Medicine for improving neurological dysfunction after cerebral ischemia

Abstract

The invention provides a medicine for improving neurological dysfunction after cerebral ischemia, and relates to the technical field of biological medicine and molecular biology, and the medicine comprises the following steps: S1, preparing materials; s2, cell injury model and intervention test; s3, cell activity and oxidative stress index detection. The invention provides application of combination of baicalein and ferulic acid in preparation of a medicine for improving neurological dysfunction after cerebral ischemia, the embodiment of the invention proves that combination of baicalein and ferulic acid can improve neurological dysfunction after cerebral ischemia by reducing cerebral infarction area, resisting oxidation, inhibiting inflammatory response and the like, and test data shows that compared with a model group, the combination of baicalein and ferulic acid has the advantages that the combination of baicalein and ferulic acid has a good application prospect. White cerebral infarction areas of the rats in the administration group are reduced; after the palmitic acid treatment, the baicalein and ferulic acid combined administration group can promote the proliferation of SH-SY5Y nerve cells; after baicalein and ferulic acid are combined for treatment, the MDA level in damaged cells is remarkably reduced, and the SOD and GSH levels are remarkably increased, so that the baicalein and ferulic acid have the effects of resisting oxidation, inhibiting inflammation and protecting nerve cells.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine and molecular biology, and in particular to a medicine for improving neurological dysfunction after cerebral ischemia. Background Art

[0002] Ischemic stroke is a sudden neurological deficit caused by local cerebral ischemia and permanent cerebral infarction (such as positive DWI examination). The main causes of ischemic stroke are atherosclerosis, arteritis, trauma, etc., which cause cerebrovascular lesions to form plaques that block blood vessels. It is also related to emboli produced by various cardiovascular diseases such as atrial fibrillation entering the blood and blocking brain blood vessels.

[0003] A central event during cerebral ischemia is the generation of reactive oxygen species (ROS), which can cause damage to lipids, proteins, and DNA. During reperfusion after ischemia, increased oxygen supply leads to excessive production of ROS, and neuronal cell death caused by oxidative stress can be observed in stroke patients. In addition, inflammatory responses also play a role in cerebral ischemia. Inflammation may occur in replication-competent glial cells, thereby exacerbating neurodegeneration or destroying the structural and functional connections between neurons and glial cells, leading to the influx of peripheral inflammatory factors and aggravating neuronal cell damage. These evidences indicate that oxidative stress and inflammation are important factors affecting brain damage mediated by ischemic stroke and provide potential targets for therapeutic approaches for brain-damaged cells.

[0004] Traditional Chinese medicine has unique advantages in treating such complex diseases, and most of them have shown good therapeutic effects on cerebral ischemia at different time periods. However, the effects of baicalin or ferulic acid alone are limited and single, and there are few studies on the combination of the two. Therefore, we provide a drug for improving neurological dysfunction after cerebral ischemia. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings existing in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: comprising the following steps:

[0007] S1. Prepare materials;

[0008] S2, cell injury models and intervention experiments;

[0009] S3, detection of cell activity and oxidative stress indicators;

[0010] S4, analysis of inflammatory proteins and signaling pathways;

[0011] S5. Animal model experiments and cerebral infarction assessment.

[0012] By adopting the above technical scheme: in the step S1, the materials are prepared, SH-SY5Y neurons are used for the palmitic acid-induced neuronal cell injury model, palmitic acid, baicalein, ferulic acid, CCK-8 reagent, MDA, SOD, and GSH detection kits are prepared and collected.

[0013] By adopting the above technical scheme: in the step S2 cell injury model and intervention test, SH-SY5Y neural cells were seeded in 96-well plates or 6-well plates according to requirements and cultured to 70% to 80% confluence. Except for the control group, other groups were added with 100 μL 50 μM palmitic acid for 2 hours to induce neural cell damage.

[0014] By adopting the above-mentioned technical scheme: in the cell injury model and intervention experiment in step S2, after palmitic acid treatment, different concentrations of baicalin (5, 50 and 100 μM), ferulic acid (5, 50 and 100 μM) and a combination of baicalin and ferulic acid (5 μM baicalin + 5 μM ferulic acid, 10 μM baicalin + 10 μM ferulic acid) were given, and the action lasted for 12 hours.

