Application of ginsenoside Ro in preparation of medicine for treating cerebral ischemia-reperfusion injury

Abstract

The invention discloses an application of ginsenoside Ro in preparation of a medicine for treating cerebral ischemia-reperfusion injury. According to the application disclosed by the invention, by preparing a middle cerebral artery occlusion / reperfusion (MCAO) mouse model, the ginsenoside Ro is found to be capable of reducing the cerebral infarction volume of the model mouse, improving the pathological injury of brain tissues and increasing the cerebral blood flow at the ischemic side. Meanwhile, the ginsenoside Ro up-regulates the expression of brain tissue tight junction proteins ZO-1 and Occludin, inhibits the expression of matrix metalloproteinase MMP-9 / 2, reduces the leakage amount of Evans blue, and improves the blood brain barrier (BBB) dysfunction. Besides, by preparing an oxygen glucose deprivation / reperfusion (OGD / R) induced mouse brain microvascular endothelial cell line 3 (bEnd.3) cell model, it is found that the ginsenoside Ro can improve the damage of the barrier function of the bEnd.3 cell induced by the OGD / R, it is proved that the ginsenoside Ro can be used for the novel medical application of treating the cerebral arterial thrombosis, and the ginsenoside Ro has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of ginsenoside Ro in the preparation of drugs for treating cerebral ischemia-reperfusion injury. Background Technology

[0002] Stroke is a serious acute cerebrovascular disease that severely endangers human health, and is the second leading cause of disability and death worldwide. Ischemic stroke is the main type of stroke, accounting for 87% of all strokes. Intravenous thrombolysis is currently one of the most effective measures to restore cerebral blood flow in patients with acute ischemic stroke. The commonly used drug is recombinant tissue plasminogen activator (alteplase), but this drug has a narrow therapeutic window (≤ 4.5 h) and carries the potential risk of inducing cerebral hemorrhage. Thrombolytic therapy and reperfusion can restore blood supply and salvage ischemic brain tissue, but reperfusion can further aggravate the degree and extent of brain damage, a condition known as cerebral ischemia-reperfusion injury. Acute ischemic stroke caused by large vessel occlusion can be treated with mechanical thrombectomy within 24 hours of stroke symptom onset. Compared with alteplase, mechanical thrombectomy can improve the functional outcome of patients. However, mechanical thrombectomy requires specialized equipment and physicians trained in endovascular surgery, placing extremely high demands on medical resources. Therefore, in-depth exploration of the pathological processes and mechanisms of action in cerebral ischemia-reperfusion injury, and subsequent drug intervention, is of great significance for improving reperfusion injury caused by the restoration of cerebral blood flow.

[0003] The pathological mechanisms of ischemic stroke are complex, and one of its prominent pathological features is the dysfunction of the blood-brain barrier (BBB). The BBB, located at the blood-brain interface, is a biological barrier that maintains the central nervous system and precisely regulates the homeostasis of the brain's microenvironment. Endothelial cells are one of the most important components of the BBB, responding to ischemia, hypoxia, and potentially harmful chemicals released from the vascular system. Following ischemia, endothelial cells undergo cytoskeletal rearrangement, increased transcellular transport, and alterations in tight junction proteins, exacerbating BBB damage and secondary brain injury. Therefore, exploring the key links and molecular mechanisms involved in the disruption of the brain's microvascular endothelial barrier and identifying targets for the prevention and treatment of BBB damage in ischemic stroke are of great significance for the development of drugs for the prevention and treatment of ischemic stroke.

[0004] Ginseng is the root of the ginseng plant (Panax ginseng), belonging to the Araliaceae family. It is warm in nature, sweet and slightly bitter in taste, and enters the spleen and lung meridians. It has the effects of greatly replenishing vital energy, restoring pulse and consolidating the body, tonifying the spleen and lungs, promoting body fluid production and nourishing blood, and calming the mind and improving intelligence. Ginsenoside Ro is a bioactive natural substance isolated from ginseng root, possessing anti-inflammatory, antioxidant, antithrombotic, antitumor, and lipid-lowering pharmacological activities. However, the application of ginsenoside Ro in ischemic stroke has not yet been reported. Summary of the Invention

[0005] Purpose of the invention: The present invention aims to provide the application of ginsenoside Ro in the preparation of drugs for treating cerebral ischemia-reperfusion injury.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution for the application of ginsenoside Ro:

[0007] This invention provides the application of ginsenoside Ro in the preparation of drugs for treating cerebral ischemia-reperfusion injury.

