Application of isodehydroicariin in the preparation of drugs against herpes simplex virus type Ⅰ

Abstract

The present invention discloses the use of isodehydroicariin in the preparation of an anti-herpes simplex virus type I drug. Specifically, isodehydroicariin inhibits the proliferation of herpes simplex virus type I, which is manifested by inhibiting the total protein expression of herpes simplex virus type I, reducing the number of herpes simplex virus type I particles, and reducing the titer of herpes simplex virus type I. The present invention expands the medical field use of isodehydroicariin, discloses for the first time that it can inhibit the reproduction of herpes simplex virus type I, and can be used to prepare drugs for preventing and treating herpes simplex virus.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of isodehydroicariin in the preparation of an anti-type I herpes simplex virus drug. Background Art

[0002] Herpes simplex virus (HSV) is one of the important members of the herpesviridae family, including type Ⅰ (HSV-1) and type Ⅱ (HSV-2), which is a type of enveloped double-stranded DNA virus. HSV-1 infection is extremely common in the population, with an infection rate of up to 80%-90%. HSV-1 mainly infects mucosal epithelial cells and generally lurks in trigeminal neurons during the initial infection. When the body's resistance decreases or under certain stimulation conditions, the latent HSV-1 in the body is activated and enters the lytic cycle, causing a variety of clinical diseases. The most common clinical symptoms are acute oral ulcers, pharyngitis, and tonsillitis. HSV-1 infection of the eye can cause herpetic keratoconjunctivitis. Very few HSV-1 infections can cause herpetic meningitis with a high mortality rate. Epidemiological studies have shown that HSV-1 infection may be associated with the occurrence of Alzheimer's disease. In addition, studies have shown that in some cases, HSV-1 infection can promote co-infection with human immunodeficiency virus HIV.

[0003] At present, the drugs used in clinical treatment of HSV-1 infection are mainly nucleoside analogs, such as acyclovir, valacyclovir, penciclovir and famciclovir. The specific mechanism of action is: after the drug is taken up by cells infected with HSV-1, it is phosphorylated by the virus-encoded thymidine kinase and intracellular kinase, and then competes with nucleotides to inhibit viral DNA polymerase, so that the synthesis of the viral DNA chain is terminated and the replication of the virus is inhibited, thereby achieving the purpose of antiviral treatment. Although these drugs have significant antiviral efficacy in the treatment of acute HSV-1 infection, they also have problems such as low solubility, short half-life, unsustainable efficacy and large side effects. In addition, long-term use can induce viral mutations and lead to the emergence of drug-resistant virus strains. Therefore, it is very necessary to develop new, efficient, low-toxic and multi-target drugs for the prevention and treatment of HSV-1.

[0004] Isoanhydroicaritin (IAI) is a characteristic isopentenyl flavonoid isolated from the dried roots of Sophora flavescens. 21 H 20 N6, molecular weight is 368.38, molecular structure is as follows:

[0005]

[0006] Studies have shown that isodehydroicariin from Sophora flavescens has physiological activities such as antibacterial and anti-tumor. In mouse skin melanoma cell B16-F10 and zebrafish models, isodehydroicariin in Sophora flavescens acts as a tyrosinase inhibitor with anti-melanogenesis effect, regulating the production of melanin (reference: Front. Pharmacol. 15:1422310. doi: 10.3389 / fphar.2024.1422310). However, there are no reports on the correlation between isodehydroicariin and herpes simplex virus infection and treatment. Summary of the invention

[0007] The purpose of the present invention is to provide the use of isodehydroicariin in the preparation of anti-herpes simplex virus type Ⅰ drugs in view of the deficiencies of the prior art, so as to provide a new drug option for the prevention and treatment of herpes simplex virus type Ⅰ infection.

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

[0009] In a first aspect, the present invention provides the use of isodehydroicariin in the preparation of an anti-herpes simplex virus type Ⅰ drug.

[0010] Furthermore, isodehydroicariin inhibits the proliferation of herpes simplex virus type 1.

