CN115414370A - Application of sodium taurocholate in preparing medicine for treating or preventing influenza virus infection - Google Patents
Application of sodium taurocholate in preparing medicine for treating or preventing influenza virus infection Download PDFInfo
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- CN115414370A CN115414370A CN202211262150.0A CN202211262150A CN115414370A CN 115414370 A CN115414370 A CN 115414370A CN 202211262150 A CN202211262150 A CN 202211262150A CN 115414370 A CN115414370 A CN 115414370A
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- influenza virus
- sodium taurocholate
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
Abstract
The invention belongs to the field of biological medicines, and particularly discloses application of sodium taurocholate in preparation of a medicine for treating or preventing influenza virus infection. The applicant finds that sodium taurocholate can improve the weight reduction of mice caused by the infection of influenza viruses, obviously inhibit the proliferation of the influenza viruses in the mice, improve the lung tissue lesion caused by the infection of the influenza viruses and has an obvious effect of resisting the infection of the influenza viruses. At the same time, sodium taurocholate can inhibit the proliferation of a plurality of influenza A viruses, including human influenza virus H 1 N 1 (PR 8), avian influenza Virus H 3 N 2 Avian influenza virus H 9 N 2 The sodium taurocholate has broad spectrum, and the sodium taurocholate has simple synthesis process, is economic and rapid, is easy for large-scale production, and can be developed into anti-influenza virus medicaments.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of sodium taurocholate in preparation of a medicine for treating or preventing influenza virus infection.
Background
Influenza virus (influenza virus) is called influenza virus for short, and includes human influenza virus and animal influenza virus, and can cause infection and disease of various animals such as human, fowl, pig, horse and bat, etc., and the human influenza virus is divided into four types of A, B, C and D. Among them, influenza a viruses spread rapidly, easily from person to person, and even across species. The prevention and treatment of influenza is still difficult to date due to frequent gene recombination and site mutation among influenza viruses. The antiviral drug can directly act on the virus to make the virus lose the capability of infecting a host; it may also act on the receptor protein on the surface of host cell to prevent virus from adsorbing to the cell surface and making it unable to enter into the cell; it may also act on specific target in host cell to inhibit the release and replication of virus after entering host cell, so as to reach antiviral effect. Because different viruses infect hosts in different ways, antiviral drugs are selected differently for different viruses.
There are two main classes of anti-influenza drugs currently used clinically: neuraminidase inhibitors (NAIs), which are analogs of sialic acid, inhibit the release of newly synthesized influenza virions by binding to NA protein, including oseltamivir (trade name tamiflu) and zanamivir, ponamivir; m2 ion channel inhibitors, which block the viral M2 ion channel and thereby prevent the influenza virus de-shelling process, include amantadine and rimantadine. The medicine can be used for treating early stage influenzaThe drug has the disadvantages of controlling influenza, and the M2 ion channel inhibitor not only has the possible side effects of neurotoxicity, respiratory failure induction and the like, but also has the drug resistance to the current epidemic influenza virus strain. Several resistant strains have been shown to develop worldwide during the last two decades, such as resistant influenza A virus (H) 3 N 2 ) Seasonal A influenza virus (H) resistant to oseltamivir 1 N 1 ) And amantadine-resistant influenza A virus (H) 3 N 2 ) Influenza A virus (H) resistant to amantadine 1 N 1 ). Therefore, the prevention and treatment of influenza virus still faces huge challenges, and the development of novel anti-influenza drugs is imminent.
