Application of pyrroloquinoline quinone or derivative thereof in preparation of anti-aging and anti-cancer dual-effect medicine

Abstract

The invention discloses application of pyrroloquinoline quinone or derivatives thereof in preparation of anti-aging and anti-cancer dual-effect medicines, and belongs to the technical field of biological medicines. According to the application of the pyrroloquinoline quinone or the derivative thereof in preparing the anti-aging and anti-cancer dual-effect medicine, the pyrroloquinoline quinone or the derivative thereof can further regulate and control the activity of telomerase by regulating and controlling expression of KLF4, so that the telomere length in a plurality of cancer cells is reduced while the telomere length in normal cells is prolonged, and the anti-aging and anti-cancer dual effects are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of pyrroloquinoline quinone or its derivatives in the preparation of anti-aging and cancer-inhibiting dual-action drugs. Background Art

[0002] Aging and cancer are two major challenges in medicine today. Telomeres play a key role in maintaining the stability of chromosome ends and the ability of cells to proliferate. Their activity is closely linked to the development and progression of both aging and cancer. Rapid telomere shortening can easily trigger premature cellular aging, while continued telomere shortening can easily lead to tumor development. The cell's telomere mechanism maintains a delicate balance between these two extremes, avoiding either extreme.

[0003] Pyrroloquinoline quinone (PQQ) has garnered widespread attention in the scientific community in recent years. PQQ has been shown to possess potent antioxidant properties, capable of scavenging free radicals and protecting cells from oxidative damage. PQQ also benefits cellular energy metabolism and mitochondrial function, helping to increase cell viability. Furthermore, PQQ has been found to promote neuroprotection and neural regeneration, which are important for preventing neurodegenerative diseases. However, there is still considerable room for further research into the mechanisms of action, effects, and quantitative studies of PQQ in specific cell types, particularly regarding its dual anti-aging and anti-cancer effects. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the use of pyrroloquinoline quinone or its derivatives in the preparation of anti-aging and anti-cancer dual-action drugs, so as to exert the anti-aging and anti-cancer dual effects of pyrroloquinoline quinone or its derivatives.

[0005] In a first aspect, the present invention provides a use of pyrroloquinoline quinone or a derivative thereof in the preparation of a dual-action anti-aging and anti-cancer drug.

[0006] Compared with the existing technology, the pyrroloquinoline quinone or its derivatives in the present invention can further regulate the activity of telomerase by regulating the expression of KLF4, thereby extending the telomere length in normal cells while reducing the telomere length in various cancer cells, achieving the dual effects of anti-aging and cancer inhibition.

[0007] Furthermore, the dual effects of anti-aging and anti-cancer are manifested in at least one of the following:

[0008] A) Increases telomere length in normal cells and decreases telomere length in cancer cells;

[0009] B) Promote telomerase activity in normal cells and inhibit telomerase activity in cancer cells;

[0010] C) Increase the mitochondrial membrane potential of normal cells and inhibit the mitochondrial membrane potential of cancer cells.

[0011] Furthermore, pyrroloquinoline quinone or its derivatives regulate telomere length or telomerase activity by at least one of the following means:

[0012] A) Increase the expression level of KLF4 gene in normal cells and decrease the expression level of KLF4 gene in cancer cells;

[0013] B) Increase the expression level of PARP1 gene in normal cells and decrease the expression level of PARP1 gene in cancer cells;

[0014] C) Increase the expression level of the β-cantenin gene in normal cells and decrease the expression level of the β-cantenin gene in cancer cells;

[0015] D) Increase the expression level of CMYC genes in normal cells and decrease the expression level of CMYC genes in cancer cells.

[0016] Furthermore, the cancer cells include liver cancer cells HePG2, lung cancer cells A549 or colon cancer cells HCT116.

[0017] Normal cells include human umbilical cord mesenchymal stem cells Hu-MSC, human bronchial epithelial cells Beas-2B, human liver cells LO2 or human fibroblasts HSF.

[0018] In a second aspect, the present invention provides a dual-action anti-aging and anti-cancer drug, which comprises at least one of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives as active ingredients.

[0019] Compared with the existing technology, the active ingredients of the drug of the present invention include at least one of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives. This active substance can selectively regulate KLF4-mediated telomerase expression, achieve the dual effects of anti-aging and cancer suppression, and maintain the balance of the cell's telomere mechanism between aging and tumors.

[0020] Furthermore, the pyrroloquinoline quinone derivative includes at least one of a pharmaceutically acceptable salt of pyrroloquinoline quinone and a pharmaceutically acceptable ester of pyrroloquinoline quinone.

[0021] Furthermore, the pharmaceutically acceptable salt of pyrroloquinoline quinone includes at least one of the disodium salt, dipotassium salt, dicalcium salt, dimagnesium salt, hydrochloride, sulfate, tartrate, oxalate, maleate, fumarate, and citrate of pyrroloquinoline quinone.

[0022] Furthermore, the pharmaceutically acceptable ester of pyrroloquinoline quinone includes at least one of methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester and hexyl ester of pyrroloquinoline quinone.

[0023] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0024] Furthermore, the dosage forms of the drug include tablets, capsules, injections, aerosols, suppositories, films, pills, patches, subcutaneous implants, external liniments, oral solutions, ointments or nanoformulations. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG1( a ) is a graph showing the effect of PQQ on HePG2 cell viability at different action times in Example 1.

[0026] FIG1( b ) is a graph showing the effect of PQQ on LO2 cell viability at different action times in Example 1.

