Use of levorotatory polylactic acid and its copolymer in preparation of products for bidirectional regulation of blood sugar and improvement of insulin resistance

Abstract

This invention belongs to the field of pharmaceutical technology and relates to the application of polylactic acid (PLA) and its copolymers in the preparation of products that bidirectionally regulate blood glucose and improve insulin resistance. Specifically, this invention relates to the use of polymers in the preparation of pharmaceuticals, said polymers comprising PLA or copolymers containing repeating PLA units, wherein the repeating PLA units are...; the pharmaceuticals are used for one or more of the following purposes: bidirectional regulation of blood glucose, improvement of insulin resistance, protection of mitochondrial function, repair of nervous system damage, inhibition of liver inflammatory responses, and reduction of hepatocyte damage.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of polylactic acid (PLA) and / or copolymers containing repeating L-lactic acid units in the preparation of articles that bidirectionally regulate blood glucose and improve insulin resistance. Background Technology

[0002] Diabetes mellitus, a prevalent metabolic disease worldwide, can be classified into type 1 diabetes, type 2 diabetes, and other special types based on its pathogenesis. According to statistics from the International Diabetes Federation, there are over 500 million people with diabetes globally, and both its incidence and mortality rates are continuously rising. Steroid-induced diabetes, a representative of special types of diabetes, is induced by long-term use of glucocorticoids, with a clinical incidence of approximately 20%. The core pathological feature of diabetes is disordered glucose metabolism; if not effectively managed in the long term, it can lead to serious chronic complications, severely impacting patients' quality of life and the efficiency of the healthcare system.

[0003] Currently, clinical treatment strategies for different types of diabetes vary significantly, but existing protocols all have limitations to varying degrees. Type 1 diabetes patients require lifelong exogenous insulin replacement therapy. This treatment heavily relies on continuous monitoring of blood glucose levels and precise dosage adjustments, but the risk of hypoglycemia remains, which can lead to coma or even death in severe cases. Type 2 diabetes patients primarily control their blood glucose with oral hypoglycemic agents, but these drugs often have side effects, and treatment efficacy varies from person to person. For steroid-induced diabetes, current treatment aims to strike a balance between hormone therapy for the underlying disease and blood glucose management. However, the hyperglycemic effect of glucocorticoids often counteracts the effects of hypoglycemic agents, resulting in a lack of truly targeted treatment options.

[0004] Insulin resistance (IR) refers to a significant decrease in the sensitivity / responsiveness of target tissues such as muscles, fat, and liver to physiological concentrations of insulin. Normal doses of insulin are unable to effectively promote glucose uptake, utilization, and storage, leading to a decline in blood glucose regulation efficiency. In the early stages of IR, pancreatic β cells compensate by secreting more insulin to forcibly maintain normal blood glucose levels. However, prolonged high insulin load leads to β cell dysfunction and insufficient insulin secretion, eventually progressing to diabetes.

[0005] The main causes of insulin resistance include obesity, where visceral fat releases inflammatory factors such as free fatty acids (FFA), TNF-α, IL-6, and resistin, which directly interfere with insulin signaling, inducing lipotoxicity and oxidative stress, and are core drivers of insulin resistance. In addition, aging, polycystic ovary syndrome (PCOS), glucocorticoids, thyroid dysfunction, and metabolic-associated fatty liver disease (MAFLD) can also contribute to insulin resistance.

[0006] Current treatment for insulin resistance primarily involves insulin sensitizers. Mechanistically, insulin sensitizers such as thiazolidinediones directly improve insulin resistance. Additionally, metformin and other hypoglycemic agents also indirectly improve insulin resistance. However, thiazolidinediones are generally used in combination with insulin. Currently, there are no drugs with clearly defined boundaries for treating insulin resistance.

[0007] Currently, there is an urgent clinical need to develop a safe, effective treatment regimen that can improve insulin resistance without requiring frequent dosing. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides the use of polymers and polymer microspheres for bidirectional regulation of blood glucose and improvement of insulin resistance. These polymers and polymer microspheres exhibit good effects in bidirectional regulation of blood glucose, improvement of insulin resistance, protection of mitochondrial function, protection of nerves, and inhibition of high-lipid-induced liver inflammation. They are safe and effective, can be administered once every few months, improving patient compliance, and provide a new strategy for the treatment of insulin resistance, diabetes, mitochondrial dysfunction-related diseases (including neurodegenerative diseases), and metabolic-associated fatty liver disease (MAFLD) or metabolic-associated steatohepatitis (MASH).

[0009] In one aspect, this application provides the use of a polymer in the preparation of a pharmaceutical product, the polymer comprising polylactic acid (PLA) or a copolymer containing repeating L-lactic acid units, wherein the repeating L-lactic acid units are... This drug is used for one or more of the following purposes: bidirectional regulation of blood sugar, improvement of insulin resistance, protection of mitochondrial function, repair of nervous system damage, inhibition of liver inflammatory response, and reduction of hepatocyte damage.

[0010] Alternatively, the copolymer containing repeating L-lactic acid units may include one or more of the following: L-polylactic acid-glycolic acid copolymer, L-polylactic acid-polyethylene glycol copolymer, polyethylene glycol-L-polylactic acid-glycolic acid copolymer, L-polylactic acid-chitosan copolymer, and lactide-caprolactone copolymer.

[0011] Alternatively, the weight-average molecular weight of polylactic acid (PLA) or copolymers containing repeating L-lactic acid units can be 1,000–200,000 Da, for example 5,000–100,000 Da, for example 3,000–80,000 Da, for example 3,000–10,000 Da, 10,000–20,000 Da, 20,000–40,000 Da, 40,000–60,000 Da, or 60,000–80,000 Da. The weight-average molecular weight can be determined by gel permeation chromatography (GPC).

[0012] Alternatively, in a copolymer containing L-lactic acid repeating units, the proportion of L-lactic acid repeating units is greater than or equal to 5%, for example, 5% to 75%, such as 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, or 65% to 75%, calculated by molar ratio.

[0013] Alternatively, the configuration of polylactic acid (PLA) or copolymers containing repeating L-lactic acid units can be linear, branched, or other configurations.

[0014] In some embodiments, the drug comprises microspheres, polylactic acid (PLA), or a copolymer containing repeating L-lactic acid units present in the microspheres.

[0015] In some embodiments, the microspheres comprise L-polylactic acid (PLA) microspheres or copolymer microspheres containing repeating L-lactic acid units. In some embodiments, the microspheres comprise one or more of the following: L-PLA microspheres, L-PLA-glycolic acid copolymer microspheres, L-PLA-polyethylene glycol copolymer microspheres, polyethylene glycol-L-PLA-glycolic acid copolymer microspheres, L-PLA-chitosan copolymer microspheres, and lactide-caprolactone copolymer microspheres.

[0016] In this document, polymer microspheres refer to spherical particles formed from or primarily comprising one or more polymers, with a diameter on the nanometer or micrometer scale. "Primarily comprising" means that the microspheres contain at least 5 wt% of the polymer, for example, at least 10 wt%, at least 30 wt%, at least 50 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt%, at least 95 wt%, or at least 98 wt% of the polymer. In addition to the aforementioned polymers, polymer microspheres may also contain other substances, such as trace amounts of polyvinyl alcohol remaining from the manufacturing process.

