Application of piperidine acid in preparation of medicine for preventing and / or treating non-alcoholic steatohepatitis

Abstract

The invention relates to the field of biological medicines, and discloses application of piperidine acid in preparation of a medicine for preventing and / or treating non-alcoholic steatohepatitis. The invention discovers that the piperidine acid can prevent and / or treat the non-alcoholic steatohepatitis for the first time. Specifically, non-targeted metabonomics detection finds that the content of the piperidine acid in an excrement sample of an NASH patient and a mouse model is reduced, and in-vivo experiments show that supplement of the piperidine acid can reduce the levels of ALT and AST in serum of an NASH mouse, reduce the content of triglyceride in the liver, and improve the expression of the NASH mouse. And the fat change, liver cell balloon-like change, inflammatory cell infiltration and fibrosis degree in the liver are improved in histology. In-vitro experiments show that piperidine acid can improve lipid deposition, inflammatory cell proliferation and fibrosis of liver organs. Moreover, the piperidine acid is an intestinal flora metabolite originally existing in a living body, and has no obvious toxicity to the living body; the oral administration is convenient and fast; commercial reagents are available and can be conveniently obtained.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and more specifically, to the use of pipecolic acid in the preparation of drugs for preventing and / or treating non-alcoholic fatty hepatitis. Background Art

[0002] The incidence of nonalcoholic steatohepatitis (NASH) continues to increase worldwide. NASH can eventually progress to cirrhosis or hepatocellular carcinoma. Statistics show that NASH is the second leading cause of liver transplantation in the United States. The pathogenesis and clinical manifestations of NASH are complex and highly heterogeneous, and the research and development of related therapeutic drugs has important clinical value.

[0003] NASH patients often show imbalance in the intestinal flora, and related experiments have confirmed the impact of changes in the intestinal flora on NASH. The metabolites of the intestinal flora are one of the pathways connecting the flora and the host, and participate in the pathophysiological process of NASH in various ways. Targeting the intestinal flora to find a treatment for NASH is currently a hot topic.

[0004] Pipecolic acid is a metabolite of lysine under the action of intestinal flora. Existing studies have shown that pipecolic acid can reduce the inflammatory factors produced by mouse bone marrow-derived macrophages by inhibiting mTORC1 signal transduction, mediating the anti-inflammatory effect of early exercise. In addition, studies have shown that pipecolic acid can stimulate GABA receptors by promoting GABA release and inhibiting GABA reuptake. There are no relevant reports on the role of pipecolic acid in NASH. Summary of the invention

[0005] The purpose of the invention of this application is to explore the role of pipecolic acid in NASH. To this end, the present invention uses CDAHFD feed and WD feed to construct two NASH models: CDAHFD feed is added to C57 mice at 6 weeks of age for 9 weeks, and pipecolic acid is supplemented by gavage at the same time; WD feed is added to C57 mice at 6 weeks of age for 22 weeks, and pipecolic acid is supplemented by gavage at 1 mol / kg / d from the 3rd week. ALT and AST levels were detected by serum enzymology, and fatty degeneration, inflammation and fibrosis of the liver were evaluated by histopathological staining. Liver organoid models were constructed using hepatocytes, macrophages, hepatic stellate cells, and sinusoidal endothelial cells, OA+PA was used to induce NASH, and histopathological staining was used to evaluate the therapeutic effect of pipecolic acid.

[0006] In a first aspect, the present application provides the use of pipecolic acid in the preparation of a drug for preventing and / or treating non-alcoholic fatty hepatitis.

[0007] Furthermore, the medicine includes medicines for reducing the levels of ALT and AST in serum, reducing the TG content in the liver, and alleviating fatty degeneration, ballooning degeneration, inflammatory cell infiltration and fibrosis of the liver.

[0008] In a second aspect, the present application provides a drug for preventing and / or treating non-alcoholic steatohepatitis, wherein the drug comprises pipecolic acid as an active ingredient.

[0009] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0010] Furthermore, the pharmaceutical dosage form includes an oral solution.

[0011] In summary, this application has the following beneficial effects:

[0012] The present invention discovered for the first time that pipecolic acid can prevent and / or treat non-alcoholic fatty liver disease. Specifically, the present invention found that the content of pipecolic acid in fecal samples of NASH patients and mouse models was reduced through non-targeted metabolomics detection. In vivo experiments showed that pipecolic acid supplementation can reduce the levels of serum ALT and AST in NASH mice, reduce the content of triglycerides in the liver, and histologically improve fatty changes, ballooning changes of hepatocytes, inflammatory cell infiltration and fibrosis in the liver. In vitro experiments showed that pipecolic acid can improve lipid deposition, inflammatory cell proliferation and fibrosis in liver organoids. In addition, pipecolic acid is a metabolite of intestinal flora that originally exists in the body, and has no obvious toxicity to the organism; it is convenient and quick to administer by oral administration; there are commercial reagents that can be easily obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 :Mouse liver section staining results;

