A copper-doped bio-glass and its use in the treatment of chronic liver damage and hepatic osteodystrophy

Abstract

This invention belongs to the field of biomedical inorganic nanomaterials technology, and discloses a copper-doped bioglass and its application in the treatment of chronic liver injury and hepatic osteodystrophy. The bioglass is prepared by a sol-gel method, with copper as a dopant (Cu). 2+ It is doped in a form with a doping amount of 0.1-5 wt%, has a mesoporous structure, a particle size of approximately 163 nm, and can slowly release Cu in environments with pH 7.4 or 5.5. 2+ The treatment duration is 48-96 hours. This invention is the first to use the aforementioned material for the simultaneous treatment of chronic liver injury (such as metabolic-associated steatohepatitis) and its secondary hepatic osteopathy, achieving liver-targeted therapy via intravenous injection. Its mechanism of action includes: regulating glucose and lipid metabolism in the liver, enhancing mitochondrial function, and inhibiting inflammation and fibrosis; simultaneously upregulating the liver-derived factor LCAT, promoting osteogenic differentiation via the liver-bone axis, thereby synergistically improving liver injury and bone loss. This invention overcomes the limitations of separate liver and bone treatment, providing a safe and effective integrated treatment strategy.

Description

Technical Field

[0001] This invention relates to the field of biomedical inorganic nanomaterials technology, specifically to a copper ion-doped bioactive glass and its preparation method, as well as its cross-organ regulatory application in the treatment of chronic liver injury such as metabolic-associated steatohepatitis (MASH) and non-alcoholic fatty liver disease (NAFLD), and secondary hepatic osteodystrophy (HOD). The material is delivered to the liver via intravenous injection, utilizing Cu... 2+ Sustained release and the bioactivity of the bioglass itself simultaneously improve liver metabolic dysfunction and bone loss, belonging to the interdisciplinary field of nanomedicine and regenerative medicine. Background Technology

[0002] Hepatic osteodystrophy (HOD) is a metabolic bone disease secondary to chronic liver disease, clinically characterized by low bone mass, impaired bone quality, and increased risk of fractures. The incidence of HOD is as high as 52.2% in patients with metabolic-associated steatohepatitis (MASH). Current treatment has significant limitations: antifibrotic or metabolic drugs have limited effects on improving osteoporosis, while anti-resorption drugs are difficult to alleviate liver inflammation and fibrosis, leading to a split treatment dilemma of "treating the liver but not the bone" or "treating the bone but not the liver."

[0003] Copper ions (Cu) 2+ Cu, as a key trace element, can regulate glucose and lipid metabolism, activate superoxide dismutase, and inhibit TGF-β1 signaling, thus possessing theoretical therapeutic value for liver damage and bone repair. However, free Cu... 2+ The therapeutic window is narrow, and overdose can exacerbate oxidative damage via the Fenton reaction, posing a high risk of systemic toxicity. Bioglass (BG), as an inorganic active material, produces Ca as a degradation product. 2+ and SiO4 4- It possesses both osteogenic and anti-inflammatory properties and has been used for bone regeneration and soft tissue repair. Literature reports that drugs with particle sizes of 50-200 nm can accumulate in the liver through the endothelial fenestrations of the sinusoids. However, there are currently no studies using copper-doped bioglass for the treatment of chronic liver injury and secondary bone diseases.

[0004] The shortcomings of existing technologies are: (1) lack of integrated treatment plans to simultaneously intervene in both the liver and bones; (2) insufficient safety of free copper ions; and (3) the indications for bioglass have not been extended to the field of metabolic liver diseases. Therefore, it is necessary to develop nanomaterials based on copper-doped bioglass to achieve Cu 2+ Safe and sustained-release, by regulating the liver-bone axis to improve liver metabolic dysfunction and bone loss, will fill a treatment gap in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the primary objective of this invention is to provide copper-doped bioglass (CuBGs) nanoparticles. This material is precisely prepared via a sol-gel method, exhibiting a uniform nanospherical morphology and mesoporous structure, with copper as the dopant (Cu). 2+ Stable doping in glass networks enables slow, controlled release over 48-96 hours in physiological and pathological microenvironments, fundamentally overcoming the problems of toxicity and short half-life of free copper ions.

[0006] A second objective of this invention is to provide a novel pharmaceutical use for the aforementioned copper-doped bioglass, namely, its application in the preparation of medicaments for treating chronic liver injury (particularly MASH / NAFLD). This medicament can be delivered to the liver via intravenous injection, utilizing Cu… 2+ The sustained release and synergistic effect of the bioglass carrier effectively improve hepatic steatosis, inflammation and fibrosis through multiple mechanisms (including but not limited to: regulating hepatocyte glucose and lipid metabolism reprogramming, enhancing mitochondrial oxidative phosphorylation function, scavenging reactive oxygen species, inhibiting inflammatory pathways and hepatic stellate cell activation).

[0007] A third objective of this invention is to provide another novel pharmaceutical application of the aforementioned copper-doped bioglass: its use in the preparation of drugs for treating hepatic osteodystrophy (HOD). This application does not directly target bone, but rather works by repairing liver function and upregulating the expression and secretion of the liver-derived factor lecithin cholesterol acyltransferase (LCAT), which then acts on bone tissue via blood circulation. This indirectly and effectively promotes osteoblast differentiation, increases bone formation, and ultimately improves bone density and bone microstructure. This achieves a cross-organ synergistic treatment, moving from "treating the liver" to "protecting the bones."

[0008] To achieve the objectives of this invention, the invention includes the following technical solutions: A copper-doped bioglass, prepared by a sol-gel method, wherein copper is in the form of Cu. 2+ The copper-doped bioglass is incorporated into a glass network structure, with a copper doping concentration ranging from 0.1 wt% to 5 wt%. This copper-doped bioglass exhibits sustained-release properties, continuously releasing Cu within 48 to 96 hours in buffer solutions at pH 7.4 or pH 5.5. 2+ And there was no burst release phenomenon.

[0009] Furthermore, the copper-doped bioglass described above has spherical particles with a particle size of 163.67 ± 15.26 nm; the particles have a mesoporous structure; and X-ray photoelectron spectroscopy reveals Cu 2p characteristic peaks at binding energies of 935 eV and 955 eV.

[0010] This invention also discloses a method for preparing the above-mentioned copper-doped bioglass, comprising the following steps: (1) Preparation of precursor solution: The copper source and the complexing agent are mixed in a solvent to form a stable copper complex precursor solution; (2) Sol-gel reaction: The silicon source, calcium source and the precursor solution obtained in step (1) are mixed under alkaline catalytic conditions to carry out co-hydrolysis and polycondensation reaction to form a sol, which is then aged to obtain a wet gel. (3) Post-processing: The wet gel is centrifuged, washed and dried to obtain the dry gel precursor; (4) Calcination: The dry gel precursor is calcined in an air atmosphere and cooled to obtain the copper-doped bioglass.

