Use of astilbin in preparation of drugs for improving secondary brain injury after cerebral hemorrhage

Abstract

The application discloses a use of astilbin in preparation of a medicine for improving secondary brain injury after cerebral hemorrhage. The application is proved by a behavior experiment, a Perls staining experiment, a Nissl staining experiment, a Tunel staining experiment, an Evans blue penetration experiment, an ELISA experiment and an immunofluorescence staining experiment. The experiment proves that astilbin can improve the prognosis of secondary brain injury such as edema, iron deposition and neuron necrosis and apoptosis after cerebral hemorrhage by promoting Treg cells to be enriched in the brain tissue around the brain hematoma, secreting anti-inflammatory factors and creating an environment for inhibiting inflammation, and reduce the nerve function injury caused by cerebral hemorrhage. The application provides an experimental basis and a theoretical basis for astilbin to treat secondary brain injury after cerebral hemorrhage.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, and in particular to the use of astilbin in the preparation of a drug for improving secondary brain injury after cerebral hemorrhage. BACKGROUND

[0002] Intracerebral hemorrhage (ICH) is a type of stroke, which is caused by blood vessel rupture and intracerebral hematoma formation. Due to the high mortality rate of the disease, it has always been an important public health problem. 15-20% of strokes are classified as ICH, and 40% of ICH patients die within 30 days without intervention. The high mortality rate in the acute phase has attracted widespread attention to ICH. The volume of ICH is an important predictor of 30-day mortality. 30cm 3 is a widely accepted boundary, and patients with less than 30cm 3 have a lower 30-day mortality rate. ICH can be divided into primary and secondary. Primary hypertensive arteriolar sclerosis and amyloid angiopathy are common causes of primary ICH. Secondary ICH is usually caused by vascular malformations, trauma, and coagulation dysfunction. The primary injury of ICH is mechanical injury caused by hematoma compression, such as intracranial pressure and brain hernia. Secondary brain injury (SBI) mainly refers to the deterioration of the disease and the aggravation of brain tissue damage due to secondary changes on the basis of primary brain injury, including brain edema, blood-brain barrier (BBB) damage, and neuronal cell death caused by hematoma and its released substances (such as hemoglobin, thrombin, hematin, and iron ions); its physiological and pathological mechanisms: mitochondrial damage, oxidative stress, and inflammatory response.

[0003] In recent years, the innovative application of traditional Chinese medicine has become a trend, and the anti-inflammatory effect of many Chinese medicines in different disease processes has become a research hotspot. Astilbin is a kind of dihydroxy flavonoids, which is widely present in many Chinese herbal medicines and daily diet such as Smilax, Hypericum perforatum, grape, and grape wine. In recent years, astilbin has attracted attention due to its anti-inflammatory and antioxidant effects. The anti-inflammatory mechanism of flavonoids has been studied in different disease models such as fatty liver, hyperuricemia, colitis, and osteoarthritis. According to the study of the tissue distribution of astilbin in rats, astilbin can cross the BBB and play a role in the brain. Few studies have focused on the intracranial effect of astilbin. So far, no one has studied the anti-inflammatory effect of astilbin in the ICH model.

[0004] Regulatory T cells (Treg) are a subset of CD4+ T cells defined by the characteristic transcription factor Foxp3, and Treg cells expressing Foxp3 play a key role in maintaining immune homeostasis and preventing autoimmunity. It has been reported that there is a large accumulation of Treg cells in the mouse brain after ischemic stroke, which enhances the recovery of neurological function in the chronic stage of ischemic brain injury. Brain Treg cells are expanded in head and neck lymph nodes and driven to infiltrate the brain by chemokines CCL-1 and CCL-20. Recent studies have shown that using single-cell RNA sequencing and flow cytometry, Treg cells were found to infiltrate the brain 1-5 weeks after experimental stroke in mice. Mechanistically, Treg cell-derived osteopontin acts through integrin receptors on microglia to enhance microglial repair activity, thereby promoting oligodendrocyte generation and white matter repair. Anti-inflammatory effects of Treg cells in ICH models have also been reported. CD4+CD25+ Treg cells were isolated from the axillary, inguinal lymph nodes and spleen of mice and injected into mice via the tail vein. Studies have shown that tail vein injection of Treg cells can significantly improve short-term and long-term neurological function, reduce perihematoma edema and BBB permeability.