[0008] This invention provides the application of ginsenoside Ro in the preparation of drugs for preventing cerebral ischemia-reperfusion injury.

[0009] The present invention provides a pharmaceutical composition for treating cerebral ischemia-reperfusion injury, comprising ginsenoside Ro.

[0010] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and is formulated as an injection, tablet, or capsule.

[0011] This invention provides the application of ginsenoside Ro in the preparation of drugs that upregulate the expression of tight junction proteins ZO-1 and Occludin.

[0012] This invention provides the application of ginsenoside Ro in the preparation of drugs that improve cerebral infarction, neurological dysfunction and blood-brain barrier (BBB) ​​dysfunction.

[0013] This invention provides the application of ginsenoside Ro in the preparation of drugs for treating OGD / R-induced bEnd.3 cell barrier function impairment.

[0014] This invention provides the application of ginsenoside Ro in the preparation of drugs that inhibit the expression of MMP-9 and MMP-2 proteins.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention is the first to confirm the application of ginsenoside Ro in the preparation of drugs for treating ischemic stroke, indicating that ginsenoside Ro can be used for a new indication of treating cerebral ischemia-reperfusion injury. The current experimental results provide strong support and basis for the broad application of the new indication of ginsenoside Ro.

[0016] 2. This invention evaluated the therapeutic effect of ginsenoside Ro on ischemic stroke. Ginsenoside Ro reduced the infarct volume in MCAO / R mice, alleviated neurological dysfunction, and improved BBB dysfunction. Furthermore, ginsenoside Ro also reduced barrier function damage in bEnd.3 cells under OGD / R conditions.

[0017] 3. Ginsenoside Ro is used to prepare drugs for treating cerebral ischemia-reperfusion injury, opening up new medical applications for ginsenoside Ro in the treatment of ischemic stroke. Attached Figure Description

[0018] Figure 1 Ginsenoside Ro was used to reduce cerebral infarction volume in MCAO / R mice. A shows a representative photograph of TTC staining in mouse brain tissue, and B shows the quantitative analysis of cerebral infarction volume in mice (mean ± SD, n = 6). ## P < 0.01 vs. Sham group, * P < 0.05, ** P < 0.01 vs. Model group).

[0019] Figure 2 Ginsenoside Ro was used to alleviate brain tissue pathological damage in MCAO / R mice. A shows a representative image of H&E staining morphology in mouse brain tissue; B shows the neurological deficit score of the mice (mean ± SD, n = 6). ## P < 0.01 vs. Shamgroup, ** P < 0.01 vs. Model group).

[0020] Figure 3 Ginsenoside Ro was used to restore cerebral blood flow in MCAO / R mice. A shows Doppler ultrasound images of cerebral blood flow in mice, and B shows the quantitative analysis of cerebral blood flow in mice. (mean ± SD, n = 6, ## P < 0.01 vs. Sham group, ** P < 0.01 (v. Model group).

[0021] Figure 4 Ginsenoside Ro was used to reduce Evans blue leakage in the brain tissue of MCAO / R mice. A is a representative photograph of Evans blue staining in mouse brain tissue; B is the quantitative analysis of Evans blue leakage. (mean ± SD, n = 6, ...) ## P < 0.01 vs. Sham group, ** P < 0.01 vs. Model group).

[0022] Figure 5Ginsenoside Ro increased the expression of tight junction proteins ZO-1 and Occludin in the brain tissue of MCAO / R mice. A shows representative Western blot bands and quantitative analysis of ZO-1; B shows representative Western blot bands and quantitative analysis of Occludin; C shows representative immunofluorescence images of ZO-1; D shows quantitative analysis of immunofluorescence intensity of ZO-1; E shows representative immunofluorescence images of Occludin; and F shows quantitative analysis of immunofluorescence intensity of Occludin (mean ± SD, n = 6). ## P < 0.01 vs. Sham group, * P < 0.05, ** P < 0.01 vs. Model group).

