Use of pyridostigmine in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease

Abstract

This application discloses the application of pyridostigmine in the preparation of drugs for the prevention and treatment of non-alcoholic fatty liver disease (NAFLD). Validation was conducted using animal models of NAFLD induced by three different etiologies: methionine-choline deficiency diet, high-fat diet, and leptin gene knockout. Pyridostigmine significantly improved vagal nerve activity in the model animals (e.g., improving high-frequency and baroreflex sensitivity in heart rate variability), effectively corrected serum lipid metabolism disorders (reducing LDL cholesterol), alleviated hepatocyte damage (reducing ALT and AST), and at the histopathological level, reduced hepatic steatosis, decreased inflammatory cell infiltration, and inhibited collagen deposition, thereby achieving the prevention and treatment of NAFLD.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and in particular to the use of pyridostigmine in the preparation of medicaments for the prevention and treatment of non-alcoholic fatty liver disease. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide, encompassing a spectrum from simple hepatic steatosis to non-alcoholic steatohepatitis, and can further progress to liver fibrosis, cirrhosis, and even liver cancer. Epidemiological surveys show that the global prevalence of NAFLD in adults is as high as approximately 32.4%, and the prevalence in Chinese adults is also as high as 29.6%, with a continuously rising trend, posing a serious burden on public health.

[0003] The exact pathogenesis of NAFLD is not yet fully understood, but it is generally believed to be closely related to multiple factors, including insulin resistance, lipid metabolism disorders, oxidative stress, mitochondrial dysfunction, and chronic inflammation. Clinical treatments for NAFLD remain limited, primarily based on lifestyle interventions (such as dietary control and increased exercise), lacking specific targeted drugs that have been rigorously validated through evidence-based medicine and can definitively reverse the progression of liver disease. Therefore, developing safe and effective drugs for the prevention and treatment of NAFLD is a crucial issue that urgently needs to be addressed in the field of liver disease research.

[0004] In recent years, the role of the autonomic nervous system in regulating metabolic homeostasis has received increasing attention. Studies have shown that autonomic nervous system imbalances, particularly those characterized by decreased vagal tone and increased sympathetic activity, are closely related to the development of metabolic diseases such as obesity, insulin resistance, and type 2 diabetes. The liver, as a core metabolic organ, is also precisely regulated by the autonomic nervous system. Evidence suggests that impaired vagal activity exists in NAFLD patients and animal models. This autonomic dysfunction may promote the occurrence and progression of NAFLD by affecting hepatic lipid synthesis, breakdown, inflammatory signaling pathways, and tissue repair processes. However, the feasibility and specific effects of effectively regulating the autonomic nervous system, especially enhancing vagal activity, through pharmacological means as a novel strategy for intervening in NAFLD have not yet been fully explored and applied. Summary of the Invention

[0005] This application provides an example of the application of pyridostigmine in the preparation of a drug for the prevention and treatment of non-alcoholic fatty liver disease, thereby solving the problems mentioned in the background art.

[0006] This application provides examples of the use of pyridostigmine in the preparation of medicaments for the prevention and / or treatment of non-alcoholic fatty liver disease.

[0007] In one possible implementation, the drug is used to enhance vagal nerve activity in the test subject.

[0008] In one possible implementation, the increase in vagal nerve activity is manifested as an improvement in at least one of the following indices in the subject's heart rate variability: high frequency (HF), standard deviation of sinus RR interval (SDNN), root mean square of the difference between adjacent RR intervals (RMSSD), and baroreflex sensitivity (BRS).

[0009] In one possible implementation, the drug is used to improve liver function in the test subject.

[0010] In one possible implementation, the improvement of liver function in the subject includes at least one of the following: It improves lipid metabolism in subjects, manifested as a reduction in low-density lipoprotein cholesterol (LDL-C) levels; The study aimed to reduce liver injury markers in the subjects, specifically by lowering the levels of at least one of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0011] In one possible implementation, the drug is used to alleviate pathological changes in the liver tissue structure of the test subject.

