CN118045083B

CN118045083B - New application of L-proline in preparing medicine for preventing and treating non-alcoholic fatty liver disease

Info

Publication number: CN118045083B
Application number: CN202410446501.6A
Authority: CN
Inventors: 刘大斌; 周云飞; 孟宏学
Original assignee: Harbin Medical University
Current assignee: Harbin Medical University
Priority date: 2024-04-15
Filing date: 2024-04-15
Publication date: 2024-06-18
Anticipated expiration: 2044-04-15
Also published as: CN118045083A

Abstract

The invention relates to a new application of L-proline in preparing a medicine for preventing and treating non-alcoholic fatty liver disease, belonging to the technical field of medicines. In order to solve the problem that effective clinical means for treating NAFLD are lacking in the current stage, the invention provides a new application of L-proline in preparing medicines for preventing and treating nonalcoholic fatty liver diseases. The invention proves that the L-proline can inhibit the development of nonalcoholic fatty liver disease NAFLD by targeted activation of the fatty cell squalene epoxidase SQLE; oral L-proline activates squalene epoxidase SQLE expression in adipocytes to reduce diet-induced obesity, reduce white fat weight, and alleviate high fat diet-induced NAFLD disease; oral administration of L-proline activates squalene epoxidase SQLE expression in adipocytes to reduce ceramide Cer18:1/18:1 concentration in blood, and alleviate NAFLD progression.

Description

New application of L-proline in preparing medicine for preventing and treating non-alcoholic fatty liver disease

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a novel application of L-proline in preparing a medicine for preventing and treating nonalcoholic fatty liver diseases.

Background

Non-alcoholic fatty liver disease (Non-alcohlic FATTY LIVER DISEASE, NAFLD) and metabolic disorder syndrome are causal to each other, and are also called metabolic-related fatty liver disease, and clinically have close relation with metabolic diseases such as diabetes and atherosclerosis cardiovascular diseases. Non-alcoholic fatty liver disease is also an important cause of liver cancer occurrence, obesity-related simple fatty liver (Steatosis) can be directly developed into liver cancer without going through a hepatitis stage, and immunotherapy has little effect on liver cancer patients induced by non-alcoholic fatty liver disease.

Adipose tissue is an important cholesterol storage organ of the organism, and the role of the adipose tissue in the occurrence and development processes of metabolic fatty liver diseases is being increasingly emphasized by scientific researchers. A plurality of domestic and foreign researches prove that the cytokines and fatty acids which are derived from fat cells in the obese state can influence the metabolic homeostasis of an organism and influence the occurrence and development of metabolic related fatty liver diseases through an adipose tissue-liver tissue axis. However, targeting adipocytes to treat metabolic-related fatty liver disease is only in the conceptual phase, and no related drugs or therapeutic strategies have entered the clinical trial phase.

The discovery of new therapeutic targets and development of therapeutic modalities for nonalcoholic fatty liver disease is an unmet clinical need.

Disclosure of Invention

In order to solve the problem that effective clinical means for treating the non-alcoholic fatty liver disease are lacking in the current stage, the invention provides a new application of L-proline in preparing medicaments for preventing and treating the non-alcoholic fatty liver disease.

The technical scheme of the invention is as follows:

application of L-proline in preparing medicine for preventing and treating nonalcoholic fatty liver disease is provided.

Further, the prevention and treatment of non-alcoholic fatty liver disease is to inhibit the progress of non-alcoholic fatty liver disease by activating the expression of squalene epoxidase in adipocytes.

Further, the prevention and treatment of nonalcoholic fatty liver disease is to reduce diet-induced obesity, reduce white fat weight and alleviate high fat diet-induced nonalcoholic fatty liver disease by activating squalene epoxidase expression in adipocytes.

Further, the prevention and treatment of the nonalcoholic fatty liver disease is to reduce the concentration of ceramides Cer18:1/18:1 in blood by activating the expression of squalene epoxidase in fat cells, and relieve the progress of the nonalcoholic fatty liver disease.

Further, in the medicine for preventing and treating non-alcoholic fatty liver disease, L-proline is the only active ingredient.

