CN113995751B - Application of epalrestat in the preparation of medicine for preventing and treating ischemic stroke - Google Patents

Abstract

The invention relates to an application of epalrestat in preparing a medicine for preventing and treating ischemic stroke (cerebral infarction), belonging to the technical field of medicines. The invention provides application of epalrestat in preparing a medicament for preventing and treating ischemic stroke, aiming at cerebral stroke patients who are in early ischemic stroke, miss a treatment time window and cannot be treated by thrombolysis. The epalrestat is used for treating cerebral arterial thrombosis, so that the tight junction protein of brain microvascular endothelial cells can be up-regulated by inhibiting the expression of aldose reductase, the apoptosis of the endothelial cells is reduced, the neurological dysfunction after cerebral arterial thrombosis is improved, and the integrity of a blood brain barrier after cerebral arterial thrombosis is maintained; in addition, epalrestat can effectively reduce infiltration of peripheral inflammatory cells and relieve inflammatory reaction in ischemic brain tissues. The invention provides a new effective way for preventing and treating ischemic stroke and provides a theoretical basis for epalrestat to treat clinical ischemic stroke.

Description

Application of epalrestat in preparation of medicine for preventing and treating ischemic stroke

technical field

The invention belongs to the technical field of medicine, and in particular relates to the application of epalrestat in the preparation of medicines for preventing and treating ischemic stroke.

Background technique

Stroke, also known as apoplexy, is an acute cerebrovascular disease. It is a disease that causes brain tissue damage due to sudden rupture of blood vessels in the brain or blockage of blood vessels that prevents blood from flowing into the brain, including ischemic and hemorrhagic strokes. The incidence of hemorrhagic stroke is higher than that of hemorrhagic stroke, accounting for 60-70% of the total number of strokes. Ischemic stroke is a complex process involving multiple systems. After ischemia, neurons will be damaged, and the permeability of the blood-brain barrier will increase, causing cerebral edema, resulting in increased intracranial pressure, forming brain herniation, and leading to disease. deterioration.

In my country, there are mainly three types of drugs for the treatment of ischemic stroke: (1) drugs for improving cerebral circulation: thrombolytic drugs (t-PA, urokinase), antiplatelet drugs (aspirin), anticoagulant drugs (heparin) and Other drugs (butylphthalide, etc.); (2) Neuroprotective drugs: neuroprotective agents (edaravone, citicoline, gangliosides, etc.); (3) Others: Danshen, Chuanqiong, etc. traditional Chinese medicine. In the case of vascular occlusion, early thrombolytic therapy with intravenous thrombolytic drugs such as t-PA is currently the standard therapy for ischemic stroke, and timely restoration of blood flow in stroke patients can effectively alleviate neurological deficits. For patients meeting national and international eligibility guidelines, administration of t-PA within 4.5 hours of ischemic onset improves outcomes within 3 to 6 months.

However, thrombolytic therapy has major limitations, such as a narrow treatment time window, and reperfusion injury after thrombolytic recanalization, which can aggravate ischemic brain injury. In addition, brain tissue damage after ischemia is a complex process, which includes not only the direct damage to nerve cells caused by ischemia and hypoxia, but also the indirect damage to brain tissue induced by various inflammatory cells and inflammatory mediators on the basis of direct damage. . More and more studies have confirmed that immune involvement plays an important role in the pathogenesis of ischemic stroke. At present, there is still a lack of effective prevention and treatment drugs for ischemic brain injury in patients with ischemic stroke who have missed the treatment time window and cannot use thrombolytic therapy.

Contents of the invention

The present invention provides the application of epalrestat in the preparation of medicines for the prevention and treatment of ischemic stroke, aiming at ischemic stroke patients in the early stage of the onset of ischemic stroke and who have missed the treatment time window and cannot be treated with thrombolysis.

Technical scheme of the present invention:

Epalrestat is an aldose reductase inhibitor drug that reversibly inhibits the aldose reductase that converts glucose into sorbitol in polyol metabolism related to the pathogenesis of diabetic complications, and is used to prevent , Improve and treat peripheral nerve disorders complicated by diabetes.

The invention provides a new drug application of epalrestat, that is, the application of epalrestat in the preparation of drugs for preventing and treating ischemic stroke.

Further, the clinical type of ischemic stroke is cerebral thrombosis, lacunar infarction or cerebral embolism.

Further, epalrestat is the only active ingredient in the drug for preventing and treating ischemic stroke.

Further, the medicament for preventing and treating ischemic stroke also includes pharmaceutically acceptable adjuvants and/or carriers.

Further, the content of epalrestat in the drug for preventing and treating ischemic stroke is 0.1-99wt%.

Further, the dosage form of the drug for preventing and treating ischemic stroke includes oral dosage form and parenteral dosage form.

Further, the dosage form of the drug for preventing and treating ischemic stroke is a parenteral administration dosage form, specifically an injection dosage form, a respiratory dosage dosage form, a skin dosage dosage form, a mucosal dosage dosage form or an oral cavity dosage dosage form. drug form.

Further, the dosage form of the drug for preventing and treating ischemic stroke is an oral dosage form, specifically granules, tablets, capsules, pills, drop pills or oral liquid preparations.