[0012] In one possible implementation, the reduction of pathological changes in the liver tissue structure of the test subjects includes at least one of the following: It reduces metabolic damage to the liver, manifested as a reduction in lipid deposition in liver tissue and / or a reduction in hepatocyte steatosis; It reduces inflammatory damage to the liver, manifested by reducing the infiltration of inflammatory cells in liver tissue; It inhibits the process of liver fibrosis by reducing collagen deposition in liver tissue.

[0013] In one possible implementation, the non-alcoholic fatty liver disease includes at least one of simple fatty liver, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, and liver cancer.

[0014] In one possible implementation, the drug is administered to a mammal.

[0015] One or more technical solutions provided in the embodiments of this application have at least the following technical effects: This application discloses a novel use of pyridostigmine in the preparation of drugs for the prevention and treatment of non-alcoholic fatty liver disease (NAFLD). The protocol was systematically validated in three NAFLD animal models induced by different mechanisms: methionine-choline deficiency (MCD), high-fat diet (HFD), and leptin gene knockout (ob / ob), demonstrating significant model universality and effect stability. Specific technical effects are reflected in the following three aspects: First, pyridostigmine can clearly enhance vagal nerve activity and significantly improve key indicators such as HF and BRS, suggesting that it may exert its effects by regulating autonomic nerve function; second, the drug can effectively improve liver function, correct serum lipid metabolism disorders (such as reducing LDL-C), and alleviate hepatocellular damage (reducing ALT and AST); third, pyridostigmine can also reverse pathological changes in liver tissue structure, including reducing hepatic steatosis, decreasing inflammatory cell infiltration, and inhibiting collagen deposition, thereby achieving multiple interventions at the tissue level against metabolic damage, inflammatory response, and fibrosis in the progression of NAFLD. In summary, this study is the first to elucidate the use of pyridostigmine in preventing and treating NAFLD by enhancing vagal nerve activity, providing a new strategy with a clear mechanism, sufficient experimental evidence, and promising translational potential for the treatment of NAFLD, which currently lacks targeted drugs in clinical practice. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The effects of pyridostigmine on vagal nerve activity indices in MCD diet-induced NAFLD mice, including HF ( Figure 1 A in SDNN Figure 1 B in the middle), RMSSD ( Figure 1 C) and BRS ( Figure 1 (D in the middle) Figure 2 The effects of pyridostigmine on serum markers in MCD mice, including LDL-C ( Figure 2 A) ALT ( Figure 2 B in the middle) and AST ( Figure 2 (C in the middle) Figure 3 The gross morphology of pyridostigmine in MCD mice ( Figure 3 A) Liver weight to body weight ratio ( Figure 3 B) Liver tissue structure (HE staining) Figure 3 C in Figure 3D), lipid deposition (Oil Red staining, Figure 3 E in Figure 3 (in F) and fibrosis (Masson staining, Figure 3 G in Figure 3 The influence of H in; Figure 4 The effects of pyridostigmine on vagal nerve activity indices in HFD diet-induced NAFLD mice, including HF ( Figure 4 A in SDNN Figure 4 B in the middle), RMSSD ( Figure 4 C) and BRS ( Figure 4 (D in the middle) Figure 5 The effects of pyridostigmine on serological parameters in HFD mice, including LDL-C ( Figure 5 A) ALT ( Figure 5 B in the middle) and AST ( Figure 5 (C in the middle) Figure 6 The gross morphology of pyridostigmine in HFD mice ( Figure 6 A) Liver weight to body weight ratio ( Figure 6 B) Liver tissue structure (HE staining) Figure 6 C in Figure 6 D), lipid deposition (Oil Red staining, Figure 6 E in Figure 6 (in F) and fibrosis (Masson staining, Figure 6 G in Figure 6 The influence of H in; Figure 7 The effects of pyridostigmine on vagal nerve activity indices in ob / ob mice, including HF ( Figure 7 A in SDNN Figure 7 B in the middle), RMSSD ( Figure 7 C) and BRS ( Figure 7 (D in the middle) Figure 8 The effects of pyridostigmine on serum parameters in ob / ob mice, including LDL-C ( Figure 8 A) ALT ( Figure 8 B in the middle) and AST ( Figure 8 (C in the middle) Figure 9 The gross morphology of pyridostigmine in ob / ob mice ( Figure 9 A) Liver weight to body weight ratio ( Figure 9 B) Liver tissue structure (HE staining) Figure 9 C in Figure 9 D), lipid deposition (Oil Red staining, Figure 9 E in Figure 9 (in F) and fibrosis (Masson staining, Figure 9 G in Figure 9 The influence of H in it. Detailed Implementation

[0018] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0019] This application provides examples of the use of pyridostigmine in the preparation of medicaments for the prevention and / or treatment of non-alcoholic fatty liver disease. The medicaments are administered to mammals.

