阅读说明：本技术 烟酸在制备慢性乙型肝炎治疗药物中的用途 (Application of nicotinic acid in preparing medicine for treating chronic hepatitis B ) 是由 唐松青 吴春凤 李雪 陈欣悦 樊苏萍 朱海珍 于 2019-11-29 设计创作，主要内容包括：本发明涉及一种烟酸在制备慢性乙型肝炎治疗和\或辅助治疗药物中的应用。本发明能够显著降低HBV的S抗原、Core蛋白的表达水平,阻止HBV成熟病毒颗粒释放至细胞外,从而使得细胞内HBV的cccDNA、Total DNA和pgRNA等病毒源性的核酸物质发生积累,并促进MAPK信号通路发生活化,增强抗病毒的炎症细胞因子TNF-α产生,最终导致细胞内HBV的S抗原、Core蛋白以及HBV的遗传物质cccDNA发生显著较低,从而达到明显的抗病毒长期慢性感染的作用。(The invention can obviously reduce the expression level of S antigen and Core protein of HBV and prevent mature virus particles of HBV from being released to the outside of cells, thereby accumulating viral nucleic acid substances of cccDNA, Total DNA, pgRNA and the like of HBV in the cells, promoting the activation of MAPK signal channels and enhancing the generation of antiviral inflammatory cytokine TNF- α, finally leading the generation of S antigen, Core protein of HBV in the cells and genetic substance cccDNA of HBV to be obviously lower, and further achieving the obvious effect of resisting long-term chronic infection of virus.)

1. Application of nicotinic acid in preparing medicine for treating and/or adjunctively treating chronic hepatitis B is provided.

2. The use according to claim 1, wherein nicotinic acid inhibits HBV replication.

3. The use of claim 1, wherein niacin inhibits HBV exocytosis.

4. The use of claim 1, wherein niacin promotes the production of the antiviral inflammatory cytokine TNF- α.

5. The use according to claim 1, wherein niacin inhibits S antigen, Core protein of HBV and genetic material cccDNA production of HBV.

6. The use as claimed in claim 1, wherein the nicotinic acid is used as one or only effective component of the chronic hepatitis B treatment and \ or adjuvant treatment medicine.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of nicotinic acid in preparation of a medicine for treating chronic hepatitis B.

Background

Chronic Hepatitis B is a major infectious disease caused by persistent chronic infection with Hepatitis B Virus (HBV). As a hepadnavirus, HBV persisting chronic infection can further promote the conversion of chronic hepatitis B to serious malignant diseases such as cirrhosis and liver cancer. At present, about 2.57 hundred million people worldwide suffer from chronic hepatitis B, and more than one third of the patients come from China. However, the existing medical treatment for chronic hepatitis B can only achieve the curative effect of controlling HBV from the function, but cannot radically and completely cure the chronic hepatitis B patients. One of the main reasons is that viral cccDNA in hepatocytes infected with HBV in patients cannot be completely cleared, resulting in the risk of relapse of patients once they are taken off.

HBV infection can induce acute hepatitis b and chronic hepatitis b. After more than 90% of adults are infected by HBV, acute infection is mainly induced, and HBV viruses in the bodies of patients can be thoroughly eliminated by the immune system of the bodies quickly; after only a small percentage of adults are infected with HBV, chronic infection can develop, causing chronic hepatitis B. On the contrary, more than 95% of neonates and infants are infected by HBV and then cause chronic hepatitis B. Once chronic hepatitis B is formed, the chronic hepatitis B cannot be completely cured at present.

Research shows that in chronic hepatitis B patients, a low-level inflammation microenvironment triggered by HBV can acclimate Kupffer cells colonized by liver to be converted into M2 type macrophages, and further can mediate the formation of an immunosuppression microenvironment and subsequent HBV specific CD⁸⁺Exhaustion of T cells leads to the failure of the immune system to completely eliminate HBV.

At present, interferon and nucleoside analogues are mainly used for clinically treating chronic hepatitis B patients, but the treatment has the problems of drug resistance, large side effect and the like, and cannot achieve the ideal treatment target of thoroughly eliminating the viral cccDNA, and once the drug is stopped, the patients have the risk of reoccurrence, so that a new treatment drug is explored, and the application value is very important.

