Application of juglone as heat shock protein 90 inhibitor in treatment of glioma

Abstract

The invention discloses application of juglone as a heat shock protein 90 inhibitor in treatment of glioma. It is found that juglone can inhibit ATP enzyme activity of heat shock protein 90 and promote pyroptosis of glioma cells, and the mechanism of juglone is that juglone can target an ATP enzyme binding domain of heat shock protein 90, promote STAT3 degradation and cause mitochondrial active oxygen generation, and then Caspase-3-GSDME pathway is activated to mediate the occurrence of pyroptosis of glioma cells. Therefore, the invention provides a theoretical basis for development of juglone for drugs for inhibiting glioma focus growth, and also provides a new thought and a new strategy for treatment of brain glioma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of juglone in the treatment of glioma by targeting HSP90-STAT3 degradation to induce glioma cell pyroptosis. Background Art

[0002] Heat shock protein 90 (HSP90) is the most abundant type of heat shock protein. Its expression level can be as high as 4%-6% under stress conditions, and about 600 client proteins have been found in mammals. Generally speaking, HSP90 plays a chaperone function by forming a complex with client proteins to maintain the stability and normal function of proteins in the body. The structure of HSP90 is relatively complex, including an ATPase domain (core region) in the amino-terminal region, an intermediate domain, and a carboxyl-terminal domain. More and more evidence confirms that STAT3 is one of the client proteins of HSP90. Both play an important regulatory role in tumor cell growth and survival, are important components of many cancer gene pathways, and have become one of the targets for the development of anti-tumor drugs. Therefore, the development of targeted inhibition of HSP90 function to cause degradation of client protein STAT3 has become a hot spot in current anti-tumor treatment.

[0003] Gliomas are primary malignant tumors originating from the central nervous system (CNS), accounting for about 85%-90% of all primary central nervous system tumors. At present, the recurrence rate of glioma patients is high and the prognosis is poor, and the 5-year mortality rate is second only to lung cancer and pancreatic cancer. Although surgical resection combined with chemotherapy can prolong the survival of glioblastoma patients to a certain extent, the median survival time after standardized treatment is still very short (9 to 14 months), and the survival rate within 5 years is only 0.05%-4.70%. An important reason is that it has innate or acquired resistance to chemotherapy drugs, which makes it difficult to achieve satisfactory results. Cell pyroptosis is a newly discovered form of programmed cell death in recent years. It is usually participated by Caspase family proteins and executed by Gasdermin family proteins. It manifests as continuous swelling of cells until the cell membrane ruptures, leading to the release of cell contents, especially a series of inflammatory factors, and then activating a strong inflammatory response. Based on this, it is urgent to seek drugs that induce cell pyroptosis to participate in the treatment of gliomas, which has great social significance and clinical application value.

[0004] Juglans mandshurica Maxim., also known as walnut, walnut, its green peel (also known as Qinglongyi) is a traditional Chinese medicine and Manchu medicine. Juglans mandshurica is one of the folk prescriptions accumulated in my country for hundreds of years to treat tumors, which is circulated among the people and in the traditional Chinese medicine community. Juglone, as the main active ingredient in Juglans mandshurica, has strong anti-tumor activity against a variety of tumor cells, but there has been no research report on the application of juglone as an HSP90 inhibitor in the treatment of gliomas. Summary of the invention

[0005] The purpose of the present invention is to address the above problems and provide a method for treating brain glioma using juglone as a heat shock protein 90 inhibitor.

[0006] In order to achieve its purpose, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a drug for treating brain glioma, wherein the drug comprises juglone or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0008] Furthermore, the juglone or a pharmaceutically acceptable salt thereof is added with pharmaceutical excipients to prepare a preparation.

[0009] Furthermore, the pharmaceutical excipients include one or more of diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, and other excipients.

[0010] Furthermore, the preparation is in the form of tablets, capsules, oral liquids, injections, lyophilized preparations for injection or powder injections.