[0015] By adopting the above technical scheme: in the detection of cell activity and oxidative stress indicators in step S3, the CCK-8 method is used, 10μL CCK-8 reagent is added to the cell culture medium, and after incubation at 37°C for 1 hour, the cell activity is measured by the absorbance at 450nm to evaluate the protective effect of baicalein and ferulic acid on palmitic acid-induced damage.

[0016] By adopting the above technical scheme: in the detection of cell activity and oxidative stress indicators in step S3, MDA, SOD, and GSH kits are used, and the operation is carried out according to the instructions to measure the optical density (OD value) of each well, and the activity of MDA, SOD, and GSH in each group is calculated. The MDA level indicates lipid peroxidation, and SOD and GSH are antioxidant indicators for evaluating the oxidative stress state of cells.

[0017] By adopting the above technical scheme: in the analysis of inflammatory proteins and signal pathways in step S4, after the cell culture is completed, the culture medium is aspirated, the cells are washed three times with PBS, lysis buffer is added to extract proteins, the supernatant is taken after centrifugation at 4°C and 12000r / min for 15 minutes, and Western Blot is used to detect the phosphorylation levels of HIF-1α, GPX4, and p38 in the cells.

[0018] By adopting the above technical scheme: in the analysis of inflammatory proteins and signal pathways in step S4, Western Blot results showed that palmitic acid induced the phosphorylation of p38, and the expression levels of HIF-1α and GPX4 were reduced; while baicalein combined with ferulic acid treatment can significantly restore the expression of HIF-1α and GPX4 and inhibit the activation of p38.

[0019] By adopting the above technical scheme: in the animal model experiment and cerebral infarction assessment in step S5, male SD rats were fasted overnight before surgery, intraperitoneally anesthetized with 2% sodium pentobarbital, the internal carotid artery of the rats was blocked by suture embolism for 1 hour and then removed, and blood was reperfused to restore blood supply, and the neurological function scores of the rats were evaluated after surgery to screen the experimental animals with successful modeling.

[0020] By adopting the above technical scheme: in the animal model experiment and cerebral infarction assessment in step S5, rats were randomly divided into a normal group, a model group and a drug intervention group (baicalein 50 mg / kg + ferulic acid 50 mg / kg), and the drugs were administered by gavage daily for 7 consecutive days. The cerebral infarction area was detected by TTC staining, and fresh brain tissue sections were placed in TTC dye solution. After incubation at 37°C in the dark for 30 minutes, the distribution of normal tissue (red) and infarction area (white) was observed, and the infarction area was calculated using image analysis software. The results showed that the infarction area of ​​the drug intervention group was significantly reduced.

[0021] Compared with the prior art, the advantages and positive effects of the present invention are:

[0022] The invention provides an application of baicalin combined with ferulic acid in preparing a medicine for improving neurological dysfunction after cerebral ischemia. The embodiments of the invention confirm that baicalin combined with ferulic acid can improve neurological dysfunction after cerebral ischemia by reducing cerebral infarction area, anti-oxidation, inhibiting inflammatory response, etc. Experimental data show that compared with the model group, the white infarction area of ​​the brain of the rats in the drug administration group is reduced; after palmitic acid treatment, the baicalin combined with ferulic acid administration group can promote the proliferation of SH-SY5Y nerve cells; after baicalin combined with ferulic acid treatment, the MDA level in the damaged cells is significantly reduced, and the SOD and GSH levels are significantly increased, thereby exerting the effects of both on anti-oxidation, inhibiting inflammation and protecting nerve cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The structural formulas are baicalein (A) and ferulic acid (B);

[0024] Figure 2 The results of the effects of baicalin, ferulic acid, and baicalin combined with ferulic acid on the proliferation and toxicity of palmitic acid-induced SH-SY5Y cells;

[0025] Figure 3 The results of the effects of baicalin, ferulic acid, and baicalin combined with ferulic acid on antioxidant-related indices of SH-SY5Y cells modeled with palmitic acid, where Figure A is a comparison of the effects on malondialdehyde (MDA) levels, Figure B is a comparison of the effects on superoxide dismutase (SOD) levels; Figure C is a comparison of the effects on reduced glutathione (GSH) levels;

[0026] Figure 4Comparison of the effects of baicalin, ferulic acid, and baicalin combined with ferulic acid on the antioxidant and anti-inflammatory related proteins in palmitic acid-induced SH-SY5Y cells;

