CN110694069A

CN110694069A - Medicine for preventing and treating fulminant hepatitis

Info

Publication number: CN110694069A
Application number: CN201911087217.XA
Authority: CN
Inventors: 殷文伟; 贾方; 任红
Original assignee: Second Affiliated Hospital of Chongqing Medical University
Current assignee: Second Affiliated Hospital of Chongqing Medical University
Priority date: 2019-11-08
Filing date: 2019-11-08
Publication date: 2020-01-17
Anticipated expiration: 2039-11-08
Also published as: CN110694069B

Abstract

The invention relates to the technical field of medicine, in particular to a medicine for preventing and treating fulminant hepatitis. The research of the invention shows that the NOD1 receptor agonist can remarkably up-regulate the expression of liver tissue A20. A20 (also called TNFAIP3) is an anti-inflammatory signaling molecule, and A20 can achieve anti-apoptosis effect by down-regulating NF-kB signaling pathway, thereby protecting liver cells. In addition, the NOD1 receptor agonist can also obviously reduce the serum ALT level and the liver tissue cl-caspase3 level of a fulminant hepatitis mouse, relieve the liver injury of the fulminant hepatitis mouse and reduce the liver cell apoptosis. Thus, NOD1 receptor agonists are believed to be useful in the treatment of explosive liver injury.

Description

Medicine for preventing and treating fulminant hepatitis

Technical Field

The invention relates to the technical field of medicine, in particular to a medicine for preventing and treating fulminant hepatitis.

Background

Fulminant hepatitis is currently considered as the initiation link and common pathway of various serious liver diseases, has sudden onset and poor prognosis, can further cause serious liver failure and multi-organ dysfunction once not processed correctly, is very difficult to treat, and has high clinical mortality. Sepsis, triggered by LPS, a cell wall component of gram-negative bacteria, plays a critical role in fulminant hepatitis, one of the common critical cases of ICU. When intestinal permeability is increased due to various reasons, bacteria in the intestinal tract can enter intestinal wall blood vessels to further reach the liver to cause inflammatory reaction.

LPS is a component of the outer wall of bacterial cells and is also the most main disease treatment factor of the bacterial cells, LPS binding protein is further combined with liver CD14 and TLR4 receptors to activate liver kupffer cells, and the kupffer cells can release a large amount of inflammatory factors after being activated, wherein the most main factor is early secreted TNF-alpha which can be combined with hepatocyte TNF-alpha receptors to activate downstream inflammation and apoptosis signal pathways of the TNF-alpha receptors, so that serious liver injury is caused. The acute fulminant hepatitis induced by LPS/D-GalN is similar to clinical acute fulminant hepatitis, and has been widely used in the research of fulminant hepatitis mechanism and treatment method.

Once fulminant hepatitis is formed and the condition of the hepatitis rapidly worsens, various complications (hepatic encephalopathy, respiratory cycle failure, renal failure, blood coagulation disorder, infection and gastrointestinal hemorrhage) generated by the fulminant hepatitis are mainly clinically supported and treated at present, but the disease progresses rapidly, so that the conservative treatment means is limited, and the clinical prognosis is extremely poor. Liver transplantation remains the ultimate treatment for severe fulminant hepatitis, but is very difficult to implement because of the limited conditions of transplantation. Therefore, development of new therapeutic methods is urgently required.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a drug for preventing and treating fulminant hepatitis, which comprises NOD1 receptor activator and has protective effect on fulminant hepatitis.

The invention provides the use of an NOD1 receptor activator in the preparation of a formulation for increasing the level of A20.

In the present invention, the increase in a20 level includes an increase in the transcription level of the a20 gene and/or an increase in the a20 protein level.

The invention also provides the use of an NOD1 receptor activator for the preparation of a formulation for reducing the level of cl-caspase 3.

In the invention, the cl-caspase3 is cl-caspase3 in liver tissues of fulminant hepatitis animals; said reducing the level of cl-caspase3 comprises reducing the level of cl-caspase3 protein.

In the invention, the NOD1 receptor activator is C14-Tri-LAN-Gly.

The invention also provides application of the NOD1 receptor activator in preparing a medicament for preventing and/or treating fulminant hepatitis.

In the present invention, the prevention and/or treatment comprises: reducing mortality, inhibiting ALT levels, alleviating pathological changes in liver tissue, and/or reducing apoptosis in liver cells.

In the invention, the NOD1 receptor activator is C14-Tri-LAN-Gly.