Beneficial effects of the present invention:

The invention provides a new drug application of epalrestat, that is, the application of epalrestat in the preparation of drugs for preventing and treating ischemic stroke. The use of epalrestat after ischemic stroke can inhibit the expression of aldose reductase in cerebral vascular endothelial cells, thereby up-regulating the expression of tight junction protein in cerebral microvascular endothelial cells, reducing endothelial cell apoptosis, and improving ischemia In addition, epalrestat can effectively reduce the infiltration of peripheral inflammatory cells and reduce the inflammatory response in ischemic brain tissue. The present invention provides a new and effective treatment method for patients with ischemic stroke who are in the early stage of onset of ischemic stroke and who have missed the treatment time window and cannot be treated with thrombolysis, and provide epalrestat for the treatment of clinical ischemic stroke A theoretical basis is provided.

Description of drawings

Figure 1 and Figure 2 are sequentially the TTC staining diagram of the ischemic brain tissue and the volume comparison diagram of the necrotic tissue in Example 1;

Fig. 3 is the mouse neurobehavioral score result figure in the cerebral ischemia process of embodiment 2;

Figure 4 and Figure 5 are sequentially the EB staining diagram of the ischemic brain tissue and the comparison diagram of the EB content in the ischemic brain tissue in Example 3;

Fig. 6 and Fig. 7 are the photographs of the fluorescent staining of the macrophages (F4/80) around the brain microvascular endothelial cells (CD31) in Example 4 and the comparison chart of the average fluorescence intensity of the macrophages;

Fig. 8 is a diagram showing the results of measuring infiltrating neutrophils in ischemic brain tissue by flow cytometry in Example 4;

Figure 9 is a comparison chart of the percentage of infiltrated neutrophils in ischemic brain tissue in Example 4;

Figure 10, Figure 11 and Figure 12 are the TJs protein map, the Occludin protein expression comparison chart and the ZO-1 protein expression comparison chart in the ischemic brain tissue measured by Western Blot experiment in Example 5;

Figure 13 and Figure 14 are sequentially the co-staining photos of endothelial cells (CD31) and Occludin in the ischemic brain tissue of Example 6 and the comparison chart of the average fluorescence intensity of endothelial cells (CD31) and Occludin co-staining;

Figure 15 and Figure 16 are sequentially the co-staining photos of endothelial cells (GFAP) and ZO-1 in the ischemic brain tissue of Example 6 and the comparison chart of the average fluorescence intensity of endothelial cells (CD31) and ZO-1 co-staining;

Figure 17 and Figure 18 are sequentially the co-stained photos of Occludin around astrocytes (GFAP) in the ischemic brain tissue of Example 6 and the comparison chart of the average fluorescence intensity of Occludin around astrocytes (GFAP);

Figure 19 and Figure 20 are the co-stained photos of ZO-1 around astrocytes (GFAP) and the comparison of the average fluorescence intensity of ZO-1 around astrocytes (GFAP) in the ischemic brain tissue of Example 6 ;

Figure 21, Figure 22 and Figure 23 are the diagrams of Bax, Bcl2 and Cleaved-casp3 apoptosis-related proteins in ischemic brain tissue measured by Western Blot experiment in Example 7, the comparison chart of Cleaved-casp3 protein expression, and the ratio of Bcl2 to Bax protein comparison chart;

Figure 24, Figure 25 and Figure 26 are the TJs protein map, the expression comparison chart of ZO-1 protein and the comparison chart of Occludin protein expression in the endothelial cells under the action of OGD determined by Western Blot experiment in Example 8;

Figure 27 is a comparison chart of the statistical results of the permeability of the blood-brain barrier in vitro under the action of Example 8 OGD;

Figure 28, Figure 29 and Figure 30 are the protein maps of Bax, Bcl2 and Cleaved-Casp3 in endothelial cells under the action of OGD measured by Western Blot in Example 9, the comparison chart of the expression of Cleaved-casp3 protein, and the comparison of the ratio of Bcl2 and Bax proteins picture;

Fig. 31 and Fig. 32 are the results of flow cytometry determination of the expression of PI and AnnexinV in endothelial cells in Example 9 and the comparison of the expression levels of PI and AnnexinV in endothelial cells in each group;

Figure 33 and Figure 34 are the photos of the co-staining of endothelial cells (CD31) and LC3B protein in Example 10 and the comparison chart of the average fluorescence intensity of the co-staining of endothelial cells (CD31) and LC3B;

Figure 35 and Figure 36 are the co-staining photos of endothelial cells (CD31) and Beclin1 protein in Example 10 and the comparison chart of the average fluorescence intensity of endothelial cells (CD31) and Beclin1 co-staining;

Figure 37 and Figure 38 are the autophagy-related protein maps of LC3B-I and LC3B-II in bEnd.3 cells measured by Western Blot experiment in Example 11 and the comparison chart of the ratio between LC3B-I and LC3B-II;

Figure 39 is a photo of co-staining of endothelial cells (CD31) and Beclin1 in Example 11;

Figure 40 is a comparison chart of average fluorescence intensity co-stained with endothelial cells (CD31) and Beclin1 in Example 11;

Figure 41 is a photo of co-staining of endothelial cells (CD31) and LAMP-1 in Example 11;