[0027] FIG1(c) shows the effect of PQQ on Hu-MSC cell viability at different action times in Example 1, and the effect of PQQ on Beas-2B cell viability after 24 hours of action.

[0028] FIG1(d) is a graph showing the effect of PQQ on A549 cell viability at different action times in Example 1.

[0029] FIG1(e) is a graph showing the effect of PQQ on HCT116 cell viability at different action times in Example 1.

[0030] Figure 2 Graph showing the effect of PQQ on telomere length in Example 2: three normal cells, Hu-MSC, HSF, and Beas-2B, and three cancer cells, A549, HePG2, and HCT116.

[0031] Figure 3 Graph showing the effect of PQQ on Tert expression levels in three normal cells, LO2, HSF, and Beas-2B, and two cancer cells, HePG2 and HCT116, in Example 3.

[0032] FIG4( a ) is a graph showing the effect of PQQ on the expression level of KLF4 in HePG2 and HCT116 cancer cells in Example 4. ...

[0033] FIG4( b ) is a graph showing the effect of PQQ on the expression level of KLF4 in two normal cells, Hu-MSC and Beas-2B, in Example 4.

[0034] Figure 5 This is a graph showing the effect of PQQ on the expression levels of PARP1 in three normal cells, Beas-2B, LO2, and Hu-MSC, and two cancer cells, A549 and HCT116, in Example 4.

[0035] Figure 6Graph showing the effect of PQQ on the expression levels of β-cantenin in two normal cells, LO2 and Beas-2B, and three cancer cells, A549, HePG2, and HCT116, in Example 4.

[0036] Figure 7 This is a graph showing the effect of PQQ on the expression levels of CMYC in two normal cells, LO2 and Hu-MSC, and in HCT116 cancer cells in Example 4.

[0037] Figure 8 Graph showing the effect of PQQ on the mitochondrial membrane potential of LO2 cells and HePG2 cancer cells in Example 5.

[0038] Figure 9 Graph showing the effect of PQQ on the cell membrane potential of LO2 cells and HePG2 cancer cells in Example 6. DETAILED DESCRIPTION

[0039] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0040] In a first aspect, an embodiment of the present invention provides a use of pyrroloquinoline quinone or a derivative thereof in the preparation of a dual-action anti-aging and anti-cancer drug.

[0041] The pyrroloquinoline quinone or its derivatives in the present invention can achieve differential regulation of normal and cancer cells by selectively regulating KLF4-mediated telomerase expression. On the one hand, pyrroloquinoline quinone or its derivatives can significantly extend the telomere length of normal cells, increase telomerase activity, and enhance mitochondrial membrane potential and cell membrane potential, thereby delaying cell aging and maintaining normal cellular physiological functions. On the other hand, pyrroloquinoline quinone or its derivatives can significantly shorten the telomere length of cancer cells, reduce telomerase activity, and inhibit mitochondrial membrane potential, thereby limiting the growth and replication potential of cancer cells and inducing apoptosis.

[0042] Furthermore, based on the above embodiments, the dual effects of pyrroloquinoline quinone or its derivatives in the embodiments of the present invention on anti-aging and anti-cancer performance are manifested in at least one of the following aspects:

[0043] A) Increases telomere length in normal cells and decreases telomere length in cancer cells;

[0044] B) Promote telomerase activity in normal cells and inhibit telomerase activity in cancer cells;

[0045] C) Increase the mitochondrial membrane potential of normal cells and inhibit the mitochondrial membrane potential of cancer cells.

[0046] Telomere length is an important marker of cell aging and carcinogenesis, and telomerase activity is a key factor in maintaining telomere length. The pyrroloquinoline quinone or its derivatives in the present invention can affect the telomere length in cells by regulating the activity of telomerase, thereby achieving the dual effects of anti-aging and cancer inhibition.

[0047] Furthermore, mitochondrial membrane potential is an important indicator of mitochondrial function, and its changes are closely related to cellular energy metabolism and apoptosis. The pyrroloquinoline quinone or its derivatives in the present invention can increase the mitochondrial membrane potential of normal cells to maintain their normal function, while reducing the mitochondrial membrane potential of cancer cells, opening the mitochondrial permeability transition pore and releasing cytochrome C, which in turn activates caspase family proteins and triggers apoptosis in cancer cells.

[0048] Furthermore, based on the above embodiments, in the embodiments of the present invention, pyrroloquinoline quinone or its derivatives regulate telomere length or telomerase activity by at least one of the following methods:

[0049] A) Increase the expression level of KLF4 gene in normal cells and decrease the expression level of KLF4 gene in cancer cells;

[0050] B) Increase the expression level of PARP1 gene in normal cells and decrease the expression level of PARP1 gene in cancer cells;

[0051] C) Increase the expression level of the β-cantenin gene in normal cells and decrease the expression level of the β-cantenin gene in cancer cells;

[0052] D) Increase the expression level of CMYC genes in normal cells and decrease the expression level of CMYC genes in cancer cells.

[0053] Tert is the catalytic subunit of telomerase, and its expression level is directly related to the activity of telomerase, thereby affecting telomere length. KLF4 can directly bind to the promoter region of the Tert gene, regulate its expression, and then regulate the activity of telomerase. PARP1 regulates the activation and transcription of Tert by promoting the direct localization of KLF4 in the Tert promoter and further recruiting β-cantenin to the Tert promoter, thereby affecting the telomere length of the cell. In addition, CMYC is also closely related to the activation of telomerase. The pyrroloquinoline quinone or its derivatives in the present invention can increase the expression level of KLF4, PARP1, β-cantenin and CMYC gene mRNA in normal cells and reduce the expression level of KLF4, PARP1, β-cantenin and CMYC gene mRNA in cancer cells, thereby achieving the dual effect of anti-aging and cancer suppression.

