CN101642454A - Application of berberine in medicament for treating hepatitis B virus infection - Google Patents
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Abstract
The invention discloses the application of berberine in medicament for treating hepatitis B virus infection. In the invention, adopting an HepG2.2.15 cell model for the in vitro selection of anti-hepatitis B virus medicine, preliminarily selecting the effect of the medicament by measuring the difference of HBeAg and HBsAg expression volumn in supernatant fluid of the cell after administration, andfurther determining the effectiveness of the medicine by studying the inhibitory effect of the HBV DNA reproduction level (ccc DNA) in the HepG2.2.15 cell by the medicament. Meanwhile, the influenceon the reproduction of the hepatitis B virus by the transcriptional level of the medicine is studied according to the activity inhibitory effect to HBV viral promoters by the medicine, and key HBV cis-regulatory elements sensitive to the antivirus medicine are found. A pivotal cellular signal transduction pathway possibly participating the transcription reproduction for inhibiting viruses by the medicine is analyzed and verified on the basis, the antivirus molecular mechanism of the medicament is further studied, and the medicament is indicated to be developed and applied to clinical treatmentas an anti-hepatitis B effective medicament.
Description
Technical field
The present invention relates to medical technical field, be specifically related to the application of a kind of berberine in preparation treatment or prevention hepatitis B virus infection medicine.
Background technology
HBV is the prototype virus of Hepadnaviridae (Hepadanaviridae).The HBV genome is an incomplete double-stranded circular DNA that is about 3200 pairs of bases (kb).It has following characteristics: 1. each ORF district is the virus antigen albumen coded sequence in the HBV DNA minus strand, cis-regulating element in the genome all is in each coding region, comprised 4 promoteres and 2 enhancers, these 4 promoteres respectively to genome C and PreC, L albumen, M albumen, S albumen, and the mRNA of X protein play regulating and controlling effect.Envelope protein S be the S gene code albumen (hbs antigen, HBsAg).It is receptor related that HBsAg and virus are adsorbed onto surface of hepatocytes, can change the host range of HBV by genetic recombination.HBsAg content in serum is the highest, can stimulate body to produce neutralizing antibody.Viral core protein PreC albumen and e antigen HBeAg are two kinds of albumen of HBV core gene coding.Do not infect essential albumen though HBeAg is not HBV, the collaborative HBx albumen of result of study explanation HBc albumen has in recent years also play a part certain in HCC develops.P albumen is a basic protein that contains a plurality of functional areas---class DNA polymerase protein, also is a kind of important structure albumen of HBV.The viral DNA polymerase is endogenic, can repair short chain and breach in the viral genome, forms the DNA of complete two strands.Hbv replication needs the reverse transcription process, and its DNA polymerase has the reverse transcription activity.HBx albumen is the minimum frame coding of reading of mammal hepadnavirus HBV genome, forms by 145～154 aminoacid, and be that necessary albumen is infected in virus foundation.Also there is experimental evidence proof HBx albumen to participate in the HBV virus replication.2. the HBV genome structure is tight, and is overlapped between encoding gene, wherein the P gene fully and S and PreS gene overlap, X gene is overlapped with it, makes the HBV genome can make full use of its limited length, becomes one of the highest virus of code efficiency.There is polymorphism in HBV genome between 3. different strains, is the higher DNA viruses of variation frequency.Copy feature through replicative intermediate RNA reverse transcription is the Basic of Biology of hepadnavirus behavior.
The topmost characteristics of hbv replication are will be through a reverse transcription process.(covalently closed circular DNA cccDNA) transcribes formation pregenome RNA (pregenome RNA) to the genomic replication initiation of HBV in covalently closed circular DNA; Synthesize minus-strand dna through the pregenome RNA reverse transcription again, and follow the amplification of cccDNA; Synthetic then positive chain DNA.Newly-generated double-stranded DNA can maturation be cccDNA again, participates in genomic duplicating again.
Because the transcriptive process,reversed that duplicates the archaeal dna polymerase process pregenome RNA that will utilize self of HBV, and viral dna polymerase lacks the check and correction function, as if therefore, the variation frequency of HBV will exceed about 4 orders of magnitude than other DNA viruses, more approach RNA viruses.