Figure 42 is a comparison chart of average fluorescence intensity co-stained with endothelial cells (CD31) and LAMP-1 in Example 11;

Fig. 43 is the photo of co-staining of endothelial cells (CD31) and P62 in embodiment 11;

Figure 44 is a comparison chart of average fluorescence intensity co-stained with endothelial cells (CD31) and P62 in Example 11;

Figure 45 and Figure 46 are the comparison charts of the AR protein map and the expression level of AR protein in the brain tissue after ischemia measured by Western Blot experiment in Example 12;

Figure 47 is a co-staining photo of endothelial cells (CD31) and AR protein in ischemic brain tissue in Example 12;

Fig. 48 is a comparison diagram of average fluorescence intensity co-stained with endothelial cells (CD31) and AR in ischemic brain tissue in Example 12;

Figure 49 and Figure 50 are the graphs of AR protein in different endothelial cells of the embodiment 12 OGD and the comparison charts of the expression of AR protein in sequence;

Fig. 51, Fig. 52 and Fig. 53 are the comparison diagram of epalrestat changing the protein expression of p-AKT and p-mTOR in endothelial cells, the comparison diagram of p-AKT protein expression and the expression of p-mTOR protein in Example 13 comparison chart;

Figure 54, Figure 55 and Figure 56 are the comparison chart of p-AKT and p-mTOR protein expression in endothelial cells changed by epalrestat, AKTi and mTORi in Example 13, the comparison chart of p-AKT protein expression and p-mTOR Protein expression comparison chart;

Fig. 57, Fig. 58 and Fig. 59 are the comparison diagram of the protein expression of ZO-1 and Occludind in the endothelial cells changed by epalrestat, AKTi and mTORi in Example 13, the comparison diagram of the protein expression of ZO-1 and the expression of Occludind protein comparison chart;

Figure 60, Figure 61 and Figure 62 are the comparison charts of the protein expression of Bax, Bcl2 and Cleaved-Casp3 in endothelial cells changed by epalrestat, AKTi and mTORi in Example 14, and the comparison chart of the protein expression of Cleaved-Casp3 and Bcl2 /Bax ratio comparison chart;

Figure 63, Figure 64 and Figure 65 are the comparison charts of the protein expression of LC3B-I, LC3B-II and Beclin-1 in endothelial cells changed by epalrestat, AKTi and mTORi in Example 14, and the protein expression of Beclin-1 Comparison chart and ratio comparison chart of Beclin-1.

Detailed ways

The technical solution of the present invention will be further described below in conjunction with the examples, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered by the present invention within the scope of protection. The process equipment or devices not specifically indicated in the following examples all adopt conventional equipment or devices in the art. If not specified, the raw materials used in the examples of the present invention, etc. can be commercially available; if not specified, this The technical means used in the embodiments of the invention are conventional means well known to those skilled in the art.

The following examples will verify the prevention and treatment effect of epalrestat on ischemic stroke through in vivo experiments and in vitro experiments.

Example 1

This example investigates the effect of epalrestat on cerebral ischemic injury in the process of cerebral ischemia.

The experimental animals used in the present invention are all purebred C57BL/6 male mice with a body weight of 19-21 g, purchased from Liaoning Changsheng Biotechnology Co., Ltd.

Modeling, grouping, and administration methods: establish the mouse permanent middle cerebral artery occlusion model (pMCAL) and the sham operation group sham, respectively, and divide the pMCAL model and the sham operation (sham) group into normal (ischemic) groups - labeling The normal group, the epalrestat treatment group-Eps group and the sham group were intragastrically administered immediately after the establishment of the model. The dosage of the Eps group was 100 μg/g, and the Normal group was intragastrically administered with the same amount of normal saline.

Brain tissue sections were taken at the time points of ischemia time of 12h, 1d, 3d and 5d respectively, and TTC staining was performed on the brain tissue sections according to the conventional TTC staining method in this field (TTC is called 2,3,5-triphenyl chloride Tetrazolium).

The results of the TTC staining experiment are shown in Figure 1. The mouse brain tissue was divided into 5 slices with a thickness of 2 mm per slice. The red area is normal brain tissue, and the white area is cerebral ischemic necrosis tissue. The volume of brain necrosis tissue in each group of mice was counted as shown in Figure 2. Compared with the normal group, the volume of brain necrosis tissue in the cerebral ischemia 1d, 3d and 5d treatment groups decreased, and there was a statistical difference (*P <0.05, **P<0.01), which proves that epalrestat treatment can reduce cerebral ischemic injury.

Example 2

This example investigates the effect of epalrestat on the neurobehavioral function of mice during cerebral ischemia.

