Application of lycium barbarum polysaccharide in preparation of medicine for inhibiting methylamphetamine-induced nerve injury

Abstract

The invention discloses application of lycium barbarum polysaccharide in preparation of a medicine for inhibiting methylamphetamine-induced nerve injury, and relates to the technical field of biological medicines. It is found and proved that lycium barbarum polysaccharide can inhibit methylamphetamine-induced nerve injury, and the effect is remarkable. From the aspect of cell survival, the survival rate of the microglial cells pretreated by the lycium barbarum polysaccharide after METH stimulation is obviously improved. In the molecular mechanism, the lycium barbarum polysaccharide can effectively lower the protein expression level of the METH-induced pyroptosis marker, the content of inflammatory factors IL-1beta and IL-18 can be reduced, and the release of lactic dehydrogenase is reduced. The result shows that the lycium barbarum polysaccharide can alleviate inflammatory response by inhibiting NLRP3 / ASC pathway mediated microglial cell pyroptosis, thereby inhibiting METH-induced nerve injury. The invention provides a new direction for developing METH neurotoxicity intervention medicines.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of Lycium barbarum polysaccharide in the preparation of drugs that inhibit methamphetamine-induced nerve damage. Background Technology

[0002] Methamphetamine (METH) neurotoxicity can lead to microglia death and exacerbate inflammation, with pyroptosis involved in METH-induced microglia damage. Currently, specific inhibitors and intervention strategies targeting METH-induced microglia pyroptosis have limited effectiveness. Pyroptosis is a form of programmed cell death activated by the NLRP3 inflammasome and is closely related to the release of inflammatory factors such as IL-1β and IL-18. The NLRP3 / ASC pathway is a key pathway regulating pyroptosis, but its role in METH-induced microglia pyroptosis remains unclear.

[0003] Lycium barbarum polysaccharides (LBP) have been reported to have antioxidant and anti-inflammatory activities, but their application in inhibiting methamphetamine-induced nerve damage has not been reported. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Lycium barbarum polysaccharides in the preparation of drugs that inhibit methamphetamine-induced nerve damage, thereby addressing the problems existing in the prior art. This invention has found that Lycium barbarum polysaccharides can inhibit METH-induced nerve damage, providing a new direction for the development of drugs to intervene in METH neurotoxicity.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of Lycium barbarum polysaccharide in the preparation of drugs that inhibit METH-induced nerve damage.

[0007] Furthermore, the wolfberry polysaccharide inhibits METH-induced nerve damage by suppressing microglia pyroptosis.

[0008] Furthermore, the wolfberry polysaccharide inhibits METH-induced nerve damage by reducing neuroinflammatory responses.

[0009] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0010] Furthermore, the excipients include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, or colorants.

[0011] Furthermore, the dosage form of the drug is powder, tablet, granule, capsule, pill, or oral liquid.

[0012] The present invention discloses the following technical effects:

[0013] This invention discovers and confirms that Lycium barbarum polysaccharides (LBP) can significantly inhibit METH-induced neuronal damage. From a cell survival perspective, BV2 microglia pretreated with 300 μg / mL and 600 μg / mL LBP showed significantly increased survival rates after METH stimulation. At the molecular level, LBP effectively downregulates the protein expression levels of METH-induced pyroptosis markers (NLRP3, ASC, Caspase1), and also reduces the levels of inflammatory cytokines IL-1β and IL-18, and decreases lactate dehydrogenase (LDH) release. This indicates that LBP can inhibit METH-induced neuronal damage by suppressing NLRP3 / ASC-mediated microglia pyroptosis and alleviating inflammatory responses. This invention provides a new direction for developing drugs to intervene in METH neurotoxicity. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 The figure shows the results of detecting the survival rate of BV2 microglia in each group using the CCK-8 assay; where * indicates P<0.05 compared with the con group;

[0016] Figure 2 This is a graph showing the results of detecting the survival rate of BV2 microglia in each group using the TUNEL assay.

[0017] Figure 3 Western blot plots and relative protein expression statistics of pyroptosis markers ASC, NLRP3, Caspase1, and inflammatory factors IL-1β and IL-18 are shown. In the figure, A represents the Western blot plot; B and F represent the relative protein expression statistics of NLRP3, ASC, Caspase1, IL-1β, and IL-18, respectively. * indicates P < 0.05 compared to the con group; # indicates P < 0.05 compared to the methamphetamine group.

[0018] Figure 4 The graph shows the detection results of lactate dehydrogenase (LDH) activity in BV2 microglia in each group; where * indicates P<0.05 compared with the con group; # indicates P<0.05 compared with the methamphetamine group;

[0019] Figure 5 The image shows the ELISA results of IL-1β levels in cell supernatants of each group; where * indicates P<0.05 compared to the con group; # indicates P<0.05 compared to the methamphetamine group.