[0014] Figure 2 :Statistical results of percentage of fibrotic tissue area in mice;

[0015] Figure 3 : ALT, AST, and AKP levels in mouse serum;

[0016] Figure 4 : The levels of IL6 and TNFα in mouse serum;

[0017] Figure 5 :Triglyceride content in fresh liver of mice;

[0018] Figure 6 :Mouse liver section staining results;

[0019] Figure 7 : The levels of ALT, AST and ALP in mouse serum;

[0020] Figure 8: The levels of IL6 and TNFα in mouse serum;

[0021] Fig. 9 :3D liver organoid slice staining results;

[0022] Fig.10 : Statistical analysis results;

[0023] Fig.11 : Triglyceride content in 3D liver organoids;

[0024] Fig.12 : IL6 content in the supernatant of 3D liver organoids. DETAILED DESCRIPTION

[0025] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0026] It is worth noting that the materials and methods used in the examples are all existing methods unless otherwise specified.

[0027] Example 1: Pharmacodynamic study on the effect of pipecolic acid on improving non-alcoholic fatty liver disease (NAFLD)-related liver fibrosis in mice. C57BL6 / J mice were fed with a 60% high-fat, 0.1% methionine choline-deficient diet (CDAHFD, Research Diet A06071302) for 9 weeks to establish an animal model of NAFLD-related liver fibrosis, and the improvement effect of pipecolic acid on liver fibrosis was evaluated from serum biochemical indicators and liver pathology.

[0028] 1.1 Experimental methods

[0029] 1.1.1 Animal grouping

[0030] Five-week-old C57BL6 / J male mice were housed in a standard SPF barrier environment, maintained at a constant temperature and humidity, with a 12-hour day and night cycle, and free access to food and water. After one week of adaptive feeding, the 18 mice were evenly divided into a control group, a liver fibrosis model group, and a pipecolic acid treatment group.

[0031] 1.1.2 Model establishment

[0032] A 60% high-fat, 0.1% methionine, choline-deficient diet (CDAHFD, Research Diet A06071302) was used to induce a NAFLD-related liver fibrosis model. The control group was fed with ordinary feed, and the other groups were fed with CDAHFD feed for 9 consecutive weeks.

[0033] 1.1.3 Method of administration

[0034] When feeding CDAHFD diet, the pipecolic acid treatment group was gavaged with pipecolic acid 1 mol / kg / d during the same period, and the pipecolic acid was dissolved in pure water.

[0035] 1.1.4 Tissue processing

[0036] After the experiment, the mice were bled and dissected. Blood was collected from the mice's eyeballs, and the serum was separated to detect alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), IL6, and TNFα in the mouse serum. After the mice were killed, the liver was quickly removed and washed with saline. Fresh mouse livers were taken to detect the triglyceride content in the liver. Part of the liver tissue was taken and immersed in 4% paraformaldehyde solution for fixation and paraffin embedding, and H&E staining and Sirius red staining were performed.

[0037] 1.2 Experimental Results

[0038] 1.2.1 Slice staining results

[0039] The results of liver section staining of mice in each group are shown in Figure 1 H&E staining was at 100X, and Sirius red staining was at 100X.

[0040] according to Figure 1 It can be seen that the results of H&E staining and Sirius red staining showed that the liver tissue structure of the mice in the control group fed with ordinary feed was normal, the hepatocytes were evenly arranged, and there was no fatty degeneration, ballooning, punctate necrosis, inflammatory cell infiltration and fibrosis. The liver fibrosis model group fed with CDAHFD feed caused the mice to have different degrees of fatty degeneration of hepatocytes, ballooning, punctate necrosis, inflammatory cell infiltration and collagen fiber hyperplasia. After the supplementation of pipecolic acid, the above pathological changes were alleviated to varying degrees. Figure 2 The statistical results of the percentage of fibrotic tissue area in each group of mice are shown. The above results show that pipecolic acid can effectively improve CDAHFD diet-induced liver fibrosis.

[0041] 1.2.2 Liver function levels of mice in each group

[0042] The levels of ALT, AST and AKP in the serum of mice in each group were as follows Figure 3 The results of serological liver function indicators showed that CDAHFD diet caused a significant increase in ALT, AST, and AKP (p < 0.01), and the intervention of pipecolic acid significantly reduced the increase in ALT, AST, and AKP caused by CDAHFD diet (p < 0.01), indicating that pipecolic acid can effectively improve liver damage induced by CDAHFD diet.