This example observes the state and neurobehavioral changes of mice after cerebral ischemia, and reflects the severity of cerebral ischemia in mice. According to the experimental plan, mice are modeled with pMCAL, and the modeling, grouping, and administration methods are the same as in Example 1. same. The neurological score of normal mice is 0, and the more severe the symptoms related to ischemia, the greater the score. The neurobehavioral scoring criteria are shown in Table 1:

Table 1

Neurological Svevrity Scores neurobehavioral score Normal neurological function 0 marks Mild neurological deficit (flexion of right forelimb when tail is raised) 1 point Moderate neurological deficit (circles to the right while walking) 2 minutes Moderate neurological deficit (slope to the right) 3 points No spontaneous walking, decreased consciousness 4 points ischemia related death 5 points

By comparing the neurobehavioral scores of each group of mice after ischemia shown in Figure 3, it was found that the neurobehavioral performance of the mice in the ischemia group was significantly worse than that of the sham operation (Sham) group; The neurobehavioral score was higher than that of the epalrestat treatment group, with a statistical difference (*P<0.05), which indicated that epalrestat treatment could restore part of the neurological function of the mice and improve the neurological dysfunction caused by ischemic injury.

Example 3

This example investigates the effect of epalrestat on the blood-brain barrier function during cerebral ischemia.

The modeling, grouping, and administration methods of this embodiment are all the same as in Example 1, and the mice are injected with Evans Blue (EB) dye into the tail vein at the time point when the ischemia time is 12h, 1d, 3d, and 5d, and after 2h The brain tissue was removed by perfusion to observe the penetration of the blue dye in the brain tissue. The results are shown in Figure 4. The blue is the damaged area of the blood-brain barrier, and the white is the normal brain tissue; cerebral ischemia damage leads to the destruction of the integrity of the blood-brain barrier, and the EB dye can enter the brain parenchyma through the damaged blood-brain barrier and destroy the ischemic area. dyed blue.

Through the extraction of trichloroacetic acid, the content of EB in the brain tissue of each group was detected by ultraviolet spectrophotometer, and the statistical analysis data are shown in Figure 5. The content of EB in the brain of the normal group was higher than that of the sham group; compared with the normal group, the cerebral ischemia 3d The content of EB in the ischemic brain tissue of the epalrestat treatment group and 5d group was significantly reduced, with a statistical difference (*P<0.05), which indicated that epalrestat treatment could reduce the damage of the blood-brain barrier in the process of cerebral ischemic injury degree.

Example 4

This example investigates the effect of epalrestat on the infiltration of perivascular inflammatory cells during cerebral ischemia.

In this example, the modeling, grouping, and administration methods are the same as in Example 1. The mice in the sham group, the Normal-3d group, and the Eps-3d group with an ischemia time of 3 days were taken, and the macrophages in the brain were detected by immunofluorescence staining. Figure 6 shows that the macrophages around the brain microvascular endothelial cells are marked by immunofluorescence staining, CD31-labeled vascular endothelial cells are green, F4/80-labeled macrophages are red, and DAPI-labeled nuclei are blue; statistical analysis As shown in Figure 7, the number of perivascular macrophages in the ischemic brain tissue of the epalrestat treatment group was less than that of the normal group, with a statistical difference (**P<0.01).

Flow cytometry was used to detect the infiltration of neutrophils in ischemic brain tissue, and the results are shown in Figure 8, CD11b ⁺ CD45 ^high Ly6G ⁺ labeled neutrophils infiltrated in the brain; the percentage of neutrophils in each group was counted as As shown in Figure 9, compared with the normal group, the number of neutrophils (CD11b ⁺ CD45 ^high Ly6G ⁺ ) in the ischemic brain tissue of the epalrestat treatment group was lower, with a statistical difference (**P<0.01).

The above results show that epalrestat treatment can maintain the integrity of the blood-brain barrier after ischemic injury, effectively reduce the infiltration of peripheral inflammatory cells, and reduce the inflammatory response in ischemic brain tissue.

Example 5

The blood-brain barrier is mainly composed of brain microvascular endothelial cells (CD31), astrocytes (GFAP) and pericytes. The expression of tight junction proteins TJs in brain microvascular endothelial cells is the structural basis for maintaining the selective permeability of the blood-brain barrier. This example investigates the effect of epalrestat on the expression of tight junction proteins TJs: Occludin and ZO-1 in the ischemic cerebral hemisphere during cerebral ischemia.

The modeling, grouping, and administration methods of this example were the same as in Example 1. The protein was extracted from the brain tissue by perfusion at different times of ischemia, and the effect of epalrestat on the expression of TJs in the ischemic brain tissue was detected by Western Blot experiment. The results are shown in Figure 10, the expression of TJs (Occludin and ZO-1) in the ischemic brain tissue; the statistical analysis is shown in Figure 11 and Figure 12, compared with the Sham group, the expression of ZO-1 and Occludin in the brain of the normal group decreased; the expression of Occludin in ischemic brain tissue of mice treated with epalrestat for 1d and 3d of cerebral ischemia was higher than that of normal group, with statistical difference (*P<0.05, **P<0.01); cerebral ischemia 12h , 1d and 3d epalrestat treatment group in the ischemic brain tissue of mice with ZO-1 expression was higher than the normal group, with statistical difference (*P<0.05, **P<0.01). The results indicated that during cerebral ischemia, epalrestat treatment could up-regulate the expression of TJs in ischemic brain tissue.

Example 6

This example investigates the effect of epalrestat on the expression of tight junction proteins TJS: Occludin and ZO-1 on endothelial cells (CD31) and around astrocytes (GFAP) during cerebral ischemia.