[0054] Furthermore, based on the above embodiments, the present invention further selects cancer cells including liver cancer cells HePG2, lung cancer cells A549, or colon cancer cells HCT116. Normal cells include human umbilical cord mesenchymal stem cells Hu-MSC, human bronchial epithelial cells Beas-2B, human liver cells LO2, or human fibroblasts HSF.

[0055] In a second aspect, an embodiment of the present invention provides a dual-action anti-aging and anti-cancer drug, which includes at least one of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives as an active ingredient.

[0056] Compared with the prior art, the beneficial effects of the dual-action anti-aging and anti-cancer drug provided by the embodiment of the present invention are the same as the beneficial effects of the use of pyrroloquinoline quinone or its derivatives in the preparation of the dual-action anti-aging and anti-cancer drug mentioned above, which will not be described in detail here.

[0057] Furthermore, based on the above embodiments, the embodiments of the present invention further select pyrroloquinoline quinone derivatives including at least one of pyrroloquinoline quinone pharmaceutically acceptable salts and pyrroloquinoline quinone pharmaceutically acceptable esters.

[0058] In some embodiments, the pharmaceutically acceptable salt of pyrroloquinoline quinone can be selected from at least one of the disodium salt, dipotassium salt, dicalcium salt, dimagnesium salt, hydrochloride, sulfate, tartrate, oxalate, maleate, fumarate, and citrate of pyrroloquinoline quinone.

[0059] In some embodiments, the pharmaceutically acceptable ester of pyrroloquinoline quinone includes at least one of methyl, ethyl, propyl, butyl, pentyl, and hexyl esters of pyrroloquinoline quinone.

[0060] In other embodiments, the dual-action anti-aging and anti-cancer drugs described above further include pharmaceutically acceptable excipients. These excipients can be those commonly used in various formulations, such as isotonicity agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, and lubricants. These excipients can also be emulsifiers, solubilizers, or antibacterial agents compatible with the active ingredient, pyrroloquinoline quinone or pyrroloquinoline quinone derivatives, to improve the stability, solubility, or release rate of the drug, thereby enhancing the drug's efficacy.

[0061] Furthermore, the dosage forms of the above-mentioned dual-action anti-aging and anti-cancer drugs include tablets, capsules, injections, aerosols, suppositories, films, pills, patches, subcutaneous implants, external liniments, oral solutions, ointments or nanoformulations.

[0062] It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0063] Example 1

[0064] Effects of PQQ on the vitality of normal cells and cancer cells

[0065] Human umbilical cord mesenchymal stem cells (Hu-MSC), human bronchial epithelial cells (Beas-2B), human liver cells (LO2), human hepatocellular carcinoma cells (HePG2), human lung cancer cells (A549) and human colon cancer cells (HCT116) were selected as experimental subjects, among which Hu-MSC, Beas-2B, and LO2 represent normal cells, and HePG2, A549, and HCT116 represent cancer cells.

[0066] The above six types of cells were respectively cultured at 5×10 3 Each well was inoculated with 100 μL of complete culture medium and cultured in a 37°C incubator with 5% CO 2 .

[0067] The complete culture media used for the above 6 cell types are as follows:

[0068] The complete culture medium used for LO2, Beas-2B, HePG2 and A549 cells was DMEM complete culture medium containing 15% fetal bovine serum and 1% penicillin-streptomycin solution.

[0069] The complete culture medium used for HCT116 cells was 1640 complete culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody solution.

[0070] The complete culture medium used for Hu-MSC cells is a complete culture medium consisting of 475 ml serum-free basal medium, 25 ml human platelet lysate, and 5 ml penicillin-streptomycin-amphotericin B mixture.

[0071] The above six cell types were set up as blank control groups without drug administration and PQQ drug administration groups respectively. The PQQ drug administration groups were divided into PQQ drug administration group 1, PQQ drug administration group 2, PQQ drug administration group 3, PQQ drug administration group 4, PQQ drug administration group 5, PQQ drug administration group 6, PQQ drug administration group 7, PQQ drug administration group 8, PQQ drug administration group 9 and PQQ drug administration group 10 according to the PQQ drug administration concentration, and 6 replicate wells were set up in each group.

[0072] When the growth density of the above six cell types reached 70%, each PQQ-administered group was treated with PQQ. The specific process was as follows:

[0073] The blank control and PQQ-treated groups were simultaneously treated with discarded culture medium and washed once with PBS. 100 μL of complete culture medium was added to each well of the blank control group, and PQQ-treated groups 1 to 10 were treated with PQQ at concentrations of 7 μmol / L, 14 μmol / L, 35 μmol / L, 70 μmol / L, 105 μmol / L, 140 μmol / L, 280 μmol / L, 350 μmol / L, 490 μmol / L, and 700 μmol / L, respectively. HePG2 and LO2 cells were treated for 4, 18, and 24 hours, respectively; Hu-MSC, A549, and HCT116 cells were treated for 4 and 24 hours, respectively; and Beas-2B cells were treated for 24 hours.

[0074] The cell viability was detected by CCK8 assay, and the results are shown in Figures 1(a) to (e).

