Application of PLGA (poly (lactic-co-glycolic acid)) to treatment of lean non-alcoholic fatty liver disease by taking Marco + macrophages as target

Abstract

The invention discloses application of PLGA (poly (lactic-co-glycolic acid)) to treatment of lean non-alcoholic fatty liver disease by taking Marco + macrophages as targets, and belongs to the technical field of biological medicines. The invention proves that the tail vein injection PLGA specifically targets abnormally increased Marco + macrophages in the liver of lean non-alcoholic fatty liver, effectively inhibits the inflammatory response and fibrosis process mediated by the Marco + macrophages, remarkably reduces liver lipid deposition, relieves insulin resistance and recovers the fat storage function of adipose tissue. The invention provides a new strategy for treating the lean non-alcoholic fatty liver disease by targeting the PLGA to the Marco + macrophages for the first time, expands the clinical transformation potential of the PLGA in the treatment of the metabolic liver disease, and provides a new scheme for the intervention and treatment of the lean non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to PLGA with Marco + The invention relates to the application of macrophages as targets in the treatment of lean non-alcoholic fatty liver disease, and belongs to the field of biomedicine technology. Background Art

[0002] Lean nonalcoholic fatty liver disease (Lean MAFLD) is a hepatic manifestation of metabolic syndrome. Its diagnostic criteria are hepatic steatosis involving ≥5% of hepatocytes and a patient with a normal body mass index (BMI < 25 kg / m2 in Western populations). 2 , East Asian population BMI < 23 kg / m 2 ). Epidemiological studies have shown that lean MAFLD accounts for 7%-23% of global cases and has more serious clinical outcomes, including progressive liver fibrosis, cardiovascular events, and significantly increased all-cause mortality. Its pathological core lies in lipotoxicity driven by abnormal cholesterol metabolism, rather than the traditional belief of triglyceride (TG) accumulation. Even a slight accumulation of cholesterol can activate oxidative stress, mitochondrial dysfunction, and inflammatory response, and a high-cholesterol diet is significantly associated with the onset of lean MAFLD. However, the molecular mechanism of cholesterol metabolism disorders and the progression of fibrosis in lean MAFLD has not yet been elucidated, and there is a clear gap in targeted intervention strategies.

[0003] Macrophages are the core targets for regulating liver inflammation and fibrosis, among which Marco + Macrophage subsets participate in metabolic homeostasis by secreting anti-inflammatory factors and regulating tissue repair. + Macrophages have both pro-inflammatory and pro-fibrotic functions in obesity-related MAFLD, but their phenotypes are significantly heterogeneous in lean MAFLD. The latest single-nucleus RNA sequencing data suggest that macrophages in the liver tissue of lean MAFLD patients have a high expression of macrophages. + The proportion of macrophages is abnormally elevated and positively correlated with the expression of liver fibrosis markers (α-SMA and Col1α1). However, existing research has mostly focused on macrophage-mediated inflammatory pathways, and there is still a lack of systematic analysis of its non-inflammatory regulatory mechanisms (such as cholesterol metabolic reprogramming) and targeted intervention strategies.

[0004] Poly(lactic-co-glycolic acid) (PLGA) is an FDA-approved biodegradable nanocarrier that can specifically induce Marco + Macrophage apoptosis. Existing studies have confirmed that PLGA nanoparticles can effectively improve tissue inflammation and fibrosis by targeting and eliminating pathologically activated macrophages in models of myocardial infarction, autoimmune encephalomyelitis, and multi-organ fibrosis. Its mechanism of action includes charge-mediated cell membrane interactions and programmed cell death induced by lysosomal escape. Based on Marco +Macrophages play a key pathological role in lean MAFLD. The targeted delivery characteristics of PLGA provide an innovative technical path for precisely regulating the liver immune microenvironment and blocking the fibrosis process driven by abnormal cholesterol metabolism. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a new target and potential treatment plan for the clinical treatment of lean non-alcoholic fatty liver disease.

[0006] To achieve the above object, the present invention provides PLGA with Marco + Application of macrophage targeting in the treatment of lean nonalcoholic fatty liver disease.