[0017] In some embodiments, the microspheres are formed of polylactic acid (PLA) or a copolymer containing repeating L-lactic acid units, or mainly comprise polylactic acid or the copolymer containing repeating L-lactic acid units.

[0018] Alternatively, the microspheres have an average particle size of at least 1 μm, with selectable average particle sizes ranging from 1 to 200 μm, such as 20 to 60 μm, 15 to 55 μm, or 20 to 40 μm. The average particle size can be measured using a laser particle size analyzer.

[0019] Alternatively, the drug may contain a pharmaceutically acceptable carrier or excipient.

[0020] Alternatively, the drug can be formulated as an oral or injectable dosage form.

[0021] Furthermore, injectable dosage forms are classified according to their physical state, including liquid injections, powders for injection, and tablets for injection. They are also classified according to the injection site, including but not limited to intradermal injections, subcutaneous injections, intramuscular injections, and intravenous injections. Injectable dosage forms can be sterile water or oil suspensions for injection, or sterile solutions for injection. Suitable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspension media.

[0022] Furthermore, oral dosage forms include, but are not limited to, tablets, pills, granules, powders, soft / hard capsules, and oral liquids. Tablets typically use lactose and corn starch as carriers, and lubricants such as magnesium stearate may also be added. Capsules typically use lactose and dried corn starch as diluents. Oral liquids usually involve mixing the active ingredient with suitable emulsifiers and suspending agents. If desired, sweeteners, flavorings, or colorings may also be added to the above oral dosage forms.

[0023] As an alternative, the drug can be formulated into a sustained-release preparation.

[0024] In some embodiments, the drug is present in the form of an injectable (e.g., an intramuscular injection). Alternatively, the injectable comprises a liquid matrix (e.g., physiological saline) and a complex solvent (e.g., sodium carboxymethyl cellulose). In some embodiments, the injectable contains 30–70 mg / mL of polylactic acid (PLA) or a copolymer containing repeating PLA units. In some embodiments, PLA or a copolymer containing repeating PLA units is present in microspheres.

[0025] In some embodiments, the drug is used to prevent and / or treat diabetes or its complications by bidirectionally regulating blood glucose. In some embodiments, diabetes may be selected from type 1 diabetes, type 2 diabetes, and steroid-induced diabetes. In some embodiments, bidirectional regulation of blood glucose includes, without insulin dependence, lowering an individual's blood glucose level, lowering an individual's glycated hemoglobin level, improving an individual's glucose tolerance, and / or promoting an individual's muscle GLUT4 mRNA and protein expression. In some embodiments, the individual is a diabetic patient. In some embodiments, the drug does not lower the blood glucose level of a normal individual.

[0026] In some implementations, the medication is used to prevent and / or treat insulin resistance syndrome. In some implementations, insulin resistance syndrome is characterized by one or more of the following conditions: obesity, hyperinsulinemia, dyslipidemia, hypertension, fatty liver, polycystic ovary syndrome (PCOS), metabolic syndrome, or atherosclerosis. In some implementations, improving insulin resistance includes: lowering an individual's fasting blood glucose level, lowering an individual's blood insulin level, and / or lowering an individual's insulin resistance index.

[0027] In some implementations, the drug is used to prevent and / or treat diseases associated with mitochondrial dysfunction. In some implementations, diseases associated with mitochondrial dysfunction include diabetes or its complications (e.g., type 1 and type 2 diabetes, diabetic cardiomyopathy), neurodegenerative diseases (e.g., Alzheimer's disease), cardiovascular diseases, cancer, fatty liver disease, or schizophrenia. In some implementations, protecting mitochondrial function includes increasing intracellular ATP levels and increasing intracellular mitochondrial DNA levels.

[0028] In some implementations, the drug is used to prevent and / or treat neurodegenerative diseases (such as Alzheimer's disease). In some implementations, the drug is used to improve an individual's cognitive function by repairing damage to the nervous system.

[0029] In some embodiments, the drug is used to prevent and / or treat metabolic-associated fatty liver disease or metabolic-associated steatohepatitis. In some embodiments, inhibiting the liver inflammatory response or reducing hepatocellular damage includes: reducing the expression levels of pro-inflammatory cytokines (such as IL-6, IL-1β, or TNF-α) in serum, or reducing the levels of ALT or AST in serum.

[0030] In one aspect, this application provides an injectable preparation comprising polymer microspheres; the polymer comprising poly(L-lactic acid) or a copolymer comprising repeating L-lactic acid units, wherein the repeating L-lactic acid units are... .

[0031] Alternatively, the polymer, polymer microspheres, or injection may be any of the polymers, polymer microspheres, or injections described above.

[0032] Alternatively, the injection can be used for any of the purposes described above.

[0033] In one aspect, this application provides a health food product comprising polymer microspheres; the polymer includes poly(L-lactic acid) or a copolymer containing repeating L-lactic acid units, wherein the repeating L-lactic acid units are... .

[0034] Alternatively, the polymer or polymer microspheres may be any of the polymers or polymer microspheres described above.

[0035] As an optional method, this health supplement is used to maintain healthy blood sugar levels.

[0036] In one aspect, this application provides a method for treating a disease, the method comprising administering to an individual in need an effective amount of any polymer, polymer microspheres or drug as defined above, the disease being any disease as defined above.

[0037] Alternatively, the polymers, microspheres, or drugs described above can be administered via oral, intraperitoneal, intravenous, subcutaneous, or intramuscular injection routes.

[0038] Alternatively, the polymer, microspheres, or drug described above may be applied once or multiple times within a cycle. This cycle may be one week, one month, or two months. In some embodiments, the polymer, microspheres, or drug may be applied once daily, once weekly, or once every eight weeks.

[0039] In this application, "effective amount" means, within reasonable medical judgment, an amount sufficient to treat or prevent a patient's disease while avoiding serious side effects with a sufficiently low risk-reward ratio. The effective amount in this application can vary depending on the target population, whose age, weight, basic health condition, severity of obesity, and the specific combination of drugs administered to the target population all influence the determination of the effective amount. Therefore, those skilled in the art can determine the effective amount of a polymer or drug combination based on, for example, amounts found to be effective in animal or clinical studies, physician experience, and / or recommended dosage ranges or dosing guidelines. Based on the above considerations, the effective amount can vary considerably depending on the specific polymer or drug combination and the dosage unit used, combined with the dosing regimen, treatment duration, the target population's age and weight, and the nature and severity of the treated condition.

[0040] Alternatively, the therapeutically effective amount of the aforementioned polymer, microspheres, or drug is 25 to 500,000 mg / kg body weight, for example 200 to 10,000 mg / kg body weight, for example 500 to 5,000 mg / kg body weight, for example 10 to 250 mg / kg body weight, for example 100 to 250 mg / kg body weight.

[0041] Alternatively, the therapeutically effective dose of the aforementioned polymer, microspheres, or drug is 25 to 1600 mg / kg body weight daily, for example, 170 to 1000 mg / kg body weight, or 300 to 800 mg / kg body weight.

[0042] Alternatively, the therapeutically effective dose of the aforementioned polymer, microspheres, or drug is 5 to 300 mg / kg body weight for a single administration, for example, 10 to 250 mg / kg body weight, or for example, 100 to 250 mg / kg body weight.

