CN114796208B - Application of rapamycin in preparing medicament for preventing or treating crucian hematopoietic necrosis - Google Patents

Abstract

The invention provides an application of rapamycin in preparing a medicament for preventing or treating crucian hematopoietic necrosis, wherein the dose of rapamycin is 500nmol/L, 10 mu mol/L or 20 mu mol/L, rapamycin inhibits CyHV-2 virus infection replication in crucian GiCF cells, and rapamycin obviously inhibits phosphorylation of mTOR and expression quantity of ORF121 protein at protein level or inhibits replication of CyHV-2 by inhibiting expression quantity of phosphorylation-mediated PI3K/AKT/mTOR signal path partial genes of mTOR; the rapamycin of the invention inhibits the phosphorylation of mTOR, inhibits the expression quantity of PI3K/AKT/mTOR signal path part genes, and inhibits the copy number of CyHV-2 virus and the expression quantity of virus part genes, thereby inhibiting the replication of CyHV-2.

Description

Application of rapamycin in the preparation of drugs for preventing or treating hematopoietic organ necrosis in crucian carp

Technical Field

The invention belongs to the technical field of agriculture, fishery and disease prevention and control, and specifically relates to an application of rapamycin in preparing a medicine for preventing or treating crucian carp hematopoietic organ necrosis.

Background Art

Cyprinid herpesvirus 2 (CyHV-2) is a fish herpesvirus that can infect crucian carp or goldfish and cause hematopoietic organ necrosis. CyHV-2 was first discovered in Japan in 1992, and in 2013, it was first found in my country (Xu J, Zeng L, Zhang H, et al. Cyprinid herpesvirus 2 infection emerged in cultured gibel carp, Carassius auratus gibelio in China [J]. Veterinary Microbiology, 2013, 166 (1-2): 138-144.). CyHV-2 can infect crucian carp or goldfish growing at different stages, and the mortality rate after onset can reach up to 100%. CyHV-2 virus infection can cause a series of symptoms such as loss of appetite, slow swimming, bleeding on the body surface, bleeding on the gills, bleeding at the base of the pectoral fins and pelvic fins, and eventually lead to death. Existing studies have confirmed that CyHV-2 infection of hosts can cause acute infection and persistent infection, and persistent infection can be transformed into acute infection under certain conditions (Chai W, Qi L, Zhang Y, et al. Evaluation of Cyprinid Herpesvirus2Latency and Reactivation in Carassius gibel[J]. Microorganisms, 2020, 8(3): 445.). This complex and changeable infection situation makes the virus easy to spread and makes virus prevention and control more difficult.

In the front-line breeding process, the following measures are mainly used to prevent and control viruses: First, pay attention to water disinfection, including the disinfection of source water, breeding water and breeding tail water. Equipment such as filter sand tanks and ultraviolet disinfection can be introduced to reduce the toxicity of source water. Chlorine dioxide, glutaraldehyde, trichloroisocyanuric acid and other disinfectants can also be used to disinfect the water bodies of breeding ponds. Secondly, choose healthy seedlings and carry out real-time monitoring during the peak period of virus outbreaks in spring and autumn, so as to detect and deal with them early. In addition, strengthening the selection and breeding of disease-resistant seedlings is also an effective way to resist viruses from the source. Furthermore, microbial preparations can be used for daily water quality management and prevention.