Nicotinic acid, also known as nicotinic acid, belongs to vitamin B3 and is one of 13 vitamins essential to human body. The nicotinic acid content in human blood is about 0.1-0.3 mg/dl, and the nicotinic acid content in liver tissue can reach 15mg/100g liver tissue. The niacin in food is mainly transported to the liver for utilization and metabolism after being digested and absorbed. The niacin content in meat foods is very high, while in vegetables and fruits is very low, especially in breast milk, which is only about 0.15 mg/dl. For infants, especially for newborn infants, breast milk is the only food source for taking niacin, and the low content of niacin in breast milk leads to insufficient intake of niacin for infants, which causes deficiency of niacin in liver tissue cells of infants (adults can meet the requirement of niacin in vivo through the intake of carnivorous food). The phenomenon of nicotinic acid deficiency in liver tissue cells of infants is closely related to that more than 95 percent of infants infected by HBV are easy to convert into chronic hepatitis B. However, it is still unclear whether or not nicotinic acid can be used as an effective pharmaceutical ingredient for treating chronic hepatitis B.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of nicotinic acid in preparing the chronic hepatitis B treatment medicine.

In order to solve the problems, the technical scheme of the invention is as follows:

the invention discloses application of nicotinic acid in preparing medicaments for treating and/or assisting in treating chronic hepatitis B.

On the basis of the technical scheme, the invention can be further improved as follows.

Furthermore, the nicotinic acid is used as an inhibitor for inhibiting HBV replication in the preparation of medicaments for treating and/or assisting chronic hepatitis B.

Furthermore, the nicotinic acid is used as an inhibitor for inhibiting the release of HBV extracellular cells in the preparation of medicaments for treating and/or assisting in treating chronic hepatitis B.

Furthermore, the nicotinic acid is used as an accelerant for promoting the generation of an antiviral inflammatory cytokine TNF- α in the preparation of medicaments for treating and/or assisting in treating chronic hepatitis B.

Further, the nicotinic acid is used as an inhibitor for inhibiting the generation of an S antigen, a Core protein and genetic substance cccDNA of HBV in the preparation of medicaments for treating chronic hepatitis B and/or adjuvant treatment.

Furthermore, the nicotinic acid is used as one of the effective components or the only effective component of the chronic hepatitis B treatment and \ or adjuvant treatment medicine.

Advantages of the invention over other methods may be realized in one or more of the following aspects:

1. preferably, the nicotinic acid can inhibit the release of HBV mature virus particles to the outside of cells;

2. preferably, nicotinic acid enhances the activation of MAPK signaling pathways in HBV persistently infected cells;

3. preferably, the nicotinic acid can promote the generation of an inflammatory cytokine TNF- α with antiviral effect in HBV continuously infected cells;

4. preferably, the nicotinic acid can obviously reduce the S antigen, the Core protein and the genetic substance cccDNA of HBV in HBV continuous infected cells, and is expected to radically cure chronic hepatitis B.

The invention has the beneficial effects that the nicotinic acid is firstly clarified to act on the hepatic cells continuously and chronically infected by HBV, the generation of S antigen and Core protein of HBV virus can be inhibited, and HBV mature virus particles are prevented from being released to the outside of the cells, so that viral nucleic acid substances such as cccDNA, Total DNA and pgRNA of HBV in the cells are accumulated, MAPK signal channels are promoted to be activated, the generation of antiviral inflammatory cytokine TNF- α is enhanced, and finally the S antigen, the Core protein and genetic substance cccDNA of HBV in the cells are remarkably reduced, so that the obvious antiviral effect is achieved.

Drawings

FIG. 1A is a diagram for quantitative PCR detection of the relative expression amount of cccDNA of HBV in HLCZ01(HLCZ01-HBV) cells persistently infected with HBV;

FIG. 1B is a diagram showing the detection of the relative expression amount of HBV pgRNA in HLCZ01-HBV cells by quantitative PCR;

FIG. 1C is a diagram showing the quantitative PCR detection of the relative expression amount of total HBV DNA in HLCZ01-HBV cells;

FIG. 1D is a diagram showing the quantitative PCR detection of the HBV DNA content in the supernatant of HLCZ01-HBV cell culture;

FIG. 1E shows the detection of the HBV S antigen content in the supernatant of HLCZ01-HBV cell culture by ELISA;

FIG. 1F shows the detection of the expression levels of S antigen and Core protein of HLCZ01-HBV in HBV cells for Western Blot;

FIG. 1G is a diagram for quantitative PCR detection of the relative expression level of cccDNA of HBV in HepG2.2.15 cells;

FIG. 1H is a diagram showing the quantitative PCR detection of the pgRNA relative expression level of HBV in HepG2.2.15 cells;

FIG. 1I is a diagram for quantitative PCR detection of the relative expression level of HBV total DNA in HepG2.2.15 cells;

FIG. 1J shows the quantitative PCR detection of HBV DNA content in HepG2.2.15 cell culture supernatant;

FIG. 1K is a graph showing the detection of HBV S antigen content in HepG2.2.15 cell culture supernatant by ELISA;

FIG. 2A is a diagram showing the detection of relative expression of HLCZ01-HBV intracellular TNF- α by quantitative PCR;