[0023] Figure 6 Ginsenoside Ro reduced the expression of matrix metalloproteinases MMP-9 / 2 in the brain tissue of MCAO / R mice. A shows representative bands and quantitative analysis of MMP-9 using Western blotting; B shows representative bands and quantitative analysis of MMP-2 using Western blotting (mean ± SD, n = 6). ## P < 0.01 vs. Sham group, * P < 0.05, ** P < 0.01 vs. Model group).

[0024] Figure 7 To assess cell viability using the MTT assay, ginsenoside Ro significantly improved OGD / R-induced bEnd.3 cell damage. A shows the effect of ginsenoside Ro on bEnd.3 cell viability under normal conditions, and B shows the effect of ginsenoside Ro on bEnd.3 cell viability under OGD / R conditions (mean ± SD, n = 6). ## P < 0.01 vs. Sham group, * P < 0.05, ** P < 0.01 (v. Model group).

[0025] Figure 8 Ginsenoside Ro significantly improved OGD / R-induced endothelial barrier disruption in bEnd.3 cells. A shows the quantitative analysis of transendothelial resistance in bEnd.3 cells, and B shows the quantitative analysis of Evans blue permeation in bEnd.3 cells (mean ± SD, n = 6). ## P < 0.01 vs. Sham group, *P < 0.05, ** P < 0.01 vs. Model group).

[0026] Figure 9 Ginsenoside Ro was used to increase the expression of OGD / R-induced tight junction proteins ZO-1 and Occludin in bEnd.3 cells. A shows representative Western blot bands and quantitative analysis of ZO-1; B shows representative Western blot bands and quantitative analysis of Occludin; C shows representative immunofluorescence images of ZO-1; D shows quantitative analysis of immunofluorescence intensity of ZO-1; E shows representative immunofluorescence images of Occludin; and F shows quantitative analysis of immunofluorescence intensity of Occludin (mean ± SD, n = 6). ## P < 0.01 vs. Sham group, ** P < 0.01 vs. Model group).

[0027] Figure 10 Ginsenoside Ro significantly reduced the expression of matrix metalloproteinases MMP-9 / 2 in OGD / R-induced bEnd.3 cells. A shows representative bands and quantitative analysis of MMP-9 by Western blotting, and B shows representative bands and quantitative analysis of MMP-2 by Western blotting (mean ± SD, n = 6). ## P < 0.01 vs. Sham group, * P < 0.05, ** P < 0.01 (v. Model group). Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0029] This invention, through the establishment of an MCAO / R mouse model, revealed that ginsenoside Ro can reduce the infarct volume, improve brain tissue pathological damage, and increase cerebral blood flow on the ischemic side of the brain. Simultaneously, ginsenoside Ro significantly reduced Evans blue leakage in the brain tissue induced by MCAO / R, upregulated the expression of tight junction proteins in brain tissue, inhibited the expression of matrix metalloproteinases MMP-9 / 2, and improved BBB dysfunction. Furthermore, through the establishment of an OGD / R model, it was found that ginsenoside Ro can improve OGD / R-induced bEnd.3 cell barrier function damage, confirming the novel medicinal use of ginsenoside Ro in the treatment of ischemic stroke, with broad application prospects.

[0030] Example 1: The effect of ginsenoside Ro on improving MCAO / R-induced brain injury in mice