Sodium taurocholate (Sodium taurocholate hydrate) is a conjugated bile acid Sodium salt formed by linking the carboxyl group of cholic acid (cholic acid,3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oic acid) and the amino group of taurine (taurine) through an amide bond, is a main component used by bile drugs (including animal gallstones) (1991), widely exists in the bile of vertebrates such as cattle, sheep, snakes and the like, can participate in fat emulsification and dissolve and absorb fat. Sodium taurocholate has a high degree of hydrophilicity and when used in combination with other large drugs, it has the effect of promoting absorption of the drug through the intestinal tract, and studies by Khatun Z et al (2013) have found that taurocholic acid enhances the penetration of heparin-docetaxel conjugates in the lower intestine by extracellular and/or paracellular pathways. In addition, yan et al (2014) research finds that bile acid or salt thereof and derivatives thereof can inhibit the transportation of Hepatitis B Virus (HBV), hepatitis c virus (HDV) and sodium taurocholate cotransporter polypeptide (NTCP), and the NTCP is not only a bile salt transporter for transporting bile salt from portal blood to hepatocytes, but also a functional receptor for infecting hosts with HBV and HDV in vivo and is very important for virus invasion. When NTCP is combined with PRE-S1 lipopeptide on HBV, it can block NTCP from taking up bile salt substrate of taurocholate, etc. so as to inhibit infection of HBV and HDV, and it shows that the bile acid and its derivative mainly depend on NTCP to block infection of HBV and HDV, and because of the existence of said special functional receptor, partial bile salt can play the role of inhibiting hepatitis B virus and hepatitis C virus, but the influenza virus has no such receptor.
At present, the research on sodium taurocholate is limited to the participation in fat emulsification in vivo and the promotion of the absorption of macromolecular drugs in vivo and the application to Na + The function of taurocholic acid cotransporter (NTCP) entering into the physiological transport matrix of liver microtubules and resisting influenza virus is not reported.
The invention content is as follows:
the invention aims to provide application of sodium taurocholate in preparing a medicament for treating or preventing influenza virus infection, wherein the structural formula of the sodium taurocholate is as follows:
in order to achieve the purpose, the invention adopts the following technical measures:
the application of the sodium taurocholate in preparing the medicine for treating or preventing the infection of the influenza virus comprises that the sodium taurocholate is used as the only effective component or one of the effective components to prepare the medicine for inhibiting the proliferation of the influenza virus; or sodium taurocholate is used as the only effective component or one of the effective components to prepare the medicine for relieving the symptoms after the infection of the influenza virus; or sodium taurocholate is used as the only effective component or one of the effective components to prepare the medicine for treating or preventing the infection of the influenza virus;
in the above application, the symptoms after influenza virus infection include: lung tissue damage, weight loss.
In the above applications, the virus includes human influenza virus H 1 N 1 (PR 8), avian influenza Virus H 3 N 2 Avian influenza virus H 9 N 2 。
Compared with the prior art, the invention has the following advantages:
(1) The sodium taurocholate can obviously inhibit the proliferation of influenza viruses.
(2) The sodium taurocholate can obviously inhibit the expression level of influenza virus mRNA.
(3) The sodium taurocholate has low cytotoxicity and can inhibit the proliferation of influenza viruses in a dose-dependent manner.
(4) The sodium taurocholate can improve weight loss caused by influenza virus infection, increase the survival rate of mice, and has an obvious effect of resisting influenza virus infection.
(5) The sodium taurocholate in the invention can improve lung tissue lesion caused by influenza virus infection.
(6) The sodium taurocholate can inhibit the proliferation of a plurality of influenza A viruses including human influenza virus H 1 N 1 (PR 8), avian influenza Virus H 3 N 2 Avian influenza virus H 9 N 2 The sodium taurocholate has broad spectrum, and the sodium taurocholate has simple synthesis process, is economic and rapid, is easy for large-scale production, and can be developed into anti-influenza virus medicaments.
Drawings
FIG. 1 is a Western Blotting schematic of the effect of sodium taurocholate on influenza virus proliferation.
Figure 2 is a graphical representation of the viral titer of sodium taurocholate on the proliferation of influenza virus.
FIG. 3 is a schematic representation of the effect of sodium taurocholate on viral mRNA.
FIG. 4 is a schematic of dose-dependent inhibition of influenza virus proliferation by sodium taurocholate.
FIG. 5 is a graph showing the effect of intragastric sodium taurocholate on the body weight of mice infected with influenza virus.
FIG. 6 is a schematic diagram showing the effect of intragastric sodium taurocholate on the survival rate of mice infected with influenza virus.
