model group of mice were significantly increased, and the activities of alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly increased, indicating that the model group of mice had liver and kidney dysfunction.

[0184] Figure 16

[0185] ​​​​​After 28 days of low-dose and high-dose MSP4-SeNPs gavage treatment, compared with the model group, the activities of AST, ALT and ALP in the serum of mice in each MSP4-SeNPs group were significantly reduced, and the contents of GLU, GSP, TG, TP and UA were also significantly reduced, and the effect of H-MSP4-SeNPs was better. It can be seen that L-MSP4-SeNPs and H-MSP4-SeNPs can improve glucose metabolism, lipid metabolism, liver function and protein metabolism to varying degrees, and can alleviate the liver and kidney dysfunction caused by diabetes, indicating that Morel polysaccharide selenium nanoparticles can resist liver and kidney dysfunction caused by diabetes, and Morel polysaccharide selenium nanoparticles shows a dose-dependent effect.

[0186] (7) Effect of MSP4-SeNPs on liver biochemical indicators of type II diabetic mice

[0187] After the experiment, the liver tissues of mice in each group were taken, and the liver biochemical indicators of mice in different groups were detected, and the results are shown in Figure 17 , wherein Figure 17 A is the liver glycogen content of mice in different groups, B is the total protein (TP) content of mice in different groups, C is the MDA content in the liver of mice in different groups, D is the activity of GSH-Px in the liver of mice in different groups, E is the activity of SOD in the liver of mice in different groups, F is the activity of HK in the liver of mice in different groups, and G is the activity of PK in the liver of mice in different groups.

[0188] When insulin resistance occurs, hepatic gluconeogenesis increases, liver glycogen synthesis decreases and decomposition is enhanced, and liver glucose output increases, which promotes the increase of fasting and postprandial blood glucose. As can be seen from Figure 17 A, compared with the control group of normal mice, the glycogen content in the liver of mice in the model group showed a significant downward trend, indicating that insulin resistance leads to a decrease in liver glycogen synthesis, and the blood glucose accumulated in the blood of mice is difficult to be synthesized into liver glycogen, thereby causing an increase in blood glucose in the body.

[0189] Compared with the model group, after the intervention of high and low dose MSP4-SeNPs (H-MSP4-SeNPs, L-MSP4-SeNPs), the synthesis of liver glycogen in mice was significantly promoted. This indicates that MSP4-SeNPs can improve insulin resistance by enhancing cell insulin sensitivity, promote liver glycogen synthesis and accumulation, and thus reduce blood glucose concentration in the body, achieve the effect of reducing blood glucose, and the high-dose group shows a more significant effect of reducing blood glucose.

[0190] As can be seen from Figure 17 B, compared with the control group of normal mice, the total protein content in the liver of mice in the model group was significantly increased, indicating that the liver reserve function of mice in the model group was impaired, and there was a risk of liver disease.

[0191] Compared with the model group, the total protein content of the mouse liver was significantly reduced after high and low dose MSP4-SeNPs (H-MSP4-SeNPs, L-MSP4-SeNPs) intervention, indicating that Morel polysaccharide nano selenium can improve the liver metabolic abnormalities caused by insulin resistance.

[0192] From Figure 17 As can be seen from C and D, compared with the normal mice in the control group, the MDA level in the liver of the model mice was significantly increased and the GSH-Px activity was significantly decreased, indicating that the liver of the model mice was damaged by oxidative stress. In the high-fat diet model, MDA was significantly increased and glutathione peroxidase (GSH-Px) activity was decreased, suggesting that oxidative stress exacerbates lipid metabolism disorder through lipid peroxidation, and lipid metabolism disorder exacerbates oxidative stress. Oxidative stress and lipid metabolism disorder together constitute the core features of metabolic syndrome.

[0193] Compared with the model group, the accumulation of MDA in the liver of mice was significantly reduced and the activity of GSH-Px was significantly increased after high and low dose MSP4-SeNPs (H-MSP4-SeNPs, L-MSP4-SeNPs) intervention, indicating that Morel polysaccharide nano selenium can improve oxidative stress damage and thus improve metabolic syndrome, and high dose MSP4-SeNPs (H-MSP4-SeNPs) is particularly outstanding in improving oxidative stress damage and can be used for the preparation of drugs for improving metabolic syndrome caused by insulin resistance.

[0194] Those skilled in the art will readily understand that the above description is only the preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An application of Morchella polysaccharide nano-selenium, characterized in that: The Morchella polysaccharide nano-selenium is MSP4-SeNPs, which is used to prepare a preparation for improving insulin resistance.

2. The use according to claim 1, characterized in that The Morchella polysaccharide nano-selenium is used for preparing a preparation for improving cellular insulin resistance, and the concentration of the Morchella polysaccharide nano-selenium is 1.5 to 25 μg / mL or 1.5 to 25 μg / mg.

3. The use according to claim 2, characterized in that The concentration of the Morchella polysaccharide nano-selenium is 1.5-6.25 μg / mL or 1.5-6.25 μg / mg.

4. The use according to claim 1, wherein The Morchella polysaccharide nano-selenium is used for preparing medicine for treating glucose metabolism disorder syndrome caused by insulin resistance.

5. The use according to claim 4, characterized in that The Morchella polysaccharide nano-selenium is used for preparing medicine for treating type II diabetes with insulin resistance.

6. The use according to claim 1, wherein The Morchella polysaccharide nano-selenium is used for preparing a medicine for improving metabolic syndrome of insulin resistance.

7. The use according to claim 5 or 6, characterized in that The concentration of Morchella polysaccharide nano-selenium in the medicine is 0.08-0.4 mg / kg or 0.08-0.4 mg / L.

8. The use according to any one of claims 1 to 7, characterized in that The Morchella polysaccharide nano-selenium is a spherical MSP4-SeNPs complex with an average particle size of 67.45±0.52 nm and a selenium content of 52.6±3.21 mg / kg.

9. The use according to claim 8, characterized in that The Morchella polysaccharide MSP4 is composed of mannose, glucose and galactose and has a molecular weight of 2.98×10 5 The α-type polysaccharide of Da is an α-type polysaccharide with a 1,4-glycosidic bond as the main chain and branches at O-6, O-3 and O-2.

10. The use according to claim 9, characterized in that The MSP4-SeNPs complex was prepared according to the following method: The Morchella polysaccharide MSP4 solution was mixed with the mixed solution of ascorbic acid-sodium selenite at a mass ratio of 1:1, and reacted with magnetic stirring at 38°C for 2h to make SeO3 2- The reduced nano-selenium SeNPs interact with the OH and NH of Morchella polysaccharide MSP4 to form a stable spherical structure, obtaining an MSP4-SeNPs complex. The concentration of MSP4 in the Morchella polysaccharide solution is 7 mg / mL, and the molar ratio of ascorbic acid to sodium selenite in the ascorbic acid-sodium selenite mixed solution is 2:1.

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