At present, the infection mechanism of CyHV-2 is still unclear. Methods such as disinfecting the breeding environment and improving fish immunity are not effective. There is no commercial vaccine for industrial prevention and control. It is difficult to develop a targeted drug for CyHV-2. Studies have shown that improving and compounding existing Chinese medicine formulas can prevent and control the CyHV-2 virus. For example, a study by He Jie et al. found that adding bile acid to feed can promote fish growth and improve the fish's antiviral ability (He Jie. Effects of adding bile acid to feed on growth, oxidative damage repair, endogenous bile acid metabolism and CyHV-2 immune protection of silver crucian carp [D]. Suzhou University, 2018.); Su et al. found that berberine hydrochloride (BBH) can inhibit CyHV-2 virus replication in vivo and in vitro (Su M, Tang R, Wang H, et al. Suppression effect of plant-derived berberine on cyprinidherpesvirus 2 proliferation and its pharmacokinetics in Crucian carp (Carassius auratus gibelio) [J]. Antiviral Research, 2021, 186: 105000.); Shen Zhaoyuan et al. found that 1% andrographis paniculata had a good anti-CyHV-2 effect (Shen Zhaoyuan, Lu Jianfei, Xu Dan, Lv Liqun. Effects of Chinese herbal feed additives on the infection of type II carp herpesvirus in silver carp[J]. Fisheries Science, 2018, 37(03): 289-294.). However, compound Chinese herbal preparations have not yet been commercialized, and the specific antiviral mechanism remains to be studied.

Mammalian target of rapamycin (mTOR), as an important serine-threonine protein kinase downstream of PI3K/AKT, can regulate cell angiogenesis and promote cell metabolism. It also participates in cell autophagy and apoptosis, and plays an indispensable role in many diseases. In recent years, the PI3K/AKT/mTOR pathway has attracted much attention and is known as the "star pathway". mTOR inhibitors have been shown to have promising therapeutic prospects in a series of studies targeting respiratory syncytial virus (RSV) (Huynh H, Levitz R, Huang R, et al. mTOR kinase is a therapeutic target for respiratory syncytial virus and coronaviruses[J]. Scientific Reports, 2021, 11(1): 24442.) and white spot syndrome virus (WSSV) (Su MA, Huang YT, Chen IT, et al. An invertebrate Warburg effect: a shrimp virus achieves successful replication by altering the host metabolome via the PI3K-Akt-mTOR pathway[J]. PLoS Pathogens, 2014, 10(6): e1004196.), and its representative drug is rapamycin. Rapamycin specifically inhibits mTORC1, has a chemical formula of C ₅₁ H ₇₉ NO ₁₃ , and a chemical structure of:

.

Summary of the invention

In view of the deficiencies in the prior art, the object of the present invention is to provide an application of rapamycin in the preparation of a drug for preventing or treating hematopoietic organ necrosis in crucian carp.

To achieve the above object, the solution of the present invention is:

The invention provides an application of rapamycin in preparing a drug for preventing or treating crucian carp hematopoietic organ necrosis. The rapamycin is a drug for inhibiting mTOR gene phosphorylation (i.e., an mTOR inhibitor) with a dosage of 500 nmol/L, 10 μmol/L or 20 μmol/L.

As a preferred embodiment of the present invention, rapamycin can effectively inhibit the infection and replication of CyHV-2 virus in crucian carp GiCF cells, that is, the rapamycin drug is used in the GiCF cell model infected with CyHV-2, which can well reduce the copy number of CyHV-2 virus and achieve the effect of hindering viral infection and replication.

As a preferred embodiment of the present invention, rapamycin significantly inhibits the phosphorylation of mTOR and the expression of ORF121 protein at the protein level to inhibit the replication of CyHV-2.

As a preferred embodiment of the present invention, rapamycin inhibits the replication of CyHV-2 by inhibiting the expression of some genes in the PI3K/AKT/mTOR signaling pathway mediated by the phosphorylation of mTOR. Among them, some genes include AKT2, IRS1, PDPK1 and EIF4EBP2.

A medicine for preventing or treating crucian carp hematopoietic organ necrosis, wherein the active ingredient of the medicine is the above-mentioned rapamycin.

As a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

As a preferred embodiment of the present invention, the dosage form of the drug is any dosage form acceptable in pharmacological therapy.

As a preferred embodiment of the present invention, the dosage form of the drug is powder.

Due to the adoption of the above scheme, the beneficial effects of the present invention are:

The present invention uses rapamycin drugs on GiCF cell models infected with CyHV-2, effectively reduces the expression of the immediate early gene ORF121 of CyHV-2, effectively reduces the expression of the PI3K/AKT/mTOR signaling pathway, can well reduce the copy number of the CyHV-2 virus, and achieves the effect of hindering viral infection and replication, thereby providing a novel method for treating CyHV-2 virus, and is expected to become a candidate drug for preventing and treating crucian carp hematopoietic organ necrosis. Therefore, the present invention provides a new method for treating crucian carp hematopoietic organ necrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the detection of cytotoxicity after rapamycin treatment by CCK-8 method in Example 1 of the present invention.