FIG. 7 is a schematic diagram showing the effect of sodium taurocholate in intragastric administration on lung tissue lesion of infected mice.
FIG. 8 is a schematic view showing that sodium taurocholate inhibits influenza A virus proliferation in a broad spectrum.
Detailed Description
The test methods and conditions in the examples of the present invention are conventional methods unless otherwise specified.These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples. The technical scheme of the invention is a conventional scheme in the field if no special description exists; the reagents or materials are commercially available, unless otherwise specified. The invention is mainly based on human influenza virus H, limited to space 1 N 1 (PR 8) is taken as an example to illustrate the application of sodium taurocholate in preparing a medicament for treating influenza virus infection, wherein the sodium taurocholate can inhibit the proliferation of influenza virus. Proved by experiments, the sodium taurocholate also has the avian influenza virus H 3 N 2 Avian influenza virus H 9 N 2 Has the same function.
Example 1:
effect of sodium taurocholate on cell activity:
and (3) determining the influence of the sodium taurocholate on the cell activity by using a CCK-8 kit, and screening out the optimal experimental concentration. The method comprises the following specific steps:
caco-2 cells in 96-well plate are grown to about 80%, sodium taurocholate of different concentrations (0.186 μm, 0.465 μm, 0.930 μm, 1.395 μm, 1.860 μm, 3.720 μm) is incubated, CO is added at 37 deg.C 2 The culture box is used for 24h, and then CCK-8 (10 mu L/hole) is added under the condition of keeping out light. After culturing in a 37 ℃ CO2 incubator for 2 hours, OD was measured using a microplate reader 450 nm Absorbance of (b).
The results show that 1.395 μ M sodium taurocholate incubated caco-2 cells activity was 96.66% (table 1), and 1.395 μ M sodium taurocholate was used for subsequent experimental studies since 96.66% was not statistically different from 100%.
TABLE 1 Effect of sodium taurocholate on cell Activity
Example 2:
preliminary verification shows that sodium taurocholate has an inhibiting effect on in-vitro proliferation of influenza A virus:
1. western blot experiment (Western Blotting)
The results of Western Blotting experiments using influenza virus (A/Puerto Rico/8/1934H1N1 (PR 8)) to infect caco-2 cells were as follows:
H 1 N 1 infecting caco-2 cells for 1h, incubating the compound for 12h, 24h and 36h, adding cell lysate to lyse the cells, centrifuging to obtain supernatant, adding 5x protein loading, boiling for 10min, and centrifuging for later use. Preparing 10% polyamide gel, spotting, performing electrophoresis, performing constant pressure 80V on concentrated gel and 30min on the concentrated gel, performing constant pressure 120V on the separation gel for 1-1.5h, and stopping electrophoresis when bromophenol blue comes out of the separation gel. When the membrane is transferred, the membrane transfer liquid is precooled in advance, a blackboard is placed below the membrane, and the lower layer sponge, the filter paper, the gel, the NC membrane, the filter paper and the upper layer sponge are sequentially paved. Putting into a film-rotating groove, and rotating for 1h at constant pressure of 100V. Then taking out the NC membrane, sealing for 1h, incubating the primary antibody for 2h or overnight at 4 ℃, and then washing with 1x TBST for 6 times, 5min each time. After incubation, HRP-labeled secondary antibodies from the corresponding sources were washed 6 times for 5min at 1h,1x TBST. Color development was performed using Thermo's ECL color development kit.
The results showed that, as a result of verifying the above results by Western Blotting experiment, compounds were incubated for 12, 24, 36H after infection with influenza virus, and H was detected 1 N 1 The NP protein expression of the influenza virus, sodium taurocholate, can significantly inhibit the influenza virus proliferation (figure 1).
