Application of rosmarinic acid in bovine herpesvirus type I and bovine viral diarrhea virus infection

Abstract

The invention relates to an application of rosmarinic acid in bovine herpesvirus type I and bovine viral diarrhea virus infection. Rosmarinic acid (RA) shows obvious inhibition ability on two key pathogens, namely bovine herpesvirus type I (BHV-1) and bovine viral diarrhea virus (BVDV), which cause bovine respiratory disease syndrome (BRDC). According to the application, the bovine herpesvirus type I (BHV-1) and the bovine viral diarrhea virus (BVDV) are taken as common targets, the rosmarinic acid (RA) is revealed to be capable of simultaneously inhibiting the dual activities of the bovine herpesvirus type I and the bovine viral diarrhea virus, and the rosmarinic acid (RA) has a remarkable inhibition capability, so that the prevention and treatment effect of'one drug with double effects' is realized, and the application prospect is wide. And a more efficient strategy is provided for prevention and control of infection of the two pathogens.

Description

TECHNICAL FIELD

[0001] The application relates to traditional Chinese medicine, in particular to application of rosmarinic acid in preparation of a medicine for preventing and / or treating bovine herpes virus type I and bovine viral diarrhea virus infection. BACKGROUND

[0002] Bovine herpes virus type I (BHV-1) and bovine viral diarrhea virus (BVDV) are important pathogens causing bovine respiratory disease complex (BRDC), leading to high mortality and production performance decline of cattle, and widely exist in the world. Especially in the developed areas of cattle industry, the infection of the two viruses has become an important factor restricting the development of cattle industry. BHV-1 and BVDV can cause severe respiratory symptoms and other systemic complications. Among them, BHV-1 is an important member of the alphaherpesvirus subfamily of the herpesviridae family, is a double-stranded DNA virus, mainly spreads through the respiratory tract, has strong infectivity, and can cause acute infection and latent infection of cattle. BVDV is a member of the Flaviviridae family - Pestivirus genus, is an enveloped single-stranded positive-strand RNA virus, and can cause persistent infection and immunosuppression of cattle herd. At present, there is still no specific medicine for preventing and treating the two infectious diseases, and vaccination is the core strategy for preventing the two diseases. However, the current domestic vaccines for preventing the two diseases are all inactivated vaccines, such as bovine infectious rhinotracheitis inactivated vaccine (C1 strain), bovine viral diarrhea / mucosal disease-bovine infectious rhinotracheitis bivalent inactivated vaccine (type 1, NM01 strain + LN01 / 08 strain), etc., and there are problems of low protection rate and short immune duration. In view of the universality and harmfulness of the infection of the two viruses, it is of great value to find an effective antiviral strategy for the prevention and control of the disease. In addition, as two important pathogens of BRDC, the existing research and prevention strategies often study BHV-1 and BVDV independently, and there are problems of complex drug use, large toxic and side effects, etc.

[0003] Rosmarinic acid (RA) is a water-soluble polyphenolic natural compound, which widely exists in Labiatae plants. Recent studies have shown that RA has significant anti-inflammatory, antioxidant, neuroprotective and metabolic regulation effects. In addition, RA also shows good antibacterial and antiviral activity. Related studies have found that RA shows significant inhibition effect on various bacteria such as Staphylococcus aureus and Escherichia coli, and also shows potential application prospect in anti-dengue virus and influenza virus. However, whether RA has the effect of resisting BHV-1 and BVDV has not been reported. SUMMARY

[0004] The purpose of the present application is to provide a rosmarinic acid for application in bovine herpes virus type I and bovine viral diarrhea virus infection, which has high protection rate, long action time, simple drug use and small toxic and side effects.

[0005] The technical solution of the present application is: Use of rosmarinic acid in the preparation of a medicament for preventing and / or treating bovine herpes virus type I (BHV-1) and bovine viral diarrhea virus (BVDV) infection.

[0006] A pharmaceutical composition for preventing and / or treating bovine herpes virus type I (BHV-1) and bovine viral diarrhea virus (BVDV) infection, comprising a therapeutically effective amount of rosmarinic acid and a pharmaceutically acceptable carrier.