[0031] Experimental methods

[0032] 1.1 Preparation of MCAO / R mouse model

[0033] A MCAO / R mouse model was established using the suture occlusion method. Male C57BL / 6J mice weighing 20-22 g were selected. Mice were anesthetized by intraperitoneal injection of 3% sodium pentobarbital, fixed in a supine position, and a midline incision was made in the neck. The right common carotid artery, external carotid artery, and internal carotid artery were isolated and exposed. A ligation suture was placed proximally in the common carotid artery, and the external carotid artery was ligated distally. The distal end of the external carotid artery was then freed. A segment of the internal carotid artery was freed deep along the internal carotid artery, and a ligation suture was placed proximally in the external carotid artery. The proximal end of the common carotid artery and the distal end of the internal carotid artery were clamped using small, non-invasive vascular clamps. A small incision was made at the distal end of the external carotid artery using microsurgical scissors, and a suture occluded through the bifurcation of the common carotid artery into the internal carotid artery. The suture segment was inserted until slight resistance was encountered, at which point the tip of the suture segment penetrated approximately 1 mm into the anterior cerebral artery. After insertion, the ligation suture previously placed in the external carotid artery was tightened. One hour after MCAO, the external carotid artery was isolated and exposed, the fixation sutures were loosened, the suture plugs were removed, and the ligation sutures of the common carotid artery were loosened to achieve reperfusion. The neck wound was then routinely sutured. The rectal temperature of the mice was maintained at 37 °C during the operation. After the operation, the mice were placed in a feeding box with clean bedding and had free access to water and food.

[0034] Following MCAO surgery, mice were randomly divided into six groups: sham group, model group, low-dose ginsenoside Ro (20 mg / kg), medium-dose ginsenoside Ro (40 mg / kg), high-dose ginsenoside Ro (80 mg / kg), and positive control group (Human Urinary Kallindinogenase, HUK). The sham group received the same treatment as the other groups except for the absence of a suture plug. Two hours after reperfusion, mice in the Sham and MCAO / R groups were given an equal volume of saline, while the ginsenoside Ro groups were administered different doses of ginsenoside Ro (20 mg / kg, 40 mg / kg, and 80 mg / kg) by gavage. The positive control group received an intraperitoneal injection of HUK (0.0025 PNA / kg). Behavioral evaluations were performed 24 hours later, and brain tissue samples were collected.

[0035] 1.2 TTC staining to determine cerebral infarction volume

[0036] After establishing the brain model using the suture occlusion method, brain tissue samples were collected from each group of mice and frozen at -20 ℃. The frozen brain tissue was then sliced ​​into five coronal slices approximately 2 mm thick on an ice box, and the slices were quickly placed in a 2% TTC solution. Subsequently, they were incubated in a 37 ℃ oven in the dark for 30 min. The slices were then photographed on black cardboard, and the infarct volume was measured using ImageJ image analysis software. The calculation formula is as follows: Infarct volume percentage = Right hemisphere infarct volume / Total brain volume × 100%

[0037] 1.3 Neurological function scores and brain tissue pathological damage

[0038] Neurological deficit is one of the important indicators for evaluating ischemic stroke injury. The degree of neurological deficit in MCAO / R mice was assessed using the Longa Neurological Function Scale. Neurological deficit was divided into five grades: 0 points: normal, no neurological deficit; 1 point: right (paralyzed side) forepaw cannot be fully extended, but there is no obvious circling when walking, mild neurological deficit; 2 points: mouse circles to the right, walks slowly, moderate neurological deficit; 3 points: mouse leans to the right, slowly turns small circles, severe neurological deficit; 4 points: unable to walk spontaneously, loss of consciousness.

[0039] 1.4 Cerebral blood flow

[0040] Twenty-four hours after reperfusion, the mice were anesthetized, placed prone on a temperature-controlled blanket, and their scalps were disinfected with iodine. The scalps were then cut open, and the hair on the skull was removed. Doppler flowmetry was used to measure cerebral blood flow in the mice.

[0041] Experimental results

[0042] 1.1 Ginsenoside Ro reduces cerebral infarction volume in MCAO / R model mice

[0043] The effect of ginsenoside Ro on cerebral infarction volume in MCAO / R mice was investigated. Figure 1 As shown in Figures AB, the brain tissue of mice in the Sham group appeared red, with no obvious infarct area. The brain tissue of mice in the Model group showed severe white infarct foci on the ischemic side. Administration of ginsenoside Ro (40 mg / kg, 80 mg / kg) and HUK significantly reduced the cerebral infarct volume in MCAO / R mice (P < 0.01).