2. Virus titer determination (TCID) 50 )
H 1 N 1 Infecting caco-2 cells for 1h, incubating the compounds for 12, 24, 36h, collecting cell supernatant, diluting it with 1% FBS-containing DMEM medium at 10 fold-by-10 serial dilutions -7 Seeded in single layer 96-well MDCK cells, repeated 8 wells per dilution, 5% CO at 37 ℃ 2 Culturing for 48h under the condition, and setting a normal cell control group. Observing the cytopathic effect (CPE) every 12h, recording the number of lesion holes, and calculating the TCID of the virus according to a Reed-Muench formula 50 。
By TCID 50 The results show that compound 12, 24, 36h after infection with influenza virus, and virus titer of supernatant of infected cells was detected, and sodium taurocholate can significantly reduce virus titer (fig. 2).
Example 3:
effect of sodium taurocholate on viral mRNA:
to investigate the effect of sodium taurocholate on mRNA, a fluorescent quantitation assay was performed as follows:
first, RNA was inverted into cDNA, cells were sufficiently lysed using Trizol reagent, transferred to an EP tube of RNase-free, sufficiently inverted, and ice-washed for 10min. Add 200. Mu.L of chloroform to each tube, vortex, and centrifuge by standing. The supernatant was transferred to a new EP tube of RNase-free, and an equal volume (about 500. Mu.L) of isopropanol was added thereto, followed by gently mixing, standing, centrifugation and aspiration of the supernatant. Add 900. Mu.L of absolute ethanol, vortex and centrifuge, discard the supernatant. And drying the super clean bench during working. Adding DNA digestion system, water bath at 37 deg.C for 1 hr, adding 5 μ L of DNA Stop Solution to each tube, flicking, instantly separating, and water bath at 65 deg.C for 15min. A small amount was taken for RNA concentration. After a reverse transcription system is prepared, the cDNA product is obtained by transient dissociation, reverse transcription is carried out for 60min at 42 ℃ on a PCR instrument, and then inactivation is carried out for 15min at 72 ℃.
The cDNA synthesized in the previous step was then used directly in the fluorescent quantitative PCR, three replicates per sample, using the fluorescent quantitative enzyme SYBR Green mix.
The reaction steps are as follows: pre-denaturation at 95 ℃ for 10min, and then denaturation at 95 ℃ for 15s; annealing at 60 ℃ for 30s; extension at 72 ℃ for 30s for 40 cycles. The whole reaction uses QuantStudio TM 6Flex System real-time fluorescent quantitative PCR instrument. The calculation method of the relative quantitative result: for all mRNA measurements, GAPDH was used as an internal reference gene and the relative expression was calculated using the formula 2- Δ Δ Ct.
The results show that the mRNA expression of the mRNA in 12h, 24h and 36h after infection of the influenza virus is detected, and the mRNA level of the NP protein of the sodium taurocholate incubated in the step is very obviously lower than that of the sodium taurocholate not incubated in the step infected with the influenza virus (figure 3).
Example 4:
sodium taurocholate is dose-dependent on the inhibitory effect on influenza a virus:
is infected with H 1 N 1 Incubating sodium taurocholate with different concentrations (0.465 μm, 0.930 μm, 1.395 μm) for 24h, collecting cell protein, performing Western Blotting experiment, and detecting influenza virus NP proteinAnd (4) expressing the situation.
The results showed that the cells were infected with influenza A virus (H) 1 N 1 ) After incubation with sodium taurocholate at various concentrations, it was found that the lower the expression of NP protein with increasing sodium taurocholate concentration, the more significant the inhibitory effect on influenza virus proliferation (fig. 4) without cytotoxicity.
Example 5:
effect of sodium taurocholate on body weight and survival rate of mice after influenza virus infection:
the experimental animals were 8 week old female C57BL/6 Specific Pathogen Free (SPF) mice, 20 in total, randomly divided into 2 groups of 10 animals each.
The first group is an intragastric PBS control group, and the second group is an intragastric sterile sodium taurocholate group.
Infection with influenza virus H 1 N 1 And (3) gavage sodium taurocholate for the first 7 days, gavage once every other day after the first 7 days, gavage 5 times at intervals, observing the state of the mice for 14 consecutive days after the infection of the influenza virus, measuring the weight condition and observing the survival rate of the mice.
The gavage concentration of the sodium taurocholate is 250 mu g/kg, and the gavage dosage of each mouse is 200 mu L/mouse.
