Application of neferine in preparation of anti-hepatitis B virus drug

A technology of hepatitis B virus and liensinine, which is applied in the field of medicine and biology, to achieve remarkable drug effects, easy promotion, and easy results

Pending Publication Date: 2022-07-29
康珞生物科技(武汉)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, whether it can inhibit the hepatitis B virus has been unknown

Method used

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  • Application of neferine in preparation of anti-hepatitis B virus drug
  • Application of neferine in preparation of anti-hepatitis B virus drug
  • Application of neferine in preparation of anti-hepatitis B virus drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0024] The changes of HBsAg and HBeAg levels in the supernatant of the cells treated with lysinine were detected by enzyme-linked immunosorbent assay (ELISA)

[0025] Step 1. Inoculate liver cancer cells in six-well plates, and start adding drug stimulation 1 day after HepG2.2.15 inoculation, or 1 day after Huh7 transfection with 1.1 / 1.3 x HBV plasmid. Add 0.5 μM, 1.5 μM, 2.0 μM and 2.5 μM to the medium in turn, and add 30 nM ETV to the medium of the positive control. The treatment time is 3 days, and the medium is changed every day. 20°C for use.

[0026] Among them, the 1.1 x HBV plasmid used is pCH9-3091, which contains 1.1 times the HBV genome of the adw genotype, and its upstream is promoted by a human cytomegalovirus (HCMV) type 1 early-mid protein (IE1) promoter, which is a strong promoter After the plasmid is transfected into mammalian eukaryotic cells, the HBV RNA transcript can be transcribed with high efficiency. This plasmid was a gift from Dr. Michael Nassal, Un...

Embodiment approach 2

[0040] Detection of intracellular viral mRNA levels by Northern blotting

[0041] Step 1. Wash the cells of each group used to detect HBsAg and HBeAg in the supernatant three times with PBS, then add 1 mL of Trizol solution to each well of the six-well plate, digest at room temperature for 5 minutes, and shake Trizol together with the cells. Transfer to a new centrifuge tube and pipette repeatedly until no obvious precipitation occurs.

[0042] Step 2. Add 1 / 5 volume of chloroform, shake vigorously until the solution becomes milky white, and let stand for 5 minutes at room temperature.

[0043] Step 3. Centrifuge at 12000g for 15min at 4°C. After centrifugation, the mixture is divided into 3 layers: the colorless supernatant containing RNA, the genomic DNA in the middle, and the colored lower organic phase. Transfer the supernatant carefully to another. in a new centrifuge tube.

[0044] Step 4. Add an equal volume of isopropanol, mix well, and let stand at room temperature ...

Embodiment approach 3

[0062] Detection of viral DNA levels in intracellular HBV nucleocapsids by Southern blotting

[0063] Step 1. Inoculate HepG2.2.15 in a 10cm petri dish, and inoculate Huh7 in a 6cm petri dish. After 1 day of HepG2.2.15 inoculation, or 1 day after Huh7 was transfected with 1.1 / 1.3x HBV plasmid, drug stimulation was started. Add 0.5 μM, 1.5 μM, 2.0 μM and 2.5 μM lysinine, and 30 nM ETV as a positive control to the culture medium, the treatment time is 3 days, the medium is changed every day, and the cells are cryopreserved at -80 °C after 3 days. use;

[0064] Step 2. Add 2.5 μM lysinine and 30 nM ETV as a positive control to the culture medium of the cells with the same conditions in Step 1 for 4 days, change the medium every day and collect the cells from the first day to the fourth day of treatment. Frozen at -80℃ for later use;

[0065] Step 3. Add cell lysate to each sample, add 1mL of lysate to cells in a 10cm dish, add 500μL of lysate to a 60mm dish, shake for 30min at ...

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Abstract

According to the application of the neferine in preparation of the anti-hepatitis B virus medicine, the neferine targets hepatitis B virus RNA and interferes with synthesis of the hepatitis B virus RNA, so that transcription of pgRNA, preS / S RNA and X RNA of the virus is reduced, reverse transcription synthesis of cDNA in a virus nucleocapsid in a cell and synthesis of virus protein are reduced, and therefore, the anti-hepatitis B virus medicine is obtained. Therefore, the synthesis and secretion of antigens HBsAg and HBeAg are reduced. According to the application disclosed by the invention, an in-vitro cell model is applied, the neferine is found to have an inhibiting effect on the HBV for the first time, and a new clue is provided for subsequent in-depth research and clinical medication of resisting the HBV.

Description

technical field [0001] The invention relates to the technical field of medicine and biology, and in particular relates to the application of lichenine in the preparation of anti-hepatitis B virus medicines. Background technique [0002] Hepatitis B Virus (HBV) infection and its resulting chronic hepatitis B constitute a worldwide public health threat. 30% of the world's people, that is, about 2 billion people, have been infected with HBV, and more than 350 million, these hepatitis B patients are also more likely to further develop liver cirrhosis and even liver cancer, and about 800,000 people worldwide die from it every year. China is a high incidence area of ​​hepatitis B virus infection. About 600 million people have been infected with hepatitis B virus. There are about 86 million virus carriers and more than 28 million chronic hepatitis B patients, the number ranking first in the world. In order to control the development of hepatitis B epidemic, the current focus is on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/4725A61P31/20
CPCA61K31/4725A61P31/20
Inventor 胡康洪
Owner 康珞生物科技(武汉)有限公司
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