[0043] In this application, "individual" includes mammals, including but not limited to humans, non-human primates (e.g., apes, monkeys, chimpanzees, etc.), domestic animals (e.g., dogs or cats), farm animals (e.g., horses, cattle, goats, sheep, pigs, etc.), and laboratory animals (e.g., mice, rats, rabbits, etc.).

[0044] In this application, "prevention" means the delay of the onset of one or more symptoms of a particular disease, condition, or disorder, and / or a reduction in the frequency and / or severity of their occurrence; "treatment" means any application of a therapy that partially or completely relieves, improves, alleviates, or inhibits one or more symptoms, features, and / or causes of a particular disease, condition, and / or disorder, delays its onset, reduces its severity, and / or reduces its incidence. Attached Figure Description

[0045] Figure 1 The fasting blood glucose, glycated hemoglobin, oral glucose tolerance test (OGTT), and blood lactate levels of each group of type 1 diabetic (T1DM) mice in Example 1 are shown, along with representative photos of the appearance of each group of mice.

[0046] Figure 2 The fasting blood glucose, glycated hemoglobin, oral glucose tolerance test (OGTT), and blood lactate levels of each group of type 2 diabetes (T1DM) mice in Example 1 are shown, along with representative photos of the appearance of each group of mice.

[0047] Figure 3 The fasting blood glucose, glycated hemoglobin, oral glucose tolerance test (OGTT), and blood lactate levels of each group of steroid-induced diabetic mice in Example 1 are shown, along with representative photos of the appearance of each group of mice.

[0048] Figure 4 The results show the GLUT4 protein level and relative mRNA expression level in each group of mice in the three diabetes models in Example 1.

[0049] Figure 5 The changes in blood glucose levels in normal mice after injection of PLLA microspheres and glibenclamide, respectively, are shown in Example 1.

[0050] Figure 6 The results of Example 2 show the blood glucose levels, insulin levels, and insulin resistance index of normal mice, various groups of obesity-insulin resistance syndrome model mice, and drug-induced insulin resistance model mice.

[0051] Figure 7 The figures show the relative ATP and mtDNA content in normal C2C12 cells and various groups of palmitic acid-induced lipotoxic injury model cells in Example 3.

[0052] Figure 8 The figure shows the percentage of time spent exploring the new arm in normal mice and in each group of Alzheimer's disease model mice in Example 4.

[0053] Figure 9 The expression levels of key pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in the serum of normal mice and mice with metabolic-associated fatty liver disease induced by high-fat diet (HFD) in each group, as well as the serological indicators ALT and AST levels, are shown in Example 5.

[0054] Figure 10 Representative photographs of liver appearance in normal mice, high-fat diet (HFD) induced metabolic-associated fatty liver disease model mice, and T8a group mice treated with the drug are shown in Example 5. Detailed Implementation

[0055] The embodiments of the present invention will be described in detail below with reference to the examples. Unless otherwise specified, the conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0056] In the following preparation examples, the weight-average molecular weight of the polymer was determined by gel permeation chromatography (GPC), and the average particle size of the microspheres was determined by a laser particle size analyzer.

[0057] Microsphere preparation example

[0058] 1. PLLA microspheres (Mw=3 kDa)

[0059] Preparation method: 10 g of polylactic acid (PLLA, weight average molecular weight 3,000 Da) was dissolved in 150 mL of dichloromethane. After complete dissolution, the polymer solution was slowly added to 2000 mL of a 0.5% (w / w) aqueous solution of polyvinyl alcohol (PVA). Emulsification was carried out at 1200 rpm for 10 min. The stirring speed was reduced to 500 rpm, and stirring was continued for 3 h to evaporate and remove the dichloromethane. The mixture was then freeze-dried to obtain PLLA microspheres.

[0060] Characterization methods: The particle size range was determined using a laser particle size analyzer, and the particle size range was 15–55 μm. Microspheres with a particle size of 20–40 μm were then collected and used for drug delivery in the example.

[0061] 2. PLLA microspheres (Mw=20 kDa)

[0062] Preparation method: 12 g of polylactic acid (PLLA, weight average molecular weight 20,000 Da) was dissolved in 150 mL of dichloromethane. After complete dissolution, the polymer solution was slowly added to 2000 mL of a 0.5% (w / w) aqueous solution of polyvinyl alcohol (PVA). Emulsification was carried out at 2000 rpm for 10 min. The stirring speed was reduced to 500 rpm, and stirring was continued for 3 h to evaporate and remove the dichloromethane. The mixture was then freeze-dried to obtain PLLA microspheres.

[0063] Characterization methods: The particle size range was determined using a laser particle size analyzer, and the particle size range was 10–50 μm. Microspheres with a particle size of 20–40 μm were then collected and used for drug delivery in the example.

[0064] 3. PLLA microspheres (Mw=80 kDa)

[0065] Preparation method: 8 g of polylactic acid (PLLA, weight average molecular weight 80,000 Da) was dissolved in 150 mL of dichloromethane. After complete dissolution, the polymer solution was slowly added to 2000 mL of a 0.5% (w / w) aqueous solution of polyvinyl alcohol (PVA). Emulsification was carried out at 4000 rpm for 10 min. The stirring speed was reduced to 500 rpm, and stirring was continued for 3 h to evaporate and remove the dichloromethane. The mixture was then freeze-dried to obtain PLLA microspheres.

[0066] Characterization methods: The particle size range was determined using a laser particle size analyzer, and the particle size range was 15–45 μm. Microspheres with a particle size of 20–40 μm were then collected through uniform sieving and used for drug delivery in the example.

[0067] 4. PLGA microspheres (Mw=20 kDa)

[0068] 12 g of polylactic acid-glycolic acid copolymer (PLGA, weight average molecular weight 20,000 Da, lactic acid to glycolic acid molar ratio 75:25) was dissolved in 150 mL of dichloromethane. After complete dissolution, the polymer solution was slowly added to 2000 mL of 0.5% (w / w) aqueous polyvinyl alcohol (PVA). Emulsification was carried out at 2200 rpm for 10 min. The stirring speed was reduced to 500 rpm, and stirring was continued for 3 h to evaporate and remove the dichloromethane. The mixture was then freeze-dried to obtain PLGA microspheres.

[0069] Characterization methods: The particle size range was determined using a laser particle size analyzer, and the particle size range was 8–45 μm. Microspheres with a particle size of 20–40 μm were then collected and used for drug delivery in the example.

[0070] 5. PDLA microspheres (Mw=20 kDa)

[0071] 12 g of dextrorotatory polylactic acid (PDLA, weight average molecular weight 20,000 Da) was dissolved in 150 mL of dichloromethane. After complete dissolution, the polymer solution was slowly added to 2000 mL of a 0.5% (w / w) aqueous solution of polyvinyl alcohol (PVA). Emulsification was carried out at 2000 rpm for 10 min. The stirring speed was reduced to 500 rpm, and stirring was continued for 3 h to evaporate and remove the dichloromethane. The mixture was then freeze-dried to obtain PDLA microspheres.

[0072] Characterization methods: The particle size range was determined using a laser particle size analyzer, and the particle size range was 13–55 μm. Microspheres with a particle size of 20–40 μm were then collected and used for drug delivery in the example.