[0007] Rosmarinic acid (RA) shows significant inhibitory ability to two key pathogens of bovine respiratory disease complex (BRDC), bovine herpes virus type I (BHV-1) and bovine viral diarrhea virus (BVDV).

[0008] The technical effect of the present application is that the present application takes bovine herpes virus type I (BHV-1) and bovine viral diarrhea virus (BVDV) as common targets, reveals the dual drug antiviral activity of rosmarinic acid (RA) in simultaneously inhibiting the two viruses, realizes the prevention and treatment effect of "one drug with double effect", and provides a more efficient strategy for the prevention and control of mixed infection of the two pathogens. Rosmarinic acid (RA) shows significant inhibitory ability to two key pathogens (BHV-1 and BVDV) of BRDC. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 Effect of RA on the viability of BT cells; Figure 2 Effect of RA on the replication of BHV-1, (A) qRT-PCR detection of BHV-1 gB gene level; (B) Western blot detection of BHV-1 VP8 protein expression; (C) VP8 protein gray scale analysis; (D, E) IFA detection of BHV-1 VP8 protein; Figure 3 Effect of RA on the replication of BVDV, (A) qRT-PCR detection of BVDV 5'UTR gene level; (B) Western blot detection of BVDV E2 protein expression; (C) E2 protein gray scale analysis; Figure 4 Effect of RA on the clinical score of BHV-1 infected New Zealand white rabbits; Figure 5 Effect of RA on the viral load in the nasal wash and lung of BHV-1 infected New Zealand white rabbits, (A) Viral copy number in nasal wash; (B) Viral copy number in lung; Figure 6The effect of RA on lung pathological damage in BHV-1 infected New Zealand white rabbits; Figure 7 The effect of RA on BVDV 5'UTR gene levels. (AD) represent the expression of the BVDV 5'UTR gene in the lungs, duodenum, jejunum, and spleen, respectively. Figure 8 The effect of RA on BVDV E2 protein expression. (AD) represent the expression of BVDV E2 protein in the lungs, duodenum, jejunum, and spleen, respectively. Figure 9 The effect of RA on pathological damage to the lungs and duodenum of BVDV-infected mice. (B) Histopathological examination of the lungs; (C) Histopathological examination of the duodenum. Detailed Implementation

[0010] 1. Materials and Methods 1.1. Virus, cell and laboratory animal sources Bovine nasal turbinate epithelial cells (BT), BHV-1 isolate DQ2023, and CP BVDV (NADL strain, No. VR-534) were all provided by Heilongjiang Bayi Agricultural Reclamation University (preserved by the Heilongjiang Provincial Engineering Technology Research Center for Bovine Disease Prevention and Control). Female SPF-grade BALB / c mice aged 6-8 weeks and healthy New Zealand white rabbits aged 2-3 months were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The experimental animal production license number is SCXK(Liaoning)2020-0001. Animal experiments were approved by the Experimental Animal Ethics Committee of Heilongjiang Bayi Agricultural Reclamation University, approval number: DWKJXY2023029.

[0011] 1.2. Main Reagents RA (purity >98%) was purchased from Chengdu Mansite Biotechnology Co., Ltd. GAPDH antibody was purchased from Wuhan Sewell Biotechnology Co., Ltd. (CAS No.: GB15002). BHV-1 VP8 monoclonal antibody and BVDV E2 monoclonal antibody were preserved by the Heilongjiang Provincial Engineering Technology Research Center for Bovine Disease Prevention and Control. Goat anti-mouse IgG antibody was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (CAS No.: ZB-2305).

[0012] 1.3. RA for BT cell viability detection When the BT cell fusion degree reached 80%, different concentrations of RA (40-320 μg / mL) were added to treat the cells for 48 h, and 6 repeated holes were set for each concentration. After washing the cells with PBS, 100 μL of DMEM culture medium containing 10% CCK-8 was added to each hole, and incubated at 37°C for 1.5 h in the dark. The multifunctional enzyme label instrument was used to measure the optical density (OD) value of each hole at 450 nm main wavelength.