[0075] As shown in Figure 1, after treatment with PQQ, in HePG2, A549, and HCT116 cancer cells, intervention with 35 μmol / L or higher concentrations for 4 hours and 18 hours in HePG2 cells, and intervention with 105 μmol / L, 350 μmol / L, and 700 μmol / L concentrations for 24 hours in HePG2 cells significantly reduced cell viability (p<0.05); treatment with 35 μmol / L or higher concentrations for 4 hours in A549 cells, and treatment with 14 μmol / L, 35 μmol / L, 105 μmol / L, and 700 μmol / L for 24 hours in A549 cells significantly inhibited cell viability (p<0.05); treatment with 280 μmol / L or higher concentrations for 4 hours in HCT116 cells, and treatment with 35 μmol / L or higher concentrations for 24 hours in HCT116 cells, all had a significant inhibitory effect on cell viability (p<0.05). The above results indicate that PQQ has an inhibitory effect on the viability of cancer cells.

[0076] In LO2 cells, PQQ intervention at a concentration of 490 μmol / L for 4 hours, at a concentration of 14 μmol / L and above (except 105 μmol / L) for 18 hours, and at concentrations of 7 μmol / L, 14 μmol / L, and 35 μmol / L for 24 hours significantly increased LO2 cell viability (p<0.05); however, PQQ intervention at a concentration of 700 μmol / L for 24 hours significantly decreased LO2 cell viability (p<0.05), indicating that appropriate PQQ concentrations have protective and proliferation-promoting effects on LO2 cells.

[0077] In Hu-MSC cells, there was no significant change after 4 hours of PQQ intervention. After 24 hours of PQQ intervention, 280 μmol / L and 350 μmol / L PQQ interventions significantly reduced the cell viability of Hu-MSC (p<0.05), indicating that appropriate concentrations of PQQ have no harm to the activity of Hu-MSC cells, but too high a concentration of PQQ and too long an action time will inhibit the viability of Hu-MSC cells.

[0078] In Beas-2B cells, when PQQ was intervened for 24 hours, PQQ intervention at concentrations of 490μmol / L and 700μmol / L could significantly reduce Beas-2B cell viability (p<0.05), indicating that PQQ should not be taken excessively at one time, but appropriate concentrations do not harm cells.

[0079] The above results indicate that appropriate PQQ concentration and intervention time have an inhibitory effect on the cell viability of cancer cells, while protecting or promoting the proliferation of normal cells.

[0080] Example 2

[0081] Effects of PQQ on telomere length in normal and cancer cells

[0082] Human umbilical cord mesenchymal stem cells (Hu-MSC), human bronchial epithelial cells (Beas-2B), human fibroblasts (HSF), human liver cancer cells (HePG2), human lung cancer cells (A549) and human colon cancer cells (HCT116) were selected as experimental subjects, among which Hu-MSC, Beas-2B and HSF represent normal cells, and HePG2, A549 and HCT116 represent cancer cells.

[0083] The above six types of cells were respectively cultured at 2×10 6 Cells were seeded into 6-well plates (6 6-well plates for each cell type), 1.5 mL of complete medium was added to each well, and the cells were cultured in a 37°C incubator with 5% CO2. The complete medium used for Hu-MSC, Beas-2B, HePG2, A549, and HCT116 cells was the same as in Example 1, and HSF cells were cultured using HSF-specific complete medium.

[0084] The above six cell types were set up as blank control group without drug administration and PQQ administration group respectively. The PQQ administration group was divided into PQQ administration group 1, PQQ administration group 2, PQQ administration group 3, PQQ administration group 4 and PQQ administration group 5 according to the PQQ administration concentration, and 4 replicate wells were set up in each group.

[0085] When the growth density of the above six cell types reached 70%, each PQQ-administered group was treated with PQQ. The specific process was as follows:

[0086] The blank control and PQQ-treated groups were simultaneously treated with discarded culture medium and washed once with PBS. Each well in the blank control group was treated with 1.5 mL of complete culture medium. PQQ-treated groups 1 through 5 were treated with PQQ at concentrations of 14 μmol / L, 35 μmol / L, 70 μmol / L, 105 μmol / L, and 140 μmol / L, respectively. A549 cells were treated for 4 hours, while other cells were treated for 24 hours.

[0087] Real-time quantitative PCR (RT-qPCR) technology was used to detect the relative length of telomeres, and the results are shown in Figure 3.

[0088] Depend on Figure 2 The results showed that after PQQ treatment, the telomere length of Hu-MSC, HSF, and Beas-2B cells showed significant increases compared to the control group (p < 0.05). After 24 hours of treatment with 140 μmol / L PQQ, the average telomere length of Hu-MSC cells increased by 31.5%. After 24 hours of treatment with 14 μmol / L PQQ, the average telomere length of HSF cells increased by 67.6%. After 24 hours of treatment with 14 μmol / L, 35 μmol / L, 70 μmol / L, and 140 μmol / L PQQ, the average telomere length of Beas-2B cells increased by 29.4%, 27.8%, 35.0%, and 43.2%, respectively.

[0089] In contrast, in A549, HePG2, and HCT116 cancer cells, the application of PQQ resulted in a significant decrease in telomere length compared to the control group (p<0.05). After 4 hours of treatment of A549 cells with PQQ at concentrations of 14μmol / L, 35μmol / L, 70μmol / L, 105μmol / L, and 140μmol / L, the average telomere length of A549 cells was shortened by 18.1%, 14.2%, 26.4%, 22.2%, and 24.6%, respectively; after 4 hours of treatment of HePG2 cells with PQQ at concentrations of 14μmol / L, 35μmol / L, 70μmol / L, 105μmol / L, and 140μmol / L, the average telomere length of A549 cells was shortened by 18.1%, 14.2%, 26.4%, 22.2%, and 24.6%, respectively. After 24 hours, the telomere lengths of HePG2 cells were shortened by an average of 15.2%, 21.3%, 29.7%, 23.9% and 36.7%, respectively; after 24 hours of treatment with PQQ at concentrations of 14μmol / L, 35μmol / L, 70μmol / L, 105μmol / L and 140μmol / L, the telomere lengths of HCT116 cells were shortened by an average of 54.1%, 49.2%, 49.1%, 49.8% and 64.7%, respectively.