[0005] Meanwhile, quercetin can regulate immunity by promoting the differentiation of T cells into Treg cells, secreting anti-inflammatory factors to inhibit inflammation and reduce inflammatory damage, and this mechanism has been verified in various disease models. Studies on the immune regulation of quercetin in a mouse model of colitis have shown that quercetin can effectively induce Treg cells to secrete cytokines such as TGF-β1 and IL-10 in vitro, thereby participating in immune regulation. For rheumatoid arthritis mice, compared with methotrexate alone, the combination of quercetin and methotrexate significantly inhibited the differentiation of T cells into Th1 and Th17 cells, while significantly enhancing the differentiation into Treg cells, thereby reducing the damage of rheumatoid arthritis. For autoimmune myasthenia gravis (MG) in rats, quercetin can reduce the severity of MG by reducing autoantigen-specific antibodies, upregulating Treg cells and downregulating Th17 cells. However, it is not clear whether Treg cells promoted by quercetin to differentiate from T cells can promote Treg cells to enter the brain tissue around the brain hematoma after ICH, thereby playing an anti-inflammatory role. SUMMARY

[0006] The present application focuses on exploring the mechanism of action of the flavonoid quercetin in treating ICH and its secondary damage, and evaluating the efficacy of quercetin on secondary brain injury (SBI) after ICH. Experimental results have shown that quercetin can reduce SBI after ICH by enriching Treg cells through the BBB.

[0007] The technical scheme of the present application is:

[0008] The present application provides the use of astragalin in the preparation of a drug for improving secondary brain injury after cerebral hemorrhage.

[0009] In some embodiments, astragalin reduces apoptosis of brain cells after cerebral hemorrhage, reduces necrosis of neurons after cerebral hemorrhage, reduces iron deposition in brain tissue after cerebral hemorrhage, and improves the prognosis of neurological function induced by cerebral hemorrhage.

[0010] In some embodiments, astragalin reduces secondary brain injury after cerebral hemorrhage by regulating Treg cells to pass through the blood-brain barrier through chemotaxis.

[0011] In some embodiments, astragalin promotes the accumulation of Treg cells characterized by the transcription factor Foxp3 in the damaged brain tissue around the hematoma, and plays an immunosuppressive role of Treg cells.

[0012] In some embodiments, astragalin promotes the expression of CCL-1 and CCL-20 chemokines, and at the same time expresses anti-inflammatory factors IL-10, IL-35, and TGF-β1, which promotes the formation of an anti-inflammatory microenvironment in the brain tissue around the hematoma, thereby inhibiting the inflammatory response of the damaged brain tissue and reducing secondary brain injury.

[0013] The present application research shows that astragalin improves the prognosis of SBI such as edema, iron deposition, and neuronal necrosis and apoptosis after ICH by promoting the chemotaxis of Treg cells in the damaged brain tissue around the hematoma, secreting anti-inflammatory factors, and creating an anti-inflammatory environment, thereby reducing neurological damage caused by ICH. The present application provides an experimental basis and theoretical basis for the treatment of SBI after ICH by astragalin. BRIEF DESCRIPTION OF DRAWINGS

[0014] The present application will be further described below in conjunction with the drawings and examples:

[0015] Figure 1 is an ICH model and comparison with the control group, scale: 1mm: a. Sham group brain coronal section, right cerebral cortex to striatal area with blood needle tract left after sham operation; b. Brain coronal section 24 hours after ICH modeling, right brain striatal area injection of autologous arterial blood to form a circular hematoma;

[0016] Figure 2 is the data of the stepping experiment: a. forelimb stepping experiment, n=6; b. hindlimb stepping experiment, n=6; P<0.0001 vs Sham; P<0.05 vs ICH + DMSO; P<0.01 vs ICH + DMSO; P<0.05 vs ICH + PBS; P<0.01 vs ICH + PBS;

[0017] Figure 3 is the results of the tape test: n=6;####P<0.0001 vs Sham; ****P<0.0001 vs ICH + DMSO; @@@@ P<0.0001 vs ICH + PBS;

[0018] Figure 4 is the results of the rotarod test: n=6;####P<0.0001 vs Sham; ****P<0.0001 vs ICH + DMSO; @@@@ P<0.0001 vs ICH + PBS;

[0019] Figure 5 is the results of the modified Garcia score test: n=6;####P<0.0001 vs Sham; ****P<0.0001 vs ICH + DMSO; @@@@ P<0.0001 vs ICH + PBS;

[0020] Figure 6 Figure 7 is the results of the Prussian blue staining test: a. The general appearance of the hematoma and the surrounding damaged brain tissue was observed under a microscope at 100x. The black square was enlarged to 400x to observe the typical performance of iron deposition in the damaged brain tissue around the hematoma; b. Four 400x fields of view were taken from the adjacent damaged brain tissue around the hematoma on each brain section. Image J software was used to calculate the iron deposition area / total area around the hematoma; n=6, data was expressed as mean + SD,####P<0.0001 vs Sham; ****P<0.0001 vs ICH + DMSO; @@@@ P<0.0001 vs ICH + PBS, the concentration of the quinquefolin administered by intraperitoneal injection was 100 mg / kg;