[0013] 1.4. Experimental rabbit grouping, treatment and sample collection 60 healthy 2-3 month old New Zealand white rabbits were randomly divided into Control group, BHV-1 group, RA (15 mg / kg) + BHV-1 group, RA (30 mg / kg) + BHV-1 group, RA (60 mg / kg) + BHV-1 group, and RA (60 mg / kg) group, with 10 rabbits in each group. BHV-1 was inoculated by eye drops, and was given intragastrically 24 h before inoculation (-1 d), twice a day. The clinical score statistics were performed at 09:00 am every day, and the specific scoring criteria are shown in Table 1. On the 3rd day after infection, nasal swabs were collected, and euthanasia was performed, followed by collection of rabbit lung tissue for pathological analysis and viral load detection.

[0014] Table 1 Clinical Scoring Sheet Table 1 Clinical Scoring Sheet Scoring criteria Body temperature Respiratory symptoms Mental status Nose and eye discharge 0 points ≤39.5℃ Normal respiration Normal condition None 1 point Increase of 0.5°C Mild breathing difficulty Head down, not often moving Serous 2 points Increase of 1.0°C Breathing difficulty, with cough Lying down, not moving Purulent 3 points Increase of 1.5°C Gurgling or sharp breathing sound Difficulty standing Large amount of purulent 4 points Increase of 2.0°C None None None 1.5. Experimental mouse grouping, treatment and sample collection BALB / c mice were randomly divided into 6 groups, 10 in each group, namely: Control group, BVDV group, RA (25 mg / kg) + BVDV group, RA (50 mg / kg) + BVDV group, RA (100 mg / kg) + BVDV group, and RA (100 mg / kg) group. According to the BVDV-infected mouse model established by the research group in the early stage, the mice were given intragastrically 24 h before inoculation (-1 d), twice a day. On the 7th day after infection, euthanasia was performed, and the lung, duodenum, jejunum and spleen of the mice were collected for pathological analysis and viral load detection.

[0015] 1.6. Real-time fluorescent quantitative PCR (qRT-PCR) detection BHV-1 DNA was extracted from cells and tissues using the TIANGEN Genomic DNA Extraction Kit (DP304). BVDV RNA was extracted using the TRIZOL method and reverse transcribed into cDNA. The PCR amplification program was set as follows: 95℃ pre-denaturation for 30 s, followed by 45 cycles of 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. Negative and positive controls were included in the experiment, and absolute quantification of BHV-1 was performed using a standard curve. GAPDH was used as an internal control, and 2- △△Ct The method was used to determine the relative expression level of BVDV mRNA.

[0016] 1.7. Western blot detection Total cellular protein was extracted from BT cells using RIPA lysis buffer. Protein concentration was determined using the BCA protein assay. Subsequently, protein samples were separated by SDS-PAGE gel electrophoresis and successfully transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk. After 2 h, the PVDF membrane was incubated with primary antibody. The following day, the PVDF membrane was washed four times with TBST for 10 min each time. After incubating the membrane with secondary antibody for 1 h, the immunoblotting detection system was used to detect the immunoreaction bands.

[0017] 1.8. Indirect immunofluorescence (IFA) Cells were fixed with 4% paraformaldehyde for 15 min and washed three times with PBS. Each well was treated with 0.5% Triton X-100 for 20 min to permeate the cell membrane. After washing three times with PBS, IBRV VP8 antibody was added, and the cells were incubated overnight at 4°C. The next day, fluorescent secondary antibody was added, and the cells were incubated at 37°C for 1 h. After washing three times with PBST, the cells were stained with DAPI and observed and photographed under a fluorescence microscope.

[0018] 1.9. Data Analysis Experimental data were statistically analyzed using SPSS 17.0, with each experiment repeated three times. All data are expressed as mean ± standard deviation (SD). Differences in the means of normally distributed data were assessed using one-way ANOVA (Tukey's multiple comparison test) and Student's t-test. p <0.05 and ** p <0.01 represents significant and highly significant differences, respectively.

