Preparation method and application of inonotus hispidus melanin
By preparing and purifying Cialis melanin in coccyclovita, the problem of insufficient research and application in this field was solved, and the significant inhibitory effect of melanin on breast cancer cells was achieved, demonstrating its potential for development as an anti-breast cancer drug.
Patent Information
- Application Number
- CN202510086857.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-20
- Publication Date
- 2025-06-06
AI Technical Summary
In the prior art, there are few researches and applications on melanin of coccycadobacteria, and there is a lack of effective preparation methods and application methods, especially in anti-breast cancer.
The method of preparing coccyclovir melanin includes collecting and drying coccyclovir fruiting bodies, crushing and filtering through a screen, ultrasonic reaction using NaOH solution, adding an acid solution to water bath heat after centrifugation, followed by multiple centrifugation, washing and purification, and finally obtaining the purified melanin sample by running water dialysis and freeze-drying.
This method successfully obtained purified Cialis melanin and demonstrated that it can significantly inhibit the growth, proliferation and migration of human breast cancer cells, induce apoptosis and pyroptosis, thus showing the potential for development as an anti-breast cancer drug.
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Figure CN120093799A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of bioengineering, in particular to a preparation method and application of tricholoma melanin. Background Art
[0002] Inonotus hispidus (Bull.) P. Karst. is a precious medicinal fungus, included in the Chinese Medicinal Fungi List, also known as Inonotus hispidus, belonging to the genus Inonotus, family Inonotus, order Inonotus, order Inonotus, family Inonotus, family Inonotus. As a traditional Chinese medicine "mulberry yellow", Inonotus hispidus has pharmacological values such as antioxidant, anti-tumor, hypoglycemic, liver protection, and immunity enhancement. In modern pharmacology, the medicinal components of Inonotus hispidus are gradually being explored, such as polysaccharides, polyphenols, flavonoids, terpenes and other active substances [6-8]. Melanin is also one of the active ingredients of Inonotus hispidus, but there are few reports on the relevant research on Inonotus hispidus melanin. Correspondingly, the application of Inonotus hispidus melanin is also rarely involved. Summary of the invention
[0003] The purpose of the present invention is to provide a preparation method and application of Porifera pubescens melanin, which provides a preparation method of Porifera pubescens melanin and proves that Porifera pubescens melanin can significantly inhibit the growth, proliferation and migration of human breast cancer cells; and induce apoptosis and pyroptosis of human breast cancer cells; further proves that Porifera pubescens melanin has the potential to be developed into an anti-breast cancer drug.
[0004] The present invention is achieved through the following technical solution: A method for preparing the melanin of Pleurotus eryngii comprises the following steps:
[0005] S1, collecting the fruiting bodies of the tricholoma, drying and crushing them, and then filtering them through a sieve;
[0006] S2, weigh the powder and add NaOH solution, and place it in ultrasound for full reaction;
[0007] S3, centrifuging the solution in step 2 in a centrifuge and collecting the supernatant;
[0008] S4, adding an acidic solution to the supernatant collected in S3 until the pH is 1.3-1.7, and heating in a water bath for 8-11 hours;
[0009] S5, after centrifugation again, the precipitate is collected;
[0010] S6, washing the precipitate with distilled water until the pH value is in the middle, and then adding chloroform, ethyl acetate, anhydrous ethanol, 70%-75% ethanol and distilled water in sequence for purification;
[0011] S7, dissolving the precipitate with NaOH solution, adjusting the pH value to neutral with HCl solution, collecting the supernatant by centrifugation, and finally dialysis with running water and freeze-drying to obtain the purified melanin sample of the fruiting body of the tricholoma.
[0012] Further, in S1, the fruiting bodies of the Pleurotus eryngii are dried and crushed and then sieved through a sieve of 80 meshes.
[0013] Furthermore, in S2, calculated by weight fraction ratio, the weight of the powder added to every 30 parts of NaOH solution is 0.9-1.1 parts, wherein the mass concentration of NaOH is 1.5 mol / L.
[0014] Furthermore, in S2, the reaction time in ultrasound is 50-65 minutes.
[0015] Furthermore, the rotation speed of the centrifuge in S3 is 10000 rpm / min, and the centrifugation time is 4-6 minutes.
[0016] Furthermore, the acidic solution added in S4 is a hydrochloric acid solution.
[0017] Furthermore, the mass concentration of the NaOH solution added to S7 is 0.1 mol / L, and the mass concentration of the hydrochloric acid solution is 0.1 mol / L.