[0011] The second aspect of the present invention provides the use of juglone as a targeted inhibitor of heat shock protein 90 in the preparation of a drug for treating brain glioma.

[0012] In the above application technology scheme, the brain glioma cells are glioblastoma lines U87MG and LN229.

[0013] In the above application technology scheme, juglone induces pyroptosis of glioblastoma cells and thus inhibits cell growth.

[0014] In the above-mentioned application technology scheme, juglone targets and binds to the ATPase domain of HSP90, accelerates the ubiquitin-proteasome degradation of the client protein STAT3, aggravates the production of mitochondrial ROS (mtROS) in glioma cells, and then activates the Caspase-3-GSDME pathway to mediate the occurrence of glioma cell pyroptosis and inhibit the growth of glioma cells.

[0015] The beneficial effects of the present invention are as follows: the present invention study confirms that juglone can significantly reduce the viability of glioma cells, but shows lower toxicity to normal human astrocytes, and its mechanism is that juglone targets and binds to the ATPase domain of HSP90 and destroys the interaction with STAT3, leading to the degradation of STAT3 ubiquitin-proteasome pathway. The reduction of STAT3 exacerbates the release of ROS in glioma mitochondria, thereby triggering Caspase-3-GSDME-mediated pyroptosis of glioma cells. The application of the present invention is expected to become a new drug for the treatment of brain glioma diseases, with high safety and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of juglone activating Caspase-3-GSDME pathway to induce pyroptosis of glioma cells in an embodiment of the present invention, wherein A is the cell survival rate of U87 MG, LN229 and HA1800 after adding juglone, B is the cell morphological change of U87 MG and LN229 after adding juglone, C is the effect of juglone treatment for different time on the expression of cell pyroptosis-related proteins in U87 MG and LN229, and D is the effect of Caspase-3 specific inhibitor on GSDME shearing in U87 MG and LN229 induced by juglone;

[0017] Figure 2 Schematic diagram of juglone-induced mtROS promoting pyroptosis of glioma cells in an embodiment of the present invention, wherein A is the effect of juglone treatment for different time periods on the mtROS content in U87 MG and LN229, and B is the effect of Mito-Tempo on the activation of GSDME and Caspase-3 in U87 MG and LN229 induced by juglone;

[0018] Figure 3 Schematic diagram of juglone inhibiting STAT3 signal and promoting pyroptosis of glioma cells in an embodiment of the present invention, wherein A is the effect of juglone treatment for different time periods on the expression levels of STAT3 and p-STAT3 (Tyr705) proteins in U87 MG and LN229, B is the effect of overexpression of STAT3 (OE-STAT3) on the cleavage of GSDME-N-terminal protein and cleavedCaspase-3 protein in glioma cells induced by juglone, and C is the effect of overexpression of STAT3 (OE-STAT3) on the amount of mtROS generated in glioma cells induced by juglone;

[0019] Figure 4: is a schematic diagram of juglone targeting HSP90 binding to promote STAT3 ubiquitin-proteasome degradation in an embodiment of the present invention, wherein A is the direct binding of juglone to HSP90, B is the effect of juglone on the thermal stability of HSP90 at different temperatures, C is the effect of juglone treatment on HSP90 and its N-terminal fragment after trypsin cleavage at different concentrations, D is the intermolecular force analysis of the binding of juglone to the ATP-binding region of the amino terminal of HSP90, E is the effect of juglone at different concentrations on the ATPase activity of HSP90, and F is a co-immunoprecipitation experiment (Co-IP) to verify that juglone weakens the mutual binding between HSP90 and STAT3;

[0020] Figure 5 : is a schematic diagram of juglone inhibiting the growth of glioma in mice in an embodiment of the present invention, wherein A is a schematic diagram of the processing flow of the experiment of juglone inhibiting the growth of glioma in vivo, B is a comparison of the body weight of mice in the juglone treatment group and the blank control group, C is the effect of the juglone treatment group and the control group on the volume and weight of nude mouse xenograft tumors, D is hematoxylin and eosin (H&E) staining to detect cell death in glioma tissue caused by juglone, and E is immunohistochemical staining to detect the reduction of Ki67 expression in glioma tissue caused by juglone;

