Application of Hydroxychloroquine Sulfate in the Preparation of Drugs for Preventing and Treating Porcine Reproductive and Respiratory Syndrome

Abstract

The present invention discloses the use of hydroxychloroquine sulfate in the preparation of a medicament for preventing and treating porcine reproductive and respiratory syndrome. The present invention discloses that hydroxychloroquine sulfate has a significant effect against porcine reproductive and respiratory syndrome virus. Its maximum non-toxic dose is 100 μM, which can effectively inhibit the infection and proliferation of porcine reproductive and respiratory syndrome virus and has a good therapeutic effect on PRRSV infection in piglets. It can be used as a medicament for preventing and treating porcine reproductive and respiratory syndrome and has good application prospects in the prevention and treatment of porcine reproductive and respiratory syndrome.

Description

Technical Field

[0001] The present invention relates to the technical field of antiviral, and more specifically, to the application of hydroxychloroquine sulfate in the preparation of a medicament for preventing and treating porcine reproductive and respiratory syndrome. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ear disease, is an acute and highly contagious viral disease characterized by reproductive disorders and respiratory symptoms in swine herds. It is caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). In addition, PRRSV can suppress the immune system and increase the risk of secondary infections, such as Streptococcus suis and Haemophilus parasuis. PRRSV is a single-stranded positive-sense RNA virus belonging to the genus Arterivirus of the family Arteriviridae. The diameter of the internal nucleocapsid of the virus is about 25-30 nm. Most virus particles are spherical or oval, showing icosahedral symmetry and appearing smooth under an electron microscope. Genome sequencing results show that PRRSV can be divided into two types: PRRSV-I (European type, prototype strain Lelystad virus) and PRRSV-II (North American type, prototype strain VR-2332 virus). The PRRSV genome is about 15.4 kb and has more than 11 open reading frames (ORFs), encoding at least 16 non-structural proteins and 8 structural proteins. PRRSV results in decreased production efficiency, increased mortality, and increased treatment and prevention costs. Since the discovery of PRRSV more than 30 years ago, it has continuously threatened the global pig industry and caused huge economic losses.

[0003] Currently, the prevention and control of PRRSV still mainly rely on vaccine research and development and biosecurity measures. There are few direct and effective antiviral drugs. Other antiviral drugs for livestock are basically ineffective in the treatment of PRRSV. Traditional antibiotics can only control secondary infections. The prevention and control of PRRSV mainly rely on vaccines and biosecurity measures. However, due to the genetic diversity of PRRSV and the complexity of the immune response, the effect of PRRSV vaccines is not ideal. Therefore, in the current situation, it is particularly important to develop effective anti-PRRSV drugs.

[0004] Hydroxychloroquine Sulphate (HCQ Sulfate) is an antimalarial drug used to treat autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, inflammation, and skin diseases. It is also an inhibitor of autophagy and toll-like receptor (TLR) 7 / 9. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide the use of hydroxychloroquine sulfate in the preparation of a medicament for preventing and treating porcine reproductive and respiratory syndrome.

[0006] The first object of the present invention is to provide the use of hydroxychloroquine sulfate in the preparation of a medicament for preventing and treating porcine reproductive and respiratory syndrome.

[0007] The second object of the present invention is to provide the use of hydroxychloroquine sulfate in the preparation of a medicament for preventing and treating porcine reproductive and respiratory syndrome virus.

[0008] In order to achieve the above object, the present invention is realized by the following technical solutions:

[0009] The present invention claims the following uses:

[0010] The use of hydroxychloroquine sulfate in the preparation of a medicament for preventing and treating porcine reproductive and respiratory syndrome.

[0011] The use of hydroxychloroquine sulfate in the preparation of a medicament for preventing and treating porcine reproductive and respiratory syndrome virus.

[0012] Preferably, the medicament further comprises a pharmaceutically acceptable excipient.

[0013] Preferably, the prevention and treatment of porcine reproductive and respiratory syndrome is to inhibit or avoid the occurrence of cytokine storm.

