Use of piperidinic acids in the preparation of a medicament for the prevention and / or treatment of nonalcoholic steatohepatitis

Abstract

The application relates to the field of biological medicine, and discloses application of piperidinic acid in preparation of a medicine for preventing and / or treating non-alcoholic steatohepatitis. The application first discovers that piperidinic acid can prevent and / or treat non-alcoholic steatohepatitis. Specifically, the application finds, through non-targeted metabolomics detection, that the content of piperidinic acid in fecal samples of NASH patients and mouse models is reduced, in-vivo experiments show that supplementing piperidinic acid can reduce the serum ALT and AST levels of NASH mice, reduce the content of triglyceride in the liver, and improve the degree of fatty degeneration, hepatocyte ballooning, inflammatory cell infiltration and fibrosis in the liver in histology. In-vitro experiments show that piperidinic acid can improve lipid deposition, inflammatory cell proliferation and fibrosis of liver organoids. Moreover, piperidinic acid is a metabolite of intestinal flora originally existing in a living body, has no obvious toxicity to the living body, is convenient and fast to administer through an oral mode, and can be conveniently obtained as a commercial reagent.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, more particularly, it relates to the application of pipecolic acid in the preparation of a drug for preventing and / or treating non-alcoholic steatohepatitis. BACKGROUND

[0002] The incidence of non-alcoholic steatohepatitis (NASH) is increasing worldwide. NASH can eventually progress to cirrhosis or hepatocellular carcinoma, and statistics show that NASH has become 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 development of related therapeutic drugs has important clinical value.

[0003] NASH patients often show intestinal flora imbalance, and related experiments have confirmed the influence of intestinal flora changes on NASH. The metabolic products of intestinal flora are one of the ways to connect flora and host, and participate in the pathophysiological process of NASH in various ways. Targeting intestinal flora to find a treatment for NASH is a current research hotspot.

[0004] Pipecolic acid is a metabolic product of lysine under the action of intestinal flora. Existing studies have shown that pipecolic acid can reduce the production of inflammatory factors by mouse bone marrow-derived macrophages by inhibiting mTORC1 signal transduction, and mediate the anti-inflammatory effect produced by early exercise. In addition, studies have shown that pipecolic acid can promote GABA release and inhibit GABA reuptake to play an agonistic role in GABA receptors. The role of pipecolic acid in NASH has not been reported. SUMMARY

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

[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 steatohepatitis.

[0007] Further, the medicine comprises a medicine for reducing the levels of ALT and AST in serum, reducing the TG content in liver, and reducing the steatosis, ballooning, inflammatory cell infiltration and fibrosis of liver.

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

[0009] Further, the medicine further comprises a pharmaceutically acceptable excipient.

[0010] Further, the medicine dosage form comprises an oral liquid.

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

[0012] The present application firstly discovers that piperideic acid can prevent and / or treat non-alcoholic steatohepatitis. Specifically, the present application discovers that the content of piperideic acid in fecal samples of NASH patients and mouse models is reduced through non-targeted metabolomics detection, and in vivo experiments show that supplementing piperideic acid can reduce the levels of ALT and AST in serum of NASH mice, reduce the content of triglyceride in liver, and improve the steatosis, ballooning of hepatocytes, inflammatory cell infiltration and fibrosis degree in liver in histology. In vitro experiments show that piperideic acid can improve lipid deposition, inflammatory cell proliferation and fibrosis of liver organoids. Moreover, piperideic acid is a metabolite of intestinal flora originally existing in the body, and has no obvious toxicity to the organism; it is convenient and fast to administer by oral administration; and it is a commercial reagent and can be easily obtained. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 : Staining results of mouse liver sections;

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

[0015] Figure 3 : Contents of ALT, AST and AKP in serum of mice;

[0016] Figure 4 : Contents of IL6 and TNFα in serum of mice;

[0017] Figure 5 : Content of triglyceride in fresh liver of mice;

