Application of plumbagin in medicine for promoting glioma cell apoptosis and medicine

Abstract

The invention relates to the field of biological medicine. In particular to a medicine for promoting glioma cell apoptosis through plumbagin and application of the plumbagin in medicines. The plumbagin disclosed by the invention can be used for remarkably down-regulating expression of glycolysis rate-limiting enzyme gene hexokinase II so as to inhibit lactic acid generation and extracellular acidification rate, and inhibiting proliferation and migration of glioma cells by interfering glycolysis metabolism reprogramming, and a novel medicine required by a targeted therapy strategy can be obtained.

Description

Technical Field

[0001] This invention relates to the field of biomedicine; specifically, to a drug that promotes apoptosis of glioma cells through prunella vulgaris, and the application of prunella vulgaris in pharmaceuticals. Background Technology

[0002] Gliomas are the most common malignant tumors of the central nervous system. They are highly malignant and aggressive, resulting in extremely poor prognosis for patients. Statistics show that the 5-year survival rate for glioma patients is less than 10%, posing a serious threat to human life and health.

[0003] Gliomas are the most common primary tumors. High-grade gliomas are rapidly progressing intracranial tumors, encompassing anaplastic oligodendrogliomas, anaplastic astrocytomas (both classified as grade III in the World Health Organization [WHO] classification system), and glioblastomas (WHO grade IV).

[0004] Despite advancements in diagnostic and treatment technologies in recent years, glioma treatment remains primarily based on surgical resection and radiotherapy / chemotherapy. However, due to metabolic reprogramming and abnormal activation of signaling pathways in gliomas, the efficacy of these treatments is limited. Metabolic reprogramming, similar to the Warburg effect, refers to tumor cells relying primarily on glycolysis for energy even in aerobic environments. This metabolic pattern leads to a significant accumulation of lactate, which not only provides energy and raw materials for tumor cell synthesis but also reshapes the tumor microenvironment, promoting tumor invasion and metastasis. Abnormal activation of signaling pathways, such as ROS-p38MAPK axis dysregulation, is closely related to the imbalance of reactive oxygen species (ROS) homeostasis and the abnormal activation of the p38 mitogen-activated protein kinase (p38MAPK) pathway, both jointly regulating tumor cells. Therefore, surgical resection alone often fails to achieve the desired clinical outcomes due to high recurrence rates, and radiotherapy and chemotherapy alone can develop resistance over time. These factors further complicate glioma treatment.

[0005] Plumbagoside (PLB) is a natural compound isolated from the traditional medicinal plant *Plumbago stenoptera*, extracted from the roots of plants in the *Plumbago* genus. As a vitamin K3 analog and pro-oxidant, plumbagoside possesses various biological activities, including antioxidant, anti-inflammatory, antifungal, anti-atherosclerotic, and analgesic activities. However, no publicly available technologies or research have been conducted regarding the application of plumbagoside in the treatment of glioma. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a drug that promotes apoptosis in glioma cells and the application of pistachiosin in the drug.

[0007] Among them, the present invention provides a drug that inhibits the proliferation and / or migration of glioma cells and promotes apoptosis of glioma cells by means of prunella vulgaris.

[0008] Specifically, the aforementioned drug, prunella vulgaris, is used to inhibit hexokinase II, which is involved in glycolysis in glioma cells, thereby reducing lactate production and inducing the accumulation of reactive oxygen species in mitochondria, which in turn activates the p38 mitogen-activated protein kinase pathway, inhibits glioma cell proliferation and / or migration, and promotes glioma cell apoptosis.

[0009] The aforementioned drugs, specifically, also contain one of the following: solvents, disintegrants, antioxidants, binders, fillers, thickeners and / or diluents, or combinations thereof.

[0010] The aforementioned drug specifically refers to soluble lyophilized ...

[0011] The present invention also provides the use of pistachiool as a pharmaceutical ingredient for inhibiting the proliferation and / or migration of glioma cells in the preparation of a drug for promoting apoptosis of glioma cells.

[0012] As described above, the pistachio extract is specifically used to inhibit hexokinase II, which is involved in glycolysis in glioma cells, reduce lactate production and induce the accumulation of reactive oxygen species in mitochondria, thereby activating the p38 mitogen-activated protein kinase pathway and inhibiting the proliferation and / or migration of glioma cells.

