A method for constructing a carrier-free nadh nanoparticle for restoring the function of senescent hepatocytes, product and application

Abstract

This invention discloses a method for constructing carrier-free NADH nanoparticles to restore the function of aging hepatocytes, the product, and its applications, belonging to the pharmaceutical field. The method includes preparing carrier-free nano-coordination polymer NADH-Gd using a reverse microemulsion method, followed by a two-step lipid modification process: first, DOPA modification, then DPPC, cholesterol, and DSPE-PEG. 5k NADH-PEG was synthesized by modification, with DSPE-PEG added during the modification process. 5k NADH-Gal is ultimately synthesized from NADH-Gal. NADH-Gal exhibits excellent hepatocyte targeting and can replenish NAD+ in senescent hepatocytes in the MASH environment. + The content is increased by adjusting... Aldh18a1 The expression of [a specific substance] regulates proline metabolism. Improved proline metabolism can restore mitochondrial function by promoting mitophagy and mitochondrial biosynthesis, thereby alleviating hepatocyte aging. Simultaneously, it reduces monocyte recruitment and hepatic stellate cells (HSCs), ultimately providing an effective treatment for MASH. This invention provides a novel strategy for the treatment of MASH.

Description

Technical Field

[0001] This invention relates to a method for constructing carrier-free NADH nanoparticles for restoring the function of aging liver cells, as well as the product and its application, belonging to the pharmaceutical field. Background Technology

[0002] Existing studies analyzing differentially expressed genes in the livers of healthy individuals and patients with metabolic dysfunction-associated steatohepatitis (MASH) revealed that the upregulated differentially expressed genes were mainly concentrated in aging-related pathways, indicating a close association between aging and MASH. Long-term excessive free fatty acids lead to an abnormal increase in electron flux in the mitochondrial electron transport chain, increased generation of reactive oxygen species (ROS), which in turn damages mitochondrial function, accelerates hepatocyte aging, and promotes MASH progression. Immunofluorescence analysis of MASH mouse livers showed elevated expression of aging markers p21, p53, and p16 proteins, most of which were co-localized with hepatocytes. Furthermore, MASH mice exhibited an increased number of senescence-associated secretory phenotypes (SASPs) in their aging livers compared to normal mice, all of which indicate that MASH mice exhibit significant hepatocyte senescence. However, the role of hepatocyte senescence in the progression of metabolic dysfunction-associated steatotic liver disease (MASLD) and the efficacy of reversing hepatocyte senescence in MASH treatment remain unclear.

[0003] Multiple studies have shown that the aging process is related to nicotinamide adenine dinucleotide (NAD) in cells and tissues. + The decrease in NAD levels is associated with this. Despite NAD supplementation... + The precursor may alleviate MASLD in mice by improving liver mitochondrial function and reducing oxidative stress, but NAD supplementation... + The potential mechanisms by which hepatocyte mitochondrial function is restored and hepatocyte aging is reversed remain unclear. Recent research indicates that amino acids, rather than glucose, are the primary substrates for the hepatocyte mitochondrial tricarboxylic acid (TCA) cycle and lipid production. Furthermore, NAD+... + As an essential cofactor in the TCA cycle, it participates in the metabolism of carbohydrates, lipids, and amino acids. Therefore, we hypothesize that NAD supplementation... +It is possible that NAD⁺ precursors can restore mitochondrial activity in aging hepatocytes by regulating amino acid metabolism, thereby promoting MASH treatment. However, preliminary clinical trial results showed that oral administration of NAD⁺ precursors had limited effect on reducing liver fat in obese individuals. This may be because gut microbiota dysbiosis in obese individuals affects the metabolism of NAD⁺ precursors, leading to reduced bioavailability of NAD⁺ in the liver. Due to NAD⁺… + NAD is a negatively charged hydrophilic molecule that is difficult for cells to take up directly. Therefore, previous studies have designed various nanocarriers such as calcium phosphate (CaP), metal-organic framework (MOF) nanoparticles, and nanomicelles to load NAD. + Alternatively, NADH can be used to deliver it intracellularly. However, these nanocarriers still suffer from drawbacks such as poor biodegradability and limited loading capacity. Therefore, developing a hepatocyte-targeted delivery system with simple structure, high loading capacity, and biodegradability is crucial to enhancing the efficacy of NAD therapy for MASH. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a method for constructing carrier-free NADH nanoparticles to restore the function of aging liver cells and their application.

