CN113559096A

CN113559096A - Application of melatonin in preparation of medicine for directly inhibiting Coxsackie virus B group 3

Info

Publication number: CN113559096A
Application number: CN202111010830.9A
Authority: CN
Inventors: 王欢; 刘正芸
Original assignee: Zunyi Medical University
Current assignee: Zunyi Medical University
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2021-10-29

Abstract

The application discloses an application of melatonin in preparation of a medicine for directly inhibiting Coxsackie virus B group 3 in the technical field of medicine preparation, and particularly shows that the melatonin can directly inhibit replication of CVB3 at a gene level and reduce virus titer by researching an in-vitro CVB3 infected cell model experiment, so that the melatonin is proved to have an obvious effect of resisting CVB3 and can be used for preparing a medicine for resisting CVB 3.

Description

Application of melatonin in preparation of medicine for directly inhibiting Coxsackie virus B group 3

Technical Field

The invention relates to the technical field of medicine preparation, and relates to application of melatonin in preparation of a medicine for directly inhibiting Coxsackie virus B group 3.

Background

Viral Myocarditis (VMC) refers to a myocardial disease caused by infection with cardiotropic virus, mainly changing by degeneration, necrosis, interstitial inflammatory cell infiltration and fiber exudation of myocardial cells, and is the most common infectious cardiomyopathy in clinic, and can be complicated by severe arrhythmia, heart failure, cardiogenic shock and sudden death. The causative agent is mainly coxsackievirus group B (CVB), of which CVB3 has a strong psychotropic property among six serotypes. CVB3 belongs to the family Picornaviridae, Enterovirus, the non-enveloped single positive strand RNA virus. 10-20% of infected patients can progress to dilated cardiomyopathy, and a few critically ill patients can develop heart failure and even death. At present, the clinical treatment of viral myocarditis mainly aims at improving the symptomatic support treatment of left cardiac insufficiency, and medicines for specific treatment are not available, and specific antiviral treatment is recommended. Therefore, new effective anti-CVB 3 medicines are continuously developed, and the medicine has important significance for treating the viral myocarditis.

In 1959, foreign scholars isolated a hormone from the body of the pine cone of a cow, which was capable of fading the skin on the back of the frog, so named Melatonin (Meldonin, MT) and the chemical component was N-acetyl-5-methoxytryptamine. MT is an endogenous indoleamine widely distributed in plants, unicellular organisms, algae, bacteria, invertebrates and vertebrates and has important physiological effects in the human body, such as regulation of biological clock rhythm, regulation of metabolism, oxidation resistance, regulation of reproductive function, and the like.

At present, researches report that melatonin can reduce myocardial damage of mice, but the action mechanism of the melatonin is that the expression of apoptosis indexes including clear caspase3, clear caspase9 and Bax protein in myocardial tissues of the mice is down-regulated, and the expression of Bcl-2 protein is up-regulated, so that the melatonin plays a role in protecting the mice with viral myocarditis, but whether the replication of CVB3 can be inhibited or not is not directly related.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the application of melatonin in preparing anti-coxsackie virus B group 3 type medicaments.

One of the purposes of the invention is to provide the application of melatonin or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers or excipients in preparing medicaments for resisting coxsackie virus B group 3.

The invention also aims to provide application of melatonin or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient in preparing a medicament for inhibiting replication of coxsackie virus B group 3.

Diseases caused by infection of the coxsackievirus group B3 include: human viral myocarditis, aseptic encephalitis, and systemic infection of newborns.

Furthermore, the melatonin inhibiting concentration is 0.5-2 mM.

The invention also aims to provide a medicament, namely an anti-coxsackie virus B group 3 medicament prepared by using melatonin as an active ingredient.

The molecular formula of the melatonin is as follows: c₁₃H₁₆N₂O₂(commercially available).

The invention is also directed to the above formula C₁₃H₁₆N₂O₂The following experiments were performed with melatonin:

detecting the influence of melatonin on cytopathic effect (CPE) after CVB3 infection by taking a Hela (Hela) Cell infected by CVB3 as a research model; effect on virus titer in supernatant; effect on CVB3 mRNA. The detection result shows that the melatonin can inhibit CPE caused by CVB3 in Hela cells, reduce virus titer and inhibit the transcription level of CVB3 mRNA.

