CN115531364A - Microbial metabolite preparation for preventing or treating rotavirus infection and application thereof - Google Patents
Microbial metabolite preparation for preventing or treating rotavirus infection and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
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Abstract
The present invention provides a pharmaceutical composition for preventing, inhibiting or treating rotavirus infection or diarrhea caused by rotavirus infection in a subject, the pharmaceutical composition comprising homoserine. The invention also provides the use of homoserine in the preparation of a pharmaceutical composition for the prevention, inhibition or treatment of rotavirus infection or diarrhoea caused by rotavirus infection in a subject.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a microbial metabolite preparation for preventing or treating rotavirus infection and application thereof. More specifically, the present invention relates to a microbial metabolite preparation comprising homoserine and its use.
Background
The infantile diarrhea caused by rotavirus infection is a common pediatric disease, the rotavirus enteritis in the infantile diarrhea has the morbidity of up to 50 percent, more than 100 million people are killed by rotavirus infection all over the world every year, and the infantile diarrhea is one of the diseases which are mainly prevented and treated by WHO and China. Rotavirus can cause intestinal infection of children patients and also can cause a series of symptoms such as dehydration, vomiting, fever, diarrhea and the like. The treatment by adopting active and effective measures has important significance for treating children patients with rotavirus enteritis. At present, broad-spectrum antiviral drugs are mostly adopted in the process of treating rotavirus diarrhea, but the treatment effect is not ideal, and the clinical treatment requirements cannot be met.
The most effective way for preventing and treating rotavirus infection is vaccination at present, and the pentavalent rotavirus vaccine of the Minshadong company is approved by the national drug administration on the market in 2018, 4 months and 12 days, so that the pentavalent rotavirus vaccine becomes the first multivalent rotavirus vaccine in China. However, the spread and dissemination of vaccines still requires a long time. Furthermore, although the use of vaccines can significantly reduce the number and symptoms of critically ill children, infection of infants by rotavirus is still not completely eliminated due to the extremely rapid mutation and recombination rates of rotavirus. Therefore, the development of clinical therapeutic drugs to accelerate the recovery of children still remains an urgent task in rotavirus research.
Disclosure of Invention
The invention provides an amino acid medicament aiming at the current situation that rotavirus is lack of specific drugs clinically, and experiments show that the addition of the amino acid medicament can obviously reduce the copy number of viruses in rotavirus susceptible cells HT-29 and MA-104 in vitro and inhibit rotavirus infection.
The present invention provides one kind of amino acid homoserine capable of inhibiting rotavirus infection obviously in vitro (FIG. 1). Homoserine (C) 4 H 4 NO 4 ) Is a microbial metabolite, an intermediate product of threonine, methionine and cystathionine biosynthesis, is present in bacterial peptidoglycan, and is also present in human blood, urine and feces. Homoserine is an active variant of serine and is not suitable for protein formation. Experiments show that homoserine can remarkably inhibit rotavirus infection in vitro, and homoserine with the final concentration of 20mM can remarkably reduce the infection of monkey rotavirus RRV in host cells, namely human colon cells HT-29 and monkey kidney cells MA-104And this antiviral ability is dose dependent.
The invention also detects the antiviral ability of homoserine in mice, and finds that homoserine can obviously inhibit rotavirus infection in vivo. Compared with a control group, the content of rotavirus in intestinal tissues of the mice injected with the homoserine in the abdominal cavity is obviously reduced, the intraperitoneal injection of the homoserine medicament shows good in-vivo rotavirus resistance, and the homoserine injection has obvious curative effects on replication of tissue virus and alleviation of diarrhea symptoms, and the mice have no obvious adverse reaction.
Based on the above studies, in one aspect, the present invention provides a pharmaceutical composition for preventing, inhibiting or treating rotavirus infection or diarrhea caused by rotavirus infection in a subject, the pharmaceutical composition comprising homoserine.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutical composition is an oral or injectable formulation.
In some embodiments, the rotavirus comprises, but is not limited to, a rotavirus EW strain and a rotavirus RRV strain.
In some embodiments, the subject is a human or an animal.
In some embodiments, the subject is an infant or a young child.
In another aspect, the invention provides the use of homoserine in the preparation of a pharmaceutical composition for the prevention, inhibition or treatment of rotavirus infection in a subject.