Basic research for HBV mainly is to utilize cell culture model or animal infection modal at present.The cell model commonly used of research HBV mainly contains two classes: primary hepatocyte system, Bel7402.The HepG2.2.1.5 cell strain is most widely used HBV cell model, and this cell strain has been widely used in the in-vitro screening of anti-HBV medicine.
According to statistics, whole world hepatitis B virus infection person accumulative total reaches 2,000,000,000, and 3.5 hundred million hepatitis B virus carrierss are arranged at present approximately, and annual infected number reaches 5,000 ten thousand, dies from the number about 1,000,000 of hepatitis B virus infection relevant disease (hepatic fibrosis or hepatocarcinoma).China's hepatitis B virus carriers is 1.5 hundred million, and chronic hepatitis patient has 3,400 ten thousand, and the number of dying from the hepatitis B virus infection relevant disease every year has 270,000.Although China has implemented hepatitis b precaution vaccination, owing to huge virus carrier, immunologic tolerance, Drug resistance, the infected be lifelong viral communication source, virus by reasons such as mother-to-baby transmissions, the situation is tense to make hepatitis B control.Hepatitis B still is one of main infectious disease of harm China population health in quite long one period from now on.
Therefore find that new anti-hepatic-B virus medicine also is emphasis of research in the current treating hepatitis B.The present invention is based on above-mentioned background, carried out large-scale drug screening, find that finally berberine has the effect that good anti-hepatitis virus duplicates, can be used as a kind of new anti-hepatic-B virus medicine and be applied to clinical.
Rhizoma Coptidis is the dry rhizome of ranunculaceae plant Rhizoma Coptidis Coptis chinensis Franch., Coptis deltoidea C.Y.Cheng et Hsiao Coptis deltoideaC.Y.Cheng et Hsiao or Coptis Teeta Wall Coptis teeta Wall..Has heat clearing and damp drying, effects such as eliminating fire and detoxication.Be used for that damp and hot feeling of fullness, vomiting acid regurgitation, dysentery, jaundice, unconsciousness due to high fever, hyperactivity of heart-fire, dysphoria and insomnia, heat in blood are told nosebleed, conjunctival congestion, have a toothache, quenched one's thirst, the carbuncle furuncle; External treatment eczema, eczema, auditory meatus are suppurated.Berberine is a kind of important alkaloid, is that China uses Chinese medicine for a long time.Can from plants such as Rhizoma Coptidis, Cortex Phellodendri, Radix Berberidis, extract.It has significant bacteriostasis.The bibliographical information alkaloid compound has the effect of resisiting influenza virus in the recent period, berberine is the main effective ingredient of Rhizoma Coptidis, the report that its anti-HBV effect is not also arranged at present, therefore the present invention has found that first berberine has good anti-hepatitis B activity, shows that it has good exploitation prospect.
Summary of the invention
The objective of the invention is to be to provide a kind of berberine in the new purposes for the treatment of or preventing to use in the hepatitis B virus infection medicine as preparation.Estimate in the face of the berberine anti-HBV effect from cell and molecular layer, for its further Application and Development lays a good foundation.
In order to realize above-mentioned task, the present invention by the following technical solutions:
Because berberine has certain infection and inflammatory effect, simultaneously be used for the enteritis disease clinically good application foundation is arranged, and often be attended by inflammatory reaction in the viral caused infectious disease generating process, so this medicine all has alleviation and auxiliary treatment effect preferably to symptoms such as the caused inflammation of virus in the treatment viral disease.
Berberine mainly is to be used for the enteritis disease with tablet at present, and adopts pharmaceutics technology commonly used, berberine can be made thin membrane coated tablet, capsule, granule, dispersant, and is oral.In the medicine of treatment hepatitis B virus infection and complication thereof, can add this medicine composition or in therapeutic process drug combination use this medicine.
Utilizing the cell model HepG2.2.15 cell (buying in Chinese representative microbial preservation center) of the research hbv replication of having generally acknowledged at present is object of study, in conjunction with relevant hepatocyte, as HepG2 cells such as (buying in Chinese representative microbial preservation center), methods such as employing transfection are carried out drug screening and functional evaluation.