[0027] Figure 5 Schematic diagram of the effect of baicalin combined with ferulic acid on the cerebral infarction volume of MCAO rats, where A is the TTC staining image of the rat brain and B is the cerebral infarction area image. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

[0030] like Figure 1-2 As shown, the present invention provides a technical solution: a drug for improving neurological dysfunction after cerebral ischemia, in the cell injury model and intervention test in step S2, according to the requirements, SH-SY5Y neural cells are inoculated in a 96-well plate or a 6-well plate, and cultured to 70% to 80% confluence, and except for the control group, other groups are added with 100 μL 50 μM palmitic acid for 2 hours to induce neural cell damage;

[0031] In the cell injury model and intervention test in step S2, after palmitic acid treatment, different concentrations of baicalin (5, 50 and 100 μM), ferulic acid (5, 50 and 100 μM) and a combination of baicalin and ferulic acid (5 μM baicalin + 5 μM ferulic acid, 10 μM baicalin + 10 μM ferulic acid) were given and the effect lasted for 12 hours.

[0032] In the present embodiment, SH-SY5Y cells were inoculated in 96-well plates. After reaching 70% to 80% fusion, the control group replaced with fresh culture medium. After 2 hours of modeling with 100 μL 50 μM palmitic acid in other groups, 100 μL of 5, 50 and 100 μM baicalein (labeled as H in this study), 5, 50 and 100 μM ferulic acid (labeled as A in this study) or a mixture of 5 μM baicalein and 5 μM ferulic acid, 10 μM baicalein and 10 μM ferulic acid were used to treat cells for 12 hours. Subsequently, 10 μL CCK-8 reagent was added to the SH-SY5Y culture medium and incubated in a 37°C constant temperature incubator for 1 hour. Finally, the absorbance value of each well was measured at 450 nm.

[0033] Wherein: Preparation of baicalin and ferulic acid 1 mg of baicalin was dissolved in 0.3700 mL of 75% medical alcohol to obtain a mother solution with a concentration of 10 mM, and then diluted to an appropriate concentration in the cell culture medium for the experiment. 1 mg of ferulic acid was dissolved in 0.5150 mL of 75% medical alcohol to obtain a mother solution with a concentration of 10 mM, and then diluted to an appropriate concentration in the cell culture medium for the experiment.

[0034] Example 2

[0035] like Figure 1-3 As shown, the present invention provides a technical solution: a drug for improving neurological dysfunction after cerebral ischemia, in the detection of cell activity and oxidative stress indexes in step S3, the CCK-8 method is used, 10 μL CCK-8 reagent is added to the cell culture medium, and after incubation at 37°C for 1 hour, the cell activity is measured by the absorbance at 450nm to evaluate the protective effect of baicalein and ferulic acid on palmitic acid-induced damage;

[0036] In the step S3 cell activity and oxidative stress index detection, MDA, SOD, GSH kits are used according to the instructions to measure the optical density (OD value) of each well, and the activities of MDA, SOD and GSH in each group are calculated. The MDA level indicates lipid peroxidation, and SOD and GSH are antioxidant indicators for evaluating the oxidative stress state of cells.

[0037] In this embodiment, the staff can operate according to the instructions of the MDA, SOD, and GSH kits, use a multifunctional microplate reader to detect the optical density OD value of each well, and calculate the activity of MDA, SOD, and GSH in each group.

[0038] Example 3

[0039] like Figure 1-4 As shown, the present invention provides a technical solution: a drug for improving neurological dysfunction after cerebral ischemia, in the analysis of inflammatory proteins and signal pathways in step S4, after the cell culture is completed, the culture medium is aspirated, and the cells are washed with PBS for 3 times, and then a lysis solution is added to extract the protein, and the supernatant is taken after centrifugation at 4°C and 12000r / min for 15 minutes, and the phosphorylation levels of HIF-1α, GPX4, and p38 in the cells are detected by Western Blot;

[0040] In the analysis of inflammatory proteins and signaling pathways in step S4, Western Blot results showed that palmitic acid induced the phosphorylation of p38 and reduced the expression levels of HIF-1α and GPX4; while baicalein combined with ferulic acid treatment could significantly restore the expression of HIF-1α and GPX4 and inhibit the activation of p38.