[0044] 1.2 Ginsenoside Ro improves brain tissue pathological damage in MCAO / R mice

[0045] The effects of ginsenoside Ro on pathological damage in the brain tissue of MCAO / R mice were investigated using H&E staining. Figure 2As shown in Figure A, compared to the Sham group, the Model group mice exhibited vacuolation, pyknosis of cell nuclei, a decrease in the number of nerve cells, and widening of perivascular cavities in their brain tissue. In contrast, the pathological damage in the brain tissue of mice treated with ginsenoside Ro (40 mg / kg, 80 mg / kg) and the HUK group was significantly reduced. These results indicate that ginsenoside Ro has an ameliorative effect on the pathological structural damage induced by MCAO / R in cerebral infarction in mice. Figure 2 As shown in Figure B, no significant neurological deficits were observed in the Sham group mice, while the neurological function scores of the MCAO / R group mice were significantly elevated. Administration of ginsenoside Ro (40 mg / kg, 80 mg / kg) after ischemia significantly improved the neurological behavioral scores of the model mice. These experimental results indicate that ginsenoside Ro can improve MCAO / R-induced brain injury.

[0046] 1.3 Ginsenoside Ro improves cerebral blood flow in MCAO / R mice

[0047] like Figure 3 As shown, ginsenoside Ro (40 mg / kg, 80 mg / kg) can increase cerebral blood flow in MCAO / R mice, and the efficacy of ginsenoside Ro at 80 mg / kg is comparable to that of the positive control drug HUK, indicating that ginsenoside Ro can effectively restore cerebral blood flow on the ischemic side of MCAO / R mice.

[0048] Example 2: Ginsenoside Ro improves MCAO / R-induced BBB dysfunction in mice

[0049] Experimental methods

[0050] 2.1 Evans blue permeability assay for BBB permeability in MCAO / R mice

[0051] Twenty-two hours after reperfusion, mice in each group were injected with 2% Evans blue solution via the tail vein. Two hours later, the heart was perfused with 1×PBS until the perfused fluid was no longer blue. The brain was then harvested and photographed. The ischemic side of the brain was harvested, and residual blood vessels were washed away in physiological saline. The water was blotted dry with filter paper and weighed. Pre-chilled formamide and brain tissue were homogenized at a ratio of 1 mL: 0.1 g. The mixture was centrifuged at 4 °C and 12,000 rpm for 20 min. The supernatant was collected, and the absorbance was measured at 620 nm. Quantitative analysis was performed according to a standard curve.

[0052] 2.2 Western blot detection of tight junction proteins and matrix metalloproteinases in mouse brain tissue

[0053] Brain tissue was collected and added with an appropriate amount of lysis buffer containing 1% PMSF, and incubated on ice for 30 min. The lysis buffer was collected and centrifuged for 10 min (4℃, 12000 rpm). An appropriate amount of supernatant was used for protein quantification. The remaining supernatant was added with an appropriate amount of 5× loading buffer, boiled at 100℃ for 5 min, and stored at -80℃. An appropriate concentration of SDS-PAGE gel was prepared to separate the target protein. Wet transfer was performed (200 mA, 3 h), and the protein bands were incubated slowly at room temperature with 5% BSA for 2 h. After blocking, the corresponding primary antibody was added, and the gel was incubated overnight at 4℃. The next day, the protein bands were removed, washed three times with TBST (10 min / wash), and incubated slowly at room temperature with the corresponding secondary antibody for 1.5 h. Finally, ECL developing solution was added under dark conditions, and the bands were exposed using a BIO-RAD Chemi Doc™ XRS gel imaging system. Using β-actin as an internal control, the bands were statistically analyzed using Image Lab software. The relative protein expression level was calculated as: target protein gray value / internal control gray value.

[0054] 2.3 Immunofluorescence technique was used to detect the expression level of tight junction proteins in mouse brain tissue.

