CN113730551B

CN113730551B - Application of MMI-0100 short peptide compound in preparation of medicine for treating cholestatic liver disease

Info

Publication number: CN113730551B
Application number: CN202111023996.4A
Authority: CN
Inventors: 张蓓蓓; 张波; 颜超; 郑葵阳; 李静; 徐娜; 刘继鑫; 谈仁秀; 黄芝月
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2021-09-02
Filing date: 2021-09-02
Publication date: 2022-11-25
Anticipated expiration: 2041-09-02
Also published as: CN113730551A

Abstract

The invention discloses an application of an MMI-0100 short peptide compound in preparation of a cholestatic liver disease treatment drug. The amino acid sequence of the MMI-0100 short peptide compound is as follows: YARAAARQARAKALARQLGVAA. After MMI-0100 drug treatment, the fibrosis of the liver of a mouse is relieved, infiltration of inflammatory cells is reduced, the hyperplasia degree of bile ducts is relieved, the mRNA expression level of liver fibrosis gene alpha-SMA is reduced, and the results show that MMI-0100 has a good treatment effect on cholestatic liver injury.

Description

Application of MMI-0100 short peptide compound in preparation of cholestatic liver disease treatment drug

Technical Field

The invention relates to an application of an MMI-0100 short peptide compound in preparation of a cholestatic liver disease treatment drug, and belongs to the technical field of new application of the MMI-0100 short peptide compound as a drug.

Background

Cholestasis refers to a disorder of bile formation and/or flow, resulting in a disorder of bile flow, and is one of the common complications of liver diseases. Various factors causing bile flow disorder can cause cholestasis, the common factors mainly comprise parasite, virus and bacterial infection, autoimmunity, medicament or poison, alcohol, calculus, genetic metabolism and the like, and clinically common cholestatic diseases mainly comprise Primary Biliary Cholangitis (PBC), primary Sclerosing Cholangitis (PSC) and cholestasis caused by various viral hepatitis. In most cases, cholestasis is a benign lesion with low morbidity or mortality, but long-term cholestasis can eventually lead to cirrhosis and even liver failure, with a poor patient prognosis. Currently, only two drugs, obeticholic acid (OCA) and ursodeoxycholic acid (UDCA), are approved by the FDA in the united states and used in the clinical treatment of cholestatic liver disease. However, obeticholic acid has some hepatotoxicity and UDCA is only therapeutically effective in some patients. Therefore, the search and development of effective drugs for cholestatic liver diseases has been a research focus in recent years.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides the application of the MMI-0100 short peptide compound in preparing the medicine for treating cholestatic liver diseases; by adopting a 3, 5-diethoxycarbonyl-1, 4-dihydro-2, 4, 6-trimethylpyridine (DDC) induced PSC mouse model, the treatment effect of MMI-0100 on cholestatic liver injury is observed, so as to provide an experimental basis for a new drug treatment means for cholestatic liver diseases.

The invention is realized by the following technical scheme that the MMI-0100 short peptide compound is applied to the preparation of the medicine for treating cholestatic liver diseases.

As a preferred scheme of the application of the MMI-0100 short peptide compound in the preparation of the medicine for treating cholestatic liver diseases, the medicine comprises the following components in percentage by weight: the amino acid sequence of the MMI-0100 short peptide compound is as follows: YARAAARQARAKALARQLGVAA.

The experimental protocol was as follows:

in order to realize the purpose, the invention is realized by the following technical scheme:

(1) MMI-0100 short peptide compounds (YARAAARQARAKALARQLGVAA) were synthesized by Gill Biochemical (Shanghai) Inc.

(2) And (3) medicine intervention: establishing a liver cholestasis model induced by DDC irradiation feed feeding. After 1w of DDC feeding, MMI-0100 is intraperitoneally injected every day for treatment, the injection amount of each mouse is 500ug/Kg, and 1w is continuously injected.

(3) HE and Masson staining detected liver pathology.