[0014] More preferably, the occurrence of cytokine storm is related to IL-1β and / or IL-6.

[0015] Preferably, the prevention and treatment of porcine reproductive and respiratory syndrome virus is to inhibit the infection of porcine reproductive and respiratory syndrome virus.

[0016] Preferably, the prevention and treatment of porcine reproductive and respiratory syndrome virus is to inhibit the proliferation of porcine reproductive and respiratory syndrome virus.

[0017] Preferably, the effective use concentration of hydroxychloroquine sulfate is 1-100 μΜ.

[0018] More preferably, the effective use concentration of hydroxychloroquine sulfate is 100 μΜ.

[0019] Preferably, the effective use dose of hydroxychloroquine sulfate is 6-7 mg / kg.

[0020] More preferably, the effective use dose of hydroxychloroquine sulfate is 6.5 mg / kg.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention discloses that hydroxychloroquine sulfate has a significant effect against porcine reproductive and respiratory syndrome virus. Its maximum non-toxic dose is 100 μM, which can effectively inhibit the infection and proliferation of porcine reproductive and respiratory syndrome virus, and has a good therapeutic effect on PRRSV infection in piglets. It can be used as a drug for preventing and treating porcine reproductive and respiratory syndrome, and has good application prospects in the prevention and treatment of porcine reproductive and respiratory syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the experimental result of using the CCK-8 kit to determine the toxicity of hydroxychloroquine sulfate to PAM cells and Marc-145 cells.

[0024] Figure 2 It is the result of detecting the expression of PRRSV N protein after treating Marc-145 cells with different concentrations of hydroxychloroquine sulfate by IFA.

[0025] Figure 3 It is the result of detecting the expression of PRRSV N protein after treating PAM and Marc-145 cells with different concentrations of hydroxychloroquine sulfate by Western Blot.

[0026] Figure 4 It is the result of using real-time fluorescence quantitative PCR to determine the level of PRRSV Nsp9 mRNA after treating PAM and Marc-145 cells with different concentrations of hydroxychloroquine sulfate.

[0027] Figure 5 It is the result of using real-time fluorescence quantitative PCR to determine the gene expression of inflammatory factors IL-1β and IL-6 after treating PAM cells infected with PRRSV with different concentrations of hydroxychloroquine sulfate to inhibit the infection.

[0028] Figure 6 It is the effect of hydroxychloroquine sulfate treatment on the survival rate, body temperature and body weight of piglets infected with PRRSV.

[0029] Figure 7 It is the ELISA test result of PRRSV antibody in piglets infected with PRRSV after hydroxychloroquine sulfate treatment. The abscissa is the number of days after infection with PRRSV.

[0030] Figure 8 It is the RT-qPCR test result of Nsp9 in piglets infected with PRRSV after hydroxychloroquine sulfate treatment. The abscissa is the number of days after infection with PRRSV.

[0031] Figure 9 It is the effect of hydroxychloroquine sulfate treatment on the histopathology of the lungs of piglets infected with PRRSV. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the test methods used in the following embodiments are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0033] Hydroxychloroquine sulfate, CAS 747-36-4, was purchased from Chengdu Purigen Bioscience and hereinafter referred to as HCQ in the examples and appendix.

[0034] PRRSV: Highly pathogenic PRRSV strain XHGD (GenBank accession number EU624117), stored in the group of Zhang Guihong in the College of Veterinary Medicine, South China Agricultural University.

[0035] PRRSV N antibody: JNT, China; no. JN0401.

[0036] The XH-GD plasmid is a plasmid containing the full-length cDNA of the highly pathogenic PRRSV strain XHGD, namely pOKQ-XH. See the prior art: Wu Xinwei, Xiong Yongzhong, Qin Hongyang, etc. Construction of an infectious clone of porcine reproductive and respiratory syndrome virus strain XH [J]. Chinese Journal of Preventive Veterinary Medicine, 2011, 33(2): 97-100. DOI: 10.3969 / j.issn.1008-0589.2011.02.04, stored in the group of Zhang Guihong in the College of Veterinary Medicine, South China Agricultural University.