1) at first carries out preliminary assessment.Adopt 2 antigens---the Preliminary screening of how much carrying out medicine of HBeAg and HBsAg expression in the ELISA detection hepatitis B virus duplication process;
Table 1 medicine is to HBeAg and HBsAg expression in the HepG2.2.15 cell conditioned medium liquid
The result shows that medicine has obvious inhibitory action to HBeAg and HBsAg expression in the HepG2.2.15 cell conditioned medium liquid, with the cell matched group tangible significant difference (p＜0.05) is arranged relatively.
2) after Preliminary screening, active drug is repeated screening, detect HBeAg and HBsAg expression earlier, adopt of the influence of real-time PCR detection of drugs again virus cccDNA in the hepatitis B virus duplication.
Table 2 berberine is to the influence of hepatitis B virus duplication China virus cccDNA expression
Berberine has the obvious suppression effect to virus titer as can be seen, and drug effect and concentration have dependency simultaneously, show the increase along with drug level, and its inhibitory action to virus titer is obvious more, with matched group significant difference (p＜0.05) is arranged relatively.
3) select the maximum no cytotoxicity dosage of medicine to carry out the discussion of mechanism of drug action:
A) reaffirming of drug effectiveness used the influence of real-time PCR detection of drugs to virus quantity cccDNA in the hepatitis B virus duplication, uses Western Blot to detect duplicate (variation of HBcAg content) of virus simultaneously.
B) investigate the influence of medicine to HBV virus replication promoter, adopt the method for transient transfection (transienttransfection), HBV4 that will have the fluorescence report gene is duplicated promoter (S1P, S2P, CP, HBX) (Wu Jianguo professor seminar of virusology National Key Laboratory makes up, respectively promoter is connected on the PGL-3 carrier that has the GFP reporter gene, these plasmids all are ammonia benzyl resistances, list of references Naazneen Moolla, Michael Kew and Patrick Arbuthnot.Regulatory elements ofhepatitis B virus transcription, Journal of Viral Hepatitis, 2002,9,323-331) and total length promoter FP transfection in the HepG2 cell of monolayer adherence, be replaced with the pastille culture medium behind the transfection 8h, after continuing to cultivate 36h, cell lysis is measured fluorescent value (luciferase activity), carry out statistical analysis, find out medicine the promoter related situation that influences.(Wu Jianguo professor seminar of virusology National Key Laboratory makes up, respectively promoter is connected on the PGL-3 carrier that has the GFP reporter gene, these plasmids all are ammonia benzyl resistances, list of references Naazneen Moolla, Michael Kew and Patrick Arbuthnot.Regulatory elements of hepatitis B virus transcription, Journal of Viral Hepatitis, 2002,9,323-331).
C) medicine is to the influence of coherent signal path in the hbv replication process.
Continue to carry out in a deep going way the study on mechanism of active drug---on the one hand, from the signal path angle carry out that medicine inquires into to the influence of coherent signal path promoter level that the medicine resisting HBV virus duplicates and signal path between dependency.On the other hand, according to the promoter The selection result, seeking influences upstream gene and the albumen that different promoters is expressed.The apparent in view promoter of drug influence is carried out methods such as truncate, design primer, evaluation, clone and make up different big or small promoter fragments, the zone of action that screening of medicaments is possible and site then.As medicine the HBX of hbv replication there is tangible downward modulation inhibitory action, can adopts methods such as RT-PCR, EMSA to continue the research medicines whether to the effect situation that changes regulatory factor HNF HNF-4 α mRNA and protein expression over to of HBX; Use for reference the result of present interferon research comparative maturity in treatment hepatitis B virus duplication process simultaneously, some the downstream albumen regulated from alpha-interferon inhibition virus replication or the angle of gene are started with, investigate of the influence of different active drugs to these protein expression levels, meanwhile, by consulting lot of documents, can start with from the albumen that the HBV virus replication is had clearly effect, adopt the influence of the method preliminary assessment active drug of RT-PCR, adopt of the influence of the method comparative drug of Western Blot then its protein expression difference.
For other present anti-hepatic-B virus medicines, the present invention compared with prior art has the following advantages and effect: 1, this medicine is a kind of pure natural medical, has advantages such as toxic and side effects is little.2, Orally-administrable, easy to use.3, have antiinflammatory action concurrently,, improve therapeutic quality and give drug compliance, alleviate the treatment misery so can improve the toleration in the therapeutic process.4, drug resource is abundant, has reduced the medicine cost, and can according to medicine not the same-action purpose be prepared into the different dosing dosage form.