Further, the medicine for preventing and treating the nonalcoholic fatty liver disease comprises L-proline and a pharmaceutically acceptable carrier.

Further, the content of the L-proline in the medicine for preventing and treating the nonalcoholic fatty liver disease is 0.1-99wt%.

Further, the medicament for preventing and treating the nonalcoholic fatty liver disease is in the form of granules, tablets, capsules, pills or oral liquid.

Furthermore, the medicine for preventing and treating the nonalcoholic fatty liver disease is orally administered, and the daily oral administration of the medicine for preventing and treating the nonalcoholic fatty liver disease is 250mg/kg body weight.

The invention has the beneficial effects that:

In two diet-induced non-alcoholic fatty liver disease animal models, the L-proline is supplemented by drinking water, so that the L-proline can inhibit the development of the non-alcoholic fatty liver disease through targeted activation of the fat cell squalene epoxidase (SQLE). The invention proves that the oral administration of L-proline can activate the expression of squalene epoxidase in fat cells to reduce diet-induced obesity, reduce white fat weight and relieve high-fat diet-induced nonalcoholic fatty liver disease; oral administration of L-proline activates squalene epoxidase expression in adipocytes to reduce the blood ceramide Cer18:1/18:1 concentration, and alleviate the progression of nonalcoholic fatty liver disease. The invention also uses the hepatocyte-specific SQLE knockout mice to prove that the targeted activation of the adipocyte SQLE and the inhibition of the hepatocyte SQLE are effective and feasible strategies for treating metabolic related fatty liver diseases with higher safety.

Drawings

FIG. 1 is a schematic diagram of a method of feeding NAFLD model mice on a high fat high cholesterol diet in example 1;

FIG. 2 is a graph showing comparison of weight gain rates of two groups of mice in example 1;

FIG. 3 is a graph showing the comparison of liver weights, blood glutamic oxaloacetic transaminase (AST) and alanine Aminotransferase (ALT) measurements of two groups of mice in example 1, in order from left to right;

FIG. 4 is a graph showing the results of insulin resistance test experiments for two groups of mice in example 1;

FIG. 5 is a comparison of hematoxylin-eosin staining (HE) and oil red staining results for two groups of mice in example 1;

FIG. 6 is a graph showing comparison of ceramide content in blood of two groups of mice in example 1;

FIG. 7 is a graph showing comparison of mRNA expression levels of MARCH6 gene in white adipose tissue (eWAT) of two mice in example 1;

FIG. 8 is a graph showing comparison of protein expression levels of HIF 2. Alpha./ACER 2 gene from two groups of mice in example 1, white adipose tissue (eWAT) SQLE and downstream thereof;

FIG. 9 is a graph showing comparison of SQLE protein expression levels in two groups of mouse 3T3L1 cells and LO2 cells in example 1;

FIG. 10 is a schematic of the feeding method of NAFLD model mice with methionine choline deficiency high fat diet in example 2;

FIG. 11 is a comparison photograph of HE staining results of two groups of mice in example 2;

FIG. 12 is a schematic of the high fat high cholesterol diet feeding method of the wild type mice and Sqle ko/Alb-Cre mice of example 3;

FIG. 13 is a comparison of HE and oil red staining results for three groups of mice in example 3;

Fig. 14 is a graph showing, in order from left to right, the weight gain, liver/body weight ratio, serum ALT, serum cholesterol, and serum Triglyceride (TG) of the three groups of mice in example 3.

Detailed Description

The following embodiments are used for further illustrating the technical scheme of the present invention, but not limited thereto, and all modifications and equivalents of the technical scheme of the present invention are included in the scope of the present invention without departing from the spirit and scope of the technical scheme of the present invention. The process equipment or apparatus not specifically noted in the following examples are all conventional equipment or apparatus in the art, and the raw materials and the like used in the examples of the present invention are commercially available unless otherwise specified; unless specifically indicated, the technical means used in the embodiments of the present invention are conventional means well known to those skilled in the art.