FIG. 2 is a graph showing cell viability after treatment with different concentrations of rapamycin in Example 1 of the present invention.

FIG3 is a Western Blot detection diagram after rapamycin treatment in Example 3 of the present invention.

FIG. 4 is a graph showing the detection of viral copy number after rapamycin treatment in Example 3 of the present invention.

Figure 5 is a graph showing changes in the expression levels of each gene after treatment with rapamycin of the present invention (among which: A. changes in the expression levels of ORF121 gene and key genes in the PI3K/AKT/mTOR signaling pathway after treatment with rapamycin; B. changes in the expression levels of some genes in the PI3K/AKT/mTOR signaling pathway after treatment with rapamycin; C. changes in the expression levels of some genes in CyHV-2 after treatment with rapamycin).

DETAILED DESCRIPTION

The invention provides an application of rapamycin in preparing a medicine for preventing or treating crucian carp hematopoietic organ necrosis.

The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Material

(1) Experimental strains and cells

The caudal fin of Carassius auratus gibelio cell line GiCF (The caudal fin of Carassius auratus gibelio) was constructed in this laboratory and frozen in liquid nitrogen. The culture medium used for cell recovery is M199 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in a constant temperature incubator at 27°C. The CyHV-2 isolate YC-01 (hereinafter referred to as CyHV-2) infected cells at 25°C. After all the cells died, the cell supernatant was collected and filtered with a 0.22μm syringe filter to obtain pure virus liquid, which was stored at -80°C for future use.

(2) Experimental reagents

M199 medium, fetal bovine serum, and penicillin-streptomycin 100× were purchased from Gibco; CellCounting Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; Count&Viability kit was purchased from Merck KgaA; rapamycin was purchased from MCE; DMSO was purchased from Sigma; Trizol was purchased from Invitrogen; chloroform was purchased from Shanghai Collins Reagent Co., Ltd.; anhydrous ethanol, methanol, and isopropanol were purchased from Sinopharm Chemical Reagent Co., Ltd.; DEPC-treated water, PBS, and Tween-20 were purchased from Sangon Biotech (Shanghai) Co., Ltd.; viral genomic DNA/RNA extraction kit (DP315) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; TB Premix ExTaq ^TM II (RR820A) and PrimeScript ^TM RT Master Mix (RR036A) were purchased from TaKaRa; anti-mTOR monoclonal antibody and anti-Phospho-mTOR monoclonal antibody were purchased from Cell Signaling Technology; anti-β-actin monoclonal antibody was purchased from Proteintech; anti-ORF121 polyclonal antibody was prepared in our laboratory; Goat anti-Mouse IgG (H+L)-HRP was purchased from Bioworld; Goat anti-Rabbit IgG H&L (HRP) was purchased from Abcam; PVDF membrane and ultra-sensitive ECL chemiluminescence kit were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.; Trans-Blot Turbo 5×Transfer Buffer was purchased from Bio-RAD.

Embodiment 1:

(1) Cytotoxicity assay

The mTOR inhibitor rapamycin was dissolved in DMSO at 10 mmol/L and diluted to the indicated concentrations with 10% M199 medium before administration.

GiCF cells were plated in 96-well plates one day in advance, with 5×10 ³ cells per well, and cells were seeded in quadruplicate. The treatment concentrations of rapamycin were 500nmol/L, 10μmol/L, and 20μmol/L, and the solvent group DMSO was used as a control. The cells were cultured at 27°C. Cytotoxicity was detected at 24h, 48h, and 72h after rapamycin treatment: the culture medium containing CCK reagent (10μL CCK reagent per well) was replaced, and the cells were cultured for 3h at 27°C in a constant temperature incubator, and the absorbance at 450nm was detected using a multi-function microplate reader Synergy ^TM 2.

