CN114452249A

CN114452249A - Application of toona sinensis milk gel in preparation of medicines for resisting low-risk HPV infection and preventing and treating low-risk HPV related diseases

Info

Publication number: CN114452249A
Application number: CN202210243495.5A
Authority: CN
Inventors: 吴梦瑶; 龚云; 何娟娟; 赵威; 彭冬冬; 张小冬; 姜德建
Original assignee: Qianjin Pharmaceutical Co ltd
Current assignee: Qianjin Pharmaceutical Co ltd
Priority date: 2022-03-11
Filing date: 2022-03-11
Publication date: 2022-05-10

Abstract

The invention discloses application of toona sinensis milk gel in preparation of medicines for resisting low-risk HPV infection and preventing and treating low-risk HPV related diseases. According to the research of the invention, the toona sinensis milk gel can obviously reduce the expression of related genes and proteins of HPV6 and HPV11, inhibit the proliferation of HPV6 and HPV11 viruses, reduce the virus infectivity, recover the abnormal cell cycle distribution induced by low-risk HPV infection, and play a role in recovering and treating the abnormal cell development induced by the low-risk HPV infection. The toona sinensis milk gel can obviously reduce the pH value of the vagina and the virus titer of cervical tissues after low-risk HPV infection, relieve the pathological changes of the cervical tissues, and relieve the epithelial cell proliferation, necrosis and inflammatory cell infiltration conditions of the cervical tissues caused by HPV6 and HPV11 infection. Therefore, the toona sinensis milk gel can be used for preventing low-risk HPV virus infection and treating diseases caused by the low-risk HPV virus. For example, it can be used for preventing and treating condyloma acuminatum, and can be used in early stage of condyloma acuminatum formation.

Description

Application of toona sinensis milk gel in preparation of medicines for resisting low-risk HPV infection and preventing and treating low-risk HPV related diseases

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of ailanthus altissima latex gel in preparing medicines for resisting low-risk HPV infection and preventing and treating low-risk HPV related diseases.

Background

Human Papilloma Virus (HPV) is a spherical double-stranded DNA Virus with a genome sequence of approximately 7900bp in length. Currently, over 160 different subtypes of HPV virus have been detected, primarily by transmission of sexual activity to the genitalia and its surrounding skin.

Research reports that different HPV subtypes can cause different clinical diseases and can be roughly divided into low-risk subtypes and high-risk subtypes according to the severity of the diseases. The low-risk HPV, also called non-cancer related HPV, mainly comprises HPV6, 11, 13, 42, 43, 44, 53 and 54, etc., and the infection of the HPV mainly causes benign skin diseases such as verruca vulgaris, condyloma acuminatum and recurrent respiratory papilloma, etc., and can not directly induce malignant tumors such as cervical cancer, penile cancer, etc. High-risk HPV, also called malignant tumor related types, including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 61, etc. it is believed that HPV infection mainly causes malignant epithelial tumors such as cervical cancer and penile cancer. HPV viruses are already the most common genital tract viruses, but most HPV viruses are eliminated by the immune system of the body of the infected person, and only very few subtypes persistently infect the host and eventually progress to condyloma acuminata, precancerous lesions, or even malignant tumors. In recent years, the morbidity and mortality of HPV infection-related diseases have increased worldwide, especially in subfira countries, year by year, and the age of the disease has a tendency to be younger.

In the prevention and treatment process of sexually transmitted diseases, the low-risk human papilloma viruses HPV6 and HPV11 are two important pathogenic viruses, specifically infect keratinocytes in the epithelium of the anogenital area, cause abnormal proliferation of cells and generate fleshy neoplasms. Due to the difference in the site of infection and the physiological state of the skin, there are different pathological changes, most typically condyloma acuminatum, which most often occurs around the genitals and anus. The skin lesions of condyloma acuminatum may initially be only small single reddish pimples, often without subjective symptoms, and then gradually increase in volume and number. The disease is easy to relapse, and the relapse is generally considered to be caused by incomplete lesion excision and low local immunity of patients and is mainly related to the failure of effective elimination of HPV virus particles. Although most studies suggest that a combination of traditional physical means of removing lesions and adjunctive antiviral therapy will be effective in controlling the development and recurrence of condyloma acuminatum, there is still a lack of highly effective antiviral therapeutic agents, and therefore, there is only a reduction in the recurrence rate and a lack of early stage condyloma acuminatum control agents. And the existing medicines for inhibiting high-risk HPV such as rebarsen and the like are only high-risk HPV inhibitors and lack medicines specially aiming at low-risk HPV.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides the application of the ailanthus altissima gel in preparing the medicines for resisting low-risk HPV infection and preventing and treating low-risk HPV related diseases.

The invention aims to provide application of a traditional Chinese medicine composition with the effective components of ailanthus bark, sophora flavescens, moutan bark, frankincense and borneol in preparing a medicine for resisting low-risk HPV virus and/or treating diseases caused by the low-risk HPV virus.

The application of a traditional Chinese medicine composition in preparing a medicine for resisting low-risk HPV virus comprises the effective components of cortex ailanthi, radix sophorae flavescentis, cortex moutan, frankincense and borneol.

Preferably, the anti-low-risk type HPV virus is an anti-low-risk type HPV virus that inhibits replication and/or proliferation.

Preferably, the low-risk type HPV virus resistance is low-risk type HPV virus infection inhibition.

Preferably, the anti-low-risk type HPV virus is used for restoring low-risk type HPV host cell environment pH imbalance and/or relieving cervical tissue lesion caused by low-risk type HPV virus infection.

More preferably, the expression of mRNA of the low-risk HPV is inhibited, the expression of protein of the low-risk HPV is inhibited or the cell cycle disorder of the low-risk HPV host is recovered.

More preferably, said inhibiting infection by low risk HPV virus comprises reducing infectivity of low risk HPV virus and/or reducing titer of low risk HPV virus in tissue.

The application of a traditional Chinese medicine composition in preparing a medicine for treating diseases caused by low-risk HPV virus is characterized in that the effective components of the traditional Chinese medicine composition are ailanthus bark, radix sophorae flavescentis, moutan bark, frankincense and borneol.

Preferably, the treatment of the disease caused by the low-risk type HPV virus is one or more of alleviating pathological changes of cervical tissues, recovering low-risk type HPV host cell cycle disorder or recovering low-risk type HPV host cell environment pH disorder.

Preferably, the disease is condyloma acuminatum.

More preferably, the condyloma acuminatum is early condyloma acuminatum.

Preferably, the low-risk type HPV virus is one or more of HPV6, HPV11, HPV13, HPV42, HPV43, HPV44, HPV53 and/or HPV 54.

More preferably, the low risk type HPV virus is HPV6 and/or HPV 11.