(4) CK19 and Ki67 immunohistochemistry detected hepatobiliary hyperplasia.

(5) And detecting the expression of the hepatic fiber ring related gene in the hepatic tissue by real-time fluorescent quantitative PCR (qRT-PCR).

The invention has the beneficial technical effects that:

in DDC diet-induced cholestatic liver injury of mice, bile ducts are obviously proliferated, a large number of inflammatory cells infiltrate around the bile ducts, and hepatic fibrosis is obvious. After MMI-0100 drug treatment, the fibrosis of the liver of a mouse is relieved, infiltration of inflammatory cells is reduced, the hyperplasia degree of bile ducts is relieved, the mRNA expression level of liver fibrosis gene alpha-SMA is reduced, and the results show that MMI-0100 has a good treatment effect on cholestatic liver injury.

Drawings

FIG. 1 is a comparison of the macroscopic observations of the livers of the groups of mice;

fig. 2 is the histopathological results (HE staining) of the livers of the mice of each group, note: p < 0.05 compared to model group;

FIG. 3 shows the Masson staining of liver tissue and the expression of liver alpha-SMA mRNA in each group of mice, note: p < 0.05 compared to model group, compared to control group ^# P＜0.05；

FIG. 4 is an immunohistochemical assay of CK19 expression in liver tissue of mice in each group, note: p < 0.05 compared to model group;

fig. 5 is an immunohistochemical assay of Ki67 expression in liver tissue of each group of mice, note: p < 0.05 compared to model group.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention is further illustrated by the following examples.

Example 1

1. Histopathological changes of the liver

1. Operation of

15 Balb/c mice were randomly assigned to control, model (DDC), and treatment (DDC + MMI-0100), 5 mice per group. Except for the control group, two groups of mice of the model group and the treatment group were given 0.1% DDC irradiation feed to establish a cholestasis model, and the treatment group mice were treated with MMI-0100 by intraperitoneal injection every day after DDC feeding 1w, with an injection amount of drug per mouse of 500ug/Kg, and were continuously injected 1w while being given a normal diet. The last day of drug injection, mice were sacrificed by drug cervical dislocation and livers were collected.

Detection of liver pathology: separating the complete liver of a mouse at low temperature, observing pathological changes of the liver of the mouse by naked eyes, taking tissues with the size of about 5mm multiplied by 2mm, fixing the tissues in 10 percent paraformaldehyde for 24 hours, carrying out alcohol gradient dehydration, embedding paraffin, continuously slicing the tissues with the thickness of 4 mu m, carrying out HE (high intensity staining) and Masson (Masson) staining, observing pathological changes of the liver under a microscope, scoring the inflammatory activity of the liver tissues by Knodell Score histological activity indexes, and carrying out semi-quantitative analysis on the fibrosis degree of the liver by Image-Pro Plus.

Immunohistochemical staining: CK19 and Ki67

Deparaffinizing the liver tissue slices, washing with PBS for 5min for 3 times; placing the slices into 0.01mmol/L citrate buffer solution for antigen heat restoration for 15min, and washing with PBS for 3 times, 5min each time; dropwise addition of H ₂ O ₂ After incubation at room temperature for 10min, washing with PBS for 3 times, 5min each time; the rabbit anti-CK 19 polyclonal antibody (1; PBS is washed for 3 times, after 10min each time, DAB color development kit (Beijing Zhonghua Jinqiao company, cat number: PV-9001) is used for color development, and the specific steps are as follows: adding reagent 1 dropwise, incubating at 37 deg.C for 10min, washing with PBS for 3 times, each time for 10min; dropwise adding reagent 2 (horseradish enzyme labeled anti-rabbit IgG polymer), incubating for 30min at 37 ℃, washing for 3 times with PBS (10 min each time); adding DAB for color development, performing hematoxylin counterstaining, returning blue with tap water, dehydrating with gradient alcohol, sealing, observing under a lens, and performing semi-quantitative analysis with Image-Pro Plus.