[0037] Example 1 Cytotoxicity of hydroxychloroquine sulfate on PAM and Marc-145 cells

[0038] I. Experimental method

[0039] 1. Preparation of hydroxychloroquine sulfate stock solution

[0040] Prepare a 10 mM aqueous solution of hydroxychloroquine sulfate as the hydroxychloroquine sulfate stock solution. After aliquoting, wrap it with tin foil and store it at -20°C for later use.

[0041] 2. Treatment of PAM cells with hydroxychloroquine sulfate

[0042] Dilute the hydroxychloroquine sulfate stock solution with RPMI Medium 1640 medium containing 10% FBS (containing penicillin 100 U / mL, streptomycin 50 μg / mL, amphotericin 0.25 μg / mL) so that the concentration of hydroxychloroquine sulfate reaches 1, 10, 50, 100, 150, 200, 250, 500, 1000, 1500 μM to obtain RPMI Medium 1640 medium containing different concentrations of hydroxychloroquine sulfate.

[0043] PAM cells were seeded in 96-well plates. After the cells adhered to the plate, the culture medium in the plate was discarded, and RPMI Medium 1640 medium containing different concentrations of hydroxychloroquine sulfate was added, 100 μL / well. A blank cell control group (RPMI Medium 1640 medium) was set, and 6 replicates were made for each treatment concentration.

[0044] After culturing in a 37 °C, 5% CO2 incubator for 48 h, the supernatant was discarded, the cells were washed twice with PBS, fresh culture medium was added, and then CCK-8 reagent was added, 10 μL / well. After continuous incubation in the dark for 1 h, the absorbance of each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a reference wavelength of 450 nm.

[0045] 3. Treatment of Marc-145 cells with hydroxychloroquine sulfate

[0046] The stock solution of hydroxychloroquine sulfate was diluted with DMEM medium (without antibiotics) containing 10% fetal bovine serum (FBS) to make the concentration of hydroxychloroquine sulfate reach 1, 10, 50, 100, 150, 200, 250, 500, 1000, 1500 μM, and DMEM medium containing different concentrations of hydroxychloroquine sulfate was obtained.

[0047] Marc-145 cells were seeded in 96-well plates. After the cells adhered to the plate, the culture medium in the plate was discarded, and DMEM medium containing different concentrations of hydroxychloroquine sulfate was added, 100 μL / well. A blank cell control group (DMEM medium without hydroxychloroquine sulfate) was set, and 6 replicates were made for each treatment.

[0048] After culturing in a 37 °C, 5% CO2 incubator for 48 h, the supernatant was discarded, the cells were washed twice with PBS, fresh culture medium was added, and then CCK-8 reagent was added, 10 μL / well. After continuous incubation in the dark for 1 h, the absorbance of each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a reference wavelength of 450 nm.

[0049] II. Experimental results

[0050] The results are as Figure 1 shown. In PAM and Marc-145 cells, hydroxychloroquine sulfate had no toxicity to the cells at a concentration of 100 μM.

[0051] Example 2 Time-resolved immunofluorescence assay to detect the effect of hydroxychloroquine sulfate on PRRSV N protein

[0052] I. Experimental method

[0053] 1. Treatment of PRRSV-infected Marc-145 cells with hydroxychloroquine sulfate

[0054] Marc-145 cells were seeded in 12-well plates. After the cells adhered to the wall, the culture medium was discarded. PRRSV (MOI = 0.1) was inoculated at 10 μL / well. After culturing at 37°C for 2 h, the virus supernatant was discarded, and the cells were washed twice with PBS. Then, DMEM medium containing 1 μM, 10 μM, and 100 μM hydroxychloroquine sulfate was added at 2 mL / well. Meanwhile, a cell group was set as a negative control (Mock: DMEM medium without inoculating PRRSV + hydroxychloroquine sulfate), and a virus-infected positive control group (PRRSV: DMEM medium inoculated with PRRSV + without hydroxychloroquine sulfate). The cells were cultured in an incubator at 37°C and 5% CO2 for 48 h.

