Application of catechin derivative WM-21 in preparation of neuroprotection and anti-neuroinflammation drugs

Abstract

The invention discloses an application of a catechin derivative WM-21 in preparation of neuroprotection and anti-neuroinflammation drugs, and belongs to the technical field of biomedicines.The catechin derivative WM-21 is (2R, 3S)-2-(3, 4-dihydroxyphenyl)-6-((E)-styrene) chroane-3, 5, 7-triol (C22H19O6), and the chemical structural formula of the catechin derivative WM-21 is shown in the formula (I); the catechin derivative WM-21 provided by the invention has a neuroprotective effect on a plurality of nerve cells, has certain application potential in preparation of neuroprotective drugs, and is beneficial to prevention or alleviation of nervous system diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a catechin derivative WM-21 in the preparation of neuroprotective and anti-neuroinflammatory drugs. Background Art

[0002] As the population ages, the incidence of neurological diseases increases year by year. Neuronal damage and neuroinflammation are common pathological features of many neurological diseases, including Alzheimer's disease, Parkinson's disease, depression, etc. Neuroinflammation can lead to neuronal damage and death, thereby aggravating the symptoms of neurological diseases. Therefore, finding effective neuroprotective and anti-neuroinflammatory drugs is of great clinical significance.

[0003] Catechins are natural polyphenol compounds and the main active ingredients of tea. They have strong antioxidant effects. Existing literature reports that catechin compounds have multiple effects such as anti-inflammatory, antiviral, antibacterial, anti-tumor, and neuroprotective effects. They can be used for the prevention and treatment of obesity, hyperlipidemia, hepatitis, infection, tumors, and Alzheimer's disease. However, since natural catechin compounds have the disadvantages of low in vivo pharmacological activity and bioavailability, artificial catechin derivatives obtained by structural modification of catechins using medicinal chemistry methods have certain advantages.

[0004] (2R,3S)-2-(3,4-dihydroxyphenyl)-6-((E)-phenylethylene)chromene-3,5,7-triol (C 22 H 19 O6)(WM-21) is one of the derivatives of catechins. So far, there are no reports on the application of WM-21 in neuroprotection and anti-neuroinflammatory drugs. Experiments have shown that WM-21 can protect neurons from cell damage induced by amyloid or hydrogen peroxide, increase the vitality of neurons, inhibit the expression of apoptotic proteins, and improve the antioxidant capacity of neurons, thereby playing a neuroprotective role; at the same time, WM-21 can reduce the secretion of pro-inflammatory cytokines by microglia, inhibit the expression of pro-inflammatory related proteins and the abnormal activation of microglia, thereby inhibiting neuroinflammation. In summary, the neuroprotective and anti-neuroinflammatory activities of WM-21 will be used in drug preparation, and will make important contributions to the fields of drug development and disease prevention and treatment of nervous system diseases. Summary of the invention

[0005] The present invention provides the use of a catechin derivative WM-21 in the preparation of neuroprotective and anti-neuroinflammatory drugs, which can effectively solve the above problems.

[0006] The specific technical solutions of the present invention are as follows:

[0007] Application of catechin derivative WM-21 in the preparation of neuroprotective drugs, wherein the catechin derivative WM-21 is (2R, 3S)-2-(3,4-dihydroxyphenyl)-6-((E)-phenylethylene)chromene-3,5,7-triol (C 22 H 19 O6), whose chemical structure is shown in formula (I).

[0008]

[0009] Human neuroblastoma cells SH-SY5Y were selected to establish H2O2-induced SH-SY5Y oxidative damage model and amyloid protein (Aβ)-induced SH-SY5Y apoptosis model, respectively. Different concentrations of WM-21 (0.5μM, 1μM, 2μM) were used for 24h. The changes in cell density and cell morphology of SH-SY5Y cells under the action of WM-21 were observed under a microscope. The CCK8 experiment was used to detect the effect of WM-21 on SH-SY5Y cell viability. The flow cytometer was used to detect the effect of WM-21 on SH-SY5Y cell apoptosis after Annexin V-FITC / PI staining. The protein immunoblotting experiment was used to detect the effect of WM-21 on the expression of apoptosis-related proteins Bcl-2 and Bax in SH-SY5Y cells. The immunofluorescence staining experiment was used to detect the effect of WM-21 on the expression of Cytochrome C.

[0010] The application of the catechin derivative WM-21 in the preparation of anti-neuroinflammatory drugs. Mouse microglial cells BV-2 were selected to establish a lipopolysaccharide (LPS)-induced BV-2 overactivation model, and different concentrations of WM-21 (1μM, 2μM, 4μM) were used for 24 hours. The enzyme-linked immunosorbent assay was used to detect the content of IL-1β, IL-6, and TNF-α in the supernatant of BV-2 cells, and the protein immunoblotting experiment was used to detect the expression of iNOS and COX-2 in BV-2 cells; the immunofluorescence staining experiment was used to detect the expression of Iba1 in BV-2 cells.

[0011] The catechin derivative WM-21 is used in the preparation of drugs or health products for improving the antioxidant capacity of various neural cells. Human neuroblastoma cells SH-SY5Y were selected to establish a H2O2-induced SH-SY5Y oxidative damage model, and different concentrations of WM-21 (0.5μM, 1μM, 2μM) were used for 24 hours. The ROS level of SH-SY5Y cells was detected by a reactive oxygen species detection kit, the GPX level of SH-SY5Y cells was detected by a total glutathione peroxidase detection kit, the MDA level of SH-SY5Y cells was detected by a lipid oxidation (MDA) detection kit, and the SOD activity of SH-SY5Y cells was detected by a total SOD activity detection kit.