[0019] 2. Results 2.1. Effects of RA on BT cell viability like Figure 1As shown, RA (320, 640 μg / mL) treatment significantly inhibited BT cell activity compared with the untreated group. However, RA (40, 80, 160 μg / mL) did not produce significant toxic effects on BT cells.

[0020] 2.2. RA inhibits BHV-1 replication in BT cells As shown in Fig. 2A, RA treatment significantly reduced viral gB gene expression compared with the BHV-1 group in a dose-dependent manner. Figure 2 A). Western blot analysis also found that RA treatment significantly reduced viral VP8 protein expression compared with the BHV-1 group (B, C). Figure 2 D, E). Thus, it was found that RA inhibited the expression of VP8 protein of BHV-1 in a dose-dependent manner. Figure 2

[0021] 2.3. RA inhibits BVDV replication in BT cells As shown in Fig. 3A, RA significantly inhibited BVDV mRNA gene expression compared with the BVDV group, and RA (160 μg / mL) was the most effective. Figure 3 A). Analysis of BVDV E2 protein levels also found that RA inhibited BVDV replication in a dose-dependent manner (B, C). Figure 3

[0022] Thus, it was found that RA inhibited the replication of BHV-1 and BVDV in vitro at a concentration of 40 μg / mL to 160 μg / mL.

[0023] 2.4. Protective effect of RA on BHV-1 -infected New Zealand white rabbits 2.4.1. Effect of RA on clinical scores of BHV-1 -infected New Zealand white rabbits Rising body temperature and increased nasal discharge are clinical manifestations of BHV-1 -infected New Zealand white rabbits. In this study, some New Zealand white rabbits showed symptoms such as rising body temperature on the first day of BHV-1 infection, and the clinical score reached a peak on the third day. Although RA (40 μg / mL) did not significantly reduce the clinical score compared with the BHV-1 group, the clinical score of the animals in this group showed a decreasing trend. Figure 3 However, RA (80 μg / mL) and RA (160 μg / mL) significantly reduced the clinical manifestations of New Zealand white rabbits on the second and third days (Fig. 2B).

[0024] ​​2.4.2. Effect of RA on the viral gB gene level in the nasal swabs and lung tissues of BHV-1 infected New Zealand white rabbits The results are shown in Figure 5 A. RA treatment significantly reduced the viral copy number in the nasal swabs of New Zealand white rabbits at day 3 compared to the BHV-1 group. The detection of the BHV-1 gB gene level in the lung tissues also found that RA reduced the viral copy number in the lung tissues in a dose-dependent manner (B). Figure 5 B). Thus, RA inhibited the gB gene expression of BHV-1 in a dose-dependent manner. RA reduced the viral load in the nasal swabs and lung tissues of BHV-1 infected animal model.

[0025] 2.4.3. Effect of RA on the lung histopathological lesions of BHV-1 infected New Zealand white rabbits The results are shown in Figure 6 The lung tissues of the Control group New Zealand white rabbits were covered with a smooth serosa, and no obvious abnormalities were observed. The alveolar walls of the BHV-1 group New Zealand white rabbits were moderately thickened in a large area, the alveolar septa were widened, and a large number of alveoli were compensatory dilated. In addition, a small amount of granulocyte infiltration was observed in the alveolar wall (orange arrow), and a large number of lymphocyte focal infiltration was observed around the bronchioles and blood vessels (cyan arrow). Although RA (40 μg / mL) treatment did not significantly improve the thickening of the alveolar wall, it reduced the inflammatory cell infiltration. However, RA (80 μg / mL) and RA (160 μg / mL) treatment significantly improved the lung pathological damage induced by BHV-1. Thus, RA reduced the lung pathological damage of BHV-1 infected animal model.