[0013] As described above, the drug further includes excipients that can be formulated with peony root extract; specifically, one of the following: solvent, disintegrant, antioxidant, binder, filler, thickener / or diluent, or a combination thereof.

[0014] As described above, the plumbagoside is a soluble plumbagoside freeze-dried powder.

[0015] The present invention also provides a drug that inhibits hexokinase 2, a glycolytic enzyme in glioma cells, by means of prunella vulgaris extract.

[0016] Specifically, the aforementioned drug, pterostilbene, inhibits hexokinase II, reduces lactate production, and induces the accumulation of mitochondrial reactive oxygen species, thereby activating the p38 mitogen-activated protein kinase pathway, inhibiting glioma cell proliferation and / or migration, and promoting glioma cell apoptosis.

[0017] Beneficial effects: This invention discloses the mechanism of action of prunella vulgaris in gliomas, enabling the development of novel drugs for targeted therapy strategies.

[0018] This invention discloses the inhibitory effect of plumbagoside on glioma, and how it reduces lactate production and induces the accumulation of mitochondrial reactive oxygen species by inhibiting hexokinase II, which is used for glycolysis in glioma cells, thereby activating the p38 mitogen-activated protein kinase pathway, inhibiting glioma cell proliferation and / or migration, and promoting glioma cell apoptosis.

[0019] The pathway involved, "inhibition of hexokinase II, reduction of lactate production and induction of mitochondrial reactive oxygen species accumulation, thereby activating p38 mitogen-activated protein kinase," is the core molecular network through which pterostilbene exerts its anti-tumor effect. This invention provides new candidate drugs and targets for metabolic targeted therapy of gliomas, and has significant basic research value and clinical translational potential.

[0020] The drug disclosed in this invention can compensate for the shortcomings of the original treatment plan for glioma, which is mainly based on surgical resection, radiotherapy and chemotherapy, or replace the original treatment plan for glioma, which is mainly based on surgical resection, radiotherapy and chemotherapy, and instead provide a new drug with a targeted therapy strategy. Attached Figure Description

[0021] Figure 1 Effects of OE-HK2 and PLB treatments on tumor growth and histological morphology; Figure 1 A shows the changes in tumor volume at different time points in different treatment groups; Figure 1 B shows the hematoxylin-eosin (HE) staining results of tumor tissues from different treatment groups.

[0022] Figure 2 Immunohistochemistry of the effects of different treatments on the expression of key proteins in tumor tissues; Figure 2 A shows the immunohistochemical staining results of Cleaved caspase-3, HK2, Ki67, and P-P38 in tumor tissues from different treatment groups. Figure 2 B represents the statistical results of the percentage of positive areas for Cleaved caspase-3, HK2, Ki67, and P-P38 in tumor tissues of different treatment groups.

[0023] Figure 3 Analysis of the effects of different treatments on iron content in tumor tissue; Figure 3 A shows the iron staining results of tumor tissues from different treatment groups; Figure 3 B represents the statistical results of the percentage of iron-positive area in tumor tissues from different treatment groups.

[0024] Figure 4 Effects of OE-HK2 and PLB treatment on the expression of tumor cell-related proteins and genes; Figure 4A represents the Western blot (WB) results of ACSL4, GPX4, HK2, and other proteins in cells from different treatment groups. Figure 4 B represents the quantitative analysis results of ACSL4, GPX4, HK2, and other proteins in cells from different treatment groups after standardization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Figure 4 C represents the qRT-PCR detection results of genes such as HK2, PFKM, and LDHA in cells of different treatment groups.

[0025] The P-values ​​(P) mentioned in the figures refer to the statistical P-value (P), which is the probability of observing the current sample data or a more extreme result when the null hypothesis is true. Detailed Implementation

[0026] Example 1: The main ingredient of the drug is plumbagoside, a naphthoquinone compound extracted from plants of the genus Plumbago (such as Plumbago spp. and Plumbago aurea).

[0027] Plumbagoside can be soluble lyophilized powder.

[0028] The drug also contains excipients, such as solvents, disintegrants, antioxidants, binders, fillers, thickeners and / or diluents, or combinations of these excipients. In different routes of administration (oral tablets, intravenous infusions, intramuscular injections, etc.), pistachiosin and the excipients are formulated using medically feasible methods. Administration is then carried out in combination with different excipients.