[0011] The present invention also discloses the use of the above-mentioned copper-doped bioglass in the preparation of a medicament for treating chronic liver injury; wherein the chronic liver injury is metabolic-associated steatohepatitis (MASH) or non-alcoholic fatty liver disease (NAFLD).

[0012] Furthermore, in the above-described uses, the drug is a drug capable of achieving functions such as (a)-(e): (a) Reduce serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST); (b) Reduce triglyceride accumulation in liver tissue; (c) Downregulates the mRNA expression levels of interleukin-1β and tumor necrosis factor-α in the liver; (d) Inhibition of F4 / 80 positive macrophage infiltration in the liver; and (e) Reduces the expression of α-smooth muscle actin α-SMA, thereby improving hepatocyte ballooning degeneration and inhibiting hepatic stellate cell activation.

[0013] Furthermore, in the above-mentioned uses, the drug can upregulate the mRNA expression of key glycolytic enzyme genes in the liver, including Pklr, Pfkm, Pgm1, Eno1, Pdhb, and Pgam1; and improve the expression of insulin resistance signaling pathway-related molecules Ppargc1a, Slc27a1, and Pparα, thereby alleviating glucose intolerance and inhibiting hepatic lipid production.

[0014] Furthermore, in the above-mentioned uses, the drug can be taken up by hepatocytes and significantly co-localize with mitochondria within hepatocytes; the drug inhibits the NF-κB signaling pathway and inflammatory response by enhancing the mRNA expression of oxidative phosphorylation-related genes Ndufa4l2 and Atp6vod2 and improving the clearance efficiency of reactive oxygen species (ROS).

[0015] The present invention also discloses the use of the above-mentioned copper-doped bioglass in the preparation of a drug for treating hepatic osteodystrophy, wherein the hepatic osteodystrophy is caused by metabolic-associated steatohepatitis (MASH).

[0016] Furthermore, in the above-described uses, the drug is a drug capable of achieving functions (a)-(c): (a) Increase bone mineral density (BMD), bone volume (BV), and bone volume / tissue volume ratio (BV / TV) of the femur; (b) Reduce trabecular separation (Tb.Sp); and (c) Upregulate the mRNA expression of osteoblast markers Runx2 and Sp7 in bone tissue.

[0017] The present invention also discloses the use of the above-mentioned copper-doped bioglass in the preparation of a medicament for the simultaneous treatment of chronic liver injury and hepatic osteodystrophy.

[0018] In some embodiments: Cu / L-ascorbic acid complexes were prepared as the copper source using a microemulsion-assisted sol-gel method. Tetraethyl orthosilicate, calcium nitrate, and the precursor were co-hydrolyzed and condensed under alkaline conditions. After centrifugation, washing, drying, and calcination at 700℃ for 4 hours, bio-glass nanoparticles with copper doping levels of 0.1–5 wt% were obtained. Scanning electron microscopy (SEM) showed that the CuBGs had a particle size of 163.67 ± 15.26 nm, transmission electron microscopy confirmed the internal mesoporous structure, and X-ray photoelectron spectroscopy detected Cu at 935 eV and 955 eV. 2+ Characteristic peaks. In vitro release experiments confirmed its sustained release of Cu for 48-96 hours in simulated physiological and pathological microenvironments. 2+ No initial burst release.

[0019] In vitro and in vivo pharmacodynamic evaluations showed that in a primary hepatocyte PA-induced MASH model, treatment with 0.1 mg / mL CuBGs for 24 hours resulted in dose-dependent decreases in ALT and AST levels in the culture medium and intracellular TG content, a reduction in the area of ​​Oil Red O staining, and a significant downregulation of IL-1β and TNF-α mRNA expression. Transwell co-culture experiments showed that CuBGs treatment upregulated Runx2 and Sp7 mRNA expression in MC3T3-E1 cells, while CuBGs alone had no direct effect on osteoblasts.

[0020] In the HFMCD-induced MASH-HOD mouse model, intravenous injection of CuBGs (10 mg / kg, every 48 hours) for 2 weeks significantly improved the liver / body weight ratio, decreased liver triglyceride and cholesterol levels, inhibited serum ALT and AST activities, downregulated liver F4 / 80 and α-SMA expression, and consistently reduced CD36 transcription and translation levels. μCT analysis showed that MASH model mice had approximately 30% less bone mineral density, 54% less bone mass, a 32% less bone volume / tissue volume ratio, and 32% more trabecular separation. Cu-BG treatment significantly increased bone mineral density, improved trabecular parameters, increased femoral copper content, and upregulated Runx2 and Sp7 expression in bone tissue, while TRAP staining showed no inhibition of osteoclast activity.

[0021] Transcriptome sequencing analysis revealed that 1451 differentially expressed genes in the liver of the CuBGs-treated group were upregulated and 1336 were downregulated, significantly enriched in the insulin resistance signaling pathway and oxidative phosphorylation. qRT-PCR validation showed that CuBGs upregulated the expression of key insulin resistance pathway molecules Ppargc1a, Slc27a1, and Pparα, and enhanced the transcription of oxidative phosphorylation-related genes Ndufa4l2 and Atp6vod2. Random blood glucose and glucose tolerance assays confirmed that CuBGs improved glucose intolerance in MASH-HOD mice. Confocal microscopy showed that FITC-labeled CuBGs had significantly higher co-localization with mitochondria than undoped BGs.

[0022] Mechanistic studies have confirmed that CuBGs treatment significantly increases LCAT protein levels in the liver, serum, and bone tissue. When primary hepatocytes from hepatocyte-specific LCAT knockout mice were co-cultured with MC3T3-E1 cells, the osteogenic effect of CuBGs was completely eliminated, demonstrating that this effect depends on LCAT-mediated liver-bone axis regulation.

[0023] Compared with the prior art, the present invention has the following outstanding advantages: 1. First time achieving cross-organ synergistic repair between liver and bone This invention is the first to apply bioglass to the treatment of chronic liver disease and secondary bone disease, simultaneously intervening in two target organs with a single material. Animal experiments have demonstrated that CuBGs treatment significantly reduced the liver injury index (ALT / AST) in MASH-HOD mice, while simultaneously increasing bone mineral density and significantly improving trabecular bone parameters (BV / TV, Tb.N, Tb.Th). This addresses the challenge of splitting clinical diagnosis and treatment.

[0024] 2. The safe sustained-release system overcomes the limitations of copper ion toxicity. Cu was chemically bonded using a silica network. 2+In vitro and in vivo data showed that: (1) repeated administration for 15 days (10 mg / kg, q48h) did not cause histopathological damage to major organs such as the heart, spleen, and kidneys; (2) serum ALT, AST, and creatinine levels were not statistically different from those in the normal control group; (3) Cu 2+ Slow-release formulation avoids burst-release toxicity.