[0043] 1.2.3 Levels of inflammatory factors in mice in each group

[0044] The levels of IL6 and TNFα in the serum of mice in each group were as follows Figure 4 The results of serum inflammatory factors showed that CDAHFD diet led to increased levels of IL6 and TNFα in serum (p < 0.01), and pipecolic acid supplementation significantly reduced the increase of IL6 and TNFα caused by CDAHFD diet (p < 0.01), indicating that pipecolic acid can effectively improve the inflammatory state induced by CDAHFD.

[0045] 1.2.4 Triglyceride levels in liver of mice in each group

[0046] The content of triglyceride in fresh liver of mice in each group is as follows Figure 5 The results of triglyceride quantification showed that CDAHFD diet led to a significant increase in triglyceride content in the liver (p < 0.01), and pipecolic acid supplementation significantly reduced the triglyceride content in the liver (p < 0.01), indicating that pipecolic acid can effectively reduce liver fat deposition caused by CDAHFD.

[0047] Example 2: Pharmacodynamic study of pipecolic acid in improving non-alcoholic steatohepatitis (NASH) in mice

[0048] C57BL6 / J mice were fed a high-fat, high-fructose, high-cholesterol (WD) diet (Research diet, D09100310) for 22 weeks to establish the NASH animal model, and the improvement effect of pipecolic acid on NASH was evaluated based on serum biochemical indicators and liver pathology.

[0049] 2.1 Experimental methods

[0050] 2.1.1 Animal grouping

[0051] Five-week-old C57BL6 / J male mice were housed in a standard SPF barrier environment, maintained at a constant temperature and humidity, with a 12-hour day and night cycle, and free access to food and water. After one week of adaptive feeding, the 18 mice were evenly divided into a control group, a NASH model group, and a pipecolic acid treatment group.

[0052] 2.1.2 Model establishment

[0053] A high-fat, high-fructose, high-cholesterol (Western Diet, WD) diet (Research diet, D09100310) was used to induce the NASH model. The control group was fed with ordinary feed, and the other groups were fed with WD feed for 22 consecutive weeks.

[0054] 2.1.3 Method of administration

[0055] After 3 weeks of feeding WD diet, the pipecolic acid treatment group began to be gavaged with pipecolic acid 1 mol / kg / d, and pipecolic acid was dissolved in purified water.

[0056] 2.1.4 Tissue processing

[0057] After the experiment, the mice were bled and dissected. Blood was collected from the eyeballs of the mice, and the serum was separated to detect alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), IL6, and TNFα in the serum of the mice. After the mice were killed, the liver was quickly removed and washed with saline. Part of the liver tissue was soaked in 4% paraformaldehyde solution for fixation and paraffin embedding, and H&E staining and Sirius red staining were performed.

[0058] 2.2 Experimental Results

[0059] 2.2.1 Slice staining results

[0060] The results of liver section staining of mice in each group are shown in Figure 6 As shown. H&E staining is 100X, Sirius red staining is 100X. Figure 6 It can be seen that the results of H&E Sirius red staining showed that the liver tissue structure of the control mice fed with ordinary feed was normal, the hepatocytes were evenly arranged, and there was no fatty degeneration, ballooning, punctate necrosis, inflammatory cell infiltration and fibrosis. The NASH model fed with WD feed caused the mouse liver to have different degrees of pathological changes such as fatty degeneration of hepatocytes, ballooning, punctate necrosis, inflammatory cell infiltration and collagen fiber hyperplasia. After supplementation with pipecolic acid, the above pathological changes were alleviated to varying degrees, indicating that pipecolic acid can effectively improve NASH induced by WD feed.

[0061] 2.2.2 Liver function levels of mice in each group

[0062] The levels of ALT, AST and ALP in the serum of mice in each group were as follows Figure 7 As shown. The results of serological liver function indicators showed that WD diet caused a significant increase in ALT and AST (p < 0.01), and the intervention of pipecolic acid significantly reduced the increase in ALT and AST caused by WD diet (p < 0.01). There was a large difference in the results of ALP within the NASH group, but the experimental results still showed that pipecolic acid had a trend of improving the increase in ALP caused by WD diet. This shows that pipecolic acid can effectively improve liver damage induced by WD diet.

[0063] 2.2.3 Levels of inflammatory factors in mice in each group

[0064] The levels of IL6 and TNFα in the serum of mice in each group were as follows Figure 8 The results of serum inflammatory factors showed that WD diet led to an increase in the levels of IL6 and TNFα in serum (p < 0.01), and the supplementation of pipecolic acid significantly reduced the increase in IL6 and TNFα caused by WD diet (p < 0.01), indicating that pipecolic acid can effectively improve the inflammatory state induced by WD diet.