Preferably, the traditional Chinese medicine composition comprises the following components in parts by weight: and (3) bark of Chinese toon: 35-45; flavescent sophora root: 25-35; moutan bark: 25-35; frankincense: 25-35; borneol: 0.8-1.2, wherein the radix sophorae flavescentis, the cortex moutan radicis and the frankincense are equal in amount.

More preferably, the traditional Chinese medicine composition comprises the following components in parts by weight: and (3) bark of Chinese toon: 40; flavescent sophora root: 30, of a nitrogen-containing gas; moutan bark: 30, of a nitrogen-containing gas; frankincense: 30, of a nitrogen-containing gas; borneol: 1.

more preferably, the traditional Chinese medicine composition is prepared into gel.

More preferably, the preparation method of the gel of the traditional Chinese medicine composition is characterized by comprising the following steps:

s1: adding 2-9 times of water into the toona sinensis leaves, adding water, decocting for 1-3 times, decocting for 1-2.5 hours each time, mixing decoctions, filtering, and concentrating the filtrate under reduced pressure to obtain toona sinensis leaf bark clear paste with the relative density of 1.05-1.10;

s2: adding 6-12 times of water into the cortex toonae sinensis, decocting for 1-3 times, soaking for 0.5-1.5 hours before the first decoction, decocting for 0.5-3 hours each time, combining decoction liquids, filtering, concentrating filtrate under reduced pressure to obtain radix sophorae flavescentis clear paste with the relative density of 1.10-1.15, cooling, adding ethanol to enable the alcohol content to reach 60-70%, standing for 8-24 hours, filtering, recovering ethanol from filtrate until no alcohol smell exists, combining the filtrate with the cortex toonae sinensis extract, and concentrating to obtain clear paste with the relative density of 1.05-1.10;

s3: adding 6-10 times of water into frankincense, distilling for 5-9 hours, and collecting volatile oil;

s4: distilling the moutan bark by using a steam distillation method, collecting distillate with the mass being 4-8 times of the amount of the medicinal materials, standing for crystallization, filtering, and standing the paeonol crystal; redistilling the aqueous solution, collecting distillate with the mass 2-4 times of the medicinal materials, standing for crystallization, filtering, and combining paeonol crystals;

s5: adding medicinal sodium carboxymethylcellulose and medicinal glycerol into the mixed fluid extract of cortex Ailanthi and radix Sophorae Flavescentis, stirring, and standing to swell completely;

s6: and (3) adding the borneol into the volatile oil of the frankincense, dissolving the paeonol crystals in ethanol, adding ethylparaben and polysorbate-80, stirring to dissolve, adding the dissolved solution into the swollen solution obtained in the step S5, adding water, uniformly mixing, standing for 12-36 hours to fully swell, uniformly stirring, and subpackaging to obtain the gel.

More preferably, the dosage of the sodium carboxymethylcellulose and the pharmaceutical glycerin is respectively 0.6-1.5 parts by weight of the borneol, and the dosage of the ethylparaben and the polysorbate-80 are respectively 0.01-0.1 part by weight of the borneol; the obtained gel is 0.1-0.2 part by weight of the total weight of the traditional Chinese medicine raw materials.

Most preferably, the Chinese medicinal composition is a toona sinensis milk gel.

Compared with the prior art, the invention has the following beneficial effects:

the toona sinensis milk gel is a pure traditional Chinese medicine preparation which is independently developed and uniquely produced in the Qianjin pharmaceutical industry, is prepared from 5 traditional Chinese medicines of toona sinensis bark, radix sophorae flavescentis, tree peony bark, frankincense and borneol, has the effects of clearing heat, drying dampness, removing blood stasis and promoting granulation, is scientific and reasonable in combination of clearing and tonifying. According to the characteristics of the main function and main treatment, the invention researches the pharmacodynamics of the toona sinensis milk gel in vivo and in vitro anti-low risk HPV. In vitro experiments observed the effect of the toona sinensis milk gel on the cell cycle of HPV6 and HPV11 viruses and 293 FT; in vivo test, a BALB/C mouse model infected by low-risk HPV virus extracted from 293FT cells containing HPV6 genome and HPV11 genome is used for evaluating the medicinal effect of the ailanthus altissima gel on resisting HPV6 and HPV11 in vivo by detecting the virus titer of cervical tissues, HPV6E 6mRNA, HPV6E7mRNA, HPV11E 6mRNA, HPV11E7mRNA and the protein expression of HPV6 and HPV 11.

According to the research of the invention, the toona sinensis milk gel can obviously reduce the expression of related genes and proteins of HPV6 and HPV11, inhibit the proliferation of HPV6 and HPV11 viruses, reduce the virus infectivity, recover the abnormal cell cycle distribution induced by low-risk HPV infection, and play a role in recovering and treating the abnormal cell development induced by the low-risk HPV infection. The toona sinensis milk gel can obviously reduce the pH value of the vagina and the virus titer of cervical tissues after low-risk HPV infection, relieve the pathological changes of the cervical tissues, and relieve the epithelial cell proliferation, necrosis and inflammatory cell infiltration conditions of the cervical tissues caused by HPV6 and HPV11 infection. Therefore, the toona sinensis milk gel can be used for preventing low-risk HPV virus infection and treating diseases caused by the low-risk HPV virus. For example, the medicine can be used for preventing and treating condyloma acuminatum, can play a role in the early stage of condyloma acuminatum formation, avoids the pain of excision of the neoplasm after the formation of the condyloma acuminatum occurs and the possibility of relapse, and provides a choice for clinical medicines for preventing and treating low-risk HPV and early condyloma acuminatum.

Drawings

FIG. 1 is a graph of the morphological effect of Ailanthus altissima gel on 293FT cells after 24h of culture, wherein A is a blank control group; b is 1000 mug/mL ailanthus altissima gel; c is toona sinensis milk gel of 500 mu g/mL; d is 62.5 mu g/mL toona sinensis milk gel.

FIG. 2 is a graph of the morphological effect of Ailanthus altissima gel on 293FT cells after 48h of culture, wherein A is a blank control group; b is 1000 mug/mL toona sinensis milk gel; c is toona sinensis milk gel of 500 mu g/mL; d is 62.5 mu g/mL toona sinensis milk gel.

FIG. 3 is a graph of the morphological effect of Ailanthus altissima gel on 293FT cells after 72h of culture, wherein A is a blank control group; b is 1000 mug/mL ailanthus altissima gel; c is toona sinensis milk gel of 500 mu g/mL; d is 62.5 mu g/mL toona sinensis milk gel.

FIG. 4 shows the effect of the test groups on 293FT cell proliferation 48h after virus infection, wherein A is vehicle control/cell maintenance solution; b is HPV6 and HPV 11; c is 125 mu g/mL of toona sinensis milk gel intervention; d is 100 mu mol/L positive drug intervention.