[0025] 3. Multidimensional intervention in the MASH pathological process Cu-BG works synergistically through three mechanisms: (1) metabolic reprogramming: upregulating the expression of key glycolytic enzymes PKLR and PFKM, inhibiting gluconeogenesis, and improving insulin resistance; (2) anti-oxidative stress: enhancing the expression of mitochondrial oxidative phosphorylation-related genes (such as Ndufa4l2 and Atp6vod2), and significantly reducing ROS levels; (3) anti-fibrosis. The above three mechanisms synergistically correct the key pathological changes in the pathogenesis of MASH. It promotes liver repair and rebuilds the liver-bone axis homeostasis, increases LCAT expression, and alleviates MASH-induced bone loss.

[0026] 4. Significant potential for clinical translation. The material synthesis process is simple and controllable, with low raw material costs. Particles with a diameter of 50-200 nm can achieve passive targeting through the endothelial pores of the liver sinusoids (100-150 nm). The intravenous administration regimen is compatible with clinical infusions, and significant efficacy can be observed with a short-term treatment of 2 weeks, providing an industrially viable solution for the unmet clinical need of MASH-HOD. Attached Figure Description

[0027] Figure 1 The following are the material characterization and in vivo distribution results of the copper-doped bioglasses (CuBGs) of this invention: Wherein, Figure 1 A is a schematic diagram of the synthesis process of CuBGs; Figure 1 B and Figure 1 D are scanning electron microscope images of undoped bioglass (BGs) and CuBGs, respectively; Figure 1 C and Figure 1 E represents the particle size distribution statistics of BGs and CuBGs, respectively; Figure 1 F and Figure 1 H represents the transmission electron microscope images of BGs and CuBGs, and their corresponding elemental surface distribution diagrams (Si, Ca, O, Cu). Figure 1 G and Figure 1 I represents the energy dispersive X-ray spectra of BGs and CuBGs, respectively; Figure 1 J represents the full X-ray photoelectron spectrum of BGs and CuBGs. Figure 1 K represents the high-resolution X-ray photoelectron spectrum of the Cu2p orbital in BGs and CuBGs. Figure 1 L and Figure 1M represents in vivo and ex vivo fluorescence imaging of major organs of mice 8 hours after intravenous injection of Cy5-labeled BGs and CuBGs. Figure 2 The results of the functional experiments of CuBGs at the cellular level of this invention are as follows: Figure 2 A and Figure 2 B represents the detection results of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the primary hepatocyte model; Figure 2 C represents the intracellular triglyceride content; Figure 2 D is the Oil Red O staining image; Figure 2 E represents quantitative analysis of Oil Red O; Figure 2 F and Figure 2 G represents the mRNA expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α); Figure 2 H represents the distribution of FITC-labeled BGs and CuBGs in primary hepatocytes; Figure 2 I is a schematic diagram of the co-culture system of hepatocytes and osteoblasts; Figure 2 J and Figure 2 K represents the expression of Runx2 and Sp7 in osteoblasts under a co-culture system; Figure 3 The effect of CuBGs of this invention on improving liver function in animal models: Figure 3 A shows a schematic diagram of the animal drug administration experiment process and a digital photograph of the injection solution; Figure 3 B represents the mouse body weight change curve; Figure 3 C represents the liver weight of mice 2 weeks after drug administration; Figure 3 D represents the liver / body weight ratio; Figure 3 E represents the liver triglyceride content; Figure 3 F shows digital photographs of liver tissue, H&E, and Oil Red O stained images; Figure 3 G and Figure 3 H represents serum ALT and AST levels; Figure 3 Image I is an immunostaining image of F4 / 80 and α-SMA; Figure 3 J represents the expression of Cd36, a lipid metabolism-related gene. Figure 3 K and Figure 3 L represents CD36 immunohistochemical staining images and quantitative analysis; Figure 4 The effect of CuBGs of this invention on the bone tissue phenotype of MASH-HOD mice: Figure 4 A is a 3D reconstruction image of the femur using μCT; Figure 4 B represents the bone mineral density test result; Figure 4 C to Figure 4 E represents bone volume (BV), tissue volume (TV), and the bone volume / tissue volume ratio (BV / TV). Figure 4F represents immunohistochemical staining for osteocalcin; Figure 4 G represents the immunohistochemical quantification of osteocalcin; Figure 4 H represents the mRNA expression of osteogenic-related genes Sp7 and Runx2; Figure 4 I represents Runx2 protein expression; Figure 4 J represents Runx2 immunohistochemical staining; Figure 4 K represents the immunohistochemical quantification of Runx2; Figure 4 L represents the TRAP-stained image; Figure 4 M represents the quantitative analysis of TRAP-positive cells; Figure 5 Transcriptomic and metabolic mechanism analysis of CuBGs in treating MASH-HOD according to this invention: Figure 5 A represents a heatmap of gene expression clustering; Figure 5 B is a volcano diagram of differentially expressed genes between the HFMCD-CuBGs group and the HFMCD-NS group; Figure 5 C represents the KEGG enrichment analysis result; Figure 5 D represents the qRT-PCR results of insulin resistance-related genes; Figure 5 E represents blood glucose level; Figure 5 F and Figure 5 G represents the result of the glucose tolerance test; Figure 5 H represents the statistical analysis of differentially expressed genes between the HFMCD-CuBGs group and the HFMCD-BGs group; Figure 5 I represents KEGG enrichment pathway analysis; Figure 5 J represents the expression of genes related to oxidative phosphorylation; Figure 5 K represents the colocalization image of BGs and CuBGs with mitochondria; Figure 6 To verify the mechanism by which CuBGs of this invention alleviate bone damage through the LCAT-mediated liver-bone axis: Figure 6 A represents Lcat mRNA expression in liver tissue; Figure 6 B to Figure 6 E represents the detection of LCAT protein in the liver; Figure 6 F represents serum LCAT detection; Figure 6 G to Figure 6 J represents LCAT protein expression in bone tissue; Figure 6 K is a schematic diagram of the LCAT knockout model co-culture system; Figure 6 L and Figure 6 M represents the expression of genes related to osteogenic differentiation; Figure 7 The following are the biosafety evaluation results of CuBGs of this invention: Figure 7 A represents the dosing regimen and changes in body weight; Figure 7 B to Figure 7 E represents the ratio of major organs to body weight; Figure 7 F and Figure 7 G represents serum ALT and AST; Figure 7 H represents serum creatinine; Figure 7 I is an H&E staining image of the major organs; Figure 8 Supplementing results for cell experiments: Figure 8 A is a Bodipy staining image; Figure 8 B represents lipid metabolism gene expression; Figure 8 C is TUNEL staining; Figure 8 D represents quantitative analysis of apoptotic cells; Figure 9 Supplementing characterization results for the liver injury model: Figure 9 A represents the ratio of white adipose tissue to body weight; Figure 9 B represents the epididymal fat / body weight ratio; Figure 9 C represents the heart-to-body weight ratio; Figure 9 D represents the kidney-to-body weight ratio; Figure 9 E represents the total cholesterol (TC) content in the liver; Figure 9 F to Figure 9 G represents the expression of genes related to liver inflammation; Figure 9 H represents a positive quantitative result from F4 / 80 immunostaining. Figure 9 I arrive Figure 9 J represents the expression of genes related to liver fibrosis; Figure 9 K represents a positive quantitative result of α-SMA immunostaining; Figure 9 L represents the expression of genes related to liver lipid metabolism; Figure 10 Supplementing analysis results for bone metabolism: Figure 10 A represents the number of trabeculae (Tb.N); Figure 10 B represents the trabecular bone thickness (Tb.Th); Figure 10 C represents trabecular separation (Tb.Sp); Figure 10 D represents bone copper content; Figure 10 E represents H&E staining of bone tissue; Figure 10 F represents the expression of osteoclast-related genes; Figure 11 Supplementing transcriptome analysis results: Figure 11 A is the principal component analysis diagram; Figure 11 B is a volcano diagram of differentially expressed genes between the HFMCD-BGs group and the HFMCD-NS group; Figure 11 C represents KEGG enrichment analysis of upregulated genes; Figure 11 D is for verification of glucose metabolism genes; Figure 11 E and Figure 11 F represents the result of reactive oxygen species (ROS) detection; Figure 12 Additional verification results for Lcat knockout: Figure 12 A represents gene expression before and after Lcat knockout; Figure 12B represents protein expression before and after Lcat knockout. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1 Synthesis and characterization of CuBGs, and their biosafety and biodistribution in mice.