FIG. 2B is a graph showing the relative expression of TNF- α in HepG2.2.15 cells by quantitative PCR;

FIG. 2C shows the measurement of TNF- α content in the supernatant of HLCZ01-HBV cell culture by ELISA;

FIG. 2D shows the ELISA assay for TNF- α in HepG2.2.15 cell culture supernatant;

FIG. 3A is a graph showing the detection of phosphorylation levels of HLCZ01-HBV intracellular P38, ERK1/2 and the total protein level thereof for Western Blot;

FIG. 3B shows the detection of phosphorylation levels of P38 and ERK1/2 in HepG2.2.15 cells and the total protein level thereof by Western Blot;

FIG. 4A is a graph showing the relative expression of cccDNA of HLCZ01-HBV in cells treated with nicotinic acid for different time periods in quantitative PCR assay;

FIG. 4B is a graph showing the relative expression of HBV pgRNA in HLCZ01-HBV cells treated with nicotinic acid for different periods of time for quantitative PCR assay;

FIG. 4C is a graph showing the relative expression level of total HBV DNA in HLCZ01-HBV cells treated with nicotinic acid for different periods of time in quantitative PCR assay;

FIG. 4D is a Western Blot to detect the expression of S antigen, Core protein in HLCZ01-HBV cells treated with nicotinic acid for different times.

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

The invention designs a technical scheme of application of nicotinic acid in preparation of medicaments for treating and/or assisting in treatment of chronic hepatitis B.

Nicotinic acid, also known as nicotinic acid, anti-pellagra factor, molecular formula: c₆H₅NO₂The chemical name 3-picolinic acid belongs to vitamin B3.

Niacin is found primarily in animal gut and muscle tissue, and is present in lower amounts in vegetables, fruits, and breast milk. Nicotinic acid is part of the daily diet and has no obvious toxic and side effects.

The inventor finds that the high and low content of the nicotinic acid in the food is closely related to the occurrence of chronic hepatitis B. For example, developed countries have a higher niacin content in their diet than developing countries, where the incidence of chronic hepatitis b is lower than in developing countries; the nicotinic acid content in most provinces in the north of China is higher than that in the south, and the incidence rate of chronic hepatitis B is lower in the north of China than in the south of China; the nicotinic acid content in food of adults is obviously higher than that of infants, and the incidence rate of chronic hepatitis B is obviously lower in the adults than that of the infants.

Furthermore, the nicotinic acid is used as an inhibitor for inhibiting HBV replication in the preparation of medicaments for treating and/or assisting chronic hepatitis B.

Furthermore, the nicotinic acid is used as an inhibitor for inhibiting the release of HBV extracellular cells in the preparation of medicaments for treating and/or assisting in treating chronic hepatitis B.

Furthermore, the nicotinic acid is used as an accelerant for promoting the generation of an antiviral inflammatory cytokine TNF- α in the preparation of medicaments for treating and/or assisting in treating chronic hepatitis B.

Further, the nicotinic acid is used as an inhibitor for inhibiting the generation of an S antigen, a Core protein and genetic substance cccDNA of HBV in the preparation of medicaments for treating chronic hepatitis B and/or adjuvant treatment.

Furthermore, the nicotinic acid is used as one of the effective components or the only effective component of the chronic hepatitis B treatment and \ or adjuvant treatment medicine.

The specific embodiment of the invention is as follows:

the invention designs the application of nicotinic acid in preparing a medicament for treating chronic hepatitis B, which inhibits S antigen and/or Core protein of HBV and genetic material cccDNA of HBV.

Furthermore, the usage amount of the nicotinic acid in the medicine is 5-10 mM/L.

The inventor designs an experimental method for researching the influence of nicotinic acid on HBV virus:

reagent: nicotinic acid was purchased from sigma-Aldrich, antibody was purchased from Cell Signaling Technology (CST), viral DNA extraction kit was purchased from tiangen biochemistry (beijing) limited, RNA extraction kit and reverse transcription kit were purchased from nanjing novispan biotechnology limited, fluorescent quantitative PCR reagent was purchased from roche biotechnology limited, ELISA kit was purchased from Biolegend.

HLCZ01 cells and HepG2.2.15 cells infected with HBV for a long time are derived from cells of liquid nitrogen frozen stock of Hunan university and are cultured in DMEM cell culture medium.