(2) Cell viability assay

GiCF cells were plated in 6-well plates one day in advance, with 10 ⁶ cells per well, and cells were inoculated in triplicate. Rapamycin treatment concentrations were 500nmol/L, 10μmol/L, and 20μmol/L, and the solvent group DMSO was used as a control. Cells were cultured at 27°C for 72h. All cells in the well plate were collected and resuspended in PBS. 50μL of cells were mixed with 450μL of Muse Count&Viability reagent, incubated at room temperature in the dark for 5min, and then detected using the MUSE all-purpose cell state analyzer.

Embodiment 2:

Rapamycin inhibition assay

In vitro inhibition experiments were first pretreated with 500 nmol/L, 10 μmol/L and 20 μmol/L rapamycin (DMSO group as control) for 2 h, and then infected with CyHV-2 (MOI = 1). After 72 h of virus infection, cells were collected to detect the expression of some genes in the PI3K/AKT/mTOR signaling pathway at the mRNA and protein levels, as well as the number of CyHV-2 copies in the cell supernatant.

(1) RNA extraction

Collect the drug-treated cells, add 1mL Trizol and mix well. Add 200μL chloroform and vortex for 15s, let stand for 3min, and centrifuge at 12000×g for 15min at 4℃. Take the supernatant and add an equal amount of isopropanol, let stand for 10min, and centrifuge at 12000×g for 10min at 4℃. Discard the supernatant, add 1mL of 75% ethanol (prepared with DEPC-treated water), and centrifuge at 7500×g for 5min at 4℃. Add 20μL DEPC-treated water to dissolve RNA.

(2) Fluorescence quantitative PCR detection (RT-qPCR)

The reverse transcription reaction was performed by adding 500 ng RNA to every 10 μL system, and the experiment was performed with reference to the PrimeScript ^TM RT Master MixKit, as shown in Tables 1 and 2.

Table 1 Reverse transcription reaction system

Table 2 Reverse transcription reaction conditions

The RT-qPCR reaction system was based on TB Green Premix Ex Taq II (Tli RNaseH Plus) Kit. The relative expression of each gene was calculated by the 2 ^-ΔΔCt method (the internal reference gene was β-actin). The primer sequences used for RT-qPCR are shown in Table 3.

Table 3 RT-qPCR primers

Table 4 RT-qPCR reaction system

Table 5 RT-qPCR reaction conditions

Embodiment 3:

(1) Virus copy number detection

The recombinant plasmid pDsRed-express-N1-ORF121 was constructed by our laboratory, and its plasmid concentration was measured by NanoDrop2000. Copy number (copies/μL) = 6.02×10 ²³ (copies/mol)×plasmid concentration (ng/μL)×10 ^-9 /(recombinant plasmid base number×660g/mol). RT-qPCR analysis was performed using 10-fold gradient dilution of the plasmid as a template, and a standard curve was established using the cycle number Ct and the logarithm of the copy number as the horizontal and vertical coordinates, respectively. The primers used are listed in Table 3. The genomic DNA in 200 μL of the virus supernatant was extracted using a viral genomic DNA/RNA extraction kit, and a total of 30 μL was eluted. Using the viral genomic DNA as a template, RT-qPCR was performed using the same primers as the standard curve, and the Ct was brought into the standard curve to convert the viral copy number.

(2) Western Blot

For cell samples, 60 μL 2×SDS-PAGE loading buffer was added to each well of the six-well plate, and cells were collected with a cell scraper. The prepared samples were boiled in a water bath for 15 min, centrifuged at 12000×g for 3 min, and the supernatant was taken for SDS-PAGE. After the gel run, the protein was transferred to the PVDF membrane using the semi-dry transfer method. The semi-dry transfer buffer was prepared as distilled water: anhydrous ethanol: Trans-BlotTurbo 5×Transfer Buffer = 3:1:1. The transfer condition was constant current 2.5A transfer for 4.5 min. The PVDF membrane after transfer was blocked with 5% skim milk at room temperature for 2 h, then incubated with the primary antibody at 4°C overnight, and washed with PBST 5 times, each time for 5 min. Then incubated with the secondary antibody at room temperature for 1.5 h, washed with PBST 5 times, each time for 5 min. ECL colorimetric solution was used for color development, and the ChemiDoc ^TM Imaging System imaging system was used for photography.