[0030] To develop bioactive glasses with the potential to enhance efficacy in MASH-HOD treatment, we used a Cu / L-ascorbic acid complex as a precursor and prepared highly dispersed CuBGs (copper-doped bioglasses) via a sol-gel method. The synthesis process is as follows: Figure 1 As shown in the schematic diagram.

[0031] Specific preparation method 1. Raw materials and reagents Silicon source: Tetraethyl orthosilicate (TEOS), analytical grade.

[0032] Calcium source: calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), analytical grade.

[0033] Copper source and complexing agent: copper chloride dihydrate (CuCl2·2H2O), analytical grade; L-ascorbic acid (AA), analytical grade.

[0034] Catalyst: Concentrated ammonia (NH3·H2O, 25-28%), analytical grade.

[0035] Solvents: Anhydrous ethanol (EtOH), analytical grade. Deionized water.

[0036] 2. Preparation steps (1) Preparation of copper complex precursor solution 1.705 g (0.01 mol) of copper chloride dihydrate was weighed and dissolved in 50 mL of deionized water. The solution was magnetically stirred at 80°C to obtain an aqueous solution of copper chloride. 3.5224 g (0.02 mol) of L-ascorbic acid was weighed and dissolved in 50 mL of deionized water. The solution was magnetically stirred at room temperature to obtain an aqueous solution of L-ascorbic acid. The aqueous solution of L-ascorbic acid was added dropwise to the aqueous solution of copper chloride. The mixture was stirred at 80°C for 24 h until the solution turned a clear light blue, indicating the formation of a stable copper-ascorbic acid complex. The mixture was centrifuged at 7000 rpm for 20 min, and the upper suspension containing the Cu / ascorbic acid complex was collected as the copper precursor. This solution was labeled as solution A.

[0037] (2) Sol-gel reaction Hexadecyltrimethylammonium bromide (CTAB, 0.56 g) was dissolved in a mixture of ethyl acetate (8 mL) and deionized water (26 mL). After complete dissolution, ammonia (1 mol / L, 5.6 mL) was added and reacted for 15 min. Subsequently, tetraethyl orthosilicate (TEOS, 2.88 mL) and calcium nitrate tetrahydrate (1.83 g) were added sequentially and reacted for 30 min. Under continuous vigorous stirring, 5 mL of solution A prepared in step (1) was slowly added dropwise to the above mixture, and stirring was continued for 4 h. It was observed that the system gradually changed from clear to milky white, and finally formed a homogeneous, stable, slightly pale blue sol.

[0038] (3) Aging, drying and calcination Finally, the sol was centrifuged at 8000 rpm for 20 min, and the colloidal product was collected. It was washed twice, successively with deionized water and ethanol, and then vacuum-dried overnight at 60°C. The dried gel block was ground into a fine powder and placed in an alumina crucible. The crucible was placed in a muffle furnace and calcined in air. The calcination program was as follows: the temperature was increased from room temperature to 700°C at a rate of 3°C / min, and then calcined at 700°C for 4 hours. After calcination, the power was turned off, and the material was allowed to cool naturally to room temperature with the furnace. Copper-doped bioglass was finally obtained.

[0039] The same method was used to prepare copper-free bioglass.

[0040] A series of comprehensive characterizations were then performed to confirm successful preparation. Scanning electron microscopy (SEM) images showed that both BGs (bioglass) and CuBGs exhibited uniform spherical morphology. Figure 1 BD), where the particle sizes of BGs and CuBGs are 131.46 ± 11.23 nm and 163.67 ± 15.26 nm, respectively. Figure 1CE). Transmission electron microscopy (TEM) analysis further confirmed the presence of mesoporous structures within the particles. Figure 1 FH), this characteristic facilitates drug loading and enhances bioactivity, consistent with published studies. Subsequently, to verify the successful copper doping, we investigated the elemental composition and chemical state of copper. Energy-dispersive X-ray spectroscopy (EDS) confirmed the presence of key elements silicon (Si), calcium (Ca), and oxygen (O) in the BGs, and the successful incorporation of copper in CuBGs. Figure 1 GI). Full-spectrum X-ray photoelectron spectroscopy (XPS) further confirmed this finding, revealing characteristic peaks for silicon (Si), calcium (Ca), oxygen (O), and copper (Cu). Figure 1 J). High-resolution XPS analysis of the copper 2p region showed that at approximately 935 electron volts (Cu 2p... 3 / 2 ) and 955 electron volts (Cu 2p 1 / 2 Four characteristic peaks appeared at the location, confirming that Cu... 2+ It is the main oxidation state in CuBGs ( Figure 1 K). EDS analysis showed that the actual copper doping content in the CuBGs obtained in this embodiment was 2.96 wt% (based on CuO), within the range of 0.1–5 wt% as defined in claim 1. Those skilled in the art will understand that the final copper doping content can be adjusted within the range of 0.1 wt% to 5 wt% by adjusting the amount of copper chloride dihydrate added during precursor preparation (e.g., 0.0004 g to 0.02 g) or the volume of solution A added in step (2) (0.2 mL to 10 mL). Experimental verification showed that the products obtained by the above adjustments all maintained good spherical morphology, mesoporous structure, and CuO content after 48–96 hours. 2+ The sustained-release properties indicate that the preparation method of the present invention is universally applicable across the entire doping range.