[0041] In this embodiment, the staff can inoculate the cells in a 6-well plate, and when the cells grow to about 70% to 80%, the control group replaces the fresh culture medium, and the other groups add 1mL 50μM palmitic acid and put it in the incubator for 2h; after 2h of modeling, the model group adds 1mL 50μM palmitic acid, and the drug administration group adds 1mL 10μM baicalein, 10μM ferulic acid, 5μM baicalein and 5μM ferulic acid, 10μM baicalein and 10μM ferulic acid, respectively, and continue to be placed in the incubator for 12h. The original culture medium is aspirated, washed 3 times with pre-cooled PBS, and the protease inhibitor is added to the lysate in proportion to lyse the cells, 4℃, 12000r / min, centrifuged for 15min, and the supernatant is taken. Western Blot is used to detect protein expression.

[0042] Example 4

[0043] like Figure 1-5 As shown, the present invention provides a technical solution: a drug for improving neurological dysfunction after cerebral ischemia, in the animal model experiment and cerebral infarction evaluation in step S5, male SD rats are fasted overnight before surgery, intraperitoneally anesthetized with 2% sodium pentobarbital, the internal carotid artery of the rats is blocked by suture embolism for 1 hour and then removed, blood is reperfused to restore blood supply, neurological function scores of the rats are evaluated after surgery, and experimental animals with successful modeling are screened;

[0044] In the animal model experiment and cerebral infarction assessment in step S5, rats were randomly divided into a normal group, a model group and a drug intervention group (50 mg / kg baicalein + 50 mg / kg ferulic acid), and the drugs were administered by gavage daily for 7 consecutive days. The cerebral infarction area was detected by TTC staining. Fresh brain tissue sections were placed in TTC dye solution and incubated at 37°C in the dark for 30 minutes. The distribution of normal tissue (red) and infarction area (white) was observed, and the infarction area was calculated using image analysis software. The results showed that the infarction area in the drug intervention group was significantly reduced.

[0045] In this embodiment, it was found through CCK-8 detection that baicalin combined with ferulic acid can increase the activity of SH-SY5Y cells and reduce palmitic acid-induced cell damage. Oxidative stress is one of the main pathogenesis of ischemic stroke, which is manifested by increased MDA levels and decreased SOD and GSH activities. The combination of baicalin and ferulic acid can significantly inhibit oxidative stress response, reduce MDA levels, and increase SOD and GSH activities. In addition, the expression of hypoxia-inducible factor 1 (HIF-1α) and antioxidant enzyme GPX4 increased after combined medication, and inhibited the phosphorylation of p38. Animal experiments showed that compared with the MCAO model group, the cerebral infarction area of ​​rats in the drug intervention group was reduced. In summary, baicalin combined with ferulic acid has a significant protective effect on cerebral ischemic injury.

[0046] The present invention adopts a palmitic acid-induced SH-SY5Y nerve cell injury model in an in vitro experiment, adds different concentrations of baicalin, ferulic acid and a combination thereof to the cells, and after 12 hours of treatment, uses the CCK-8 method to detect cell activity. The experimental results show that baicalin combined with ferulic acid significantly increases cell activity and reduces palmitic acid-induced cell damage, indicating that it can effectively protect nerve cells from damage.

[0047] Secondly, for the evaluation of the oxidative stress state of cells, MDA, SOD and GSH kits were used to detect oxidative stress-related indicators. The results showed that ischemic injury caused an increase in MDA levels and a decrease in SOD and GSH activities, but after combined treatment with baicalin and ferulic acid, MDA levels were significantly reduced and SOD and GSH activities were restored. This shows that the drug can reduce cell damage by inhibiting oxidative stress response. In addition, Western Blot experimental results showed that palmitic acid-induced p38 phosphorylation was significantly inhibited, and HIF-1α and GPX4 protein expression was significantly increased, indicating that the drug has significant efficacy in anti-oxidation and anti-inflammatory aspects.

[0048] Finally, the in vivo experiment used the MCAO rat model, divided the rats into groups and gave them a combined treatment of baicalin and ferulic acid. After 7 consecutive days of drug intervention, the cerebral infarction area of ​​the rats was detected by TTC staining. The results showed that the cerebral infarction area of ​​the drug intervention group was significantly reduced, further verifying the protective effect of baicalin combined with ferulic acid on ischemic brain injury. In summary, the present invention significantly improves neurological dysfunction after cerebral ischemia by inhibiting nerve cell damage, oxidative stress and inflammatory response, and has good clinical application prospects.

