Application of protoporphyrin IX in preparation of products for resisting porcine reproductive and respiratory syndrome virus

Abstract

The invention relates to the technical field of medicine application, in particular to application of protoporphyrin IX in medicine for inhibiting porcine reproductive and respiratory syndrome virus. The PpIX has a remarkable antiviral effect on porcine reproductive and respiratory syndrome virus infection, can be used for preparing anti-PRRSV drugs, provides a reliable basis for treatment and prevention and control of diseases caused by the virus, and provides a new thought and an effective coping strategy for prevention and control work of PRRSV. The research also discovers the optimal action mode of PpIX for inhibiting PRRSV proliferation and the period of PRRSV replication, and a theoretical basis is provided for prevention and control of PRRS.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical application technology, specifically to the application of protoporphyrin IX in drugs that inhibit porcine reproductive and respiratory syndrome virus. Background Technology

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the genus Arterivirus of the family Arteriviridae. It is an enveloped, single-stranded, positive-sense RNA virus. PRRSV can invade the pig's body through the respiratory tract, infecting alveolar macrophages and causing porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease. This is a highly contagious acute infectious disease. The main characteristics of the disease are loss of appetite and fever in sows, especially in late pregnancy, often accompanied by abortion, and possible stillbirth and mummified fetuses. At the same time, infected piglets exhibit diverse and varying degrees of respiratory symptoms. In addition to causing the above typical clinical manifestations, PRRS also affects the pig's immune function, making it susceptible to mixed or secondary infections with other pathogens, thus increasing the complexity of the disease and the difficulty of prevention and control.

[0003] Currently, my country primarily uses vaccination to prevent PRRS, but live attenuated vaccines pose a risk of virus shedding, and inactivated vaccines have poor immunization efficacy. PRRS is antibody-dependent; low levels of antibodies can actually promote viral infection and proliferation. Therefore, the development of drugs for PRRS prevention and control has been a hot research topic.

[0004] Protoporphyrin IX (PpIX), CAS No.: 553-12-8, has the chemical structure shown in Formula 1 below.

[0005] PpIX is an intermediate in the heme biosynthesis pathway and is generally used clinically as a photosensitizer in photodynamic therapy for cancer. PpIX is also widely used in anticancer, antibacterial, and antifungal treatments. Whether PpIX can inhibit PRRSV proliferation has not yet been reported.

[0006] (Formula 1). Summary of the Invention

[0007] The purpose of this invention is to provide the application of protoporphyrin IX in drugs that inhibit porcine reproductive and respiratory syndrome virus.

[0008] This invention is the first to discover that PpIX can significantly reduce the infection of porcine reproductive and respiratory syndrome virus (PRRSV), thereby more effectively controlling PRRS and mitigating its impact on the livestock industry and agricultural economy.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: Application of protoporphyrin IX in the preparation of anti-porcine reproductive and respiratory syndrome virus products.

[0010] The product is preferably a drug or feed additive.

[0011] Preferably, the anti-porcine reproductive and respiratory syndrome virus is used for the prevention or treatment of porcine reproductive and respiratory syndrome virus infection, or to inhibit the proliferation of porcine reproductive and respiratory syndrome virus.

[0012] Preferably, the anti-porcine reproductive and respiratory syndrome virus (PRRSV) is used to inhibit viral infection during the adsorption, internalization, or replication phases of PRRSV.

[0013] Preferably, the anti-porcine reproductive and respiratory syndrome virus (PRRSV) is used to reduce the PRRSV titer, reduce the PRRSV N protein content, or kill the PRRSV.

[0014] Preferably, the dosage form of the drug is a gastrointestinal dosage form.

[0015] Preferably, the gastrointestinal dosage form is a tablet or granule.

[0016] For PAM-KNU cells, the concentration of PpIX in the product does not exceed 3 μmol / L.