[0021] Figure 6 Schematic diagram of the relationship between juglone-induced pyroptosis of glioma cells in mice and HSP90-mediated ubiquitination of STAT3 in an embodiment of the present invention, wherein A is the changes in Caspase-3 and GSDME protein cleavage and p-STAT3 (Tyr705) and total STAT3 protein expression in glioma tissues caused by juglone, B is that juglone increases the ubiquitination and degradation of STAT3 in tumor tissues, and C is that juglone inhibits the direct interaction between HSP90 and STAT3 in tumor tissues. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme of the present invention will be further described in detail below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0023] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0024] Embodiment 1:

[0025] Application of juglone in the preparation of an anti-glioma growth drug, application of juglone or a pharmaceutically acceptable salt thereof in the preparation of an anti-glioma growth drug, the structural formula of juglone is as follows:

[0026]

[0027] Juglone or its pharmaceutically acceptable salt is added with pharmaceutical excipients to prepare a preparation.

[0028] Pharmaceutical excipients include one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, and other excipients.

[0029] The dosage form of the preparation is tablet, capsule, oral solution, injection, lyophilized injection or powder injection.

[0030] Juglone or its pharmaceutically acceptable salt can be administered to patients in need of such treatment in the form of a composition by oral administration, nasal inhalation, rectal administration or parenteral administration. When used for oral administration, it can be made into conventional solid preparations such as tablets, powders, granules, capsules, etc., and liquid preparations such as water or oil suspensions, etc.; when used for parenteral administration, it can be made into solutions for injection, water or oil suspensions, etc. Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient is mixed with one or more carriers and then made into the desired dosage form.

[0031] Embodiment 2:

[0032] See also Figure 1 This embodiment provides a technical solution based on the first embodiment: juglone activates Caspase-3-GSDME pathway to induce glioma cell pyroptosis.

[0033] U87 MG and LN229 cells are both commonly used cell lines in brain glioma research. U87 MG is derived from human glioblastoma tissue and can continue to grow and proliferate under in vitro culture conditions, providing an important model for studying the biological characteristics and treatment of glioblastoma. The LN229 cell line is also derived from human glioblastoma tissue and is widely used in in vitro experimental studies of brain glioma.

[0034] 1. The CCK8 method was used to detect the cell survival rate of glioma cells after juglone acted on them.

[0035] Two glioblastoma cell lines (U87 MG, LN229) or human astrocyte cell line (HA1800) were seeded in 96-well plates at 5×10 3 / well density was inoculated in a 96-well plate. After 24 hours, different concentrations of juglone (0μM-100μM) or different treatment times (12 hours, 24 hours, 48 ​​hours) were added, and CCK8 (10μL per well, incubated at 37 degrees for 1 hour) was added to detect cell viability. Juglone can induce a decrease in the survival rate of glioblastoma cells in a concentration- and time-dependent manner ( Figure 1 A) IC of two cell lines at 24 hours 50 The values ​​were 8.82 μM and 15.03 μM, respectively. 50 The highest concentration of 15 μM will be used in subsequent experiments. Compared with the two glioma cells, the toxicity of juglone to human normal astrocytes HA1800 was ( Figure 1 A).

[0036] 2. Optical microscopy observation was used to detect the changes in cell morphology after juglone acted on glioma cells.

[0037] Press 5×10 3 / well density inoculated in a 96-well plate. After 24 hours, juglone (15 μM) was added for different treatment times (0 hours, 3 hours, 6 hours, 12 hours and 24 hours). Through optical microscopy, we unexpectedly found that some cells in the 12-hour and 24-hour treatment groups were swollen and enlarged, accompanied by bubble-like protrusions ( Figure 1 B), it was preliminarily concluded that juglone-induced glioma cell death may involve cell pyroptosis.

[0038] 3. Western blotting method was used to detect the expression of cell pyroptosis-related proteins after juglone acted on glioma cells.