FIG. 5 shows the effect of each assay group on 293FT cell proliferation 72h after virus infection, where A is vehicle control/cell maintenance; b is HPV6 and HPV 11; c is 125 mu g/mL of toona sinensis milk gel intervention; d is 100 mu mol/L positive drug intervention.

FIG. 6 is a PI single stain flow cytometer analysis of 293FT cell cycle, where A is HPV6 and HPV11 group; b is a solvent control group; c is a toona sinensis milk gel low dose group; d is the dosage group in the toona sinensis milk gel; e is a toona sinensis milk gel high dose group; f is a positive medicine group.

FIG. 7 shows the status of HPV6 and HPV11 colonization examination in the model control group 48h after molding.

FIG. 8 shows the status of HPV6 and HPV11 colonization examination in the model control group before the last administration.

FIG. 9 is a pathological section of vaginal tissue (HE X100) wherein A is a blank control group; b is a model control group; c is podophyllotoxin group; d is a toona sinensis milk gel low dose group; e is the dosage group in the toona sinensis milk gel; f is the toona sinensis milk gel high dose group.

Fig. 10 is a pathological section of cervical tissue (HE × 100), in which a is a blank control group; b is a model control group; c is podophyllotoxin group; d is a toona sinensis milk gel low dose group; e is the dosage group in the toona sinensis milk gel; f is the toona sinensis milk gel high dose group.

FIG. 11 is a melt curve of the cDNA of the Real-time PCR assay, wherein A is HPV6E 6; b is HPV6E 7; c is HPV11E 6; d is HPV11E 7.

FIG. 12 shows the effect of Ailanthus altissima gel on mouse cervical tissue HPV6E 6mRNA, HPV6E7mRNA, HPV11E 6mRNA and HPV11E7mRNAComparison with blank control group⁺⁺P is less than or equal to 0.01; comparison with model control group^*P≤0.05，^**P≤0.01。

FIG. 13 is a gray scale band of HPV6 and HPV11 proteins from 6 replicates, wherein A is a blank control; b is a model control group; c is podophyllotoxin group; d is a toona sinensis milk gel low dose group; e is the dosage group in the toona sinensis milk gel; f is the toona sinensis milk gel high dose group.

FIG. 14 is a graph of the effect of Ailanthus altissima gel on HPV6 and HPV11 protein expression in cervical tissues of mice, compared to a placebo⁺⁺P is less than or equal to 0.01; comparison with model control group^*P≤0.05，^**P≤0.01。

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

The materials used in the examples are as follows:

the test substance: toona sinensis milk gel, batch number: 20191205, respectively; specification: 4 g/piece; the preparation formulation is as follows: a gel formulation; the characteristics are as follows: a tan gel; the validity period is as follows: to 2022.05; produced by Qianjin pharmaceutical industry, Inc. The toona sinensis milk gel is prepared according to a preparation method of 'national food and drug administration standard YBZ 12212009'.

Positive control: podophyllotoxin, lot No.: M0521B, specification: 1 g/count, expiration date to: to 2022.07; manufactured by melem biotechnology limited, suzhou.

Cell machine plasmid: 293FT cells (purchased from Chong Xinzhou Biotechnology, Inc., Shanghai); pcDNA3.1-EGFP-HPV6-E6/E7, pcDNA3.1-EGFP-HPV11-E6/E7 (sent to general biology (Anhui) systems, Inc. for synthesis); pCMV-HPV6-L1-flag-L2-6 His, pCMV-HPV11-L1-flag-L2-6 His (sent to Megaku Biotechnology Ltd., Wuhan, for synthesis).

Experimental animals: 100 SPF-grade female BALB/C mice with the weight of 15.6-18.8 g are purchased from Schbefu (Beijing) biotechnology limited company, and experimental animals are provided with production license numbers: SCXK (Jing) 2019-: SYXK (Xiang) 2020-.

The main reagents are as follows: DMEM medium (Hyclone, cat # AF 29584966); fetal Bovine Serum (FBS) (Sciencell, cat # M009-6); lipo-fectamine 2000(Invitrogen corporation, cat # 2117484); Opti-MEM^TM(Thermo Fisher company, Cat. 31985070); CellTiter-Glo luminescence cell viability assay kit (Biyuntian Bioreagent company, cat # C0065M); cell cycle detection kit (Invitrogen, cat # 2117484); DH 5. alpha. competent cells (Tiangen Biochemical technology (Beijing) Ltd., cat # CB 101); endotoxin-free plasmid macroextraction kit (Tiangen Biochemical technology (Beijing) Ltd., cat # DP 117); RIPA lysate (strong) (bi yun day, cat No. P0013B); high Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, cat # 4368814); RNA extraction kit (Vazyme, cat # RC 101); PowerUP SYBR Green Master Mix (Thermo Fisher Scientific, A25742); SDS-PAGE gel rapid preparation kit (Biyunyan, cat # P0012 AC); prestained Protein Ladder, (10-180kDa) (Thermo, cat # 26617); ECL chemiluminescence kit (bi yun day, cat No. P0018A); PVDF membrane (Merck Millipore, cat # IPVH 00010); HRP-Goat Anti-Rabbit IgG (H + L) (Abclonal, cat # AS 014); HRP-Goat Anti-Mouse IgG (H + L) (Abclonal, cat # AS 003); beta-actin Antibody (Abclonal, cat # AC 026); HPV6E7 Antibody (LSBio, cat # LS-C144095); HPV11E7 Antibody (Abcam, cat No. ab 100967); estradiol benzoate injection (Sichuan gold pharmaceutical industry, Inc., Cat. 20190312).

The main apparatus is as follows: an electrophoresis apparatus power supply (Beijing Liuyi, model DYY-6C/D); a double vertical electrophoresis apparatus (Tanon, model VE-180); transfer electrophoresis apparatus (tank) (Tanon, model VE-186); horizontal decolorization shaker (Haimanychijier model ZD-9550); a bench-top high-speed high-capacity refrigerated centrifuge (ThermoFisher, model ST 8R); microplate readers (Molecular Devices, model Spectra Max i3 x); metal constant temperature bath (BIOER, model HB-20)2) (ii) a A chemiluminescent imaging system (Tanon, model 5200 Multi); real-time fluorescent quantitative PCR instrument (Thermo Fisher, USA, model 7500); PCR instrument (ABI, usa, model 9700); a trace nucleic acid detector (Thermo Fisher, model NanoDrop Lite); a desk type low speed centrifuge (Kaida Industrial development Co., Ltd., Hunan province, model TD 4); biological microscopes (Chongqing Ott optical instruments, Inc., model DM 2000); paraffin microtomes (Leica, germany, model RM 2235); tissue embedding machines (Leica, germany, model EG1150H + C); a full-automatic dehydrator (Leica, germany, model ASP 200S); biomicroscopy (Leica, germany, model CX 31); inverted fluorescence microscope (Leica, germany, model DMi 8); flow cytometry (BD, model C6); CO 2₂Incubator (Thermo Fisher, model 3111); medical decontamination platforms (Suzhou von Willebrand laboratory animal facilities Co., Ltd., model CJ-1F).