[0017] The present invention has the following beneficial effects: This invention is the first to discover that PpIX has a significant antiviral effect against porcine reproductive and respiratory syndrome virus (PRRSV) infection, and can be used to prepare anti-PRRSV drugs. This provides a reliable basis for the treatment and prevention of diseases caused by this virus, and offers new ideas and effective strategies for PRRSV control. This study also discovered the optimal mechanism of action of PpIX in inhibiting PRRSV proliferation and the PRRSV replication cycle, providing a theoretical basis for PRRS control. Attached Figure Description

[0018] Figure 1 Results of safety concentration determination of different concentrations of PpIX in PAM-KNU cells; Figure 2 The results are for the determination of PpIX's anti-PRRSV activity. Figure 3 Results show the optimal mechanism of action of PpIX in inhibiting PRRSV proliferation; Figure 4 The result of PpIX suppressing the PRRSV replication cycle. Detailed Implementation

[0019] The technical solution of this application will be specifically described below with reference to specific embodiments. The described embodiments are merely some embodiments of this invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without innovative effort are within the scope of protection of this invention.

[0020] Example 1: Determination of safe concentrations of different concentrations of PpIX in PAM-KNU cells Immortalized porcine alveolar macrophages (PAM-KNU) were evenly seeded in 96-well plates, 100 μL per well. When the cell density reached 80%, the original culture medium was discarded, and 100 μL of different concentrations of PpIX (diluted with 1640 culture medium containing 10% FBS, 100 U / mL penicillin, and 50 μg / mL streptomycin) were added to each well, with concentrations of 1, 2, 3, and 4 μM. A cell control group and a blank control group were also set up, with 3 replicates in each group. After incubation at 37 ℃ for 24 h, 10 μL of CCK8 was added to each well, and the cells were cultured for another 2 h. The absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated using the cell viability calculation formula to determine the cell survival rate of PpIX under different concentration conditions and to determine the maximum safe concentration of PpIX.

[0021] Cell viability = [OD(drug-treated) - OD(blank)] / [OD(normal) - OD(blank)] × 100%.

[0022] Wherein, OD (drug-treated) is the absorbance of the wells in the drug-treated group after drug treatment, OD (blank) is the absorbance of the wells in the blank group without cells, and OD (normal) is the absorbance of the wells in the normal cell control group.

[0023] Experimental results are as follows Figure 1 As shown, compared with normal control cells, PpIX concentrations of 3 μM and below had no significant effect on PAM-KNU cell viability.

[0024] Example 2: Determination of PpIX's anti-PRRSV activity 2.1 Detection of viral cDNA copy number by real-time PCR PAM-KNU cells were seeded in 24-well plates. After the cells reached a confluent monolayer, the nutrient solution was discarded, and cell maintenance medium containing different concentrations of PpIX (1, ​​2, and 3 μM) was added. The plates were then incubated in a CO2 incubator at 37 °C for 24 h. The cells were washed twice with PBS, and 1 MOI of PRRSV was added. The plates were incubated at 37 °C for another 24 h. The supernatant and cells were collected separately. RNA was extracted from the collected cells and reverse transcribed. Using the reverse-transcribed cDNA as a template and PRRSV-NF and PRRSV-NR as primers, quantitative real-time PCR was performed to detect the PRRSV copy number in the samples. A standard curve was plotted using plasmid pXJ41-N as a template and PRRSV-NF and PRRSV-NR as primers. The results are as follows: Figure 2 As shown in (A), the viral cDNA copy number after PpIX treatment was significantly downregulated compared with the viral control group without PpIX.

[0025] The primers used for quantitative real-time PCR are: PRRSV-NF: 5'-AATAACAACGGCAAGCAGCAG-3' PRRSV-NR: 5'-CCTCTGGACTGGTTTTGTTGG-3' 2.2 TCID 50 Determining viral titer MARC-145 cells were seeded in 96-well plates and cultured. When the cell density reached approximately 90%, the cell supernatant from step 2.1 was serially diluted 10-fold. -1 Up to 10 -10 Discard the cell supernatant from the 96-well plate. Add the virus solution of each dilution to the 96-well plate, performing six replicates for each gradient. Observe the lesion condition daily. Once the lesion stabilizes, calculate the TCID of the virus using the Reed-Muench method. 50 The result is as follows Figure 2 As shown in (B), the viral titer was significantly reduced after PpIX treatment compared to the viral control group.