[0011] Furthermore, isodehydroicariin inhibited the expression of total herpes simplex virus type 1 protein.

[0012] Furthermore, isodehydroicariin reduced the number of herpes simplex virus type 1 particles.

[0013] Furthermore, isodehydroicariin reduced the titer of herpes simplex virus type 1.

[0014] In a second aspect, the present invention provides an anti-herpes simplex virus type I drug, comprising isodehydroicariin, and a pharmaceutically acceptable carrier and / or excipient.

[0015] Furthermore, the dosage form of the drug is any pharmaceutically acceptable dosage form.

[0016] In a third aspect, the present invention provides an in vitro method for inhibiting the proliferation of herpes simplex virus type 1 in cells for non-therapeutic purposes, comprising the following steps: treating the cells with isodehydroicariin.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention discloses for the first time that isodehydroicariin is a Chinese medicine monomer compound that has the ability to resist the replication of herpes simplex virus type I, and verifies the inhibitory effect of isodehydroicariin on virus proliferation in cells by establishing an HSV-1 infected Hela cell model, observing the cell state after virus infection, virus protein immunoblotting experiment (Western blot), virus electron microscope photos, and virus titer determination. Therefore, the present invention expands the medical field use of isodehydroicariin, which can not only resist melanin production, but also inhibit the reproduction of herpes simplex virus type I, and can be used to prepare drugs for preventing and treating herpes simplex virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are the results of the cytotoxicity assay of different concentrations of isodehydroicariin on Hela cells provided in Example 1.

[0020] Figure 2 The effects of different concentrations of isodehydroicariin provided in Example 2 on the state of Hela cells infected with HSV-1.

[0021] Figure 3 The effects of different concentrations of isodehydroicariin provided in Example 3 on the expression level of viral late protein ICP34.5 in Hela cells infected with HSV-1.

[0022] Figure 4 The effect of isodehydroicariin provided in Example 4 on the expression level of total viral protein in Hela cells infected with HSV-1.

[0023] Figure 5 The effect of isodehydroicariin provided in Example 5 on the number of viral particles in Hela cells infected with HSV-1.

[0024] Figure 6 The effect of isodehydroicariin provided in Example 6 on the viral titer in Hela cells infected with HSV-1; wherein A: MOI=0.05, B: MOI=5. DETAILED DESCRIPTION

[0025] The following will be clearly and completely described in conjunction with the embodiments of the present invention and the technical effects produced, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without paying creative work all belong to the scope of protection of the present invention. The reagents or materials used in the following examples can all be obtained through commercial channels unless otherwise specified.

[0026] Example 1: MTT assay to detect the toxic effect of isodehydroicariin on Hela cells

[0027] Dissolve isodehydroicariin (IAI) in DMSO solution prepared in complete culture medium for later use;

[0028] Hela cells were inoculated in a 96-well culture plate, with 200 μL of fresh complete medium in each well. When the confluence of the cell wells reached more than 85%, isodehydroicariin was added at final concentrations of 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM, respectively, and six replicate wells were made for each concentration. The plates were placed in an incubator at 37°C with a volume fraction of 5% CO2 and continued to be cultured for 48 hours. Then 10 μl of MTT solution (5 mg / ml) was added to each well and cultured in the cell culture incubator for 4 hours. The culture was terminated and the culture medium in the well was carefully discarded. 100 μl of dimethyl sulfoxide (DMSO) was added to each well and shaken for 10 minutes to fully dissolve the crystals. The absorbance of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay, and zero wells and control wells were set to ensure the accuracy of the results.

[0029] The results are as follows Figure 1 As shown in the figure, isodehydroicariin has no effect on the growth of Hela cells at a concentration of 5 μM. Therefore, isodehydroicariin with a concentration less than 5 μM was selected for subsequent antiviral studies.