[0020] Figure 6 The image shows the ELISA results of the inflammatory factor IL-18 content in the cell supernatant of each group; where * indicates P<0.05 compared with the con group; # indicates P<0.05 compared with the methamphetamine group. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] The following examples use

[0027] Example 1

[0028] 1. Experimental Materials

[0029] BV-2 microglia were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. They were seeded in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin (Gifco product) and cultured in a constant temperature incubator at 37°C with 5% carbon dioxide.

[0030] The wolfberry polysaccharide was purchased from Solarbio, product number SP9311.

[0031] 2. Experimental Methods

[0032] 2.1 Modeling and Experimental Grouping

[0033] To observe the effects of different concentrations of Lycium barbarum polysaccharides on BV2 microglia, BV-2 cells were randomly divided into 5 groups:

[0034] Control group (con group): BV2 microglia were cultured normally without any special treatment (the solvent control was used instead of wolfberry polysaccharide or METH treatment).

[0035] Methamphetamine group (METH group): BV2 microglia were stimulated for 24 h by adding 1.0 mM METH to the culture medium.

[0036] METH+LBP (100 μg / mL) group: 100 μg / mL of Lycium barbarum polysaccharide was added to the culture medium, and 1.0 mM of METH was added to the culture medium after 2 h to stimulate BV2 microglia for 24 h.

[0037] METH+LBP (300 μg / mL) group: 300 μg / mL of Lycium barbarum polysaccharide was added to the culture medium, and 1.0 mM of METH was added to the culture medium after 2 h to stimulate BV2 microglia for 24 h.

[0038] METH+LBP (600 μg / mL) group: 600 μg / mL of Lycium barbarum polysaccharide was added to the culture medium, and 1.0 mM of METH was added to the culture medium after 2 h to stimulate BV2 microglia for 24 h.

[0039] 2.2 CCK-8 assay for cell viability

[0040] Cell viability in each group was detected using the CCK-8 assay, as follows:

[0041] 1) Remove the culture medium from each group of BV2 microglia, wash twice with PBS, place in a 96-well plate, and add 10 μL of LCK-8 and 100 μL of serum-free DMEM medium to each well.

[0042] 2) Place the 96-well plate in a 37°C incubator and incubate in the dark for 2 hours. Measure the OD value at a wavelength of 450 nm using an ELISA reader.

[0043] 3) Calculate the cell viability using the formula:

[0044] Cell viability % = (Measured OD value - Control OD value) / (Standard OD value - Blank OD value) × 100%.

[0045] 2.3 TUNEL staining method for detecting cell death

[0046] The TUNEL fluorescence system (EMD Millipore, Bielerica, USA) was used to visualize cellular DNA degradation in each group (for detecting dead cells). Cells were counterstained with 4',6-diamidinyl-2-phenylindole (DAPI) and analyzed using a fluorescence microscope. The METH+LBP group was selected using METH+LBP (600 μg / mL).

[0047] Subsequently, LBP, mcc950, and METH+mcc950 groups were added for indicator testing:

[0048] LBP group: BV2 microglia were cultured for 24 h with 600 μg / mL of Lycium barbarum polysaccharide added to the culture medium.

[0049] mcc950 group: 5 μM mcc950 (a specific inhibitor of NLRP3) was added to the culture medium and BV2 microglia were cultured for 24 h.

[0050] METH+mcc950 group: 5 μM mcc950 was added to the culture medium, and 1.0 mM METH was added to the culture medium 2 h later to stimulate BV2 microglia for 24 h.

[0051] 2.4 Biomarkers of pyroptosis: Detection of the protein composition of NOD-like receptor thermoprotein domain-associated protein 3 (NLRP3), apoptosis-associated speckle-like protein (ASC), cysteine-aspartate specific protease-1 (Caspase 1), and inflammatory factors interleukin-1β (IL-1β) and interleukin-18 (IL-18).

[0052] After treatment, total cellular protein was extracted from each group of cells, followed by protein denaturation and electrophoretic separation. Western blot was then used to detect pyroptosis markers ASC, NLRP3, Caspase1, and inflammatory factors IL-1β and IL-18 proteins. The METH+LBP group used the METH+LBP (600 μg / mL) method.

[0053] Methods for extracting total cellular protein:

[0054] 1) Pour out the culture medium and invert the bottle onto absorbent paper to allow the paper to absorb the culture medium.

[0055] 2) Add 3 mL of pre-cooled PBS (0.01 M pH 7.2-7.3) at 4°C to each flask of cells; lay flat and gently shake for 1 min to wash the cells, then discard the washing solution; repeat the above operation twice, washing the cells a total of three times to remove the culture medium; after discarding the PBS, place the culture flask on ice.