[0018] Figure 6 : Staining results of mouse liver sections;

[0019] Figure 7 : Contents of ALT, AST and ALP in serum of mice;

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

[0021] Figure 9 : 3D liver organoid section staining results;

[0022] Figure 10 : Statistical analysis results;

[0023] Figure 11 : The content of triglyceride in 3D liver organoid;

[0024] Figure 12 : The content of IL6 in 3D liver organoid supernatant. DETAILED DESCRIPTION

[0025] The technical solutions and effects of the present application are further described in detail below in conjunction with examples. It can be understood that the specific examples described herein are merely used to explain the present application, and are not a limitation on the present application.

[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 of piperidinic acid on improving non-alcoholic fatty liver (NAFLD) related liver fibrosis mice

[0028] 1.1 Experimental method

[0029] 1.1.1 Animal grouping

[0030] 5-week-old C57BL6 / J male mice were raised in a standard SPF barrier environment, kept at constant temperature and humidity, 12-hour alternating day and night, and free to eat and drink. After 1 week of adaptive feeding, 18 mice were evenly divided into a control group, a liver fibrosis model group, and a piperidinic acid treatment group.

[0031] 1.1.2 Model establishment

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

[0033] 1.1.3 Dosing method

[0034] The piperidinic acid treatment group was given piperidinic acid 1 mol / kg / d by gavage at the same time when fed with CDAHFD feed. The piperidinic acid was dissolved in pure water.

[0035] 1.1.4 Tissue processing

[0036] After the experiment, the mice were bled and dissected. The mice were bled by eyeball, and the serum was separated to detect the alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), IL6, TNFα in the serum of the mice. After the mice were sacrificed, the liver was quickly taken out and washed with physiological saline. Fresh mouse liver was taken to detect the triglyceride content in the liver. Part of the liver tissue was soaked in 4% paraformaldehyde solution for fixation and paraffin embedding, and H&E staining, Sirius red staining were performed.

[0037] 1.2 Experimental results

[0038] 1.2.1 Staining results of liver sections

[0039] The staining results of liver sections of mice in each group are shown in Figure 1 . Among them, H&E staining is 100X, and Sirius red staining is 100X.

[0040] According to Figure 1 It can be seen that the results of H&E staining and Sirius red staining show that the liver tissue structure of the control group of mice fed with ordinary feed is normal, the liver cells are arranged uniformly, and there is no fatty degeneration, ballooning degeneration, punctate necrosis, inflammatory cell infiltration focus and fibrosis. The CDAHFD feed fed to the liver fibrosis model group caused different degrees of pathological changes such as liver cell fatty degeneration, ballooning degeneration, punctate necrosis, inflammatory cell infiltration focus and collagen fiber proliferation in the liver of mice. After supplementing piperidinic acid, the above pathological changes were reduced to different degrees. Figure 2 The statistical results of the percentage of fibrotic tissue area of mice in each group are shown. The above results show that piperidinic acid can effectively improve the liver fibrosis induced by CDAHFD feed.

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

[0042] The contents of ALT, AST and AKP in the serum of mice in each group are shown in Figure 3 . The results of serological liver function indicators suggest that CDAHFD feed can significantly increase ALT, AST and AKP (p<0.01), and the intervention of piperidinic acid can significantly reduce the increase of ALT, AST and AKP caused by CDAHFD feed (p<0.01), indicating that piperidinic acid can effectively improve the liver damage induced by CDAHFD feed.

[0043] 1.2.3 Inflammatory factor levels of mice in each group

[0044] The content of IL6 and TNFa in serum of mice in each group is shown in Table 1. Figure 4 The results of serum inflammatory factors show that CDAHFD feed causes the increase of IL6 and TNFa levels in serum (p<0.01), and the supplementation of piperine significantly reduces the increase of IL6 and TNFa caused by CDAHFD feed (p<0.01), indicating that piperine can effectively improve the inflammatory state induced by CDAHFD.