The materials and the method for animal experiments of the invention are as follows:

A. Human blood specimen

The human blood sample is presented by a team of doctor's Li Dongdong at the clamshell medical college of first affiliated hospital. Written informed consent was obtained for all subjects and the study protocol was approved by the ethics committee of the scientific clinical study of the first affiliated hospital of the concha's medical college.

White adipose tissue of the abdomen of normal and obese people was collected by professor Meng Hongxue of the affiliated tumor Hospital at the university of Harbin medical science. Written informed consent was obtained for all subjects, and the study protocol was approved by the ethical committee of the scientific clinical study of tumor affiliated hospitals at the university of halbine medical science.

B. Transgenic mouse model

NAFLD mouse model:

wild type mice (WT) at 6 weeks of age were fed a high fat high cholesterol diet. At 8 weeks of age, mice were randomized into two groups and treated with water or L-proline (water was set to 300ppm concentration with water changing drinking water every 2 days). After 12 weeks of L-proline treatment, mice were sacrificed to examine the therapeutic effect of L-proline.

Wild type mice (WT) fed 8 weeks old lack a high fat diet (MCD) for methionine choline. At 9 weeks of age, mice were randomized into two groups and treated with water or L-proline (water was set to 300ppm concentration with water changing drinking water every 2 days). After 4 weeks of L-proline treatment, mice were sacrificed to examine the therapeutic effect of L-proline.

Mice were fed a 6 week old Sqle ko/Alb-Cre and wild type littermates (WT) high fat high cholesterol diet. At 12 weeks of age Sqle ko/Alb-Cre mice were randomized into two groups and treated with water or L-proline (water was set to 300ppm concentration with water changes every 2 days). After 12 weeks of L-proline treatment, mice were sacrificed to examine the therapeutic effect of L-proline.

Assessment of NAFLD: liver histology was assessed at the time of sacrifice using HE staining.

Inflammation evaluation: serum ALT and AST were used to determine liver injury.

Evaluation of metabolic syndrome: oil red staining (oil red), insulin resistance test and glucose resistance test.

Hepatocyte-specific Sqle knockout mouse model:

Sqle knockout mice were constructed by BIOCYTOGEN company (Beijing, china) based on CRISPR/Cas9 technology.

To drive the Sqle hepatocyte-specific knockout, sqle ko mice were hybridized with B6.CgTg (Alb-cre) 21Mgn/JNju mice (university of Nanjing, china). Sqle ko/Alb-Cre mice were verified by PCR genotyping.

WT and Sqle-related transgenic mice were free to ingest high fat and high cholesterol (HFHC: 27.4% fat, 40.1% carbohydrate, 24.2% protein, 2% cholesterol) diet (BiotechHD co., ltd. Beijin, china) at different time points, forming a diet-induced NAFLD model.

All animal studies were conducted according to guidelines approved by the ethical committee of animal experiments in the fourth hospital, affiliated to the university of halbine medical science.

Liver cholesterol level detection method: 2mg of tissue was harvested and liver cholesterol levels were measured by cholesterol/cholesterol ester quantification kit (ab 65359, abcam) according to the manufacturer's instructions. All experiments were performed in triplicate. Results show no mean ± SEM.

Liver Triglyceride (TG) levels: 100mg of tissue was harvested and liver triglyceride levels were detected by triglyceride quantification kit (ab 65336, abcam) according to the manufacturer's instructions. All experiments were performed in triplicate. Results show no mean ± SEM.

C. biological function analysis

Serum cholesterol, TG, ALT and AST assays

A5. Mu.L sample of blood was taken and tested for serum cholesterol, TG, ALT and AST levels using ELISA kits. Wherein cholesterol levels are detected using a cholesterol/cholesterol ester quantification kit (ab 65359, abcam). Triglyceride (TG) levels were detected using the triglyceride quantification kit (ab 65336, abcam). ALT and AST levels were detected using the glutamic pyruvic transaminase activity detection kit (BC 155, soy baby China) and the glutamic pyruvic transaminase activity detection kit (BC 1565, soy baby China), respectively.