The results show that sodium taurocholate can relieve H after intragastric administration 1 N 1 Influenza infection resulted in weight loss in mice (table 2, figure 5), while being able to increase the survival of influenza infected mice (figure 6).
TABLE 2 Effect of sodium taurocholate on mouse body weight after influenza Virus infection
Example 6:
effect of sodium taurocholate on lungs of mice infected with influenza virus:
the experimental animals were 8 week old female C57BL/6 Specific Pathogen Free (SPF) mice, 18 in total, randomized into 3 groups of 6 animals each. The first group is not infected with H 1 N 1 A control group of influenza viruses, a second groupInfection H 1 N 1 Influenza virus gavage PBS control group, infection H in the third group 1 N 1 And (3) a sodium taurocholate group for intragastric administration of influenza virus. The dosage used was the same as in example 5.
Infection with influenza virus H 1 N 1 And in the first 7 days, the gastric sodium taurocholate is perfused once every other day after infection, the mice are dissected on the 7 th day after infection of influenza virus, lung tissue samples are collected and fixed by tissue fixing liquid, and histopathological sections are prepared for histopathological analysis.
The results show that on day 7 after the mice are infected with the influenza virus, severe inflammatory cell infiltration is observed in the lungs of the mice in the virus control group, and the inflammatory response of the gastric gavage sodium taurocholate group is obviously weakened (figure 7), so that the sodium taurocholate can relieve the pathological damage of the lungs of the mice infected with the influenza virus.
Example 7:
sodium taurocholate has broad spectrum on the inhibition of influenza A virus:
in order to prove the broad-spectrum effect of sodium taurocholate on influenza virus, the sodium taurocholate on avian influenza virus H is explored 3 N 2 And avian influenza virus H 9 N 2 The effects of influenza virus. Respectively with H 3 N 2 And H 9 N 2 Infecting cells, then incubating sodium taurocholate, respectively collecting proteins after 12h, 24h and 36h, performing Western Blotting experiment, and detecting the expression condition of the influenza virus NP protein.
The results show that sodium taurocholate is on H 3 N 2 、H 9 N 2 There was also a significant inhibition of influenza virus proliferation (figure 8).
Claims (3)
1. Application of sodium taurocholate in preparing medicine for preventing and treating influenza virus infection is provided.
2. The use of claim 1, wherein the symptoms after influenza virus infection comprise: lung tissue damage, weight loss.
3. According to claim1, the influenza virus is human influenza virus PR8 and avian influenza virus H 3 N 2 Avian influenza virus H 9 N 2 。
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Citations (4)
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|---|---|---|---|---|
| WO1990003172A2 (en) * | 1988-09-20 | 1990-04-05 | Fisons Plc | Bile acids for treatment of viral infections |
| WO1998052585A1 (en) * | 1997-05-23 | 1998-11-26 | Lorus Therapeutics Inc. | Immunomodulating, bile-derivable compositions for the treatment of viral disorders |
| CN103800343A (en) * | 2014-02-27 | 2014-05-21 | 华南农业大学 | Application of chenodeoxycholic acid in preparing medicine for preventing and treating bird flu of poultry |
| CN106031731A (en) * | 2015-03-18 | 2016-10-19 | 中国医学科学院基础医学研究所 | New uses of tauroursodeoxycholic acid |
-
2022
- 2022-10-14 CN CN202211262150.0A patent/CN115414370A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990003172A2 (en) * | 1988-09-20 | 1990-04-05 | Fisons Plc | Bile acids for treatment of viral infections |
| WO1998052585A1 (en) * | 1997-05-23 | 1998-11-26 | Lorus Therapeutics Inc. | Immunomodulating, bile-derivable compositions for the treatment of viral disorders |
| CN103800343A (en) * | 2014-02-27 | 2014-05-21 | 华南农业大学 | Application of chenodeoxycholic acid in preparing medicine for preventing and treating bird flu of poultry |
| CN106031731A (en) * | 2015-03-18 | 2016-10-19 | 中国医学科学院基础医学研究所 | New uses of tauroursodeoxycholic acid |
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| Title |
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