Example 1 HPV6 and HPV11 pseudovirus preparation

First, experiment method

HPV6 and HPV11 pseudovirus construction

(1) Construction of structural Gene expression plasmid

Genes of structural proteins L1 and L2 of HPV6 and HPV11 which are optimized by codons are synthesized and cloned into a vector pCMV-MCS, and structural gene expression plasmids are named as pCMV-HPV6-L1-flag-L2-6 His and pCMV-HPV11-L1-flag-L2-6 His respectively.

(2) Construction of plasmids

The report plasmids pcDNA3.1-EGFP-HPV6-E6, pcDNA3.1-EGFP-HPV6-E7, pcDNA3.1-EGFP-HPV11-E6 and pcDNA3.1-EGFP-HPV11-E7 were synthesized. Hereinafter, pcDNA3.1-EGFP-HPV6-E6/E7 means pcDNA3.1-EGFP-HPV6-E6 and pcDNA3.1-EGFP-HPV6-E7, and pcDNA3.1-EGFP-HPV11-E6/E7 means pcDNA3.1-EGFP-HPV11-E6 and pcDNA3.1-EGFP-HPV 11-E7.

(3) Conventional transformation and mass extraction of plasmids

Taking out 4-tube DH5 alpha competent cells from a refrigerator at the temperature of-80 ℃, putting the cells on ice, after dissolving, respectively adding 0.5-1 mu L of precooled target plasmid into the 4-tube competent cell suspension, gently mixing the cells by using a pipette, adding the cells into an electric shock cup by using an electric rotating instrument for 2.5kv and 5s, then adding 200mu L of preheated LB liquid culture medium into each centrifugal tube, mixing the cells uniformly, putting the cells at the temperature of 37 ℃, and performing shaking culture on a shaking table at the speed of 200rpm for 1h to resuscitate the cells. And (3) mixing the contents of the centrifuge tube uniformly, sucking the transformed competent cells, adding the competent cells to an LB solid culture medium containing corresponding antibiotics, and culturing at 37 ℃ for 12-15 h. Selecting a single colony on a plate to inoculate (or directly taking 100-200 mu L of bacterial liquid corresponding to the synthetic plasmid) into 2mL of LB liquid culture medium containing corresponding antibiotics to culture, then inoculating into 200mL of LB liquid culture medium to carry out amplification culture, extracting plasmids by using an endotoxin-free plasmid macroextraction kit, measuring the purity and concentration of the plasmids by using a Nanodrop, and storing at-20 ℃ for later use.

Preparation of HPV6 and HPV11 pseudoviruses

(1) Structural gene expression plasmid and report plasmid are cotransfected to 293FT cells

The day before transfection, at 1X 10⁶The 293FT cells are paved in a 6-well plate culture plate at the density of mL, the cells are cultured overnight at 37 ℃, transfection experiments can be carried out when the cells grow to 60-70%, structural gene expression plasmids (pCMV-HPV6-L1-flag-L2-6 His and pCMV-HPV11-L1-flag-L2-6 His) and report plasmids (pcDNA3.1-EGFP-HPV6-E6/E7 and pcDNA3.1-EGFP-HPV11-E6/E7) are respectively co-transfected to 293FT cells by using Lipo-fectamine 2000, and simultaneously, single transfection report plasmids are respectively set as negative control groups. Before 0.5h of transfection, a DMEM complete culture medium in a culture well plate is replaced by a serum-free DMEM culture medium, starvation treatment is carried out on cells, and the dosage is reduced by half; firstly, a transfection reagent Lipofectamine 2000, a control group plasmid and an experimental group plasmid (pcDNA3.1-EGFP-HPV 6-E6: pCMV-HPV6-L1-flag-L2-6 His ═ 1: 1, pcDNA3.1-EGFP-HPV 6-E7: pCMV-HPV6-L1-flag-L2-6 His ═ 1: 1, cDNA3.1-EGFP-HPV 11-E6: pCMV-HPV11-L1-flag-L2-6 His ═ 1: 1, pcDNA3.1-EGFP-HPV 11-E7: pCMV-11-L1-L2-6 His ═ 1: 1, two plasmids in each group are respectively diluted by Opti-flag-3 mu g and placed in an Opti-5 min and then mixed at the temperature; mixing Lipofectamine 2000 with the control group plasmid and the experimental group plasmid respectively, slightly reversing and mixing on a vortex vibration instrument, and standing at room temperature for about 20 min; adding the transfection mixed solution into a culture plate which is changed into a serum-free culture medium, slightly and uniformly mixing, placing the mixture in a culture box for 4-6 hours, sucking off the serum-free culture medium,and (4) changing the culture medium into a DMEM complete culture medium for continuous culture, harvesting pseudoviruses after 48-72 hours, and performing virus titer determination.

(2) Viral titer determination

293FT cells at 1.5X 10⁴The cells were plated in 96-well plates at 37 ℃ with 5% CO at a density of/mL₂The culture was carried out overnight in an incubator. Respectively carrying out 10 treatments on the HPV6 and the HPV11 pseudoviruses prepared in the step (2) by using DMEM culture medium^-1、10^-2、10^-3、10^-4And 10^-5And (3) performing multiple dilution, namely adding 100 mu L of each gradient virus diluent into 293FT cells, wherein each dilution gradient has 8 multiple wells, putting a 96-well plate into an incubator at 37 ℃ for incubation for 72h, and then observing under an inverted fluorescence microscope, wherein more than one diseased cell appears and is determined as a positive well. Calculation of cell culture half-infection amount (TCID) by Reed-Muench method₅₀) I.e. the dilution of the virus when it infects half of the cells.

Second, experimental results

The results of the virus titer determinations are shown in table 1:

table 1 titer determination of HPV6 and HPV11 pseudoviruses statistics of CPE hole appearance (n ═ 8)

According to the formula, the following results are obtained:

logTCID₅₀log (dilution of percentage of death higher than 50%) + PD × dilution factor

＝-2.8

Looking up an inverse logarithm table: 10-2.8 ═ TCID₅₀End point dilution ratio 1: 630.95

That is, the HPV6 and 11 pseudoviruses were assayed to contain 630 TCIDs per 0.1mL inoculum size₅₀Units.