[0029] The leucovorin and excipients are mixed in parts by weight, with leucovorin in the range of 1 to 100 parts (e.g., 1 part, 10 parts, 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, or 90 parts) and excipients in the range of 99 to 0 parts, together constituting 100% (100 parts).

[0030] The dosage is determined based on the patient's condition and the active ingredient.

[0031] Example 2: I. Experimental preparation.

[0032] Four-week-old female BALB / c nude mice were used in the experiment and purchased from Jicui Pharmaceutical Co., Ltd. This animal experiment followed ethical guidelines for laboratory animals and was approved by the Ethics Committee of Sichuan University (Approval No.: 20211018A).

[0033] Preparation of leucovorin drug: Accurately weigh 9.8 mg of lyucovorin lyophilized powder, dissolve it in 1 mL of dimethyl sulfoxide (DMSO) to prepare a 50 mmol / L stock solution, dispense it into 1.5 mL EP tubes and store at -20℃ for later use.

[0034] Cells in the logarithmic growth phase and in good condition were used in all experiments.

[0035] Tumor model establishment: Under sterile conditions, tumor homogenate was first prepared by mixing the excised tumor cells with sterile saline at a ratio of 1g:4mL and then grinding them into a single-cell suspension using a tissue homogenizer. 0.2mL of the suspension was inoculated subcutaneously into the right forelimb axilla of mice to establish a tumor model. Once the tumor reached a volume of 5mm, lentiviral vector was injected into the tumor tissue intratumorally under sterile conditions, with 0.1mL injected into each mouse, completing the viral intervention. The mice's condition and tumor growth were closely monitored post-surgery.

[0036] A control group was also set up in the experiment.

[0037] 2. Glioma cell analysis.

[0038] Tumor tissue was obtained from a mouse-based tumor model through surgical resection or biopsy. RNA was extracted from the tumor tissue, and the differences in RNA expression among the samples in each experimental group were analyzed.

[0039] The study validated the function of the hexokinase II gene, confirming its significant regulatory role in the proliferation, migration, and invasion of glioma cells. Specifically, comparative experiments were conducted with four groups: the NC group (HK2 overexpression control group), the OE-HK2 group (HK2 overexpression group), the si-NC group (HK2 silencing control group), and the si-HK2 group (HK2 silencing group). The results showed that compared to the NC group, the cell migration rate in the OE-HK2 group was significantly increased, while the si-HK2 group exhibited the opposite trend, with a significantly decreased cell migration rate. Furthermore, compared to the NC group, the cell proliferation capacity in the OE-HK2 group was significantly enhanced, while the cell proliferation capacity in the si-HK2 group was significantly inhibited.

[0040] Since hexokinase II has a significant regulatory effect on the proliferation, migration and invasion of glioma cells, and the hexokinase II gene is a core regulatory node in the glycolysis pathway, the hexokinase II gene, as a key rate-limiting enzyme gene in glycolysis, is significantly downregulated after treatment with plumbagoside. Therefore, the drug prepared with plumbagoside has a significant effect on promoting apoptosis of glioma cells.

[0041] 3. Observation and analysis of tumor tissue.

[0042] Experimental groups: DMSO was the control group; OE-HK2 was the HK2 overexpression group; PLB was the prunella vulgaris group; and OE-HK2+PLB was the prunella vulgaris group treated with prunella vulgaris to treat the HK2 overexpression genome. The control group used the same concentration of dimethyl sulfoxide (DMSO) as the experimental groups.

[0043] Tumor tissue sampling: Tumor tissue is obtained through surgical resection or puncture biopsy. After fixation, dehydration and clearing, paraffin embedding, trimming, and sectioning, samples are prepared. These samples are then stained with hematoxylin and eosin (HE staining), mounted with neutral resin, and used for observation. Alternatively, sections are stained with iron and mounted with neutral resin for further observation.

[0044] 1. Effects of pampasodilator (PLB) and hexokinase II (HK2) genes on tumor growth and histopathology: Tumor volume measurement results show (see attached) Figure 1 The tumor volume gradually increased at 2 weeks, indicating successful modeling. At 2 weeks, the DMSO group showed significantly lower tumor volume than the OE-HK2 group (P<0.05); the OE-HK2 group showed significantly higher tumor volume than the OE-HK2+PLB group (P<0.05); and the PLB group showed significantly lower tumor volume than the OE-HK2+PLB group (P<0.05). "w" represents the duration, which can be understood as one week.