[0021] Figure 7 is Nissl staining to evaluate the number of surviving neurons: a. Microscopic appearance of Nissl staining of brain tissue damaged around the hematoma in each group. The left side is the overall situation around the hematoma, and the scale bar is 200 μm. The right side is an enlarged view of the black square area, and the arrow points to the surviving neurons in the damaged brain tissue around the hematoma. The scale bar is 50 μm. b. The number of Nissl staining positive surviving neurons per 400x microscopic field of brain tissue around the hematoma. n=6, data is expressed as mean + SD,####P<0.0001 vs Sham; ****P<0.0001 vs ICH+DMSO;@@@@P<0.0001 vs ICH+PBS;△P<0.05 vs ICH+astilbin 60mg / kg;

[0022] Figure 8 is Tunel staining to count the number of apoptotic neuron cells in the damaged brain tissue around the hematoma: a. Neun staining of surviving and apoptotic neuron cytoplasm, DAPI staining of cell nucleus, Tunel staining with Neun and DAPI, Tunel positive can distinguish apoptotic neuron cells, scale bar=50 μm; b. Statistics of Tunel staining positive apoptotic neuron cells in each group, n=6, data is expressed as mean + SD,####P<0.0001 vs Sham; ****P<0.0001 vs ICH+DMSO;@@@@P<0.0001 vs ICH+PBS, the concentration of astilbin injected into the astilbin administration group is 100 mg / kg;

[0023] Figure 9 is the result of dry-wet method to determine the water content of the ipsilateral brain, contralateral brain and cerebellum of each group of mice: a. The percentage of the water content of the ipsilateral brain to the total weight of the contralateral brain of the 8 groups of mice; b. The percentage of the water content of the contralateral brain to the total weight of the ipsilateral brain of the 8 groups of mice; c. The percentage of the water content of the cerebellum to the total weight of the cerebellum of the 8 groups of mice, n=6, data is expressed as mean + SD,####P<0.0001 vs Sham; ***P<0.001 vs ICH+DMSO; **P<0.01 vs ICH+DMSO;@@@@P<0.0001 vs ICH+PBS;

[0024] Figure 10BBB injury was observed by Evans blue extravasation: a. The brain tissue around the hematoma was observed for Evans blue dye extravasation, scale bar: 1 mm; b. The ratio of Evans blue brain homogenate OD value to the contralateral brain, n = 6, data were expressed as mean + SD, ####P<0.0001 vs Sham; ****P<0.0001 vs ICH + DMSO; △△△△P<0.0001 vs ICH + astilbin 60 mg / kg;

[0025] Figure 11 : a. The content of T cell chemotactic factor CCL-1 in peripheral blood, n = 6; b. The content of T cell chemotactic factor CCL-20 in peripheral blood, n = 6; data were expressed as mean + SD, ####P<0.0001 vs Sham; ****P<0.0001 vs ICH + DMSO; @P<0.05 vs ICH + PBS; @@ P<0.01 vs ICH + PBS; △△P<0.01 vs ICH + astilbin 60 mg / kg; △△△△P<0.0001 vs ICH + astilbin 60 mg / kg;

[0026] Figure 12: a. The content of anti-inflammatory factor IL-10 in peripheral blood; b. The content of anti-inflammatory factor IL-35 in peripheral blood; c. The content of anti-inflammatory factor TGF-β1 in peripheral blood, n = 6, data were expressed as mean + SD, ####P<0.0001 vs Sham; ***P<0.001 vs ICH + DMSO; ****P<0.0001 vs ICH + DMSO; @@@P<0.001 vs ICH + PBS; @@@@P<0.0001 vs ICH + PBS; △△△△P<0.0001 vs ICH + astilbin 60 mg / kg;

[0027] Figure 13The results of immunofluorescence staining of Foxp3 protein: a. Observe the staining of hematoma and brain tissue around hematoma under low power microscope, and select brain tissue around hematoma under high power microscope; DAPI is used for nuclear staining; after fusion, cells expressing Foxp3 protein are shown; b. Under a microscope at 400x, the number of Foxp3 immunofluorescence staining positive cells in brain tissue around hematoma in each group is counted; n = 6, the data is expressed as mean + standard deviation (mean + SD), **** P < 0.0001 vs ICH + DMSO; @@@@ P < 0.0001 vs ICH + PBS, the concentration of the administration of reynoutria glycoside in the reynoutria glycoside group is 100 mg / kg;

[0028] Figure 14 The mechanism of reynoutria glycoside in playing an anti-inflammatory role in chemotaxis of Treg cells. DETAILED DESCRIPTION

[0029] The above scheme is further described below in combination with specific embodiments.