[0055] 2. Time-resolved immunofluorescence assay

[0056] (1) After 48 h, the culture medium was removed, and Marc-145 cells were fixed with 80% acetone at 37°C for 10 minutes, and then washed three times with PBS.

[0057] (2) 200 μl of 0.25% Triton X-100 was added to each well and incubated for 10 minutes to permeabilize the cell membrane, and then the cells were washed three times with PBS.

[0058] (3) 200 μl of 3% bovine serum albumin was added to each well and incubated at 37°C for 1 hour to block non-specific binding, and then the cells were washed three times with PBS.

[0059] (4) 200 μl of PRRSV-N antibody (dilution ratio 1:500) was added to each well and incubated at 4°C for 12 hours, and then the cells were washed three times with PBS.

[0060] (5) 200 μl of Alexa Fluor 488 (dilution ratio 1:1000) was added to each well and incubated in the dark at 37°C for 1 hour.

[0061] (6) Then, the fixed cells were stained with DAPI in the dark for 10 minutes, and the cells were observed using a Leica DMI 4000B fluorescence microscope (Leica, Wetzlar, Germany).

[0062] II. Experimental results

[0063] As Figure 2 shown, compared with the virus-infected positive control group, the amount of PRRSV N protein was significantly reduced after treatment with hydroxychloroquine sulfate. When the concentration of hydroxychloroquine sulfate was 100 μM, more than 95% of the PRRSV N protein could be inhibited, and basically no PRRSV N protein appeared in the field of view.

[0064] Example 3: Detection of the effect of hydroxychloroquine sulfate on PRRSV N protein by Western Blot

[0065] I. Experimental method

[0066] 1. Treatment of PRRSV-infected PAM cells or Marc-145 cells with hydroxychloroquine sulfate

[0067] Inoculate PAM cells and Marc-145 cells into 6-well plates respectively, and then, according to the method of Example 2, perform "treatment of PRRSV-infected Marc-145 cells with hydroxychloroquine sulfate"; at the same time, replace Marc-145 cells with PAM cells and perform treatment of PRRSV-infected PAM cells with hydroxychloroquine sulfate.

[0068] 2. Western Blot detection

[0069] Use a cell scraper to scrape the treated cells on the above 6-well plate into a 1.5 mL centrifuge tube, centrifuge at 12000 g for 3 min at 4°C, and discard the supernatant. Lyse the precipitate thoroughly with NP40 lysis buffer on ice. After 30 min, centrifuge at 13000 g for 30 min at 4°C, aspirate the supernatant into a new 1.5 mL centrifuge tube, add protein loading buffer, heat at 100°C for 10 min, and then perform Western Blot detection.

[0070] First, perform SDS-PAGE on a Bio-Lab electrophoresis apparatus at a voltage of 80 V for 30 min, and then adjust the voltage to 120 V for 60 min; after transferring the membrane, block it with 5% skim milk powder for 1 h, wash the membrane with TBST, 5 min each time, for a total of 3 times;

[0071] Incubate with primary antibodies: Use anti-N protein antibody and anti-GAPDH antibody, and incubate overnight at 4°C; recover the primary antibodies, wash the membrane with TBST, 5 min each time, for a total of 3 times;

[0072] Incubate with secondary antibody: Incubate with a fluorescent secondary antibody of IRDye800CW Goat anti-mouse (diluted 1:10000) in the dark in a 37°C incubator for 1 h,

[0073] Discard the secondary antibody, quickly wash the membrane in the dark on a shaker with TBST, 5 min each time, for a total of 3 times;

[0074] Finally, analyze the expression of the target protein using the Odyssey system of the dual-color laser analysis instrument of LI-COR company.