[0020] In the embodiments of this application, the drug is used to enhance the vagus nerve activity of the test subjects.

[0021] In the embodiments of this application, the improvement of vagal nerve activity is manifested as an improvement in at least one of the following indicators in the subject's heart rate variability: high frequency (HF), standard deviation of sinus RR interval (SDNN), root mean square of the difference between adjacent RR intervals (RMSSD), and baroreflex sensitivity (BRS).

[0022] In the embodiments of this application, the drug is used to improve the liver function of the test subjects.

[0023] In this embodiment of the application, improving the liver function of the test subjects includes at least one of the following: It improves lipid metabolism in subjects, manifested as a reduction in low-density lipoprotein cholesterol (LDL-C) levels; The study aimed to reduce liver injury markers in the subjects, specifically by lowering the levels of at least one of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0024] In this embodiment of the application, the drug is used to alleviate pathological changes in the liver tissue structure of the test subjects.

[0025] In this embodiment of the application, alleviating the pathological changes in the liver tissue structure of the test subjects includes at least one of the following: (1) Reduce liver metabolic damage, manifested as reducing lipid deposition in liver tissue and / or reducing hepatocyte steatosis; (2) Reduces inflammatory damage to the liver, manifested by reducing the infiltration of inflammatory cells in liver tissue; (3) It inhibits the process of liver fibrosis, which is manifested by reducing collagen deposition in liver tissue.

[0026] In the embodiments of this application, non-alcoholic fatty liver disease includes at least one of simple fatty liver, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, and liver cancer.

[0027] In the description of the embodiments of this application, the term "test subject" specifically refers to mammals, preferably humans. The mice used in the experiment are mammalian models, and the results can be reasonably extrapolated to human subjects. The preferred dosage of pyridostigmine is 5 mg / kg / day. This dosage has been verified by preliminary experiments to have no obvious toxicity and to exert its efficacy. In practical applications, the dosage range (e.g., 2-8 mg / kg / day) can be adjusted according to the weight, age, and severity of the disease (such as different disease stages such as simple fatty liver and liver fibrosis) of the test subject. The administration method can be gavage, intraperitoneal injection, intravenous injection, etc., all of which are within the protection scope of this application.

[0028] Non-alcoholic fatty liver disease (NAFLD) encompasses various disease types, including simple fatty liver, non-alcoholic steatohepatitis, and liver fibrosis. This embodiment comprehensively verifies the preventive and therapeutic effects of pyridostigmine using three classic animal models to ensure the stability and applicability of the technical solution.

[0029] The reagents and instruments used in this embodiment are as follows: SPF-grade C57BL / 6 male mice (weighing 18-22g) were provided by the Animal Center of the School of Medicine, Xi'an Jiaotong University; methionine-choline (MCD) deficient diet, control diet (NFD, 10% fat content), and high-fat diet (HFD, 45% fat content) were purchased from Shanghai Shuyu Biotechnology Co., Ltd.; pyridostigmine was purchased from a regular pharmacy; the data acquisition and analysis system from Taimeng was used for electrocardiogram and vagal nerve activity index detection; serological indicators (low-density lipoprotein cholesterol LDL-C, alanine aminotransferase ALT, aspartate aminotransferase AST) and liver pathological examination (HE staining, Masson staining, and Oil Red O staining) were performed by Wuhan Sewell Biotechnology Co., Ltd.; and phenformin solution was used for baroreflex sensitivity (BRS) detection.

[0030] Animal housing conditions: All mice were housed in an SPF-grade animal facility with a temperature of 22-25℃ and humidity of 50-60%. The circadian rhythm was 12 hours of light / 12 hours of darkness. They had free access to food and water and were acclimatized for one week before the experiment began.