100 times TCID₅₀HPV6 and HPV11 pseudovirion: stock solutions of HPV6 and HPV11 pseudoviruses were cultured in DMEM medium at 1: diluting by 6.3 times to obtain the final product.

Example 2 Effect of Ailanthus altissima latex gel on morphology and proliferation of 293FT cells

First, experiment method

293FT cells at 1.5X 10⁴The cells were plated in 96-well plates at 37 ℃ with 5% CO at a density of/mL₂The culture was carried out overnight in an incubator. After the cells grow into monolayer cells, washing the cells with 0.9% NaCl solution for 2-3 times, and respectively diluting a mother solution (40mg/mL) of a test product (ailanthus milk gel) with 10% FBS-containing DMEM culture medium for 6 concentrations (1000, 500, 250, 125, 62.5 and 31.25 mu g/mL); setting 6 concentrations (100, 50, 25, 12.5, 6.25 and 3.125. mu. moL/L) of positive drug (podophyllotoxin), adding 100. mu.L of each group of drug into 96-well plate, setting blank control group, setting 3 multiple wells for each group, placing at 37 deg.C and 5% CO₂The culture box is used for culturing, the morphology of the cells is observed under an inverted fluorescence microscope after 24 hours, 48 hours and 72 hours respectively, the CellTiter-Glo luminescence method is adopted to measure the activity of the cells after 72 hours, and the IC of the test sample and the positive medicine is calculated₅₀And TC₀(IC₂₀)。

Second, experimental results

As shown in Table 2, the IC of the test article on 293FT cells₅₀333.80 μ g/mL, TC₀129.20 mu g/mL, has little difference in toxicity to 293FT cells in the concentration range of (100-3.125 mu mol/L) with the positive drug, and does not detect the IC of the positive drug to 293FT cells in the test concentration range₅₀And TC₀. FIGS. 1 to3 show the effect of adding toona sinensis latex gel solution (concentration 1000-31.25. mu.g/mL, partial pictures) on 293FT cell morphology after 24-72 h treatment, the cell morphology of the blank control group is round, and the water caltrops are clear. Compared with a blank control group, the toona sinensis milk gel dry-treatment group has obvious changes in cell morphology, mainly takes cell shedding and shrinkage as main points, and prompts that cell proliferation is inhibited to a certain extent after medicine intervention.

TABLE 2 toxicity results of Ailanthus altissima gel and Podophyllotoxin on 293FT cells

Example 3 Effect of Ailanthus altissima gel on the infectivity of HPV6 and HPV11 pseudoviruses

First, experiment method

293FT cells at 1.5X 10⁴The cells were plated in 96-well plates at 37 ℃ with 5% CO at a density of/mL₂The culture was continued overnight in an incubator until a monolayer of cells was formed. When the cells were grown to about 80%, the culture medium was discarded and 100-fold of TCID prepared in example 1 was added₅₀100 μ L of HPV6 and HPV11 pseudovirus liquid per well. After further culturing for 2h, the medium was replaced with 100. mu.L/well of different concentrations of drug-containing medium (concentration was set according to cytotoxicity), and the preparation method of the drug-containing medium is shown in example 2, wherein the toona sinensis milk gel was diluted to 6 concentrations (125, 62.5, 31.25, 15.62, 7.81 and 3.90. mu.g/mL), the positive drug was diluted to 6 concentrations (100, 50, 25, 12.5, 6.25 and 3.125. mu.mol/L), and the vehicle control group was set: adding DMEM medium, repeating 8 wells each, and standing at 37 deg.C under 5% CO₂And after incubation for 24-72 h in the incubator, observing and recording the cytopathic conditions of the experimental group and the control group under an inverted fluorescence microscope.

Second, experimental results

After 48h of virus infection, cells showed obvious pathological changes, and as shown in FIG. 4, the cells of HPV6 and HPV11 group showed different degrees of cytopathic changes compared with the vehicle control group. The stem prognosis cytopathic degree of the toona sinensis milk gel is obviously reduced, which shows that the toona sinensis milk gel has certain capacity of inhibiting virus proliferation; as shown in figure 5, the virus inhibition effect of the toona sinensis milk gel is reduced after 72h of culture, and the difference of cytopathic effect between the HPV6 and HPV11 groups and the toona sinensis milk gel intervention group is not large, and the positive drug has certain capacity of inhibiting virus proliferation in 72 h. It is known that continuous administration is required to enhance the drug action in order to maintain the antiviral ability of the ailanthus altissima gel. In conclusion, the toona sinensis milk gel has a certain inhibition effect on the proliferation of HPV6 and HPV11 viruses.

Example 4 Effect of Ailanthus altissima milk gel on 293FT cell cycle

First, experiment method

293FT cells at 1.5X 10⁶The density of each well was laid in a 6-well plate and placed at 37 ℃ in 5% CO₂Culturing in an incubator overnight, when the cells grow to about 80%, washing the cells with 0.9% NaCl solution for 2-3 times, and adding 100 times of TCID prepared in example 1₅₀100 μ L of HPV6 and HPV11 pseudovirus liquid per well. After further culturing for 2h, the medium was replaced with 100. mu.L/well of different concentrations of drug-containing medium (concentration was set according to cytotoxicity), and the preparation method of the drug-containing medium was shown in example 2, wherein the toona sinensis milk gel was diluted to3 concentrations (low dose: 32.3. mu.g/mL, medium dose: 64.6. mu.g/mL, high dose: 129.20. mu.g/mL) of diluted concentration of positive drug (3.12. mu. mol/L), and a virus control group (HPV6 and HPV11 groups) and a vehicle control group were set. The cell culture plate was placed at 37 ℃ in 5% CO₂After incubating in the incubator for 48 hours, centrifuging at 1000rpm for 3min to collect cells, washing twice with precooled normal saline, and centrifuging; adding PI 10 μ L to final concentration of 20 μ g/mL (diluted with 190 μ L Binding Buffer (1 ×)), wrapping with tinfoil paper, dyeing at 4 deg.C in dark for 30min, and centrifuging; adding 200 μ L Binding Buffer (1 ×) for washing, and centrifuging; the cells were resuspended in 200. mu.L of physiological saline, and cell cycle data were collected and analyzed by flow cytometry (single flow cytometry run, cell count of 1 sample was about 10)⁶)。

Second, experimental results

The results of the analysis of the change in the 293FT cell cycle cells at each stage by PI single stain flow cytometry are shown in FIG. 6. G in cells 48h after HPV6 and HPV11 infection (FIG. 6, A)₀/G₁The proportion of cells in phase is significantly lower than that in the vehicle control group (figure 6, B), the proportion of cells in S phase is higher than that in the vehicle control group, G₂the/M cell ratio was not significantly different from the vehicle control group. Ailanthus altissima gel low dose (32.3. mu.g/mL), medium dose (64.6. mu.g/mL) and high dose (129.20. mu.g/mL) groups 293FT cell cycles (FIGS. 6C, D and E), G₀/G₁The proportion of cells in the stage is obviously higher than that of the HPV6 and HPV11 groups, the proportion of cells in the S stage is lower than that of the HPV6 and HPV11 groups, and G₂The cell ratio/M was lower than that of group HPV6 and HPV 11. Ailanthi emulsion gel group G₀/G₁The ratio of cells in phase is lower than that in the vehicle control group, and compared with that in the positive group (FIG. 6F)₀/G₁There was no significant difference in the proportion of cells in phase.