[0055] (1) Infusion, fixation, dehydration, embedding, sectioning

[0056] Mice in each group were sacrificed 24 h after MCAO / R surgery. The heart was perfused with pre-cooled PBS, and the brain was harvested. The brain tissue was blotted dry with filter paper and fixed with 4% paraformaldehyde for 48 h, followed by dehydration with 40% sucrose solution until the brain tissue settled. The olfactory bulb and hindbrain were removed. The portion of the brain tissue near the olfactory bulb was placed vertically into an embedding cassette lined with OCT gel. Frozen tissue was stored at -80 °C. The brain tissue was cut into 5 μm sections using a cryostat for later use.

[0057] (2) Staining and photographing

[0058] Brain tissue sections were equilibrated at room temperature for 30 min in a humidified chamber, then rinsed three times (5 min each time) in a glass bath containing pre-chilled PBS, and dried with lint-free paper. The brain tissue was circled with a histochemical pen, and rapid blocking buffer was added and incubated at room temperature for 30 min, then dried with lint-free paper. The corresponding primary antibodies (ZO-1 dilution 1:2000, Occludin dilution 1:1000, CD31 dilution 1:50) were then added and incubated overnight at 4 °C. The sections were rinsed three times with pre-chilled PBS and dried with lint-free paper. The corresponding secondary antibodies were added, and the sections were incubated at room temperature in the dark for 1.5 h, then rinsed three times and dried with lint-free paper. The sections were mounted with DAPI-containing anti-fluorescence quencher and photographed using an LSM700 laser confocal microscope.

[0059] Experimental results

[0060] 2.1 Ginsenoside Ro reduces Evans blue leakage in MCAO / R-induced mouse brain tissue

[0061] To investigate the effect of ginsenoside Ro on the brain ductal bleeding (BBB) ​​in MCAO / R model mice, this study measured the Evans blue leakage in the right-sided damaged brain tissue of the mice. Figure 4 As shown in Figures AB, compared with the Sham group, the Evans blue leakage in the brain tissue of MCAO / R mice was significantly increased. Administration of ginsenoside Ro (40 mg / kg, 80 mg / kg) and HUK significantly reduced the Evans blue leakage in the brain tissue of MCAO / R mice. These results indicate that ginsenoside Ro has a protective effect against MCAO / R-induced BBB damage.

[0062] 2.2 Ginsenoside Ro restores the expression of tight junction protein in the brain tissue of MCAO / R model mice

[0063] To investigate the effect of ginsenoside Ro on MCAO / R-induced brain tissue barrier dysfunction in mice, Western blotting was used to detect the expression of ZO-1 and Occludin in mouse brain tissue. Figure 5 As shown in Figures AB, compared to the Sham group, the expression levels of ZO-1 and Occludin in the brain tissue of MCAO / R mice were significantly decreased (P < 0.01). After ischemia, administration of ginsenoside Ro (40 mg / kg, 80 mg / kg) significantly increased the expression of ZO-1 and Occludin (P < 0.01). Simultaneously, immunofluorescence results ( Figure 5 CF studies showed that administration of ginsenoside Ro (40 mg / kg, 80 mg / kg) after ischemia significantly increased the expression levels of ZO-1 and Occludin in the brain tissue of MCAO / R mice. These results indicate that ginsenoside Ro exerts a BBB-protective effect by increasing the expression levels of tight junction proteins.

[0064] 2.3 Ginsenoside Ro reduces the expression of matrix metalloproteinases in the brain tissue of MCAO / R model mice.

[0065] MMP-9 / 2 is a key molecule promoting the degradation of tight junction proteins and the basement membrane of cells after cerebral ischemia-reperfusion, and it is also an important factor involved in the inflammatory response. Western blotting was used to investigate the effect of ginsenoside Ro on MMP-9 / 2 protein expression in the brain tissue of MCAO / R mice. Figure 6As shown in Figure AB, compared with the Sham group, the expression levels of MMP-9 and MMP-2 in the brain tissue of Model group mice were significantly increased (P < 0.01). Compared with the MCAO / R group, the expression of MMP-9 / 2 in the ginsenoside Ro (40 mg / kg, 80 mg / kg) group and HUK group was significantly decreased (P < 0.01). These results indicate that ginsenoside Ro exerts a BBB protective effect by reducing the expression level of matrix metalloproteinases.

