Application of phycocyanin in inhibition of cell ferroptosis and maintenance of morphological stability of cell mitochondria

Abstract

The invention discloses application of phycocyanin to inhibition of cell ferroptosis and maintenance of morphological stability of cell mitochondria, and belongs to the technical field of biological medicines. Specifically, the invention provides any one of the following applications of phycocyanin: 1, application of phycocyanin in preparation of a medicine for reducing the expression level of a cell ferroptosis marker; 2, application of the compound in preparation of a pharmaceutical composition for inhibiting cell ferroptosis by being combined with a chemical reagent; 3, application in preparation of drugs for maintaining the morphological stability of cell mitochondria; 4, application in preparation of drugs for increasing cell mitochondrial membrane potential; and 5, application in preparation of drugs for improving the cell respiration rate and / or glycolysis. The novel application of phycocyanin in inhibition of cell ferroptosis and maintenance of cell mitochondrial morphological stability is provided, and the application prospect and economic value are good.

Description

Technical Field

[0001] The invention relates to the technical field of crude drug medicine, and in particular to the application of phycocyanin in inhibiting cell ferroptosis and maintaining the stability of cell mitochondrial morphology. Background Art

[0002] Ferroptosis is an iron-dependent, novel type of programmed cell death that is different from apoptosis and autophagy. The main mechanism of ferroptosis is that under the action of divalent iron or esteroxygenase, unsaturated fatty acids highly expressed on the cell membrane are catalyzed to cause lipid peroxidation, thereby inducing cell ferroptosis. Studies have found that ferroptosis is closely related to a variety of diseases such as endocrine, tumors, and metabolism. Inhibiting ferroptosis can effectively reduce the occurrence and development of diseases. Therefore, how to effectively inhibit ferroptosis has become one of the hot issues in current research, with important research value and application prospects.

[0003] Mitochondria are a type of organelles covered by two membranes that exist in most eukaryotic cells. They are structures that produce energy in cells and are the main place for cells to perform aerobic respiration. They are called "power houses". Their diameter is about 0.5 to 1.0 microns. Except for Entamoeba histolytica, Giardia lamblia, and several microsporidia, most eukaryotic cells have mitochondria to a greater or lesser extent, but the mitochondria they possess differ in size, number, and appearance. Mitochondria have their own genetic material and genetic system, but their genome size is limited, and they are semi-autonomous organelles. In addition to providing energy for cells, mitochondria are also involved in processes such as cell differentiation, cell information transmission, and cell apoptosis, and have the ability to regulate cell growth and cell cycle. Therefore, it is of great significance to maintain the stability of the mitochondrial morphology of cells.

[0004] Phycocyanin (C-PC) is a dark blue powder isolated from Spirulina. It is mainly found in cyanobacteria, red algae and cryptophyceae. Phycocyanin is usually divided into C-phycocyanin and R-phycocyanin. The former is mainly found in cyanobacteria, while the latter is mainly found in red algae. Cryptophyceae has both. Its function is to absorb light (orange-yellow) energy and transmit light energy. It is not only a protein, but also an excellent natural food pigment and a good health food. However, there are no reports on the application of phycocyanin in cell ferroptosis and maintaining the stability of cell mitochondrial morphology. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to provide a new use of phycocyanin.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The first aspect of the present invention proposes any of the following uses of phycocyanin:

[0008] (1) Application in the preparation of drugs for increasing the expression level of cell ferroptosis marker proteins;

[0009] (2) Use of the drug in combination with chemical reagents in the preparation of a pharmaceutical composition for inhibiting cell ferroptosis;

[0010] (3) Application in the preparation of drugs for maintaining the stability of cell mitochondrial morphology;

[0011] (4) Application in the preparation of drugs for increasing cell mitochondrial membrane potential;

[0012] (5) Use in the preparation of drugs for improving cellular respiration rate and / or glycolysis.

[0013] Preferably, in (1), the cell ferroptosis marker protein is one or more of GPX4 and xCT.

[0014] (GPX4 and xCT protein expressions decrease when cell ferroptosis occurs, so GPX4 and xCT can be used as important markers to identify cell ferroptosis)

[0015] Preferably, in (2), the chemical reagent is selected from any one of Fer-1 and NAC; the concentration of Fer-1 is 6 to 10 mM, more preferably 8 mM; the concentration of NAC is 3 to 8 mM, more preferably 5 mM.

[0016] Preferably, in (1) to (5), the concentration of the phycocyanin is 2.5 to 50 mM, more preferably 5 or 10 mM.