The invention researches the pharmacodynamics of the toona sinensis milk gel in vitro against the low-risk HPV virus. In vitro test results show that after 293FT cells are treated by the toona sinensis milk gel (1000-31.25 mug/mL) for 24-72 hours, the cell morphology in a stem cell pretreatment group of the toona sinensis milk gel is obviously changed compared with a solvent control group, the cell proliferation is inhibited to a certain extent, and the IC50 of the toona sinensis milk gel on the 293FT cells is 333.80 mug/mL; the results in the antiviral test show that: the Chinese toon milk gel solution can reduce the infectivity of HPV6 and HPV11 pseudovirus and inhibit the proliferation of HPV6 and HPV11 in the tested concentration range. Cell cycle results show that: the gel treatment of the toona sinensis milk can obviously regulate the cell cycle distribution induced by HPV6 and HPV11 pseudovirus infection and make the cell cycle distribution of infected cells close to normal. In conclusion, the toona sinensis milk gel has a certain treatment effect on cells infected by low-risk HPV.

Example 5 Effect of Ailanthus altissima gel on mouse model of HPV6 and HPV11 pseudovirus infection BALB/C

HPV E6 and HPVE7 are two oncogene fragments of HPV, resulting in genetic lesions that cause uncontrolled cell cycle in infected cells, resulting in anti-apoptosis, and ultimately in abnormal cell proliferation and tumorigenesis, while HPV E6mRNA and HPVE 7mRNA are viral oncogene transcripts whose expression leads to malignant transformation of host cells. Therefore, the invention researches the pharmacodynamics of resisting the low-risk HPV in the toona sinensis milk gel. In vivo test, a BALB/C mouse model infected by low-risk HPV virus extracted from 293FT cells containing HPV6 genome and HPV11 genome is used, and the pharmacodynamic effect of resisting HPV6 and HPV11 in the toona sinensis milk gel body is evaluated by detecting the virus titer of cervical tissue, HPV6E 6mRNA, HPV6E7mRNA, HPV11E 6mRNA, HPV11E7mRNA, HPV6 protein expression and HPV11 protein expression.

First, experiment method

1. Grouping and administration of drugs

Selecting 100 quarantine qualified SPF female BALB/C mice, injecting 0.1 μ g/female benzoic acid estradiol per animal subcutaneously 1 time per day for 3 days, and collecting 25 μ L of 100 times TCID prepared in example 1 in afternoon on 3 days₅₀Injecting the mixture of HPV6 and HPV11 pseudovirus solution and 15 muL of 4% CMC into the vagina of a mouse for infection, and injecting 40 muL of 4% CMC into another 16 blank control mice for treatment; after 48h of pseudovirus infection, slightly anaesthetizing the mice with ether, pouring 50 mu L of 15mg/mLD-Luciferase into the vaginas of the mice to promote luc luminescence in vaginal epithelial cells of the mice, and utilizing the internal bodies of the mice after 3minThe expression of luciferase in mice was detected by visible light imaging system (IVIS, Caliper Life Science). Selecting 80 BALB/C mice with obvious vaginal redness and swelling, and randomly dividing the mice into a model control group, a podophyllotoxin group and a toona sinensis milk gel low and medium high dose group according to the body weight, wherein each group comprises 16 mice. Each group of animals was vaginally administered 1 time per day for 20 consecutive days.

2. Dosage design

The clinical planned dose of the toona sinensis milk gel is 1 (4g) per day, and the equivalent dose of the toona sinensis milk gel converted into the mouse by a body surface area method is 4 g/day 0.0026 and approximately 0.01 g/day; according to the previous experiments, 1 time, 2 times and 4 times of equivalent dose of the experiment are set as the low, medium and high dose groups of the toona sinensis milk gel, namely 0.01, 0.02 and 0.04 g/day. The concentration of the podophyllotoxin is 5mg/mL, and the maximum loading capacity of the vagina of the mouse is 0.2mL, so that the dose of the podophyllotoxin is set to be 1 mg/mouse. See table 3 for details.

TABLE 3 grouping and dose design

3. Detecting the index

(1) Vaginal appearance score

Behavioral activity and vulvar condition of mice were observed and the vaginal opening was scored 10 and 20 days after administration, respectively, wherein the vaginal opening was scored for redness and swelling: none (score 0), mild (score 1), moderate (score 2) and severe (score 3); vaginal opening purulent secretion scoring standard: none (0 min), small (1 min), medium (2 min) and large (3 min).

(2) pH value detection

Vaginal secretions were sampled with sterile cotton swabs 10 and 20 days after administration, respectively. The material-taking part is the cervical orifice or the posterior fornix of the cervix of the mouse, the secretion is collected by using a disposable sterile vaginal swab, the circumference is rolled for 3 times, and the pH value of the vaginal secretion is detected by adopting 0.1-grade precise pH test paper.

(3) Histopathological examination

After the last administration, 8 animals are randomly selected to be dislocated and euthanized, and the vagina and the uterine tissue are dissected and taken to be observed by an HE staining method to observe pathological tissue changes of the vagina, the uterus and the cervix of the mice. Grading standard: NSL, counted as "0" point; calculating as 1 point; 2 is counted as "+"; and +++, is counted as 3.

(4) Cervical tissue virus titer detection

Taking a certain amount of tissue homogenate, centrifuging to take supernatant, inoculating 293T cells in a 96-well plate, and using up the whole culture medium (1-10) on day 2^-9) Diluting virus liquid by 10 gradients, adding 100 microliter diluted virus liquid into each hole, and setting 3 repetitions for each gradient; cells were observed after 48h for GFP expression, if 10^-8Gradient wells with 2, 3 and 4 cells GFP (+) respectively, averaged to give a 3X 10 viral titer⁸TU/mL. The hemagglutination inhibition assay was used to measure the hemagglutination inhibition titer (the maximal dilution factor for complete inhibition of erythrocyte agglutination) of cervical suspensions as log₂(maximum dilution factor), reflecting the viral titer in the cervix.