[0090] The above results indicate that PQQ has an effect of extending the telomere length of normal cells and shortening the telomere length of cancer cells.

[0091] Example 3

[0092] Regulation of telomerase activity in normal and cancer cells by PQQ

[0093] Human bronchial epithelial cells (Beas-2B), human liver cells (LO2), human fibroblasts (HSF), human hepatocellular carcinoma cells (HePG2) and human colon cancer cells (HCT116) were selected as experimental subjects, among which Beas-2B, LO2 and HSF represent normal cells, and HePG2 and HCT116 represent cancer cells.

[0094] The above five types of cells were respectively cultured at 2×10 6 Cells were seeded into 6-well plates (4 6-well plates for HePG2 and LO2 cells, and 6 6-well plates for other cell types). 1.5 mL of complete medium was added to each well and the cells were cultured in a 37°C incubator with 5% CO2. The complete medium used for Beas-2B, LO2, HePG2, and HCT116 cells was the same as in Example 1. HSF cells were cultured using the HSF-specific complete medium.

[0095] The above five cell types were set up as blank control group without drug administration and PQQ administration group, among which the PQQ administration groups of HePG2 and LO2 cells were divided into PQQ administration group 1, PQQ administration group 2, and PQQ administration group 3 according to the PQQ administration concentration; the PQQ administration groups of HCT116, HSF, and Beas-2B cells were divided into PQQ administration group 1, PQQ administration group 2, PQQ administration group 3, PQQ administration group 4, and PQQ administration group 5 according to the PQQ administration concentration, and 4 replicate wells were set up in each group.

[0096] When the growth density of the above five cell types reached 70%, each PQQ-administered group was treated with PQQ. The specific process was as follows:

[0097] The culture medium of the blank control group and the PQQ-treated group was discarded at the same time, and after washing with PBS, 1.5 mL of complete culture medium was added to each well of the blank control group. The HePG2 and LO2 cells in the PQQ-treated groups 1 to 3 were added with 14 μmol / L, 35 μmol / L, and 70 μmol / L concentrations of PQQ for 24 hours, respectively; the HCT116, HSF, and Beas-2B cells in the PQQ-treated groups 1 to 5 were added with 14 μmol / L, 35 μmol / L, 70 μmol / L, 105 μmol / L, and 140 μmol / L concentrations of PQQ for 24 hours, respectively.

[0098] The expression level of Tert was analyzed by RT-qPCR using primers and probes specific for the mRNA of telomerase Tert. As the catalytic subunit of telomerase, the expression level of Tert is directly related to the activity of telomerase. Figure 3 shown.

[0099] Depend on Figure 3 PQQ treatment significantly increased Tert mRNA expression in LO2, HSF, and Beas-2B cells. Specifically, in LO2 cells, treatment with 70 μmol / L PQQ for 24 hours increased Tert expression by an average of 25.3%. In HSF cells, treatment with 14 μmol / L PQQ for 24 hours increased Tert expression by an average of 24.8%. In Beas-2B cells, treatment with 35 μmol / L, 70 μmol / L, 105 μmol / L, and 140 μmol / L PQQ for 24 hours increased Tert expression by an average of 45.3%, 47%, 34%, and 30.3%, respectively.

[0100] The expression level of Tert was significantly reduced in HePG2 and HCT116 cancer cells. Specifically, in HePG2 cells, after being treated with PQQ at concentrations of 14μmol / L, 35μmol / L, and 70μmol / L for 24 hours, the expression level of telomerase Tert in HePG2 cells decreased by an average of 28.1%, 45.3%, and 50.2%, respectively; in HCT116 cells, after being treated with PQQ at concentrations of 14μmol / L, 70μmol / L, 105μmol / L, and 140μmol / L for 24 hours, the expression level of telomerase Tert in HCT116 cells decreased by an average of 41.4%, 42.6%, 44.2%, and 33.3%, respectively.

[0101] The above results indicate that PQQ promotes the telomerase activity of normal cells and inhibits the telomerase activity of cancer cells.

[0102] Example 4

[0103] PQQ regulates the expression of KLF4, PARP1, β-catenin and related gene CMYC in normal and cancer cells

[0104] Human umbilical cord mesenchymal stem cells (Hu-MSC), human bronchial epithelial cells (Beas-2B), human liver cells (LO2), human hepatocellular carcinoma cells (HePG2), human lung cancer cells (A549) and human colon cancer cells (HCT116) were selected as experimental subjects, among which Hu-MSC, Beas-2B, and LO2 represent normal cells, and HePG2, A549, and HCT116 represent cancer cells.

[0105] 1. PQQ regulates KLF4 expression in normal cells and cancer cells

[0106] Hu-MSC, Beas-2B, HePG2, and HCT116 cells were cultured at 2×10 6 Each well was inoculated with 1.5 mL of complete medium and cultured in a 37° C. incubator with 5% CO 2 . The complete medium used for the above four cell types was as described in Example 1 .