[0005] Technical solution: The method for constructing carrier-free NADH nanoparticles for restoring the function of aging liver cells according to the present invention includes the following steps: (1) NADH and GdCl3·6H2O were synthesized into a carrier-free nano-coordination polymer NADH-Gd by reverse microemulsion method; (2) NADH-Gd was first modified by DOPA using a two-step lipid modification method, and then modified by DPPC, cholesterol, DSPE-PEG and DSPE-PEG-Gal to synthesize NADH-Gal.

[0006] Furthermore, the microemulsion used in step (1) includes Triton X-100, CO-520, n-hexanol and cyclohexane.

[0007] Further, the DSPE-PEG and DSPE-PEG-Gal mentioned in step (2) are respectively DSPE-PEG 5k and DSPE-PEG 5k -Gal.

[0008] Furthermore, the mass ratio of NADH-Gd, DPPC, cholesterol, DSPE-PEG and DSPE-PEG-Gal in step (2) is 1:4:2:4:4.

[0009] The above-described method of the present invention prepares carrier-free NADH nanoparticles.

[0010] The present invention relates to the application of the carrier-free NADH nanoparticles described above in the preparation of drugs for treating metabolic dysfunction-related steatohepatitis.

[0011] Furthermore, the carrier-free NADH nanoparticles can reduce lipids, resist aging, and alleviate fibrosis.

[0012] Furthermore, the anti-aging effect is achieved by reducing the levels of inflammatory factors TNF-α, IL-1β, IL-6, and TGF-β.

[0013] Furthermore, the carrier-free NADH nanoparticles can alleviate mitochondrial function.

[0014] Furthermore, the mitigation of mitochondrial function involves activating mitochondrial biosynthesis and mitophagy.

[0015] This invention utilizes the interaction between metal ions and phosphate groups, specifically the direct interaction between NADH and gadolinium ions (Gd). 3+ By coordinating with galactose, a carrier-free nanopolymer (NADH-Gd, with an NADH mass fraction of approximately 42.3%) was synthesized. After modification with galactose (Gal), the NADH-Gal nanoparticles exhibited excellent biocompatibility, accumulating in the liver after intravenous injection, particularly being taken up by hepatocytes. This targeted delivery of NADH is achieved by recognizing the desialylate glycoprotein receptor (ASGPR) specifically expressed on the surface of hepatocytes. Analysis of high-throughput transcriptomic data from normal and aging hepatocytes revealed that NADH-Gal, upon uptake, replenishes intracellular NAD. + Horizontal, thus raising Aldh18a1 The expression of [a specific substance] promotes proline metabolism. After the improved proline metabolism exerts its antioxidant effect, it activates AMPK phosphorylation, which in turn promotes mitochondrial biosynthesis mediated by PGC1-α / NRF1 / TFAM and mitophagy mediated by PINK1 / Parkin / p62 to restore mitochondrial function, thereby alleviating hepatocyte aging. At the same time, it reduces the recruitment of monocytes and the activation of HSCs, thereby delaying the progression of liver fibrosis and ultimately effectively treating MASH.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The carrier-free NADH nanoparticles NADH-Gal disclosed in this invention exhibit excellent hepatocyte targeting, and can replenish NAD in senescent hepatocytes in the MASH environment. + The content is increased by adjusting... Aldh18a1The expression of [a specific substance] regulates proline metabolism. Improved proline metabolism can restore mitochondrial function by promoting mitophagy and mitochondrial biosynthesis, thereby alleviating hepatocyte aging. Simultaneously, it reduces monocyte recruitment and HSC activation, ultimately providing an effective treatment for MASH. This invention provides a new strategy for the treatment of MASH. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the application mechanism of NADH-Gal in MASH treatment according to an embodiment of the present invention.