[0007] The present invention determined that Marco + The present invention proves that tail vein injection of PLGA targeted intervention of Marco + Macrophages, significantly inhibit liver fat deposition, relieve insulin resistance, and restore fat storage capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Figure 2 Pathological changes of liver and adipose tissue in mice fed a normal diet (NCD), a high-fat diet (HFD), and a high-cholesterol diet (PD). A is the weight gain curve of mice under the three diet models; B is the ratio of liver weight to body weight of the three groups of mice; C is the ratio of fat weight to body weight of the three groups of mice; D is the H&E staining, picrosirius red staining, and F4 / 80 staining of liver sections of the three groups of mice. + Immunohistochemical staining; EH is the quantitative score of MAFLD activity in mouse liver in terms of steatosis, hypertrophy, inflammation and fibrosis.

[0009] Figure 2 Marco in clinical samples and mouse models + Changes in the number of macrophages. A is the Marco immunofluorescence staining of liver sections from normal people, non-lean non-alcoholic fatty liver patients, and lean non-alcoholic fatty liver patients in clinical samples; B is the Marco + Quantitative results of cell ratio; C is Marco immunohistochemical staining of liver sections of NCD mice and PD mice; D is Marco + Quantitative results of cell proportion.

[0010] Figure 3 The mouse model was established by feeding a high-cholesterol diet and undergoing intervention (tail vein injection of saline or PLGA solution). A shows the treatment regimen of PD-fed mice; B shows H&E staining of heart, spleen, lung, and kidney tissues.

[0011] Figure 4 The liver histopathological results of mice fed a high cholesterol diet and intervened (tail vein injection of normal saline or PLGA solution). A is a representative image of liver tissue. The bar graph shows the ratio of liver weight to body weight. B is the F4 / 80 + and Marco + Cell immunohistochemical staining image results and quantitative analysis; C is the levels of AST and ALT in mouse serum; D is H&E staining, Oil Red O staining, and Sirius Red staining of mouse liver sections; E is the quantitative score of MAFLD activity in mouse liver in terms of steatosis, hypertrophy, inflammation, and fibrosis.

[0012] Figure 5 Figure 5 is the insulin resistance test results of mice fed a high-cholesterol diet and intervened (tail vein injection of normal saline or PLGA solution). AC are the results of glucose tolerance test, insulin tolerance test and pyruvate tolerance test in mice.

[0013] Figure 6 Figure 3 Protein expression levels of insulin resistance-related pathways in the livers of mice fed a high-cholesterol diet and intervened (tail vein injection of saline or PLGA solution). A is the protein immunoblotting results of P-IRS1, IRS1, P-AKT, and AKT in mouse liver tissue; B is the quantitative analysis of the protein expression ratio of P-IRS1 / IRS1; C is the quantitative analysis of the protein expression ratio of P-AKT / AKT.

[0014] Figure 7 The pathological results of epididymal white adipose tissue in mice fed a high-cholesterol diet and intervened (tail vein injection of normal saline or PLGA solution). A is a representative image of epididymal white adipose tissue. The bar graph shows the ratio of epididymal white adipose tissue weight to body weight. B is H&E staining of epididymal white adipose tissue sections and F4 / 80 + Immunohistochemical staining; C is the analysis of adipocyte diameter in epididymal white adipose tissue; D is the statistical analysis of F4 / 80 positive area in epididymal white adipose tissue.

[0015] Figure 8Figure 3 shows the mRNA expression levels of fatty acid metabolism-related genes in the liver tissue and epididymal white adipose tissue of mice fed a high-cholesterol diet and intervened (tail vein injection of normal saline or PLGA solution). A is the mRNA expression level of fatty acid synthesis-related genes in mouse liver; B is the mRNA expression level of fatty acid uptake-related genes in mouse liver; C is the mRNA expression level of fatty acid decomposition-related genes in mouse liver; D is the mRNA expression level of fatty acid synthesis-related genes in epididymal white adipose tissue of mouse; E is the mRNA expression level of fatty acid uptake-related genes in epididymal white adipose tissue of mouse; F is the mRNA expression level of fatty acid decomposition-related genes in epididymal white adipose tissue of mouse.