[0045] 1.2.4 Liver triglyceride levels of mice in each group

[0046] The content of triglyceride in fresh liver of mice in each group is shown in Table 2. Figure 5 The results of triglyceride quantification show that CDAHFD feed causes a significant increase in the content of triglyceride in liver (p<0.01), and the supplementation of piperine significantly reduces the content of triglyceride in liver (p<0.01), indicating that piperine can effectively reduce the liver fat deposition caused by CDAHFD.

[0047] Example 2: Pharmacodynamic study of piperine on non-alcoholic steatohepatitis (NASH) mice

[0048] C57BL6 / J mice were fed with high-fat high-fructose high-cholesterol (Western Diet, WD) feed (Research diet, D09100310) for 22 weeks to replicate NASH animal models, and the improvement of piperine on NASH was evaluated from serum biochemical indicators and liver pathological conditions.

[0049] 2.1 Experimental method

[0050] 2.1.1 Animal grouping

[0051] 5-week-old C57BL6 / J male mice were raised in a standard SPF barrier environment, with constant temperature and humidity, 12-hour alternating day and night, and free feeding and drinking water. After 1 week of adaptive feeding, 18 mice were evenly divided into control group, NASH model group, and piperine treatment group.

[0052] 2.1.2 Model establishment

[0053] NASH models were induced by high-fat high-fructose high-cholesterol (Western Diet, WD) feed (Research diet, D09100310), and the control group was fed with ordinary feed, and the rest of the groups were fed with WD feed for 22 weeks.

[0054] 2.1.3 Dosing method

[0055] After 3 weeks of WD feed, the piperine treatment group started to be given piperine by gavage at 1 mol / kg / d, and piperine was dissolved in pure water.

[0056] 2.1.4 Tissue processing

[0057] After the end of the experiment, the mice were bled and dissected. The mice were bled by eyeball, and the serum was separated. The serum of the mice was detected for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), IL6, TNFα. After the mice were sacrificed, the liver was quickly removed and washed with normal saline. Part of the liver tissue was soaked in 4% paraformaldehyde solution for fixation and paraffin embedding, and H&E staining, Sirius red staining were performed.

[0058] 2.2 Experimental results

[0059] 2.2.1 Staining results of liver sections

[0060] The staining results of liver sections of mice in each group are shown in Figure 6 . Among them, H&E staining is 100X, and Sirius red staining is 100X. As can be seen from Figure 6 , H&E and Sirius red staining results show that the liver tissue structure of the control mice fed with ordinary feed is normal, the liver cells are arranged uniformly, and there is no fatty degeneration, ballooning, point necrosis, inflammatory cell infiltration focus and fibrosis. WD feed feeding of NASH model causes different degrees of liver cell fatty degeneration, ballooning, point necrosis, inflammatory cell infiltration focus and collagen fiber proliferation in mice. After the supplementation of piperidinic acid, the above pathological changes are reduced to different degrees, indicating that piperidinic acid can effectively improve NASH induced by WD feed.

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

[0062] The contents of ALT, AST and ALP in the serum of mice in each group are shown in Figure 7 . The results of serological liver function indicators suggest that WD feed causes a significant increase in ALT and AST (p<0.01), and the intervention of piperidinic acid significantly reduces the increase of ALT and AST caused by WD feed (p<0.01). The results of ALP show that there is a large difference within the NASH group, but the experimental results still show that piperidinic acid has a trend of improving the increase of ALP caused by WD feed. It is shown that piperidinic acid can effectively improve the liver injury induced by WD feed.

[0063] 2.2.3 Inflammatory factor levels of mice in each group

[0064] The contents of IL6 and TNFα in the serum of mice in each group are shown in Figure 8 . The results of serum inflammatory factors show that WD feed causes an increase in the levels of IL6 and TNFα in the serum (p<0.01), and the supplementation of piperidinic acid significantly reduces the increase of IL6 and TNFα caused by WD feed (p<0.01), indicating that piperidinic acid can effectively improve the inflammatory state induced by WD feed.