[0075] II. Experimental results

[0076] As Figure 3 shown, hydroxychloroquine sulfate inhibits the expression of PRRSV N protein in a dose-dependent manner, and hydroxychloroquine sulfate can inhibit the expression of more than 95% of PRRSV N protein at 100 μM.

[0077] PRRSV N protein is a structural protein of porcine reproductive and respiratory syndrome. This protein is expressed early in virus infection and is present in relatively large amounts, serving as a marker for detecting porcine reproductive and respiratory syndrome virus. If the expression of N protein is inhibited, the virus cannot complete genome packaging, resulting in the inability to normally assemble or release virus particles. Combining the results of Examples 2 and 3, hydroxychloroquine sulfate can inhibit the expression of PRRSV N protein, indicating that hydroxychloroquine sulfate can inhibit PRRSV infection and replication.

[0078] Example 4 RT-qPCR Detection of the Effect of Hydroxychloroquine Sulfate on Nsp9 mRNA

[0079] I. Experimental Method

[0080] 1. Treatment of PRRSV-infected PAM cells or Marc-145 cells with hydroxychloroquine sulfate

[0081] Seed PAM cells and Marc-145 cells into 6-well plates respectively, and perform "treatment of PRRSV-infected PAM cells or Marc-145 cells with hydroxychloroquine sulfate" according to the method of Example 3.

[0082] 2. RT-qPCR Detection

[0083] Collect the cell supernatant, extract the genome of PRRSV using the AxyPrep Body fluid Viral DNA / RNA Miniprep Kit (Axygen), and then reverse transcribe the RNA into cDNA.

[0084] Using the upstream and downstream primers of Nsp9 mRNA and the GAPDH internal reference gene in Table 1, with SYBR Green I as the fluorescent dye, perform RT-qPCR detection. Use the Bio-Rad CFX96 qRT-PCR detection system to detect the relative expression of Nsp9 mRNA. The reaction conditions are as follows: activate the hot start enzyme at 95°C for 2 min; denature at 95°C for 15 s, anneal at 60°C for 1 min, extend at 72°C for 30 s, and perform 40 cycles of reaction. Collect the fluorescence signal at this step.

[0085] Table 1

[0086]

[0087] II. Experimental Results

[0088] As Figure 4 shown, compared with the positive control group infected with the virus, at 100 μM, hydroxychloroquine sulfate could inhibit the synthesis of DNA of more than 95% of PRRSV Nsp9 mRNA, and the difference was very significant (P<0.001). The PRRSV Nsp9 gene is highly conserved and is a necessary gene for virus detection. The results indicate that HCQ can inhibit the synthesis of PRRSV mRNA.

[0089] Example 5 Effect of hydroxychloroquine sulfate treatment on inflammatory factors in PRRSV-infected PAM cells

[0090] I. Experimental method

[0091] 1. Treatment of PRRSV-infected PAM cells with hydroxychloroquine sulfate

[0092] Inoculate PAM cells into 24-well plates, and treat PRRSV-infected PAM cells with hydroxychloroquine sulfate according to the method of Example 3.

[0093] 2. RT-qPCR detection

[0094] Detect the relative expression of IL-1β, IL-2, and IL-6 mRNA genes. The specific method is the same as that in Example 4, and the primers are shown in Table 2.

[0095] Table 2

[0096]

[0097] II. Experimental results

[0098] As Figure 5 shown, hydroxychloroquine sulfate inhibited the upregulation of IL-1β and IL-6 induced by PRRSV infection in PAM cells in a dose-dependent manner within the concentration range of 1-100 μM. Compared with the control group, hydroxychloroquine sulfate significantly inhibited IL-1β and IL-6 at 100 μM, inhibiting the occurrence of inflammation.

[0099] After PRRSV infection, the levels of pro-inflammatory factors IL-1β and IL-6 increased significantly, which is one of the core markers of cytokine storm. The results showed that hydroxychloroquine sulfate could limitedly inhibit the cytokine storm caused by PRRSV infection.