[0039] Press 1×10 5 / well density was inoculated in a 6-well plate and cultured overnight. Juglone (15μM) was added for treatment at different times (0 hours, 3 hours, 6 hours, 12 hours and 24 hours). The cells were collected and centrifuged, the samples were lysed by PIPA, and the loading buffer was added and boiled at 100 degrees for 10 minutes. The protein was then electrophoresed and transferred to a PVDF membrane. The membrane was incubated in the corresponding primary antibody at 4 degrees overnight, washed three times with TBST the next day, and the secondary antibody of the corresponding species was added and incubated for 90 minutes. Developed in a gel imager. The results showed that when the two glioma cells were treated with juglone (15μM) for 12h-24h, GSDME in the cells was cleaved to form GSDME-N-terminal protein, and Caspase-3 and PARP-1 in the cells were also cleaved and activated ( Figure 1 C), indicating that in glioma, juglone induces cell pyroptosis mainly through the Caspase-3-GSDME pathway.

[0040] 4. Western blotting was used to verify that juglone-induced glioma cell pyroptosis was dependent on Caspase-3 protein.

[0041] To further clarify that Caspase-3 is the upstream molecule activated by GSDME, the Caspase-3 specific inhibitor Av-DVED-CHO was used. Figure 1 As shown in D, juglone alone can significantly induce the cleavage of GSDME and produce GSDME-N-terminal protein. However, in the presence of Caspase-3 specific inhibitor, GSDME did not undergo obvious cleavage. Therefore, the above results can confirm that inducing Caspase-3-GSDME-dependent cell pyroptosis is the main way for juglone to exert its anti-glioma effect.

[0042] Embodiment three:

[0043] See also Figure 2 This embodiment provides a technical solution based on the second embodiment: pyroptosis of glioma cells induced by juglone is related to the production of mtROS.

[0044] 1. Flow cytometry was used to detect the content of mtROS after juglone acted on glioma cells.

[0045] The cells were treated with juglone (15 μM) for 0, 1, 3, and 6 hours. The cells were collected by centrifugation. After washing with PBS, 1 mL of MitoSOX working solution was added and incubated at 37 degrees for 20 minutes. The cells were centrifuged and the supernatant was discarded. The cells were washed with PBS at least twice, and finally resuspended with 1 mL of PBS and detected by flow cytometry. The results are shown in Figure 2 As shown in A, it was found that juglone could significantly induce the production of mtROS within 1 hour, and the amount of mtROS generated increased accordingly as time went on.

[0046] 2. Western blotting was used to verify that juglone-induced glioma cell pyroptosis was dependent on mtROS.

[0047] To further investigate the role of mtROS in juglone-induced pyroptosis in glioma cells, the mitochondrial ROS-specific scavenger Mito-Tempo was used. 5 The cells were inoculated at a density of 100 μg / well in a 6-well plate and cultured overnight. Mito-Tempo (50 μM) was added for pretreatment for 1 hour, and then juglone (15 μM) was added for treatment for 24 hours. Cell proteins were collected and the cleavage levels of GSDME, Caspase-3, and PARP-1 were detected by Western blotting. The results are shown in Figure 2As shown in B, compared with the control group, the expression levels of GSDME-N-terminal protein and cleaved Caspase-3 protein in glioma cells treated with juglone were significantly increased; after the application of Mito-TEMPO, it was found that the clearance of mtROS inhibited the activation of Caspase-3 and GSDME caused by juglone, indicating that mtROS is upstream of juglone-induced cell pyroptosis.

[0048] Embodiment 4:

[0049] See also Figure 3 This embodiment provides a technical solution based on the third embodiment: juglone inhibits STAT3 signaling to promote glioma cell pyroptosis.

[0050] 1. Western blotting was used to verify that juglone reduced the expression of total STAT3 protein and phosphorylated STAT3 (Tyr705).