[0031] Technical Background and Experimental Objectives Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic progressive liver disease, encompassing nonalcoholic fatty liver, nonalcoholic steatohepatitis, and advanced liver fibrosis, cirrhosis, and even liver cancer. As the core organ regulating energy and glucose metabolism, the liver's ability to utilize and transport excess nutrients declines, leading to excessive lipid deposition and inducing persistent low-grade liver inflammation, thus driving the progressive development of NAFLD. Therefore, exploring treatments that reduce excessive lipid deposition, decrease liver inflammation, and improve liver function is crucial for the prevention and treatment of NAFLD and can provide new insights for clinical diagnosis and treatment.

[0032] Recent clinical studies have shown that autonomic nervous system imbalance, characterized by decreased vagal nerve activity and increased sympathetic tone, accompanies the entire development of NAFLD and may be a key factor increasing the risk of NAFLD. Previous research has confirmed that increasing vagal nerve activity through electrical stimulation can effectively inhibit glucose and lipid metabolism disorders caused by diseases such as obesity, diabetes, and metabolic syndrome, reduce inflammatory responses, and improve the energy metabolism function of target organs. Based on this, this application hypothesizes that increasing vagal nerve activity may exert a preventive and therapeutic effect on NAFLD by reducing excessive lipid deposition in the liver and lowering liver inflammation.

[0033] Currently, drug-mediated strategies to enhance vagal nerve activity have received widespread attention in the scientific community. Previous studies have confirmed that the reversible cholinesterase inhibitor pyridostigmine (PYR) can improve cardiac, renal, and peripheral vascular function in cardiovascular disease model animals through mechanisms such as inhibiting oxidative stress, reducing inflammatory responses, and decreasing apoptosis. Recent research by our team has further discovered that pyridostigmine can improve energy metabolism in the heart of obese animals by increasing vagal nerve activity, reducing lipid deposition in the heart, decreasing the accumulation of lipotoxic products, and improving energy metabolism in the heart of obese experimental animals. Based on this, this application proposes a core hypothesis: pyridostigmine is expected to become a potential therapeutic drug, improving NAFLD by increasing vagal nerve activity, reducing excessive lipid deposition in the liver, and decreasing liver inflammation. To verify the above hypothesis, this embodiment conducts experimental studies using three classic NAFLD animal models, as detailed below.

[0034] Example 1: Validation of the preventive and therapeutic effects and mechanism of pyridostigmine on MCD diet-induced NAFLD in mice. 1.1 Experimental Grouping and Model Establishment Eighteen 8-week-old C57BL / 6 mice were randomly divided into three groups of six each: Normal control group (Control group): fed with the standard control diet for 4 consecutive weeks, without medication, only with an equal volume of physiological saline by gavage.

[0035] NAFLD model group (MCD group): The patient was fed the MCD diet for 4 weeks to replicate the simple fatty liver and steatohepatitis models. During the period, the patient was given an equal volume of physiological saline by gavage.

[0036] Pyridostigmine intervention group (MCD+PYR group): The patient was fed the MCD diet for 4 weeks and pyridostigmine (5 mg / kg) was administered by gavage daily for 4 weeks.

[0037] 1.2 Sample Collection and Testing Methods 1.2.1 Detection of vagal nerve activity indicators: After the last administration, mice were fasted for 12 hours and maintained anesthesia with 1% isoflurane. Electrodes were inserted into the skin of the right forelimb, right hindlimb, and left hindlimb. Electrocardiogram data for 20 minutes were collected using the data acquisition and analysis system of Taiying Company. Stable data for 5 minutes within the middle 15-20 minutes were selected to analyze HF, SDNN, and RMSSD. Subsequently, the mice were fixed in a supine position, and electrocardiogram and blood pressure were recorded. The left femoral vein was isolated, and phenylephrine solution was administered in a pulse. Changes in electrocardiogram and blood pressure were monitored simultaneously. The ratio of the change in RR interval to the change in systolic blood pressure was calculated, which is the BRS.

