Preparation method and application of sesamin targeted carrying nanoparticles

Abstract

The invention discloses a preparation method and application of sesamin targeted carrying nanoparticles, and the preparation method comprises the following steps: reacting ketal thiol TK, 2-succinimido-1, 1, 3, 3-tetramethylurea tetrafluoroborate, N, N-diisopropylethylamine and iramitide SS-31 peptide to prepare an SS31 peptide-TK compound, then reacting with chitosan, and simultaneously preparing a sodium alginate-lactobionic acid compound, so as to obtain the sesamin targeted carrying nanoparticles. Finally, mixing the two solutions to obtain a covalent complex; the method comprises the following steps: baking pork slices, extracting with ethanol, filtering, carrying out rotary evaporation, extracting, dialyzing and purifying, and freeze-drying; and mixing the products of the first two steps, dissolving in ethanol, adding sesamin, and carrying out electrostatic self-assembly and freeze drying to obtain the visual sesamin targeted nanoparticles. According to the invention, specific positioning of liver mitochondria, ROS stimulation response intelligent drug release and real-time visualization of food-borne fluorescence labeling are realized, and the application prospect in prevention and treatment of liver diseases is wide.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical nanotechnology, and more particularly to a method for preparing and applying sesamin-targeted delivery nanoparticles. Background Technology

[0002] The aging of bodily tissues and organs caused by population aging has gradually become one of the major scientific issues in the field of population and health in the 21st century, posing a severe challenge to the entire life sciences and medical field. The liver, the largest solid organ in the body, accounting for approximately 2% of total body weight, has metabolic functions such as storage, secretion, and decomposition. It is also one of the organs most susceptible to damage during the aging process; liver aging can alter biological characteristics and lead to related diseases. The antagonistic role of mitochondria in aging indicates that mitochondria are not only energy suppliers but also play a crucial role in aging and age-related diseases. The accumulation of oxidative damage in mitochondria is the free radical theory basis of aging and a driving factor for mitochondrial dysfunction and the observed decline in organ function during aging. Some natural active substances, endogenous compounds, and synthetic products have been shown to reduce the production of reactive oxygen species and protect the human body from chronic diseases. Therefore, it is essential to develop liver-targeted products with hepatoprotective effects to mitigate age-related oxidative stress damage.

[0003] As people's demands for nutrition and health continue to increase, in addition to providing the body with essential nutrients and energy, foods containing bioactive components are becoming increasingly popular among consumers. Food functional factors are closely related to precision nutrition, serving as a crucial material basis for achieving precision nutrition and an important means of nutritional regulation. However, many food functional factors suffer from drawbacks such as poor solubility, instability, and low bioavailability, which represent bottlenecks in their industrial application in the food sector. Therefore, the stable and precise targeted release of functional active ingredients is currently a hot research topic in the industry.

[0004] Sesame, also known as flaxseed, is considered a tonic in traditional medicine. It possesses unique properties in biological, chemical, and physiological aspects due to its unique chemical components, such as sesamin. Sesamin is one type of sesamin, found in high concentrations in sesame seeds and sesame oil. In 1890, a Scottish scientist first isolated sesamin (SES) from the acetic acid extract of sesame oil, and its structure and configuration were later determined by German scientists.

[0005] Domestic and international studies have shown that sesamin possesses physiological functions such as anti-cancer, neuroprotective, improvement of hyperlipidemia and hypertension, and antioxidant and anti-inflammatory effects. The liver-protective effect of sesamin is related to its antioxidant stress response, anti-inflammatory, and anti-lipid peroxidation effects. Sesamin enhances the body's antioxidant capacity and regulates iNOS expression, improving AlCl3 and D-galactose-induced liver damage in rats; it resists lipid peroxidation, protecting rats from chronic liver injury; it inhibits the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in rat serum, while significantly enhancing SOD activity, reducing the content of the oxidative metabolite malondialdehyde (MDA), improving the body's antioxidant capacity, and providing some protection against alcoholic liver injury; it scavenge free radicals in the body, regulates blood lipid levels, and prevents and treats non-alcoholic fatty liver disease.