[0018] Figure 2 Figure 1 shows the characterization results of NADH-Gal in an embodiment of the present invention; wherein, A. Schematic diagram of NADH-Gal preparation; B. TEM image of NADH-Gd; C. UV-Vis absorption spectrum of free-NADH(NADH) / NADH-Gd / NADH-PEG; D. TEM image of NADH-PEG; E. Hydrated particle size of NADH-PEG during 7 days of incubation in PBS, water, and DMEM / F12 culture medium; F. NADH release curves of NADH-Gd and NADH-PEG at different pH values; G. TEM image of NADH-Gd at pH=5.6; H. TEM image of NADH-Gal; I. Cell viability of AML12 cells after treatment with NADH / NADH-PEG / NADH-Gal; J. Confocal image of AML12 cells uptake of NADH-PEG and NADH-Gal within 8 h, red: Dil.

[0019] Figure 3 The embodiment of the present invention demonstrates the ability of NADH-Gal to alleviate hepatocyte senescence and reduce HSC activation in vitro; wherein, AB. SA-β-gal staining and Oil Red staining of AML12 cells induced by FFA-DOX with or without NADH / NADH-PEG / NADH-Gal treatment; C. NAD in AML12 cells after different treatments. + / NADH ratio; D. Changes in SASP factor expression in AML12 cells after different treatments; E. Effects of different treatments on Col1 expression in HSC-T6 cells demonstrated by co-culture experiments.

[0020] Figure 4This invention relates to an embodiment of NADH-Gal, which alleviates mitochondrial function in senescent hepatocytes and its mechanism of action. The embodiment includes: A. Fluorescence images of AML12 cells stained with JC-1 after different treatments; B. Changes in intracellular ATP levels in AML12 cells after different treatments; C. Venn diagram of differentially expressed genes and mitochondrial-related genes; D. GO enrichment analysis based on mitochondrial-related differentially expressed genes; E. Heatmap of upregulated mitochondrial-related differentially expressed genes; F. String analysis of upregulated mitochondrial-related differentially expressed genes; G. AML12 cells treated with FFA-DOX and NADH-Gal. Aldh18a1 Expression of H. Schematic diagram of the therapeutic mechanism after NADH-Gal treatment; IK. Glutamate / proline / glutathione content in different groups; L. Expression of P5CS (Aldh18a1), p-AMPK, mitophagy (p62 / PINK1 / Parkin) and mitochondrial biogenesis (PGC1-α / Nrf1 / TFAM) related genes in different groups.

[0021] Figure 5 Examples of embodiments of the present invention include: in vivo targeting behavior of NADH-Gal; fluorescence enrichment images of AB. MCD mice 3h / 6h / 12h after tail vein injection of DiD-NADH-PEG and DiD-NADH-Gal; T1-weighted MRI images of CD. mice 6 hours before and after injection of NADH-PEG and NADH-Gal, with quantitative analysis of T1-weighted MRI signal intensity by relative signal-to-noise ratio (ΔSNR); colocalization analysis of NADH-PEG and NADH-Gal with hepatocytes and macrophages in the liver of EF. MCD mice, with DiD-NPs: DiD-NADH-PEG or DiD-NADH-Gal; fluorescence enrichment images of GH. CDAHFD mice 3h / 6h / 12h after tail vein injection of DiD-NADH-PEG and DiD-NADH-Gal; and colocalization analysis of NADH-PEG and NADH-Gal with hepatocytes and macrophages in the liver of IJ. MCD mice.

[0022] Figure 6 This invention provides an evaluation of the in vivo therapeutic effect and mechanism of NADH-Gal in embodiments thereof; wherein: A. Gross liver images of MCD mice before and after treatment; B. Liver NAD in different groups of MCD mice. +C. Changes in the NADH ratio; D. Serum ALT / AST / TG and liver TG levels in MCD mice after different treatments; E. Oil Red staining images of liver H&E in MCD mice before and after treatment; F. Expression of p21 and albumin (hepatocytes) in the liver of MCD mice before and after different treatments; G. Masson staining images of liver in MCD mice before and after treatment; + Immunofluorescence images of macrophages; changes in the expression of Aldh18a1, glutamate, proline, glutathione, mitochondrial biogenesis and mitophagy-related genes in the livers of healthy, MCD and NADH-Gal-treated mice.