In yet another aspect, the present invention provides the use of homoserine in the preparation of a pharmaceutical composition for the prevention or treatment of diarrhea caused by rotavirus infection in a subject.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention discovers that rotavirus infection can obviously reduce the content of homoserine in the intestinal tract;
2. homoserine can effectively inhibit rotavirus from being replicated and amplified in a host cell by using in vitro;
3. the use of the in vivo medicine effectively inhibits the diarrhea phenotype caused by rotavirus infection and effectively inhibits the infection of intestinal rotavirus;
4. homoserine has biocompatibility and safety, and the homoserine drug is used in rotavirus infection, so that the survival of mice is not influenced.
Drawings
FIG. 1 shows the chemical structure of homoserine;
fig. 2 is the results of mass spectrometry in situ of tissues of rotavirus infection group and control group in the example of the present invention, and the significance of two groups of data was calculated statistically by using student T test: p <0.05; **: p <0.01; * **: p <0.001;
fig. 3 is a graph showing the expression level of viral genes in rotavirus infected rhesus monkey kidney cells in an example of the present invention, and the significance of each group of data was statistically calculated using student T test: p <0.05; **: p <0.01; * **: p <0.001;
fig. 4 is a graph showing the expression level of viral genes in human colorectal cancer cells infected with rotavirus in an example of the present invention, and the significance of two groups of data was statistically calculated using student T test: p <0.05; **: p <0.01; * **: p <0.001,ns means that there was no significant difference between the two sets of data
Fig. 5A is a graph of the ratio of viral gene expression and mouse diarrhea at 2 days of rotavirus infection in an example of the invention, statistically calculated using student T-test for the significance of each group of data: p <0.05; * *: p <0.01; * **: p <0.001;
fig. 5B is a graph of the ratio of viral gene expression and mouse diarrhea at 4 days of rotavirus infection in an example of the invention, statistically calculated using student T-test for significance in each group of data: p <0.05; * *: p <0.01; * **: p <0.001,ns means that there was no significant difference between the two sets of data.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
Example 1 Mass Spectrometry detection of substances that changed in rotavirus infection group relative to control group
By a tissue in situ mass spectrometry technology, intestines of C57 mice in an infection group (2 days of infection and 3 mice) of infected mouse rotavirus EW strain (from the university of St.Louis Washington, USA, refer to Nature.2017Jun 29 (7660): 667-670, doi:10.1038/nature22967.Epub 2017Jun 21.) 10DD50 (1 DD50 represents that 50 percent of wild mice have diarrhea after the virus infection of the dose, and the 10DD50 dose is 10 times of 1DD 50) are embedded with intestinal tissues of the control group (3) of mice, frozen sections are cut, and the sections are subjected to tissue in situ mass spectrometry to obtain the contents of different metabolites in the intestinal tissues, so as to find the metabolites with obvious variation difference. Some of the results are shown in the table below (the metabolite content is expressed as the relative content of the mean values of the infection groups):
as shown in fig. 2, by comparing the results of the tissue in situ mass spectrometry of the infected group and the control group, we found that the content of homoserine in the intestinal tract was significantly reduced after infection, and the relative content of homoserine in the control group was about four times that of the infected group. Thus, in the following examples, homoserine was selected to study its effects on rotavirus.
Example 2 in vitro inhibition of homoserine in Rotavirus-infected rhesus monkey kidney cells
Host cell rhesus monkey kidney MA-104 cells were as follows 5 x 10 5 Homoserine (MCE, HY-W002292) was added at concentrations of 5mM, 10mM, and 20mM to DMEM medium for culturing host cell rhesus monkey kidney MA-104 cells as homoserine-treated group, while the control group was supplemented with no metabolite in the medium, but only water for dissolving the metabolite. Homoserine treatment group and control group were 5% CO 2 And culture conditions at 37 ℃ were followed by addition of monkey rotavirus RRV (virus titer 5 x 10) 6 FFU/mL, from st louis washington university, usa, see nature.2017jun 29;546 (7660) 667-670, doi 10.1038/nature22967.Epub 2017Jun 21.), infection at a multiplicity of infection of 0.1, 12 hours later cellular RNA was extracted, and Trizol reagent (Tiangen Biochemical technology (Beijing) Ltd., product number: DP 424), adding chloroform and isopropanol, RNA extraction was performed according to the instruction manual, by RT-qPCR method using SYBR green qPCR enzyme (hiscript iii RT SuperMix for qPCR + gDNA wiper R323-01; SYBR qPCR Master Mix Q311-03) for PCR detection of rotavirus genes NSP2 and VP7, wherein the PCR process is strictly performed according to the product instruction manual, and the influence of homoserine on the replication condition of rotavirus genes NSP2 and VP7 infecting host cells in vitro is observed.