The modeling, grouping, and administration methods of this example are the same as those in Example 1. Immunofluorescence is used to detect the expression of tight junction proteins on endothelial cells and around astrocytes, and the brain tissue of each group is prepared after 3 days of cerebral ischemia. Frozen sections were double-stained with Occludin and ZO-1, CD31 and GFAP, respectively.

The expression of Occludin in ischemic brain microvascular endothelial cells was detected by immunofluorescence staining. The results are shown in Figure 13. CD31-labeled brain microvascular endothelial cells were green, Occludin staining was red, and co-expression of CD31 and Occludin was yellow. Statistical data analysis results are shown in Figure 14, the average fluorescence intensity of CD31 and Occludin co-expression in the ischemic brain tissue of the epalrestat treatment group was significantly higher than that of the normal group, with a statistical difference (**P<0.01).

The expression of ZO-1 in ischemic cerebral hemisphere microvascular endothelial cells was detected by immunofluorescence staining. The results are shown in Figure 15. CD31-labeled cerebral microvascular endothelial cells were green, ZO-1 staining was red, and the co-expression of CD31 and ZO-1 was yellow. Statistical data analysis results are shown in Figure 16, the average fluorescence intensity of CD31 and ZO-1 co-expression in the ischemic brain tissue of the epalrestat treatment group was significantly higher than that of the normal group, with a statistical difference (**P<0.01).

The above results indicate that epalrestat can increase the expression levels of TJs: Occludin and ZO-1 on microvascular endothelial cells in ischemic brain tissue.

The Occludin around the astrocytes in the ischemic brain hemisphere was detected by immunofluorescence staining, and the results were shown in Figure 17. The GFAP-labeled astrocytes were green, the Occludin staining was red, and the nuclei were blue. Statistical data analysis results are shown in Figure 18, the average fluorescence intensity of Occludin expression around GFAP in the ischemic brain tissue of the epalrestat treatment group was significantly higher than that of the normal group, with a statistical difference (***P<0.001).

The situation of ZO-1 around the astrocytes in the ischemic brain hemisphere was detected by immunofluorescence staining, and the results were shown in Figure 19, GFAP-labeled astrocytes were green, ZO-1 staining was red, and the nuclei were blue. Statistical data analysis results are shown in Figure 20, the average fluorescence intensity of ZO-1 expression around GFAP in the ischemic brain tissue of the epalrestat treatment group was significantly higher than that of the normal group, with a statistical difference (**P<0.01).

The above results show that epalrestat can increase the expression levels of TJs around astrocytes in ischemic brain tissue: Occludin and ZO-1, and reduce the loss of TJs in endothelial cells after cerebral ischemia.

Example 7

This example investigates the effect of epalrestat on apoptosis-related proteins in ischemic brain tissue during cerebral ischemia.

The modeling, grouping, and administration methods of this example were the same as those in Example 1. The brain tissue proteins of each group were collected at different times of cerebral ischemia, and the expression of apoptosis-related proteins in the ischemic brain tissue was detected by Western Blot experiment. The results are shown in Figure 21. With the prolongation of cerebral ischemia time, the expressions of pro-apoptotic proteins cleaved-casp3 and Bax in ischemic brain tissue gradually increased, while the expression of anti-apoptotic protein Bcl2 gradually decreased.

Statistical analysis As shown in Figure 22, compared with the normal group, the expression of cleaved-casp3 in the ischemic brain tissue of the epalrestat treatment group was reduced, and there was a statistical difference between 3d and 5d of cerebral ischemia (**P<0.01 , ***P<0.001). Statistical analysis of the ratio of Bcl2 and Bax as shown in Figure 23, the value of Bcl2/Bax in the ischemic brain tissue of the cerebral ischemia 3d epalrestat treatment group was significantly higher than that of the normal group, with a statistical difference (*P<0.05). The results indicated that epalrestat treatment could effectively reduce the production of apoptotic protein in ischemic brain tissue after cerebral ischemia.

Example 8

This example investigated the effect of epalrestat on the integrity of the blood-brain barrier in vitro and the expression of TJs protein in endothelial cells under OGD conditions.

An in vitro oxygen-glucose deprivation (OGD) model was established to simulate cerebral ischemia conditions, and bEnd.3 (mouse brain microvascular endothelial cell line) cells were cultured under OGD (Control) and Eps conditions for different time: 0h, 2h, 4h, 6h And 8h, the expression of compact protein TJs: Occludin and ZO-1 in bEnd.3 cells was detected by Western Blot experiment. The results are shown in Figure 24, the expression of compact protein in bEnd.3 cells; statistical analysis is shown in Figure 25 and Figure 26, OGD conditions can reduce the expression of ZO-1 and Occludin proteins on bEnd.3 cells; OGD acts for 4h and 6h After treatment, the protein expressions of ZO-1 and Occludin in endothelial cells in the epalrestat group were significantly higher than those in the Control group, with statistical differences (*P<0.05).

The in vitro blood-brain barrier model was established using bEnd.3 cells, and the permeability of the blood-brain barrier was detected after OGD for 4 hours. The statistical results are shown in Figure 27. Compared with the non-OGD group, the BSA-FITC diffusion rate increased after OGD 4h; the use of epalrestat can effectively reduce the BSA-FITC diffusion rate, which is statistically different from the OGD group (*P< 0.05).