[0030] Example 2: Observation of the effect of isodehydroicariin on the state of Hela cells infected with herpes simplex virus type Ⅰ under a microscope

[0031] Hela cells were inoculated in 6-well culture plates. The next day, when the confluence of the cell wells reached more than 85%, the culture medium was discarded, washed twice with PBS (phosphate buffer), and HSV-1 diluted with DMEM (MOI=1) was added, 0.6ml / well, and incubated at 37°C for 2h. During this period, it was gently shaken every 30 minutes to allow the virus to fully contact the cells. Then the virus supernatant was discarded, washed twice with PBS, and isodehydroicariin with a final concentration of 1μM, 2μM, and 5μM was added, 2mL / well. At the same time, a normal control group (DMSO only) and an HSV-1 control group were set up. The cells continued to be cultured at 37°C, and the cell state was observed under a microscope and photographed 24h after infection.

[0032] The results are as follows Figure 2 As shown in the figure, it can be seen that the cells in the Mock group (only DMSO was added) grew and replicated normally; in the HSV-1 plus DMSO group, the cells showed obvious apoptosis; in the HSV-1 plus IAI group, the cell state gradually improved with the increase of IAI concentration. This shows that IAI can inhibit the replication of HSV-1 in Hela cells and is concentration-dependent.

[0033] Example 3: Western Blot analysis of the inhibitory effect of different concentrations of isodehydroicariin on late protein synthesis of herpes simplex virus type Ⅰ

[0034] Hela cells were inoculated in 6-well culture plates. The next day, when the confluence of the cell wells reached more than 85%, the culture medium was discarded, washed twice with PBS (phosphate buffer), and HSV-1 diluted with DMEM (MOI=1) was added, 0.6ml / well, and incubated at 37°C for 2h, during which time it was gently shaken every 30 minutes. Then the virus supernatant was discarded, washed twice with PBS, and isodehydroicariin with a final concentration of 1μM, 2μM, and 5μM was added, 2mL / well. At the same time, a normal control group (only DMSO was added) and an HSV-1 control group were set up. The cells continued to be cultured at 37°C, and the cells in each well were collected and lysed 24h after infection. The cell lysate was separated by SDS-PAGE, transferred to the membrane, and the expression of HSV-1 late protein was detected by anti-ICP34.5 (ICP34.5 protein is a viral late protein encoded by the γ134.5 gene of HSV-1) and anti-tubulin.

[0035] The results are as follows Figure 3 As shown: In the HSV-1 plus IAI group, the expression level of the viral late protein ICP34.5 gradually decreased with the increase of IAI concentration, and the effect was most obvious when the concentration reached 5μM.

[0036] Example 4: Western Blot analysis of the inhibitory effect of isodehydroicariin on the synthesis of total protein of herpes simplex virus type Ⅰ

[0037] The methods of virus infection (MOI=1) and IAI drug treatment (5 μM) were as described in Example 2. 24 h after infection, cells in each well were collected and lysed, and the cell lysate was separated by SDS-PAGE and transferred to a membrane. The expression of HSV-1 total protein was detected using anti-HSV-1 and anti-tubulin antibodies, respectively.

[0038] The results are as follows Figure 4 As shown in the figure, compared with the DMSO group, IAI significantly inhibited the expression of viral proteins of HSV-1. It can be seen that IAI can inhibit the replication of HSV-1 in Hela cells, and isodehydroicariin can be used to inhibit the expression of total viral proteins in Hela cells infected with HSV-1.

[0039] Example 5: Electron microscopic observation of the inhibitory effect of isodehydroicariin on the replication of herpes simplex virus type Ⅰ

[0040] The methods of virus infection (MOI=1) and IAI drug treatment (5μM) were as described in Example 2. Then the cells were harvested and washed with 1×PBS, centrifuged at 1000rpm for 5min, and fixed with 6% (w / v) glutaraldehyde for 2h. The fixed cells were washed twice with 0.1mol PBS and fixed with 1% osmium acid for 1 hour. After fixation with osmium acid, the specimens were washed twice with distilled water, followed by dehydration with ethanol and propylene oxide with volume fractions of 30%, 70%, 90%, and 100% for 10-15min each (including dehydration with ethanol and propylene oxide with volume fractions of 100% twice), and the specimens were soaked with a mixture of 1:1 propylene oxide and EPON embedding solution for 2h, and then the samples were picked into capsules and placed in an oven (60℃, 24h). The slices were sliced ​​with a German Leica UC7 ultrathin slicer with a thickness of 500nm. The cells were stained with uranyl acetate for 10 min and with lead stain for 8 min. The distribution and number of virus particles in the cells were observed using an H600 electron microscope.