In the examples of the present invention, in order to verify the preventive effect of the NOD1 receptor activator on fulminant hepatitis, a model of fulminant hepatitis animals was made after the NOD1 receptor activator was administered. The modeling drugs are LPS and D-GalN. That is, NOD1 receptor activators were administered to the animals prior to modeling with LPS and D-GalN. The results show that NOD1 receptor activator can increase the A20 level of the organism and inhibit the cl-caspase3 level, thereby protecting the liver tissue when fulminant hepatitis occurs. Namely, the invention provides the application of the NOD1 receptor activator in preparing the medicine for protecting the liver tissue of the animals with the explosive liver injury. In one embodiment of the present invention, the prevention of fulminant hepatitis is the prevention of fulminant hepatitis induced by the combination of LPS and D-GalN.

According to the research condition of the invention, the NOD1 receptor agonist can protect liver tissues and reduce the damage of fulminant infection to the liver. Although studies show that the NOD1 receptor agonist can inhibit the action of cl-caspase3 level by increasing A20 level, the actual mechanism of the A-caspase 3 level cannot be clarified, and no evidence exists in previous studies that the NOD1 receptor agonist has the effect of protecting liver. On the other hand, since current experimental data does not determine whether administration of NOD1 receptor agonist after administration of LPS will exacerbate the liver injury condition, the treatment of the present invention does not include treatment of the explosive liver injury induced by LPS, and the mechanism of the same is still under further investigation.

The invention also provides a medicament for preventing and/or treating fulminant hepatitis, which comprises an NOD1 receptor activator.

The invention also provides a medicament for preventing and/or treating fulminant hepatitis, which is used for administering the medicament.

The research of the invention shows that the NOD1 receptor agonist can remarkably up-regulate the expression of liver tissue A20. A20 (also called TNFAIP3) is an anti-inflammatory signaling molecule, and A20 can achieve anti-apoptosis effect by down-regulating NF-kB signaling pathway, thereby protecting liver cells. In addition, the NOD1 receptor agonist can also obviously reduce serum ALT level and cl-caspase3 level of a fulminant hepatitis mouse, relieve liver injury of the fulminant hepatitis mouse and reduce hepatocyte apoptosis. Thus, NOD1 receptor agonists are believed to be useful in the treatment of explosive liver injury.

Drawings

FIG. 1 is a flow chart of an animal model construction method provided in an embodiment of the present invention;

FIG. 2 is serum ALT levels compared to model group for C14-Tri-LAN-Gly stem prognosis;

FIG. 3 is a 36 hour survival curve of C14-Tri-LAN-Gly after stem prediction versus model set;

FIG. 4 liver HE staining of treatment groups;

FIG. 5 Tunnel staining of liver tissue in each treatment group;

FIG. 6 is C14-Tri-LAN-Gly stem prognosis vs. model group for cl-caspase3 and A20 protein expression and A20mRNA expression;

FIG. 7 shows the expression of protein and mRNA levels of A20 in the normal administration group compared with the normal control group.

Detailed Description

The invention provides a medicine for preventing and treating fulminant hepatitis, and a person skilled in the art can use the content of the medicine for reference and appropriately improve the process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

Among them, C14-Tri-LAN-Gly, whose biological component is iE-DAP, is a ligand of intracellular pattern recognition receptor NOD1, purchased from invitrogen, product number tlrl-ctlg, CAS 1863978-69-1.

The invention is further illustrated by the following examples:

examples

Male C57BL/6 mice were purchased from Chongqing university of medicine animal laboratories, and housed between Chongqing university of medicine animal laboratories, SPF-class housings. Male C57BL/6, week-old 6-8 weeks, body weight 18-21g, divided into 4 groups including normal control group, model group, normal administration group and treatment group.

Normal control groups were given equal amounts of PBS in the same injection;

the model group is injected with LPS7.5 mug/kg and D-GalN 500mg/kg in the abdominal cavity;

C14-Tri-LAN-Gly 5 μ g/mouse is injected into the abdominal cavity in the normal administration group;

the treatment group was administered C14-Tri-LAN-Gly 5. mu.g/mouse 6 hours before the administration of the model group (LPS/D-GalN).

Blood and liver tissue were collected 6 hours after LPS/D-GalN injection for subsequent analysis. Another two groups of mice are taken and set as a model group and a treatment group, the state of the mice is observed every 2 hours after the LPS/D-GalN injection, and the survival status of the two groups of mice is observed after 36 hours to prepare a 36-hour mouse survival curve.

1. Histological analysis

Liver tissues were fixed with 4% paraformaldehyde and embedded in paraffin, then the tissues were cut into 5 μm thick slices, one for HE staining, and one for Tunnel staining of the slices according to the Roche apoptosis detection kit POD detection procedure.