[0045] Figure 1 The p-values ​​(P) involved are: *P<0.05, **P<0.001, ***P<0.001, ****P<0.0001.

[0046] Hematoxylin-eosin (HE) staining results showed that: in the DMSO group, cell proliferation was active, nuclei were enlarged, nucleoli were prominent, cytoplasm was abundant and deeply stained, cells were dense and disordered, large areas of tissue necrosis were accompanied by hemorrhage and a large amount of inflammatory cell infiltration; in the OE-HK2 group, cell proliferation was active, mitotic figures were slightly increased, cells were dense and disordered, and large areas of right-side tissue necrosis were accompanied by a large amount of inflammatory cell infiltration; in the PLB group, cell division was reduced, some tissue areas were necrotic and accompanied by hemorrhage and inflammatory cell infiltration; in the OE-HK2+PLB group, mitotic figures were increased, and large areas of left-side tissue necrosis were accompanied by a large amount of inflammatory cell infiltration (cell morphology: indicated by black arrows, inflammatory cells: indicated by red arrows).

[0047] 2. Effects of PLB and HK2 genes on immunohistochemistry: Immunohistochemical staining results revealed (see attached document) Figure 2): Detection of key proteins in tumor tissues from different treatment groups revealed that, regarding HK2, the OE-HK2 group had the highest staining intensity, confirming HK2 gene overexpression; the staining intensity of the PLB group and the DMSO group was comparable; the staining intensity of the OE-HK2+PLB group was lower than that of the OE-HK2 group. Regarding Cleaved caspase-3 (activated form of caspase-3 protease) and P-P38 (phosphorylated mitogen-activated protein kinase p38 antibody), the staining intensity of the PLB group was significantly higher than that of the DMSO group, while the staining intensity of the OE-HK2+PLB group was lower than that of the PLB group, and there was no significant difference between the OE-HK2 group and the DMSO group. Regarding cell proliferation activity-related proteins (Ki67), the results were the opposite: the staining intensity of the PLB group was lower than that of the DMSO group, and the staining intensity of the OE-HK2 group was higher than that of the DMSO group, while the staining intensity of the OE-HK2+PLB group was higher than that of the PLB group.

[0048] Further quantitative analysis revealed that: regarding cleaved caspase-3 and P-P38, the DMSO group was significantly higher than the OE-HK2 group, the OE-HK2 group was significantly lower than the OE-HK2+PLB group, and the PLB group was significantly higher than the OE-HK2+PLB group (P<0.05); regarding HK2 and Ki67, the results showed the opposite trend (P<0.05).

[0049] Figure 2 A shows the immunohistochemical staining results of Cleaved caspase-3 (activated caspase-3 protease), HK2, Ki67 (cell proliferation activity-related protein), and P-P38 (phosphorylated mitogen-activated protein kinase p38 antibody) in tumor tissues from different treatment groups. Figure 2 The p-values ​​(P) involved are: *P<0.05, **P<0.001, ***P<0.001, ****P<0.0001.

[0050] 3. Effects of PLB and HK2 genes on iron staining: Results obtained via fluorescence microscopy (see attached reference) Figure 3 Iron deposition was less in the DMSO group, significantly increased in the PLB group with denser blue signals, and minimal in the OE-HK2 group. Iron deposition was significantly less in the OE-HK2+PLB group compared to the PLB group. Quantitative analysis of the positive area showed that the iron-positive area in the OE-HK2 group was significantly higher than that in the DMSO group; the OE-HK2 group was significantly lower than that in the OE-HK2+PLB group; and the PLB group was significantly higher than that in the OE-HK2+PLB group (P<0.05), consistent with the staining results.

[0051] Figure 3The p-values ​​(P) involved are: *P<0.05, **P<0.001, ***P<0.001, ****P<0.0001.