[0051] In order to further explore the possible mechanism of action of juglone on core proteins, we first detected the effect of juglone on the expression level of STAT3 protein. 5 / well density was inoculated in a 6-well plate and cultured overnight. Juglone (15μM) was added for different treatment times (0 hours, 3 hours, 6 hours, 12 hours and 24 hours). The cells were collected and centrifuged, and the samples were lysed using PIPA. After adding the loading buffer, they were boiled at 100 degrees for 10 minutes. The proteins were then electrophoresed and transferred to a PVDF membrane. The membranes were incubated in the corresponding primary antibodies at 4 degrees overnight, washed three times with TBST the next day, and the secondary antibodies of the corresponding species were added and incubated for 90 minutes. The gel was developed in a gel imager. The results are shown in Figure 3 As shown in A, juglone reduced the protein level of STAT3 in U87 MG and LN229 cells in a time-dependent manner. At the same time, we tested whether juglone inhibited STAT3 phosphorylation. The results showed that juglone strongly inhibited the activation of p-STAT3 (Tyr705) in glioma cells ( Figure 3 A).

[0052] 2. Western blotting method was used to verify the role of STAT3 signaling pathway in the anti-glioma pyroptosis effect of juglone.

[0053] In order to study the role of the STAT3 signaling pathway in the anti-glioma effect of juglone, we used two types of glioma cells transfected with overexpressed STAT3 (OE-STAT3) and empty vector (OE-NC) plasmids, respectively. 24 hours after transfection, the culture medium in the wells was aspirated, and 2 mL of fresh culture medium was added to continue culturing for 24 hours. Then, 500 μg / mL of G418 was added for screening. After the cell state stabilized, the culture medium containing G418 was replaced every 3 days. After the cells formed a large number of colonies, they were digested and inoculated into new culture dishes for subsequent experiments. Figure 3 As shown in B, compared with cells transfected with empty vector (OE-NC), transfection of STAT3 significantly increased the total STAT3 protein level in glioma cells. Subsequent Western blotting results also confirmed that cells overexpressing STAT3 significantly reduced the cleavage of GSDME-N-terminal protein and cleaved Caspase-3 protein caused by juglone ( Figure 3 B).

[0054] 3. Flow cytometry was used to verify the role of STAT3 signaling pathway in juglone-induced mtROS.

[0055] In order to study the role of STAT3 signaling pathway in juglone-induced mtROS, we further found by flow cytometry that overexpression of STAT3 significantly weakened the massive generation of mtROS induced by juglone ( Figure 3 C) These results indicate that inhibition of STAT3 activation is a key upstream target of juglone-induced pyroptosis in glioma cells.

[0056] Embodiment five:

[0057] See also Figure 4 This embodiment provides a technical solution based on the fourth embodiment: juglone targets and binds to the ATPase domain of HSP90 protein, thereby weakening the mutual binding with STAT3.

[0058] 1. Protein pull-down binding assay was used to detect the direct binding of juglone to HSP90.

[0059] The activated Sepharose 6B beads were divided into two equal parts, and the coupling buffer and 200 μL of 25mM juglone solution were added in turn. 200 μL of DMSO was added to the control group, and the beads were incubated overnight at room temperature on a rotating incubator. The next day, the excess unbound drugs were washed away, and the Sepharose 6B beads were blocked with 1M ethanolamine solution overnight. After blocking, the Sepharose 6B beads were washed with PBS and continued to be incubated with cell lysate samples or human recombinant HSP90 protein at 4 degrees overnight. Then, they were washed 5 times with 1 mL of RIPA lysis buffer, 5× loading buffer was added to the supernatant, and it was boiled at 100 degrees for 10 minutes for immunoblot analysis. The experimental results showed that juglone could directly bind to human recombinant HSP90 protein and HSP90 in cell lysate ( Figure 4 A).

[0060] 2. The cellular endogenous protein thermal shift analysis (CETSA) experiment was used to verify the direct binding of juglone to HSP90 protein.