[0023] Figure 7 This invention provides an evaluation of the in vivo therapeutic effect and mechanism of NADH-Gal in embodiments of the present invention; wherein: A. Gross liver images of CDAHFD mice before and after treatment; B. Liver NAD in different groups of CDAHFD mice. + C. Changes in the NADH ratio; D. Serum ALT / AST / TG and liver TG levels in CDAHFD mice after different treatments; E. Oil Red staining images of liver H&E in CDAHFD mice before and after treatment; F. Expression of p21 and albumin (hepatocytes) in the liver of CDAHFD mice before and after different treatments; G. Masson staining images of liver in CDAHFD mice before and after treatment; + Immunofluorescence images of macrophages; changes in the expression of Aldh18a1, glutamate, proline, glutathione, mitochondrial biogenesis and mitophagy-related genes in the livers of healthy, CDAHFD, and NADH-Gal-treated mice. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0025] Example 1: Construction of NADH-Gal S1: Synthesis of NADH-Gd nanoparticles 75 μL of an aqueous solution containing NADH (0.264 M, reduced nicotinamide adenine dinucleotide) and 75 μL of an aqueous solution containing GdCl3·6H2O (0.681 M, CAS:13450-84-5) were added dropwise to 2.5 mL of oil phase (0.3635 mL Triton X-100 CAS:9002-93-1, 0.066 mL CO-520 (polyoxyethylene (5) nonylphenyl ether) CAS:68412-54-4, 0.3695 mL n-hexanol, 1.701 mL cyclohexane). After mixing thoroughly, 10 μL of triethylamine was added, and the mixture was sonicated in a water bath (160 W) for 10 min. After adding 2.5 mL of ethanol as a demulsifier, the mixture was centrifuged at 14800 rpm for 5 min to obtain the precipitate, namely NADH-Gd. The precipitate was washed three times with ethanol to obtain purified NADH-Gd.

[0026] S2: NADH-Gal nanoparticles were obtained by two-step modification of surface lipids. First, add 0.1 mL of chloroform solution containing DOPA (1 mg / mL). -1 Add to 5 mL of an ethanol solution containing NADH-Gd (prepared in S1) (0.2 mg / mL) -1 The mixture was then sonicated (160 W) for 20 minutes. The solution was then centrifuged (14800 rpm, 5 min), and the precipitate was washed three times with ethanol and dispersed in 5 mL of chloroform. DPPC, cholesterol, and DSPE-PEG were then added. 5k (Xi'an Ruixi Biotechnology Co., Ltd.) and DSPE-PEG 5k -Gal (Chongqing Yusi Pharmaceutical Technology Co., Ltd.), NADH-Gd (quantified using NADH), DPPC, cholesterol, DSPE-PEG 5k and DSPE-PEG 5k The mass ratio of NADH-Gal was 1:4:2:4:4. After stirring overnight, chloroform was removed by rotary evaporation, and 5 mL of neutral water was added for ultrasonic hydration to obtain NADH-Gal. Other steps were the same, except that DSPE-PEG was not added. 5k -Gal was prepared by NADH-PEG, NADH-Gd, DPPC, cholesterol, and DSPE-PEG. 5k The mass ratio is 1:4:2:8.

[0027] The preparation process of NADH-Gal is as follows: Figure 2 As shown in A; the application mechanism in MASH treatment is as follows: Figure 1 As shown.