[0012] The drug of the present invention further comprises a pharmaceutically acceptable carrier. The drug administration routes include: oral administration, injection, respiratory administration and skin administration.

[0013] Beneficial effects of the present invention:

[0014] The catechin derivative WM-21 of the present invention has a neuroprotective effect on a variety of nerve cells, and has a certain application potential in the preparation of neuroprotective drugs, which is helpful to prevent or alleviate nervous system diseases.

[0015] WM-21 has anti-neuroinflammatory effects, which is one of the important characteristics of many nervous system diseases. Inhibiting neuroinflammation can reduce early nerve damage and enhance the immune function of microglia. It will have certain application potential in the preparation of anti-neuroinflammatory drugs, which will help prevent and treat diseases related to chronic neuroinflammation. In addition, WM-21 also has the effect of significantly improving the antioxidant capacity of various nerve cells, so it can be used to prepare drugs that have neuroprotective and anti-neuroinflammatory effects, as well as improve the antioxidant capacity of various nerve cells. The present invention may have potential therapeutic effects on a variety of nervous system diseases, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and other diseases, and provide a basis for further development of new neuroprotective and anti-neuroinflammatory drugs, and provide more options for the treatment of clinical nervous system diseases. Future research of the present invention includes further exploration of the mechanism of action of WM-21, optimization of drug formulations, and clinical trials to determine its effectiveness and safety in clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The morphology of SH-SY5Y cells induced by H2O2 was observed under a microscope;

[0017] Figure 2 The morphology of SH-SY5Y cells induced by amyloid protein Aβ42 was observed under a microscope;

[0018] Figure 3 CCK8 assay was used to detect the viability of SH-SY5Y cells induced by H2O2;

[0019] Figure 4 CCK8 assay was used to detect the viability of SH-SY5Y cells induced by amyloid protein Aβ42;

[0020] Figure 5 Annexin V / PI staining was used to detect apoptosis of SH-SY5Y cells induced by H2O2 oxidative damage;

[0021] Figure 6 Annexin V / PI staining was used to detect apoptosis and necrosis of SH-SY5Y cells induced by Aβ42 oxidative damage;

[0022] Figure 7 Western blotting was used to detect the expression of Bcl-2 and Bax in SH-SY5Y cells induced by H2O2;

[0023] Figure 8 Western blotting was used to detect the expression of Bcl-2 and Bax in SH-SY5Y cells induced by Aβ42;

[0024] Fig. 9 Immunofluorescence was used to detect the expression of Cytochrome C in SH-SY5Y cells induced by H2O2;

[0025] Fig.10 Immunofluorescence was used to detect the expression of Cytochrome C in SH-SY5Y cells induced by Aβ42;

[0026] Fig.11 Enzyme-linked immunosorbent assay was used to detect the contents of IL-1β, IL-6, and TNF-α in the supernatant of BV-2 cells;

[0027] Fig.12 Immunofluorescence was used to detect the expression of Iba1 in BV-2 cells;

[0028] Fig.13 Western blotting was used to detect the expression of iNOS and COX-2 in BV-2 cells;

[0029] Fig.14 The ROS level of SH-SY5Y cells was detected by reactive oxygen species detection kit;

[0030] Fig.15 The total glutathione peroxidase detection kit was used to detect the GPx level in SH-SY5Y cells;

[0031] Fig.16 The lipid oxidation (MDA) detection kit was used to detect the MDA level of SH-SY5Y cells;

[0032] Fig.17 The total SOD activity detection kit was used to detect the SOD activity of SH-SY5Y cells. DETAILED DESCRIPTION

[0033] Example 1

[0034] Combined with cell morphology observation, CCK-8 experiment, WB and Annexin V-FITC / PI fluorescence staining experiments, the neuroprotective effect of WM-21 on various nerve cells was tested.

[0035] Principle method:

[0036] 1.1 Detection of neuroprotective effects of WM-21

[0037] 1.1.1 SH-SY5Y cell culture

[0038] Human neuroblastoma cell SH-SY5Y is a commonly used cell line for evaluating the neuroprotective effect of compounds in vitro. Its culture conditions are: 5% CO2, 37°C; 90% basic DMEM (Dulbecco's Modified Eagle Medium) medium + 10% FBS (Fetal Bovine Serum) + 1% double antibody (Penicillin-Streptomycin Solution). When the cell density reaches 90%, it is necessary to pass the cells, and the passaging ratio is 1:4. Wash the cells once with PBS (Phosphate Buffered Saline, phosphate buffered saline), add 1mL of trypsin containing EDTA (Ethylenediaminetetraacetic acid) to the culture flask to digest the cells, and add 1mL of complete culture medium to stop digestion after standing at 37°C for 5min. Use a pipette to gently blow the cells to make them fall off, transfer the cell suspension to a centrifuge tube, and centrifuge at 500rpm for 3min to obtain cell clumps. Discard the supernatant, add complete culture medium, gently blow and mix the cell clumps, and according to the subculture ratio, take an appropriate amount of cell suspension, transfer it to the culture bottle pre-added with fresh complete culture medium, and gently blow and mix it. Place it in a 37℃ constant temperature cell culture incubator. Observe the growth of cells on the second day. If the culture medium turns yellow or there are too many non-adherent cells, replace it with fresh culture medium.