Description of drawings
Fig. 1 berberine is to the HepG2 cytotoxic effect, and the variable concentrations berberine is to the HepG2 cytotoxicity, and the result shows that the concentration medicine does not have obvious cytotoxicity.
Fig. 2 berberine is to the influence of HBeAg output in the HepG2 2.15 cell conditioned medium liquid, and the result shows that berberine can obviously reduce HBeAg output.
Fig. 3 berberine is to the influence of HBsAg output in the HepG2 2.15 cell conditioned medium liquid, and the result shows that berberine can obviously reduce HBsAg output.
Fig. 4 real-time PCR detects the influence of different pharmaceutical to cccDNA expression in the HepG2 2.15 cell conditioned medium liquid, and the result shows that berberine can obviously reduce cccDNA output.
Fig. 5 A berberine is to the influence of some cell signal paths, and the plasmid of unlike signal path is transferred in the HepG2 cell, and transfection added medicine after 6 hours, drug effect after 48 hours detection of drugs to the influence of these signal paths.
Fig. 5 B berberine is to the influence of some cell signal paths, and the plasmid of unlike signal path is transferred in the HepG2 cell, and transfection added medicine after 6 hours, drug effect after 48 hours detection of drugs to the influence of these signal paths.
Fig. 6 berberine is transferred to the different promoters plasmid in the HepG2 cell HBV promoter influence, and transfection added medicine after 6 hours, drug effect after 48 hours detection of drugs to the influence of these signal paths.
Fig. 7 transfection process sketch map, cell adopt liposome transient transfection procedure chart.
The specific embodiment
Embodiment 1: berberine is to the cytotoxicity of HepG2 cell and HepG2 2.15 cells
With trypsin well-grown HepG2 cell and HepG2 2.15 cells are dispersed into the individual cells suspension, by 1 * 10
5/ ml concentration branch is planted in 96 orifice plates, and every hole 0.1ml puts 37 ℃, 5%CO
2Cultivate 24h in the incubator.After treating that cell grows up to monolayer, abandon the culture fluid supernatant, change the pastille that contains variable concentrations and keep liquid, each concentration is established 3 multiple holes, and establishes the normal cell contrast.Continue to cultivate 48h, abandon culture supernatant, every hole adds the DMEM culture medium 100 μ l that the MTT (tetramethyl azo azoles salt) that contains 100ug/ml does not contain serum, puts CO
2After continuing in the incubator to cultivate 2～3h, abandon the MTT supernatant, PBS washes 3 times, and every hole adds lysate (DMSO: the ethanol volume ratio is 1: 1) 100 μ l, vibrates 5～10 minutes, and the place reads the OD value at microplate reader 570nm wavelength, and calculates cell survival rate according to following formula.
Cell survival rate (%)=(experimental port OD value/matched group OD value) * 100%
The result shows that medicine does not have significant cytotoxicity in the concentration range of 0.32ug/ml-1000ug/ml.Embodiment 2: the external influence to HBsAg and HBeAg in HepG2 2.15 cells of berberine
1) gets bag and reacted bar, be fixed on the plastic frame, add specimen 50 μ l/ holes by micropore, cloudy, property contrast 50 μ l (or 1)/each two hole, hole, stay a hole not add simultaneously and make blank, add immediately and survey HBsAg or HBeAg enzyme conjugates 50 μ l (or)/hole, blank does not add.
2) put 37 ℃ of thermostat water baths, temperature is bathed 30min.
3) carefully remove liquid in the hole, pat dry; With distilled water cleaning mixture is done 1: 20 times of dilution, fill with each hole, discard behind the stop 30s, so cyclic washing is 6 times, all pats dry at every turn.Avoid cross-contamination between the hole as far as possible.
4) add substrate solution 50 μ l (or)/hole, add colour developing liquid 50 μ l (or)/hole immediately, 37 ℃ of colour developing 10min.
5) every hole adds 50 μ l (or) 2MH
2SO
4Stop buffer, cessation reaction.
6) use the blank school of 450nm wavelength zero point immediately, measure the OD value of yin and yang attribute contrast and specimen respectively; When exceptional value occurs, should adopt dual wavelength to measure, reference wavelength is 650nm.