result

(1) Cytotoxicity assay

The treatment concentrations of rapamycin were 500 nmol/L, 10 μmol/L and 20 μmol/L. Taking the solvent group DMSO as the control, the cells were treated with the above concentrations for 24 h, 48 h and 72 h. As shown in Figure 1, at all tested concentrations, the cell survival rate was more than 95%, indicating that the concentrations of 20 μmol/L and below were not toxic to the cells.

(2) Cell viability assay

The results of the cell viability test are shown in FIG2 . After administration of different concentrations of rapamycin (DMSO, 500 nmol/L, 10 μmol/L and 20 μmol/L) for 72 h, the percentage of live cells was greater than 96%, indicating that the drug concentration selected by the present invention is a relatively safe experimental concentration.

(3) Rapamycin inhibits the replication of CyHV-2

GiCF cells were treated with rapamycin for 2 hours and then infected with CyHV-2 at MOI = 1. After 72 hours of virus infection, the cells were collected, and the phosphorylation of mTOR and the changes in ORF121 were detected by WB. The expression of some genes in the PI3K/AKT/mTOR signaling pathway and the copy number of CyHV-2 in the cell supernatant were detected by RT-qPCR. As shown in Figure 3, compared with the control group, rapamycin significantly inhibited the phosphorylation of mTOR and the expression of ORF121 protein at the protein level. As shown in Figure 4, the copy number of CyHV-2 virus decreased significantly. As shown in Figure 5, the ORF121 gene was significantly inhibited at the transcriptional level (Figure 5 A), and the other four immediate early genes (ORF54, ORF141, ORF147, ORF155) in the CyHV-2 genome, as well as an early gene (ORF24) and a late gene (ORF7) were all inhibited (Figure 5 C), that is, inhibiting the phosphorylation of mTOR inhibited the replication of CyHV-2. Genes of the PI3K/AKT/mTOR signaling pathway: AKT2, IRS1, PDPK1, and EIF4EBP2 were inhibited at the mRNA level (Figure 5 A and B), indicating that rapamycin mediates the PI3K/AKT/mTOR signaling pathway by inhibiting the phosphorylation of mTOR, thereby inhibiting the replication of CyHV-2.

In summary, the rapamycin of the present invention has a significant inhibitory effect on CyHV-2, and the inhibitory effect is dose-dependent. After rapamycin action, the phosphorylation of mTOR is inhibited, the expression of some genes in the PI3K/AKT/mTOR signaling pathway is inhibited, the copy number of CyHV-2 virus and the expression of some viral genes are inhibited, indicating that rapamycin mediates the PI3K/AKT/mTOR signaling pathway by inhibiting the phosphorylation of mTOR, thereby inhibiting the replication of CyHV-2.

The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Sequence Listing

<110> Shanghai Ocean University

<120> Application of rapamycin in the preparation of drugs for preventing or treating hematopoietic organ necrosis in crucian carp

<141> 2022-05-27

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Claims (4)

1. An application of rapamycin in the preparation of a drug for preventing or treating hematopoietic organ necrosis in crucian carp, the dose of rapamycin is 500 nmol/L, 10 μmol/L or 20 μmol/L. 2. The application according to claim 1, characterized in that: the rapamycin inhibits CyHV-2 virus infection and replication in crucian carp GiCF cells. 3. The application according to claim 2, characterized in that: the rapamycin inhibits the phosphorylation of mTOR and the expression of ORF121 protein at the protein level to inhibit the replication of CyHV-2. 4. The application according to claim 2, characterized in that: the rapamycin inhibits the replication of CyHV-2 by inhibiting the phosphorylation of mTOR and mediating the expression of some genes of the PI3K/AKT/mTOR signaling pathway; The partial genes are AKT2, IRS1, PDPK1 and EIF4EBP2.