[0026] 2.3 Western blotting to detect the expression level of PRRSV N protein PAM-KNU cells were seeded in 24-well plates. After the cells reached a confluent monolayer, the nutrient solution was discarded, and cell maintenance medium containing different concentrations of PpIX (1, ​​2, 3 μM) was added. The plates were then incubated in a CO2 incubator at 37 °C for 24 h. The supernatant was discarded, and the cells were washed twice with PBS. 1 MOI of PRRSV was added, and the plates were incubated at 37 °C for 24 h. The samples were collected, the supernatant was discarded, and the cells were washed 2-3 times with PBS. An appropriate amount of cell lysis buffer was added, and the cells were lysed for 20 min. The samples were collected into 1.5 mL EP tubes, centrifuged at 12000 r / min at 4 °C for 10 min, and the supernatant was collected. An appropriate amount of loading buffer was added, and the tubes were boiled for 10-15 min to identify protein expression.

[0027] After SDS-PAGE gel electrophoresis, the samples were transferred to an NC membrane, blocked with 5% skim milk at room temperature for 2 h, incubated overnight at 4 °C with mouse anti-PRRSV-N antibody as the primary antibody, and then incubated with HRP-labeled goat anti-mouse secondary antibody at room temperature for 1 h. The samples were then developed using ECL Plus ultrasensitive luminescent solution, exposed using a BIO-RAD gel imaging system, and finally analyzed for grayscale using β-actin as a reference. Figure 2 As shown in (C) and (D), the N protein content of PRRSV in cells was significantly reduced after PpIX treatment.

[0028] Example 3: Optimal Mechanism of Action of PpIX in Inhibiting PRRSV Proliferation 3.1 Preventive effect PAM-KNU cells were seeded into 24-well plates. After the cells reached a confluent monolayer, different concentrations of PpIX (1, ​​2, and 3 μM) were added to the cell culture plates, and the plates were incubated at 37 °C for 4 h. The cells were then infected with 1 MOI of PRRSV, and incubated at 37 °C for 2 h. The supernatant was discarded, and the cells were washed twice with PBS. Cell maintenance medium was added, and the plates were incubated at 37 °C for 48 h. The cells and supernatant were collected, and the PRRSV cDNA copy number was detected by real-time PCR. Figure 3 As shown in (A), after PpIX pretreatment, the PRRSV cDNA copy number was significantly lower than that of the control group, and this was in a dose-dependent manner.

[0029] 3.2 Direct killing effect PAM-KNU cells were seeded into 24-well plates. After the cells reached a confluent monolayer, 1 MOI PRRSV was prepared using different concentrations of PpIX (1, ​​2, 3 μM). The cells were incubated at 4 °C for 1 h, then added to cell culture plates and incubated at 37 °C for 2 h. The supernatant was discarded, and the cells were washed twice with PBS. Cell maintenance medium was added, and the cells were incubated at 37 °C for 48 h. The cells and supernatant were collected, and the PRRSV cDNA copy number was detected by real-time PCR. Figure 3As shown in (B), after PpIX treatment with direct killing effect, the PRRSV cDNA copy number was lower than that of the control group.

[0030] 3.3 Therapeutic effects PAM-KNU cells were seeded into 24-well plates. After the cells reached a confluent monolayer, 1 MOI of PRRSV was added, and the cells were incubated at 37 °C for 2 h. The supernatant was discarded, and the cells were washed twice with PBS. Different concentrations of PpIX (1, ​​2, and 3 μM) were added, and the cells were incubated at 37 °C for 48 h. The cells and supernatant were collected, and the PRRSV cDNA copy number was detected by real-time PCR. Figure 3 As shown in (C), PpIX was used for treatment, and the PRRSV cDNA copy number was lower than that in the control group.

