Application of gossypol in inhibiting HPV16 infection

Abstract

The present invention provides an application of gossypol in inhibiting HPV16 infection, belonging to the field of medical technology. Through long-term and extensive experiments, the inventors found that gossypol can inhibit the infection of human epithelial cells by HPV16 by downregulating the Wnt / β-catenin signaling pathway, and the inhibitory effect is time- and concentration-dependent. The present invention opens up a new application field of gossypol, which can be used for the preparation of drugs for blocking the persistent infection of high-risk HPV, helps to promote the early prevention and treatment of HPV-related tumors such as cervical cancer, and has certain theoretical significance and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of gossypol in inhibiting HPV16 infection. Background Art

[0002] Persistent infection with high-risk human papillomavirus (hrHPV) is the main risk factor for HPV-related tumors such as cervical cancer and head and neck squamous cell carcinoma. However, there is no effective drug clinically, which is a major public health problem affecting the health of the Chinese population, especially women of childbearing age. HPV belongs to the papillomavirus family and is a small non-enveloped DNA virus that can infect epithelial cells. More than 100 subtypes have been discovered so far. Infection with high-risk HPV (such as HPV16, etc.) is associated with 5% of human tumors, including cervical, vulvar, vaginal, anal, penile, and oropharyngeal tumors.

[0003] HPV infects host cells mainly by relying on two viral capsid proteins to mediate the binding, internalization, and transportation of the virus and cells, namely the major capsid protein L1 and the minor capsid protein L2. HPV is a virus that is extremely rich in human species specificity and tissue specificity and has a high tissue tropism for stratified squamous epithelium such as the cervix. Viral particles enter tissues through tiny wounds in the epithelial cells of the female genital tract. Only by infecting the basal cells of the stratified squamous epithelium can the complete life cycle be completed, generating new HPV viral particles to enter the reinfection process. Through the cycle of infection - release - reinfection, persistent infection is caused. During persistent infection, the DNA of high-risk HPV is often integrated into the genome of host cells, which can ultimately lead to the malignant transformation of host cells. However, there is currently no effective drug for persistent HPV infection. The clinical strategy is mainly follow-up observation, which causes great psychological burden to patients and is also prone to over-treatment, increasing the social health and economic pressure. Therefore, it is necessary to research and develop inhibitors of HPV infecting host cells to provide intervention means for blocking the disease process of persistent HPV infection developing into cervical intraepithelial neoplasia and cervical cancer.

[0004] Gossypol is a polyphenolic compound and is an extract of the seeds and root bark of Gossypium herbaceum, Gossypium arboreum, or cotton in the Malvaceae family. The content of gossypol varies with the cotton variety, the climate and soil of the planting area, and the water content; at different maturity stages of cotton, the content of gossypol is also constantly changing; the content of gossypol in cotton seeds increases with the maturity of cotton, and its content is the highest after cotton matures. The chemical structure of gossypol is 1,1’,6,6’,7,7’-hexahydroxy-3,3’-dimethyl-5,5’-diisopropyl-2,2’-binaphthalene-8,8’-dialdehyde, and the molecular formula is C 30 0H 30 O 8, with a molecular weight of 518.55, has two optical isomers. Because it contains six hydroxyl groups and two aldehyde groups, it is soluble in most organic solvents. As a natural product with a polyhydroxy binaphthaldehyde structure, gossypol has attracted much attention due to its wide range of biological activities. Studies have found that gossypol has various biological effects such as anti-inflammatory, antioxidant, anti-parasitic, anti-fertility, and anti-tumor effects. At the same time, gossypol also has a certain antiviral effect. However, whether gossypol can inhibit the infection of high-risk HPV (such as HPV16) has not been reported in this regard. Summary of the Invention

[0005] The object of the present invention is to propose the application of gossypol in inhibiting HPV16 infection. The inventors have found through a large number of long-term experiments that gossypol can inhibit the infection of host cells by HPV16 pseudovirus by down-regulating the Wnt / β-catenin pathway, and the inhibitory effect is time- and concentration-dependent. Therefore, gossypol can be used to prepare an inhibitor for HPV16 infection.

[0006] The technical solution of the present invention is realized as follows:

[0007] The present invention provides the application of gossypol in inhibiting HPV16 infection.