[0038] 1.2.2 Serological marker detection: After anesthetizing mice, the eyeballs were removed and blood was collected. Whole blood was placed in EP tubes and allowed to stand at room temperature for 30 minutes. The tubes were then centrifuged at 4°C and 3000 r / min for 10 minutes. Serum was collected and the levels of LDL-C, ALT and AST were detected using a fully automated biochemical analyzer.

[0039] 1.2.3 Liver histological examination: The mice were dissected and the livers were separated. The liver weight and mouse body weight were weighed, and the liver weight-to-body weight ratio was calculated. About 1g of liver lobule tissue was taken, fixed in formaldehyde solution, and after dehydration, embedding, and sectioning, HE staining (to observe hepatocyte steatosis and inflammatory cell infiltration), Oil Red O staining (to quantitatively analyze lipid deposition), and Masson staining (to observe collagen deposition and fibrosis). The results were scored and quantitatively analyzed using a pathological image analysis system.

[0040] 1.3 Experimental Results and Analysis 1.3.1 Effects of pyridostigmine on vagal nerve activity in MCD mice: such as Figure 1 As shown, compared with the control group, the HF (high HF) of mice in the MCD model group was significantly lower. Figure 1 A in SDNN Figure 1 B in the middle), RMSSD ( Figure 1 C) and BRS ( Figure 1The levels of vagal nerve activity (D) were significantly reduced, indicating a clear impairment of vagal nerve activity in the NAFLD model group mice. After intervention with pyridostigmine (MCD+PYR group), all the above vagal nerve activity indicators were significantly improved. This result demonstrates that pyridostigmine can effectively reverse the decrease in vagal nerve activity induced by MCD diet in NAFLD mice, providing direct experimental evidence for its prevention and treatment of NAFLD by enhancing vagal nerve function.

[0041] 1.3.2 Effects of pyridostigmine on liver function in MCD mice: such as Figure 2 As shown, compared with the Control group, the serum LDL-C (…) in the MCD model group mice was lower. Figure 2 A) ALT ( Figure 2 B in the middle) and AST ( Figure 2 The levels of LDL-C (C) in the mice were significantly elevated, indicating significant lipid metabolism disorder and hepatocellular damage in the model mice. After intervention with pyridostigmine (MCD+PYR group), all of the above indicators were significantly reversed: serum LDL-C, ALT, and AST levels were all significantly reduced. These results demonstrate that pyridostigmine can effectively improve lipid metabolism and alleviate liver damage in NAFLD model mice, thereby playing a protective role in liver function.

[0042] 1.3.3 Effects of pyridostigmine on the structural and pathological changes of liver tissue in MCD mice: such as Figure 3 As shown, in terms of the gross morphology and weight of the mice ( Figure 3 In the control group (A), the liver-to-body weight ratio of mice in the MCD model group was significantly lower (A). Figure 3 (B in the original text) After intervention with pyridostigmine, the ratio recovered somewhat, but the difference did not reach a statistically significant level. At the histopathological level, HE staining revealed... Figure 3 C in Figure 3 In the D group, the livers of mice in the MCD group showed severe hepatocellular steatosis and significant inflammatory cell infiltration, and their NAFLD pathological scores were significantly higher than those in the control group. Oil Red O staining results ( Figure 3 E in Figure 3 The F-staining results showed that the amount of lipid deposition in the liver was significantly increased in the MCD group; Masson staining results (F-staining results) Figure 3 G in Figure 3The H) further indicated a significant increase in collagen deposition, suggesting that the model mice already exhibited obvious hepatic metabolic damage, inflammatory damage, and an early tendency towards fibrosis. After pyridostigmine intervention (MCD+PYR group), the degree of hepatic steatosis was significantly reduced, inflammatory cell infiltration decreased, and the NAFLD pathological score was significantly lowered; simultaneously, both hepatic lipid deposition and collagen deposition were significantly inhibited. These results collectively demonstrate that pyridostigmine can effectively alleviate hepatic metabolic and inflammatory damage in NAFLD model mice and inhibit the progression of hepatic fibrosis, thus confirming its protective effect on the liver at the histopathological level.