[0018] Furthermore, the duration of running water dialysis is 46-50 hours.
[0019] The application of the Porites pubescens melanin in drugs for treating breast cancer is described. The Porites pubescens melanin is applied to breast cancer.
[0020] Specifically, the tricholoma melanin is used for human triple-negative breast cancer cells MDA-MB-231, human triple-negative breast cancer cells MDA-MB-468 and human breast cancer cells MCF-7; for inhibiting the growth, proliferation and migration of human breast cancer cells; and for inducing apoptosis and pyroptosis of human breast cancer cells.
[0021] Compared with the previous technology, the beneficial effects of the present invention are:
[0022] 1. A method for extracting melanin from Porites tricholoma is provided, which provides the possibility for the subsequent preparation of breast cancer drugs.
[0023] 2. It was proved that the melanin of Porifera pubescens can significantly inhibit the growth, proliferation and migration of human breast cancer cells; and induce apoptosis and pyroptosis of human breast cancer cells; further proving that the melanin of Porifera pubescens has the potential to be developed into an anti-breast cancer drug. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1Schematic diagram of IHFM inhibiting the viability of breast cancer cells in a time- and concentration-dependent manner;
[0025] Figure 2 Schematic diagram of IHFM's ability to inhibit breast cancer cell proliferation in a concentration-dependent manner;
[0026] Figure 3 Schematic diagram of IHFM's ability to inhibit breast cancer cell migration in a concentration-dependent manner;
[0027] Figure 4 Schematic diagram of the effects of different cell death inhibitors on the viability of IHFM-induced breast cancer cells;
[0028] Figure 5 Schematic diagram of the effect of different concentrations of IHFM on breast cancer cell morphology;
[0029] Figure 6 Schematic diagram of IHFM-induced apoptosis of breast cancer cells;
[0030] Figure 7 Schematic diagram of IHFM-induced pyroptosis of breast cancer cells. DETAILED DESCRIPTION
[0031] The present invention is described in detail below in conjunction with the accompanying drawings:
[0032] like Figure 1 As shown: Collect the fruiting bodies of the thick-haired fibrous fungus, dry them in an oven, crush them with a grinder, and filter them through an 80-mesh sieve. Weigh 30g of powder and slowly add it to 900mL of 1.5mol / L NaOH, and place it in an ultrasonic wave for full reaction for 1h. Then centrifuge it in a centrifuge for 5min (10000rpm / min), collect the supernatant, adjust the pH value of the supernatant to 1.5 with concentrated hydrochloric acid, bathe it in a water bath at 80℃ for 10h, and collect the precipitate by centrifugation. Wash the precipitate with distilled water until the pH value is neutral, and then add chloroform, ethyl acetate, anhydrous ethanol, 75% ethanol and distilled water in sequence for purification. Then dissolve the precipitate with 0.1mol / L NaOH, adjust the pH value to neutral with 0.1mol / L HCl, collect the supernatant by centrifugation, dialyze it with running water for 48h, and freeze-dry it to obtain the purified thick-haired fibrous fungus fruiting body melanin sample (IHFM).
[0033] The application of the Porites pubescens melanin in drugs for treating breast cancer is described. The Porites pubescens melanin is applied to breast cancer.
[0034] Specifically, the tricholoma melanin is used for human triple-negative breast cancer cells MDA-MB-231, human triple-negative breast cancer cells MDA-MB-468 and human breast cancer cells MCF-7; for inhibiting the growth, proliferation and migration of human breast cancer cells; and for inducing apoptosis and pyroptosis of human breast cancer cells.
[0035] The following is explained in conjunction with specific embodiments:
[0036] Example 1
[0037] Culture of human breast cancer cells
[0038] (1) Cell recovery
[0039] Add a small amount of cell culture medium to a 10 mL sterile centrifuge tube in advance; take out the cryotube from the -80°C refrigerator, place it in a 37°C constant temperature water bath, thaw it quickly within 1 min, and transfer it to a centrifuge tube for centrifugation (1000 rpm / min); discard the supernatant, add new culture medium to resuspend, blow the cells thoroughly and transfer them to a cell culture bottle with culture medium, and place it at 37°C and 5% CO 2 Culture in an incubator, observe cell growth and replace culture medium the next day.