[0056] 3) Add 10 μL of benzyl sulfonyl fluoride (PMSF) (100 mM) to 1 mL of lysis buffer, shake well and place on ice.

[0057] 4) Add 400 μL of lysis buffer containing PMSF to each flask of cells and lyse on ice for 30 min. To ensure complete cell lysis, the culture flask should be shaken frequently.

[0058] 5) After lysis, use a clean scraper to scrape the cells onto one side of the culture flask, and then use a pipette to transfer the cell debris and lysis buffer into a 1.5 mL centrifuge tube.

[0059] 6) Centrifuge at 12000 rpm for 5 min at 4℃.

[0060] 7) Transfer the supernatant after centrifugation to 0.5 mL centrifuge tubes and store at -20°C.

[0061] Protein denaturation method: Mix the extracted protein supernatant with 5× protein loading buffer, incubate in a metal bath for 5 minutes, check if the sample is clear, then pipette to check if it is viscous. If it is not clear or viscous, the boiling time needs to be extended. After denaturation, cool to room temperature and then store at -20℃.

[0062] Electrophoretic separation method:

[0063] 1) Preparation of electrophoretic gel

[0064] After washing and drying the glass plate, fix it on the gel casting machine and begin preparing the separating gel (preparation system shown in Table 1). Pour the separating gel into the gaps in the glass plate to an appropriate height, then cover the separating gel with double-distilled water until the gel is completely polymerized. Pour out the double-distilled water and blot dry with filter paper. Then add the stacking gel (preparation system shown in Table 2) to an appropriate height and insert the comb teeth. Remove the comb teeth after the stacking gel has completely polymerized.

[0065] Table 1 Separating Gel Formulation System

[0066]

[0067] Table 2. Concentrated Gel Formulation System

[0068]

[0069] 2) Electrophoretic separation

[0070] Fix the prepared gel onto the electrophoresis tank and pour the electrophoresis buffer into the reservoir. Use a micropipette to add the prepared protein samples and protein standards to the sample wells. After adding the samples, perform electrophoresis at a constant voltage of 80V until the bromophenol blue indicator forms a line at the junction of the stacking and separating gels. Then, switch to a constant voltage of 120V until the bromophenol blue reaches the bottom of the gel. This process takes approximately 1.5 hours.

[0071] Western blot detection method:

[0072] 1) Transfer: Remove the gel and cut off the target band. Rinse with distilled water. Cut a PVDF membrane and filter paper to the same size as the PAGE gel. Soak the PVDF membrane in methanol for a few seconds, then soak it and the filter paper in the electrotransfer buffer. Arrange the plates in the following order: black plate - fiber pad - filter paper - gel - PVDF membrane - filter paper - fiber pad - white plate. Clamp the plates and place them in the transfer apparatus, with the black plate facing the black negative electrode. Fill the transfer tank with electrotransfer buffer and begin the transfer. Transfer conditions are shown in Table 3.

[0073] Table 3 Transfer conditions

[0074]

[0075] 2) Sealing: Take out the electro-converted PVDF membrane, wash it once with TBST, and seal it with 5% skim milk / TBST on a shaker at room temperature for 1 hour.

[0076] 3) Washing: Rinse with TBST for 2 minutes.

[0077] 4) Binding of primary antibody to target protein: Dilute primary antibody with 1% BSA / PBST (1:1000), seal the membrane with a hybridization bag, and incubate overnight at 4°C.

[0078] 5) Washing: Wash 3 times with PBST, 10 min each time.

[0079] 6) Secondary and primary antibody incubation: Place the PVDF membrane in horseradish peroxidase-labeled secondary antibody (1:5000) diluted with 5% skim milk / PBST and incubate on a shaker at room temperature for 1 h.

[0080] 7) Washing: Wash 3 times with PBST, 10 min each time.

[0081] 8) Color development: Drop the color developing solution onto the PVDF film after it has been drained of moisture, allow it to react for 1 minute, and use a luminometer to automatically detect the exposure time to obtain the developed image.

[0082] 2.5 Detection of lactate dehydrogenase (LDH) activity

[0083] After treatment of each group of cells, the cell culture supernatant was collected, and the activity of lactate dehydrogenase (LDH) was detected using a microplate reader at a wavelength of 450 nm. LDH activity was calculated according to the formula:

[0084] LDH activity (U / L) = (Measured OD value - Background blank control OD value) / (Standard tube OD value - Standard blank tube OD value) × Standard concentration × Sample dilution factor.

[0085] The METH+LBP group was selected from the METH+LBP (600 μg / mL) group.