[0041] To assess the biosafety of BGs and CuBGs in vivo, we administered a 15-day treatment regimen to C57BL / 6 mice, with 10 mg / kg administered intravenously every 48 hours. -1 BGs or CuBGs ( Figure 7 (A is marked with a black line at the top). During treatment, compared with the saline (NS) control group, the body weight of mice treated with BGs and CuBGs remained essentially unchanged. Figure 7 (Lower A). No significant differences were observed in the tissue / body weight ratios of liver, heart, spleen, and kidney between mice fed a normal diet (ND) and mice fed ND and treated with BGs or CuBGs. Figure 7BE). Treatment with BGs and CuBGs did not alter serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, indicating that liver function was not affected. Figure 7 F and G). Similarly, serum creatinine (SCR) levels, used to assess renal function, remained unchanged. Figure 7 H). Furthermore, histopathological analysis (H&E staining) showed that no structural damage or inflammatory infiltration was observed in the major organs of mice treated with BGs and CuBGs. Figure 7 I). In summary, administration of CuBGs did not induce histopathological changes in major organs, and the tissue structure was completely comparable to that of the saline-treated control group. This confirms that CuBGs have excellent systemic biocompatibility and in vivo safety.

[0042] Next, we used an in vivo imaging system (IVIS) to assess the distribution of Cy5-labeled BGs / CuBGs after intravenous injection into mice. Figure 1 As shown in L and M, 8 hours after tail vein injection of BGs / CuBGs, fluorescent signals (Cy5) of BGs / CuBGs were mainly detected in the lungs, liver, and bones, with a small distribution in the spleen and kidneys.

[0043] Example 2 CuBGs alleviate liver damage and promote osteoblast differentiation through the liver-bone axis.

[0044] To investigate the effect of CuBGs on alleviating liver injury in vitro, we established a metabolic dysfunction-associated steatohepatitis (MASH) cell model by treating mouse primary hepatocytes with 0.5 mM palmitic acid (PA) for 24 hours. This cell model reproduced the core characteristics of MASH: significant lipid droplet accumulation and the progression of hepatocyte injury and inflammation. Figure 2 AG). We treated these primary hepatocytes with BGs or CuBGs at dose-dependent concentrations. Alanine aminotransferase (ALT) levels in the culture medium were measured. Figure 2 A) Aspartate aminotransferase (AST, Figure 2 B) and intracellular triglycerides (TG, Figure 2 C). Both CuBGs and BGs reduced lipid accumulation and hepatocyte damage in the MASH cell model in a dose-dependent manner. At a concentration of 0.1 mg / mL, both compounds were effective and safe, but CuBGs showed superior efficacy in alleviating PA-induced lipotoxicity compared to BGs. Therefore, this concentration was used in all subsequent studies. Furthermore, Oil Red O staining and Bodipy staining showed that CuBGs and BGs inhibited intracellular lipid accumulation (C). Figure 2 D and E, Figure 8A). We examined the expression of genes related to lipid metabolism in these three groups, including fatty acid uptake (significantly inhibiting the major regulator of fatty acid transport, CD36), lipogenesis, fatty acid oxidation, and triglyceride transport. Key findings indicate that CuBGs effectively reduced lipid accumulation in primary hepatocytes by inhibiting lipid uptake and de novo lipogenesis while enhancing fatty acid oxidation to accelerate lipid clearance. Figure 8 B). Simultaneously, CuBGs significantly attenuated the expression of key MASH-related inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Figure 2 F and G), demonstrating dual therapeutic effects on both metabolic and inflammatory markers of MASH. To determine whether CuBGs could enter cells, we used confocal fluorescence microscopy to observe the intracellular localization of FITC-labeled CuBGs (FITC & CuBGs) in primary hepatocytes. Figure 2 As shown in H, the confocal images demonstrate that FITC & CuBGs can cross the cell membrane of primary hepatocytes and distribute throughout the cytoplasm. Since CuBGs are taken up by the cells, we assessed their potential cytotoxicity using TUNEL staining. Figure 8 C). Quantitative analysis showed no significant difference in TUNEL-positive cells among the PA treatment groups ( Figure 8 (D), demonstrating that CuBGs have excellent biocompatibility.

[0045] Inter-organ regulation plays a crucial role in maintaining systemic homeostasis, with the liver-bone axis being a key mechanism for alleviating bone damage in hepatic osteodystrophy (HOD). To investigate whether CuBGs improve bone damage in vitro through the liver-bone axis, we established a MASH-HOD co-culture model. Primary hepatocytes and mouse skull-derived osteoblast precursor cells (MC3T3-E1) were co-cultured under PA-induced lipotoxicity conditions. Figure 2 I). PA treatment specifically triggered hepatic lipid accumulation and inflammatory damage in primary hepatocytes, reproducing key pathological features of MASH. This system, as a functional surrogate model of skeletal deterioration in MASH-HOD, allowed us to explore the protective effects of CuBGs on bone. Results showed that CuBGs treatment alone failed to promote osteogenic differentiation in PA-treated MC3T3-E1 cells. However, when primary hepatocytes were co-cultured with MC3T3-E1 cells in transfected dishes to simulate liver-bone dialogue, significant upregulation of osteoblast marker genes (Runx2 and Sp7) was observed, confirming the existence of liver-bone axis regulation. Figure 2 J and K). Notably, supplementing hepatocytes with CuBGs in the perforation system further enhanced their osteogenic effect on MC3T3-E1 cells. Figure 2 J and K).

[0046] In summary, CuBGs alleviated hepatic fat accumulation and inflammation by regulating lipid metabolism and promoted osteogenic differentiation in the MASH-HOD cell model through the liver-bone axis.

[0047] Example 3 CuBGs improve liver injury in the MASH-HOD mouse model.

[0048] To verify the in vivo manifestations of MASH-induced bone loss, we established a MASH-HOD mouse model by feeding C57BL / 6 mice a high-fat, methionine / choline-deficient (HFMCD) diet. After 4 weeks of HFMCD diet, the liver / body weight ratio of this group of mice significantly increased. Figure 3 D), this is a key indicator for assessing liver injury, which may be related to the inhibition of lipid efflux from hepatocytes by HHFMCD feeding, leading to the accumulation of triglycerides in liver tissue. Figure 3 E). Simultaneously, serum liver injury markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly elevated ( Figure 3 G and Figure 3 H). Hematoxylin-eosin (H&E) and Oil Red O staining results showed that a large number of lipid droplets accumulated in the liver tissue of MASH mice. Figure 3 F). Sustained lipid accumulation caused severe lipotoxic damage to liver tissue, leading to ballooning degeneration of hepatocytes, infiltrating hepatic macrophages, and activated hepatic stellate cells (HSCs). These pathological changes may induce liver inflammation and promote the fibrotic process. MASH group mice exhibited tissue inflammation in their livers—manifested as elevated F4 / 80 (immune cell infiltration) expression (F). Figure 3 I and Figure 9 F), and liver fibrosis—characterized by increased expression of α-smooth muscle actin (α-SMA). Figure 3 I and Figure 9 I).