[0040] (2) Cell passaging
[0041] When the cells grow to 80-90%, discard the old culture medium, wash with PBS and discard; add 500 μL of trypsin for digestion, and after the cells fall off, use serum-containing RPMI 1640, DMEM and MEM culture medium to terminate the digestion; centrifuge and discard the supernatant, resuspend the cells with new culture medium, mix well, transfer to a new culture bottle, add culture medium, and continue culturing.
[0042] (3) Cell cryopreservation
[0043] Select cells with good growth status for cryopreservation, replace with fresh culture medium the night before cryopreservation, discard the old culture medium the next day, wash with PBS; add trypsin for digestion, centrifuge and discard the supernatant; add cryopreservation solution, mix by pipetting, and dispense into sterile cryopreservation tubes, and mark them. Cool down in a gradient, first cool at 4℃ for 30min, cool at -20℃ for 2h, and cool at -80℃ overnight, and transfer to liquid nitrogen the next day for long-term storage.
[0044] Example 2
[0045] Introduction to the detection method of human breast cancer cells
[0046] Human breast cancer cell viability assay
[0047] (1) Plating: Take MDA-MB-231, MDA-MB-468, and MCF-7 cells in the logarithmic growth phase, discard the culture medium, digest with trypsin, and dilute the cell density to 5×10 4 200 μL of PBS was added to the outer circle of a 96-well plate and placed in a CO 2 Incubate in the incubator for 24 h.
[0048] (2) Drug loading: After the cells adhered to the wall, the original culture medium was discarded, and a control group (CK group) without drug loading, a zero adjustment group without cells, and an IHFM group (IHFM group) were set. The concentrations of the melanin of the tricholoma were set to 0.125, 0.25, 0.5, 1, 2, and 4 mg / mL, respectively, with 5 replicates in each group and at least 3 biological replicates. The 96-well plate was placed in an incubator and cultured for 24 h, 48 h, and 72 h.
[0049] (3) MTT reaction: After the cell culture is completed, the culture medium is removed. 10 μL of 5 mg / mL MTT solution and 90 μL of complete culture medium are added to each well and incubated for another 4 h. The culture medium containing MTT is discarded, and 150 μL of dimethyl sulfoxide (DMSO) is added to each well to fully dissolve the purple-brown formazan. After mixing, the mixture is placed in an ELISA reader to detect the absorbance at 490 nm.
[0050] (4) Survival rate analysis: Human breast cancer cell viability assay
[0051] (1) Breast cancer cells in the logarithmic growth phase were digested with trypsin, and a serum-containing culture medium was added to terminate the digestion; the cells were centrifuged in a centrifuge and the supernatant was discarded; the cell density was diluted to 1000 cells / mL, and 1 mL was inoculated per well in a 6-well plate and cultured overnight.
[0052] (2) After the cells adhered, the original culture medium was removed and a control group and an IHFM group were set up. 2 mL of complete culture medium was added to the control group, and the concentrations of the IHFM group were set to 0.25, 0.5, 1, 2, and 4 mg / mL, respectively. The final volume was 2 mL, and 3 replicates were set for each group. Fresh culture medium was replaced every 3 days and cultured in an incubator for 8-12 days.
[0053] (3) Observe the cell clone formation under an inverted microscope every day. When the number of colonies is greater than 50, it means that clones have been formed and the culture can be terminated.
[0054] (4) Slowly discard the culture medium, rinse with PBS first and then add 4% paraformaldehyde to fix the cells. After fixation, add an appropriate amount of 0.1% crystal violet for staining. Then wash with PBS until there is no obvious purple background. After drying at room temperature, take pictures and record.
[0055] (5) Use Image J software to analyze and calculate the cell clone formation rate according to formula (2-2). Clone formation rate / % = number of clones (IHFM组) / Number of clones (对照组) ×100.
[0056] Detection of migration ability of human breast cancer cells
[0057] (1) Take a sterile 6-well plate and mark the back with a line.
[0058] (2) The breast cancer cells were diluted to a density of 10,000 cells / mL, 2 mL was inoculated into each well, and the cells were cultured in a cell culture incubator.
[0059] (3) When the cells grow to about 80-90%, discard the culture medium, rinse with PBS, and then use a 10μL white pipette tip to scratch. The pipette tip should be vertical and not tilted during scratching. Place under an inverted microscope to record the width of the scratch on the original cell at 0h.
[0060] (4) The scratched cells were rinsed with PBS, and a control group and an IHFM group were set up. The control group was added with a culture medium containing 2.5% serum, and the IHFM group was added with a 2.5% serum culture medium of different concentrations (0.25, 0.5, 1, 2, and 4 mg / mL IHFM), and the cells were cultured in an incubator for 48 h.