[0006] However, simply applying sesamin solution is not very effective in improving liver damage because sesamin is a fat-soluble substance with poor solubility in water (2.5 μg / mL), which limits its dissolution rate and release efficiency, and also has certain toxic side effects.

[0007] Therefore, in view of the shortcomings of the above-mentioned existing technologies, a method for preparing visual ROS-stimulated liver mitochondrial-targeted sesamin nanoparticles and their application are provided. This method solves the problems of low bioavailability, poor targeting and insufficient controllability of drug release of sesamin, and thus significantly improves its efficacy and safety in the prevention and treatment of liver diseases. Summary of the Invention

[0008] In view of this, the present invention provides a method for preparing and applying sesamin-targeted transport nanoparticles.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] A method for preparing sesamin-targeted nanoparticles includes the following steps:

[0011] Step 1: Preparation of ROS-stimulated liver mitochondrial-targeted delivery wall material

[0012] Ketothiols TK, 2-succinimide-1,1,3,3-tetramethylurea tetrafluoroborate, N,N-diisopropylethylamine and aramidin SS-31 peptide were mixed and stirred to prepare the SS31 peptide-TK complex.

[0013] 2-Succinimide-1,1,3,3-tetramethylurea tetrafluoroborate and chitosan were added to the SS31 peptide-TK complex, and the reaction was stirred to obtain the chitosan-TK-SS31 peptide complex.

[0014] Lactobionic acid, dicyclohexylcarbodiimide and dimethylaminopyridine were reacted to prepare lactobionic acid carboxyl activated solution, and then sodium alginate was added and stirred to obtain sodium alginate-lacobionic acid complex.

[0015] The sodium alginate-lactobionic acid complex was mixed with the chitosan-TK-SS31 peptide complex to obtain a chitosan-TK-SS31 peptide-sodium alginate-lactobionic acid covalent complex, which is a ROS-stimulated liver mitochondrial-targeted delivery wall material.

[0016] Step 2: Preparation of food-derived fluorescent nanoparticles

[0017] Pork samples were sliced ​​and baked in an electric oven at 180℃, 230℃ or 280℃ for 30 minutes. After cooling, ethanol was added and stirred for 1 day. The filtrate was filtered and the solvent was removed by rotary evaporation until it became viscous. Deionized water was added and the mixture was extracted with ethyl acetate. The aqueous phase was retained and purified by dialysis using a dialysis bag with a molecular weight of 500 Da for 72 hours. The filtrate was then freeze-dried to obtain food-derived fluorescent nanoparticle powder.

[0018] Step 3: Visualization of the preparation of ROS-stimulated liver mitochondrial-targeted sesamin nanoparticles

[0019] The ROS-stimulated liver mitochondrial-targeting delivery wall material prepared in step 1) was mixed and stirred with the food-derived fluorescent nanoparticles prepared in step 2), dissolved in ethanol, and then sesamin was added. The complex was formed through electrostatic self-assembly, and then freeze-dried to obtain visualized sesamin-targeting nanoparticles.

[0020] Preferably, in step 1), the molar ratio of ketithiolide, 2-succinimide-1,1,3,3-tetramethylurea tetrafluoroborate, N,N-diisopropylethylamine and aramidin is 1:(1.2-1.5):(2-3):(0.8-1.2).

[0021] Preferably, in step 1), the molar ratio of lactobionic acid, dicyclohexylcarbodiimide and dimethylaminopyridine is 1:(1.1-1.3):(0.1-0.3).

[0022] Preferably, in step 3), the mass ratio of ROS-stimulated liver mitochondrial targeted delivery wall material, food-derived fluorescent nanoparticles and sesamin is (5-10):(1-2):(2-5).

[0023] Preferably, in step 2), the amount of ethanol used is 2L of ethanol per 100g pork sample.

[0024] Preferably, in step 3), the conditions for electrostatic self-assembly are: stirring reaction at 25°C for 2-4 hours.

[0025] A method for preparing sesamin-targeted nanoparticles.

[0026] Application of sesamin-targeted nanoparticles in the preparation of drug delivery systems with ROS-stimulated responsiveness and liver mitochondrial targeting.

[0027] Application of sesamin-targeted nanoparticles in the preparation of drugs for the prevention or treatment of liver diseases.