[0017] The second aspect of the present invention provides a drug for inhibiting cell ferroptosis, wherein the active ingredient of the drug includes phycocyanin and / or the chemical reagent described above.

[0018] Preferably, the drug also includes a pharmaceutically acceptable salt, specifically an organic salt or an inorganic salt.

[0019] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0020] Preferably, the pharmaceutically acceptable excipient is selected from one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

[0021] Preferably, the drug is prepared into a pharmaceutically acceptable dosage form.

[0022] Preferably, the dosage form is tablet, pill, paste or granule.

[0023] The beneficial effects of the present invention are:

[0024] 1. The present invention proposes a new use of phycocyanin in inhibiting cell ferroptosis and maintaining the stability of cell mitochondrial morphology, which has good application prospects and economic value.

[0025] 2. From a mechanistic point of view, the study found that the ferroptosis signaling pathway was abnormally activated in the ovarian tissue of PCOS mice, and the mitochondrial membrane potential in ovarian granulosa cells was significantly reduced. Phycocyanin can reverse these abnormal phenomena. Phycocyanin and its related drug preparations may become a new therapeutic method to inhibit cell ferroptosis and maintain the stability of cell mitochondrial morphology, and have great potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The immunoblot images (left) and corresponding protein quantitative statistical graphs (right) of oxidative stress and ferroptosis marker protein expression in ovarian tissues of all groups of mice in Example 1 of the present invention are shown;

[0027] Figure 2 The immunofluorescence images of ovarian tissue oxidative stress NRF2 and ovarian tissue iron death marker GXP4 protein in all groups of mice in Example 1 of the present invention are shown;

[0028] Figure 3 It is a quantitative statistical graph of the cell viability of human ovarian granulosa cells in all groups of Example 2 of the present invention;

[0029] Figure 4 The immunofluorescence oxidative stress and ferroptosis markers of human ovarian granulosa cells in all groups of Example 2 of the present invention are immunoblot images (left) and corresponding protein quantitative statistical images (right);

[0030] Figure 5 The morphological diagram of mitochondria observed by transmission electron microscopy in human ovarian granulosa cells of all groups in Example 2 of the present invention (left figure) and the quantitative statistical diagram of the corresponding mitochondrial swelling degree and mitochondrial relative area (right figure);

[0031] Figure 6 The statistical graph of the mitochondrial membrane potential of human ovarian granulosa cells in all groups of Example 2 of the present invention using the JC-1 probe (left) and the corresponding statistical graph of the mitochondrial membrane potential red / green fluorescence intensity (right);

[0032] Figure 7 Statistical diagrams of mitochondrial energy metabolism (left) and glycolysis (right) of human ovarian granulosa cells in all groups of Example 2 of the present invention observed by transmission electron microscopy;

[0033] Figure 8These are immunofluorescence images of the oxidative stress marker NRF2 (left) and the ferroptosis marker GPX4 (right) of human ovarian granulosa cells in all groups of Example 2 of the present invention;

[0034] Fig. 9 The statistical results of immunofluorescence intensity of NRF2 (left) and GPX4 (right) of oxidative stress marker and ferroptosis marker in human ovarian granulosa cells of all groups in Example 2 of the present invention are shown;

[0035] Fig.10 The immunoblot images of the ferroptosis protein of human ovarian granulosa cells in all groups of Example 3 of the present invention improved by Fer-1, DFO, NAC, H2O2 or phycocyanin (left A, C, E, G) and the corresponding Image J quantitative statistical graphs (right B, D, F, H);

[0036] In the figure, “*” represents p<0.05; “**” represents p<0.01; “***” represents p<0.001. “#” represents p<0.05; “##” represents p<0.01; “###” represents p<0.001; “&” represents p<0.05; “&&” represents p<0.01; “+” represents presence, and “-” represents absence. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are 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 are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0039] If no specific technology or conditions are specified in the examples, they can be carried out according to the technology or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples are repeated more than three times, and the results are averaged.

[0040] Embodiment 1:

[0041] Construct an animal model to study the effect of phycocyanin (C-PC) on cell ferroptosis marker proteins:

[0042] Construction of PCOS mouse model: 8-week-old ICR mice were used as research subjects (10 mice per group). After 1 week of adaptive feeding, ICR mice were subcutaneously injected with dehydroepiandrosterone (DHEA, 6 mg / 100 g body weight, 0.1 ml / mouse) for 3 consecutive weeks to induce PCOS. PCOS model mice were gavaged with phycocyanin (300 mg / kg, 0.1 ml / mouse, sterile ddH2O saline) for 4 consecutive weeks. The control group mice received the same dose of drug treatment for the same period of time. After treatment, ovarian and orbital blood were collected. At the end of the experiment, the mice were killed for subsequent analysis.