[0008] As a further improvement of the present invention, the application of gossypol in inhibiting the infection of host cells by HPV16.

[0009] As a further improvement of the present invention, the application of gossypol in preparing a drug for inhibiting HPV16 infection.

[0010] As a further improvement of the present invention, the chemical structural formula of gossypol is shown in Formula I:

[0011]

[0012] As a further improvement of the present invention, the inhibitory effect of gossypol is time- and concentration-dependent.

[0013] First, the inventors found through an effect experiment of gossypol on the infection of human immortalized keratinocyte cell line HaCat cells by HPV16-GFP pseudovirus that gossypol can inhibit the infection of HaCat cells by HPV16-GFP pseudovirus.

[0014] Second, the inventors found through a time-effect experiment of gossypol on the infection of human immortalized keratinocyte cell line HaCat cells by HPV16-GFP pseudovirus that the inhibition of the infection of HaCat cells by HPV16-GFP pseudovirus by gossypol is time-dependent.

[0015] Third, through the time-effect experiment of gossypol acting on primary human cervical epithelial cells infected with HPV16-GFP pseudovirus, the inventors found that the inhibition of HPV16-GFP pseudovirus infection of primary human cervical epithelial cells by gossypol is time-dependent.

[0016] Fourth, through the dose-effect experiment of gossypol acting on human immortalized keratinocyte cell line HaCat cells infected with HPV16-GFP pseudovirus, the inventors found that the inhibition of HPV16-GFP pseudovirus infection of HaCat cells by gossypol is concentration-dependent.

[0017] Fifth, through the dose-effect experiment of gossypol acting on primary human cervical epithelial cells infected with HPV16-GFP pseudovirus, the inventors found that the inhibition of HPV16-GFP pseudovirus infection of primary human cervical epithelial cells by gossypol is concentration-dependent.

[0018] Sixth, through the experiment on human immortalized keratinocyte cell line HaCat cells in vitro, the inventors found that gossypol can down-regulate the Wnt / β-catenin signaling pathway.

[0019] It can be determined therefrom that gossypol can inhibit the infection of host cells by HPV16 pseudovirus by down-regulating the Wnt / β-catenin pathway, and the inhibitory effect is time- and concentration-dependent. Therefore, gossypol can be used to prepare an inhibitor for HPV16 infection.

[0020] The present invention has the following beneficial effects:

[0021] 1. Through long-term and extensive experiments, the inventors found that gossypol can inhibit the infection of host cells by HPV16 pseudovirus by down-regulating the Wnt / β-catenin pathway, and the inhibitory effect is time- and concentration-dependent. Therefore, gossypol can be used to prepare an inhibitor for HPV16 infection.

[0022] 2. The present invention discovers a new application of gossypol. China is a major cotton-producing country, and the annual extraction amount of gossypol is about 17,500 tons, which provides a reference value for improving the development and utilization of gossypol; at the same time, gossypol is a natural plant component, with good bioavailability, relatively small toxic and side effects, and wide sources. Studying the new uses of gossypol and its derivatives has broad application prospects in the medical field and is of great significance to China.

[0023] 3. The present invention provides a new method and new idea for the treatment of high-risk HPV persistent infection, and promotes the progress of medicine. Description of the Drawings

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 For Example 1 of the present invention, under an immunofluorescence microscope, gossypol was observed to inhibit the infection of human immortalized keratinocyte cell line HaCat cells by HPV16-GFP pseudovirus.

[0026] Figure 2 For Example 1 of the present invention, flow cytometry fluorescence sorting detected that gossypol inhibited the infection of human immortalized keratinocyte cell line HaCat cells by HPV16-GFP pseudovirus. Compared with the results of the control group, * indicates a significant difference with P < 0.05, and ** indicates a highly significant difference with P < 0.01.

[0027] Figure 3 For Example 2 of the present invention, it is a time-effect diagram of gossypol inhibiting the infection of human immortalized keratinocyte cell line HaCat cells by HPV16-GFP pseudovirus. Compared with the results at 0 h, * indicates a significant difference with P < 0.05, and ** indicates a highly significant difference with P < 0.01.

[0028] Figure 4 For Example 3 of the present invention, it is a time-effect diagram of gossypol inhibiting the infection of primary human cervical epithelial cells by HPV16-GFP pseudovirus. Compared with the results at 0 h, * indicates a significant difference with P < 0.05, and ** indicates a highly significant difference with P < 0.01.