Insulin resistance test (ITT) and glucose resistance test (GTT)

For glucose tolerance test, mice were transferred to clean cages without food or stool at the top or bottom of the cages and fasted overnight. The mice were then intraperitoneally injected with 0.75U insulin/kg body weight (ITT) or 1g glucose/kg body weight (GTT) in water. Blood from the tail vein was obtained 30, 60 and 90 minutes before and after injection, and blood glucose was measured using a glucometer.

D. statistical analysis

All statistical tests were performed using SPSS or GraphPad software. Data are expressed as mean ± SEM. Multiple sets of comparisons were analyzed by univariate ANOVA. A Mann-Whitney U test or student t test was performed to compare the variables of the two groups. P values <0.05 were statistically significant.

Example 1

This example demonstrates by animal experiments that oral L-proline is capable of activating adipocyte SQLE expression and alleviating NAFLD progression.

In the high fat high cholesterol diet induced obesity related NAFLD model, wild mice were fed water or L-proline by the method shown in figure 1.

Fig. 2 is a graph showing comparison of weight gain rates of two groups of mice in example 1, fig. 3 is a graph showing comparison of liver weights, blood AST and ALT measurement results of two groups of mice in example 1 in order from left to right, and fig. 4 is a graph showing insulin resistance test results of two groups of mice in example 1; the figure shows that oral administration of L-proline can significantly reduce the weight gain rate and liver weight of mice, can significantly relieve insulin resistance, and suggests that L-proline may relieve high-fat diet induced NAFLD.

FIG. 5 is a comparison of HE and oil red staining results for two groups of mice in example 1; the figure shows that under high fat high cholesterol diet conditions, the control group can develop NAFLD disease, the pathology is shown by typical liver steatosis levels exceeding 10% (HE) and excessive fat accumulation (oil red staining). Whereas oral L-proline significantly reduced liver steatosis levels (less than 5%, HE staining) and fat accumulation levels (oil red staining), confirming that L-proline can effectively alleviate this type of NAFLD disease progression.

FIG. 6 is a graph showing comparison of the ceramide levels in blood of two groups of mice in example 1, FIG. 7 is a graph showing comparison of MARCH6 gene mRNA expression levels in white fat eWAT tissue of two groups of mice in example 1, and FIG. 8 is a graph showing comparison of protein expression levels in white fat eWAT tissue SQLE and its downstream HIF2α/ACER2 gene of two groups of mice in example 1; experiments prove that the oral administration of L-proline can reduce the content of ceramide Cer18:1/18:1 in blood, inhibit the mRNA expression level of MARCH6 gene in white fat eWAT tissue, and raise the protein expression level of SQLE and its downstream HIF2 alpha/ACER 2 gene, but has no obvious effect on the SQLE mRNA expression of adipose tissue and the SQLE protein expression level of liver.

FIG. 9 is a graph comparing the SQLE protein expression levels in the two groups of mouse 3T3L1 cells and LO2 cells of example 1, showing that oral L-proline can significantly reduce SQLE protein expression levels in 3T3L1 cells, but has no significant effect on the expression of SQLE protein expression levels in LO2 cells. This suggests that L-proline may interfere with the function of adipocytes SQLE, alleviating NAFLD progression.

Taken together, it was demonstrated that oral L-proline was able to activate expression of SQLE in adipocytes, reduce blood ceramide (Cer 18:1/18:1) levels and alleviate NAFLD progression, thus suggesting that targeted activation of SQLE expression in adipocytes is a novel strategy for NAFLD therapy.

Example 2

In methionine choline deficient high fat diet induced lean NAFLD model, wild mice were fed water or L-proline by the method shown in FIG. 10.

FIG. 11 is a comparison photograph of HE staining results of two groups of mice in example 2; the figure shows that the control group can develop NAFLD disease in the absence of high fat diet feed, and the pathology is shown by serious degree of steatosis. While oral administration of L-proline significantly reduced liver steatosis levels, confirming that L-proline is effective in alleviating NAFLD disease progression induced by this type of high fat diet.

Example 3

In the embodiment, experiments of a liver cell specific Sqle knockout mouse model prove that oral administration of L-proline and simultaneous inhibition of expression of liver cells SQLE can inhibit NAFLD from progressing and reduce blood ALT and cholesterol levels.