[0049] Hepatic osteodystrophy (HOD) is bone damage caused by chronic liver injury. Microcomputed tomography (CT) results showed that, compared with the control group, MASH mice had significantly lower bone mineral density (BMD), bone volume (BV), and bone volume / total volume ratio (BV / TV, %), while trabecular bone separation (Tb.Sp) was significantly increased. In summary, MASH mice exhibited bone loss caused by metabolic dysregulation, displaying the typical pathological features of MASH-associated hepatic osteodystrophy (MASH-HOD).

[0050] To evaluate the therapeutic effect of CuBGs, we used a MASH-HOD mouse model and administered saline (NS), BGs, or CuBGs suspension (10 mg / kg) via tail vein injection every 48 hours for 2 weeks. Figure 3 A). We first monitored body weight changes every two days throughout the treatment period. The body weight curve results showed that CuBGs significantly reduced body weight loss in the HFMCD group mice ( Figure 3 B). The liver / body weight ratio also indicated that both BGs and CuBGs treatments significantly improved the MASH-induced increase in liver / body weight ratio. Figure 3 D). In addition to liver-specific pathological changes, dyslipidemia caused by the HHFMCD diet also leads to systemic organ damage, including abnormal lipid production and heart-kidney damage. Figure 9 AD). CuBGs intervention partially restored normal lipid metabolism and improved cardiac injury. Notably, although MASH induced compensatory copper accumulation in liver tissue, CuBGs administration further increased liver copper content (AD). Figure 3 D).

[0051] Subsequently, we investigated the role of CuBGs-induced copper elevation in improving three hallmark pathological features of MASH, including hepatic lipid accumulation, liver injury, inflammation, and fibrosis. CuBGs treatment significantly reduced triglyceride and cholesterol levels in the liver ( Figure 3 E and Figure 9 E). Pathological examination, including H&E and Oil Red O staining, confirmed that CuBGs treatment inhibited hepatic lipid deposition (E). Figure 3 F). Hepatic lipid accumulation may lead to steatohepatitis and liver injury. Therefore, we measured serum ALT and AST levels to assess whether CuBGs treatment improved liver injury. Significantly elevated ALT and AST activities in MASH-HOD mice were inhibited by CuBGs (F). Figure 3 G and H). Inflammation and fibrosis are characteristic pathological features of MASH-related liver injury. CuBGs can lead to downregulation of the mRNA expression of pro-inflammatory cytokines, including IL-1β and TNF-α. Figure 9 F and Figure 9 G), and inhibited macrophage infiltration (G), and inhibited macrophage infiltration ( Figure 3 I). Simultaneously, we assessed fibrosis in mice. CuBGs inhibited the increase in mRNA expression of fibrosis markers (including α-SMA and Timp-1) in the liver of MASH mice, as well as the protein expression of α-SMA (…). Figure 3 I and Figure 9 I-9K).

[0052] Lipid accumulation in the liver is a key driver of MASH (metastatic lipid metabolism). To further investigate lipid metabolism in the liver, we examined the mRNA expression levels of genes related to lipid metabolism. CuBGs regulate multiple aspects of fatty acid metabolism, including fatty acid uptake (…). Figure 3 J), as well as lipidogenesis, fatty acid oxidation and lipid transport (J), and lipidogenesis, fatty acid oxidation and lipid transport (J) Figure 9 L). CuBGs treatment significantly inhibited the transcriptional level of CD36 in liver tissue ( Figure 3 J), consistent with in vitro findings ( Figure 8 B). Immunohistochemistry of liver CD36 showed a parallel decrease in protein expression relative to mRNA levels. Figure 3 K and L confirmed consistent inhibition at the transcriptional-translational level. These data suggest that CuBGs alleviate diet-induced hepatic lipid accumulation in HHFMCD by specifically inhibiting CD36—a key mediator in the pathogenesis of diet-induced fatty liver disease.

[0053] In summary, CuBGs exert a protective effect by reducing hepatic lipid accumulation, counteracting lipotoxicity-induced damage, inhibiting inflammatory responses and fibrosis processes, thereby enhancing overall liver function.

[0054] Example 4 CuBGs treatment can prevent bone loss in the MASH-HOD mouse model.

[0055] In the three main types of osteoporosis—senile osteoporosis, sex steroid deficiency osteoporosis, and secondary osteoporosis—increasing evidence suggests that MASH (the higher histological phenotype of MAFLD) is an important pathological factor in the development of secondary osteoporosis. This potential link has sparked great interest in bone-liver axis research. In our study, μCT analysis showed that HFMCD-diet mice exhibited severe bone loss compared to mice on a normal diet. Figure 4 A). Further analysis revealed that, compared to the control group, the bone mineral density (BMD) of the MASH group mice was reduced by approximately 30% ( Figure 4 B). Furthermore, compared to the control group, the MASH group showed a 54% reduction in bone volume (BV), a 32% reduction in tissue volume (TV), and a 32% reduction in the bone-to-tissue-volume ratio (BV / TV). Figure 4 CE), while trabecular separation (Tb.Sp) increased significantly by 32% ( Figure 10 C). Similarly, compared with the control group mice, the MASH group mice also had a decrease in the number of trabeculae (Tb.N) and the thickness of trabeculae (Tb.Th). Figure 10 A and Figure 10 B). Consistent with μCT data, H&E-stained distal femoral sections showed fewer trabeculae in MASH mice, indicating accelerated bone resorption. Figure 10 E). It is worth noting that both CuBGs and BGs treatments effectively reduced bone damage, with CuBGs showing superior therapeutic effects. Figure 4 AE and 10A-C, Figure 10 E. CuBGs treatment increased bone copper content in MASH mice compared to the untreated or BGs-treated groups. Figure 10 D), which is consistent with the known CuBGs through Cu 2+ The findings are consistent with the mediated pathways that induce osteoblast differentiation.

[0056] The maintenance of bone homeostasis depends on a precisely regulated balance between bone formation by osteoblasts and bone resorption by osteoclasts. Disruption of this delicate balance, whether due to mechanical overload, inflammation, aging, or metabolic dysfunction, can lead to pathological bone loss, clinically manifesting as osteoporosis or other skeletal diseases. To investigate the impaired coupling of bone formation and resorption mediated by MASH-HOD, we analyzed key osteogenic and osteoclast biomarkers in bone samples. To assess osteoblast formation and differentiation capacity in bone tissue, we examined three representative osteogenic markers: SP7 (early osteogenic marker), Runx2 (osteoblast-specific transcription factor), and OCN (late osteogenic marker). The results showed that, compared to CuBGs and BGs treatments which significantly enhanced osteoblast formation and differentiation capacity, the expression of these osteogenic-related markers was downregulated in MASH-HOD mice. Figure 4 (FK). Notably, CuBGs showed better therapeutic effects than BGs, which we speculate may be related to the higher copper content in the bones of the CuBGs group.