Rotavirus infection of MA104 cells as host cells was detected by RT-qPCR, and the expression levels of the rotavirus genes NSP2 and VP7 relative to the housekeeping gene Gapdh are shown in the following table.
The primers used in RT-qPCR are as follows:
VP7-F:ACGGCAACATTTGAAGAAGTC(SEQ ID NO:1)
VP7-R:TGCAAGTAGCAGTTGTAACATC(SEQ ID NO:2)
NSP2-F:GAGAATCATCAGGACGTGCTT(SEQ ID NO:3)
NSP2-R:CGGTGGCAGTTGTTTCAAT(SEQ ID NO:4)
Gapdh-F:TGCACCACCAACTGCTTAGC(SEQ ID NO:5)
Gapdh-R:GGAAGGCCATGCCAGTGA(SEQ ID NO:6)
as shown in FIG. 3, NSP2 and VP7 are genes of rotavirus respectively, the expression level thereof represents the speed of virus replication and the titer of virus, gapdh is housekeeping gene, and the expression level is not affected by virus infection, thus serving as an internal reference of qPCR for uniformizing the expression levels of different genes. In the research, housekeeping genes with relatively close expression levels are usually selected as internal references according to the expression level of the target gene, and in this example, the expression levels of different genes are plotted relative to the expression level of Gapdh, so that different samples can be compared in a horizontal manner. As a result, it was found that when homoserine with different concentrations was added to the culture medium, replication of rotavirus gene infected in the cells was effectively inhibited compared to the control group in which water was added to the culture medium, and thus, addition of the metabolite could effectively inhibit replication of rotavirus in the host cells, indicating that homoserine has a good effect of resisting rotavirus infection in vitro, and that the effect is enhanced with an increase in the dosage of homoserine.
Example 3 in vitro inhibition of homoserine in human colorectal cancer cells infected with rotavirus
Human colon cancer HT-29 cells were treated as 5 x 10 cells 5 Each well was plated on a 24-well cell culture plate, homoserine (MCE, HY-W002292) was added at a concentration of 20mM (physiological concentration in the intestinal tract) to DMEM medium in which human colon cancer HT-29 cells were cultured as homoserine-treated group, and control group was added with no metabolite but only water for dissolving the metabolite in the medium, then monkey rotavirus RRV was added, infection was performed at a multiplicity of infection of 0.1, cellular RNA was extracted after 12 hours, trizol reagent (nikka bio-science (beijing) limited, product number: DP 424) was used, chloroform and isopropanol were added, RNA extraction was performed according to instruction manual, SYBR green qPCR enzyme (Mix for qPCR + gdawirer 323-01) was used by RT-qPCR method, SYBR miex gene NSP2 and SYBR miex master03) were used for qPCR, and the influence of homoserine replication was strictly observed by PCR on host cell replication and in vitro inhibition of host cell infection by rotavirus VP 2 and PCR 7 by interfering with the rotavirus VP 2 and the PCR method.
The results of detection of rotavirus-infected human intestinal HT-29 cells by RT-qPCR are shown in the following table, wherein each expression is relative to the housekeeping gene Gapdh.
As shown in FIG. 4, interferon-. Beta.is an important antiviral cytokine secreted from cells, and the level of expression represents the antiviral ability of the cells, and CXCL10 is an important interferon-stimulated gene that can be expressed by interferon induction. The result shows that after 20mM of homoserine is added into the culture medium, the virus genes are remarkably reduced, which indicates that the homoserine can effectively inhibit the replication of rotavirus in human intestinal tract cells, and has good in vitro rotavirus infection resistance, while the expression of interferon beta and downstream interferon stimulating gene CXCL10 is not remarkably changed, which indicates that the inhibition of the homoserine on the virus replication is independent of interferon.