[0073] Drug preparation method

[0074] A 0.05 mg / ml glibenclamide solution was prepared using physiological saline, and administered intraperitoneally at a dose of 0.5 mg / kg based on the mouse's body weight.

[0075] Using physiological saline as a matrix, 0.5% (w / v) sodium carboxymethyl cellulose (CMC-Na) was added as a composite solvent to prepare PLLA microsphere solutions with a concentration of 50 mg / mL. Intramuscular injection was administered at a dose of 100 mg / kg based on mouse body weight. PLLA microsphere solutions of all molecular weights, as well as PLGA and PDLA microsphere solutions, were prepared using this method.

[0076] Prepare a neutral isotonic solution with a concentration of 100 mg / mL using physiological saline, and administer it intramuscularly at a dose of 500 mg / kg based on the mouse's body weight.

[0077] Example 1: Bidirectional Blood Glucose Regulation Experiment

[0078] Animal models: (1) Type 1 diabetes (T1DM) mouse model: Male C57BL / 6 mice (8 weeks old) were selected. After fasting for 12 h, STZ (150 mg / kg) was injected intraperitoneally once. The model establishment criteria were: random blood glucose was measured 72 hours after injection, and blood glucose was ≥16.7 mmol / L and remained stable for more than 3 days.

[0079] (2) Type 2 diabetes mellitus (T2DM) mouse model: Male C57BL / 6 mice (8 weeks old) were selected, fed a high-fat diet (60% fat content) for 8 weeks, and then fasted for 12 h in the 9th week before being injected intraperitoneally with streptozotocin (STZ, 40 mg / kg) for 3 consecutive days to establish the T2DM model. The model establishment criteria were: random blood glucose ≥16.7 mmol / L for 2 weeks.

[0080] (3) Steroid-induced diabetes / drug-induced diabetes mouse model: Male C57BL / 6 mice (8 weeks old) were selected and injected intraperitoneally with dexamethasone (DEX, 5 mg / kg / d) for 3 consecutive weeks to establish a steroid-induced diabetes model. The model establishment criteria were: fasting blood glucose (FBG) > 8.0 mmol / L.

[0081] Grouping and Dosing: Table 1. Injection Administration

[0082] Note: All other groups received a single dose, except for L-lactic acid monomer, which was administered daily.

[0083] Table 2 Oral Administration

[0084] For simplicity, the dosing regimens for the T2DM model group and the steroid-induced diabetes model group are not shown in the table. Referring to the grouping method for T1DM model mice in Table 1, the T2DM model mice and the steroid-induced diabetes model mice were grouped, with the dosing regimen, route of administration, dosage, and duration remaining consistent except for mouse condition. Specifically, the T2DM model mice were divided into a T2DM model control group (M2), experimental groups (T2a, T2b, T2c, T2d), and comparative groups (C2a, C2b); the steroid-induced diabetes model mice were divided into a steroid-induced diabetes model control group (M3), experimental groups (T3a, T3b, T3c, T3d), and comparative groups (C3a, C3b).

[0085] Experimental methods: 1. Fasting blood glucose: After treatment, the mice were fasted for 12 hours. Blood samples of 2 μL were collected from the mice using the tail clipping method, and the blood glucose levels were measured using an animal blood glucose meter (purchased from Hansi Trading).

[0086] 2. Glycated hemoglobin: (1) After collecting mouse blood, let it stand at room temperature for 30-60 min, then centrifuge the standing sample for 15 min (4℃, 1000g) and collect serum; (2) After taking the ELISA kit (purchased from Shanghai Enzyme Linker) out of the refrigerator, equilibrate it at room temperature for 20 min; follow the instructions of the kit to perform serial dilutions of the standard to obtain seven standard concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 ng / mL. Add the diluted standard to the pre-coated well plate, and use the standard dilution as the 0 ng / mL concentration, for a total of eight standard concentrations.

[0087] (3) Add the sample or standard of different concentrations to the corresponding well at a rate of 100 μL / well, seal the reaction well with sealing film, and react at 37°C in the dark for 90 min. Wash the plate 3 times, and pat it dry on thick absorbent paper for the last time.

[0088] (4) Add 100 μL of biotinylated antibody working solution to each well. Cover with sealing tape and incubate at 37°C in the dark for 60 min. After incubation, wash the plate 4 times and pat dry.

[0089] (5) Add 100 μL of 1×SA-HRP working solution to each well, cover with sealing tape, react at 37℃ in the dark for 30 min, wash 4 times, and pat dry.

[0090] (6) First add 50 μL of colorimetric solution A to each well, then add 50 μL of colorimetric solution B, mix gently, cover with sealing tape, and react at 37°C in the dark for 15 min.

[0091] (7) After the colorimetric reaction is complete, add 50 μL of stop solution to each well, mix gently, and measure the absorbance at 450 nm using a preheated microplate reader within 5 min.

[0092] 3. Oral glucose tolerance test (OGTT): After treatment, the animals were fasted for 12 hours. They were then administered a glucose solution at a dose of 1 g / kg body weight by gavage, and the blood glucose levels of each group of mice were immediately measured at 0, 30, 60, 90, and 120 min after administration of the glucose solution. The area under the curve was then calculated.

[0093] 4. Blood lactate level: (1) After collecting mouse blood, let it stand at room temperature for 30-60 min, then centrifuge the standing sample for 15 min (4℃, 1000g) and collect serum.

[0094] (2) The blood lactate content was determined using the L-lactic acid content detection kit (purchased from Solarbio, catalog number BC2230). 100 μL of liquid was added to 1 mL of extraction solution one, centrifuged at 12000 g at room temperature for 10 min, and 0.8 mL of supernatant was taken. Then, 0.15 mL of extraction solution two was slowly added and slowly mixed until no bubbles were generated. After centrifugation at 12000 g at room temperature for 10 min, the supernatant was taken for testing.

[0095] (3) Preheat the spectrophotometer for more than 30 minutes, adjust the wavelength to 570 nm, and zero it with distilled water. Incubate the working solutions of Reagent 2 and Reagent 4 at 37°C for 5 minutes.

[0096] (4) Sample addition table: (Add the following reagents to a 1.5 mL EP tube)

[0097] Table 3

[0098] L-LA content (umol / mL) = C standard ΔA determination ÷ ΔA standard x (V supernatant + V extract 2) ÷ [V liquid x V supernatant ÷ (V extract 1 + V liquid)] x F

[0099] =26.125 × ΔA (measured) ÷ ΔA (standard) × F

[0100] 5. GLUT4 protein level: Experimental methods for protein extraction and Western blotting 1) Collect mouse muscle tissue, add lysis buffer containing protease and phosphatase inhibitors, and grind the tissue into a homogenate using a mortar and pestle.

[0101] 2) Centrifuge at 12000 rcf for 25 min at 4℃, and determine protein concentration using Coomassie Brilliant Blue. Separate equal volumes of protein by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to a pre-activated methanol-modified polyvinylidene fluoride (PVDF) membrane. Block the PVDF membrane with 5% skim milk at room temperature to prevent the binding of nonspecific proteins.