[0016] Among them: *, p < 0.05; **, p < 0.01; ***, p < 0.001. DETAILED DESCRIPTION

[0017] The following is a detailed description of the specific embodiments of the present invention in conjunction with the accompanying drawings, providing a detailed basis for those skilled in the art to fully understand the technical solution. The experimental methods described in the present invention are based on internationally recognized standard operating procedures, and the experimental reagents are purchased through commercial channels. All human research protocols have been approved by the Medical Ethics Committee of the hospital where the samples were obtained. Wild-type C57BL / 6J mice were purchased from the Animal Management Center of Chongqing Medical University. All animal experiments involved in this study met the standards set by the Animal Ethics Committee of Chongqing Medical University and strictly followed the "Guidelines for the Care and Use of Laboratory Animals" and the "National Laboratory Animal Quality Inspection Standards" to ensure the welfare of experimental animals and the scientific nature of the data.

[0018] The materials used in the following examples are as follows: The normal control diet (D12102C), high-fat acid diet (D12492), and high-cholesterol diet (D12109C) were purchased from Research Diets. Poly(lactic-co-glycolic acid) (PLGA) was purchased from Phosphorex. AST and ALT assay kits were obtained from Nanjing Jiancheng Biological Co., Ltd. Insulin powder was obtained from Solebol. Pyruvate was obtained from MacLean. Glucose powder was obtained from Beyotime. H&E staining kits were obtained from Beyotime, immunohistochemistry kits and DAB color development reagents were obtained from Zhongshan Jinqiao, Oil Red O kits were obtained from Solebol, and picrosirius red staining kits were obtained from Regen Biotech. Anti-MARCO antibody (AB259264) and Anti-MARCO antibody (AB231046) were from abcam, Anti-F4 / 80 antibody (Q61549), Anti-Phospho-IRS1 antibody (Ser302) (2384), Anti-IRS1 antibody (2382) and Anti-Phospho-AKT (Ser473) (P31751) were from CST, Anti-AKT antibody (60203-2-Ig) and Anti-GAPDH antibody (60004-1-Ig) were from Proteintech. Wild-type C57BL / 6J mice were purchased from the Animal Management Center of Chongqing Medical University, aged 6-8 weeks, and were housed in the Animal Experiment Center of Chongqing Medical University.

[0019] Clinical samples: Patient medical records and liver pathology samples were donated by the First Affiliated Hospital of Wenzhou Medical University. Animal Models and Treatments: Lean and non-lean nonalcoholic fatty liver disease models were established as follows: 6-8-week-old C57BL / 6J male mice (20-25 g) were fed a conventional diet (NCD, 10 kcal / % fat), a high-fat diet (HFD, 60 kcal / % fat), or a high-cholesterol diet (PD, 40 kcal / % fat, 1.25 gm / % cholesterol, 0.5 gm / % bile acid) for 8, 12, or 16 weeks. The PLGA treatment model was established as follows: 6-8-week-old C57BL / 6J mice were fed a PD diet for 12 weeks. Starting from the eighth week, they were injected three times weekly into the tail vein with poly(lactic-co-glycolic acid) (PLGA) at 0.35 mg per mouse. A control group was injected with an equal volume of saline. Mice were enrolled in batches and euthanized at the same age. All mice were housed at the Experimental Animal Center of Chongqing Medical University under a 12-h light-dark cycle and an ambient temperature of 22°C. They had free access to food. After modeling, mice in each group were euthanized by intraperitoneal injection of an overdose of sodium pentobarbital (200 mg / kg) under deep anesthesia, and the liver, epididymal white fat, heart, spleen, kidney, and lung were removed. The body weight, liver weight, and epididymal white fat weight of the mice were photographed, weighed, and recorded to observe their pathological changes.