[0107] The above four cell types were set up as blank control group without drug administration and PQQ administration group respectively. The PQQ administration group was divided into PQQ administration group 1, PQQ administration group 2, PQQ administration group 3, PQQ administration group 4 and PQQ administration group 5 according to the PQQ administration concentration, and 4 replicate wells were set up in each group.

[0108] When the growth density of the above four cell types reached 70%, each PQQ-administered group was treated with PQQ. The specific process was as follows:

[0109] The culture medium was discarded from the blank control group and the PQQ-treated group at the same time, and the cells were washed once with PBS. 1.5 mL of complete culture medium was added to each well of the blank control group, and PQQ-treated groups 1 to 5 were treated with PQQ at concentrations of 14 μmol / L, 35 μmol / L, 70 μmol / L, 105 μmol / L, and 140 μmol / L, respectively. The cell treatment time was 24 hours.

[0110] The expression level of KLF4 mRNA was analyzed by RT-qPCR using primers and probes specific for KLF4. The results are shown in Figures 4(a) to (b).

[0111] 2. PQQ regulates PARP1 expression in normal cells and cancer cells

[0112] Beas-2B, LO2, Hu-MSC, A549, and HCT116 cells were cultured at 2×10 6Cells were seeded into 6-well plates (A549 cells were plated in four 6-well plates, LO2 cells were plated in two 6-well plates, and all other cells were plated in six 6-well plates). 1.5 mL of complete medium was added to each well and the cells were cultured in a 37°C incubator with 5% CO2. The complete medium used for the five cell types was as described in Example 1.

[0113] The above five cell types were respectively set up as blank control group without drug administration and PQQ administration group. Among them, the PQQ administration groups of HCT116, Beas-2B and Hu-MSC cells were divided into PQQ administration group 1, PQQ administration group 2, PQQ administration group 3, PQQ administration group 4 and PQQ administration group 5 according to the PQQ administration concentration; the PQQ administration group of A549 cells was divided into PQQ administration group 1, PQQ administration group 2 and PQQ administration group 3 according to the PQQ administration concentration; there was only one PQQ administration group for LO2 cells, and 4 replicate wells were set for each group.

[0114] When the growth density of the above five cell types reached 70%, each PQQ-administered group was treated with PQQ. The specific process was as follows:

[0115] The culture medium of the blank control group and the PQQ-treated group was discarded at the same time, and the cells were washed once with PBS. 1.5 mL of complete culture medium was added to each well of the blank control group. PQQ-treated groups 1 to 5 of HCT116, Beas-2B, and Hu-MSC cells were added with 14 μmol / L, 35 μmol / L, 70 μmol / L, 105 μmol / L, and 140 μmol / L PQQ, respectively, for 24 hours; PQQ-treated groups 1 to 3 of A549 cells were added with 70 μmol / L, 105 μmol / L, and 140 μmol / L PQQ, respectively, for 4 hours; the PQQ-treated group of LO2 cells was added with 70 μmol / L PQQ, respectively, for 24 hours.

[0116] The expression level of PARP1 gene mRNA was analyzed by RT-qPCR using specific primers and probes for PARP1. Figure 5 shown.

[0117] 3. PQQ regulates β-catenin expression in normal cells and cancer cells

[0118] Beas-2B, LO2, HePG2, A549, and HCT116 cells were cultured at 2×10 6Cells were seeded at 100 cells / well in 6-well plates (HCT116 and Beas-2B cells were plated in 6 wells, A549 cells were plated in 4 wells, and HePG2 and LO2 cells were plated in 2 wells). 1.5 mL of complete medium was added to each well and the cells were cultured in a 37°C incubator with 5% CO2. The complete medium used for the above five cell types was as described in Example 1.

[0119] The above five cell types were set up as blank control group without drug administration and PQQ administration group respectively. Among them, the PQQ administration groups of HCT116 and Beas-2B cells were divided into PQQ administration group 1, PQQ administration group 2, PQQ administration group 3, PQQ administration group 4, and PQQ administration group 5 according to the PQQ administration concentration; the PQQ administration group of A549 cells was divided into PQQ administration group 1, PQQ administration group 2, and PQQ administration group 3 according to the PQQ administration concentration; there was only one PQQ administration group for HePG2 and LO2 cells, and 4 replicates were set for each group.

[0120] When the growth density of the above five cell types reached 70%, each PQQ-administered group was treated with PQQ. The specific process was as follows:

[0121] The culture medium was discarded from the blank control group and the PQQ-treated group at the same time, and the cells were washed with PBS. 1.5 mL of complete culture medium was added to each well of the blank control group. PQQ-treated groups 1 to 5 of HCT116 and Beas-2B cells were treated with PQQ at concentrations of 14 μmol / L, 35 μmol / L, 70 μmol / L, 105 μmol / L, and 140 μmol / L, respectively, for 24 hours; PQQ-treated groups 1 to 3 of A549 cells were treated with PQQ at concentrations of 14 μmol / L, 35 μmol / L, and 70 μmol / L, respectively, for 24 hours; the PQQ-treated group of HePG2 and LO2 cells was treated with PQQ at a concentration of 70 μmol / L for 24 hours.

[0122] The expression level of β-catenin mRNA was analyzed by RT-qPCR using specific primers and probes for β-catenin. Figure 6 shown.

[0123] 4. PQQ regulates CMYC expression in normal and cancer cells

[0124] Hu-MSC, LO2, and HCT116 cells were cultured at 2×10 6 Each well was inoculated with 1.5 mL of complete medium and cultured in a 37°C incubator with 5% CO 2 . The complete medium used for the above three cells was as described in Example 1 .