[0102] 3) Incubate the membrane with an appropriately diluted primary antibody overnight at 4°C. Rinse the membrane with TBST buffer and incubate with secondary antibody at room temperature for 1 hour.

[0103] 4) Enhanced chemiluminescence solution was used to visualize protein bands, with GAPDH used as an internal control to control for differences in protein load.

[0104] RNA extraction and Real-time PCR reaction steps

[0105] 1) Collect 20mg of muscle tissue and add 1ml of Trizol. Use a mortar and pestle to grind the tissue into a homogenate. Add 0.2ml of chloroform to every 1ml of Trizol, shake vigorously for 15s, and let stand at room temperature for 2-3 minutes.

[0106] 2) Centrifuge at 12000 rcf at 4℃ for 15 min, and carefully aspirate the uppermost layer. Add isopropanol to the aspirated supernatant at a ratio of 0.5 ml per 1 ml Trizol, and let stand at room temperature for 10 min.

[0107] 3) Centrifuge at 12000 rcf at 4℃ for 10 min, discard the supernatant, and obtain the RNA precipitate. Add 1 ml of 75% ethanol.

[0108] 4) Centrifuge at 7500 rcf at 4℃ for 5 min, repeat once, and wash the RNA precipitate. Remove all ethanol and air dry for 5-10 min.

[0109] 5) Dissolve the RNA precipitate in an appropriate amount of nuclease-free water, measure the A260 / A280 ratio, and analyze the purity and concentration of total RNA.

[0110] 6) 500 ng of total RNA was transcribed into cDNA. Gene expression was quantified using a real-time quantitative PCR system. CT values ​​were analyzed using the 2-ΔΔCT method. Primer sequences (5' ~ 3') are as follows: SLC2A4 F: 5'CATTCCCTGGTTCATTGTGG-3' (SEQ ID NO. 1) SLC2A4 R: 5'- GAAGACGTAAGGACCCATAGC-3' (SEQ ID NO. 2) GAPDH F: 5'-CATCACTGCCACCCAGAAGACTG-3' (SEQ ID NO. 3) GAPDH R: 5'-ATGCCAGTGAGCTTCCCGTTCAG-3' (SEQ ID NO. 4) mt-Nd1 F: 5'-CTAGCAGAAACAAACCGGGC-3' (SEQ ID NO. 5) mt-Nd1 R: 5'-CCGCTTAATTTTTCGTCGGG-3' (SEQ ID NO. 6) Hkb F: 5'-GCCAGCCTCTCCTGATTTTAGTGT-3' (SEQ ID NO. 7) Hkb R: 5'-GGGAACACAAAAGACCTCTTCTGG -3' (SEQ ID NO. 8) Experimental results: To verify the hypoglycemic effects of PLLA and PLGA microspheres, three different types of diabetes models were established in this embodiment. Based on the blood glucose levels in the model groups, type 1 diabetes, type 2 diabetes, and steroid-induced diabetes were all successfully modeled. Experimental results showed that administration of PLLA, PLGA, and L-lactic acid significantly reduced blood glucose and glycated hemoglobin levels and significantly improved glucose tolerance in the model group mice. PDLA microspheres did not show a significant hypoglycemic effect. Although lactic acid also has a hypoglycemic effect, it requires multiple high-dose injections, which significantly increases the risk of hyperlactatemia and is accompanied by significant side effects. Figures 1-3 At the same time, PLLA microspheres also showed the effect of promoting the expression of GLUT4 mRNA and protein in muscle. Figure 4 Therefore, it can be concluded that PLLA microspheres can exert a certain hypoglycemic effect without relying on insulin.

[0111] To further verify the bidirectional hypoglycemic effect of PLLA microspheres, the inventors injected high-dose PLLA microspheres (PLLA molecular weight 20 kDa, intramuscular injection, dose 250 mg / kg, single administration) into mice with normal blood glucose levels. Since PLLA microspheres degrade slowly in vivo, to ensure the accuracy of the results, the inventors monitored the blood glucose levels of the mice in the PLLA microsphere administration group weekly for 8 weeks after administration, and found no hypoglycemia. A control group of glibenclamide (0.5 mg / kg) was set up. Since glibenclamide takes effect within a few hours after administration, for better comparison, the inventors fasted the mice for 4 hours every 2 weeks, followed by glibenclamide injection, and then monitored the blood glucose levels of the mice at 30, 60, 90, 120, 150, and 180 minutes after injection. The results showed that glibenclamide lowered the blood glucose levels of normal mice, but PLLA microspheres did not. (See...) Figure 5 (Only the changes in blood glucose after 8 weeks of glibenclamide injection are shown). It can be concluded that PLLA microspheres have a bidirectional regulatory effect on blood glucose and are independent of insulin.

[0112] Example 2: Insulin Resistance Test

[0113] Animal models: 1. A model of obesity-insulin resistance syndrome was established by feeding male C57BL / 6 mice a high-fat diet (HFD, 60% fat calories) for 12 weeks. The model was established based on the following criteria: significantly elevated insulin levels, significantly elevated fasting blood glucose levels (8.0-11.0 mmol / L), and a two-fold increase in HOMA-IR.

[0114] 2. Drug-induced insulin resistance model: Dexamethasone (Dex) induction method, intraperitoneal injection of 1 mg / kg / day for 7 consecutive days to establish a drug-induced insulin resistance model. Model establishment criteria: significantly increased insulin level, significantly increased fasting blood glucose level (8.0-11.0 mmol / L), and a 2-fold increase in HOMA-IR.

[0115] Grouping and administration: Referring to the grouping method of T1DM model mice in Table 1 of Example 1, the obese-insulin resistance syndrome model mice and the drug-induced insulin resistance model mice were grouped together. Except for the mouse status, the administration regimen, administration route, administration dose and cycle were kept consistent.

[0116] Obesity-insulin resistance syndrome (OIS) model mice were divided into three groups: control group (M4), experimental group (T4a, T4b, T4c, T4d), and control group (C4a, C4b). Drug-induced insulin resistance model mice were divided into three groups: control group (M5), experimental group (T5a, T5b, T5c, T5d), and control group (C5a, C5b). In addition to the above groups, a normal control group (K group) was also included.

[0117] Experimental methods: 1. Fasting insulin level: (1) After collecting mouse blood, let it stand at room temperature for 30-60 min, then centrifuge the standing sample for 15 min (4℃, 1000g) and collect serum.

[0118] (2) After taking the ELISA kit (purchased from Jianglai Biotechnology) out of the refrigerator, allow it to equilibrate at room temperature for 10 min; follow the instructions of the kit to prepare the standard into 8 concentration gradients of 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.2 pg / mL and 0 pg / mL.

[0119] (3) Add 50 μL of sample or standard of different concentrations to each well, add 50 μL of universal diluent to each blank well, and then add 50 μL of Biotin-antibody working solution to each well. Cover with sealing film and incubate at 37°C for 60 min. Wash the plate 3 times, and pat dry on thick absorbent paper for the last time.

[0120] (4) Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 min, wash the plate 5 times, and finally place it on thick absorbent paper to dry.

[0121] (5) Add 90 μL TMB to each well, cover with sealing film, incubate at 37℃ in the dark for 15 min, take out the microplate, add 50 μL stop solution directly to each well, and immediately measure the OD value of each well at a wavelength of 450 nm.