[0057] After confirming the therapeutic effect of nanoparticles on bone disease in MASH-HOD mice, we further investigated whether CuBGs target osteoclasts. Osteoclasts were detected by TRAP staining on distal femoral epiphyseal sections. Figure 4 L), we assessed the number of osteoclasts and osteoclast surface coverage on the bone surface, i.e., by quantifying the number of TRAP-positive multinucleated cells adhering to the bone surface (L). Figure 4 M). The expression of osteoclast marker genes, including tartrate-resistant acid phosphatase (TRAP), osteoclast-associated immunoglobulin-like receptor (OSCAR), cathepsin K (CTSK), matrix metalloproteinase 9 (MMP9), and colony-stimulating factor 1 (CSF1), was upregulated in the bone tissue of MASH-HOD mice. Figure 10 F). However, these results clearly demonstrate that CuBGs do not inhibit osteoclast-mediated bone resorption (F). Figure 4 L and M).

[0058] HFMCD diet-induced liver injury disrupts the osteoblast-osteoclast balance, leading to impaired bone metabolism and decreased bone mineral density (MASH-HOD). Notably, CuBGs improve MASH-HOD-related bone deterioration and reduce bone loss by promoting osteoblast differentiation rather than modulating osteoclast activity.

[0059] Example 5 Transcriptome analysis reveals the therapeutic effect of CuBGs on MASH-HOD-induced liver injury. Finally, we explored how CuBGs and BGs improved MASH-HOD. Given that CuBGs and BGs are primarily located in the mouse liver, we used transcriptome sequencing to analyze changes in the three groups of mice. Cluster heatmaps showed that, compared to the normal diet (ND) group, the expression trends of some genes in the BGs and CuBGs groups were similar (…). Figure 5 A). To assess intergroup differences and intragroup sample repeatability, we performed principal component analysis (PCA) on the read count matrix. The results showed tight clustering within groups and significant separation between groups, confirming minimal sample variability and a clear treatment-specific transcriptomic profile. Figure 11 A).

[0060] Next, we used volcano plot analysis to compare statistically significant differentially expressed genes (DEGs) between the BGs-treated group and the saline-treated group of MASH-HOD mice, and found 255 upregulated DEGs and 319 downregulated DEGs. Figure 11 B). Enrichment analysis of liver genes upregulated by BGs using the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that their transcriptomic effects were enriched in metabolic processes such as carbon metabolism, amino acid biosynthesis, glycolysis / gluconeogenesis, starch and sucrose metabolism, and the glucagon signaling pathway. Figure 11 C). DEGs between the CuBGs-treated group and the saline-treated group of MASH-HOD mice were as follows: Figure 5 As shown in B, there were 1451 upregulated DEGs and 1336 downregulated DEGs. KEGG pathway enrichment analysis of the upregulated genes after CuBGs treatment showed that their enrichment was particularly significant in the cofactor biosynthesis and insulin resistance signaling pathways.

[0061] Literature reports that the pathogenesis of MASH is closely related to hyperglycemia. During MASH development, an imbalance in lipid flow leads to excessive lipid accumulation, which in turn triggers chronic inflammation, mitochondrial dysfunction, and insulin resistance. On the one hand, this can lead to glucose metabolism disorders such as type 2 diabetes; on the other hand, hyperglycemia further promotes the conversion of carbohydrates into triglycerides (TG). Simultaneously, elevated insulin levels promote the transcription of SREBP-1c and LXR, enhancing de novo lipid synthesis, thereby indirectly accelerating the progression of non-alcoholic steatohepatitis (MASH). Overall, glucose metabolism pathways can influence MASH development by regulating lipid metabolism. Given that both BGs and CuBGs can affect glucose metabolism and insulin resistance, we hypothesize that they alleviate lipid accumulation in MASH by regulating glucose metabolism and insulin sensitivity.

[0062] We performed qRT-PCR validation on key genes (Pklr, Pfkm, Pgm1, Eno1, Pdhb, and Pgam1) in the glycolysis / gluconeogenesis pathway. Compared to the normal diet control group, the expression of these glycolytic enzymes in the liver of HFMCD-fed mice was significantly downregulated. Figure 11 D). Notably, BGs treatment effectively restored the expression levels of these glucose metabolism regulators. We also validated three key molecules in the insulin resistance signaling pathway (Ppargc1a, Slc27a1, PPARα), which showed significant upregulation in the CuBGs treatment group compared to downregulation in the HFMCD group. Furthermore, BGs and CuBGs significantly improved HFMCD-induced hyperglycemia. The glucose tolerance test (GTT) confirmed glucose intolerance in MASH-HOD mice, and CuBGs / BGs treatment improved this condition. Figure 5 (F and G). The above results confirm our hypothesis that BGs and CuBGs alleviate MASH symptoms by improving blood glucose levels and glucose tolerance.

[0063] CuBGs demonstrated superior therapeutic efficacy for MASH-HOD compared to BGs alone at both cellular and animal levels. We further evaluated the different effects of CuBGs and BGs on the transcriptome of liver tissue from MASH-HOD mice. In the liver tissue of MASH-HOD mice, the CuBGs group resulted in the upregulation of 1945 DEGs and the downregulation of 1876 DEGs (…). Figure 5 H), most of which upregulated DEGs are enriched in fatty acid metabolism processes, such as non-alcoholic fatty liver disease (NAFLD) signaling, oxidative phosphorylation, and chemically carcinogenic reactive oxygen species (ROS) processes. Figure 5I). HFMCD-induced decreased oxidative phosphorylation produces a large amount of ROS, which on the one hand impairs mitochondrial function and on the other hand activates the NF-κB signaling pathway, promoting the transcription of inflammatory factors (IL-6, IL-1β), thereby exacerbating inflammation and accelerating the progression of MASH. We hypothesized that CuBGs might alleviate inflammation by enhancing oxidative phosphorylation and ROS clearance. Subsequent qRT-PCR validation confirmed that, compared with BGs treatment, CuBGs significantly enhanced the upregulation of genes related to oxidative phosphorylation (Ndufa4l2, Ndufb2, Atp6vod2) in MASH-HOD liver tissue. Figure 5 J). In vitro experiments showed that CuBGs had significantly superior ROS scavenging ability compared with BGs alone. Figure 11 E and Figure 11 F). Given that oxidative phosphorylation and ROS production mainly occur in the mitochondria, we hypothesized that CuBGs might enter the mitochondria to exert a protective effect against liver injury. After FITC labeling of CuBGs and BGs and co-staining with mitochondrial dye, immunofluorescence showed a significantly higher colocalization of CuBGs with mitochondria, indicating that they are more likely to be taken up by mitochondria and exert their effects. Figure 5 In summary, CuBGs alleviate liver damage caused by MASH-HOD through a dual mechanism: on the one hand, they enhance glucose tolerance to trigger metabolic reprogramming and reduce lipid accumulation; on the other hand, they improve oxidative phosphorylation and inhibit mitochondrial ROS production to reduce inflammation—thus improving the disease condition from multiple angles.