[0052] 4. Effects of PLB and HK2 genes on Western blot (WB) and qRT-PCR assays: Results of Western blot (WB) assay for protein immunoblotting (see attached) Figure 4 Among the proteins ACSL4, GPX4, HK2, and xCT, the expression levels of the OE-KH2 group were significantly higher than those of the DMSO group, the OE-HK2 group was significantly higher than that of the OE-HK2+PLB group, and the PLB group was significantly lower than that of the OE-HK2+PLB group. The expression level of P-P38 protein showed the opposite trend (P<0.05). Unit: kDa (kilodaltons).

[0053] qRT-PCR analysis revealed that the levels of HK2, PFKM, LDHA, SOD2, CAT, GPX4, SLC7A11, and ACSL4 genes were significantly higher in the OE-KH2 group than in the DMSO group, significantly higher in the OE-HK2 group than in the OE-HK2+PLB group, and significantly lower in the PLB group than in the OE-HK2+PLB group (P<0.05). Conversely, the levels of MAPK14 gene were significantly lower in the OE-KH2 group than in the DMSO group, significantly lower in the OE-HK2 group than in the OE-HK2+PLB group, and significantly higher in the PLB group than in the OE-HK2+PLB group (P<0.05). qRT-PCR is a method for the quantitative analysis of specific RNA sequences in a sample.

[0054] Figure 4 The p-values ​​(P) involved are: *P<0.05, **P<0.001, ***P<0.001, ****P<0.0001. The mRNA expression level shown in the figure compared with GAPDH represents the mRNA expression level compared to GAPDH; the comparison with GAPDH represents the result compared to GAPDH.

[0055] Consistency verification through in vitro and in vivo experiments: Animal experiments showed that plumbagolide significantly inhibited tumor growth and induced the accumulation of ferroptosis markers and iron deposition, while overexpression of hexokinase II could antagonize its effects, further supporting the anti-glioma effect of plumbagolide by targeting hexokinase II.

[0056] This demonstrates that plumbagolide targets and inhibits hexokinase II in regulating glycolysis metabolism. Plumbagolide significantly downregulates the expression of hexokinase II, the rate-limiting enzyme in glycolysis, inhibits lactate production and extracellular acidification, and suppresses glioma cell proliferation and migration by interfering with glycolysis reprogramming.

[0057] The above examples represent only some implementations of the present invention and do not imply that the scope of protection of the present invention is limited thereto.

Claims

1. A drug that inhibits glioma cell proliferation and / or migration and promotes glioma cell apoptosis by prunella vulgaris.

2. The drug as described in claim 1, characterized in that, Specifically, the pterostilbene is used to inhibit hexokinase II, which is involved in glycolysis in glioma cells, reduce lactate production and induce the accumulation of reactive oxygen species in mitochondria, thereby activating the p38 mitogen-activated protein kinase pathway, inhibiting glioma cell proliferation and / or migration, and promoting glioma cell apoptosis.

3. The drug as described in claim 1, characterized in that, It also contains one of the following: solvent, disintegrant, antioxidant, binder, filler, thickener / or diluent, or a combination thereof.

4. The drug as described in claim 1, characterized in that, The paeoniflorin is a soluble paeoniflorin freeze-dried powder.

5. The use of prunella vulgaris as a pharmaceutical ingredient that inhibits the proliferation and / or migration of glioma cells in the preparation of a drug for promoting apoptosis of glioma cells.

6. The application as described in claim 5, characterized in that, The pterostilbene is specifically used to inhibit hexokinase II, which is used for glycolysis in glioma cells, reduce lactate production and induce the accumulation of reactive oxygen species in mitochondria, thereby activating the p38 mitogen-activated protein kinase pathway and inhibiting the proliferation and / or migration of glioma cells.

7. The application as described in claim 5, characterized in that, The drug also includes excipients that can be formulated with peony root extract; specifically, one of the following: solvent, disintegrant, antioxidant, binder, filler, thickener / or diluent, or a combination thereof.

8. The application as described in claim 5, characterized in that, The paeoniflorin is a soluble paeoniflorin freeze-dried powder.

9. A drug that inhibits hexokinase II, a glycolytic enzyme in glioma cells, via prunella vulgaris.

10. The medicament as claimed in claim 9, characterized in that, Specifically, the p38 mitogen-activated protein kinase pathway is activated by inhibiting hexokinase II, reducing lactate production and inducing the accumulation of mitochondrial reactive oxygen species, thereby inhibiting glioma cell proliferation and / or migration and promoting glioma cell apoptosis.

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