[0026] 2.5. Protective effect of RA on BVDV infected mice 2.5.1. Effect of RA on the viral 5'UTR gene level in the lung, duodenum, jejunum and spleen of BVDV infected mice The detection of the viral 5'UTR gene level in the lung, duodenum, jejunum and spleen of BVDV infected mice at day 7 by qRT-PCR found that RA significantly inhibited the viral load in the above organs in a dose-dependent manner (A-D). Thus, RA inhibited the 5'UTR gene expression of BVDV in a dose-dependent manner. Figure 7

[0027] 2.5.2. Effect of RA on the viral E2 protein expression in the lung, duodenum, jejunum and spleen of BVDV infected mice The detection of the BVDV E2 protein level in each group sample by Western blot found that RA significantly inhibited the viral load in the lung and spleen of mice (A and B). Figure 8 ​A, B). Detection of viral E2 protein in the duodenum and jejunum also confirmed that RA inhibits BVDV replication in a dose-dependent manner. Figure 8 (C, D). This demonstrates that rosmarinic acid inhibits E2 protein expression in BVDV in a dose-dependent manner.

[0028] Rosmarinic acid can reduce viral load in the lungs, duodenum, jejunum and spleen of animal models infected with BVDV.

[0029] 2.5.3. Effects of RA on pathological damage to the lungs and duodenum of BVDV-infected mice The results are as follows Figure 9 As shown in Figure A, the lung structure of the Control group mice was clear and without obvious abnormalities. In the BVDV-infected group mice, the lung parenchyma showed numerous bronchioles and their terminal alveoli. Scattered granulocyte infiltration was observed in the alveolar walls (purple arrows). In addition, a small number of bronchioles showed epithelial cell necrosis and eosinophilic material (yellow arrows). However, all three concentrations of RA treatment effectively reduced granulocyte infiltration and improved bronchiolar epithelial cell necrosis and sloughing, with RA (160 μg / mL) showing the most significant effect.

[0030] Analysis of the duodenal pathological structure revealed that BVDV infection led to the necrosis and loss of some intestinal glandular structures, replaced by a small amount of connective tissue hyperplasia, accompanied by a small amount of inflammatory cell infiltration, mainly granulocytes and lymphocytes (purple arrows). Furthermore, BVDV infection also caused minor damage to the intestinal villi epithelium and separation from the lamina propria (green arrows). However, these BVDV-induced pathological damages were improved by RA in a dose-dependent manner. Figure 9 B). This demonstrates that rosmarinic acid can improve the pathological damage to the lungs and duodenum in animal models infected with BVDV.

[0031] 3. Discussion In recent years, BHV-1 has become a major pathogen affecting the health and productivity of cattle, and its persistent economic losses are becoming a key bottleneck restricting the sustainable development of large-scale farming. In the context of the limitations of vaccine application and the lack of specific treatment drugs, it is essential to find effective prevention and control strategies to protect the health of cattle and improve productivity. Traditional Chinese medicine is a treasure of world cultural heritage, with rich plant and animal medicinal resources. Due to its characteristics of multi-target, low toxicity and side effects, and easy access, it has become a hot spot for antiviral drug research and development. As a multifunctional phenolic compound, the wide range and diversity of its pharmacological effects make it an important research value in the field of drug development. To explore the effect of RA on BHV-1 replication, we first used the CCK8 method to detect the maximum safe concentration of RA on BT cells, and determined that 160 μg / mL RA had no significant toxic effect on BT cells. Based on the above data, we selected 40, 80 and 160 μg / mL RA for subsequent in vitro experiments. Antiviral research results showed that different concentrations of RA can significantly inhibit the replication of BHV-1, and show a dose-dependent manner. The above results show that RA has an inhibitory effect on BHV-1 replication in vitro.