[0020] Hematoxylin and eosin (H&E) staining: After mouse tissues were rinsed in phosphate-buffered saline, the same liver section from each mouse was fixed in 4% paraformaldehyde for at least 24 hours. The tissues were then embedded in paraffin and cut into 5μm sections for later use. The sections were oven-baked at 60°C for 30 minutes to secure the tissues to the mount. The sections were then dewaxed and rehydrated in xylene and 100%, 95%, 80%, and 75% ethanol, and then placed in ultrapure water for staining. The filtered hematoxylin stain solution was applied dropwise to the tissue and stained for 1-3 minutes. Microscopic observation revealed that the nuclei of the cells were stained pink-purple. After rinsing under running tap water for 1-2 minutes, the nuclei were observed to turn from pink-purple to light blue. Eosin stain solution was applied dropwise to the tissue and stained for 1-2 minutes. Excess stain was then washed off and the sections were dehydrated and transparentized with 75%, 80%, 95%, 100% ethanol, and xylene. Prepare mounting medium using a 1:1 ratio of neutral gum to xylene. Allow any remaining xylene to air dry before mounting the sections. Once fully fixed, use a tissue scanner to observe and analyze tissue pathology.

[0021] Sirius red staining: Dewax and rehydrate the sections, add filtered Sirius red stain to the tissue, stain for 20-30 minutes, remove excess stain, rehydrate, transparentize, seal, and scan the sections to detect the degree of liver fibrosis.

[0022] Oil Red O staining: After rinsing mouse liver and epididymal white adipose tissue in phosphate buffered saline, sample the same portion of liver and adipose tissue from each mouse, dry on absorbent paper, and then freeze-embed in cryoembedding medium. Cut the tissue into 5 μm sections using a cryostat. Allow the frozen sections to thaw at room temperature for 15 minutes. Fix the sections with 4% paraformaldehyde for 15 minutes. After washing with phosphate buffered saline, apply filtered Oil Red O stain and stain for 15-30 minutes in the dark. Rinse with ultrapure water to remove excess stain. Stain the nuclei with hematoxylin and blue under running water. Mount the sections with glycerol gelatin and observe and image the sections under a microscope to assess the extent of lipid deposition.

[0023] Immunohistochemical staining: After routine dewaxing and rehydration, sections were placed in citrate buffer, brought to a slight boil, and then naturally cooled to room temperature. This process was repeated three times to allow antigen retrieval. Sections were washed three times with phosphate buffer, and an appropriate amount of peroxidase blocking reagent was added to the tissue. The sections were incubated at room temperature for 15 minutes, followed by three washes with phosphate buffer. Tissues were blocked with 10% goat serum. After a 30-minute incubation, the primary antibody solution (F4 / 80, Marco) was added and incubated overnight at 4°C. Sections were washed three times with phosphate buffer, and biotin-conjugated secondary antibody was added to the tissue. The reaction was allowed to proceed at room temperature for 20 minutes, followed by three washes with phosphate buffer. DAB working solution was prepared at a ratio of DAB solvent to chromogen solution (1:1) and applied to the tissue for development, ensuring consistent development time for the control and experimental groups. Cell nuclei were stained with hematoxylin solution and blued under running water. After routine dehydration and clearing, sections were mounted with mounting medium and images were acquired using a slide scanner.

[0024] Immunofluorescence staining: After routine dewaxing and rehydration of the sections, place the sections in citrate buffer, boil until slightly boiling, and then cool naturally to room temperature. Repeat three times to repair the antigen. After washing three times with phosphate buffer, add 10% goat serum to the tissue for blocking. After incubation for 30 minutes, add the primary antibody solution (marco) to the tissue and incubate overnight in a 4°C refrigerator. After washing the sections three times with phosphate buffer, add the fluorescently labeled secondary antibody to the tissue, incubate at 37°C for one hour, and then wash three times with phosphate buffer. After sealing with an anti-fluorescence quencher containing DAPI, store at 4°C for a short period of time. Collect images and analyze them under a confocal microscope.

[0025] Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured: Mouse serum was thawed at 4°C in advance, and the test kit was allowed to return to room temperature. The assay was performed according to the Nanjing Jiancheng test kit instructions. The OD value of each well was measured on a microplate reader, and the ALT and AST activity values ​​were calculated according to the instructions.

[0026] Glucose tolerance test (GTT): Mice were fasted for 16 hours the night before the experiment. On the morning of the second day of fasting, their body weight was recorded, and their fasting blood glucose levels were measured using a portable blood glucose meter. A 1.5g / kg glucose solution was intraperitoneally injected into the mice, and blood glucose levels were measured 15, 30, 60, 90, and 120 minutes after injection.