[0039] 1.1.2 Construction of neuronal cell injury model

[0040] H2O2-induced SH-SY5Y cell injury: Take 30% H2O2 solution, dilute it to 1M with sterile water, and store it at 4°C away from light for later use. When using, dilute the 1M H2O2 solution with culture medium to a final concentration of 500μM, and give it to SH-SY5Y cells for 24h to establish the H2O2-induced SH-SY5Y cell injury model.

[0041] Aβ42-induced SH-SY5Y cell injury: Take 1 mg of Aβ42 lyophilized powder, add 0.22 mL of hexafluoroisopropanol (HFIP) and mix thoroughly, then ultrasonicate at room temperature for 1 hour until it is completely dissolved and the solution is transparent and clear to obtain Aβ42 monomer solution. Dispense into centrifuge tubes, 100 μL per tube, place the tube in a clean bench overnight with the lid open, until HFIP completely evaporates, and a clear transparent film can be seen at the bottom of the tube. Seal the tube and store it in a -80°C refrigerator for later use. When used, take the Aβ42 monomer solution, dissolve it thoroughly with 20 μL of DMSO, then add 80 μL of phenol red-free DMEM medium, shake and mix thoroughly for 10 minutes, and incubate at 37°C for 24 hours to obtain Aβ42 oligomers with a concentration of 1 mM. The Aβ42-induced SH-SY5Y cell injury model was established by diluting the Aβ42-induced SH-SY5Y cell injury model with culture medium to 20 μM and administering the Aβ42-induced SH-SY5Y cell injury model to SH-SY5Y cells.

[0042] 1.1.3WM-21 configuration and administration method

[0043] Weigh an appropriate amount of WM-21 powder, dissolve it with DMSO, prepare a 0.1M stock solution, and store it at 4°C for later use. When used, dilute the WM-21 stock solution with culture medium to a final concentration of 0.5μM, 1μM, and 2μM, respectively. For H2O2-induced SH-SY5Y cells, the administration method is: ① blank group (no treatment); ② model group, 500μM H2O2; ③ low concentration group, 500μM H2O2+0.5μM WM-21; ④ medium concentration group, 500μM H2O2+1μM WM-21; ⑤ high concentration group, 500μM H2O2+2μM WM-21; ⑥ control group, 500μM H2O2+20μM Catechin; cells in each group were treated according to the corresponding conditions, and the corresponding indicators were detected after 24h. For Aβ42-induced SH-SY5Y cells, the administration method was: ① blank group (no treatment); ② model group, 20μM Aβ42; ③ low concentration group, 20μM Aβ42+0.5μM WM-21; ④ medium concentration group, 20μM Aβ42+1μM WM-21; ⑤ high concentration group, 20μM Aβ42+2μM WM-21; ⑥ control group, 20μAβ42+20μM Catechin; cells in each group were treated according to the corresponding conditions, and the corresponding indicators were detected after 48 hours.

[0044] 1.1.4CCK8 cell viability assay

[0045] SH-SY5Y cells were inoculated into 96-well cell culture plates and given different concentrations of WM-21 after grouping. CCK8 working solution was prepared in a dark place, with basal DMEM medium:CCK8=9:1. The cell culture supernatant was discarded, and CCK8 working solution was added, 100μL / well, and incubated in a constant temperature incubator at 37°C for 10min. The absorbance OD value was measured at a wavelength of 450nm using a microplate reader. Since WST-8 in the CCK-8 working solution can be catalyzed by mitochondrial dehydrogenase to produce a water-soluble orange-yellow formazan product, the product has an obvious absorption peak at a wavelength of 450nm. In addition, the activity of mitochondrial dehydrogenase is proportional to the cell viability / number, that is, the amount of formazan product generated is also linearly related to the cell viability / number. Therefore, the viability / number of cells in each group can be calculated based on the OD value, reflecting the effect of WM-21 on the viability of SH-SY5Y cells.

[0046] 1.1.5 Annexin V / PI staining experiment

[0047] SH-SY5Y cells were inoculated into 6-well cell culture plates and given different concentrations of WM-21 after grouping. After the administration, the cell culture plates were placed in a plate centrifuge at 4°C, 500rpm, and centrifuged for 5min. The cell culture medium was removed and pre-cooled PBS was added for washing once. Annexin V-FITC apoptosis detection kit (Biyuntian, C1062L) was used to add 195μL Annexin V-FITC binding solution to each well; 5μL Annexin V-FITC was added and gently mixed; then 10μL propidium iodide (PI) staining solution was added and gently mixed; incubated at room temperature in the dark for 20min, and then placed in an ice bath. Observed and photographed under a fluorescence microscope, Annexin V-FITC is green fluorescence, and PI is red fluorescence. Among them, cells stained only with green fluorescence are apoptotic cells, cells stained with both green and red fluorescence are necrotic cells, and cells not stained with fluorescence are normal cells. According to the staining of cells in each group, the effect of WM-21 on SH-SY5Y cell apoptosis can be reflected.