Experimental results prove that the melatonin has an obvious direct anti-CVB 3 effect and can be used for preparing anti-CVB 3 medicines.

Drawings

FIG. 1 is a schematic diagram showing the effect of melatonin on Hela cell proliferation measured by the CCK8 method;

fig. 2 is a schematic diagram of the observation of the effect of melatonin on CPE induced by CVB3 in Hela cells;

FIG. 3 is a graph showing the calculation of the change in viral titer following the action of melatonin on CVB 3-infected Hela cells using the Reed-Muench formula;

FIG. 4 is a graph showing the effect of melatonin on the transcript levels of CVB3 mRNA in CVB 3-infected Hela cells as measured by fluorescent quantitative PCR.

Detailed Description

The following is further detailed by way of specific embodiments:

1. CCK8 screening melatonin acting Hela cell maximum nontoxic concentration

Hela cells in logarithmic growth phase were taken, digested with 0.25% trypsin, and seeded in 96-well plates at 2X 10⁴Per well, after the cells grew over the monolayer, the culture medium was discarded and different concentrations of melatonin (0.25mM, 0.5 mM) were added1mM, 2mM and 4mM) were added to a 96-well plate, 5 wells per dilution were inoculated as an experimental group, 5 wells were filled with medium alone, cells were used as an experimental control group, and 5 wells were filled with medium alone as a blank control, 0.1mL per well. Standing at 37 deg.C for 5% CO₂Culturing in an incubator, measuring OD value by adopting a CCK8 method after culturing for 24h, and calculating the cell survival rate.

As shown in figure 1, the method of CCK8 analysis is used for detecting the influence of the drug on the cell growth, a CVB3 specific cell model Hela is adopted, the concentrations of the selected melatonin are 0.25mM, 0.5mM, 1mM, 2mM and 4mM respectively, and the experimental data are subjected to one-factor anova, so that the result shows that the melatonin with the concentration of more than 2mM has obvious influence on the proliferation of Hela cells (p is less than 0.05).

2. Observation of the Effect of melatonin on CPE-inducing CVB3 in Hela cells

Hela cells in logarithmic growth phase were taken, digested with 0.25% trypsin, and seeded in 24-well plates at 2X 10⁵And (3) removing the culture solution after the cells grow into a monolayer, washing with PBS for 2-3 times, infecting Hela cells with CVB3 with MOI of 0.1, sucking out the virus solution after 2h, replacing the virus solution with a culture medium containing melatonin with different concentrations prepared by 2% FBS culture medium, and setting a melatonin single administration group, a CVB3 infection group and a negative culture medium control group. After 17h the CPE was observed under an optical microscope. As shown in fig. 2, CPE is a series of changes that occur in the cell after the virus replicates in the host cell, such as: initially, the nuclei change, chromatin borders, nuclei condense, and subsequently the cell membrane changes, losing adhesion, cells become rounded, fall off the flask wall, or cells fuse with each other, etc. These changes can be examined by light microscopy. The results show that: melatonin concentrations of 0.5mM, 1mM and 2mM were effective in inhibiting CPE caused by CVB3, particularly 2mM, compared to the CVB 3-infected group.

3. Observation of the Effect of melatonin on the Virus Titers of CVB3 in Hela cells

Plating, inoculating poison and adding medicine in the same way as 2, adding medicine for 17h, collecting cell supernatant, centrifuging at 4 ℃ and 12000rpm for 10min, and collecting cell supernatant again for later use.