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

[0066] NASH model was constructed by treating liver organoids with medium containing 1 mM free fatty acid, and the improvement of piperideic acid on NASH was evaluated from histopathological conditions.

[0067] 3.1 Experimental methods

[0068] 3.1.1 Experimental grouping

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

[0070] 3.1.2 Construction of liver organoids

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

[0072] 3.1.3 Construction of NASH model

[0073] 3D liver organoids were treated with medium containing 1 mM free fatty acid (FFA, PA: OA = 1:2) for 14 days to construct NASH model.

[0074] 3.1.4 Drug administration method

[0075] Different concentrations of piperideic acid were added to the medium at the same time when the free fatty acid-containing medium was used, and piperideic acid was dissolved in sterile PBS.

[0076] 3.1.5 Tissue processing

[0077] After the experiment, the supernatant of the culture medium was taken for IL6 detection. Fresh 3D liver organoid model was taken for triglyceride quantification. 3D liver organoid model was soaked in 4% paraformaldehyde solution for fixation and paraffin embedding, and H&E staining, Sirius red staining and immunofluorescence staining were performed.

[0078] 3.2 Experimental results

[0079] 3.2.1 Section staining results

[0080] The section staining results of 3D liver organoids in each group are shown in Figure 9 H&E staining is 400X, Sirius red staining is 400X, and immunofluorescence staining is 400X.

[0081] According to Figure 9 As can be seen, the results of H&E staining and sirius red staining show that the cell structure of 3D liver organoids in normal medium is normal and arranged regularly. After treatment with free fatty acid-containing medium, the content of lipid droplets in cells within 3D liver organoids increases, the area of necrosis increases, inflammatory cells proliferate, and collagen deposition increases, indicating that the medium containing free fatty acids induces steatosis, inflammation and fibrosis of 3D liver organoids. After adding piperideic acid, the above pathological changes are reduced and show a positive correlation with the concentration of piperideic acid. The results of immunofluorescence staining also show that the activation of hepatic stellate cells and the content of collagen fibers in 3D liver organoids increase after treatment with free fatty acid-containing medium. Figure 10 The results of statistical analysis are shown, and compared with the disease group, the collagen area of the treatment group is reduced, and the activation degree of stellate cells is reduced, and the difference is statistically significant, indicating that in human 3D liver organoids, piperideic acid can effectively improve NASH caused by free fatty acids.

[0082] 3.2.2 Triglyceride content of organoids in each group

[0083] The triglyceride content of 3D liver organoids in each group is shown in Table 3.2.2. Figure 11 The results of triglyceride quantification suggest that the triglyceride content of 3D organoids significantly increases after treatment with free fatty acid-containing medium (p<0.01), and piperideic acid intervention significantly reduces the increase in triglyceride content caused by free fatty acids (p<0.01), indicating that piperideic acid can effectively improve lipid deposition caused by free fatty acids.

[0084] 3.2.3 Inflammatory factor content in supernatant of organoids in each group

[0085] The IL6 content in the supernatant of 3D liver organoids in each group is shown in Table 3.2.3. Figure 12 The results of supernatant inflammatory factors show that free fatty acid treatment leads to an increase in IL6 levels in the supernatant (p<0.01), and the addition of piperideic acid significantly reduces the increase in IL6 caused by free fatty acids (p<0.01), indicating that piperideic acid can reduce the production of inflammatory factors induced by free fatty acids.

[0086] This specific embodiment is only an explanation of the present application, which is not a limitation of the present application, and those skilled in the art can make modifications to the present embodiment without creative contribution after reading the present specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. Piperine acid in the preparation of a drug for treating non-alcoholic steatohepatitis.

2. Use according to claim 1, wherein The drug is a drug for reducing the levels of ALT and AST in serum, reducing the content of TG in liver, and reducing the fatty degeneration, ballooning degeneration, inflammatory cell infiltration and fibrosis of liver.

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