[0048] 1.1.6 Western blotting experiment

[0049] This experiment was used to detect the effect of WM-21 on the expression of related proteins in SH-SY5Y cells. ① Extract total cell protein: collect cells from each group into a centrifuge tube, centrifuge for 5 minutes (2500rpm, 4℃), discard the supernatant, resuspend the cells with pre-cooled PBS, centrifuge again, discard the supernatant, and obtain cell pellets; add cell lysis buffer and mix thoroughly, lyse on ice for 20 minutes, centrifuge for 10 minutes (12000rpm, 4℃), take the supernatant, and obtain the total protein sample; use the BCA method to determine the protein concentration; take the same mass of protein (20-40μg) from each group, calculate the protein volume to be loaded according to the protein concentration, level with pure water, add 5× loading buffer, mix well, denature in a 95℃ metal bath for 10 minutes, and store at -20℃ for later use. ② Protein electrophoresis and transfer: Add the protein samples to be tested to the loading holes of the precast gel in turn, and add 2 μL of protein Marker to the left side of the blank group; set the concentrated gel constant voltage (80V) for about 20 minutes, and the separation gel constant voltage (120V) for about 120 minutes; take out the separation gel and place it in the semi-dry transfer buffer, and make a transfer "sandwich" in the order of thick filter paper, PVDF membrane, separation gel, and thick filter paper from bottom to top; set the transfer parameters to constant current (1.3A), and perform rapid semi-dry transfer for 7 minutes. ③ Antibody incubation: Remove the PVDF membrane, block it with 1% BSA at room temperature for 1 hour, place it on a decolorizing shaker and wash it 3 times with PBS (5 minutes / time); use 1% BSA to dilute the antibodies Bcl-2 (CST, #3498) and Bax (CST, #2772) to a specific multiple, immerse the PVDF membrane completely in the antibody working solution, incubate it at 4°C overnight, and wash it 3 times with PBST (5 minutes / time); add the secondary antibody, incubate it at room temperature for 1 hour, wash it 3 times with PBST (10 minutes / time), and immerse the PVDF membrane in PBS buffer. ④ Chemiluminescence imaging and protein quantification: Prepare chemiluminescent solution (liquid A: liquid B = 1:1) in a dark place, evenly drip it onto the PVDF membrane, and use a chemiluminescence instrument (Tanon 5200Multi) to expose and collect protein band images; use ImageJ / FIJI software to perform grayscale analysis on protein bands, homogenize the grayscale value of the internal reference protein, compare the grayscale values ​​of the target protein in each group, and use Graphpad Prism software to perform statistical processing on the quantitative data and compare the differences between groups.

[0050] 1.1.7 Immunofluorescence experiments

[0051] SH-SY5Y cells were inoculated into 35mm glass-bottom confocal culture dishes and given different concentrations of WM-21 after grouping. After the administration, the cell culture supernatant was discarded, pre-cooled PBS (1mL / dish) was added, placed on a decolorizing shaker, and washed 3 times at a low speed (ice bath, 5min / time). 0.8mL of 4% paraformaldehyde was added to each dish, fixed on ice for 25min, the paraformaldehyde was removed, pre-cooled PBS was added, placed on a decolorizing shaker, and washed 3 times at a low speed (ice bath, 5min / time). 3% BSA (200μL / dish) was added for blocking, and residual BSA was removed at room temperature after 1.5-2h. Cytochrome C antibody (Proteintech, #10993-1-AP) was diluted 500 times with 3% BSA solution, evenly added to the bottom of the dish (100μL / dish), and incubated overnight at 4°C. Place on a decolorizing shaker and wash 3 times at low speed with precooled PBS (8 min / time). Use 3% BSA to dilute the fluorescent rabbit secondary antibody (SAB, L3016) 800 times, evenly add it to the bottom of the dish (100 μL / dish), incubate at room temperature in the dark for 1 hour, place on a decolorizing shaker, and wash 3 times at low speed with PBS (8 min / time). Add 50 μL of DAPI-containing sealing agent (Biyuntian, C2003S) to each well and stand at room temperature in the dark for 30 minutes. Place the culture dish under a laser confocal microscope for photography.

[0052] 1.1.8 Statistical analysis

[0053] All data were obtained from 3 independent experiments. The experimental data were expressed as mean ± standard deviation. The experimental data were analyzed and processed using GraphPad Prism 8.0 software. The inter-group comparison was evaluated using T-Test (Student's t test). # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01 indicates that the difference is statistically significant.

[0054] in conclusion:

[0055] 1. Cell morphological observations showed that WM-21 could inhibit nerve cell damage induced by different stimuli and had a neuroprotective effect, which was stronger than that of catechins at the same concentration.

[0056] 1.1 As Figure 1 As shown, WM-21 can improve the morphological damage of SH-SY5Y cells induced by hydrogen peroxide (H2O2). Compared with the 20μM Catechin group, the improvement effect of WM-21 on cell morphological damage is more obvious;

[0057] 1.2 As Figure 2 As shown, WM-21 can improve the morphological damage of SH-SY5Y cells induced by amyloid protein (Aβ), and the improvement effect of WM-21 on cell morphological damage is more obvious compared with the 20μM catechin group.

[0058] 2. The CCK-8 cell viability detection experiment showed that WM-21 can improve the cell viability of neurons induced by different stimuli, and its promoting effect on the viability of neurons is stronger than that of catechins at the same concentration.

[0059] 2.1 As Figure 3 As shown, WM-21 can enhance the viability of SH-SY5Y cells induced by H2O2, and the promoting effect of WM-21 on cell viability is more obvious compared with the 20 μM catechin group;

[0060] 2.2 As Figure 4 As shown, WM-21 can enhance the viability of SH-SY5Y cells induced by Aβ, and the promoting effect of WM-21 on cell viability is more obvious compared with the 20 μM catechin group.

[0061] 3. Annexin V-FITC / PI fluorescence staining experiments showed that WM-21 can inhibit neuronal apoptosis induced by different stimuli.

[0062] 3.1 As Figure 5 As shown, WM-21 inhibited H2O2-induced apoptosis in SH-SY5Y cells;

[0063] 3.2 As Figure 6 As shown, WM-21 inhibited Aβ-induced apoptosis in SH-SY5Y cells.