[0041] The results are as follows Figure 5 As shown, no virus particles were found in the cells of the Mock+DMSO group; a large number of virus particles were found in the Hela cells of the HSV-1+DMSO group; and only a small number of virus particles were found in the cells of the HSV-1+IAI group. This indicates that IAI has an inhibitory effect on the HSV-1 virus, and isodehydroicariin can be used to reduce the number of virus particles in Hela cells infected with HSV-1.

[0042] Example 6: Titer assay to detect the inhibitory effect of isodehydroicariin on the replication of herpes simplex virus type Ⅰ

[0043] Hela cells were inoculated in 6-well culture plates. The next day, when the confluence of the cell wells reached more than 85%, the culture medium was discarded, washed twice with PBS (phosphate buffer), and HSV-1 diluted with DMEM (one group: MOI=0.05, the other group: MOI=5) was added, 0.6ml / well, and incubated at 37°C for 2h, shaking every 15 minutes during this period. Then the virus supernatant was discarded, washed twice with PBS, and 2mL / well of isodehydroicariin with a final concentration of 5μM was added to the two groups. The cells continued to be cultured at 37°C until 48h (MOI=0.05 group) and 24h (MOI=5 group) after infection, 600μl of sterilized skim milk was added to each well, and the virus was repeatedly frozen and thawed 3 times at -80°C and room temperature to fully release the virus from the cells, and then the virus titer was determined. The specific method is as follows: Vero cells were plated in a six-well plate, 3×10 6 The next day, the virus solution was diluted with 199V culture medium to a dilution of 10 -3 -10 -8 .

[0044] Take 600μl of the diluted virus solution and add it to the six-well plate. Place it in the incubator and culture it for 2 hours. Gently shake it every half an hour to allow the virus to fully contact the cells. After 2 hours, aspirate the virus and add 2ml of new 199V culture medium. Continue to culture for 2 days. Do not move the six-well plate during this period. After 2 days, count the number of virus plaques in each well, calculate the concentration of the original virus solution and plot it.

[0045] The results are as follows Figure 6 As shown, in HSV-1 infected cells, whether it was the MOI=0.05 group (infection for 48h) or the MOI=5 group (infection for 24h), the viral copy number of the virus group treated with 5μM isodehydroicariin was significantly reduced, indicating that this type of drug has an anti-HSV-1 replication effect, and isodehydroicariin can be used to reduce the viral titer in HSV-1 infected Hela cells.

[0046] In summary, the isodehydroicariin provided by the present invention can significantly inhibit the reproduction and replication of HSV-1, and has no obvious cytotoxic effect at therapeutic concentrations. Therefore, it has the advantages of good safety and high selectivity index, and can be used to prepare antiviral drugs for herpes simplex virus type Ⅰ (HSV-1), expand the treatment options for herpes virus-infected diseases, and has broad application prospects and important clinical significance.

[0047] It should be understood that although this specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation mode may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

[0048] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent implementation methods or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of isodehydroicariin in the preparation of an anti-herpes simplex virus type I drug, characterized in that: Isodehydroicariin inhibits the proliferation of herpes simplex virus type 1, inhibits the expression of herpes simplex virus type 1 total protein, reduces the number of herpes simplex virus type 1 particles, and reduces the titer of herpes simplex virus type 1.

2. A method for inhibiting the proliferation of herpes simplex virus type 1 in cells for non-therapeutic purposes in vitro, characterized in that: The following steps are involved: Cells were treated with isodehydroicariin.

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