[0043] Example 2: Validation of the preventive and therapeutic effects and mechanism of pyridostigmine on HFD diet-induced NAFLD mice 2.1 Experimental Grouping and Model Establishment Eighteen 8-week-old C57BL / 6 mice were randomly divided into three groups of six each: Normal feed control group (NFD group): fed a standard control diet with 10% fat content (D12450H) for 16 consecutive weeks, during which time an equal volume of physiological saline was administered by gavage.

[0044] NAFLD model group (HFD group): fed a high-fat diet with 45% fat content (D12451) for 16 consecutive weeks to replicate the NAFLD model, during which an equal volume of physiological saline was administered by gavage.

[0045] Pyridostigmine intervention group (HFD+PYR group): fed a 45% high-fat diet for 16 weeks, and administered pyridostigmine (5 mg / kg) by gavage daily for 16 weeks.

[0046] 2.2 Sample Collection and Testing Methods The methods for detecting vagal nerve activity indicators, serological indicators, and liver histology were consistent with those in Example 1, ensuring the consistency of experimental methods and the comparability of results.

[0047] 2.3 Experimental Results and Analysis 2.3.1 Effects of pyridostigmine on vagal nerve activity in HFD diet-induced NAFLD mice: such as Figure 4 As shown, compared with the normal diet group (NFD group), the HF (HF) in the HFD model group mice was significantly lower. Figure 4 A in SDNN Figure 4 B in the middle), RMSSD ( Figure 4 C) and BRS ( Figure 4The levels of HF, SDNN, RMSSD, and BRS were significantly reduced, indicating that a high-fat diet can also lead to significant vagal nerve function inhibition. After pyridostigmine intervention (HFD+PYR group), all of the above indicators were significantly improved: HF, SDNN, RMSSD, and BRS were significantly increased compared to the model group. The consistency of these results in different diet-induced NAFLD models indicates that pyridostigmine has a stable and significant vagal nerve activity-enhancing effect, further supporting its feasibility in preventing and treating NAFLD by regulating autonomic nerve function.

[0048] 2.3.2 Effects of pyridostigmine on liver function in HFD mice: such as Figure 5 As shown, compared with the normal diet group (NFD group), the serum LDL-C of mice in the HFD model group was significantly lower. Figure 5 A) ALT ( Figure 5 B in the middle) and AST ( Figure 5 The levels of LDL-C, ALT, and AST were significantly elevated, indicating that the HFD diet induced significant lipid metabolism disorders and hepatocellular damage. After pyridostigmine intervention (HFD+PYR group), all the above abnormal indicators were significantly reversed: serum LDL-C, ALT, and AST levels all decreased significantly. These results further confirm that pyridostigmine can effectively regulate lipid metabolism and reduce liver damage in different diet-induced NAFLD models, highlighting its stability and reliability in improving liver function.

[0049] 2.3.3 Effects of pyridostigmine on the structural and pathological changes of liver tissue in HFD mice: such as Figure 6 As shown, in terms of the gross morphology and weight of the mice ( Figure 6 In group A), compared with the NFD group, there was no statistically significant difference in liver weight to body weight ratio between the HFD model group and the HFD+PYR group. Histopathological observation (HE staining, Figure 6 C in Figure 6 The D-staining assay showed that the livers of mice in the HFD group exhibited severe steatosis and significant inflammatory cell infiltration, with a significantly higher NAFLD pathological score compared to the control group. Further Oil Red O staining (D) revealed... Figure 6 E in Figure 6 F in the middle) and Masson staining ( Figure 6 G in Figure 6The H1N1 (H2N1) results showed that both lipid and collagen deposition in the liver were significantly increased in the HFD group, indicating that the model had progressed to a stage with significant inflammation and early fibrosis. After pyridostigmine intervention (HFD+PYR group), the degree of hepatocyte steatosis was significantly reduced, inflammatory cell infiltration was decreased, and the NAFLD pathological score was significantly lower than that in the model group; at the same time, hepatic lipid and collagen deposition were also significantly inhibited. These results collectively indicate that in the HFD-induced NAFLD model, pyridostigmine can also effectively reduce metabolic and inflammatory damage to the liver and inhibit the fibrosis process, further validating its efficacy in preventing and treating NAFLD and protecting the stability of liver tissue structure in different etiological models.