[0049] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes and apply it to other fields. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A drug for improving neurological dysfunction after cerebral ischemia, characterized in that: The following steps are involved: S1. Prepare materials; S2, cell injury models and intervention experiments; S3, detection of cell activity and oxidative stress indicators; S4, analysis of inflammatory proteins and signaling pathways; S5. Animal model experiments and cerebral infarction assessment.

2. A drug for improving neurological dysfunction after cerebral ischemia according to claim 1, characterized in that: In the step S1, the materials are prepared by collecting SH-SY5Y neural cells for the neural cell injury model induced by palmitic acid, palmitic acid, baicalein, ferulic acid, CCK-8 reagent, and MDA, SOD, and GSH detection kits.

3. The drug for improving neurological dysfunction after cerebral ischemia according to claim 1, characterized in that: In the cell injury model and intervention test in step S2, SH-SY5Y neural cells were seeded in 96-well plates or 6-well plates according to requirements and cultured to 70% to 80% confluence. Except for the control group, other groups were treated with 100 μL 50 μM palmitic acid for 2 hours.

4. The drug for improving neurological dysfunction after cerebral ischemia according to claim 3, characterized in that: In the cell injury model and intervention test in step S2, after palmitic acid treatment, different concentrations of baicalin, ferulic acid, and a combination of baicalin and ferulic acid are given for a continuous action of 12 hours.

5. The drug for improving neurological dysfunction after cerebral ischemia according to claim 1, characterized in that: In the detection of cell activity and oxidative stress indicators in step S3, the CCK-8 method is used, 10 μL of CCK-8 reagent is added to the cell culture medium, and after incubation at 37° C. for 1 hour, the cell activity is measured by absorbance at 450 nm to evaluate the protective effect of baicalein and ferulic acid on palmitic acid-induced damage.

6. The drug for improving neurological dysfunction after cerebral ischemia according to claim 5, characterized in that: In the detection of cell activity and oxidative stress indexes in step S3, MDA, SOD, and GSH kits are used according to the instructions to measure the optical density of each well, and the activities of MDA, SOD, and GSH in each group are calculated. The MDA level indicates lipid peroxidation, and SOD and GSH are antioxidant indicators to evaluate the cellular oxidative stress state.

7. The drug for improving neurological dysfunction after cerebral ischemia according to claim 1, characterized in that: In the analysis of inflammatory proteins and signaling pathways in step S4, after the cell culture is completed, the culture medium is aspirated, the cells are washed three times with PBS, and then lysis buffer is added to extract proteins. After centrifugation at 4°C and 12000 r / min for 15 minutes, the supernatant is taken and the phosphorylation levels of HIF-1α, GPX4, and p38 in the cells are detected by Western Blot.

8. The drug for improving neurological dysfunction after cerebral ischemia according to claim 7, characterized in that: In the analysis of inflammatory proteins and signaling pathways in step S4, Western Blot results showed that palmitic acid induced the phosphorylation of p38 and reduced the expression levels of HIF-1α and GPX4.

9. The drug for improving neurological dysfunction after cerebral ischemia according to claim 1, characterized in that: In the animal model experiment and cerebral infarction assessment in step S5, male SD rats were fasted overnight before surgery, and intraperitoneally anesthetized with 2% sodium pentobarbital. The internal carotid artery of the rats was occluded by suture embolism for 1 hour and then removed. Blood was reperfused to restore blood supply. Neurological function scores of the rats were evaluated after surgery, and experimental animals with successful modeling were selected.

10. The drug for improving neurological dysfunction after cerebral ischemia according to claim 9, characterized in that: In the animal model experiment and cerebral infarction assessment in step S5, rats were randomly divided into a normal group, a model group and a drug intervention group, and the drugs were administered by gavage daily for 7 consecutive days. The cerebral infarction area was detected by TTC staining. Fresh brain tissue sections were placed in TTC dye solution and incubated at 37° C. in the dark for 30 minutes. The distribution of normal tissue and infarction area was observed, and the infarction area was calculated using image analysis software.

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