[0028] Preferably, the liver disease includes non-alcoholic fatty liver, liver fibrosis, or liver injury.

[0029] The present invention achieves the following technical effects compared to the prior art:

[0030] (1) This invention constructs a sesamin nanoparticle delivery system integrating multiple functions, providing a new technology for the precise delivery of food functional factors; the system achieves liver mitochondrial-specific localization through targeted molecular modification, significantly increases the concentration of sesamin at the lesion site, enhances the anti-oxidative damage treatment effect, and at the same time reduces exposure to normal tissues and reduces side effects; the introduction of ROS-sensitive groups endows it with intelligent drug release characteristics, and releases drugs rapidly only under oxidative stress, avoiding premature release of components and greatly improving drug utilization;

[0031] (2) This invention realizes real-time visualization of the distribution and migration of nanoparticles in vivo, providing precise data support for pharmacokinetic studies; the nanocarrier effectively solves the problems of poor solubility and low bioavailability of sesamin, and combined with its natural antioxidant and anti-inflammatory advantages, it shows broad application prospects in the prevention and treatment of liver diseases, and its natural properties are more easily accepted by patients.

[0032] (3) This invention simplifies the complexity of the delivery system through multifunctional integrated design, realizes the complementary advantages of natural active ingredients and advanced carrier technology, and expands the application of sesamin in the food and biomedicine fields. Its core experience lies in the design of carrier functions to accurately meet the needs of lesions, which provides an important reference for subsequent research on targeted delivery of food functional factors and helps to promote the integrated development of functional foods and precision medicine. Attached Figure Description

[0033] Figure 1 This is a flowchart illustrating the construction process of the ROS-responsive liver mitochondrial targeted sesamin delivery system of this invention.

[0034] Figure 2 This diagram illustrates the preparation of the food-derived fluorescent nanoparticles of this invention.

[0035] Figure 3 This is a diagram of the ROS-stimulated liver mitochondrial-targeted sesamin nanoparticles of this invention.

[0036] Figure 4 This is the FTIR characterization diagram of the present invention;

[0037] Figure 5 This is the XRD diffraction analysis pattern of the present invention;

[0038] Figure 6 This is a SEM morphology characterization image of the present invention;

[0039] Figure 7 This is a characterization diagram of sesamin nanoparticle uptake under different temperatures and H2O2 concentrations according to the present invention.

[0040] Figure 8 This is a diagram of the AML12 cell MTT assay of the present invention;

[0041] Figure 9 This is a mitochondrial membrane potential diagram of the cell according to the present invention;

[0042] Figure 10 This is a graph showing the cell viability assay of the present invention. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0044] Example 1:

[0045] 1. Preparation of ROS-stimulated liver mitochondrial-targeted delivery wall material

[0046] A ketithiolide (TK), 2-succinimide-1,1,3,3-tetramethylurea tetrafluoroborate, N,N-diisopropylethylamine, and aramidin (SS-31 peptide) were reacted with stirring to prepare the SS31 peptide-TK complex. 2-succinimide-1,1,3,3-tetramethylurea tetrafluoroborate and chitosan were then added to the complex and reacted with stirring to obtain the chitosan-TK-SS31 peptide complex. Lactobionic acid, dicyclohexylcarbodiimide, and dimethylaminopyridine were reacted to obtain a lactobionic acid carboxyl-activated solution, followed by the addition of sodium alginate and the reaction to obtain the sodium alginate-lactobionic acid complex. The sodium alginate-lactobionic acid complex and the chitosan-TK-SS31 peptide complex were mixed to obtain the chitosan-TK-SS31 peptide-sodium alginate-lactobionic acid covalent complex, which is a ROS-stimulated liver mitochondrial-targeting delivery wall material. Figure 1 As shown.