[0061] (5) After 48 hours, discard the drug-containing culture medium. Place the 6-well plate under a microscope to observe the change in scratch area at the scratch position at 0 hours, and take a photo to record the scratch width at 48 hours.
[0062] (6) Image J software was used to analyze the scratch area of each group at 0 h and 48 h, and the cell migration rate was analyzed according to the following formula (2-3).
[0063]
[0064] Screening of cell death patterns in human breast cancer cells
[0065] Breast cancer cells were pretreated with Caspase 3 inhibitor DEVD, Caspase 1 inhibitor YVAD, necrosis inhibitor Nec-1 and ferroptosis inhibitor Fer-1, and then incubated with IHFM (IC 50 Value), and the effect of IHFM on the survival rate of breast cancer cells after inhibitor pretreatment was detected.
[0066] Breast cancer cells were inoculated in a 96-well plate. When the cells grew to 80-90%, a control group, an IHFM group, an IHFM combined with inhibitor group, and a zeroing well were set up. DEVD, YVAD, Nec-1, and Fer-1 were added to the IHFM combined with inhibitor group to intervene in breast cancer cells for 8 hours, and then the inhibitor-containing medium was discarded and IHFM was added for 48 hours. After the cell culture was completed, the medium was discarded, and 10 μL of MTT and 90 μL of complete medium were added to each well. After 4 hours, the supernatant was discarded, and 150 μL of DMSO was added to dissolve the formazan. After it was fully dissolved, the absorbance at 490 nm was measured on an ELISA reader.
[0067] Survival analysis: Apoptosis detection of human breast cancer cells
[0068] (1) Place a clean and sterilized coverslip in a 6-well plate and inoculate breast cancer cells for overnight culture.
[0069] (2) When the cells grew to 60-80%, they were set as control group and IHFM group. 2 mL of complete culture medium was added to the control group, and culture medium containing 1, 2, and 4 mg / mL IHFM was added to the IHFM group, with a final volume of 2 mL. The cells were placed in a cell culture incubator and continued to be cultured.
[0070] (3) After 48 hours, discard the original culture medium. Add 500 μL of the fixative solution in the Hoechst 33258 kit to fix the cells for 10 minutes (or overnight at 4°C). After fixation, wash twice with PBS.
[0071] (4) Add 500 μL of Hoechst 33258 staining solution and stain for 5 min on a shaker. After staining, wash with PBS.
[0072] (5) Take a clean glass slide and mark it. Add a drop of anti-fluorescence quencher to each glass slide. Use clean tweezers to remove the cover slip and place it on the glass slide so that the cells are in contact with the anti-fluorescence quencher as much as possible.
[0073] (6) The prepared slide is placed under an inverted fluorescence microscope for observation, and the excitation wavelength and emission wavelength are set to about 350 nm and 460 nm, respectively, so that the cell nucleus appears blue in the field of view.
[0074] (7) Take photos of three different fields of view at random under a microscope, count the number of normal and apoptotic cells, and calculate the apoptosis rate of each group according to formula (2-4). The experiment was repeated three times.
[0075]
[0076] Observation of cell morphology of human breast cancer cells
[0077] (1) Prepare a uniform cell suspension, inoculate 2 mL per well in a 6-well plate, and culture overnight.
[0078] (2) After the cells adhered to the wall, the old culture medium was discarded and rinsed with PBS. A control group and an experimental group were set up separately. The experimental group was IHFM, and three different gradients were set (1 mg / mL and 2 mg / mL were set as the intermediate concentrations for triple-negative breast cancer cell lines and breast cancer cell lines, respectively). The culture was continued for 48 hours, with 3 replicates in each group, and the experiment was repeated 3 times.
[0079] (3) After the culture is completed, the cells are placed under an inverted microscope and photographed to record their morphology.