[0064] Example 6 CuBGs alleviate bone damage caused by MASH-HOD by promoting LCAT expression and secretion.

[0065] Hepatic osteodystrophy (HOD) is a metabolic bone disease often associated with chronic liver disease, characterized by bone loss. MASH, as a chronic liver disease, may be a potential precursor to HOD. The liver factor lecithin cholesterol acyltransferase (LCAT) has been shown to improve carbon tetrachloride-induced HOD by retrogradely transporting cholesterol from bone tissue to the liver for metabolism via the liver-bone axis.

[0066] In vitro cell experiments showed that CuBGs can promote osteogenic differentiation by regulating the liver-bone axis. Figure 2 J and 2K). However, whether this process depends on LCAT remains to be investigated. Therefore, we measured the LCAT levels in the liver, blood, and bones of mice in each group. The results showed that HHFMCD diet-induced MASH liver injury significantly inhibited Lcat RNA transcription in liver tissue (J and 2K). Figure 6 A); Consistent with this, the protein content of LCAT in the liver was also significantly reduced ( Figure 6 BE). In contrast, the transcriptional and translational levels of LCAT in the livers of MASH-HOD mice treated with CuBGs were significantly increased (BE). Figure 6 AE). Notably, LCAT levels in mouse blood showed a strong concordance with expression in the liver ( Figure 6 This confirms that LCAT is a liver-derived cytokine. We then examined whether the LCAT protein levels in the mouse femur were consistent with changes in the liver and blood. CuBGs treatment significantly increased the LCAT protein content in the bone tissue of MASH-HOD mice (F). Figure 6 These results indicate that CuBGs treatment can alleviate liver damage in MASH-HOD, enhance the expression of the liver-derived cytokine LCAT, and increase the LCAT content in mouse bone tissue. However, whether CuBGs-mediated improvement in MASH-HOD bone damage depends on LCAT requires further investigation. Therefore, we established a hepatocyte-specific Lcat knockout mouse (GJ). Figure 12 A and B), and primary hepatocytes and osteoblast precursor cells (MC3T3-E1) were isolated and co-cultured under palmitic acid (PA) conditions. Figure 6 K). Consistent with previous results ( Figure 2 J and 3K), when CuBGs are co-cultured with primary hepatocytes from wild-type mice, can enhance osteogenic differentiation of MC3T3-E1 cells (J and 3K). Figure 6 L and M). However, when CuBGs were co-cultured with primary hepatocytes from Lcat hepatocyte-specific knockout mice, their promoting effect on osteoblast differentiation disappeared (L and M). Figure 6 L and M).

[0067] In summary, CuBGs can improve liver damage in MASH-HOD, restore LCAT expression in liver tissue, and promote LCAT accumulation in bone tissue through liver-bone axis transport, thereby promoting osteoblast differentiation, alleviating bone damage in MASH-HOD, and increasing bone mineral density.

[0068] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A copper-doped bioglass, characterized in that, The copper-doped bioglass was prepared via a sol-gel method, with copper used as Cu. 2+ The copper-doped bioglass is incorporated into a glass network structure, with a copper doping concentration ranging from 0.1 wt% to 5 wt%. This copper-doped bioglass exhibits sustained-release properties, continuously releasing Cu within 48 to 96 hours in buffer solutions at pH 7.4 or pH 5.

5. 2 + And there was no burst release phenomenon.

2. The copper-doped bioglass according to claim 1, characterized in that, The copper-doped bioglass particles are spherical with a diameter of 163.67 ± 15.26 nm; the particles have a mesoporous structure; and X-ray photoelectron spectroscopy reveals Cu 2p characteristic peaks at binding energies of 935 eV and 955 eV.

3. A method for preparing copper-doped bioglass as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Preparation of precursor solution: The copper source and the complexing agent are mixed in a solvent to form a stable copper complex precursor solution; (2) Sol-gel reaction: The silicon source, calcium source and the precursor solution obtained in step (1) are mixed under alkaline catalytic conditions to carry out co-hydrolysis and polycondensation reaction to form a sol, which is then aged to obtain a wet gel. (3) Post-processing: The wet gel is centrifuged, washed and dried to obtain the dry gel precursor; (4) Calcination: The dry gel precursor is calcined in an air atmosphere and cooled to obtain the copper-doped bioglass.

4. The use of the copper-doped bioglass according to claim 1 or 2 in the preparation of a medicament for treating chronic liver injury; wherein, The chronic liver injury referred to is metabolic-associated steatohepatitis (MASH) or non-alcoholic fatty liver disease (NAFLD).

5. The use according to claim 4, characterized in that, The drug is a drug capable of achieving functions such as (a)-(e): (a) Reduce serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST); (b) Reduce triglyceride accumulation in liver tissue; (c) Downregulates the mRNA expression levels of interleukin-1β and tumor necrosis factor-α in the liver; (d) Inhibits the infiltration of F4 / 80 positive macrophages in the liver; (e) Reduces the expression of α-smooth muscle actin α-SMA, thereby improving hepatocyte ballooning degeneration and inhibiting hepatic stellate cell activation.

6. The use according to claim 4, characterized in that, The drug can upregulate the mRNA expression of key glycolytic enzyme genes in the liver, including Pklr, Pfkm, Pgm1, Eno1, Pdhb, and Pgam1; and improve the expression of insulin resistance signaling pathway-related molecules Ppargc1a, Slc27a1, and Pparα, thereby alleviating glucose intolerance and inhibiting hepatic lipid production.

7. The use according to claim 4, characterized in that, The drug can be taken up by hepatocytes and significantly co-localizes with mitochondria within hepatocytes; the drug inhibits the NF-κB signaling pathway and inflammatory response by enhancing the mRNA expression of oxidative phosphorylation-related genes Ndufa4l2 and Atp6vod2 and improving the clearance efficiency of reactive oxygen species (ROS).

8. Use of the copper-doped bioglass as described in claim 1 or 2 in the preparation of a medicament for treating hepatic osteodystrophy, wherein, The hepatic osteopathy is caused by metabolic-associated fatty liver disease (MASH).

9. The use according to claim 8, characterized in that, The drug is a drug capable of achieving functions (a)-(c): (a) Increase bone mineral density (BMD), bone volume (BV), and bone volume / tissue volume ratio (BV / TV) of the femur; (b) Reduce trabecular separation (Tb.Sp); (c) Upregulate the mRNA expression of osteoblast markers Runx2 and Sp7 in bone tissue.

10. Use of the copper-doped bioglass as described in claim 1 or 2 in the preparation of a medicament for the simultaneous treatment of chronic liver injury and hepatic osteodystrophy.

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