Example 4 in vivo inhibition of rotaviruses by homoserine
Mice of the C57 strain, 5 days old, were intraperitoneally injected at homoserine 20mM (physiological concentration in the intestine), as a treatment group, 10 ul/mouse were intraperitoneally injected with an equal amount of water as a control group, 12h later, the mice were intragastrically injected at a dose of 10DD50 (1 DD50 represents that 50% of the wild type mice became diarrheal after infection of the mice with the dose of the virus, 10DD50 is 10 times the dose of 1DD 50), mouse rotavirus EW strain (from st. Louis washington university of usa, see nature.2017jun 29 (7660): 667-670, doi. The diarrhea of the mice is observed every day and scored (the scoring standard: 0 points, the feces shape is normal, dry 1 point, the feces is not formed, golden yellow, viscous but opaque 2 points, the feces is not formed, contains transparent liquid and golden yellow feces 3 points, the feces is not formed, and is basically transparent liquid). Killing mice respectively at 2 days (figure 5A) and 4 days (figure 5B) (2 days represent the early stage of infection, 4 days represent the middle and late stage of infection, rotavirus generally has a course of disease of about one week, two time points are taken for researching the infection degree of rotavirus at different time points), utilizing an RT-qPCR method, carrying out qPCR detection on rotavirus genes NSP2 and VP7 by using SYBR green qPCR enzyme (HiScriptIII RT Supermix for qPCR + gDNA wier R323-01 SYBR qPCR Master Mix Q311-03, the qPCR process is strictly executed according to a product instruction manual, detecting the virus titer in intestinal tissues of mice and the virus titer in intestinal epithelial cells, and observing the influence on the infection in vivo of the rotavirus. None of the mice in the experiment died after homoserine injection.
The results of RT-qPCR detection at day 2 after infection are shown in the following table (5 control groups, 3 treatment groups), in which the expression levels of NSP2 and VP7 are relative to the expression level of housekeeping gene Hprt.
The results of RT-qPCR assay at day 4 post-infection are shown in the following table (5 control groups, 5 treatment groups), where the expression levels of NSP2 and VP7 are relative to the expression level of housekeeping gene Hprt.
The primers used in RT-qPCR are as follows:
VP7-F TCAACCGGAGACATTTCTGA(SEQ ID NO:7)
VP7-R TTGCGATAACGTGTCTTTCC(SEQ ID NO:8)
NSP2-F GAGAATGTTCAAGACGTACTCCA(SEQ ID NO:9)
NSP2-R CTGTCATGGTGGGTTTCAATTTC(SEQ ID NO:10)
Hprt-F ACCTCTCGAAGTGTTGGATACAGG(SEQ ID NO:11)
Hprt-R CTTGCGCTCATCTTAGGCTTTG(SEQ ID NO:12)
the results are shown in fig. 5A and 5B, where Hprt is a housekeeping gene and the expression level is not affected by viral infection, and therefore used as an internal reference for qPCR to normalize the expression levels of different genes, plotting the expression levels of different genes against the expression level of Hprt, allowing for lateral comparisons of different samples. The expression level of Hprt is lower than that of Gapdh. Since the expression level of the viral gene NSP2 was low after infection of mice, the Hprt housekeeping gene, which is expressed at a lower level than Gapdh, was selected as an internal control in this example.
The results of the grading of diarrhea conditions show that, starting from day 2 after rotavirus infection, the control mice have diarrhea symptoms, which are characterized in that the color of the feces is golden yellow, the water content is obviously increased, and the feces produced by the diarrhea are adhered to the abdomen and the skin of the lower limbs of the mice. And compared with a control mouse, the mouse with the intraperitoneal injection of homoserine after infection has obviously reduced diarrhea degree and clean lower limb and abdominal skin.
At 2 days and 4 days, rotavirus in intestinal tissues and intestinal epithelial cells of mice is detected, and qPCR results show that compared with a control group, the content of rotavirus in the intestinal tissues and the intestinal epithelial cells of the mice injected with homoserine in the abdominal cavity is obviously reduced, and the diarrhea degree is obviously relieved, so that the intraperitoneal injection of the homoserine medicament shows good rotavirus resistance in vivo, and has obvious curative effects on replication of the rotavirus and relieving diarrhea symptoms, and the mice have no obvious adverse reaction.
Therefore, homoserine can be used for preparing a pharmaceutical composition for preventing, inhibiting or treating rotavirus infection, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition is administered to a subject by intraperitoneal injection, intravenous infusion, intramuscular injection, or oral administration.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A pharmaceutical composition for preventing, inhibiting or treating rotavirus infection or diarrhea caused by rotavirus infection in a subject, wherein the pharmaceutical composition comprises homoserine.
2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is an oral or injectable formulation.
4. The pharmaceutical composition of claim 1, wherein the rotavirus comprises but is not limited to rotavirus EW strain and rotavirus RRV strain.
5. The pharmaceutical composition of claim 1, wherein the subject is a human or an animal.
6. The pharmaceutical composition of claim 1, wherein the subject is an infant or young child.
7. Use of homoserine in the preparation of a pharmaceutical composition for the prevention, inhibition or treatment of rotavirus infection in a subject.
8. Use of homoserine in the preparation of a pharmaceutical composition for the prevention or treatment of diarrhea caused by rotavirus infection in a subject.
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