[0064] 4. Western blotting (WB) showed that WM-21 could inhibit the expression of pro-apoptotic proteins in neurons induced by different stimuli and promote the expression of anti-apoptotic proteins.

[0065] 4.1 As Figure 7 As shown, WM-21 inhibited the expression of pro-apoptotic protein Bax and promoted the expression of anti-apoptotic protein Bcl-2 in SH-SY5Y cells induced by H2O2. Compared with the 20 μM catechin group, WM-21 had a stronger inhibitory effect on Bax expression and a stronger promoting effect on Bcl-2 expression.

[0066] 4.2 As Figure 8 As shown, WM-21 inhibited the expression of pro-apoptotic protein Bax and promoted the expression of anti-apoptotic protein Bcl-2 in SH-SY5Y cells induced by Aβ. Compared with the 20 μM catechin group, WM-21 had a stronger inhibitory effect on Bax expression and a stronger promoting effect on Bcl-2 expression.

[0067] 5. Immunofluorescence experiments (IF) showed that WM-21 could inhibit the expression of multiple pro-apoptotic proteins in nerve cells.

[0068] 5.1 As Fig. 9 As shown, WM-21 inhibited the expression of pro-apoptotic protein Cytochrome C in SH-SY5Y cells induced by H2O2;

[0069] 5.2 As Fig.10 As shown, WM-21 inhibited the expression of pro-apoptotic protein Cytochrome C in SH-SY5Y cells induced by Aβ.

[0070] In summary, combined with the results of cell morphology observation, CCK-8 experiment, WB and Annexin V-FITC / PI fluorescence staining, it is proved that WM-21 has a neuroprotective effect on a variety of nerve cells.

[0071] Example 2

[0072] Combined with ELISA, WB and immunofluorescence staining experiments, WM-21 was tested to have anti-neuroinflammatory effects.

[0073] Principle method:

[0074] 2.1 Detection of the inhibitory effect of WM-21 on neuroinflammation

[0075] 2.1.1 BV-2 cell culture

[0076] Mouse microglial BV-2 is a commonly used cell line for constructing microglial inflammatory response models and evaluating the anti-neuroinflammatory effects of compounds in vitro. The culture conditions are: 5% CO2, 37°C; 90% basic DMEM (Dulbecco's Modified Eagle Medium) medium + 10% FBS (Fetal Bovine Serum) + 1% double antibody (Penicillin-Streptomycin Solution). When the cell density reaches 90%, it is necessary to pass the cells, and the passaging ratio is 1:4. Wash the cells once with PBS (Phosphate Buffered Saline, phosphate buffered saline), add 1mL of trypsin containing EDTA (Ethylenediaminetetraacetic acid) to the culture flask to digest the cells, and add 1mL of complete culture medium to stop digestion after standing at 37°C for 5min. Use a pipette to gently blow the cells to make them fall off, transfer the cell suspension to a centrifuge tube, and centrifuge at 500rpm for 3min to obtain cell clumps. Discard the supernatant, add complete culture medium, gently blow and mix the cell clumps, and according to the subculture ratio, take an appropriate amount of cell suspension, transfer it to the culture bottle pre-added with fresh complete culture medium, and gently blow and mix it. Place it in a 37℃ constant temperature cell culture incubator. Observe the growth of cells on the second day. If the culture medium turns yellow or there are too many non-adherent cells, replace it with fresh culture medium.

[0077] 2.1.2 Construction of microglial activation model

[0078] The lipopolysaccharide (LPS) induction method was used to establish a microglial overactivation model. LPS (0.5 μg / mL) was prepared and administered to BV-2 cells. After culturing for 24 hours, BV-2 cells could secrete a large number of pro-inflammatory factors, including IL-1β, IL-6, TNF-α, etc., thus successfully establishing an LPS-induced BV-2 cell activation model.

[0079] 2.1.3WM-21 configuration and administration method

[0080] Weigh an appropriate amount of WM-21 powder, dissolve it in DMSO, prepare a 0.1M stock solution, and store it at 4°C for later use. When used, dilute the WM-21 stock solution with culture medium to a final concentration of 1μM, 2μM, and 4μM, respectively. For LPS-induced BV-2 cells, the administration method is: ① blank group (no treatment); ② model group, 0.5μg / mL LPS; ③ low concentration group, 0.5μg / mL LPS+1μM WM-21; ④ medium concentration group, 0.5μg / mL LPS+2μM WM-21; ⑤ high concentration group, 0.5μg / mL LPS+4μM WM-21; ⑥ control group, 0.5μg / mL LPS+40μM Catechin (catechin); cells were treated according to the corresponding conditions in each group, and neuroinflammation-related indicators were detected after 24h.

[0081] 2.1.4 Enzyme-linked immunosorbent assay (ELISA)