The above results indicate that epalrestat can up-regulate the expression of ZO-1 and Occludin on bEnd.3 cells after OGD and reduce the permeability of the blood-brain barrier model in vitro.

Example 9

This example examines the effect of epalrestat on the apoptosis of bEnd.3 cells under OGD conditions.

An in vitro oxygen-glucose deprivation OGD model was established to simulate cerebral ischemia conditions, and bEnd.3 (mouse brain microvascular endothelial cell line) cells were cultured under OGD and Eps conditions for different time: 0h, 2h, 4h, 6h and 8h, using Western Blot assay was used to detect the changes of expression of apoptosis-related proteins in bEnd.3 cells. The results are shown in Figure 28, the expression of apoptosis-related proteins in bEnd.3 cells; statistical analysis is shown in Figures 29 and 30, the expression of pro-apoptotic proteins cleaved-casp3 and Bax in endothelial cells under the action of OGD is up-regulated, and apoptosis is inhibited The expression of protein Bcl2 was down-regulated; compared with the OGD (Control) group, the expression of cleaved-casp3 protein in the endothelial cells of the OGD+Eps group was reduced, and there was a statistical difference between 4h and 6h (*P<0.05). At the same time, statistical analysis of the Bcl2/Bax ratio showed that compared with the OGD4h group, the Bcl2/Bax ratio in the endothelial cells of the OGD+Eps group was significantly increased, with a statistical difference (*P<0.05).

Flow cytometry was used to detect the apoptosis of endothelial cells after OGD 4h. The results are shown in Figure 31, the endothelial cells of each group express PI-PE and AnnexinV-FITC positive situation; statistical analysis is shown in Figure 32, compared with the OGD group, in the OGD+Eps group (AnnexinV ^- FITC) ⁺ /PI- The percentage of endothelial cells decreased significantly (*P<0.05).

The results showed that under OGD conditions, epalrestat could down-regulate the expression of pro-apoptotic proteins and up-regulate the expression of anti-apoptotic proteins in bEnd.3 cells, thereby reducing the apoptosis of endothelial cells after OGD.

Example 10

This example investigates the effect of epalrestat on the autophagy of cerebral microvascular endothelial cells after cerebral ischemic injury.

The modeling, grouping, and administration methods of this example were the same as those in Example 1, and the expression of autophagy-related proteins: LC3B and Beclin1 in cerebral microvascular endothelial cells in ischemic brain tissue was detected by immunofluorescence labeling technology. After 3 days of cerebral ischemia in each group, brain tissue was taken to prepare frozen sections, and LC3B, Beclin1 and CD31 were double-stained.

Immunofluorescence staining was used to detect the co-localization of CD31 and LC3B on brain microvascular endothelial cells by epalrestat. As shown in Figure 33, CD31-labeled brain microvascular endothelial cells were green, LC3B staining was red, and co-expression of CD31 and LC3B was yellow. The results of statistical analysis are shown in Figure 34. Compared with the Sham group, the co-expression mean fluorescence intensity of CD31 and LC3B in ischemic brain tissue increased after 3 days of cerebral ischemia (*P<0.05, **P<0.01); The mean fluorescence intensity of co-expression of CD31 and LC3B in the ischemic brain tissue of the sestat treatment group was higher than that of the normal group (*P<0.05).

Immunofluorescence staining was used to detect the co-localization of epalrestat on brain microvascular endothelial cells and Beclin1. As shown in Figure 35, CD31-labeled brain microvascular endothelial cells were green, Beclin1 staining was red, and CD31 and Beclin1 co-expression was yellow. The results of statistical analysis are shown in Figure 36. Compared with the Sham group, the co-expression mean fluorescence intensity of CD31 and Beclin1 in cerebral ischemia 3d ischemic brain tissue increased (*P<0.05, **P<0.01); The average fluorescence intensity of co-expression of CD31 and Beclin1 in the ischemic brain tissue of the Sestat treatment group was higher than that of the normal group (*P<0.05). The results indicated that after 3 days of cerebral ischemia, epalrestat could promote autophagy of endothelial cells in ischemic brain tissue.

Example 11

This example investigates the effect of epalrestat on autophagy of bEnd.3 cells under OGD conditions.

An in vitro oxygen-glucose deprivation OGD model was established to simulate cerebral ischemia conditions. After the bEnd.3 cells were exposed to OGD conditions for 4 hours, the cell proteins in Control group, Eps group, OGD group and OGD+Eps group were extracted, and Western Blot was used to detect the cells in each group Expression of autophagy-related proteins LC3B-I and LC3B-II.

The results are shown in Figure 37 and Figure 38, the ratio of LC3B-II and LC3B-I in endothelial cells under OGD conditions was significantly increased compared with the non-OGD group (*P<0.05, **P<0.01); compared with the OGD group In comparison, epalrestat could significantly up-regulate the ratio of LC3B-II and LC3B-I under OGD conditions (*P<0.05).