[0065] Example 3: Pharmacodynamic study of pipecolic acid in improving NASH human 3D liver organoid model

[0066] The NASH model was constructed by treating liver organoids with culture medium containing 1 mM free fatty acids, and the improving effect of pipecolic acid on NASH was evaluated based on tissue pathology.

[0067] 3.1 Experimental methods

[0068] 3.1.1 Experimental Grouping

[0069] A total of 5 experimental groups were set up, the group treated with ordinary culture medium was the control group, the group treated with culture medium containing free fatty acids was the disease group, and 4μM, 20μM, and 100μM pipecolic acid were added to the culture medium containing free fatty acids, which were the low-concentration treatment group, medium-concentration treatment group, and high-concentration treatment group, respectively.

[0070] 3.1.2 Construction of liver organoids

[0071] Human hepatocytes, macrophages, hepatic stellate cells, and sinusoidal endothelial cells were used to construct 3D liver organoids using DNA origami self-assembly technology.

[0072] 3.1.3 Construction of NASH model

[0073] The NASH model was established by treating 3D liver organoids with culture medium containing 1 mM free fatty acid (FFA, PA:OA=1:2) for 14 days.

[0074] 3.1.4 Method of administration

[0075] When the cells were treated with the medium containing free fatty acids, different concentrations of pipecolic acid were added to the medium. The pipecolic acid was dissolved in sterile PBS.

[0076] 3.1.5 Tissue processing

[0077] After the experiment, the culture medium supernatant was taken for IL6 detection. Fresh 3D liver organoid models were taken for triglyceride quantification. The 3D liver organoid models were fixed in 4% paraformaldehyde solution and embedded in paraffin for H&E staining, Sirius red staining, and immunofluorescence staining.

[0078] 3.2 Experimental Results

[0079] 3.2.1 Slice staining results

[0080] The staining results of 3D liver organoid slices in each group are as follows Fig. 9 H&E staining was at 400X, Sirius red staining was at 400X, and immunofluorescence staining was at 400X.

[0081] according to Fig. 9 It can be seen that the results of H&E staining and Sirius red staining show that the cell structure of 3D liver organoids in ordinary culture medium is normal and arranged regularly. After treatment with free fatty acid-containing culture medium, the cellular lipid droplet content in 3D liver organoids increased, the necrosis area increased, the inflammatory cells proliferated, and the collagen deposition increased, indicating that the culture medium containing free fatty acids induced fatty changes, inflammation, and fibrosis in 3D liver organoids. After the addition of pipecolic acid, the above pathological changes were alleviated and showed a trend of positive correlation with the concentration of pipecolic acid. The results of immunofluorescence staining also showed that the activation of hepatic stellate cells in 3D liver organoids increased and the content of collagen fibers increased after treatment with free fatty acid culture medium. Fig.10 The results of statistical analysis were presented. Compared with the disease group, the collagen area in the treatment group was reduced and the activation of stellate cells was reduced, and the differences were statistically significant, indicating that pipecolic acid can effectively improve NASH caused by free fatty acids in human 3D liver organoids.

[0082] 3.2.2 Triglyceride content of organoids in each group

[0083] The triglyceride content of 3D liver organoids in each group is as follows Fig.11 The results of triglyceride quantification indicated that the triglyceride content of 3D organoids increased significantly after treatment with free fatty acid medium (p < 0.01), and pipecolic acid intervention significantly reduced the increase in triglyceride content caused by free fatty acids (p < 0.01), indicating that pipecolic acid can effectively improve lipid deposition caused by free fatty acids.

[0084] 3.2.3 Content of inflammatory factors in the supernatant of organoids in each group

[0085] The content of IL6 in the supernatant of 3D liver organoids in each group is as follows Fig.12 The results of supernatant inflammatory factors showed that free fatty acid treatment led to an increase in the level of IL6 in the supernatant (p < 0.01), and the addition of pipecolic acid significantly reduced the increase in IL6 caused by free fatty acids (p < 0.01), indicating that pipecolic acid can reduce the production of inflammatory factors induced by free fatty acids.

[0086] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. Use of pipecolic acid in the preparation of a drug for preventing and / or treating non-alcoholic fatty hepatitis.

2. The use according to claim 1, characterized in that The drugs include drugs for reducing the levels of ALT and AST in serum, reducing the TG content in the liver, and alleviating fatty degeneration, ballooning degeneration, inflammatory cell infiltration and fibrosis of the liver.

3. A drug for preventing and / or treating non-alcoholic fatty hepatitis, characterized in that: The drug includes pipecolic acid as an active ingredient.

4. The drug according to claim 3, characterized in that The drug also includes pharmaceutically acceptable excipients.

5. The drug according to claim 3, characterized in that The pharmaceutical dosage forms include oral solutions.

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