[0082] This experiment was used to detect the effect of WM-21 on the secretion of inflammatory factors in BV-2 cells induced by LPS. Mouse interleukin 1β (IL-1β) ELISA detection kit (Jianglai Biology, JL18442), mouse interleukin 6 (IL-6) ELISA detection kit (Jianglai Biology, JL20268) and mouse tumor necrosis factor α (TNF-α) ELISA detection kit (Jianglai Biology, JL10484) were used respectively. BV-2 cells were inoculated into 6 cm cell culture dishes and given different concentrations of WM-21 after grouping. After the administration, the cell culture supernatant was collected (1000g, centrifuged for 20min). According to the instructions of the kit, standard products, biotinylated detection antibody working solution, enzyme conjugate working solution and washing solution were prepared, and the kit was prepared and equilibrated at room temperature for 10min. Add sample: add 100μL of sample or standard of different concentrations to the corresponding wells, add 100μL of universal diluent to the blank well, cover with sealing film and incubate at 37℃ for 30min. Add biotinylated antibody: remove the ELISA plate, discard the liquid, add 100μL of biotinylated antibody working solution directly to each well, cover with sealing film and incubate at 37℃ for 30min. Wash the plate: discard the liquid, add 300μL of washing solution to each well, let stand for 1min, shake off the washing solution, pat dry on absorbent paper, and repeat 3 times. Add 100μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37℃ for 30min, discard the liquid, and wash the plate 5 times. Add 90μL of substrate to each well, cover with sealing film, incubate at 37℃ in the dark for 5-10min, remove the ELISA plate, add 50μL of stop solution directly to each well, and immediately measure the OD value of each well at a wavelength of 450nm. According to the standard curve, the content of inflammatory factors (IL-1β, IL-6 and TNF-α) in each well sample was calculated.

[0083] 2.1.5 Immunofluorescence experiments

[0084] BV-2 cells were inoculated into 35mm glass-bottom confocal culture dishes and given different concentrations of WM-21 after grouping. After the administration, the cell culture supernatant was discarded, pre-cooled PBS (1mL / dish) was added, placed on a decolorizing shaker, and washed 3 times at a low speed (ice bath, 5min / time). 0.8mL of 4% paraformaldehyde was added to each dish, fixed on ice for 25min, paraformaldehyde was removed, pre-cooled PBS was added, placed on a decolorizing shaker, and washed 3 times at a low speed (ice bath, 5min / time). 3% BSA (200μL / dish) was added for blocking, and residual BSA was removed at room temperature after 1.5-2h. Iba1 antibody (CST, #17198S) was diluted 500 times with 3% BSA solution, evenly added to the bottom of the dish (100μL / dish), incubated overnight at 4°C, placed on a decolorizing shaker, and washed 3 times at a low speed with PBS (8min / time). Use 3% BSA solution to dilute fluorescent rabbit secondary antibody (SAB, L3016) 800 times, evenly add to the bottom of the dish (100 μL / dish), incubate at room temperature in the dark for 1 hour, place on a decolorization shaker, and wash 3 times at a low speed with PBS (8 min / time). Add 50 μL of DAPI-containing mounting medium (Biyuntian Biotechnology Co., Ltd., anti-fluorescence quenching mounting solution (containing DAPI), item number: C2003S) to each well and keep it at room temperature in the dark for 30 minutes. The culture was placed under a laser confocal microscope for photography.

[0085] 2.1.6 Protein immunoblotting

[0086] This experiment was used to detect the effect of WM-21 on the expression of inflammation-related proteins in BV-2 cells. ① Extract total cell protein: collect cells from each group into a centrifuge tube, centrifuge for 5 minutes (2500rpm, 4℃), discard the supernatant, resuspend the cells with pre-cooled PBS, centrifuge again, discard the supernatant, and obtain cell pellets; add cell lysis buffer and mix thoroughly, lyse on ice for 20 minutes, 12000rpm, 4℃, centrifuge for 10 minutes, take the supernatant, and obtain the total protein sample; use the BCA method to determine the protein concentration; take the same mass of protein (20-40μg) from each group, calculate the protein volume to be loaded according to the protein concentration, level with pure water, add 5× loading buffer, mix well, denature in a 95℃ metal bath for 10 minutes, and store at -20℃ for later use. ② Protein electrophoresis and transfer: Add the protein samples to be tested to the loading holes of the precast gel in turn, and add 2 μL of protein Marker to the left side of the blank group; set the concentrated gel constant voltage (80V) for about 20 minutes, and the separation gel constant voltage (120V) for about 120 minutes; take out the separation gel and place it in the semi-dry transfer buffer, and make a transfer "sandwich" in the order of thick filter paper, PVDF membrane, separation gel, and thick filter paper from bottom to top; set the transfer parameters to constant current (1.3A), and perform rapid semi-dry transfer for 7 minutes. ③ Antibody incubation: Remove the PVDF membrane, block it with 1% BSA at room temperature for 1 hour, place it on a decolorizing shaker and wash it 3 times with PBS (5 minutes / time); use 1% BSA to dilute the antibodies iNOS (CST, #13120S) and COX-2 (CST, #12282S) to a specific multiple, immerse the PVDF membrane completely in the antibody working solution, incubate it at 4°C overnight, and wash it 3 times with PBST (5 minutes / time); add the secondary antibody, incubate it at room temperature for 1 hour, wash it 3 times with PBST (10 minutes / time), and immerse the PVDF membrane in PBS buffer. ④ Chemiluminescence imaging and protein quantification: Prepare chemiluminescent solution (liquid A: liquid B = 1:1) in a dark place, evenly drip it onto the PVDF membrane, and use a chemiluminescence instrument (Tanon 5200Multi) to expose and collect protein band images; use ImageJ / FIJI software to perform grayscale analysis on protein bands, homogenize the grayscale value of the internal reference protein, compare the grayscale values ​​of the target protein in each group, and use Graphpad Prism software to perform statistical processing on the quantitative data and compare the differences between groups.

[0087] 2.1.7 Statistical analysis

[0088] All data were obtained from 3 independent experiments. The experimental data were expressed as mean ± standard deviation. The experimental data were analyzed and processed using GraphPad Prism 8.0 software. The inter-group comparison was evaluated using T-Test (Student's t test). # P<0.05,## P<0.01; compared with the model group, * P<0.05, ** P<0.01 indicates that the difference is statistically significant.