Real-time fluorescent quantitative PCR (qRT-PCR) detection of mRNA expression level in liver tissue

The total RNA of each group of liver tissues is extracted by a TRIzol method, and is subjected to PCR amplification reaction after being subjected to reverse transcription to form cDNA. Primer sequence, GAPDH: ACTCCACTCACGGCAATTC (upstream), TCTCCATGGGTGGTGAAGACA (downstream); alpha-SMA: CACAGCCCTGGTGTGCGA CAAT (upstream), TTGCTCTGGGCTTCATCCCCCCA (downstream). Amplification productThe series (20. Mu.l) were: 10 mul of 2 XSYBR qPCR premix, 1 mul of each upstream primer and downstream primer of 10 mul/L, 1 mul of cDNA and 7 mul of DEPC water; the amplification procedure was: 5min at 95 ℃; 35 cycles of 95 ℃ 10s,60 ℃ 10s and 72 ℃ 10 s. And calculating the relative expression level of the corresponding gene according to the obtained cycle threshold (Ct). GAPDH was used as an internal control, and the relative expression level of the target gene mRNA was 2 ^-ΔΔCt And (4) calculating.

2. Results

After the mice are fed with the DDC-containing feed, the hair is scattered and dull, the reaction is slow, and all the mice in a control group are normal. The liver of the control group mouse is pink, crimson and smooth in surface; after DDC induction, the liver of the mouse is swollen, the color is yellow and the color is dark (see figure 1).

The HE staining result shows that the liver lobules of the control group have complete structure, no biliary duct hyperplasia and fibrous tissue, normal liver cell morphology without degeneration and necrosis, and no inflammatory cell infiltration around the bile duct; the mouse liver cells of the model group are in punctate and focal necrosis, the bile duct is seriously proliferated, the surrounding fibrous tissues are seriously proliferated, and a large amount of inflammatory cells infiltrate around the bile duct; the MMI-0100 treated mice had intact lobules of the liver, no necrotic degeneration of hepatocytes, little hyperplasia of bile ducts and fibrous tissue, and little infiltration of inflammatory cells around the bile ducts (see FIG. 2A). The degree of inflammation of the liver was evaluated by the Knodell Score histological activity index Score, and the results showed that the grade of inflammation was significantly lower in the MMI-0100 treated group than in the model group (P < 0.05) (see FIG. 2B).

Masson staining results show that liver tissues of model mice have a large amount of collagen fiber deposition, and the liver has obvious necrotic foci; the deposition of collagen fibers in liver tissue of mice treated with MMI-0100 was significantly reduced (FIG. 3A). The percent area of fibrosis in the MMI-0100 treated group was significantly lower than that in the model group (P < 0.05) as calculated by Image-Pro Plus software (FIG. 3B). The expression of fibrosis gene in liver was detected, and compared with the model group, the expression of alpha-SMA in MMI-0100 treated group was decreased (P < 0.05) (see FIG. 3C).

The bile duct epithelial cell cytoplasm positively stained with CK19 was stained tan under microscopic observation (see FIG. 4A), and the liver of MMI-0100 treated group was expressed less CK19 than that of the model group (P < 0.05) by semi-quantitative analysis (see FIG. 4B).

The Ki67 immunohistochemical staining result shows that a large number of Ki67 positive bile duct epithelial cells are found in the liver of the model group mouse (see figure 5A), the number of Ki67 positive bile duct epithelial cells in the liver of the MMI-0100 treatment group mouse is obviously reduced, and the difference has statistical significance (P is less than 0.05) through software analysis (see figure 5B).

Claims

An application of MMI-0100 short peptide compound in preparing the medicine for treating cholestatic liver disease is disclosed.
2. Use of MMI-0100 short peptide compounds according to claim 1 for the preparation of a medicament for the treatment of cholestatic liver diseases characterized by: the amino acid sequence of the MMI-0100 short peptide compound is as follows: YARAAARQARAKALARQLGVAA.