[0028] Example 2: Performance Verification of NADH-Gal 1. Characterization of NADH-Gal In this embodiment, approximately 60 nm NADH-Gd was obtained using a reverse microemulsion method. Figure 2 B), UV-Vis spectroscopy analysis revealed that the mass fraction of NADH in NADH-Gd was 42.3% ( Figure 2 C), which was then modified with lipids to obtain approximately 70 nm NADH-PEG ( Figure 2 D), and has good stability and acid response characteristics ( Figure 2 To specifically target hepatocytes, galactose (Gal), a molecule capable of recognizing desialylated glycoproteins on the hepatocyte membrane surface, was added during the modification process, resulting in size-invariant NADH-Gal. Figure 2 H), after treating AML12 cells with free-NADH / NADH-PEG / NADH-Gal (at a concentration of 25 μM NADH) for 24 h, the MTT assay showed no significant cytotoxicity. Figure 2 I) After labeling NADH-PEG and NADH-Gal with fluorescent dye (1,1'-bis(octadecyl-3,3,3',3'-tetramethylindocyanine) perchlorate, Dil) and co-incubating with AML12 cells for 8 h, fluorescence microscopy revealed that NADH-Gal was more readily taken up by AML12 cells. Figure 2 J).

[0029] 2. Validation of NADH-Gal's ability to lower lipids, fight aging, and alleviate fibrosis in vitro. This embodiment constructs a lipid deposition-induced aging hepatocyte model through the following steps: AML12 cells were first treated with 500 μM free fatty acids (FFA) for 24 h, followed by treatment with 3 μM doxorubicin (DOX) for 24 h. Then, free-NADH / NADH-PEG / NADH-Gal at a concentration of 25 μM was added. SA-β-Gal and Oil Red O staining revealed that NADH-Gal significantly alleviated aging and reduced lipid deposition. Figure 3 AB), through NAD + The NADH detection kit (S0176S, Shanghai Beyotime Biotechnology Co., Ltd.) detected substances that can regulate NAD. + / NADH ratio ( Figure 3 C), in addition, RT-qPCR detection (system: 0.4 μL 10 μM forward primer + 0.4 μL 10 μM reverse primer + 10 μL 2×PerfectStart) ®Green Qpcr SuperMix + 9.2 μL enzyme-free water. Procedure: A two-step qPCR method was used for detection. The reaction conditions were: 94℃ pre-denaturation for 30 seconds; followed by 40-45 cycles with the following cycling parameters: 94℃ denaturation for 5 seconds, 60℃ annealing and extension for 30 seconds (data acquisition stage); after the reaction, melting curve analysis was performed to verify the specificity of the amplified products. The study showed decreased expression of cytokines including TNF-α (5'-ATGAGCACAGAAAGCATGATC-3' and 5'-TACAGGCTTGTCACTCGAATT-3'), IL-1β (5'-CCAAAAGATGAAGGGCTGCT-3' and 5'-ACAGAGGATGGGCTCTTCT-3'), IL-6 (5'-TAGTCCTTCCTACCCCAATTTCC-3' and 5'-TTGGTCCTTAGCCACTCCTTC-3'), and TGF-β (5'-TGGCCAGATCCTGTCCAAAC-3' and 5'-GTTGTACAAAGCGAGCACCG-3'), which also demonstrates that NADH-Gal can maximally reduce inflammation and decrease SASP expression. Figure 3 D), to demonstrate its ability to alleviate fibrosis, transwell assays showed that NADH-Gal could alleviate the activation of lower ventricular HSC-T6 by cytokines released from senescent upper ventricular hepatocytes. Figure 3 EG).