[0027] Insulin tolerance test (ITT): Mice were fasted for 5 hours the night before the experiment. After the fast, their body weight was recorded and their fasting blood glucose levels were measured. A 0.75U / kg insulin solution was intraperitoneally injected into the mice, and their blood glucose levels were measured 15, 30, 60, 90, and 120 minutes after injection.

[0028] Pyruvate tolerance test (PTT): Mice were fasted for 16 hours the night before the experiment. On the morning of the second day of fasting, their body weight was recorded, and their fasting blood glucose levels were measured using a portable blood glucose meter. A 1.25g / kg pyruvate solution was intraperitoneally injected into the mice, and blood glucose levels were measured 15, 30, 60, 90, and 120 minutes after injection.

[0029] Western blotting: (1) Extract tissue protein: Add an appropriate volume of liver to a tissue homogenate tube, add protein lysis buffer and phosphatase / protease inhibitors, fully disrupt with a biological sample homogenizer, rotate on a 4°C four-dimensional rotating mixer for 30 minutes, centrifuge at 12,000g for 15 minutes in a 4°C centrifuge, aspirate the supernatant, add the corresponding volume of loading buffer, and heat in a 100°C metal bath for 10 minutes to denature the protein. (2) Western blotting: Use a rapid gel kit to prepare an appropriate gel, add a protein marker to mark the molecular weight, and then add an appropriate volume of protein solution in sequence. Electrophoresis is performed at a constant voltage of 110V until the protein runs to the bottom of the gel. Cut the gel according to the molecular weight of the target protein, then activate the PVDF membrane with methanol, put it in ddH2O, and put the two into the transfer cassette in sequence. Electrophoresis is performed at a constant current of 250mA. The appropriate electrophoresis time is set according to the molecular weight of the protein. The membrane is blocked with 3% BSA solution for more than 1 hour. Add primary antibody solution (anti-p-IRS1, IRS1, p-AKT, AKT, GAPDH) and incubate overnight at 4°C. Wash the PVDF membrane with TBST three times for 10 minutes each time. Incubate with secondary antibody solution of the corresponding species at room temperature for 1 hour, then wash. Apply chemiluminescent solution evenly to the membrane, develop the membrane using a chemiluminescence analyzer, and acquire images.

[0030] Real-time fluorescence quantitative PCR (qRT-PCR): (1) Extraction of tissue RNA: Add an appropriate volume of liver and epididymal white adipose tissue to a tissue homogenate tube, add 1 mL of RNA lysis buffer, and fully disrupt with a biological sample homogenizer. Transfer the homogenate to an enzyme-free EP tube, add 200 μl of chloroform and shake thoroughly. After standing for 5 minutes, centrifuge at 12,000 rpm in a 4°C centrifuge for 15 minutes. After centrifugation, take the upper layer solution to another EP tube, add 500 μl of isopropanol, mix thoroughly, and stand for 10 minutes. Centrifuge at 12,000 rpm in a 4°C centrifuge for 10 minutes, discard the supernatant, add 75% ethanol and centrifuge again for 5 minutes, and discard the supernatant. After drying, add an appropriate amount of DEPC water and detect the concentration. (2) Reverse transcription: Prepare samples according to the conventional reverse transcription system within the research group and perform reverse transcription according to the conventional reaction procedure. (3) Real-time fluorescence quantitative PCR: The reaction procedure was as follows: 50°C, 2 min; 95°C, 5 min; 37°C, 20 s; 55°C, 2 s; 72°C, 30 s; for a total of 40 cycles.

[0031] Example 1 Construction of lean and non-lean non-alcoholic fatty liver disease models in mice

[0032] During the model construction process, the weight changes were recorded. Compared with the mice fed with NCD, the weight of the mice fed with HFD increased significantly, while the weight of the mice fed with PD did not increase significantly ( Figure 1 A). The ratio of liver weight to body weight showed that compared with the NCD group, the liver weight of the HFD group mice increased slightly, and the weight of epididymal white fat tissue increased significantly, while the liver weight of the PD group mice increased significantly, and the weight of epididymal white fat tissue even decreased significantly ( Figure 1 BC). The results of liver section staining showed that compared with the NCD group, the HFD group showed obvious fatty degeneration and liver cell damage in the liver as the feeding time increased. At 16 weeks of feeding, there was a significant increase in inflammatory cell infiltration, but no obvious fibrosis. The PD group also showed obvious fatty degeneration, liver cell damage and inflammatory cell infiltration in the liver, and obvious liver fibrosis ( Figure 1 These results suggest that a high-fat diet leads to a distinct non-lean MAFLD, whereas a high-cholesterol diet leads to a lean MAFLD.