[0031] The results showed that PpIX treatment for prevention resulted in the lowest viral cDNA copy number, with the greatest difference compared to the viral control group, and exhibited poor direct killing and therapeutic effects. These results indicate that PpIX was most effective in inhibiting viral replication through preventative measures.

[0032] Example 4: Specific steps of PpIX in inhibiting the PRRSV replication cycle 4.1 Virus Adsorption Period PAM-KNU cells were seeded into 24-well plates. The confluent cell culture plates were pre-cooled at 4 °C for 1 h. Different concentrations of PpIX (1, ​​2, 3 μM) were added to prepare 1 MOI PRRSV. The cells were incubated at 4 °C for 2 h, the supernatant was discarded, and the cells were washed twice with PBS. Cell maintenance medium was added, and the cells were incubated at 37 °C for 48 h in a CO2 incubator. The cells and supernatant were collected, and the PRRSV cDNA copy number was detected by Real-time PCR. Figure 4 As shown in (A), when PpIX acts on the adsorption process of viral infection, the PRRSV cDNA copy number is lower than that of the control group.

[0033] 4.2 Viral Internalization Period PAM-KNU cells were seeded into 24-well plates. The confluent cell culture plates were pre-cooled to 4 °C for 1 h, and 1 MOI of PRRSV was added. The plates were incubated at 4 °C for 2 h. The supernatant was discarded, and the cells were washed twice with PBS. Different concentrations of PpIX (1, ​​2, 3 μM) were added, and the plates were incubated at 37 °C for 2 h in a CO2 incubator. The supernatant was discarded, and the cells were washed twice with PBS. Cell maintenance medium was added, and the plates were incubated at 37 °C for 48 h. The cells and supernatant were collected, and the PRRSV cDNA copy number was detected using Real-time PCR. Figure 4 As shown in (B), when PpIX acts on the internalization process of viral infection, the PRRSV cDNA copy number is lower than that in the control group.

[0034] 4.3 Virus Replication Period PAM-KNU cells were seeded into 24-well plates. After the cells reached a confluent monolayer, 1 MOI of PRRSV was added, and the cells were incubated at 37 °C for 2 h in a CO2 incubator. The supernatant was discarded, and the cells were washed twice with PBS. The optimal concentration of PpIX (3 μM) was added, and the cells were incubated at 37 °C for 0 h. The supernatant and cells were collected at 0, 3, 6, 9, 12, and 24 h, and the PRRSV cDNA copy number was detected by Real-time PCR. Figure 4 As shown in (C), when PpIX acts on the viral replication process, the PRRSV cDNA copy number is lower than that of the control group.

[0035] The results showed that PpIX inhibited the adsorption, internalization, and replication of the virus.

[0036] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments. Any changes, modifications, combinations, substitutions, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. Application of protoporphyrin IX in the preparation of anti-porcine reproductive and respiratory syndrome virus products.

2. The application according to claim 1, characterized in that... The product is a drug or feed additive.

3. The application according to claim 1, characterized in that... The anti-porcine reproductive and respiratory syndrome virus is used to prevent or treat porcine reproductive and respiratory syndrome virus infection, or to inhibit the proliferation of porcine reproductive and respiratory syndrome virus.

4. The application according to claim 1, characterized in that... The anti-porcine reproductive and respiratory syndrome virus (PRRSV) is designed to inhibit viral infection during the adsorption, internalization, or replication phases of PRRSV.

5. The application according to claim 1, characterized in that... The anti-porcine reproductive and respiratory syndrome virus (PRRSV) is used to reduce the PRRSV titer, reduce the PRRSV N protein content, or kill the PRRSV.

6. The application according to claim 2, characterized in that... The drug is in the form of a gastrointestinal medication.

7. The application according to claim 6, characterized in that... The gastrointestinal drug delivery dosage forms are tablets and granules.

8. The application according to claim 1, characterized in that... For PAM-KNU cells, the mass concentration of PpIX in the product does not exceed 3 μmol / L.

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