[0047] 2. Preparation of food-derived fluorescent nanoparticles

[0048] Pork samples were taken, sliced, and baked in electric ovens at 180°C, 230°C, and 280°C for 30 minutes each. The actual heating temperature was checked and adjusted to the preset temperature using a handheld near-infrared thermometer. The baked pork samples were cooled to room temperature, and 2 L of ethanol was added and stirred for one day. The precipitate was removed by filtration to obtain an ethanol solution containing food-derived fluorescent nanoparticles (NPs). The solvent was removed by rotary evaporation until a viscous consistency was achieved. Then, 50 mL of deionized water was added, and the solution was extracted five times with 50 mL of ethyl acetate. The organic phase was discarded, and the aqueous phase was collected and purified by dialysis using a dialysis bag with a molecular weight of 500 Da for 72 hours. The dialysate was collected and freeze-dried to obtain a pale yellow NPs powder. Figure 2 As shown.

[0049] 3. Preparation of ROS-stimulated liver mitochondrial-targeted sesamin nanoparticles

[0050] The constructed ROS-stimulated liver mitochondrial-targeting delivery wall material was mixed with food-derived fluorescent nanoparticles, then dissolved in ethanol. Sesamin was added, and the mixture underwent electrostatic self-assembly. After freeze-drying, visualized sesamin-targeting nanoparticles were prepared. Figure 3 As shown.

[0051] 4. Visualization and characterization of ROS-stimulated liver mitochondrial-targeted sesamin nanoparticles

[0052] The structure and morphology of the constructed sesamin nanoparticles were characterized, and a ROS-stimulated liver mitochondrial targeted delivery wall material (chitosan oligosaccharide / TK-SS31 peptide / sodium alginate / lactobionic acid) was successfully prepared. Sesamin was then encapsulated into nanoparticles using self-assembly technology. Characterization showed that the nanoparticles exhibited uniform morphology, good stability, and met the encapsulation efficiency and drug loading targets. Figure 4-6 As shown.

[0053] 5. Stability and cellular uptake analysis

[0054] The encapsulation efficiency and drug loading of the prepared sesamin-targeted nanoparticles were measured under different temperatures and H2O2 concentrations. A multi-mode in-situ mapping analysis system was used to analyze the fluorescence signals surrounding cells treated with free sesamin and targeted sesamin, and to perform cell uptake and characterization analysis. Figure 7 As shown.

[0055] Example 2:

[0056] 1. Cytotoxicity test

[0057] With 1×10 6AML12 cells / mL were seeded in 96-well plates for 24 hours and incubated statically in a 37°C, 5% CO2 cell culture incubator. After complete cell adhesion, 100 μL of the nanoparticle sample from the examples described below was added and incubated for another 24 hours. Then, 15 μL of MTT solution was added to each well, and after 4 hours of incubation, 150 μL of dimethyl sulfoxide was added to each well, and the plates were shaken for 10 minutes. The absorbance at 490 nm was recorded. Figure 8 As shown.

[0058] 2. Cellular ROS experiment

[0059] With 1×10 5 LO2 cells were seeded at 1 / mL in 6-well plates for 24 hours and incubated statically in a 37°C, 5% CO2 cell culture incubator. After complete cell adhesion, 2 mL of the nanoparticle sample from the examples described below was added, and incubation continued for another 24 hours. Then, 1 mL of LDCFH-DA was added to the culture medium, and the cells were washed twice with PBS after 30 minutes. Measurements were then performed using a fluorescence inverted microscope. The results showed that the visualized sesamin nanoparticles effectively reduced the elevated ROS levels in cells caused by D-galactose treatment.

[0060] 3. Mitochondrial membrane potential experiment

[0061] AML cells were seeded in 6-well plates at 1×10⁵ cells / mL for 12 hours and then incubated statically in a 37°C, 5% CO₂ cell culture incubator. After complete cell adhesion, 1 mL of the nanoparticle sample from the examples described below was added, and incubation continued for another 12 hours. Following incubation, the JC-1 mitochondrial membrane potential staining kit (Solepro, Beijing) was used. The results, as shown in the figure, indicate that sesamin nanoparticles effectively improved the mitochondrial membrane potential imbalance in cells, and showed a significantly greater improvement than free sesamin treatment. Figure 9 As shown.

[0062] 4. Cellular mitochondrial respiratory chain assay

[0063] The activity assay kit for mitochondrial respiratory chain complex I / NADH-CoQ reductase (Solepro, Beijing) was used to analyze free sesamin and visualized ROS-responsive sesamin nanocarriers. The enzyme activity of complex I was measured, which can reflect not only the state of the respiratory electron transport chain (ETC) but also the state of ROS generation.