[0122] 2. Fasting blood glucose: After treatment, the mice were fasted for 8 hours. Blood samples of 2 μL were collected from the mice using the tail clipping method, and the blood glucose levels were measured using an animal blood glucose meter (purchased from Hansi Trading).

[0123] 3. Insulin resistance index: HOMA-IR = Fasting blood glucose (FBG, mmol / L) × Fasting insulin (FINS, mIU / L) / 22.5 Experimental results: This embodiment verifies the ameliorative effects of PLLA microspheres and PLGA microspheres on insulin resistance. An obesity-induced insulin resistance model (M4) and a drug-induced insulin resistance model (M5) were constructed. The results showed that, compared with the model group, intervention with PLLA microspheres, PLGA microspheres, or L-lactic acid significantly reduced fasting blood glucose levels, increased insulin secretion stimulation, and decreased the insulin resistance index in mice. Figure 6 Both insulin resistance and fasting blood glucose were improved. Comparative experiments showed no significant improvement in insulin resistance in the PDLA microsphere group. In conclusion, PLLA and PLGA microspheres of different molecular weights all have significant effects in improving insulin resistance.

[0124] Example 3: Mitochondrial Function Protection Test

[0125] The effects of drug administration on various cellular mitochondrial functions were demonstrated by detecting ATP production and mtDNA copy number in cells.

[0126] Table 4

[0127] Experimental methods

[0128] Grouping and Dosing

[0129] K: DMEM medium, treated for 24 hours

[0130] M6: DMEM basal medium contains 0.4 mM PA

[0131] T6a: DMEM basal medium containing 0.4 mM PA and 3 KD PLLA microspheres.

[0132] T6b: DMEM basal medium containing 0.4 mM PA and 20 kDa PLLA microspheres.

[0133] T6c: DMEM basal medium containing 0.4 mM PA and 80 kDa PLLA microspheres.

[0134] T6d: DMEM basal medium containing 0.4 mM PA and PLGA microspheres.

[0135] C6a: DMEM basal medium containing 0.4 mM PA and PDLA porous microspheres.

[0136] C6b: DMEM basal medium contains 0.4 mM PA and 25 mM lactic acid.

[0137] Cell culture: 1. Seed 1 mL of cell suspension into each well of a 12-well cell culture plate. Pre-culture the plate overnight at 37°C in a 5% CO2 incubator.

[0138] 2. Add the corresponding drugs mentioned above to the wells and incubate the culture plate in an incubator for 24 hours.

[0139] Experimental methods: 1. ATP production detection (1) For cells in 12-well plates, add 100 μL of lysis buffer to each well. Use a pipette to repeatedly pipette or shake the culture plate to ensure the lysis buffer fully contacts and lyses the cells. After lysis, centrifuge at 12000g for 5 min at 4℃, and collect the supernatant for subsequent assays.

[0140] (2) According to the kit instructions, set the concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 µM to plot the standard curve.

[0141] (3) Each sample requires 100 μL of ATP detection working solution. Take an appropriate amount of ATP detection reagent and dilute the ATP detection reagent with ATP detection reagent diluent at a ratio of 1:9.

[0142] (4) Add 100 μL of ATP detection working solution to the detection well or detection tube. Let it stand at room temperature for 3-5 min, then add 20 μL of sample or standard to the detection well or detection tube, mix quickly with a micropipette, and measure the RLU value with a chemiluminescence analyzer after 2 s interval.

[0143] 2. mtDNA content detection

[0144] mtDNA extraction

[0145] According to the instructions of the Animal Mitochondrial DNA Extraction Kit (PCR grade), purchased from Bio-Rad, the following procedures were performed.

[0146] (1) The cells were treated with 0.25% trypsin digestion solution, centrifuged and the supernatant was discarded. The cell pellet was retained and washed twice with PBS buffer. The cell pellet obtained was then used directly as material for animal mtDNA extraction.

[0147] (2) Add 250 μL of animal mitochondrial DNA purification solution A in an ice bath and disperse thoroughly by pipetting.

[0148] (3) Add 250 μL of animal mitochondrial DNA purification solution B at room temperature, gently invert and mix 10 times, and place on ice for 8 min.

[0149] (4) Add 350 μL of animal mitochondrial DNA purification solution C in an ice bath, gently invert and mix 6 times until a white precipitate is formed, and place on ice for 20 min.

[0150] (5) Centrifuge at 13000g for 10 min at room temperature, carefully transfer the supernatant to the centrifugal adsorption column, let stand for 5 min to allow the DNA to fully combine with the centrifugal adsorption column, centrifuge at 13000g for 1 min at room temperature, and discard the waste liquid in the collection tube.

[0151] (6) Add 500 μL of general column washing solution, centrifuge at 13000g for 1 min at room temperature, discard the waste liquid in the collection tube, and repeat this step twice.

[0152] (7) Centrifuge at 13000g for 1 min at room temperature and shake off the residual liquid.

[0153] (8) Place the centrifugal adsorption column into a new 1.5 mL plastic centrifuge tube, add 50 μL of DNA elution buffer, and incubate at room temperature for 2 min.

[0154] (9) Centrifuge at 13000g for 1 min at room temperature. The solution at the bottom of the centrifuge tube is mtDNA.

[0155] The content of mitochondrial DNA in cells was detected using real-time quantitative PCR. The mitochondrial cytochrome c oxidase 1 gene (mt-Co1) was selected as the target gene, and 18S rDNA was used as a nuclear internal reference gene for amplification. The copy number ratio of mitochondrial DNA to nuclear DNA was normalized, and the differences in mitochondrial DNA copy number among groups were compared.

[0156] Experimental results: This embodiment uses an in vitro cell model to verify the protective effects of PLLA and PLGA microspheres on mitochondrial function. C2C12 mouse skeletal muscle myoblasts were used to construct a lipotoxic injury model (M6) induced by palmitic acid to simulate mitochondrial dysfunction under pathological conditions. The results showed that, compared with the palmitic acid-treated model group, intervention with PLLA microspheres, PLGA microspheres, and lactate monomers of different molecular weights significantly increased intracellular ATP levels and significantly improved mtDNA copy number synthesis. No significant effect was observed in the PLLA microsphere group. Figure 7 The above results indicate that PLLA microspheres, PLGA microspheres, and lactate monomers can effectively antagonize lipotoxicity-induced mitochondrial damage and improve mitochondrial function by promoting energy metabolism synthesis and mitochondrial biogenesis.

[0157] Example 4: Animal Model Experiment for Neuroprotection

[0158] Aβ1–42-induced (AD) models were established, and the cognitive function of mice was evaluated after 8 weeks of drug treatment.

[0159] Methods for establishing an Aβ1–42-induced AD model in mice

[0160] Seven-week-old male C57BL / 6J mice were selected and acclimatized for one week before the experiment began.

[0161] The models are divided into the following groups:

[0162] K: Intraperitoneal injection of normal saline.

[0163] M7: Intraperitoneal injection of Aβ1–42 10 μg / day for 14 consecutive days, once daily.

[0164] T7a: The treatment for the first 14 days was the same as that for the M7 group. PLLA injections began on day 15, with the dosage, method, and timing consistent with implementation 1.