[0100] Example 6 Effect of hydroxychloroquine sulfate treatment on the survival rate, body temperature and body weight of PRRSV-infected piglets

[0101] I. Experimental method

[0102] Fifteen four-week-old piglets were randomly and evenly divided into 3 groups, with 5 piglets in each group, namely the negative group (Ctrl), the positive group (PRRSV), and the treatment group (PRRSV+HCQ).

[0103] The negative group was fed normally, the positive group was inoculated with 2×10 5 / mL TCID50 of PRRSV through trachea, and the treatment group was also inoculated with 2×10 5 / mL TCID50 of PRRSV.

[0104] After 24 hours of inoculation with PRRSV, the treatment group was continuously medicated for 12 days. Each time, hydroxychloroquine sulfate (diluted with normal saline) was injected intramuscularly, and the dose was 6.5 mg / kg.

[0105] After inoculation with PRRSV, the body weight, survival rate, body temperature of the piglets were monitored and recorded every day, and whether there were clinical symptoms such as dyspnea and cough was also observed. And blood was collected every day to collect serum for subsequent analysis.

[0106] II. Experimental Results

[0107] As Figure 6 shown, the survival rate of the treatment group was significantly higher than that of the positive group. And after inoculation with PRRSV, the average body weight of the treatment group gradually increased compared with the positive control group, and the body weight growth trend was basically the same as that of the control group and remained at a relatively stable level. The average body temperature of the treatment group and the negative control group was at the same level and remained relatively stable. The positive control group showed an increase in body temperature.

[0108] The results showed that hydroxychloroquine sulfate treatment effectively improved the survival rate of PRRSV-infected piglets and reduced the clinical symptoms of weight loss and body temperature increase caused by PRRSV infection.

[0109] Example 7 Effect of Hydroxychloroquine Sulfate Treatment on the Virus Titer in PRRSV-Infected Piglets

[0110] I. Experimental Methods

[0111] 1. Detection by Enzyme-Linked Immunosorbent Assay (ELISA)

[0112] The INGEZIM PRRS AMERICA kit from Ingnia Company was used to detect the PRRSV antibodies in the sera collected from the piglets in each group in Example 6 every day after inoculation with PRRSV.

[0113] 2. RT-qPCR Detection

[0114] (1) Construction of the Standard Curve for the Viral Copy Number of PRRSV

[0115] Preparation of Standard Samples: The standard sample used was the XH-GD plasmid. The concentration of this standard sample was measured by Nanodrop, and the copy number of the standard sample was calculated using a formula.

[0116] The calculation formula is: ;

[0117] Dilution of Standard Samples: The standard sample was serially diluted 10-fold, with 8 consecutive dilution steps. The qRT-PCR reactions were performed on each dilution of the standard sample and the blank control. The qRT-PCR reactions used the upstream and downstream primers of the Nsp9 and GAPDH reference genes in Table 1 according to the method of Example 4, with SYBR Green I as the fluorescent dye, and 3 replicates were performed for each dilution.

[0118] Drawing of the Standard Curve: Based on the copy number of each dilution and the corresponding Ct value, the standard curve can be made using...

[0119] (2)Detection of the Viral Copy Number of PRRSV

[0120] The genomic DNA of PRRSV was extracted from the whole blood collected in Example 6 using the AxyPrep Body fluid Viral DNA / RNA Miniprep Kit (Axygen), and then the RNA was reverse transcribed into cDNA.

[0121] According to the method of Example 4, the upstream and downstream primers of the Nsp9 and GAPDH reference genes in Table 1 were used, with SYBR Green I as the fluorescent dye, to perform RT-qPCR. The corresponding CT values were substituted into the standard curve formula to calculate the viral copy number of PRRSV in the serum of each group of piglets.

[0122] II. Experimental Results

[0123] Figure 7 This is the ELISA test result for PRRSV antibodies, where S is the absorbance value of the Sample, and P is the absorbance value of the Positive control. S / P = (Sample value - Negative control value) / (Positive control value - Negative control value). When S / P ≥ 0.4, the result is positive. The larger the value of S / P, the higher the concentration of PRRSV antibodies. The results showed that the treatment group could inhibit the replication of PRRSV in piglets compared with the positive group.