[0043] Western blotting was used to detect the effect of phycocyanin on the expression level of cell ferroptosis marker protein (GPX4 / xCT).

[0044] a. Extract protein from the ovarian tissues of normal mice, PCOS mice, and PCOS mice treated with phycocyanin. Add pre-cooled protein extraction reagent containing inhibitors (a mixture of protease inhibitors, PMSF, and phosphatase inhibitors) to the tissues of the three groups, and lyse on ice for 20 minutes. Centrifuge the lysate at 12,000 rpm in a pre-cooled centrifuge for 15 minutes. Discard the precipitate, take the supernatant (protein of each group) and immediately transfer it to a new centrifuge tube, add Loading Buffer at a ratio of supernatant: Loading Buffer = 4:1, mix thoroughly, boil at 100℃ for 10 minutes, and store at -80℃ for later use.

[0045] b. Western blotting to detect ferroptosis marker proteins (GPX4 / xCT). The protein samples of the three groups were separated on a 10%-15% SDS-polyacrylamide gel electrophoresis gel, then transferred to a PVDF membrane and detected with antibodies against GPX4 / XCT, a related indicator in the ferroptosis pathway.

[0046] The results are as follows Figure 1 and 2 As shown, compared with the normal group of mice, the expression level of GPX4 / xCT, a key indicator of ferroptosis, in the ovaries of PCOS group mice was significantly reduced, while the expression level of ferroptosis markers in the ovarian tissues of PCOS mice treated with phycocyanin (C-PC) was increased (i.e., indicating that cell ferroptosis was significantly improved).

[0047] Embodiment 2:

[0048] Construct a cell model to study the effect of phycocyanin (C-PC) on the maintenance of cell mitochondrial morphology:

[0049] (1) Construction of human ovarian granulosa cell inflammatory injury model in vitro: Human ovarian granulosa cell line (KGN, ATCC, RRID: CVCL_0603) was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM (4.5 g / L D-glucose, HyClone, USA) supplemented with 10% FBS (Gibco, USA), penicillin (100 IU / mL) and streptomycin sulfate (100 μg / mL). The cell culture medium was renewed every 2-3 days, and the culture was cultured in a fully humidified incubator at 37°C and 5% CO2. The cells were pretreated with 5 μM phycocyanin for 2 hours and then treated with 10 μM DHEA for 48 hours. A control group was also set up: the human ovarian granulosa cells in the control group were treated with 10 μM DHEA for 48 hours.

[0050] a. CCK-8 assay for cell viability: CCK-8 assay was used to assess cell viability. Human ovarian granulosa cells (1×10 4 ) were seeded into 96-well plates. The drugs were administered 24 hours before the addition of CCK-8 reagent (10 μL / well). One hour later, the absorbance was measured using a microplate reader and the values ​​of each group were counted. Figure 3 As shown, the cell viability was significantly decreased after DHEA treatment, and the viability of human ovarian granulosa cells was significantly increased after 5 mM phycocyanin treatment.

[0051] b. 1×10 4 Human ovarian granulosa cells KGN were inoculated in a 35mm confocal microscope cell culture dish. When the cells were completely attached to the wall, different concentrations of phycocyanin solution were added. The phycocyanin concentration gradient was set to 2.5mM, 5mM and 10mM. After the cells were pretreated with phycocyanin for 2 hours, they were treated with 10μM DHEA for 48 hours. It included a normal group, a DHEA-induced damage group, and a DHEA group after phycocyanin treatment (i.e., the phycocyanin experimental group). The proteins of each group were extracted separately (the method is the same as above), and the NRF2 protein expression level of each group was checked in turn. Figure 4 As shown, with the increase of phycocyanin concentration, the expression levels of NRF2, HO-1, GPX4, and xCT proteins increased, but the expression level of NRF2 at 5mM phycocyanin was significantly higher than that at 10mM phycocyanin, so 5mM phycocyanin was selected for subsequent experiments.

[0052] (2) Transmission electron microscopy observation of mitochondrial morphology

[0053] Step 1: Fix, slice, and stain the cells for observation under a transmission electron microscope. The cells can be fixed using a fixative such as an aldehyde and then cut into thin slices. Next, the sample is stained to enhance the contrast of the mitochondria, which helps to observe the mitochondrial structure more clearly under a transmission electron microscope.