Immunofluorescence technology was used to detect the expression of autophagy-related proteins: Beclin1 and LAMP1 in each group of endothelial cells. The results are shown in Figure 39. CD31-labeled brain microvascular endothelial cells were red, and Beclin1 staining was green. The statistical data analysis results were shown in Figure 40 As shown in Figure 41, the CD31-labeled brain microvascular endothelial cells were red, and the LAMP1 staining was green, and the statistical data analysis results were shown in Figure 42; as shown in Figure 40 and Figure 42, compared with the OGD group, Epa Sestat can promote the expression of Beclin1 and LAMP-1 in endothelial cells under OGD conditions (*P<0.05, **P<0.01).

The expression of P62 protein in the endothelial cells of each group is shown in Figure 43. CD31-labeled brain microvascular endothelial cells are red, and P62 staining is green. The results of statistical data analysis are shown in Figure 44. Compared with the OGD group, epalrestat in OGD inhibited the expression of P62 (*P<0.05).

The above results indicated that after 4 hours of OGD treatment, epalrestat could up-regulate the expression of autophagy proteins in endothelial cells and promote the occurrence of autophagy.

Example 12

AR is aldose reductase, which belongs to a type of aldehyde and ketone reductase. In diabetes, blood sugar rises, glucose metabolism increases, and AR is overactivated. Under the catalysis of AR, excess glucose is converted into sorbitol. Sorbitol accumulates in large quantities in nerve cells, changes osmotic pressure, causes cell edema and rupture, and causes energy metabolism disorders. This example examines the effect of epalrestat on the expression of AR protein in endothelial cells after ischemia.

The modeling, grouping, and administration methods of this example were the same as those in Example 1. Brain tissues of each group were collected at different times of cerebral ischemia, and the expression of AR protein in the ischemic brain tissues was detected by Western Blot experiment. The results are shown in Figure 45 and Figure 46. After 3 days of cerebral ischemia, the expression of AR protein in the ischemic brain tissue of the epalrestat treatment group was lower than that of the normal group, with a statistical difference (**P<0.01).

The co-localization of endothelial cells and AR in each group of cerebral ischemia 3d was detected by immunofluorescence technique. As shown in Figure 47, the CD31-labeled cerebral microvascular endothelial cells were green, the AR staining was red, and the co-staining of CD31 and AR was yellow. CD31 expresses AR in endothelial cells in ischemic brain tissue; statistical analysis The average fluorescence intensity of co-staining of CD31 and AR is shown in Figure 48. After 3 days of cerebral ischemia, CD31 and AR co-staining in ischemic brain tissue of the epalrestat treatment group The average fluorescence intensity of the normal group was lower than that of the normal group, with statistical difference (**P<0.01).

An in vitro oxygen-glucose deprivation OGD model was established to simulate cerebral ischemia conditions, and bEnd.3 cells were cultured at different times of OGD: 0h, 2h, 4h, 6h and 8h, and Western Blot was used to detect the changes of AR protein in endothelial cells. The results shown in Figure 49 and Figure 50 show that the expression of AR protein in endothelial cells increases with the prolongation of OGD action time; when OGD is applied for 4h and 6h, the intervention of epalrestat can significantly reduce the expression of AR in endothelial cells , with statistical difference (*P<0.05, **P<0.01).

The results indicated that after cerebral ischemia injury, epalrestat could effectively reduce the expression of AR protein on endothelial cells.

Example 13

This example investigated the effect of epalrestat on the expression of TJs protein in endothelial cells by regulating the AKT/mTOR/AR signaling pathway under OGD conditions.

An in vitro oxygen-glucose deprivation OGD model was established to simulate cerebral ischemia conditions. After the bEnd.3 cells were exposed to OGD conditions for 4 hours, the cell proteins of Control group, Eps group, OGD group and OGD+Eps group were extracted, and Western Blot was used to detect each group. Group cells express AKT, p-AKT, mTOR and p-mTOR. The results are shown in Figure 51, Figure 52 and Figure 53, the expression of phosphorylated proteins of AKT and mTOR in endothelial cells increased under the action of OGD; statistical analysis data found that compared with the OGD group, p-AKT and mTOR in the endothelial cells of the Eps+OGD group The expression level of p-mTOR protein was significantly decreased with statistical difference (*P<0.05, ***P<0.001).

AKT inhibitor bicalutamide 15μm/L and mTOR inhibitor rapamycin 10nm/L were used as controls to compare epalrestat inhibition of AKT/mTOR signaling pathway. After 4 hours of OGD treatment, endothelial cell proteins were extracted from Control group, OGD group, OGD+Eps group, OGD+AKTi group and OGD+mTOR group, and Western Blot was used to detect AKT, p-AKT, mTOR, p- Expression of mTOR and TJs proteins. The results are shown in Figure 54, Figure 55 and Figure 56. Under OGD conditions, epalrestat, bicalutamide and rapamycin can all reduce the activation degree of the AKT/mTOR signaling pathway; statistical analysis data found that compared with the OGD group , the expressions of p-AKT and p-mTOR proteins in endothelial cells of Eps+OGD group and AKTi+OGD group decreased, with statistical difference (**P<0.01, ***P<0.001); mTORi+OGD group endothelial cells The expression of p-mTOR protein in the cells was significantly lower than that of the OGD group (***P<0.001), while the expression of p-AKT protein was not significantly different (P>0.05).