[0029] Figure 5 For Example 4 of the present invention, it is a concentration-effect diagram of gossypol inhibiting the infection of human immortalized keratinocyte cell line HaCat cells by HPV16-GFP pseudovirus. Compared with the results of the control group, * indicates a significant difference with P < 0.05, and ** indicates a highly significant difference with P < 0.01.

[0030] Figure 6 For Example 5 of the present invention, it is a concentration-effect diagram of gossypol inhibiting the infection of primary human cervical epithelial cells by HPV16-GFP pseudovirus. Compared with the results of the control group, * indicates a significant difference with P < 0.05, and ** indicates a highly significant difference with P < 0.01.

[0031] Figure 7 For Example 6 of the present invention, it is an effect diagram of gossypol inhibiting the Wnt / β-catenin pathway in human immortalized keratinocyte cell line HaCat cells. Compared with the results of the control group, * indicates a significant difference with P < 0.05, and ** indicates a highly significant difference with P < 0.01. Detailed implementation mode

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] The human immortalized keratinocyte cell line HaCat cells were purchased from the American Type Culture Collection (ATCC). The culture conditions for HaCat cells are as follows: DMEM high-glucose medium (ATCC) + 10% fetal bovine serum (Gibco), at 37 °C, 5% carbon dioxide, with a pH value of 7.2 - 7.4, and cultured in a sterile constant temperature environment. Cell characteristics: These cells are derived from the normal skin around the lesion of a 62-year-old male with melanoma, positive for keratin, keratinocyte cross-linked outer membrane protein, and intermediate filament-associated protein, and grow adherently.

[0034] Gossypol (catalog number: HY-13407. CAS number: 303-45-7) was purchased from MedChemExpress (MCE), with a molecular weight of 518.55 and a molecular formula of C 30 H 30 O 8 , with a purity of 99.56%. The structural formula of the gossypol is shown in Formula I:

[0035]

[0036] Example 1: Gossypol inhibits the infection of human immortalized keratinocyte cell line HaCat cells by HPV16-GFP pseudovirus

[0037] In human immortalized keratinocyte HaCat cells, 20 μM gossypol (dissolved in the stock solution (DMSO: 33.33 mg / mL) in Examples 1 - 6) was added; after acting for 8 h, HPV16-GFP pseudovirus with an MOI of 100 was used for infection (the drug was not withdrawn during infection) for 12 h. After changing the medium and culturing for 72 hours, the infection of HPV16-GFP pseudovirus on HaCat cells was detected by immunofluorescence microscopy and flow cytometry fluorescence-activated cell sorting respectively.

[0038] It can be seen from Figure 1 that after observing under an immunofluorescence microscope, the infection rate of HPV16-GFP pseudovirus on HaCat cells can be reduced after the action of gossypol. It can be seen from Figure 2 that after detecting by flow cytometry fluorescence-activated cell sorting, the infection rate of HPV16-GFP pseudovirus on HaCat cells can be reduced after the action of gossypol (the infection rate decreased by 84.27%). It can be seen from this that gossypol inhibits the infection of human immortalized keratinocyte cell line HaCat cells by HPV16-GFP pseudovirus.

[0039] Example 2: Time-effect experiment on the inhibition of HPV16-GFP pseudovirus infection of human immortalized keratinocyte line HaCat cells by gossypol

[0040] In human immortalized keratinocyte HaCat cells, 10 μM gossypol was added. After acting for 0 h, 2 h, 4 h, and 8 h respectively, HPV16-GFP pseudovirus with an MOI of 100 was used to infect (without withdrawing the drug during infection) for 12 h. After changing the medium and culturing for 72 hours, the infection of HPV16-GFP pseudovirus on HaCat cells was detected by immunofluorescence microscopy and flow cytometry fluorescence sorting respectively.

[0041] It can be seen from Figure 3 that after gossypol acted for 2 h, 4 h, and 8 h respectively, the infection rates of HPV16-GFP pseudovirus on HaCat cells decreased by 12.97%, 55.67%, and 88.90% in sequence. It can be seen from this that the inhibitory effect of gossypol on the infection of HPV16-GFP pseudovirus on HaCat cells is time-dependent.