[0080] Example 3
[0081] (1) IHFM inhibits breast cancer cell growth in a concentration- and time-dependent manner
[0082] After treating MDA-MB-231, MDA-MB-468 and MCF-7 cells with different concentrations of IHFM (0.125, 0.25, 0.5, 1, 2, 4 mg / mL) for 24 hours, the survival rate of breast cancer cells decreased with the increase of concentration. Subsequently, the effect of IHFM action time (24h, 48h and 72h) on the proliferation activity of breast cancer cells was further investigated. The results are as follows: Figure 1 As shown ( Figure 1 A shows the effect of IHFM on the viability of MDA-MB-231 cells; B shows the effect of IHFM on the viability of MDA-MB-468 cells; C shows the effect of IHFM on the viability of MCF-7 cells; compared with the control group (24h), * P<0.05, ** P<0.01; compared with the control group (48h), # P<0.05, ## P<0.01; compared with the control group (72h), & P<0.05, && P<0.01. ), with the extension of IHFM treatment time, IHFM inhibited breast cancer cell proliferation in a time-dependent manner. According to the experimental results of MTT method, SPSS25.0 software was used to calculate the 50% inhibitory concentration (IC) of IHFM on MDA-MB-231, MDA-MB-468 and MCF-7 cells. 50 ). As shown in Table 1, when MDA-MB-231 cells were treated for 24h, 48h and 72h, IC 50The values were 1.14 mg / mL, 0.98 mg / mL, and 0.10 mg / mL, respectively; for MDA-MB-468 cells, they were 1.54 mg / mL, 0.63 mg / mL, and 0.26 mg / mL, respectively; after corresponding treatment, the IC 50 The values were 4.50mg / mL, 3.26mg / mL and 0.82mg / mL respectively. Studies have shown that IHFM inhibits the proliferation activity of breast cancer cells in a time-dependent and gradient-dependent manner. Combined with the results in Tables 2-4, IHFM is more sensitive to triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468. Previous studies have found that IHFM has no toxic effect on normal liver cells LO2 when the treatment concentration is 0.05-5mg / mL, indicating that IHFM can inhibit the activity of cancer cells, but does not have obvious toxic effects on normal cells.
[0083] Table 1 IC of breast cancer cells under different treatment conditions 50 Table 2-4 The IC 50 value of breast cancer cells under different treatments
[0084]
[0085] (2) IHFM inhibits the proliferation of breast cancer cells
[0086] Through MTT test and IC 50 The analysis results of the values showed that IHFM had different inhibitory abilities on different breast cancer cells. The proliferation ability of a single cell can be detected by plate cloning assay. Therefore, the plate cloning assay was used to detect the inhibitory effect of IHFM on the colony aggregation ability and proliferation ability of three breast cancer cells, MDA-MB-231, MDA-MB-468 and MCF-7 (see Figure 2 , Figure 2 A is the effect of IHFM on the colony formation of MDA-MB-231 cells; B is the effect of IHFM on the colony formation of MDA-MB-468 cells; C is the effect of IHFM on the colony formation of MCF-7 cells; D is the plate clone formation rate of MDA-MB-231 cells; E is the plate clone formation rate of MDA-MB-468 cells; F is the plate clone formation rate of MCF-7 cells; Compared with the control group, * P<0.05, **P<0.01. ). When the concentration of IHFM was 0.25 mg / mL, IHFM could significantly inhibit the proliferation of three breast cancer cells compared with the control group. At this concentration, the colony formation rates of IHFM on MDA-MB-231, MDA-MB-468 and MCF-7 cells were 38.58±1.37%, 10.98±0.55% and 84.24±4.16%, respectively. When the concentration of IHFM was 4 mg / mL, the clone formation rates of IHFM on MDA-MB-231, MDA-MB-468 and MCF-7 cells were 0.41±0.08%, 0.98±0.28% and 1.62±0.11%, respectively. With the increase of treatment concentration, the plate clone formation rate decreased, showing a concentration gradient dependence. The results showed that IHFM has the effect of inhibiting the proliferation of breast cancer cells, and the results are similar to those obtained by MTT method.