Vero cells in logarithmic growth phase were taken, digested with 0.25% trypsin, at 1X 10⁴Inoculating into 96-well plate at a concentration of 100 μ L per well, removing culture medium when the cells are full of monolayer, washing with PBS twice, and sequentially diluting the collected supernatant virus solution 10 times (10 times) with DMEM high-sugar medium^-1～10^-10) 8 wells were inoculated for each dilution, 8 additional wells were used as a blank, and 0.1mL of serum-free DMEM high-sugar medium was added to each well. Standing at 37 deg.C for 5% CO₂Culturing in incubator, observing and recording CPE every day for 72h, and calculating 50% cell infection dose (TCID) of virus solution by Reed-Muench formula₅₀)。

As shown in fig. 3, melatonin at 1mM and 2mM decreased titers in the cell supernatants caused by CVB3 infection compared to the virus-infected group, and the differences were statistically significant.

4. Observation of the Effect of melatonin on the transcript level of CVB3 mRNA in Hela cells

Inoculating a plate, inoculating and adding drugs in the same mode as 2, adding drugs for 17h, collecting cells and extracting total RNA, wherein the process comprises the following steps: taking out a 6-hole plate, placing the 6-hole plate on ice, washing the 6-hole plate for 2 times by PBS (phosphate buffer solution), adding 1mL of Trizol to perform lysis for 5-10min, and transferring a lysate to an EP (EP) tube; ② adding 0.2mL chloroform, shaking for 30s by a vortex shaking instrument, and standing for 15min at room temperature. Centrifuging at 12000rpm and 4 deg.C for 15 min; thirdly, placing the upper colorless aqueous phase into a new EP tube, adding isopropanol with the same volume, turning upside down and mixing uniformly, standing at room temperature for 10min,12000rpm, and centrifuging at 4 ℃ for 10 min; fourthly, abandoning the supernatant, washing the RNA sediment by 1mL of 75 percent ethanol, reversing the solution to wash the RNA sediment upside down, and centrifuging the RNA sediment for 8min at the temperature of 4 ℃ at 12000 rpm; fifthly, abandoning the supernatant, taking 30 mu LRNase-free water to dissolve RNA precipitate after ethanol remained in the precipitated RNA is volatilized, and freezing the RNA precipitate in a refrigerator at minus 80 ℃.

From the obtained RNA concentration value, the quantitative amount was 1. mu.g, and the amount of the desired RNA sample was calculated. The extracted RNA is reversely transcribed into cDNA in a 20-mu L system, and the reaction procedure is as follows: 15min at 37 ℃; the reaction system is stored at 85 ℃, 5sec and 4 ℃ as follows:

TABLE 1 real-time fluorescent quantitative PCR reverse transcription system

TABLE 2 real-time fluorescent quantitative PCR primer sequences

Taking the obtained cDNA as a template, repeating the cDNA in three times for each sample, taking beta-actin as an internal reference, and carrying out amplification in a fluorescent quantitative PCR instrument, wherein the reaction program is as follows: at 95 ℃ for 30 s; (95 ℃, 3s → 60 ℃, 30s) × 40 cycles, dissolution curve apparatus default, reaction system as in table:

TABLE 3 real-time fluorescent quantitative PCR reaction System

The experiment is repeated three times, the Graphpad prism5 software is used for carrying out data analysis and mapping, and beta-actin is used as an internal reference,

The results were calculated, and the relative levels between the groups were calculated.

As shown in FIG. 4, melatonin at 0.5mM, 1mM and 2mM was effective in reducing the level of transcription of CVB3 mRNA, directly inhibiting the replication of CVB 3.

The results of the above experiments were combined to draw conclusions: the melatonin has obvious effects of directly inhibiting CVB3 replication and reducing virus titer, and can be used for preparing medicines for preventing and treating viral myocarditis.

Claims

1. Application of melatonin or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers or excipients in preparing anti-coxsackie virus B group 3 medicines.

2. Use of melatonin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient in the preparation of a medicament for inhibiting replication of coxsackie virus group B type 3.

3. Use according to claim 2, characterized in that: diseases caused by infection of the coxsackievirus group B3 include: human viral myocarditis, aseptic encephalitis, and systemic infection of newborns.

4. Use according to claim 2, characterized in that: the melatonin inhibiting concentration is 0.5-2 mM.

5. An anti-coxsackie virus B group 3 type medicine prepared by taking melatonin as an active ingredient.