The quantitative PCR primer sequences used were as follows:

GAPDH：5’-GCACCGTCAAGGCTGAGAAC-3’

5’-TGGTGAAGACGCCAGTGGA-3’

HBV DNA：5’-CACCTCTGCCTAATCATC-3’

5’-GGAAAGAAGTCAGAAGGCAA-3’

cccDNA：5’-GTGCCTTCTCATCTGCCGG-3’

5’-GGAAAGAAGTCAGAAGGCAA-3’

pgRNA：5’-CTCAATCTCGGGAATCTCAATGT-3’

5’-TGGATAAAACCTAGCAGGCATAAT-3’

TNF-α：5’-CCTCTCTCTAATCAGCCCTCTG-3’

5’-GAGGACCTGGGAGTAGATGAG-3’

HBV absolute quantitative primer: 5'-GAGTGTGGATTCGCACTCC-3'

5’-GAGGCGAGGGAGTTCTTCT-3’

ELISA was performed according to the instructions of the TNF- α detection kit from Biolegend.

HLCZ01 cells and HepG2.2.15 cells infected with HBV for a long time are selected. HLCZ01 cells and HepG2.2.15 cells are respectively treated by nicotinic acid with different concentrations, and cccDNA, Total DNA and pgRNA of HBV inside and outside cells, and the expression conditions of S antigen and Core protein of HBV are respectively detected by quantitative PCR, ELISA and Western Blot methods. The specific experimental results are shown in FIG. 1.

The experimental results show that in HLCZ01 cells and HepG2.2.15 cells infected by HBV for a long time, cccDNA, Total DNA and pgRNA of the HBV are increased to a certain extent when the niacin concentration is increased for three days, but S antigen, Core protein and S antigen of extracellular HBV of intracellular HBV are reduced to a certain extent, and the DNA level of the extracellular HBV is also reduced to a certain extent.

Furthermore, the inventor respectively treats HLCZ01 cells and HepG2.2.15 cells with nicotinic acid at different concentrations in HLCZ01 cells and HepG2.2.15 cells infected with HBV for a long time, and respectively detects the production of TNF- α by quantitative PCR and ELISA methods, and the experimental results are shown in FIG. 2.

The experimental result shows that in HLCZ01 cells and HepG2.2.15 cells infected by HBV for a long time, the inflammatory cytokine TNF- α triggered by the HBV is obviously increased in the transcription level and the protein level along with the increase of the concentration of the nicotinic acid.

Through the experimental study, the inventor finds that the nicotinic acid can improve the generation of inflammatory cytokine TNF- α induced by HBV, thereby achieving obvious antiviral effect.

To further analyze the reason why nicotinic acid enhances the production of TNF- α triggered by HBV, we analyzed the activation of ERK1/2 and P38 in MAPK signaling pathway by treating HLCZ01 cells and HepG2.2.15 cells with nicotinic acid at different concentrations and then detecting the activation of ERK1/2 and P38 by Western Blot in HLCZ01 cells and HepG2.2.15 cells infected with HBV for a long time period, and the results are shown in FIG. 3.

The experimental results show that the phosphorylation levels of ERK1/2 and P38 are increased to some extent in HLCZ01 cells and HepG2.2.15 cells infected with HBV for a long time along with the increase of the concentration of nicotinic acid.

In order to analyze the effect of long-term effect of nicotinic acid on anti-HBV infection, the cells were treated with nicotinic acid at final concentrations of 5mM/L and 10mM/L, respectively, in HLCZ01 cells infected with HBV for a long time, and then the expression of HBV S antigen and Core protein in the cells was detected by Western Blot method. The results of the experiment are shown in FIG. 4.

The experimental result shows that the long-term treatment of nicotinic acid can obviously reduce the expression levels of the S antigen and the Core protein of HBV in HLCZ01 cells infected by HBV for a long time.

According to the experimental method and experimental research designed by the inventor, the inventor determines that the nicotinic acid can be applied to the preparation of the chronic hepatitis B treatment drug for inhibiting the S antigen and/or the Core protein of HBV and the CCcDNA of HBV genetic material, can obviously reduce the expression level of the S antigen and the Core protein of HBV and prevent HBV mature virus particles from being released to the outside of cells, thereby accumulating viral nucleic acid substances such as CCcDNA, Total DNA and pgRNA of HBV in the cells, promoting the activation of MAPK signal channels and enhancing the generation of antiviral inflammatory cytokine TNF- α, and finally leading the S antigen, the Core protein and the HBV genetic material of the cells to be obviously lower, thereby achieving the obvious antiviral effect.

The invention discovers that the nicotinic acid can be used for treating chronic hepatitis B for the first time. According to the age, sex, diet and degree of illness of patients with chronic hepatitis B and HBV load in vivo, the person skilled in the art can adjust the dosage and duration of administration of nicotinic acid and carry out chemical modification on nicotinic acid according to his general knowledge and experience in the field, and all of them are included in the scope of the present invention. For example, for chronic hepatitis B patients, the dosage range of the medicine suitable for human is 1-1000 mg per person per day, and the medicine is taken for more than two weeks to reach the optimal treatment time and the like.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.