The analysis of ZO-1 and Occludin protein expression in the cells of each group is shown in Figure 57, Figure 58 and Figure 59. Compared with the OGD group, ZO in endothelial cells in the Eps+OGD group, AKTi+OGD group and mTORi+OGD group -1 and Occludin protein expression increased, with statistical difference (*P<0.05).

The above results indicate that epalrestat and bicalutamide have similar effects under OGD conditions, and can inhibit the activation of AKT/mTOR signaling pathway by down-regulating the phosphorylation of AKT protein, thereby promoting the expression of TJs protein in endothelial cells.

Example 14

This example investigated the effect of epalrestat on endothelial cell apoptosis and autophagy by inhibiting the AKT/mTOR/AR signaling pathway under OGD conditions.

An in vitro oxygen-glucose deprivation OGD model was established to simulate cerebral ischemia conditions, and the AKT inhibitor bicalutamide 15 μm/L and the mTOR inhibitor rapamycin 10 nm/L were used as controls to compare the inhibition of epalrestat on the AKT/mTOR signaling pathway . After 4 hours of OGD treatment, endothelial cell proteins were extracted from Control group, OGD group, OGD+Eps group, OGD+AKTi group and OGD+mTORi group, and Western Blot was used to detect the expression of apoptosis and autophagy-related proteins in cells. The results are shown in Figure 60. Western Blot was used to detect Bax, Bcl2 and cleaved-casp3 apoptosis-related proteins in the cells of each group. Statistical analysis As shown in Figure 61 and Figure 62, the expression of cleaved-casp3 protein in endothelial cells under OGD conditions was significantly higher than that in the control group, with a statistical difference (**P<0.01, ***P<0.001) ;Compared with OGD group, the expression of cleaved-casp3 protein in OGD+Eps group, OGD+AKTi group and OGD+mTORi group decreased, with statistical difference (**P<0.01); and compared with OGD group, The ratio of Bcl2 and Bax protein in the control group and the three inhibitor groups was significantly increased, with statistical difference (**P<0.01, ***P<0.001). The results indicated that under the condition of OGD, epalrestat regulates the ATK/mTOR/AR signal to inhibit the apoptosis of endothelial cells, which is the same as ATK inhibitors and mTOR inhibitors.

The autophagy-related proteins of LC3BI, LC3BII and Beclin-1 in each group of cells were measured by Western Blot, the results are shown in Figure 63, and the statistical analysis results are shown in Figure 64 and Figure 65, LC3BII/LC3BI and Beclin-1 in endothelial cells under OGD conditions Compared with the control group, the expression of -1 protein was significantly increased, with a statistical difference (**P<0.01, ***P<0.001); compared with the OGD group, OGD+Eps group, OGD+AKTi group and OGD The expressions of LC3BII/LC3BI and Beclin-1 proteins in the +mTORi group were significantly increased (***P<0.001); the results indicated that under OGD conditions, epalrestat regulates ATK/mTOR/AR signaling by promoting endothelial The role of autophagy is the same as that of ATK inhibitors and mTOR inhibitors.

Claims (8)

1. The application of epalrestat in preparing the medicine for preventing and treating ischemic stroke is characterized in that the medicine is used for reducing the damage degree of a blood brain barrier in the process of cerebral ischemic injury, maintaining the integrity of the blood brain barrier after the ischemic injury, effectively reducing the infiltration of peripheral inflammatory cells and reducing the inflammatory reaction in ischemic brain tissues; the medicine is used for reducing the generation of apoptosis protein in ischemic brain tissue after cerebral ischemia, and can promote the autophagy of endothelial cells in the ischemic brain tissue.

2. The use of epalrestat according to claim 1 for the manufacture of a medicament for the prevention and treatment of ischemic stroke, wherein the clinical type of ischemic stroke is cerebral thrombosis, lacunar infarction or cerebral embolism.

3. The use of epalrestat according to claim 1 or 2 for the manufacture of a medicament for the prevention and treatment of ischemic stroke, wherein epalrestat is the only active ingredient in the medicament for the prevention and treatment of ischemic stroke.

4. The use of epalrestat according to claim 3 in the preparation of a medicament for preventing and treating ischemic stroke, wherein the medicament for preventing and treating ischemic stroke further comprises pharmaceutically acceptable excipients and/or carriers.

5. The use of epalrestat according to claim 4 in the preparation of a medicament for the prevention and treatment of ischemic stroke, wherein the epalrestat content in the medicament for the prevention and treatment of ischemic stroke is 0.1-99 wt%.

6. The use of epalrestat according to claim 5 in the preparation of a medicament for preventing and treating ischemic stroke, wherein the dosage forms of the medicament for preventing and treating ischemic stroke comprise oral dosage forms and parenteral dosage forms.

7. The use of epalrestat according to claim 6 in the preparation of a medicament for the prevention and treatment of ischemic stroke, wherein the medicament for the prevention and treatment of ischemic stroke is a parenteral dosage form, in particular an injection dosage form, a respiratory dosage form, a dermal dosage form, a mucosal dosage form or a luminal dosage form.

8. The use of epalrestat according to claim 6 in preparing a medicament for preventing and treating ischemic stroke, wherein the medicament for preventing and treating ischemic stroke is in an oral dosage form, specifically a granule, a tablet, a capsule, a pill, a drop pill or an oral liquid preparation.

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