[0061] Select cells in the logarithmic growth phase, lyse with IP lysis buffer, and collect proteins by centrifugation at 12,000g for 25 minutes at 4 degrees. Divide the total protein of the extracted cells into 9 equal portions, and put 100μL of each portion into PCR tubes. Then add 10μL of juglone to make the final concentration of 100μM, and add an equal volume of DMSO to the control group and incubate at room temperature for 1 hour. Place the PCR tube in the PCR instrument and control the gradient heating at 40 to 56 degrees for 5 minutes. Then transfer the sample to a 1.5mL EP tube and centrifuge at 12000g for 5 minutes. Take an equal volume of supernatant and add 5× loading buffer, boil at 100 degrees for 10 minutes, and perform immunoblot analysis. Western Blotting results showed that under heating conditions of 52 and 54 degrees, juglone significantly improved the thermal stability of HSP90 ( Figure 4 B).

[0062] 3. Limited protease degradation experiments were used to confirm that juglone directly binds to the ATPase region at the N-terminus of the HSP90 protein.

[0063] 0.4, 0.8, 1.2, 1.6, 2.0, 2.4 μg / mL of trypsin was added to PBS, and the recombinant HSP90 protein was partially proteolytically cleaved at 30 degrees for 15 minutes. The proteolytic fragments were separated by SDS-PAGE, and the cleaved fragments were detected by immunoblotting with HSP90 antibodies. The experimental results confirmed that juglone treatment not only made the full-length HSP90 resistant to trypsin, but also made its N-terminal fragment resistant. These results indicate that juglone induces conformational changes in HSP90 by binding to the N-terminal domain (ATPase region) of HSP90 ( Figure 4 C).

[0064] 4. Molecular docking simulation was used to verify that juglone can directly bind to the ATPase domain of HSP90.

[0065] The molecular structure file of juglone (PubChem CID: 3806) was downloaded from the PubChem compound database (http: / / www.ncbi.nlm.nih.gov / pccompound) and imported into Chem3D software (v16.0) for energy minimization. The crystal structure of HSP90 (PDB ID: 1BYQ) protein was downloaded from the PBD protein database (http: / / www.rcsb.org / pdb) and imported into Discovery Studio (v4.5) to delete the ligand and water molecules and retain the target protein monomer structure. Then, the molecular structure of juglone and protein was imported into AutoDock software (v4.2.6), polar hydrogen and charge were added, and chemical bond torsion was set. A three-dimensional space box of the protein docking site was created in the AutoGrid program. The docking was performed under the default conditions in the AutoDock program, and the one with the lowest binding free energy was selected from the docking results as the dominant conformation and saved. Finally, the dominant conformation file was opened in PyMOL (v2.3.4) for intermolecular force analysis. From the results of molecular simulation, it can be found that juglone binds well to the ATP-binding region of the amino terminal of HSP90, forming hydrogen bonds with Phe213, Ile214 and Ile 218 of HSP90 respectively ( Figure 4 D).

[0066] 5. The ATPase activity assay was used to verify that juglone inhibited the activity of the ATPase domain in HSP90 protein.

[0067] The experiment was performed with reference to the Malachite Green Phosphate Assay Kit. First, 150 μL of assay buffer was added to a sterile 1.5 mL EP tube, and recombinant HSP90 protein and ATP solution were added in sequence to prepare an assay solution with a concentration of 0.5 μM HSP90 protein and 1 mM ATP. Then, equal volumes of DMSO and juglone solutions with final concentrations of 5 μM, 10 μM, and 15 μM were added to the EP tubes, respectively. The assay solution was aspirated and added to a 96-well plate, 50 μL per well, and the plate was shaken in an ELISA reader for 10 seconds to mix thoroughly. Incubate in a 37-degree constant temperature incubator for 3 hours. Next, take out the 96-well plate, select an empty well and add the standard solution to draw a standard curve, and add 5 μL of geldanamycin acid solution to each well, put the 96-well plate into an ELISA reader and shake for 10 seconds to mix evenly, and incubate at room temperature for 10 minutes. After the sample incubation is completed, the OD value at a wavelength of 620 nm is detected. The results showed that juglone had a significant inhibitory effect on the ATPase activity of HSP90 ( Figure 4 E).