[0165] T7b: The treatment for the first 14 days was the same as that for the M7 group. PLLA injections began on day 15, with the dosage, method, and timing consistent with implementation 1.

[0166] T7c: The treatment for the first 14 days was the same as that for the M7 group. PLLA injections began on day 15, with the dosage, method, and timing consistent with implementation 1.

[0167] T7d: The treatment for the first 14 days was the same as that for the M7 group. PLGA injections began on day 15, with the dosage, method, and timing consistent with implementation 1.

[0168] C7a: The treatment for the first 14 days was the same as that for the M7 group. PDLA injections began on day 15, with the dosage, method, and timing being the same as in implementation 1.

[0169] C7b: The treatment for the first 14 days was the same as that for the M7 group. Lactic acid injections began on day 15, with the dosage, method, and timing being the same as in implementation 1.

[0170] Grouping and administration: Following the grouping method of T1DM model mice in Table 1 of Implementation 1, AD model mice were grouped, and the dosing regimen, route of administration, dosage and period of administration were kept consistent except for the mouse status.

[0171] The AD model mice were divided into three groups: AD model control group (M7), experimental groups (T7a, T7b, T7c, T7d), and comparative groups (C7a, C7b). In addition to the above groups, a normal control group (K group) was also included.

[0172] Y-maze experimental method: The Y-maze consists of three arms (30 cm long, 6 cm wide, and 15 cm high), arranged at a 120° angle, and is defined as the starting arm (A), the other arm (B), and the new arm (C).

[0173] Training phase: Close the new arm (C), place the mouse at the end of the starting arm (A), and allow it to explore freely between arm A and arm B for 8 minutes, then return it to the rearing cage to rest for 4 hours.

[0174] Formal testing phase: Remove the C-arm closure, reinsert the mouse from the end of the starting arm (A), record the exploration time of each arm, and calculate the percentage of the total exploration time in the new arm (C).

[0175] Experimental results: This study evaluated the protective and reparative effects of targeted PLLA microspheres and PLGA on neurological damage. An Alzheimer's disease (M7) mouse model was established using Aβ1–42 induction to simulate neurotoxic-induced cognitive impairment. Behavioral assessments showed that in the Y-maze test, compared to the model group, treatment with different molecular weights of PLLA microspheres, PLGA microspheres, or lactate monomers significantly increased the exploration time of mice for the new arm. No significant effect was observed in the PDLA microsphere group. Figure 8 Experimental results confirmed that PLLA microspheres and PLGA microspheres can effectively improve Aβ1–42-induced spatial working memory impairment. This indicates that PLLA microspheres and PLGA microspheres possess neuroprotective activity.

[0176] Example 5: Prevention and Treatment Trial of Metabolic Associative Fatty Liver Disease (MAFLD) / Metabolic Associative Steatohepatitis (MASH)

[0177] Animal models and grouped administration: Male C57BL / 6 mice (8 weeks old) were selected and fed a high-fat diet (60% fat content) for 8 weeks. The mice were then randomly divided into model group M, PLLA microsphere group, PLGA microsphere group, PDLA microsphere group, and lactate monomer group. The grouping method is as described in Example 1.

[0178] Model group M received an intramuscular injection of 300 μL of physiological saline.

[0179] The PLLA microsphere group, PLGA microsphere group, and PDLA microsphere group were administered once via intramuscular injection at a dose of 100 mg / kg.

[0180] The lactate monomer group was administered 500 mg / kg via intramuscular injection daily for 8 weeks.

[0181] Experimental methods: Eight weeks after drug administration, mice in each group were fasted for 12 hours, and 500 μL of blood was collected from the orbital venous plexus. The plasma was then obtained by centrifugation at 10,000 rpm for 20 min at 4°C. The levels of IL-6, TNF-α, IL-1β, AST, and ALT in the plasma were measured.

[0182] 1. Methods for detecting mouse interleukin-6 (IL-6) and TNF-α levels: The experiment was conducted according to the instructions of the test kit (purchased from Shanghai Enzyme-Linked Biotechnology): First, according to the experimental instructions, the standard was prepared into eight concentration gradients: 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.63 pg / mL, 7.81 pg / mL, and 0 pg / mL.

[0183] Sample addition: Add 100 μL of standard at different concentrations and pretreated test sample to each well, cover with sealing tape, and react at 37℃ in the dark for 1.5 h.

[0184] Washing: After incubation, add 300 μL of 1× washing buffer to each well, gently shake for 30 seconds, shake dry and pat dry on paper. Wash in this way 3 times.

[0185] Antibody incubation: Add 100 μL of biotinylated antibody working solution to each well, mix gently, cover with sealing tape, and incubate at 37°C in the dark for 1 hour. After incubation, repeat the above steps to wash 4 times.

[0186] Enzyme label incubation: Add 100 μL of 1×SA-HRP working solution to each well, cover with sealing tape, incubate at 37°C in the dark for 30 min, wash 4 times, and pat dry.

[0187] Substrate development: First add 50 μL of colorimetric reagent A to each well, then add 50 μL of colorimetric reagent B, mix gently, cover with sealing tape, and react at 37°C in the dark for 15 min.

[0188] Termination of reaction: After the colorimetric reaction is complete, add 50 μL of stop solution to each well, mix gently, and measure the absorbance at 450 nm using a preheated microplate reader within 5 min.

[0189] 2. Methods for detecting IL-1β levels in mice: The experiment was conducted according to the instructions of the test kit (purchased from Shanghai Enzyme-Linked Biotechnology): First, according to the experimental instructions, the standards were prepared into eight concentration gradients: 1500 pg / mL, 750 pg / mL, 375 pg / mL, 187.5 pg / mL, 93.75 pg / mL, 46.88 pg / mL, 23.44 pg / mL, and 0 pg / mL. The remaining steps were the same as those for the IL-6 and TNF-α kits.

[0190] 3. Methods for detecting AST levels in mice: The experiment was conducted according to the instructions for the test kit (purchased from Jianglai Biotechnology): First, according to the experimental instructions, the standard was prepared into eight concentration gradients: 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.12 ng / mL, and 0 ng / mL.

[0191] Add samples: Add 100 μL of the sample or standard of different concentrations to the corresponding well, and add 100 μL of diluent to the blank well. Incubate at 37°C for 60 minutes.

[0192] Add biotinylated antibody: Remove the microplate, discard the liquid, add 100 μL of biotinylated antibody working solution to each well, and incubate at 37°C for 60 minutes.

[0193] Washing the plate: Discard the liquid, add 300μL of washing solution to each well, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 3 times.

[0194] Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film, and incubate at 37°C for 30 minutes. Discard the liquid and wash the plate 5 times according to the washing method described above.

[0195] Add substrate: Add 90 μL of TMB to each well and incubate at 37°C in the dark for 15 minutes.

[0196] Add stop solution: Remove the microplate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450 nm.

[0197] 4. Methods for detecting ALT levels in mice: The experiment was conducted according to the instructions for the test kit (purchased from Jianglai Biotechnology): First, according to the experimental instructions, prepare the standards into eight concentration gradients: 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.12 ng / mL, 1.56 ng / mL, 0.78 ng / mL, and 0 ng / mL. The remaining steps are the same as those for the AST kit.