[0030] 3. Validation of the mechanism by which NADH-Gal alleviates mitochondrial function This embodiment utilizes Mito-Tracker staining (Mito-Tracker RedCMXRos (C1049B, Shanghai Beyotime Biotechnology Co., Ltd.) diluted to a working concentration of 200 nM with serum-free culture medium, followed by incubation of AML12 cells at 37°C for 30 min) and ATP generation assays (ATP assay kit, S0027, Shanghai Beyotime Biotechnology Co., Ltd.) to demonstrate that, compared to free NADH and NADH-PEG, NADH-Gal exerts the most significant effect in alleviating mitochondrial dysfunction. Figure 4 AB). To investigate the mechanism, transcriptome sequencing analysis was performed on normal cells, FFA-DOX-induced cells, and cells treated with NADH-Gal after modeling. We found that mitochondrial-related genes were mainly enriched in amino acid metabolism after NADH-Gal treatment. Figure 4 CD), and through analysis, the most relevant CD was found. Aldh18a1 ( Figure 3 EF). RT-qPCR and WB analysis Aldh18a1The mRNA levels (5'-GAGCTGGACGTAGACGGAAG-3' and 5'-GAGGGCTGGACACGATTTGA-3') and protein expression levels (P5CS, YN5999, dilution 1:1000, Immunoway Biotechnology) were downregulated after FFA-DOX stimulation and recovered after NADH-Gal treatment. Figure 4 G, L). In view of Aldh18a1 We found that NADH-Gal treatment decreased glutamate levels and increased proline levels, while si-Aldh18a1 (knocked down in normal AML12 cells) was also involved. Aldh18a1 NADH-Gal failed to function in the cells (genes) Figure 4 IJ). Furthermore, proline has antioxidant properties; GSH levels in senescent AML12 cells increased after treatment with NADH-Gal. Figure 4 K), thereby activating AMPK phosphorylation, and Western blotting demonstrated that NADH-Gal promotes mitochondrial biosynthesis and the expression of mitophagy-related proteins. Figure 4 L).

[0031] 4. Validation of NADH-Gal's in vivo targeting The animals used in this animal experiment were male C57BL / 6 mice, weighing approximately 18-20g and aged 5-6 weeks, purchased from the Experimental Animal Center of Yangzhou University. MASH mice were constructed by feeding them a methionine-choline deficient diet (MCD, purchased from Jiangsu Xiehe Biotechnology Co., Ltd.) and a choline-deficient L-amino acid-defined high-fat diet (CDAHFD, purchased from Jiangsu Xiehe Biotechnology Co., Ltd.). After fluorescently labeling NADH-PEG and NADH-Gal with a fluorescent dye (1,1'-octadecyl-3,3,3',3'-tetramethylindole dicarboxycyanine, 4-chlorobenzenesulfonate, DiD), mice fed the MCD diet for 5 weeks and the CDAHFD diet for 9 weeks were injected once via the tail vein with DiD-NADH-PEG and DiD-NADH-Gal (10 mg / kg of NADH, dissolved in PBS). Small animal in vivo fluorescence imaging then demonstrated that NADH-Gal was more abundant in the liver of MASH mice induced by MCD and CDAHFD. Figure 5 AB, GH), and MRI imaging (five weeks of MCD diet) also demonstrated the excellent liver targeting of NADH-Gal ( Figure 5 12 hours after DiD-NADH-Gal was injected into MASH mice via the tail vein, immunofluorescence staining of liver sections revealed that NADH-Gal was taken up more by hepatocytes and less phagocytosed by macrophages. Figure 5 EF, IJ).