The result by Fig. 2 and Fig. 3 as can be seen berberine to the content of HBeAg secreted in the HepG2.2.15 cell conditioned medium liquid and HBsAg have the obvious suppression effect (
*P＜0.05 versus untreated group).
Embodiment 2: the influence that medicine is expressed HBV cccDNA
The HBV nucleic acid amplification fluorescent quantitative detection kit that adopts the PG of Shenzhen biological engineering limited company to produce is measured the DNA amount of HBV.
1) processing of specimen
The thing processed group of getting it filled and not adding in medicine processed group mice serum 100 μ l to the 0.5ml EP pipes adds 100 μ l DNA extraction liquid 1, the vibration mixing, 13, the centrifugal 10min of 000rpm abandons supernatant, add 25 μ l DNA extraction liquid 2 again, the vibration mixing, 2, the centrifugal 10s of 000rpm, 100 ℃ of water-bath 10min, 13, the centrifugal 10min of 000rpm, supernatant-70 ℃ reservation is standby.
2) preparation before the amplification
A) take out HBV PCR reactant liquor, Taq enzyme and UNG from test kit, room temperature (about 25 ℃, below identical) is melted and the vibration mixing, and 2, the centrifugal 10s of 000rpm.Being formulated as follows of reaction system: PCR reactant liquor 37.6 μ l, Taq enzyme 0.4 μ L, UNG 0.06 μ l calculates the consumption of each reagent well, adds in the proper volume centrifuge tube abundant mixing, 2, the centrifugal 10s of 000rpm.
3) application of sample
A) after sample and positive working standard are put room temperature 30min, 2, the centrifugal 5min of 000rpm.Add specimen, positive control and the critical positive control of handling in the PCR reaction tube respectively.The PCR reaction tube is placed the PCR instrument, the discharging order of record sample.
4) reaction condition
A) amplification program: 37 ℃, 5min, 94 ℃, 1min, 95 ℃ of 5s, 60 ℃, 30s, 50 circulations, reaction system is 40 μ l.Fluorescence signal is collected and is set at the Fam fluorescein.Positive working standard is made as STAN and imports copy number, and specimen to be measured is made as UNKN, the start amplification.
5) interpretation of result
A) get 3-15 circulation fluorescence signal and carry out interpretation of result base line (baseline) as detector.Threshold setting (thresghold) is the peak of threshold line just above critical positive reference substance amplification curve (random noise line), and Ct value=40.0 are as the criterion.
B) detection of drugs in the cell in vitro experimentation to the detection method of HBV DNA in the method for HBV virus replication influence and the similar mice serum of process.
The result shows that berberine all can obviously suppress duplicating of virus under different dosing concentration, and relatively have evident difference (Fig. 4) (p＜0.05) without any the drug treating group.
Embodiment 3: medicine is to the influence of some classical signals paths
1, liposome transfection method:
The DNA that has negative charge combines with the end that cationic compound has positive charge, and DNA conjunction type lipid and cell membrane combine then, make DNA enter cell interior.The liposome transfection method can produce high transfection efficiency to some cell, and used hepatocyte HepG2 in this experiment (buying in Chinese representative microbial preservation center) etc. confirm to adopt liposome transfection better through the long-term transfection experiment of laboratory.Concrete grammar is example with the transfection operation with 24 orifice plates.The transfection overall process must strict sterile working.
1) will treat that transfectional cell is inoculated in 24 orifice plates, 37 ℃ of cultivations the previous day in transfection.Cell density before the transfection is as the criterion with 40-60%.
2) prepare following solution
A) dilution 2-20 μ l Sofast
TMTransfection reagent (available from Shanghai sun horse company) is in the RIPM culture medium of 100 μ l serum-frees, no antibiotics.Room temperature leaves standstill 3min behind the mixing.
B) dilution 1-2 μ g treats the RIPM culture medium of transfection plasmid DNA in 100 μ l serum-frees, no antibiotics.Room temperature leaves standstill behind the mixing.
3) mix a), b) two kinds of solution, room temperature leaves standstill 20min.And then add the DMEM culture medium of 800 μ l serum-frees, no antibiotics, vibration mixing (solution may be vaporific muddiness, but does not influence transfection).