[0086] 2.6 Detection of inflammatory factors

[0087] The inflammatory factors IL-1β and IL-18 in each group were detected by ELISA. The METH+LBP group was selected using the METH+LBP (600 μg / mL) method.

[0088] 3. Experimental Results

[0089] 3.1 Cell viability test results

[0090] The cell viability rates of the con group, the METH group, and the METH+LBP group after pretreatment with 100 μg / mL, 300 μg / mL, and 600 μg / mL LBP for 2 h are as follows: Figure 1 As shown in the figure. The results showed that LBP had a protective effect on BV2 cells under METH stimulation in a concentration-dependent manner. Within the concentration range of 100 μg / mL, LBP had no significant effect on cell viability. At 300 μg / mL and 600 μg / mL, the cell viability of the METH+LBP group was significantly increased compared with the METH group (P<0.05).

[0091] 3.2 TUNEL staining results for cell death detection

[0092] After treatment, cell death was detected by TUNEL staining. Dead cell nuclei showed green fluorescence, while surviving cell nuclei did not show green fluorescence and appeared blue (for cell counting). TUNEL-positive cell nuclei (green) were identified by randomly counting ten fields of view in the slide. Figure 2 As shown, compared with the con group, the apoptosis rate of the METH group was significantly increased; compared with the METH group, the cell death rate of the METH+LBP group was significantly decreased.

[0093] This indicates that pretreatment with LBP can reduce the mortality rate of METH-exposed BV2 microglia.

[0094] 3.3 Protein detection results of pyroptosis markers ASC, NLRP3, Caspase1, and inflammatory factors IL-1β and IL-18

[0095] After treatment of each group of cells, the protein levels of pyroptosis markers ASC, NLRP3, Caspase1, and inflammatory factors IL-1β and IL-18 were detected by Western blotting. The results are shown in [Figure number missing]. Figure 3 Among them, mcc950 is a specific inhibitor of NLRP3.

[0096] The results showed that, compared with the con group, the protein levels of pyroptosis markers ASC, NLRP3, Caspase1 and inflammatory factors IL-1β and IL-18 were significantly increased in the METH group (P < 0.05); compared with the METH group, the protein levels of pyroptosis markers ASC, NLRP3, Caspase1 and inflammatory factors IL-1β and IL-18 were significantly decreased in the METH+LBP group (P < 0.05).

[0097] This indicates that LBP pretreatment can reduce the protein levels of pyroptosis-specific markers ASC, NLRP3, Caspase1, and inflammatory factors IL-1β and IL-18 in BV2 microglia after METH exposure.

[0098] 3.4 Results of Lactate Dehydrogenase (LDH) Activity Detection

[0099] The activity of lactate dehydrogenase (LDH) in cells of each group was detected, and the results are shown in the table below. Figure 4 The results showed that, compared with the con group, the LDH activity in the METH group was significantly increased (P < 0.05); and compared with the METH group, the LDH activity in the METH+LBP group was significantly decreased (P < 0.05). This indicates that pretreatment of METH-exposed BV2 microglia with LBP can reduce LDH activity and thus improve cell survival.

[0100] 3.5 ELISA results of inflammatory factors IL-1β and IL-18 in each group.

[0101] After treatment, the inflammatory factors IL-1β and IL-18 in the cell supernatant were detected by ELISA. The results are shown in the figure below. Figures 5-6 The results showed that, compared with the con group, the METH group had significantly higher levels of inflammatory factors IL-1β and IL-18 (P < 0.05); compared with the METH group, the METH+LBP group had significantly lower levels of inflammatory factors IL-1β and IL-18 (P < 0.05).

[0102] This indicates that pretreatment of METH-exposed BV2 microglia with LBP can reduce the inflammatory factors IL-1β and IL-18 in BV2 microglia.

[0103] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of wolfberry polysaccharide in preparation of a medicine for inhibiting methamphetamine-induced nerve injury.

2. Use according to claim 1, characterized in that, The wolfberry polysaccharide inhibits methamphetamine-induced nerve injury by inhibiting pyroptosis of microglia-like cells.

3. Use according to claim 1, characterized in that, The wolfberry polysaccharide inhibits methamphetamine-induced nerve injury by alleviating neuroinflammation.

4. Use according to claim 1, characterized in that, The medicine also comprises a pharmaceutically acceptable excipient.

5. Use according to claim 4, characterized in that, The excipient comprises a filler, a binder, a disintegrant, an emulsifier, a flavoring agent, a preservative, or a colorant.

6. Use according to claim 4, characterized in that, The dosage form of the medicine is a powder, a tablet, a granule, a capsule, a pill, or an oral solution.

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