[0089] in conclusion:

[0090] 6. Enzyme-linked immunosorbent assay (ELISA) showed that ( Fig.11 ), WM-21 can inhibit the secretion of proinflammatory cytokines (IL-1β, IL-6, TNF-α) by microglia (BV-2) induced by lipopolysaccharide (LPS), and its inhibitory effect is stronger than that of 40μM Catechin (catechin).

[0091] 7. Immunofluorescence experiment (IF) showed that ( Fig.12 ), WM-21 can inhibit the expression of Iba1 in BV-2 cells induced by LPS and suppress the excessive activation of microglia.

[0092] 8. Protein immunoblotting (WB) showed that ( Fig.13 ), WM-21 could inhibit the expression of pro-inflammatory proteins iNOS and COX-2 in BV-2 cells induced by LPS, and its inhibitory effect was more obvious than that of 40μM catechins.

[0093] In summary, combined with the experimental results of ELISA, WB and immunofluorescence staining, it is proved that WM-21 has anti-neuroinflammatory effect.

[0094] Example 3

[0095] Combined with antioxidant-related index detection experiments such as reactive oxygen species (ROS) content detection, it was tested that WM-21 can significantly improve the antioxidant capacity of nerve cells.

[0096] Principle method:

[0097] 3.1 Antioxidant capacity test of WM-21

[0098] 3.1.1 SH-SY5Y cell culture (same as 1.1.1)

[0099] 3.1.2 Construction of neuronal oxidative damage model

[0100] Take 30% H2O2 solution, dilute it to 1M with sterile water, and store it at 4°C in the dark for later use. When using, dilute the 1M H2O2 solution to a final concentration of 500μM with culture medium, and give it to SH-SY5Y cells for 24h to establish the H2O2-induced SH-SY5Y cell oxidative damage model.

[0101] 3.1.3WM-21 configuration and administration method

[0102] Weigh an appropriate amount of WM-21 powder, dissolve it in DMSO, prepare a 0.1M stock solution, and store it at 4°C for later use. When used, dilute the WM-21 stock solution with culture medium to a final concentration of 0.5μM, 1μM, and 2μM, respectively. SH-SY5Y cells induced by H2O2 were divided into groups and administered: ① blank group (no treatment); ② model group, 500μM H2O2; ③ low concentration group, 500μM H2O2+0.5μM WM-21; ④ medium concentration group, 500μM H2O2+1μM WM-21; ⑤ high concentration group, 500μM H2O2+2μM WM-21; ⑥ control group, 500μM H2O2+20μM Catechin; cells were treated according to the corresponding conditions in each group, and oxidation and antioxidant related indicators were detected after 24h.

[0103] 3.1.4 ROS content detection

[0104] SH-SY5Y cells were inoculated in 96-well plates and administered in groups. After 24 hours, the ROS level of each group of cells was detected using a reactive oxygen species detection kit (Biyuntian, S0033M). The fluorescent probe DCFH-DA working solution (DMEM culture medium: DCFH-DA stock solution = 1000:1) was prepared, the cell culture supernatant in each well of the 96-well plate was discarded, and the DCFH-DA working solution (200μL / well) was added and incubated at 37°C for 20min. The cells were washed 3 times (5min / time) with basic DMEM culture medium, and the absorbance OD value was measured at an excitation wavelength of 488nm and an emission wavelength of 525nm using an ELISA reader, and the ROS content was calculated based on the OD value.

[0105] 3.1.5 GPx activity detection

[0106] Glutathione peroxidase detection kit (Biyuntian, S0056) can detect the activity of glutathione peroxidase (GPx) in cells by ultraviolet colorimetry. GPx can catalyze reduced glutathione (GSH) to produce GSSG, while glutathione reductase (GR) can use NADPH to catalyze GSSG to produce GSH. 340 The activity level of GPx can be calculated by detecting the reduction of NADPH.

[0107] SH-SY5Y cells were inoculated into 6-well plates and given different concentrations of WM-21 after grouping. Cells were collected into 2 mL centrifuge tubes with a cell scraper, the supernatant was discarded, and the cell pellets were resuspended with pre-cooled PBS (1 mL / sample, centrifuged (2500 rpm, 5 min, 4 ° C), the supernatant was discarded, and cell lysis solution (40-50 μL / sample) was added and mixed, lysed on ice for 20 min, centrifuged (12000 rpm, 10 min, 4 ° C), and the supernatant was taken to obtain the sample. The detection buffer (30 μL / well), the sample to be tested (20 μL / well) and the GPx detection working solution (40 μL / well) were added to the sample in turn, mixed, and incubated at room temperature for 15 min. 30 mM peroxide reagent solution (10 μL / well) was added, mixed, and A was immediately measured using an ELISA reader at 25 ° C 340 At this time, the value recorded is 0min, and A is automatically measured every 15s. 340 , obtain data at multiple time points. Calculate the glutathione peroxidase activity in the sample according to the formula provided in the kit instructions.