[0054] Step 2: After the sample is prepared, place it on a transmission electron microscope. The transmission electron microscope uses an electron beam to pass through the sample to produce a high-resolution image of the internal structure of the organelle. Researchers can adjust the parameters of the transmission electron microscope to obtain mitochondrial images at different depths and angles to fully observe its morphological structure.

[0055] Step 3: After obtaining the transmission electron microscope image, data analysis is required. Computer software is used to conduct quantitative and qualitative analysis of the mitochondrial morphology and structure, such as measuring parameters such as the size, shape, and number of mitochondria.

[0056] The results are as follows Figure 5 As shown, the swelling degree of mitochondria in human ovarian granulosa cells increased after DHEA treatment, and the mitochondrial morphology of human ovarian granulosa cells treated with 5mM phycocyanin gradually returned to normal, indicating that phycocyanin has the effect of maintaining the stability of cell mitochondrial morphology.

[0057] (3) JC-1 kit immunofluorescence detection of mitochondrial membrane potential:

[0058] The results are as follows Figure 6 As shown, the cell membrane potential of the phycocyanin-treated group increased significantly, indicating that phycocyanin has the effect of increasing the cell mitochondrial membrane potential.

[0059] (4) Hippocampal energy detection: Detect the cellular respiration rate and glycolysis status of each group.

[0060] The results are as follows Figure 7 As shown, the cell respiration rate and glycolysis were significantly improved in the phycocyanin-treated group.

[0061] (5) Immunofluorescence staining to detect cell oxidative stress and ferroptosis markers: After human granulosa cells were treated with phycocyanin, the examination indicators included: immunofluorescence detection of the expression of oxidative stress indicator NRF2, and immunofluorescence detection of the expression of ferroptosis marker GPX4.

[0062] Step 1: 1×10 4 Human ovarian granulosa cells were inoculated in a 35 mm confocal microscope cell culture dish. After the cells were completely attached to the wall, different concentrations of phycocyanin solution were added. The phycocyanin concentration gradient was set to 5 mM. After phycocyanin pretreatment of cells for 2 hours, they were treated with DHEA for 48 hours. There were normal group, DHEA-induced injury group, and DHEA group after phycocyanin treatment.

[0063] Step 2: After the cell treatment, wash the cells gently with 1×PBS to remove the residual culture medium and dead cells; fix with 4% formaldehyde at room temperature for 20 minutes. After fixation, wash with TBS-Tx to remove the residual fixative; add an appropriate amount of blocking solution to completely cover the coverslip, and block at room temperature for 20 minutes;

[0064] Step 3: Use tweezers to take out the coverslip with fixed cells and place it in a light-proof humidified box with the cell attached side facing up; aspirate the blocking solution, dilute the primary antibody with antibody diluent AbDil-Tx, take 50 μL and drop it on the coverslip, and incubate according to the instructions; after the primary antibody incubation is completed, aspirate and discard the primary antibody, and wash away the unbound primary antibody with TBS-Tx; according to the properties of the primary antibody, select the fluorescent secondary antibody, dilute the secondary antibody with AbDil-Tx at a ratio of 1:250, take 50 μL of the secondary antibody and drop it on the coverslip, incubate at room temperature for 1 hour, and wash away the unbound secondary antibody with TBS-Tx;

[0065] Step 4. After staining with the antibody to be labeled, take 50 μL of 1 μg / mL DAPI to stain the nucleus for 5 minutes. After staining, wash off the unbound DAPI with PBS; wipe the slide clean, add a drop of anti-fluorescence quenching mounting medium, turn the coverslip upside down on the mounting medium, so that the cell surface contacts the mounting medium; select the corresponding fluorescent channel, observe under the microscope, and take representative pictures.

[0066] The results are as follows Figure 8 and Fig. 9 As shown in the figure, compared with the normal group cells, the expression level of NRF2 in the DHEA-induced damage group cells was significantly decreased, and the expression level of ferroptosis marker protein GPX4 was also significantly decreased. However, the expression levels of NRF2 and ferroptosis marker protein GPX4 in the DHEA group treated with phycocyanin (C-PC) (i.e., phycocyanin experimental group) increased, indicating that phycocyanin inhibits cell ferroptosis.

[0067] Embodiment 3:

[0068] Further research on the inhibition of ferroptosis by phycocyanin (C-PC):

[0069] Human ovarian granulosa cells were seeded in a 6-well plate, and when the cells were completely attached, 8mM Fer-1 (ferroptosis inhibitor), 8mM DFO (iron chelator), 5mM NAC (N-acetyl-L-cysteine), 200μM hydrogen peroxide (H2O2) were used to pretreat the human ovarian granulosa cells for 2 hours, phycocyanin (5mM) was used to pretreat the cells for 2 hours, and then DHEA (5μM) was used to treat the cells for 1 hour. After 0.25% trypsin digestion, protein immunoblotting was performed. The results are shown in FIG. Fig.10 shown.