[0068] 6. Co-immunoprecipitation experiment (Co-IP) was used to verify that juglone weakened the interaction between HSP90 and STAT3.

[0069] After each group of cells was lysed with IP lysis buffer, the supernatant was collected by centrifugation, the protein concentration was determined by the BCA method, and 50 μL of protein lysate was retained for Input detection. Then 500 μg of protein was slowly shaken overnight at 4 degrees with sufficient HSP90 or STAT3 primary antibody. The next day, the protein-antibody complex was coupled with 25 μL proteinA / G agarose for 4 hours. After that, the agarose beads were washed 5 times with pre-cooled lysis buffer. Boil in 1×SDS loading buffer and perform immunoblot analysis. Endogenous Co-IP experiments confirmed that the binding of juglone to HSP90 significantly inhibited the mutual binding between HSP90 and STAT3 ( Figure 4 F).

[0070] Embodiment six:

[0071] See also Figure 5 This embodiment provides a technical solution based on the fifth embodiment: juglone inhibits the growth of glioma in vivo.

[0072] 1. The vernier caliper and balance method was used to detect the inhibitory effect of juglone on the growth of glioma in vivo.

[0073] In order to prove that juglone has the effect of treating glioma in vivo, we injected 1×10 6U87 MG cells were subcutaneously injected into nude mice to establish a subcutaneous xenograft tumor model. Starting from the 7th day after cell inoculation, juglone (2.0 mg / kg) was intraperitoneally injected once every 2 days, and the longest diameter (L) and shortest diameter (S) of the nude mouse tumor were measured with a vernier caliper. The volume (V) of the transplanted tumor was calculated according to the formula: V (mm 3 )=π / 6×L×S 2 After the experiment, the processing flow is as follows Figure 5 As shown in A. The body weight of mice in the juglone treatment group was not significantly different from that in the blank control group ( Figure 5 B). Our experimental results further showed that compared with the control group, the juglone treatment group could significantly inhibit the growth and weight of xenograft tumors in nude mice ( Figure 5 C).

[0074] 2. The hematoxylin and eosin (H&E) method was used to detect cell death in glioma tissue caused by juglone.

[0075] The tumor tissues of each group were embedded in paraffin, sliced ​​(5 μm), and baked at 58 degrees for 2 hours before use. The tissue sections were dewaxed with xylene, rehydrated with gradient ethanol, stained with hematoxylin / eosin, rinsed with running water, and then dehydrated with a gradient ethanol solution of 70%-100%. The slides were then placed in a transparent xylene solution. Finally, the slides were sealed with neutral resin and observed under an optical microscope for hematoxylin and eosin (H&E) staining. It was found that the juglone-treated group showed a decrease in densely arranged cells and the presence of sparse necrotic cell areas ( Figure 5 D).

[0076] 3. Immunohistochemical staining was used to detect the decreased expression of Ki67 in glioma tissue caused by juglone.

[0077] The sections of the tumor tissue were routinely dewaxed and dehydrated, and after blocking and inactivating endogenous peroxidase with 3% H2O2, they were placed in a 0.01M citrate buffer (PH6.0) and boiled. The sections were blocked with normal goat serum working solution for 1 hour. The primary antibody was added and refrigerated at 4 degrees overnight, and then rinsed with PBS. Then the biotin-labeled secondary antibody was added, incubated at 37 degrees for 30 minutes, and rinsed with PBS. DAB was used for color development, and the reaction was terminated with distilled water when the tissue turned brown. Finally, the sections were stained with hematoxylin, dehydrated with ethanol gradient, and sealed after transparency. Observation under an optical microscope revealed that compared with the control group, the number of Ki67-positive tumor cells in the juglone treatment group was significantly reduced ( Figure 5 E).

[0078] Embodiment seven:

[0079] See also Figure 6This embodiment provides a technical solution based on the sixth embodiment: juglone-induced tumor cell pyroptosis in mice is related to HSP90-mediated STAT3 ubiquitination.