[0087] (3) IHFM inhibits the migration of breast cancer cells
[0088] Cell migration is one of the main reasons for tumor metastasis and high recurrence rate. The cell scratch healing experiment was used to investigate the effect of IHFM on the migration ability of three breast cancer cells: MDA-MB-231, MDA-MB-468 and MCF-7. Figure 3 It can be seen that Figure 3 A. Effect of IHFM on the scratch healing ability of MDA-MB-231 cells; B. Effect of IHFM on the scratch healing ability of MDA-MB-468 cells; C. Effect of IHFM on the scratch healing ability of MCF-7 cells; D. Migration rate of MDA-MB-231 cells; E. Migration rate of MDA-MB-468 cells; F. Migration rate of MCF-7 cells; Compared with the control group, * P<0.05, **P<0.01. ), compared with 0h, the scratches in the control group and the IHFM treatment group were healed, indicating that the three breast cancer cells migrated; the cell scratch healing ability of the control group was the strongest, and the cell migration rate of the different concentrations of IHFM treatment groups showed concentration gradient dependence, and the higher the drug treatment concentration, the lower the cell migration rate. After calculation, in MDA-MB-231 cells, when the IHFM treatment concentration was 0, 0.5, 1 and 2 mg / mL, the cell migration rate was 71.09±0.21%, 42.14±1.37%, 39.28±0.46% and 29.72±1.31%, respectively, and there were extremely significant differences between each IHFM treatment group and the control group. In MDA-MB-468 cells, when the IHFM treatment concentration was 0, 0.5, 1 and 2 mg / mL, the migration rates of cells after 48 h were 65.06±1.13%, 23.35±1.36%, 19.81±0.39% and 8.31±1.69%, respectively, which were significantly different from those in the control group. In MCF-7 cells, the cell migration rate of the control group after 48 h was 72.19±1.29%, when the IHFM concentration was 0.25 mg / mL, the cell migration rate was 27.06±0.92%; when the IHFM treatment concentration was 2 mg / mL, the cell migration rate was 8.67±0.20%; when the IHFM treatment concentration was 4 mg / mL, the cell migration rate was 5.85±0.01%, which was significantly different from the control group. At the same concentration, IHFM had a weaker inhibitory effect on the migration ability of MDA-MB-231 cells, which may be due to the strong motility of MDA-MB-231 cells. The results showed that IHFM could significantly inhibit the migration ability of the three breast cancer cells, and the inhibitory ability was positively correlated with the concentration of IHFM.
[0089] (4) IHFM induces programmed cell death in breast cancer cells
[0090] Through the MTT method and plate cloning experiment, it was found that IHFM can significantly inhibit the viability and proliferation of three breast cancer cells, MDA-MB-231, MDA-MB-468 and MCF-7. Subsequently, the three breast cancer cells were pretreated with apoptosis inhibitor DEVD, pyroptosis inhibitor YVAD, ferroptosis inhibitor Fer-1 and necrosis inhibitor Nec-1 to further explore the cell death mode induced by IHFM. Figure 4 As shown ( Figure 4 A. MDA-MB-231 cell survival rate; B. MDA-MB-468 cell survival rate; C. MCF-7 cell survival rate; breast cancer cells were pretreated with YVAD, DEVD, Fer-1 and Nec-1 for 8 hours, and then incubated with IHFM for 48 hours. The MTT method was used to detect the changes in cell survival rate. Compared with the control group,* P<0.05, ** P<0.01; compared with the IHFM group, # P<0.05, ## P<0.01. ), the study found that in the IHFM combined with inhibitor group, the cell viability reduction of three breast cancer cells by IHFM was significantly eliminated by YVAD and DEVD (P
[0091] <0.01), while in MDA-MB-231 and MDA-MB-468 triple-negative breast cancer cells, Fer-1 and Nec-1 had no significant effect on the cell viability reduced by IHFM. The results showed that apoptosis and pyroptosis were involved in IHFM-induced cell death. In MCF-7 cells, Fer-1 significantly weakened the inhibitory effect of IHFM on MCF-7 cell proliferation, but Nec-1 had no significant effect on cell viability, indicating that ferroptosis may also be involved in IHFM-induced MCF-7 cell death. In summary, IHFM may inhibit breast cancer cell proliferation through two death modes: apoptosis and pyroptosis.
[0092] 3.5 Effect of IHFM on breast cancer cell morphology
[0093] To determine whether apoptosis and pyroptosis are involved in the anti-breast cancer effect of IHFM, three breast cancer cells, MDA-MB-231, MDA-MB-468 and MCF-7, were treated with IHFM, and the cell morphology was observed under an inverted microscope. The morphology of apoptosis is characterized by cell shrinkage and rounding, and a decrease in volume, but without damage to the cell membrane; while the morphology of pyroptosis is characterized by gradual flattening of the cells, damage to the cell membrane to form membrane pores, and vacuolar cytoplasm to release the contents. Figure 5 show( Figure 5 The red arrows indicate pyroptotic cells, and the white arrows indicate apoptotic cells. ), after 48 hours, the number of cells in the control group was large and the morphology was regular; the number of cells in the IHFM-treated group decreased, and there were some wrinkled and rounded apoptotic cells (marked by white arrows in the figure). Some of them were wrinkled, rounded and fell off, and some cells were flat, swollen, formed a large number of vesicles, and the cell membrane ruptured, which was a typical pyroptotic morphology. The results showed that IHFM can regulate the apoptosis-inducing and pyroptosis-inducing effects of three breast cancer cells. The higher the treatment concentration, the fewer the number of cells, and the more the number of apoptotic and pyroptotic cells.