(5) HPV6E 6mRNA, HPV6E7mRNA, HPV11E 6mRNA and HPV11E7mRNA detection

HPV6E 6mRNA, HPV6E7mRNA, HPV11E 6mRNA and HPV11E7mRNA in each group of cervical tissues are detected, total RNA of each group is extracted, reverse transcription is carried out to cDNA, PCR reaction is carried out, and Primer sequences are designed by using Primer Premier6 and Oligo7 software according to gene data in Genbank. The internal reference is beta-actin. Setting the expression quantity of the reference gene as 100, and representing the expression quantity of the target gene by the relative percentage of the target gene in the reference gene. The primer sequences are shown below:

HPV6E6 primer sequence (5 'to 3'):

Forward Primer(SEQ ID NO:1)：TGT TTC AGG ACC CAC AGG AG，

Reverse Primer(SEQ ID NO:2)：TCA CGT CGC AGT AAC TGT TG；

HPV6E7 primer sequence (5 'to 3'):

Forward Primer(SEQ ID NO:3)：CAG CTC AGA GGA GGA GGA TG，

Reverse Primer(SEQ ID NO:4)：AAC CGA AGC GTA GAG TCA CA；

HPV11E 6 primer sequence (5 'to 3'):

Forward Primer(SEQ ID NO:5)：CAC TAT AGA GGC CAG TGC CA，

Reverse Primer(SEQ ID NO:6)：CTT GTG TTT CTC TGC GTC GT；

HPV11E7 primer sequence (5 'to 3'):

Forward Primer(SEQ ID NO:7)：CGA ACC ACA ACG TCA CAC AA，

Reverse Primer(SEQ ID NO:8)：AGA AAC AGC TGC TGG AAT GC

(6) protein expression of HPV6E6, HPV6E7, HPV11E 6 and HPV11E7

And (3) reserving a sample, determining the protein concentration by using a BCA method, preparing separation gel and concentrated gel, loading the sample, and performing electrophoresis. Transferring the protein on the gel after electrophoresis to a polyvinylidene fluoride membrane, and performing ECL color development exposure. The experiment was repeated 6 times. The relative expression amount of the target protein is the gray value of the target protein band/the gray value of the beta-actin band.

(7) Statistical method

The experimental data of the invention are subjected to effective number pruning according to a rounding reduction rule, statistical analysis is carried out according to the specification of SOP, and the level of statistical significance is set to be less than or equal to 0.05. The software used was counted as SPSS 22.0. The measurement data adopts mean + -standard deviation

Showing that the method of Leven's test is used for checking the normality and the homogeneity of variance. If the normality and homogeneity of variance are met, carrying out statistical analysis by using One-way ANOVA (One-way ANOVA) and post Hoc LSD; if the data accords with the normality but the variances are not uniform, adopting Tamhane to carry out statistical analysis; if the normality is not met, statistical analysis is performed using Kruskal-Wallis. If the Kruskal-Wallis Test is statistically significant (P.ltoreq.0.05), a comparative analysis is carried out using the Dunnett's Test (nonparametric method) or the Mann-Whitney Test.

Second, experimental results

1. General observations (field planting inspection)

As shown in fig. 7 and 8, HPV6 and HPV11 were colonized 48h after molding by a small animal in-vivo imaging system, and the results showed that the fluorescence intensity of the model control group was high, and the fluorescence intensity of the model control group was still high before the last administration, indicating that HPV6 and HPV11 were colonized well in the vagina. Cage-side observation shows that the general conditions (autonomous activity, food intake, water intake and the like) of each group of animals are not abnormal after administration, and the vagina has no obvious secretion and red swelling.

2. Effect on vaginal pH

As shown in Table 4, compared with the blank control group, the pH value of the vagina of the model control group is obviously increased (P is less than or equal to 0.01) at the 10 th and 20 th days after the administration; compared with the model control group, the pH values of the vaginas of the podophyllotoxin group and the toona sinensis milk gel low-dose group are obviously reduced (P is less than or equal to 0.05 or P is less than or equal to 0.01) on the 10 th day of administration, and the pH values of the vaginas of the podophyllotoxin group, the toona milk gel low-dose group, the toona sinensis milk gel medium-dose group and the toona milk gel high-dose group are obviously reduced (P is less than or equal to 0.05) on the 20 th day of administration. The vaginal pH values of the low and high dose groups of the toona sinensis milk gel on the 10 th day of administration were lower than those of the podophyllotoxin group and were closer to those of the blank control group. The vaginal pH of the dose group in the toona sinensis milk gel at the 20 th day of administration was identical to that of the blank control group. The pH value of the vagina of the low, medium and high dose groups of toona sinensis milk gel at the 20 th day of administration is lower than that of the vagina at the corresponding 10 th day of administration, and the pH value of the vagina is reduced along with the increase of the administration time; while vaginal pH did not change on days 10 and 20 after podophyllotoxin administration. The recovery of vaginal pH value of the toona sinensis milk gel low and high dose group is better than that of the podophyllotoxin group.

TABLE 4 influence of Ailanthus altissima latex gel on vaginal pH value (

n＝8)

Note: comparison with blank control group⁺⁺P is less than or equal to 0.01; comparison with model control group^*P≤0.05，^**P≤0.01。

3. Histopathological examination

(1) Under-mirror observation

As shown in fig. 9 and 10, mucosal epithelial cell necrosis was only seen under the vaginal and cervical histology of individual animals in the placebo group with a small amount of inflammatory cell infiltration; necrosis of cervical and vaginal epithelial cells in the model control group, epithelial cell proliferation of part of animals, and inflammatory cell infiltration of epithelial layer and mucosa lamina propria; degeneration and necrosis of mucous epithelial cells can be seen under the colposcopy and the cervical histoscope of the podophyllotoxin group and the toona sinensis milk gel low, medium and high dose groups, and the degeneration and necrosis are accompanied by a small amount of inflammatory cell infiltration. After the model is made, the mouse is administrated with different tested substances through vagina, the pathological changes of the vagina tissue are not obvious, and the pathological changes of the cervix are relieved to different degrees. After 20 days of continuous administration, the animals in each group were observed under hysteroscopy without epithelial cell proliferation and necrosis, and without inflammatory cell infiltration.

(2) Effect on tissue lesion Scoring

As shown in Table 5, compared with the blank control group, the cervical tissue lesion score of the model control group is obviously increased (P is less than or equal to 0.01), and the vaginal tissue lesion score has an increasing trend but has no statistical difference; compared with the model control group, the cervical tissue lesion scores in the podophyllotoxin group, the toona sinensis milk gel group and the high-dose group are obviously reduced (P is less than or equal to 0.05 or P is less than or equal to 0.01). The vaginal tissue lesion scores of the podophyllotoxin group and the toona sinensis milk gel low, medium and high dose groups are not statistically different from those of the model control group.