[0042] Example 3: Time-effect experiment on the inhibition of HPV16-GFP pseudovirus infection of primary human cervical epithelial cells by gossypol

[0043] In primary human cervical epithelial cells, 10 μM gossypol was added. After acting for 0 h, 2 h, 4 h, and 8 h respectively, HPV16-GFP pseudovirus with an MOI of 100 was used to infect (without withdrawing the drug during infection) for 12 h. After changing the medium and culturing for 72 hours, the infection of HPV16-GFP pseudovirus on HaCat cells was detected by immunofluorescence microscopy and flow cytometry fluorescence sorting respectively.

[0044] It can be seen from Figure 4 that after gossypol acted for 2 h, 4 h, and 8 h respectively, the infection rates of HPV16-GFP pseudovirus on HaCat cells decreased by 4.44%, 56.36%, and 86.67% in sequence. It can be seen from this that the inhibitory effect of gossypol on the infection of HPV16-GFP pseudovirus on primary human cervical epithelial cells is time-dependent.

[0045] Example 4: Concentration-effect experiment on the inhibition of HPV16-GFP pseudovirus infection of human immortalized keratinocyte HaCat cells by gossypol

[0046] In human immortalized keratinocyte HaCat cells, 0 μM, 5 μM, 10 μM, and 20 μM gossypol were added respectively. After acting for 4 h, the HPV16-GFP pseudovirus with an MOI of 100 was used to infect (without withdrawing the drug during infection) for 12 h. After changing the medium and culturing for 72 h, the infection of the HPV16-GFP pseudovirus on HaCat cells was detected by immunofluorescence microscopy and flow cytometry fluorescence sorting respectively.

[0047] It can be seen from Figure 5 that after the action of 5 μM, 10 μM, and 20 μM gossypol, the infection rates of the HPV16-GFP pseudovirus on HaCat cells decreased by 17.17%, 58.23%, and 84.76% in sequence. It can be seen from this that the inhibitory effect of gossypol on the infection of the HPV16-GFP pseudovirus on primary human cervical epithelial cells is time-dependent.

[0048] Example 5: Concentration effect experiment of gossypol on inhibiting the infection of the HPV16-GFP pseudovirus on primary human cervical epithelial cells

[0049] In primary human cervical epithelial cells, 0 μM, 5 μM, 10 μM, and 20 μM gossypol were added respectively. After acting for 4 h, the HPV16-GFP pseudovirus with an MOI of 100 was used to infect (without withdrawing the drug during infection) for 12 h. After changing the medium and culturing for 72 h, the infection of the HPV16-GFP pseudovirus on primary human cervical epithelial cells was detected by immunofluorescence microscopy and flow cytometry fluorescence sorting respectively.

[0050] It can be seen from Figure 6 that after the action of 5 μM, 10 μM, and 20 μM gossypol, the infection rates of the HPV16-GFP pseudovirus on primary human cervical epithelial cells decreased by 8.99%, 54.71%, and 87.67% in sequence. It can be seen from this that the inhibitory effect of gossypol on the infection of the HPV16-GFP pseudovirus on primary human cervical epithelial cells is concentration-dependent.

[0051] Example 6: Gossypol in human immortalized keratinocyte HaCat cells can down-regulate the Wnt / β-catenin signaling pathway

[0052] In human immortalized keratinocyte HaCat cells, 10 μM gossypol and 0.5 μM Wnt pathway inhibitor IWP-2 were added respectively. After acting for 4 h, RT-qPCR was used to detect the expression level of β-catenin.

[0053] It can be seen from Figure 7It can be seen that, compared with the control group, the expression levels of β-catenin in HaCat cells in the gossypol group (10 μM, 4 h) and the Wnt pathway inhibitor IWP-2 group (0.5 μM, 4 h) were significantly decreased. Thus, it can be known that gossypol can down-regulate the Wnt / β-catenin signaling pathway in human immortalized keratinocyte HaCat cells.

[0054] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. 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 present invention.

Claims

1. Use of gossypol as the only active ingredient in the preparation of a drug for inhibiting HPV16 infection, wherein the HPV16 infection is the disease process of the progression of persistent HPV infection to cervical precancerous lesions and cervical cancer.

2. The use according to claim 1, characterized in that the chemical structural formula of the gossypol is as shown in Formula I: Formula Ⅰ.

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