[0064] 5. Cell viability assay

[0065] A D-galactose-induced cell senescence model was constructed to investigate the protective effects of free sesamin and visualized sesamin nanoparticles on cells. The results showed that fluorescent sesamin nanoparticles significantly improved the bioavailability of sesamin and demonstrated biocompatibility. Figure 10 As shown in Table 1.

[0066] Table 1:

[0067] Therefore, the present invention has the following advantages:

[0068] (1) Precise targeting and visualization

[0069] Through specific targeting molecular modifications, nanoparticles can specifically recognize and bind to liver cells, thereby precisely targeting liver mitochondria. This targeting capability significantly increases the concentration of sesamin in liver mitochondria, enhancing its therapeutic effect on liver mitochondrial-related diseases or oxidative stress damage. Compared to traditional non-targeted drug delivery systems, it reduces unnecessary exposure to other normal tissues and lowers potential side effects. Furthermore, using food-derived fluorescent markers, the distribution, migration, and release of nanoparticles can be observed in real-time and dynamically at the cellular level and in vivo. This not only contributes to in-depth research on the pharmacokinetics and biodistribution of nanoparticles in vivo, but also provides more precise data support for the development of functional food ingredients.

[0070] (2) ROS-responsive release

[0071] This study introduced ROS-sensitive groups into the design of nanoparticles, enabling them to respond to reactive oxygen species (ROS) in liver mitochondria. Under normal physiological conditions, the nanoparticles remain stable, with sesamin released slowly or not at all. However, when liver mitochondria are damaged by oxidative stress, leading to elevated ROS levels, the nanoparticle structure changes, triggering a rapid release of sesamin. This intelligent drug release mechanism ensures that sesamin exerts its antioxidant effect at the most needed time and site, improving drug efficacy and utilization while avoiding premature release and unnecessary systemic distribution of sesamin.

[0072] (3) Multifunctional integration

[0073] The nanoparticles integrate multiple functions, including sesamin loading, liver mitochondrial targeting, ROS-responsive drug release, and visualization imaging. This multifunctional integrated design not only simplifies the complexity of food bioactive factor delivery systems but also improves overall targeted delivery capabilities. The precise localization of liver mitochondria, antioxidant repair, and visualized evaluation of therapeutic effects provide new technologies and methods for the precise delivery of functional food factors in the future.

[0074] (4) Effective application of functional factors in food

[0075] Sesamin, as a natural antioxidant, boasts advantages such as wide availability, high safety, and good biocompatibility. Using sesamin as the core active ingredient can fully leverage its antioxidant, anti-inflammatory, and mitochondrial protective activities, offering potential applications in the prevention and treatment of liver diseases. Compared to chemically synthesized drugs, sesamin's natural properties make it more acceptable to patients, and it may have a lower risk of toxic side effects with long-term use. Furthermore, using nanoparticles as a carrier overcomes the problems of poor solubility and low bioavailability inherent in sesamin itself. Nanoparticles can improve the stability of sesamin, increase its circulation time in vivo, promote its transmembrane absorption into cells, and achieve targeted delivery to liver mitochondria. Simultaneously, the ROS-responsive characteristics and visualization capabilities of nanocarriers further enhance the therapeutic efficacy and monitoring capabilities of sesamin, achieving a complementary advantage between natural active ingredients and advanced carrier technology, and expanding the application prospects of sesamin in the food and biopharmaceutical fields.