[0094] (6) IHFM induces apoptosis of breast cancer cells
[0095] To further determine the involvement of apoptosis in the inhibitory effect of IHFM on the growth of three breast cancer cells, Hoechst 33258 staining was used to detect the nuclear morphology, and Western blot was used to detect the effect of IHFM on the expression of apoptotic proteins (e.g. Figure 6 As shown: A. Effect of IHFM on apoptosis of MDA-MB-231 cells and apoptosis rate; B. Effect of IHFM on apoptosis of MDA-MB-231 cells and apoptosis rate; C. Effect of IHFM on apoptosis of MDA-MB-231 cells and apoptosis rate; D. Western blot; E. Effect of IHFM on the expression of apoptotic proteins in MDA-MB-231 cells; F. Effect of IHFM on the expression of apoptotic proteins in MDA-MB-468 cells; Note: Compared with the control group, * P<0.05, ** P<0.01. ). Hoechst 33258 dye is a fluorescent dye that can penetrate the cell membrane and has low toxicity to cells. Figure 6 (As shown in AC), the morphology of MDA-MB-231, MDA-MB-468 and MCF-7 cells in the control group was intact, and the blue fluorescence of the cell nucleus was weak; after treatment with different concentrations of IHFM, the cell membrane permeability changed, and the Hoechst 33258 dye could penetrate. Some cell nuclei were seen to shrink and the chromatin was condensed, showing bright blue, which were apoptotic cells, and the number of apoptotic cells was proportional to the IHFM treatment concentration. When the IHFM concentration was 4 mg / mL, the apoptotic rates of MDA-MB-231, MDA-MB-468 and MCF-7 cells were 73.05±1.81%, 68.43±4.80% and 54.85±5.01%, respectively.
[0096] The results of apoptosis protein expression level detection showed that Figure 6 (DF) Compared with the control group, IHFM down-regulated the expression level of Bcl-2 protein in MDA-MB-231 and MDA-MB-468 cells, and the expression of Cleaved Caspase 3 protein was up-regulated by 2.85 and 1.62 times, respectively, which were statistically significant (P<0.01). The above results showed that IHFM induced apoptosis of three breast cancer cells.
[0097] (7) IHFM induces pyroptosis of breast cancer cells
[0098] (7-1) Effects of IHFM on the Membrane Integrity of Breast Cancer Cells
[0099] To determine whether IHFM could damage the cell membrane of breast cancer cells, the lactate dehydrogenase (LDH) release assay kit was used to detect the release of LDH in the cell supernatant (see Figure 7 ,in Figure 7 A is the amount of LDH released from the supernatant of MDA-MB-231 cells; B is the amount of LDH released from the supernatant of MDA-MB-468 cells; C is the amount of LDH released from the supernatant of MCF-7 cells; D is the Western blot band diagram; E is the expression level of Caspase 1 and GSDMD-NT proteins in MDA-MB-231 cells; F is the expression level of Caspase 1 and GSDMD-NT proteins in MDA-MB-468 cells; Compared with the control group, * P<0.05, ** P<0.01; compared with the IHFM group, # P<0.05, ## P<0.01. ). (The results are as follows Figure 7 AC), the LDH release in the supernatant of MDA-MB-231, MDA-MB-468 and MCF-7 cells in the control group was 8.07±0.27%, 7.00±0.28% and 9.84±0.32%, respectively. The LDH release was low, indicating that the membrane integrity of breast cancer cells in the control group was not destroyed; compared with the control group, the LDH release in the supernatant of MDA-MB-231, MDA-MB-468 and MCF-7 cells in the IHFM group was significantly increased, which was 5.84 times, 4.27 times and 2.90 times higher than that in the control group, respectively, indicating that IHFM would destroy the integrity of the cell membrane and release cell contents. In the three breast cancer cells induced by IHFM, after pretreatment with YVAD and DEVD, the LDH in the cell supernatant decreased to varying degrees compared with the IHFM group, indicating that in addition to apoptosis, pyroptosis may also be involved in IHFM-induced cell death to inhibit the proliferation of breast cancer cells. The above results preliminarily indicate that IHFM can destroy the integrity of breast cancer cell membranes, leading to the release of cell contents (such as LDH) into the supernatant. It also further indicates that IHFM may depend on Caspase 1 and Caspase 3 to induce pyroptosis of breast cancer cells.