[0032] 5. Verification of the in vivo therapeutic effect of NADH-Gal In this embodiment, we treated MCD-induced MASH mice by tail vein injection of NADH / NADH-PEG / NADH-Gal (10 mg / kg based on NADH concentration, dissolved in PBS): after four weeks of MCD diet, treatment was administered every three days for four consecutive weeks, and the treatment effect was observed in the mice. We found that compared with the normal control group, the liver volume of MCD-induced mice was reduced, and yellow discoloration and rough surface were observed. The liver morphology of MASH mice treated with NADH-Gal was smoother. Figure 6 A), and NAD + The NADH / NADH ratio is close to the normal level in healthy mice. Figure 6 B). Subsequent biochemical analysis showed that NADH-Gal treatment in MASH mice decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), increased serum triglycerides (TG), and decreased liver TG. Figure 6 C), showing improvement in liver damage and lipid metabolism disorders. H&E and Oil Red O staining indicated that NADH-Gal treatment significantly alleviated hepatocyte structural damage, lipid droplet accumulation, and inflammatory infiltration. Figure 6 D). The attenuation of p21 immunofluorescence and the downregulation of SASP demonstrated that NADH-Gal exhibited superior anti-aging effects compared to NADH and NADH-PEG. Figure 6 E). This reduces the activation of HSCs, and Masson staining shows a decrease in collagen fiber area (E). Figure 6 F), on the other hand, reduced the number of recruited monocytes (F). Figure 6 G), thereby reducing inflammation. Furthermore, in MASH mice, the liver... Aldh18a1 Expression was also significantly downregulated, while NADH-Gal treatment restored its expression. Figure 6 H), and decreased glutamate levels and increased proline levels (H). Figure 6 I). Restored GSH levels can reactivate mitochondrial biosynthesis and mitophagy in the MCD model ( Figure 6IJ) improved mitochondrial dysfunction in MASH mice. However, in MCD-fed mice, hepatic very low-density lipoprotein (VLDL) synthesis was inhibited, leading to the inability of TG to be transported out of the liver. Simultaneously, impaired protein synthesis caused muscle atrophy and weight loss, a phenotype significantly different from human MASH. To overcome these model limitations and further validate the therapeutic effect of NADH-Gal, we used the CDAHFD model, which supplements the MCD diet with 0.1% methionine and 60% fat, effectively avoiding the severe emaciation induced by the MCD diet. Treatment was administered via tail vein injection of NADH / NADH-PEG / NADH-Gal (10 mg / kg NADH, dissolved in PBS): after eight weeks on the CDAHFD diet, treatment was given every three days for four consecutive weeks, and the therapeutic effect was observed in mice. Ultimately, the same therapeutic effect as in the MCD model was achieved. Figure 7 (AJ). In summary, this embodiment successfully constructed NADH-Gal nanoparticles capable of alleviating hepatocyte aging, and due to the presence of Gal, they can specifically target hepatocytes. NADH-Gal can replenish NAD+ in aging hepatocytes. + , Upward Aldh18a1 This invention regulates proline metabolism, thereby improving mitochondrial function and alleviating aging in MASH, while reducing monocyte recruitment and HSC activation, ultimately providing an effective treatment for MASH. This invention offers a novel strategy for the treatment of MASH.

Claims

1. A method for constructing carrier-free NADH nanoparticles to restore the function of aging hepatocytes, characterized in that, The construction method includes the following steps: (1) NADH and GdCl3·6H2O were synthesized into a carrier-free nano-coordination polymer NADH-Gd by reverse microemulsion method; (2) NADH-Gd was first modified by sodium dioleoyl phosphatidyl phosphate (DOPA) using a two-step lipid modification method, and then modified by dipalmitoyl phosphatidylcholine (DPPC), cholesterol, distearate phosphatidyl ethanolamine-methoxy polyethylene glycol (DSPE-PEG), and distearate phosphatidyl ethanolamine-methoxy polyethylene glycol (DSPE-PEG-Gal) to synthesize NADH-Gal.

2. The construction method according to claim 1, characterized in that, The microemulsions used in step (1) include Triton X-100, CO-520, n-hexanol and cyclohexane.

3. The construction method according to claim 1, characterized in that, The DSPE-PEG and DSPE-PEG-Gal mentioned in step (2) are respectively DSPE-PEG 5k and DSPE-PEG 5k -Gal.

4. The construction method according to claim 1, characterized in that, The mass ratio of NADH-Gd, DPPC, cholesterol, DSPE-PEG and DSPE-PEG-Gal in step (2) is 1:4:2:4:

4.

5. The carrier-free NADH nanoparticles prepared by the method according to any one of claims 1 to 4.

6. The use of the carrier-free NADH nanoparticles according to claim 5 in the preparation of a drug for treating metabolic dysfunction-related steatohepatitis.

7. The application according to claim 6, characterized in that, The carrier-free NADH nanoparticles can reduce lipids, resist aging, and alleviate fibrosis.

8. The application according to claim 7, characterized in that, The anti-aging effect is achieved by reducing the levels of inflammatory factors TNF-α, IL-1β, IL-6, and TGF-β.

9. The application according to claim 6, characterized in that, The carrier-free NADH nanoparticles can alleviate mitochondrial dysfunction.

10. The application according to claim 9, characterized in that, The relief of mitochondrial function is achieved by activating mitochondrial biosynthesis and mitophagy.

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