4) discard old culture medium in the culture plate, with the DMEM culture medium cleaning cell of 2ml serum-free, no antibiotics 2 times.
5) add above Sofast
TMTransfection reagent ,-DNA mixture 1ml covers cell.
6) 37 ℃ of incubation 6h.
7) add normal growth medium, 37 ℃ of cultivations.
8) detect behind the 48-72h.
2, the mensuration of uciferase activity:
1) cell lysis: add the reporter gene cell pyrolysis liquid, fully cell lysis.For can directly adding lysate behind the attached cell exhaustion cell culture fluid; For suspension cell, centrifugal going adds lysate behind the supernatant again.Can measure luciferase immediately after the lysis, also can be frozen earlier, measure again after treating.Frozen sample need melt, and just can begin to measure after reaching room temperature;
2) melt the luciferase detectable, and reach room temperature;
3) press the instrumentation description and open fluor tester, measuring interval is made as 2s, minute is made as 10s;
4) sample thief 30-100 μ l (if sample size is enough, please add 100 μ l) during each sample determination, luciferase detectable 100 μ l mix the back and measure.With the reporter gene cell pyrolysis liquid is blank.
The results are shown in Figure 5A and Fig. 5 B, berberine has tangible rise effect to AP-1, and have tangible downward modulation effect (not compare with there being the drug treating group to CHOP, p＜0.05), showing that berberine suppresses duplicating of hepatitis B virus may to influence these two cell signal paths relevant with it, during related mechanism is further being studied.
Embodiment 4: berberine is to the influence of HBV promoter
Adopt above-mentioned liposome transfection method, (Wu Jianguo professor seminar of virusology National Key Laboratory makes up the HBV promoter plasmid that this laboratory is made up, respectively promoter is connected on the PGL-3 carrier that has the GFP reporter gene, these plasmids all are ammonia benzyl resistances, list of references Naazneen Moolla, MichaelKew and Patrick Arbuthnot.Regulatory elements of hepatitis B virus transcription, Journal of Viral Hepatitis, 2002,9,323-331) be transferred in the HepG2 cell, add medicine then, detection of drugs is to the influence of promoter expression behind 48h.
Berberine has obvious suppression downward modulation effect to duplicating promoter S1P as seen from Figure 6, shows that berberine may suppress duplicating of HBV virus by the expression that suppresses S1P.
Comprehensive above-mentioned cell model evaluation and part molecular biology experiment result show that berberine can obviously suppress the replication in vitro of HBV virus, reduces viral generation.And its influence to virus replication may be to work by the inhibition of duplicating promoter S1P.From the angle of medicine pair cell signal path influence, berberine suppresses duplicating of hepatitis B virus and may upward be in harmonious proportion relevant to CHOP downward modulation effect to AP-1 with it.Dependent interaction mechanism remains further to be furtherd investigate.
Claims (1)
1, the application of berberine in preparation treatment hepatitis B virus infection medicine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108136009A (en) * | 2015-07-24 | 2018-06-08 | 财团法人牧岩生命科学研究所 | For the pharmaceutical composition for preventing the cccDNA of hepatitis type B virus from being formed |
| CN109507430A (en) * | 2018-09-06 | 2019-03-22 | 重庆医科大学 | The function and purposes of SIRT7 |
| CN109971887A (en) * | 2018-12-21 | 2019-07-05 | 大理大学 | Ganglong capsule regulates and controls the research method that JAK-STAT transduction inhibits HBV |
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2009
- 2009-07-14 CN CN200910063160A patent/CN101642454A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108136009A (en) * | 2015-07-24 | 2018-06-08 | 财团法人牧岩生命科学研究所 | For the pharmaceutical composition for preventing the cccDNA of hepatitis type B virus from being formed |
| CN109507430A (en) * | 2018-09-06 | 2019-03-22 | 重庆医科大学 | The function and purposes of SIRT7 |
| CN109507430B (en) * | 2018-09-06 | 2022-03-11 | 重庆医科大学 | Function and application of SIRT7 |
| CN109971887A (en) * | 2018-12-21 | 2019-07-05 | 大理大学 | Ganglong capsule regulates and controls the research method that JAK-STAT transduction inhibits HBV |
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Application publication date: 20100210 |