[0066] Example 3: Ginsenoside Ro alleviates OGD / R-induced bEnd.3 cell barrier function damage

[0067] Experimental methods

[0068] 3.1 Preparation of OGD / R-induced bEnd.3 cell model

[0069] bEnd.3 cells in the logarithmic growth phase were harvested, and the high-glucose medium was replaced with a glucose-free medium. The cells were then placed in a tri-gas incubator (1% O2, 5% CO2, 94% N2) for 6 hours. The glucose-free medium was then discarded and replaced with normal medium, and the cells were cultured in a normal incubator for 3 hours. Simultaneously, the control group was replaced with normal medium and cultured in a 5% CO2 normal incubator for 9 hours.

[0070] 3.2 MTT assay for bEnd.3 cell viability

[0071] bEnd.3 cells in logarithmic growth phase were seeded into 96-well plates and cultured at 37 ℃ with 5% CO2 for 24 h. After treatment, cells in each experimental group were incubated with MTT working solution (100 μL / well) in the dark for 2–3 h. The OD value of each well was measured using a microplate reader (measurement wavelength 570 nm, reference wavelength 650 nm), and cell viability was calculated for each group.

[0072] 3.3 bEnd.3 Measurement of transendothelial resistance and determination of Evans blue leakage rate in the chamber.

[0073] bEnd.3 cells 200 μL (5 × 10⁻⁶) 4 (Number of cells / well density) was introduced into a Millicell suspended culture chamber (0.4 μm). 600 μL of complete culture medium was added to the outer chamber of a 24-well plate, and the plate was incubated for 5-7 days. Transfection and OGD / R modeling were then performed, and transendothelial resistance was measured using a resistance meter. The specific measurement steps are as follows: First, the resistance meter was calibrated. Then, the electrodes were sterilized by immersing them in 75% ethanol for 15 min and allowing them to air dry. Finally, the resistance was measured. Before measurement, the 24-well plate was removed from the incubator and placed in a clean bench for 30 min to equilibrate.

[0074] Cells were cultured in microplate chambers. After 7 days of culture, the resistivity reached approximately 170 Ω. After modeling and drug administration, the liquid inside and outside the chambers was discarded. 200 μL of Evans blue albumin solution was added to the inner chamber as a tracer, and 600 μL of 4% BSA solution was added to the outer chamber. After 1 h, the liquid in the outer chamber was collected, and 200 μL was added to each well of a 96-well plate. The OD value was measured at 620 nm using a microplate reader, and the Evans blue leakage was calculated based on the standard curve.

[0075] 3.4 Western blot detection of tight junction proteins and matrix metalloproteinases in bEnd.3 cells

[0076] Cellular protein extraction: Discard the culture medium, wash the culture dishes twice with pre-chilled PBS, aspirate the residual liquid, add 50 μL of cell lysis buffer containing 1% PMSF to each dish, lyse on ice for 5 min, scrape off the protein lysis buffer, transfer to 1.5 mL EP tubes, incubate at 4℃ for 30 min, then centrifuge (12000 rpm, 10 min, 4℃). Collect the supernatant and determine the protein content. Add 5× loading buffer to the remaining supernatant, denature the proteins, and load them onto 10% SDS-PAGE gels for analysis. After wet transfer, incubate the corresponding primary antibody overnight, then incubate with the corresponding secondary antibody, and finally develop using an ECL kit. Expose the bands using a gel imaging system. The expression level of the target protein is expressed as a relative value of the expression level of the corresponding internal control protein.

[0077] Experimental results

[0078] 3.1 Ginsenoside Ro enhances the viability of OGD / R-induced bEnd.3 cells

[0079] The effects of different concentrations of ginsenoside Ro on the viability of normally cultured and OGD / R-induced bEnd.3 cells were detected using the MTT assay. Figure 7 As shown in Figure A: Ginsenoside Ro (5 μM, 10 μM, 20 μM, 40 μM, 80 μM) was applied to bEnd.3 cells cultured under normal conditions for 24 h. The results showed that ginsenoside Ro had no significant effect on cell viability, indicating that it is non-toxic to normal bEnd.3 cells within this concentration range. Figure 7 As shown in Figure B, compared with the Control group, the viability of bEnd.3 cells induced by OGD / R was significantly decreased (P < 0.01). Ginsenoside Ro (5 μM, 10 μM, and 20 μM) all significantly inhibited the OGD / R-induced decrease in cell viability. Therefore, ginsenoside Ro at concentrations of 5 μM, 10 μM, and 20 μM were selected for subsequent experiments.