[0070] a. Phycocyanin and Fer-1 treated cells:

[0071] Through Western blotting, we found that Fer-1+DHEA treatment can effectively increase the expression level of ferroptosis marker proteins (GPX4 and xCT) (i.e., indicating the inhibition of cell ferroptosis). Furthermore, when phycocyanin is used together with Fer-1, the expression level of ferroptosis marker proteins is further increased (i.e., further inhibiting cell ferroptosis). Therefore, when phycocyanin is used in combination with Fer-1, it has a significant effect on inhibiting cell ferroptosis. (e.g., Fig.10 (As shown in A and B)

[0072] b. Phycocyanin and DFO treated cells:

[0073] Through protein immunoblotting, we found that after DFO+DHEA treatment, the expression level of ferroptosis marker protein GPX4 was reduced, but the expression level of ferroptosis marker protein xCT was increased. Therefore, DFO treatment alone cannot inhibit or promote cell ferroptosis. Furthermore, when phycocyanin was used in combination with DFO, the expression level of ferroptosis marker protein GPX4 was increased, but the expression level of xCT was significantly reduced. Therefore, the combination of phycocyanin and DFO cannot inhibit or promote cell ferroptosis. (such as Fig.10 C, D)

[0074] c. Phycocyanin and NAC treated cells:

[0075] Through protein immunoblotting, we found that phycocyanin and NAC alone + DHEA treatment can effectively increase the expression level of iron death marker proteins (GPX4 and xCT) (indicating that both can inhibit cell iron death). Furthermore, when phycocyanin was combined with NAC, the expression level of iron death marker proteins (GPX4 and xCT) in human ovarian granulosa cells treated with DHEA was further increased (i.e., it significantly inhibited cell iron death), indicating that phycocyanin has a significant effect in inhibiting cell iron death when used in combination with NAC. (e.g. Fig.10 (as shown in E and F)

[0076] d. Phycocyanin and hydrogen peroxide treatment of cells:

[0077] By Western blotting, we found that hydrogen peroxide + DHEA treatment reduced the expression levels of ferroptosis marker proteins (GPX4 and xCT), indicating the promotion of cell ferroptosis. Fig.10 G, H)

[0078] In the above animal and cell experiments, the data are expressed as mean ± standard deviation. Data difference analysis was performed by GraphPadPrism v6.02 and t-test. Animal experiments: between PCOS group and normal group mice, between PCOS group mice treated with phycocyanin and PCOS mice. Cell experiments: between DHEA induced group and normal control group, between DHEA induced group and DHEA induced group after phycocyanin pretreatment.

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Any of the following applications of phycocyanin: (1) Application in the preparation of drugs for increasing the expression level of cell ferroptosis marker proteins; (2) Use of the drug in combination with chemical reagents in the preparation of a pharmaceutical composition for inhibiting cell ferroptosis; (3) Application in the preparation of drugs for maintaining the stability of cell mitochondrial morphology; (4) Application in the preparation of drugs for increasing cell mitochondrial membrane potential; (5) Use in the preparation of drugs for improving cellular respiration rate and / or glycolysis.

2. The use according to claim 1, characterized in that: In (1), the cell ferroptosis marker protein is one or more of GPX4 and xCT.

3. The use according to claim 1, characterized in that: In (2), the chemical reagent is selected from any one of Fer-1 and NAC; the concentration of Fer-1 is 6 to 10 mM, more preferably 8 mM; the concentration of NAC is 3 to 8 mM, more preferably 5 mM.

4. The use according to claim 1, characterized in that: In (1) to (5), the concentration of the phycocyanin is 2.5 to 50 mM, more preferably 5 or 10 mM.

5. A drug for inhibiting cell ferroptosis, characterized in that: Its effective ingredients include phycocyanin and / or the chemical reagent described in claim 3.

6. The drug according to claim 5, characterized in that The drug also includes pharmaceutically acceptable salts, specifically organic salts or inorganic salts.

7. The drug according to claim 5, characterized in that The medicine also includes pharmaceutically acceptable excipients.

8. The drug according to claim 7, characterized in that The pharmaceutically acceptable pharmaceutical excipient is selected from one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

9. The drug according to claim 5, characterized in that The drug is prepared into a pharmaceutically acceptable dosage form.

10. The drug according to claim 9, characterized in that The dosage form is tablet, pill, paste or granule.

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