[0108] 3.1.6 MDA content detection

[0109] The lipid oxidation (MDA) detection kit (Biyuntian, S0131S) was used to detect the MDA content in cells. MDA is a natural product of lipid oxidation in organisms. When free radicals attack cell membrane phospholipids, peroxidation reaction is triggered to generate MDA. Under high temperature and acidic environment, MDA reacts with TBA to generate red MDA-TBA adducts. MDA-TBA adducts have maximum absorption at 535nm, which can be detected by colorimetry to indirectly determine the MDA content and evaluate the degree of oxidative damage of cells. SH-SY5Y cells were inoculated into 6-well cell culture plates and given different concentrations of WM-21 after grouping. After the administration, the cells were collected, the cell pellets were resuspended with pre-cooled PBS (1mL / sample), centrifuged (2500rpm, 5min, 4℃), the supernatant was discarded, and the cell lysis solution (40-50μL / sample) was added to mix, lysed on ice (20min), centrifuged (12000rpm, 10min, 4℃), and the supernatant was taken to obtain the sample. Add the sample to be tested (100 μL / tube) and MDA detection working solution (200 μL / tube) into a 1.5 mL centrifuge tube in sequence, mix thoroughly, heat in 100°C boiling water for 15 min, cool to room temperature in a water bath, centrifuge (1000 rcf, 10 min, room temperature), take the supernatant (200 μL / well) and place it in a 96-well plate, and use an ELISA reader to measure the absorbance at 532 nm.

[0110] 3.1.7 SOD activity detection

[0111] The total SOD activity detection kit (Biyuntian, S0101S) was used to detect the SOD activity in cells. SOD can catalyze the dismutation of superoxide anions to generate hydrogen peroxide and oxygen, and is an important antioxidant enzyme in organisms. Based on the color reaction of WST-8, WST-8 can react with superoxide anions (O2. - ) reaction produces a water-soluble formazan dye. Since SOD can catalyze the dismutation of superoxide anions, this reaction step can be inhibited by SOD. Therefore, the activity of SOD is negatively correlated with the amount of formazan dye generated. Therefore, the enzyme activity of SOD can be calculated by colorimetric analysis of the WST-8 product.

[0112] SH-SY5Y cells were inoculated into 6-well cell culture plates and given different concentrations of WM-21 after grouping. After the administration, the cell culture medium was aspirated, washed once with pre-cooled PBS, SOD sample preparation solution was added, and the cells were blown appropriately to fully lyse the cells. Centrifuged at 4°C, 12000g for 5 minutes, and the supernatant was taken as the sample to be tested. At the same time, the cells were collected and the protein concentration was detected by BCA method. In a 96-well ELISA plate, the sample to be tested (20μL), SOD detection buffer (20μL), WST-8 working solution (160μL), and reaction start working solution (20μL) were added respectively, incubated at 37°C for 30 minutes, and the absorbance was measured at 450nm using a multifunctional ELISA instrument. According to the total SOD activity calculation formula provided in the kit instructions, the SOD enzyme activity in the sample was calculated.

[0113] 3.1.8 Statistical analysis

[0114] All data were obtained from 3 independent experiments. The experimental data were expressed as mean ± standard deviation. The experimental data were analyzed and processed using GraphPad Prism 8.0 software. The inter-group comparison was evaluated using T-Test (Student's t test). # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01 indicates that the difference is statistically significant.

[0115] in conclusion:

[0116] 9. The antioxidant index detection experiment showed that WM-21 can improve the antioxidant capacity of nerve cells SH-SY5Y, and its antioxidant capacity is significantly higher than that of catechins with the same concentration.

[0117] 9.1 Reactive oxygen species (ROS) content detection experiment shows that ( Fig.14), WM-21 can inhibit the ROS level of SH-SY5Y cells induced by H2O2 and Rosup. Compared with the 20μM Catechin group, WM-21 has a stronger inhibitory effect on ROS;

[0118] 9.2 Total glutathione peroxidase content detection experiment shows that ( Fig.15 ), WM-21 can increase the level of GPx in H2O2-induced SH-SY5Y cells, and compared with the 20 μM catechin group, WM-21 has a stronger promoting effect on GPx;

[0119] 9.3 Lipid peroxide (MDA) content detection experiment shows that ( Fig.16 ), WM-21 could inhibit the MDA level in SH-SY5Y cells induced by H2O2, and the inhibitory effect of WM-21 on MDA was stronger than that of the 20 μM catechin group;

[0120] 9.4SOD activity detection experiment shows that ( Fig.17 ), WM-21 could enhance the activity of SOD in SH-SY5Y cells induced by H2O2, while 20μM catechin had no significant effect on the activity of SOD.

[0121] In summary, combined with the results of antioxidant-related index detection experiments such as reactive oxygen species (ROS) content detection, it is proved that WM-21 can significantly improve the antioxidant capacity of nerve cells.

Claims

1. The use of the catechin derivative WM-21 in the preparation of neuroprotective drugs, characterized in that: The catechin derivative WM-21 is (2R, 3S)-2-(3,4-dihydroxyphenyl)-6-((E)-phenylethylene)chromene-3,5,7-triol, and its chemical structure is shown in formula (I):

2. The use of the catechin derivative WM-21 in the preparation of an anti-neuroinflammatory drug, characterized in that: The catechin derivative WM-21 is (2R, 3S)-2-(3,4-dihydroxyphenyl)-6-((E)-phenylethylene)chromene-3,5,7-triol, and its chemical structure is shown in formula (I):

3. The use of the catechin derivative WM-21 in the preparation of a medicine or health product for improving the antioxidant capacity of various nerve cells, characterized in that: The catechin derivative WM-21 is (2R, 3S)-2-(3,4-dihydroxyphenyl)-6-((E)-phenylethylene)chromene-3,5,7-triol, and its chemical structure is shown in formula (I):

4. The use according to any one of claims 1 to 3, characterized in that The medicament further comprises a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that The drug administration routes include: oral administration, injection, respiratory tract administration and skin administration.

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