[0032] New Zealand white rabbits are ideal animal models for studying BHV-1 infection, and their pathological processes are consistent with those of natural hosts, such as cattle. After infection, they can reproduce typical respiratory lesions. To further evaluate the in vivo anti-BHV-1 effect of RA, we used BHV-1-infected New Zealand white rabbits as a model to analyze the clinical manifestations, viral load in nasal swabs and lungs, and lung pathology of New Zealand white rabbits. The results showed that BHV-1 infection began on the first day, and some New Zealand white rabbits showed increased body temperature and increased nasal discharge. However, RA treatment did not significantly reduce the clinical score of white rabbits. The clinical score reached a peak on the third day of BHV-1 infection. However, RA treatment significantly reduced the clinical score of infected New Zealand white rabbits in a dose-dependent manner. To further clarify the effect of RA on viral replication, we detected the viral load in the nasal swabs of New Zealand white rabbits on the third day. The results showed that RA reduced the viral copy number in a dose-dependent manner, indicating that RA reduced the virus shedding of New Zealand white rabbits. In addition, the viral load in the lungs and pathological changes further indicated that RA had a protective effect on BHV-1-infected New Zealand white rabbits.

[0033] Given that BVDV is a key co-pathogenic factor of BRDC, this study systematically investigated the in vitro and in vivo antiviral activity of RA against BVDV using cell models and a BVDV-infected mouse model established by our research group. In vitro results showed that RA significantly inhibited the expression of the BVDV 5'UTR gene and E2 protein, indicating that RA has an inhibitory effect on BVDV replication in vitro. In vivo mouse experiments further demonstrated that RA significantly reduced BVDV load in the lungs, duodenum, jejunum, and spleen in a dose-dependent manner. Pathological section analysis also confirmed that RA significantly ameliorated BVDV infection-induced pathological damage in the lungs and duodenum.

[0034] In summary, this experiment successfully demonstrated that RA has significant antiviral activity against the two key pathogens that cause BRDC (BHV-1 and BVDV).

[0035] 4. Bovine experiments: 4.1 Experimental subjects: cattle infected with bovine herpesvirus type I (BHV-1) and bovine viral diarrhea virus (BVDV) causing bovine respiratory disease syndrome (BRDC).

[0036] 4.2. Administration route: Oral administration.

[0037] 4.3 Dosage: The dosage of rosmarinic acid for treating BHV-1 infection is 15 mg / kg body weight / day to 60 mg / kg body weight / day, and the dosage of rosmarinic acid for treating BVDV infection is 25 mg / kg body weight / day to 100 mg / kg body weight / day.

Claims

1. The use of rosmarinic acid in the preparation of a medicament for the prevention and / or treatment of bovine herpesvirus type I (BHV-1) and bovine viral diarrhea virus (BVDV) infection.

2. The application as described in claim 1, characterized in that: The drug is used to prevent and / or treat bovine respiratory disease syndrome (BRDC) caused by bovine herpesvirus type I (BHV-1) and bovine viral diarrhea virus (BVDV).

3. The application as described in claim 1, characterized in that: The rosmarinic acid inhibited the replication of BHV-1 and BVDV in vitro at concentrations ranging from 40 μg / mL to 160 μg / mL.

4. The application as described in claim 3, characterized in that: The rosmarinic acid inhibits the expression of the gB gene and VP8 protein of BHV-1 in a dose-dependent manner, and inhibits the expression of the 5'UTR gene and E2 protein of BVDV in a dose-dependent manner.

5. The application as described in claim 1, characterized in that: The drug can reduce viral load in nasal swabs and lung tissue of BHV-1 infected animal models and alleviate lung pathological damage.

6. The application as described in claim 1, characterized in that: The drug can reduce viral load in the lungs, duodenum, jejunum and spleen of BVDV-infected animal models and improve pathological damage in the lungs and duodenum.

7. The application as described in claim 1, characterized in that: The drug is administered orally.

8. The application as described in claim 7, characterized in that: The dosage of rosmarinic acid for treating BHV-1 infection is 15 mg / kg body weight / day to 60 mg / kg body weight / day, and the dosage of rosmarinic acid for treating BVDV infection is 25 mg / kg body weight / day to 100 mg / kg body weight / day.

9. A pharmaceutical composition comprising a therapeutically effective amount of rosmarinic acid and a pharmaceutically acceptable carrier for the prevention and / or treatment of bovine herpesvirus type I (BHV-1) and bovine viral diarrhea virus (BVDV) infection.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition is an oral preparation.

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