TABLE 5 Effect of Ailanthus altissima gel on tissue lesion Scoring: (

n＝8)

(3) Effect on viral titre in cervical tissue

As shown in Table 6, compared with the blank control group, the virus titer of the cervical tissue of the model control group is obviously increased (P is less than or equal to 0.01); compared with the model control group, the cervical tissue virus titer of the podophyllotoxin group and the toona sinensis milk gel is obviously reduced (P is less than or equal to 0.01) in the low, medium and high dose groups. Compared with the podophyllotoxin group, the cervical tissue virus titer of the ailanthus altissima gel high-dose group is obviously reduced (P is less than or equal to 0.01).

TABLE 6 influence of Ailanthus altissima latex gel on viral titer in cervical tissue ((S. sup.))

n＝8)

Note: comparison with blank control group⁺⁺P is less than or equal to 0.01; comparison with model control group^**P is less than or equal to 0.01; comparison with Podophyllotoxin group^&&P≤0.01。

(4) Effect on expression of HPV6E 6mRNA, HPV6E7mRNA, HPV11E 6mRNA and HPV11E7mRNA

As shown in fig. 11 and 12, HPV6E 6mRNA, HPV6E7mRNA, HPV11E 6mRNA and HPV11E7mRNA were not detected in the blank control group tissues; the HPV6E 6mRNA, HPV6E7mRNA, HPV11E 6mRNA and HPV11E7mRNA in the cervical tissues of the model control group are obviously increased (P is less than or equal to 0.01); compared with a model control group, the podophyllotoxin group HPV6E 6mRNA, HPV6E7mRNA and HPV11E7mRNA are all obviously reduced (P is less than or equal to 0.05 or P is less than or equal to 0.01), the Chinese toon milk gel low dose group HPV6E7mRNA and HPV11E7mRNA are all obviously reduced (P is less than or equal to 0.05), and the Chinese toon milk gel medium and high dose group HPV6E 6mRNA, HPV6E7mRNA and HPV11E7mRNA are all obviously reduced (P is less than or equal to 0.05 or P is less than or equal to 0.01). The HPV11E7mRNA in the low, medium and high dose groups of the Chinese toon milk gel is lower than that in the podophyllotoxin group, and the HPV11E 6mRNA in the medium and high dose groups of the Chinese toon milk gel is lower than that in the podophyllotoxin group.

(5) Influence on expression level of HPV6 and HPV11 proteins

The results of 6 repeated experiments are shown in fig. 13 and fig. 14, fig. 13 is an experimental graph of protein concentration determined by 6 BCA method, fig. 14 is a statistical average value of protein expression, compared with blank control group, the protein expression of model control group HPV6 and HPV11 are both increased obviously (P is less than or equal to 0.01); compared with a model control group, the protein expression levels of HPV6 and HPV11 in a podophyllotoxin group are obviously reduced (P is less than or equal to 0.01), the protein expression levels of HPV11 in a low, medium and high dose group of toona sinensis milk gel are obviously reduced (P is less than or equal to 0.05 or P is less than or equal to 0.01), and the protein expression levels of HPV6 in the medium and high dose group of toona sinensis milk gel are obviously reduced (P is less than or equal to 0.05 or P is less than or equal to 0.01).

In vivo test results show that HPV6 and HPV11 establish low-risk human papilloma virus models through mouse vaginal infection, high fluorescence quantity is displayed before and at the end of administration through live imaging of small animals, the fact that HPV6 and HPV11 viruses are successfully implanted in mouse vaginas is prompted, and pathological results show that cervical tissue mucosal epithelial cells are necrotic and hyperplastic, and inflammatory cell infiltration is accompanied. After the toona sinensis milk gel is given 48 hours after molding, the vaginal pH value and the cervical tissue virus titer of a toona sinensis milk gel administration group are obviously reduced, the protein expressions of HPV6E 6mRNA, HPV6E7mRNA, HPV11E 6mRNA, HPV11E7mRNA, HPV6 and HPV11 are obviously reduced, and the lesion degree of cervical tissues is obviously reduced.

In conclusion, the toona sinensis milk gel has a certain inhibition effect on 293FT cell proliferation within the concentration range (31.25-1000 mug/mL), and the IC50 of the toona sinensis milk gel on cells is 333.80 mug/mL. The toona sinensis milk gel can effectively reduce the infectivity of low-risk HPV and treat the infection of the low-risk HPV to a certain extent. The toona sinensis milk gel can obviously reduce the pH value of the vagina and the virus titer of cervical tissues, and obviously reduce the protein expressions of HPV6E 6mRNA, HPV6E7mRNA, HPV11E 6mRNA, HPV11E7mRNA, HPV6 and HPV11, and the low-risk human papilloma virus model established by mice infected with HPV6 and HPV11 has obvious treatment effect, and the in vitro and in vivo results are consistent.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

Application of Ailanthus altissima latex gel in preparation of medicines for resisting low-risk HPV infection and preventing and treating low-risk HPV related diseases

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Claims

1. The application of a traditional Chinese medicine composition in preparing a medicine for resisting low-risk HPV virus is characterized in that the effective components of the traditional Chinese medicine composition are ailanthus bark, sophora flavescens, moutan bark, frankincense and borneol.

2. The use according to claim 1, wherein the anti-low risk type HPV virus is an inhibitory low risk type HPV virus replication and/or proliferation.

3. The use according to claim 1, wherein the anti-low risk type HPV virus is an inhibitory low risk type HPV viral infection.

4. The use according to claim 1, wherein the anti-low risk type HPV virus is for restoring low risk type HPV host cell environment pH imbalance and/or alleviating cervical tissue pathology caused by low risk type HPV virus infection.

5. The use according to claim 2, wherein the inhibition of mRNA expression, the inhibition of protein expression or the restoration of cell cycle disorders in low risk HPV host cells is one or more of low risk HPV.

6. The use according to claim 3, wherein the inhibiting of the infection by the low risk type HPV virus comprises reducing the infectivity of the low risk type HPV virus and/or reducing the titer of the low risk type HPV virus in the tissue.

7. The application of a traditional Chinese medicine composition in preparing a medicine for treating diseases caused by low-risk HPV virus is characterized in that the effective components of the traditional Chinese medicine composition are ailanthus bark, radix sophorae flavescentis, moutan bark, frankincense and borneol.

8. The use according to claim 7, wherein the treatment of the disease caused by the low risk type HPV virus is one or more of alleviating lesions of cervical tissue, restoring cell cycle disorders or restoring environmental pH disorders of low risk type HPV host cells.

9. The use of claim 7, wherein the disease is condyloma acuminata.

10. The use according to any one of claims 1 to 9, wherein the low risk type HPV virus is one or more of HPV6, HPV11, HPV13, HPV42, HPV43, HPV44, HPV53 and/or HPV 54.