[0198] Experimental results: This study established a metabolic-associated fatty liver disease (MAF) model to verify the protective effect of the target product on liver damage. In this embodiment, a high-fat diet (HFD) was used to induce an animal model of M8 MAF. The results showed that, compared with the model group, intervention with PLLA microspheres, PLGA microspheres, or lactate monomers significantly downregulated the expression levels of key pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in mouse serum. Simultaneously, the levels of serological indicators reflecting the degree of hepatocyte damage, ALT (alanine aminotransferase) and AST (aspartate aminotransferase), were significantly reduced. The PDLA microsphere group showed no significant effect. Figure 9 ).

[0199] Normal mouse livers are bright red in appearance, normal in shape, and without obvious enlargement. In the metabolic-associated fatty liver disease model group, the livers of mice were significantly enlarged, with a marked increase in volume, and abnormal color and shape. Compared with the model group, the liver enlargement in the PLLA-treated group was significantly reduced, and the size was between that of the normal control group and the model group.

[0200] The above results confirm that PLLA microspheres and PLGA microspheres can effectively inhibit the liver inflammatory response induced by high lipids, reduce hepatocyte damage, and have significant therapeutic potential and liver protective effects for metabolic-related fatty liver disease.

[0201] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Any implementation method that has the same structure and achieves the same effect as the technical concept within the scope of this application is included in the technical scope of this application. Furthermore, those skilled in the art should understand that various modifications conceivable by those skilled in the art to the implementation methods, and other ways of constructing by combining some of the constituent elements of the implementation methods, without departing from the spirit and scope of the technical solutions of the present invention, should all be covered within the scope of the claims of the present invention.

Claims

1. Use of a polymer in the preparation of a pharmaceutical, said polymer comprising polylactic acid (PLA) or a copolymer containing repeating L-lactic acid units, said repeating L-lactic acid units being... The drug is used for one or more of the following purposes: bidirectional regulation of blood glucose, improvement of insulin resistance, protection of mitochondrial function, repair of nervous system damage, inhibition of liver inflammatory response, and reduction of hepatocyte damage.

2. The use according to claim 1, wherein, The copolymer containing repeating units of L-lactic acid includes one or more of the following: L-polylactic acid-glycolic acid copolymer, L-polylactic acid-polyethylene glycol copolymer, polyethylene glycol-L-polylactic acid-glycolic acid copolymer, L-polylactic acid-chitosan copolymer, and lactide-caprolactone copolymer.

3. The use according to claim 1 or 2, wherein, The weight-average molecular weight of the L-lactic acid or copolymer containing L-lactic acid repeating units is 1,000 to 200,000 Da, preferably 5,000 to 100,000 Da, and more preferably 3,000 to 80,000 Da.

4. The use according to any one of claims 1-3, wherein, In terms of molar ratio, the proportion of L-lactic acid repeating units in the copolymer containing L-lactic acid repeating units is greater than or equal to 5%, preferably 5%-75%.

5. The use according to any one of claims 1-4, wherein, The drug comprises microspheres, in which polylactic acid or a copolymer containing repeating units of polylactic acid is present.

6. The use according to claim 5, wherein, The microspheres have an average particle size of at least 1 μm, preferably an average particle size of 1 to 200 μm, and more preferably an average particle size of 20 to 60 μm, for example 15 to 55 μm, for example 20 to 40 μm.

7. The use according to any one of claims 1-6, wherein, The drug may contain a pharmaceutically acceptable carrier or excipient; Preferably, the drug is formulated as an oral dosage form or an injectable dosage form; Preferably, the drug is formulated as a sustained-release preparation.

8. The use according to claim 7, wherein, The drug is in the form of an injectable (e.g., an intramuscular injection); Preferably, the injection comprises a liquid matrix and a complex solvent; Preferably, the liquid matrix is ​​physiological saline; Preferably, the composite solvent includes sodium carboxymethyl cellulose; Preferably, the injection contains 30-70 mg / mL of polylactic acid or a copolymer containing repeating units of polylactic acid; Preferably, the L-lactic acid or a copolymer containing repeating L-lactic acid units is present in the microspheres.

9. The use according to any one of claims 1-8, wherein, The drug is used to prevent and / or treat diabetes or its complications by bidirectional regulation of blood glucose; Preferably, the diabetes mellitus is selected from type 1 diabetes, type 2 diabetes, and steroid diabetes. Preferably, the bidirectional regulation of blood glucose includes reducing an individual's blood glucose level, reducing an individual's glycated hemoglobin level, improving an individual's glucose tolerance, and / or promoting an individual's muscle GLUT4 mRNA and protein expression without relying on insulin; preferably, the individual is a diabetic patient; Preferably, the drug does not lower the blood glucose level in normal individuals.

10. The use according to any one of claims 1-9, wherein, The drug is used for the prevention and / or treatment of insulin resistance syndrome; Preferably, the insulin resistance syndrome is manifested as one or more of the following conditions: obesity, hyperinsulinemia, dyslipidemia, hypertension, fatty liver, polycystic ovary syndrome (PCOS), metabolic syndrome, or combined with arteriosclerosis; Preferably, the improvement of insulin resistance includes: reducing an individual's fasting blood glucose level, reducing an individual's blood insulin level, and / or reducing an individual's insulin resistance index.

11. The use according to any one of claims 1-10, wherein, The drug is used for the prevention and / or treatment of diseases related to mitochondrial dysfunction; Preferably, the mitochondrial dysfunction-related diseases include diabetes or its complications (e.g., type 1 and type 2 diabetes, diabetic cardiomyopathy), neurodegenerative diseases (e.g., Alzheimer's disease), cardiovascular diseases, tumors, fatty liver, or schizophrenia. Preferably, the protection of mitochondrial function includes increasing the content of ATP in individual cells and increasing the content of mitochondrial DNA in individual cells.

12. The use according to any one of claims 1-11, wherein, The drug is used to prevent and / or treat neurodegenerative diseases (such as Alzheimer's disease). Preferably, the drug is used to improve an individual's cognitive function by repairing damage to the nervous system.

13. The use according to any one of claims 1-12, wherein, The drug is used for the prevention and / or treatment of metabolic-associated fatty liver disease or metabolic-associated steatohepatitis. Preferably, the inhibition of liver inflammatory response or reduction of hepatocyte damage includes: reducing the expression level of pro-inflammatory cytokines (e.g., IL-6, IL-1β, or TNF-α) in serum, or reducing the level of ALT or AST in serum.

14. An injectable preparation comprising polymer microspheres; the polymer comprising poly(L-lactic acid) or a copolymer comprising repeating L-lactic acid units, wherein the repeating L-lactic acid units are... ; Preferably, the polymer is as defined in any one of claims 2-4; Preferably, the polymer microspheres are as defined in claim 6; Preferably, the injectable is as defined in claim 8; Preferably, the injection is used for the purpose described in any one of claims 1 or 9-13.

15. A health food product comprising polymer microspheres; the polymer comprising poly(L-lactic acid) or a copolymer comprising repeating units of L-lactic acid, wherein the repeating units of L-lactic acid are... ; Preferably, the polymer is as defined in any one of claims 2-4; Preferably, the polymer microspheres are as defined in claim 6; Preferably, the health supplement is used to maintain healthy blood sugar levels.

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