[0125] The above three cell types were set up as blank control group without drug administration and PQQ administration group, and 4 replicate wells were set up in each group.

[0126] When the growth density of the above three cell types reached 70%, each PQQ-administered group was treated with PQQ. The specific process is as follows:

[0127] The culture medium of the blank control group and the PQQ-treated group was discarded at the same time, and after washing with PBS, 1.5 mL of complete culture medium was added to each well of the blank control group, and 70 μmol / L PQQ was added to each well of the PQQ-treated group for 24 hours.

[0128] The expression level of CMYC gene mRNA was analyzed by RT-qPCR using specific primers and probes for CMYC. Figure 7 shown.

[0129] As shown in Figure 4(a) to (b), in Hu-MSC cells, after 24 hours of intervention with PQQ at a concentration of 70 μmol / L, the mRNA expression level of KLF4 in Hu-MSC from P4 to P6 was significantly increased; while in HCT116 cancer cells, after 24 hours of treatment with PQQ at concentrations of 70 μmol / L, 105 μmol / L, and 140 μmol / L, the mRNA expression level of KLF4 in HCT116 cancer cells was significantly decreased; at the same time, in Beas-2B cells, PQQ at concentrations of 35 μmol / L, 70 μmol / L, 105 μmol / L and 140 μmol / L intervened for 24 hours, which significantly increased the mRNA expression level of KLF4 in Beas-2B cells. In Hepg2 cancer cells, PQQ at concentrations of 14 μmol / L, 35 μmol / L, 70 μmol / L, 105 μmol / L and 140 μmol / L intervened for 24 hours, which significantly decreased the mRNA expression level of KLF4 in Hepg2 cancer cells.

[0130] Depend on Figure 5The results showed that in A549 cancer cells, the expression level of PARP1 mRNA in A549 cells was significantly decreased after 4-hour treatment with PQQ at concentrations of 105 μmol / L and 140 μmol / L. In HCT116 cancer cells, the expression level of PARP1 mRNA in HCT116 cells was significantly decreased after 24-hour treatment with PQQ at concentrations of 14 μmol / L, 35 μmol / L, 70 μmol / L, and 140 μmol / L. In contrast, in Beas-2B cells, the relative expression level of PARP1 mRNA was significantly increased after 24-hour treatment with PQQ at concentrations of 35 μmol / L, 70 μmol / L, and 105 μmol / L. In addition, in LO2 cells, the relative expression level of PARP1 mRNA was significantly increased after 24-hour treatment with PQQ at concentrations of 70 μmol / L. In Hu-MSC cells, the relative expression level of PARP1 mRNA was significantly increased after 24-hour treatment with PQQ at concentrations of 105 μmol / L.

[0131] Depend on Figure 6 The results showed that: in A549 cancer cells, after 24 hours of treatment with 70 μmol / L PQQ, the relative expression level of β-cantenin mRNA in A549 cells decreased; in HCT116 cancer cells, after 24 hours of treatment with 14 μmol / L, 35 μmol / L, and 70 μmol / L PQQ, the relative expression level of β-cantenin mRNA in HCT116 cancer cells was significantly decreased; in HePG2 cancer cells, after 24 hours of treatment with 70 μmol / L PQQ, the relative expression level of β-cantenin mRNA in HePG2 cancer cells was significantly decreased. In contrast, after 24 hours of treatment with 70 μmol / L PQQ, the relative expression level of β-cantenin mRNA in LO2 cells was significantly increased. In Beas-2B cells, after 24 hours of treatment with 35 μmol / L and 105 μmol / L PQQ, the relative expression level of β-cantenin mRNA in cells was significantly increased.

[0132] Depend on Figure 7 The results showed that treatment with 70 μmol / L PQQ for 24 hours increased the relative expression level of CMYC mRNA in LO2 cells and significantly increased the relative expression level of CMYC mRNA in Hu-MSC cells from passages P3 to P5. In contrast, treatment with 70 μmol / L PQQ for 24 hours significantly decreased the relative expression level of CMYC mRNA in HCT116 cancer cells.

[0133] As shown in Figures 4 to 7 , PQQ promotes the expression of KLF4, PARP1, β-catenin, and CMYC in normal cells, and inhibits the expression of KLF4, PARP1, β-catenin, and CMYC in cancer cells.

[0134] Example 5

[0135] Effects of PQQ on mitochondrial membrane potential in normal and cancer cells

[0136] Human liver cells (LO2) and human hepatocellular carcinoma cells (HepG2) were selected as experimental subjects. 3 Each well was seeded with 100 μL of complete medium, and the cells were cultured in a 37°C incubator with 5% CO 2 . The complete medium used for the LO2 and HePG2 cells was as described in Example 1.

[0137] The above two cell types were set up as blank control group without drug administration and PQQ administration group, respectively. The PQQ administration group was divided into PQQ administration group 1, PQQ administration group 2, PQQ administration group 3, and PQQ administration group 4 according to the PQQ administration concentration, and 6 replicate wells were set up in each group.

[0138] When the cell growth density reached 70%, each PQQ-dosing group was treated with PQQ. The specific process was as follows:

[0139] The culture medium was discarded from the blank control group and the PQQ-treated group at the same time, and the cells were washed with PBS. 100 μL of complete culture medium was added to each well of the blank control group, and 14 μmol / L, 35 μmol / L, 70 μmol / L, and 105 μmol / L PQQ were added to treat the cells in PQQ-treated groups 1 to 4, respectively, for 24 hours.

[0140] The mitochondrial membrane potential was detected using the fluorescent probe JC-1. Figure 8 shown.