[0050] Example 3: Validation of the preventive and therapeutic effects and mechanism of pyridostigmine on NAFLD in ob / ob mice 3.1 Experimental Grouping and Model Establishment Eighteen 8-week-old C57BL / 6 background mice were randomly divided into three groups of six each: Normal control group (Control group): Wild-type C57BL / 6 mice were fed a regular diet for 12 weeks, during which they were given an equal volume of physiological saline by gavage.

[0051] NAFLD model group (ob / ob group): Leptin gene knockout mice were fed a regular diet for 12 weeks to replicate the NAFLD model using their spontaneous obesity and metabolic disorder characteristics. During the period, they were given an equal volume of physiological saline by gavage.

[0052] Pyridoxine intervention group (ob / ob+PYR group): ob / ob mice were given a normal diet and were given pyridoxine (5 mg / kg) by gavage daily for 12 weeks.

[0053] 3.2 Sample collection and testing methods The methods for detecting vagal nerve activity indicators, serological indicators, and liver histology were the same as in Example 1 to exclude the interference of dietary factors and to verify the preventive and therapeutic effects of pyridostigmine on spontaneous metabolic disorder-induced NAFLD.

[0054] 3.3 Experimental Results and Analysis 3.3.1 Effects of pyridostigmine on vagal nerve activity in ob / ob mice: such as Figure 7 As shown, compared with the normal control group, the HF (high HF) in the ob / ob model group mice was significantly lower. Figure 7 A in SDNN Figure 7 B in the middle), RMSSD ( Figure 7 C) and BRS ( Figure 7The levels of D in the vagus nerve were significantly reduced, indicating that mice with hereditary obesity and NAFLD also exhibited significant vagal nerve function inhibition. After intervention with pyridostigmine, all of the above indicators were significantly improved. This result further confirms that pyridostigmine can stably and effectively enhance vagal nerve activity in NAFLD models induced by different etiologies, providing cross-model consistent experimental evidence for its prevention and treatment of NAFLD by enhancing vagal nerve function.

[0055] 3.3.2 Effects of pyridostigmine on liver function in ob / ob mice: such as Figure 8 As shown, compared with the normal control group (Control group), the serum LDL-C (…) in the ob / ob model group mice was significantly lower. Figure 8 A) ALT ( Figure 8 B in the middle) and AST ( Figure 8 The levels of LDL-C, ALT, and AST were significantly elevated. After intervention with pyridostigmine (ob / ob+PYR group), all of the above abnormal indicators were significantly reversed: serum LDL-C, ALT, and AST levels were significantly lower than those in the model group. These results indicate that pyridostigmine can also effectively improve lipid metabolism disorders and reduce hepatocellular damage in a hereditary obesity combined with NAFLD model, further confirming its broad effectiveness in improving liver function.

[0056] 3.3.3 Effects of pyridostigmine on the structural and pathological changes of liver tissue in ob / ob mice: such as Figure 9 As shown, in terms of the gross morphology and weight of the mice ( Figure 9 In the A group, compared with the control group, the liver weight to body weight ratio of mice in the ob / ob model group was significantly increased ( Figure 9 (B in the text) This ratio decreased significantly after pyridostigmine intervention. Histopathological observation (HE staining, Figure 9 C in Figure 9 The D-staining assay showed that the livers of the ob / ob group mice exhibited severe steatosis and significant inflammatory cell infiltration, with a significantly higher NAFLD pathological score compared to the control group. Further Oil Red O staining (D) revealed further evidence. Figure 9 E in Figure 9 F in the middle) and Masson staining ( Figure 9 G in Figure 9The H) results showed that both lipid and collagen deposition in the liver were significantly increased in the ob / ob group. After pyridostigmine intervention (ob / ob+PYR group), the degree of hepatic steatosis was significantly reduced, the generation of inflammatory lesions was inhibited, and the NAFLD pathological score was significantly decreased; at the same time, hepatic lipid and collagen deposition were also significantly reduced. These results collectively indicate that in a hereditary obesity combined with NAFLD model, pyridostigmine can effectively reduce metabolic and inflammatory damage to the liver and inhibit the fibrosis process, further validating its hepatoprotective effect in NAFLD models with different etiologies from a histopathological perspective.