[0080] 1. Western blotting was used to detect pyroptosis of cells in glioma tissues induced by juglone.

[0081] In order to confirm whether juglone can induce cell pyroptosis in vivo, tissue lysis buffer was added to fully homogenize and centrifuged to obtain the protein supernatant in the tissue. After BCA quantification, samples were prepared and Western blotting was performed. Figure 6 As shown in A, Caspase-3 and GSDME proteins in the tumor tissue of nude mice in the control group were not cleaved. However, in the juglone treatment group, Caspase-3 and GSDME proteins in the tumor tissue were cleaved. At the same time, juglone treatment significantly reduced the expression of p-STAT3 (Tyr705) and total STAT3 proteins in glioma tissue ( Figure 6 A).

[0082] 2. Co-immunoprecipitation experiment (Co-IP) was used to verify that juglone increased the ubiquitination degradation of STAT3 in tumor tissues.

[0083] After each group of cells was lysed with IP lysis buffer, the supernatant was collected by centrifugation, the protein concentration was determined by the BCA method, and 50 μL of protein lysate was retained for input detection. Then 500 μg of protein was slowly shaken overnight at 4 degrees with sufficient STAT3 primary antibody. The next day, the protein-antibody complex was coupled with 25 μL proteinA / G agarose for 4 hours. After that, the agarose beads were washed 5 times with pre-cooled lysis buffer. Boil in 1×SDS loading buffer and perform immunoblot analysis. Endogenous Co-IP experiments confirmed that juglone significantly increased the ubiquitination degradation of STAT3 protein in tumor tissues ( Figure 6 B).

[0084] 3. Immunofluorescence staining was used to detect that juglone inhibited the direct interaction between HSP90 and STAT3 in tumor tissues.

[0085] The sections of tumor tissue were routinely dewaxed and dehydrated, boiled in 0.01M citrate buffer (PH6.0), punched with 0.1% Triton X-100 solution, and placed in normal goat serum working solution for blocking for 1 hour. Then, the corresponding species of mixed primary antibodies (1:100) were directly added, incubated at 4 degrees overnight, and after rinsing with PBS, Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) and Alexa Fluor 647-labeled goat anti-mouse IgG (H+L) secondary antibody mixture (1:1000) were added and incubated at room temperature in the dark for 2 hours. DAPI was used for nuclear staining, and the sections were sealed after rinsing with PBS. Laser confocal microscopy showed that the juglone treatment group inhibited the mutual binding between STAT3 and HSP90 ( Figure 6 C).

[0086] The above specific embodiments are only explanations of the present invention, and they are not limitations of the present invention. After reading this specification, those skilled in the art can make modifications to the embodiments without creative contribution as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. Use of a heat shock protein 90 inhibitor in the preparation of a drug for inhibiting the growth of glioma cells, wherein the heat shock protein 90 inhibitor is juglone.

2. Use of a heat shock protein 90 inhibitor in inducing pyroptosis of glioma cells, wherein the heat shock protein 90 inhibitor is juglone.

3. Use of a heat shock protein 90 inhibitor in promoting STAT3 ubiquitin-proteasome degradation, wherein the heat shock protein 90 inhibitor is juglone.

4. Use of a heat shock protein 90 inhibitor in promoting STAT3 degradation to accelerate glioma cell pyroptosis, wherein the heat shock protein 90 inhibitor is juglone.

5. A pharmaceutical preparation for treating glioma, wherein the active ingredient is a heat shock protein 90 inhibitor, characterized in that: The heat shock protein 90 inhibitor is juglone.

6. The pharmaceutical preparation according to claim 5, characterized in that: The composition also contains a pharmaceutically acceptable carrier to prepare a pharmaceutically acceptable dosage form.

7. The pharmaceutical preparation according to claim 6, characterized in that: The carrier is a solid, liquid or semi-solid auxiliary material.

8. The pharmaceutical preparation according to claim 7, characterized in that: The dosage forms include tablets, capsules and injections.

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