[0141] Depend on Figure 8 It can be seen that PQQ intervention at concentrations of 14μmol / L, 35μmol / L, 70μmol / L and 105μmol / L for 24 hours significantly improved the mitochondrial membrane potential of LO2 cells, indicating that PQQ can enhance the mitochondrial function of LO2 cells and maintain the normal metabolism and viability of cells.

[0142] In contrast, after 24 hours of intervention with 14 μmol / L PQQ, the mitochondrial membrane potential of HepG2 cancer cells was inhibited, indicating that PQQ has an inhibitory effect on the mitochondrial function of cancer cells and may induce cancer cell apoptosis.

[0143] The above results indicate that PQQ has a protective and enhancing effect on the mitochondrial membrane potential of normal cells, while it has an inhibitory effect on the mitochondrial membrane potential of cancer cells.

[0144] Example 6

[0145] Effects of PQQ on cell membrane potential of normal cells and cancer cells

[0146] Human liver cells (LO2) and human hepatocellular carcinoma cells (HepG2) were selected as experimental subjects. 3 Each well was seeded with 100 μL of complete medium, and the cells were cultured in a 37°C incubator with 5% CO 2 . The complete medium used for the LO2 and HePG2 cells was as described in Example 1.

[0147] The above two cell types were set up as blank control group without drug administration and PQQ administration group, respectively. The PQQ administration group was divided into PQQ administration group 1, PQQ administration group 2, PQQ administration group 3, and PQQ administration group 4 according to the PQQ administration concentration, and 6 replicate wells were set up in each group.

[0148] When the growth density of the above two cell types reached 70%, each PQQ-administered group was treated with PQQ. The specific process was as follows:

[0149] The culture medium was discarded from the blank control group and the PQQ-treated group at the same time. After washing once with PBS, 100 μL of complete culture medium was added to each well of the blank control group. PQQ-treated groups 1 to 4 were treated with PQQ at concentrations of 14 μmol / L, 35 μmol / L, 70 μmol / L, and 105 μmol / L, respectively, for 24 hours.

[0150] The anionic dye DIBAC4 (3) was used to detect the cell membrane potential. Figure 9 shown.

[0151] Depend on Figure 9 It can be seen that PQQ intervention at concentrations of 14μmol / L, 35μmol / L, 70μmol / L and 105μmol / L for 24 hours significantly improved the cell membrane potential of LO2 cells, and was concentration-dependent, indicating that PQQ can enhance the cell membrane potential of LO2 cells and maintain the normal physiological state of the cells.

[0152] However, there was no obvious effect on HepG2 cancer cells, indicating that PQQ had little effect on the cell membrane potential of cancer cells.

[0153] The above results indicate that PQQ has a protective and enhancing effect on the cell membrane potential of normal cells, but has no significant effect on the cell membrane potential of cancer cells.

[0154] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Application of pyrroloquinoline quinone or its derivatives in the preparation of dual-action anti-aging and anti-cancer drugs.

2. The use according to claim 1, characterized in that The dual effects of anti-aging and cancer suppression are manifested in at least one of the following: A) Increase telomere length in normal cells and decrease telomere length in cancer cells; B) Promote telomerase activity in normal cells and inhibit telomerase activity in cancer cells; C) Increase the mitochondrial membrane potential of normal cells and inhibit the mitochondrial membrane potential of cancer cells.

3. The use according to claim 2, characterized in that The pyrroloquinoline quinone or its derivatives regulate telomere length or telomerase activity in at least one of the following ways: A) Increase the expression level of KLF4 gene in normal cells and decrease the expression level of KLF4 gene in cancer cells; B) Increase the expression level of PARP1 gene in normal cells and decrease the expression level of PARP1 gene in cancer cells; C) Increase the expression level of the β-cantenin gene in normal cells and decrease the expression level of the β-cantenin gene in cancer cells; D) Increase the expression level of CMYC genes in normal cells and decrease the expression level of CMYC genes in cancer cells.

4. The use according to claim 2 or 3, characterized in that The cancer cells include liver cancer cells, lung cancer cells or colon cancer cells.

5. A dual-action drug for anti-aging and anti-cancer, characterized in that: The medicine comprises at least one of pyrroloquinoline quinone and pyrroloquinoline quinone derivatives as active ingredients.

6. The anti-aging and anti-cancer dual-action drug according to claim 5, characterized in that: The pyrroloquinoline quinone derivative includes at least one of a pharmaceutically acceptable salt of pyrroloquinoline quinone and a pharmaceutically acceptable ester of pyrroloquinoline quinone.

7. The anti-aging and anti-cancer dual-action drug according to claim 6, characterized in that: The pharmaceutically acceptable salt of pyrroloquinoline quinone includes at least one of disodium salt, dipotassium salt, dicalcium salt, dimagnesium salt, hydrochloride, sulfate, tartrate, oxalate, maleate, fumarate and citrate of pyrroloquinoline quinone.

8. The anti-aging and anti-cancer dual-action drug according to claim 6, characterized in that: The pharmaceutically acceptable ester of pyrroloquinoline quinone includes at least one of methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester and hexyl ester of pyrroloquinoline quinone.

9. The anti-aging and anti-cancer dual-action drug according to claim 5, characterized in that: The drug also includes pharmaceutically acceptable excipients.

10. The anti-aging and anti-cancer dual-action drug according to claim 5, characterized in that: The dosage forms of the drug include tablets, capsules, injections, aerosols, suppositories, films, pills, patches, subcutaneous implants, external liniments, oral solutions, ointments or nano preparations.

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