[0057] Based on systematic studies of three classic NAFLD animal models—MCD diet, HFD diet, and ob / ob diet—this application comprehensively verifies the preventive and therapeutic effects and mechanisms of action of pyridostigmine from three dimensions: vagal nerve activity, liver biochemical function, and histopathological structure. Comprehensive experimental data indicate that its preventive and therapeutic effects are specifically manifested in the following three interrelated and progressively layered biological levels: (1) Regulation and enhancement of autonomic nervous system function: Pyridoxine can significantly improve vagal nerve activity in model animals (subjects). This effect was objectively confirmed by the improvement of key assessment indicators of vagal nerve activity, including HF, SDNN, RMSSD and BRS levels.

[0058] (2) Improvement and protection of liver function: At the functional level, pyridostigmine can effectively correct the systemic lipid metabolism disorder associated with NAFLD, as evidenced by a significant reduction in serum LDL-C levels. At the same time, it can significantly reduce the levels of key serum markers of hepatocellular damage—ALT and AST, clearly demonstrating its hepatoprotective effect.

[0059] (3) Reversal and repair effects on liver tissue structure: At the histopathological level, pyridostigmine can directly alleviate the core pathological changes in the liver. These include: reducing metabolic damage (manifested as reduced hepatocyte steatosis and lipid deposition), reducing inflammatory damage (manifested as reduced inflammatory cell infiltration), and inhibiting the fibrotic process (manifested as reduced collagen deposition).

[0060] The empirical data from different etiological models above collectively demonstrate that pyridostigmine can achieve preventive and therapeutic effects on NAFLD (covering key disease stages from simple fatty liver, non-alcoholic steatohepatitis to liver fibrosis) through the core mechanism of enhancing vagal nerve activity.

[0061] In this embodiment, the dosage and administration method of pyridostigmine are only preferred options. Those skilled in the art can adjust the dosage and administration method (such as intraperitoneal injection, intravenous injection, etc.) according to the subject's weight and the severity of the disease, all of which fall within the scope of protection of this application. Furthermore, the vagus nerve activity assessment indicators, liver function indicators, and pathological changes mentioned in this application are all objective indicators recognized in clinical and experimental settings, ensuring the scientific validity and reproducibility of the technical solution.

[0062] The various embodiments in this specification are described in a progressive manner. For the same or similar parts between the various embodiments, please refer to each other. Each embodiment focuses on describing the differences from other embodiments.

[0063] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. The use of pyridostigmine in the preparation of medicaments for the prevention and / or treatment of non-alcoholic fatty liver disease.

2. The application according to claim 1, characterized in that, The drug is used to enhance vagal nerve activity in test subjects.

3. The application according to claim 2, characterized in that, The improvement in vagal nerve activity is manifested in the improvement of at least one of the following indicators in the subjects' heart rate variability: high frequency, standard deviation of sinus RR interval, root mean square of the difference between adjacent RR intervals, and baroreflex sensitivity.

4. The application according to claim 1, characterized in that, The drug is used to improve liver function in test subjects.

5. The application according to claim 4, characterized in that, The improvement in liver function of the subjects includes at least one of the following: It improves lipid metabolism in subjects, manifested by reducing the level of low-density lipoprotein cholesterol; The study reduced liver injury markers in the subjects, manifested by a decrease in at least one of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

6. The application according to claim 1, characterized in that, The drug is used to alleviate pathological changes in the liver tissue structure of the test subjects.

7. The application according to claim 6, characterized in that, The reduction of pathological changes in liver tissue structure in the test subjects includes at least one of the following: It reduces metabolic damage to the liver, manifested as a reduction in lipid deposition in liver tissue and / or a reduction in hepatocyte steatosis; It reduces inflammatory damage to the liver, manifested by reducing the infiltration of inflammatory cells in liver tissue; It inhibits the process of liver fibrosis by reducing collagen deposition in liver tissue.

8. The application according to claim 1, characterized in that, The non-alcoholic fatty liver disease includes at least one of simple fatty liver, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, and liver cancer.

9. The application according to claim 1, characterized in that, The drug is administered to mammals.

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