[0033] Example 2 investigates the relationship between lean non-alcoholic fatty liver disease and Marco + The relationship between macrophages

[0034] Immunofluorescence staining of liver samples from clinical patients revealed that Marco + The number of macrophages increased significantly ( Figure 2AB). Marco was also found in the livers of mice fed with PD for 8, 12, and 16 weeks. + Significant enrichment of macrophages ( Figure 2 CD), which indicates that the liver Marco + Macrophages may play an important role in lean MAFLD.

[0035] Example 3 Construction of PLGA targeted intervention Marco + Macrophage therapy model

[0036] Mice were treated with PLGA for 4 weeks after 8 weeks of PD diet induction and then euthanized at 12 weeks. Figure 3 A). H&E staining of important organs such as heart, spleen, lung, and kidney showed that PLGA did not induce obvious organ toxicity ( Figure 3 B).

[0037] Example 4: PLGA treatment model improves liver pathological changes in lean MAFLD

[0038] The liver weight / body weight ratio of mice in the PLGA treatment group showed a significant decrease ( Figure 4 A) Liver immunohistochemical analysis showed that PLGA effectively cleared liver Marco + macrophages, and significantly reduced the degree of macrophage infiltration ( Figure 4 B). Serum transaminase test confirmed that PLGA can effectively alleviate liver damage ( Figure 4 C). At the same time, liver H&E staining, Oil Red O staining, and Sirius Red staining results showed that PLGA treatment could significantly inhibit liver fat deposition and fibrosis progression ( Figure 4 DE). In terms of insulin resistance, mice in the PLGA-treated group showed improved glucose tolerance ( Figure 5 A) Insulin sensitivity Figure 5 B) and pyruvate metabolism capacity ( Figure 5 C) was significantly improved. Western blotting results showed that the phosphorylation levels of AKT and IRS1 in liver tissue were upregulated ( Figure 6 AC), which confirmed that PLGA treatment can effectively alleviate insulin resistance.

[0039] Example 5: PLGA treatment model improves fat pathological changes in lean MAFLD

[0040] The ratio of epididymal white adipose tissue weight to body weight of mice in the PLGA treatment group was significantly increased ( Figure 7 AB), H&E staining and immunohistochemical staining showed that the volume of adipocytes increased and the infiltration of macrophages decreased after PLGA treatment ( Figure 7 CE), indicating that the fat pathology was significantly improved.

[0041] Example 6 PLGA treatment promotes organ metabolic reprogramming in lean MAFLD

[0042] Analysis of lipid metabolism-related genes showed that lipid synthesis and uptake pathways in mouse liver tissue were inhibited and catabolism was enhanced after PLGA treatment ( Figure 8 AC), resulting in reduced lipid accumulation in the liver. In contrast, lipid uptake-related genes in adipose tissue were significantly upregulated, while lipid breakdown was reduced ( Figure 8 DF), indicating that the fat storage capacity of adipose tissue was significantly enhanced.

Claims

1. PLGA is based on Marco + Application of macrophage targeting in the treatment of lean nonalcoholic fatty liver disease.

2. The use according to claim 1, characterized in that: The pathological characteristics of lean non-alcoholic fatty liver disease include liver inflammatory infiltration, lipid deposition, insulin resistance, fibrosis, and reduced inflammatory infiltration and fat storage capacity of adipose tissue.

3. The use according to claim 2, characterized in that: Marco + Macrophages are specifically increased in lean nonalcoholic fatty liver disease.

4. The use according to claim 1, characterized in that: Targeted intervention of Marco by tail vein injection of PLGA + Macrophages significantly inhibit lipid deposition and fibrosis progression in the liver, alleviate insulin resistance, and enhance the fat storage capacity of adipose tissue.

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