In a high fat high cholesterol diet induced mouse model, wild mice were fed with water, and hepatocytes-specific Sqle knockout Sqle ko/Alb-Cre mice were fed with water or L-proline, as shown in fig. 12.

FIG. 13 is a comparison of HE and oil red staining results for three groups of mice in example 3; under high fat high cholesterol diet conditions, WT control mice groups (water) can develop NAFLD disease, pathologically manifested by typical severe liver steatosis levels (over 60%, HE staining) and excessive fat accumulation (oil red staining). Compared with WT control mice, the simple specific knockout of liver SQLE (Sqle ko/Alb-Cre) can significantly reduce liver steatosis level (5-10%, HE staining) and fat accumulation level (oil red staining) and restore severe NAFLD to mild or moderately lighter NAFLD disease level. While knocking out liver SQLE and assisting with oral administration of L-proline can further reduce liver steatosis level (0-5%, HE staining) and fat accumulation degree (oil red staining). It is proved that the simultaneous accurate targeting of L-proline to liver SQLE is a more effective NAFLD disease treatment means.

FIG. 14 is a graph showing, in order from left to right, the weight gain, liver/body weight ratio, serum ALT, serum cholesterol, and serum TG for the three groups of mice of example 3; the figure shows that simultaneous inhibition of expression of hepatocytes SQLE by oral administration of L-proline significantly alleviates the progression of NAFLD disease while significantly reducing blood ALT and cholesterol levels compared to wild-type mouse controls. The combination treatment regimen had better efficacy and safety compared to the dietary supplement of L-proline alone (FIG. 3).

Claims

Application of L-proline in preparing medicine for preventing and treating non-alcoholic fatty liver disease.
2. The use of L-proline in the preparation of a medicament for the prevention and treatment of non-alcoholic fatty liver disease according to claim 1, wherein the prevention and treatment of non-alcoholic fatty liver disease is to inhibit the progression of non-alcoholic fatty liver disease by activating the expression of squalene epoxidase in adipocytes.
3. The use of L-proline in the manufacture of a medicament for the prevention and treatment of non-alcoholic fatty liver disease according to claim 1, wherein the prevention and treatment of non-alcoholic fatty liver disease is to reduce diet-induced obesity, reduce white fat weight and alleviate high fat diet-induced non-alcoholic fatty liver disease by activating squalene epoxidase expression in adipocytes.
4. The use of L-proline in the manufacture of a medicament for the prevention and treatment of non-alcoholic fatty liver disease according to claim 1, wherein the prevention and treatment of non-alcoholic fatty liver disease is by reducing the concentration of ceramide Cer18:1/18:1 in the blood by activating the expression of squalene epoxidase in the adipocytes, alleviating the progression of non-alcoholic fatty liver disease.
5. The use of L-proline in the manufacture of a medicament for the prevention and treatment of non-alcoholic fatty liver disease according to any one of claims 1 to 4, wherein L-proline is the only active ingredient in the medicament for the prevention and treatment of non-alcoholic fatty liver disease.
6. The use of L-proline in the manufacture of a medicament for the prevention and treatment of non-alcoholic fatty liver disease according to claim 5, wherein the medicament for the prevention and treatment of non-alcoholic fatty liver disease comprises L-proline and a pharmaceutically acceptable carrier.
7. The application of L-proline in preparing a medicament for preventing and treating non-alcoholic fatty liver disease according to claim 6, wherein the content of L-proline in the medicament for preventing and treating non-alcoholic fatty liver disease is 0.1-99wt%.
8. The use of L-proline in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease according to claim 7, wherein the medicament for preventing and treating non-alcoholic fatty liver disease is in the form of granule, tablet, capsule, pill or oral liquid.
9. The use of L-proline in the preparation of a medicament for the prevention and treatment of non-alcoholic fatty liver disease according to claim 8, wherein the medicament for the prevention and treatment of non-alcoholic fatty liver disease is administered orally, 250mg/kg body weight of L-proline per day.