[0100] (7-2) IHFM promotes the expression of pyroptosis pathway proteins Caspase 1 and GSDMD-NT. The expression levels of pyroptosis proteins were further tested. The results (e.g. Figure 7DF), after IHFM treatment, the expression of Caspase1 protein in MDA-MB-231 and MDA-MB-468 cells increased, and the protein expression level of GSDMD-NT was also significantly upregulated, indicating that IHFM may activate Caspase1 protein, and activated Caspase 1 cuts GSDMD to produce GSDMD-NT protein. GSDMD-NT has a pore-punching function, destroys cell integrity, and releases cell contents (such as LDH, IL-18, IL-1β, etc.), which corresponds to the increase in LDH release in the cell supernatant in the above-mentioned IHFM group. The results show that IHFM destroys the integrity of the cell membrane of MDA-MB-231, MDA-MB-468 and MCF-7, and cell pyroptosis is involved in the anti-breast cancer effect of IHFM.
[0101] The above experiments show that IHFM can significantly inhibit the viability of three breast cancer cells, MCF-7, MDA-MB-231 and MDA-MB-468, in a time- and dose-dependent manner. 50 The results showed that IHFM was more sensitive to triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468. Breast cancer is highly invasive and has a poor prognosis. The scratch healing experiment found that IHFM can significantly inhibit the migration ability of three breast cancer cells, suggesting that IHFM has the potential to be developed as an anti-breast cancer drug.
[0102] Finally, it should be noted that the above is only 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, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for preparing melanin from Porites pubescens, characterized in that: S1, collecting the fruiting bodies of the tricholoma, drying and crushing them, and then filtering them through a sieve; S2, weigh the powder and add NaOH solution, and place it in ultrasound for full reaction; S3, centrifuging the solution in step 2 in a centrifuge and collecting the supernatant; S4, adding an acidic solution to the supernatant collected in S3 until the pH reaches 1.3-1.7, and heating in a water bath for 8-11 hours; S5, after centrifugation again, the precipitate is collected; S6, washing the precipitate with distilled water until the pH value is in the middle, and then adding chloroform, ethyl acetate, anhydrous ethanol, 70%-75% ethanol and distilled water in sequence for purification; S7, dissolving the precipitate with NaOH solution, adjusting the pH value to neutral with HCl solution, collecting the supernatant by centrifugation, and finally dialysis with running water and freeze-drying to obtain the purified melanin sample of the fruiting body of the tricholoma.
2. The method for preparing the melanin of Pleurotus eryngii according to claim 1, characterized in that: In S1, the fruiting bodies of the Pleurotus eryngii are dried, crushed, and then sieved through a sieve with 80 meshes.
3. The method for preparing the melanin of Pleurotus eryngii according to claim 1, characterized in that: In S2, calculated by weight fraction ratio, the weight of the powder added to every 30 parts of NaOH solution is 0.9-1.1 parts, wherein the mass concentration of NaOH is 1.5 mol / L.
4. The method for preparing the melanin of Porites pubescens according to claim 1, characterized in that: In S2, the reaction time in ultrasound is 50-65 minutes.
5. The method for preparing the melanin of Porites pubescens according to claim 1, characterized in that: The speed of the centrifuge in S3 is 10000 rpm / min, and the centrifugation time is 4-6 minutes.
6. The method for preparing the melanin of Pleurotus eryngii according to claim 1, characterized in that: The acidic solution added to S4 is hydrochloric acid solution.
7. The method for preparing the melanin of Pleurotus eryngii according to claim 1, characterized in that: The mass concentration of the NaOH solution added to S7 is 0.1 mol / L, and the mass concentration of the hydrochloric acid solution is 0.1 mol / L.
8. The method for preparing the melanin of Porites pubescens according to claim 1, characterized in that: The duration of running water dialysis is 46-50 hours.
9. The use of the melanin of Porites pubescens in a drug for treating breast cancer according to claim 1, characterized in that: The tricholoma melanin is applied to breast cancer.
10. The use of the melanin of Porites pubescens according to claim 9 in a drug for treating breast cancer, characterized in that: The melanin of Leptopus pubescens is used for human triple-negative breast cancer cells MDA-MB-231, human triple-negative breast cancer cells MDA-MB-468 and human breast cancer cells MCF-7; it is used for inhibiting the growth, proliferation and migration capabilities of human breast cancer cells; and inducing apoptosis and pyroptosis of human breast cancer cells.