[0080] 3.2 Ginsenoside Ro improves OGD / R-induced endothelial barrier disruption in bEnd.3 cells.

[0081] Microvascular endothelial cell permeability is an important indicator for assessing endothelial barrier function. Changes in cell permeability in vitro can be measured by transendothelial resistance (TEER) values ​​and by Evans blue-albumin assay to determine the leakage rate of substances from cell culture chambers. This experiment investigated the effect of Ro on TEER and Evans blue leakage in bEnd.3 cells under OGD / R conditions. Figure 8 As shown in Figure AB, the TEER value of cells was significantly decreased (P < 0.01) and the Evans blue leakage was significantly increased (P < 0.01) under OGD 6 h / R 3 h stimulation. The ginsenoside Ro groups (10 μM, 20 μM) and the HUK group significantly restored the OGD / R-induced TEER value of bEnd.3 cells (P < 0.01) and significantly reduced Evans blue leakage (P < 0.01). These results indicate that ginsenoside Ro has an ameliorative effect on OGD / R-induced barrier function damage in bEnd.3 cells.

[0082] 3.3 Ginsenoside Ro restores OGD / R-induced expression of tight junction protein in bEnd.3 cells.

[0083] The effects of ginsenoside Ro on the expression levels of tight junction proteins ZO-1 and Occludin in OGD / R-induced bEnd.3 cells were investigated using Western blot and immunofluorescence techniques. Figure 9 As shown in Figures AB, the expression of tight junction proteins ZO-1 and Occludin in the Model group was significantly decreased (P < 0.01). The ginsenoside Ro groups (10 μM, 20 μM) and the HUK group both significantly restored the OGD / R-induced expression levels of tight junction proteins in bEnd.3 cells (P < 0.01). Immunofluorescence results ( Figure 9 The results (CF) were consistent with those of Western blot. These results indicate that ginsenoside Ro exerts its BBB protective effect by increasing the expression level of tight junction proteins.

[0084] 3.4 Ginsenoside Ro inhibits OGD / R-induced expression of matrix metalloproteinases in bEnd.3 cells.

[0085] Western blot was used to investigate the effect of ginsenoside Ro on the expression level of matrix metalloproteinase MMP-9 / 2 in OGD / R-induced bEnd.3 cells. Figure 10As shown in Figures AB, the expression of MMP-9 and MMP-2 was significantly increased in the Model group cells (P < 0.01). The expression levels of MMP-9 and MMP-2 proteins were significantly decreased in the ginsenoside Ro groups (10 μM, 20 μM) and the HUK group. These results indicate that ginsenoside Ro exerts a BBB protective effect by reducing the expression level of matrix metalloproteinases.

Claims

1. Application of ginsenoside Ro in the preparation of drugs for treating cerebral ischemia-reperfusion injury.

2. Application of ginsenoside Ro in the preparation of drugs for preventing cerebral ischemia-reperfusion injury.

3. A pharmaceutical composition for treating cerebral ischemia-reperfusion injury, characterized in that, Including ginsenoside Ro.

4. The pharmaceutical composition according to claim 3, comprising a pharmaceutically acceptable carrier, and in the form of an injection, tablet or capsule.

5. Application of ginsenoside Ro in the preparation of drugs that upregulate the expression of tight junction proteins ZO-1 and Occludin.

6. Application of ginsenoside Ro in the preparation of drugs to improve cerebral infarction, neurological dysfunction, brain injury or blood-brain barrier dysfunction.

7. Application of ginsenoside Ro in the preparation of drugs to improve OGD / R-induced bEnd.3 cell barrier function damage.

8. Application of ginsenoside Ro in the preparation of drugs that inhibit the expression of MMP-9 and MMP-2 proteins.

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