[0076] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a sesame seed targeted carrier nanoparticle, characterized in that, Comprising the following steps: Step 1: Preparation of ROS stimulation responsive liver mitochondria targeting carrier wall material Mix ketone thioacetal TK, 2-succinimidyl-1,1,3,3-tetramethyl uronium tetrafluoroborate, N,N-diisopropyl ethylamine and iramil peptide SS-31 peptide, stir and react to obtain SS31 peptide-TK complex; Add 2-succinimidyl-1,1,3,3-tetramethyl uronium tetrafluoroborate and chitosan to the SS31 peptide-TK complex, stir and react to obtain chitosan-TK-SS31 peptide complex; React lactobionic acid, dicyclohexyl carbodiimide and dimethyl amino pyridine to obtain lactobionic acid carboxyl activation solution, then add sodium alginate and stir to obtain sodium alginate-lactobionic acid complex; Mix the sodium alginate-lactobionic acid complex and the chitosan-TK-SS31 peptide complex, and react to obtain chitosan-TK-SS31 peptide-sodium alginate-lactobionic acid covalent complex, namely ROS stimulation responsive liver mitochondria targeting carrier wall material; Step 2: Preparation of food-derived fluorescent nanoparticles Slice the pork sample, bake in an electric oven at 180℃, 230℃ or 280℃ for 30 min, add ethanol after cooling and stir for 1 day, filter to obtain filtrate; remove the solvent by rotary evaporation to a viscous state, add deionized water, extract with ethyl acetate, and then retain the water phase, purify by dialysis in a dialysis bag with a molecular weight of 500 Da for 72 hours, and freeze-dry to obtain food-derived fluorescent nanoparticle powder; Step 3: Preparation of visual ROS stimulation responsive liver mitochondria targeting sesamin nanoparticles Mix the ROS stimulation responsive liver mitochondria targeting carrier wall material prepared in step 1) and the food-derived fluorescent nanoparticles prepared in step 2), stir, dissolve in ethanol, and then add sesamin to form a complex by electrostatic self-assembly, and freeze-dry to obtain visual sesamin targeting nanoparticles.

2. The method of claim 1, wherein the sesame seed targeted nanoparticle is prepared by the steps of: a) mixing sesame seed oil with a surfactant; b) adding the mixture to a nanoparticle solution; and c) mixing the mixture of step b) with a nanoparticle solution. In step 1), the molar ratio of ketone thioacetal, 2-succinimidyl-1,1,3,3-tetramethyl uronium tetrafluoroborate, N,N-diisopropyl ethylamine and iramil peptide is 1:(1.2-1.5):(2-3):(0.8-1.2).

3. The method of claim 1, wherein the sesame seed targeted nanoparticle is prepared by the steps of: a) mixing sesame seed oil with a surfactant; b) adding the mixture to a nanoparticle solution; and c) mixing the mixture of step b) with a nanoparticle solution. In step 1), the molar ratio of lactobionic acid, dicyclohexyl carbodiimide and dimethyl amino pyridine is 1:(1.1-1.3):(0.1-0.3).

4. The method of claim 1, wherein the sesame seed targeted nanoparticle is prepared by the steps of: a) mixing sesame seed oil with a surfactant; b) adding the mixture to a nanoparticle solution; and c) mixing the mixture of step b) with a nanoparticle solution. In step 3), the mass ratio of ROS stimulation responsive liver mitochondria targeting carrier wall material, food-derived fluorescent nanoparticles and sesamin is (5-10):(1-2):(2-5).

5. The method of claim 1, wherein the sesame seed targeted nanoparticle is prepared by the steps of: a) mixing sesame seed oil with a surfactant; b) adding the mixture to a nanoparticle solution; and c) mixing the mixture of step b) with a nanoparticle solution. In step 2), the amount of ethanol used is 2L per 100g of pork sample.

6. The method of claim 1, wherein the sesame seed targeted nanoparticle is prepared by the steps of: a) mixing sesame seed oil with a surfactant; b) adding the mixture to a nanoparticle solution; and c) mixing the mixture of step b) with a nanoparticle solution. In step 3), the electrostatic self-assembly conditions are: stirring at 25℃ for 2-4 hours.

7. Sesamin targeting carrier nanoparticles prepared by the preparation method of sesamin targeting carrier nanoparticles according to any one of claims 1-6.

8. Use of the sesamin targeting carrier nanoparticles of claim 7 in the preparation of a drug delivery system with ROS stimulation responsiveness and liver mitochondria targeting property.

9. Use of the sesamin-targeted delivery nanoparticle of claim 7 in the manufacture of a medicament for preventing or treating a liver disease.

10. Use according to claim 9, characterized in that, The liver disease includes non-alcoholic fatty liver, liver fibrosis, or liver injury.

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