2. Serum detection

The blood sample was collected, centrifuged at 4000rpm at 4 ℃ for 10 minutes 1 hour after collection, and the serum was separated. ALT levels in serum were measured according to manufacturer (tokyo founded biol) instructions.

3、western blotting

Grinding the frozen liver tissue, cracking the frozen liver tissue by using RIPA lysate, extracting liver tissue protein, detecting the protein concentration, adding a protein sample buffer in proportion, performing protein gel electrophoresis by using 12% SDS-PAGE after high-temperature denaturation, transferring the protein gel electrophoresis to a PVDF membrane, sealing by using 5% skimmed milk, incubating by using a primary antibody at 4 ℃, incubating by using a secondary antibody after overnight, and finally detecting by using a chemiluminescence apparatus.

4、qPCR

Grinding the frozen liver tissue, extracting RNA by using a Trizol method, measuring the concentration of the RNA, obtaining a cDNA template by using a TAKARA reverse transcription kit according to the steps, finally performing fluorescent quantitative PCR by using a TAKARAqPCR kit to obtain a CT value of a target gene, and calculating the expression multiple of the target gene by using a delta CT method.

Results of the experiment

1. In the model group, serum ALT levels were significantly elevated 6 hours after intraperitoneal injection of LPS/D-GalN in combination (FIG. 2) and significantly reduced after pre-intervention C14-Tri-LAN-Gly (FIG. 2) by serum ALT assay. The 36 hour survival curve shows that both 5. mu.g or 10. mu.g of C14-Tri-LAN-Gly clearly indicate the survival of mice after LPS/D-GalN intervention (FIG. 3).

2. The HE staining of the liver tissue can find that the pathological changes of the liver tissue are obvious after the LPS/D-GalN modeling in hours, including a large amount of extravasated blood, necrosis and inflammatory cell infiltration. Prognosis of HE staining of liver tissue using C14-Tri-LAN-Gly predatory suggests that it significantly reduces liver histopathological changes (fig. 4). Tunnel staining also demonstrated a significant decrease in hepatocyte apoptosis following the use of C14-Tri-LAN-Gly (FIG. 5).

Western blotting results suggested that the protein expression of cl-caspase3 was significantly increased in liver tissue of mice in the treated group compared to mice in the unprotected group (FIG. 6).

The qPCR results showed that the liver tissue A20mRNA expression was significantly increased in mice protected with C14-Tri-LAN-Gly compared to unprotected mice (FIG. 6), and Western blotting likewise suggested an increase in A20 protein levels (FIG. 6). In addition, the levels of A20mRNA and protein in liver tissue of mice were significantly increased by C14-Tri-LAN-Gly (normal group) (FIG. 7). This suggests that A20 plays a key role in the protection of LPS/D-GalN-induced fulminant hepatitis by C14-Tri-LAN-Gly.

In conclusion, the invention firstly proves that C14-Tri-LAN-Gly can inhibit LPS/D-GalN induced liver inflammatory reaction and hepatocyte apoptosis by up-regulating liver A20 expression so as to protect induced fulminant hepatitis.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

Use of a NOD1 receptor activator for the preparation of a formulation for increasing the level of a 20.
2. The use of claim 1, wherein said increasing the level of A20 comprises increasing the transcription level of the A20 gene and/or increasing the A20 protein level.
Use of an NOD1 receptor activator for the preparation of an agent for inhibiting the level of cl-caspase 3.
4. The use of claim 1 wherein said cl-caspase3 is cl-caspase3 in liver tissue of fulminant hepatitides; said inhibiting a level of cl-caspase3 comprises inhibiting a level of cl-caspase3 protein.
5. The use according to any one of claims 1 to 4 wherein the NOD1 receptor activator is C14-Tri-LAN-Gly.
Use of an NOD1 receptor activator for the preparation of a medicament for the prevention and/or treatment of fulminant hepatitis.
7. Use according to claim 6, wherein the prevention and/or treatment comprises: reducing mortality, inhibiting ALT levels, alleviating pathological changes in liver tissue, and/or reducing apoptosis in liver cells.
8. The use of claim 6 or 7 wherein the NOD1 receptor activator is C14-Tri-LAN-Gly.
9. The use according to any one of claims 6 to 7, wherein the fulminant hepatitis is fulminant hepatitis induced by the combination of LPS and D-GalN.
10. A medicament for the prophylaxis and/or treatment of fulminant hepatitis comprising an NOD1 receptor activator.