[0124] Figure 8 This is the RT-qPCR test result for Nsp9 in infected piglets. The results showed that the treatment group could inhibit the replication of PRRSV in piglets compared with the positive group.

[0125] The results showed that hydroxychloroquine sulfate treatment effectively reduced viremia caused by PRRSV infection in pigs and inhibited the replication and proliferation of PRRSV in pigs.

[0126] Example 8 Effect of hydroxychloroquine sulfate treatment on the histopathology of the lungs of PRRSV-infected piglets

[0127] I. Experimental method

[0128] In each group of piglets in Example 6, on the 14th day after inoculation with PRRSV (14 dpi, D14), after recording the body weight and measuring the body temperature, each group of piglets was euthanized and necropsied.

[0129] After flushing the lungs clean, pathological observations were made and photographed. Subsequently, after collecting the lung tissues, 10% neutral buffered formalin (10% NBF) was used as the fixative and fixed for 24 - 48 h. This was to facilitate subsequent histopathological analysis.

[0130] After fixation, the samples underwent a series of dehydration steps, using ethanol with increasing concentrations, and then clearing in xylene. The dehydrated tissues were embedded in paraffin for easy sectioning. Using a microtome, thin sections approximately 4 μm thick were cut from the paraffin-embedded tissues. These sections were mounted on glass slides, then deparaffinized with xylene and rehydrated through a series of ethanol grades. For histological evaluation, the slides were stained with hematoxylin and eosin (HE) to observe cell and tissue structures in detail. After staining, the slides were examined under an optical microscope.

[0131] II. Experimental results

[0132] As Figure 9 shown, in the negative control group, the lungs of the piglets were normal in shape without obvious lesions; in the positive control group, the piglets showed enlarged lungs, manifested as diffuse enlargement, increased weight, a dark red color on the surface of the lungs, congestion of the lung lobes, hardening of the texture of the lungs, reduced elasticity, and certain pulmonary adhesions.

[0133] Compared with the positive control group, the treatment group showed milder lung lesions. In the treatment group, except for the grayish-white pathological changes in the visceral layer of the lung mucosa, there were no obvious lesions.

[0134] Histopathological analysis of the lungs showed that the positive group showed interstitial pneumonia with thickened alveolar septa, a large number of inflammatory cell infiltrations, interstitial edema and fibrosis in the interstitium, and destruction of the lung structure; the treatment group only showed mild inflammatory cell infiltration in the lungs. The results showed that hydroxychloroquine sulfate treatment could reduce the lung lesions in piglets caused by PRRSV infection.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes can be made based on the above description and ideas. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of hydroxychloroquine sulfate in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome.

2. Use of hydroxychloroquine sulfate in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome virus.

3. The application according to claim 1 or 2, characterized in that, The drug further comprises pharmaceutically acceptable application excipients.

4. The application according to claim 1, wherein The prevention and treatment of porcine reproductive and respiratory syndrome is to inhibit or avoid the occurrence of cytokine storm.

5. The application according to claim 4, wherein The occurrence of the cytokine storm is related to IL-1β.

6. The application according to claim 4, wherein, The occurrence of the cytokine storm is related to IL-6.

7. The application according to claim 2, wherein The prevention and treatment of porcine reproductive and respiratory syndrome virus is to inhibit the infection of porcine reproductive and respiratory syndrome virus.

8. The application according to claim 2, characterized in that The prevention and treatment of porcine reproductive and respiratory syndrome virus is to inhibit the proliferation of porcine reproductive and respiratory syndrome virus.

9. The application according to claim 1 or 2, characterized in that, The effective use concentration of hydroxychloroquine sulfate is 1 - 100 μΜ.

10. The application according to claim 9, wherein The effective use concentration of hydroxychloroquine sulfate is 100 μΜ.

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