[0030] 1. Materials and methods

[0031] 1.1 Experimental animals

[0032] Male C57BL / 6J mice were purchased from Zhaoyan New Drug Research Center Co., Ltd. The mice were raised in a mouse house that met the regulations, and were supplied with dry and soft bedding, feed, and double distilled water, and were raised to about 25 g. In the present application, the use of animals was reviewed and approved by the ethics committee of Suzhou University, and under the supervision of the ethics committee, we strictly followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and as much as possible, the number of experimental animals used in each experimental project was limited, and as much as possible, the operation was performed after anesthesia to alleviate the suffering of the experimental animals.

[0033] 1.2 C57BL / 6J mouse modeling

[0034] The weight of C57BL / 6J mice is about 25g, isoflurane gas anesthesia and maintenance; after anesthesia, the mouse tail is slightly heated to dilate the tail artery and better expose the arteriole; the right hand holds a 1ml insulin needle, and the tail artery is punctured with negative pressure. When blood is drawn, about 50μl of autologous arterial blood is drawn; the Hamilton micro-liquid feeding needle with a volume of 50μl sucks about 30μl of autologous blood which has not yet coagulated; after blood collection, the mouse is fixed in a prone position on the brain stereotaxic apparatus; the hair on the top of the head is removed, the scalp is cut along the midline, and the mucosa is separated to expose the range of anterior fontanel, posterior fontanel and the bony structure in between; the needle point is located 2mm right to the mouse anterior fontanel, a small hole is drilled, and the needle is vertically inserted about 3.5mm, and the autologous blood is injected at a speed of 2μl / min. The injection is completed in about 15min; the needle stays in the brain tissue for 10-15min to coagulate the hematoma; slowly retract the needle at a speed of about 0.5mm / min, and after completely retracting the needle, bone wax is used to cover the hole, the scalp is sutured, and the mouse is placed at room temperature to recover after isoflurane metabolism; part of the mice are decapitated to take out the brain after 1day to verify that the autologous blood is indeed injected into the striatal area and the hematoma is in place; the mice continue to be fed with sufficient water, and the samples are taken at the preset time point.

[0035] 1.3 Experimental design

[0036] Male C57BL / 6J mice were divided into 8 groups, namely sham operation group (Sham), ICH group, drug administration group: ICH+Rhaponticum 60mg / kg, ICH+Rhaponticum 80mg / kg, ICH+Rhaponticum 100mg / kg, ICH+CD28 superagonists (CD28SA), solvent control group: ICH+dimethylsulfoxide (dimethylsulfoxide, DMSO), ICH+phosphate buffered saline (phosphate buffered saline, PBS).

[0037] According to the fat-soluble and boiling water-soluble characteristics of astilbin, we first dissolved astilbin powder in dimethyl sulfoxide (DMSO), and then diluted it with warm (37°C) normal saline until the concentration of DMSO was not higher than 0.4%. According to the weight of the mice and the pre-set concentration gradient (60 mg / kg, 80 mg / kg, 100 mg / kg), we adjusted the concentration of the prepared drug. According to the literature, we prepared CD28SA injection by adding CD28SA to phosphate buffered saline (PBS) buffer at a concentration of 150 μg / kg. Solvent control group: DMSO: liquid DMSO was added to normal saline to dilute to a concentration of 0.4%. PBS: 80.0 g of NaCl, 2.0 g of KCl, 28.9 g of Na2HPO4 . 12H2O, 2.0 g of KH2PO4, and water to 2000 ml, and used after dilution 5 times.

[0038] 1.4 Administration

[0039] The sham operation group only inserted the needle and did not inject autologous blood or give intraperitoneal administration. The ICH group only modeled and did not give intraperitoneal administration. The astilbin administration group was given intraperitoneal injection of astilbin at concentrations of 60 mg / kg, 80 mg / kg, and 100 mg / kg. The DMSO solvent control group was given intraperitoneal injection of DMSO at a concentration of 0.4%. The CD28SA administration group was given intraperitoneal administration of CD28SA at a concentration of 150 μg / kg. The PBS solvent control group was given intraperitoneal administration of PBS at the same volume as the CH28SA administration group.

[0040] The above reagents were injected intraperitoneally into the mice after modeling, and the specific time points were adjusted according to different experiments.

[0041] 1.5 Behavioral test

[0042] We selected four time points: one day before modeling, three days, seven days, and fourteen days after modeling, to perform four tests: modified Garcia score, rotarod test, paper removal test, and stepping test, to evaluate the behavioral disorders of the mice in each group before and after modeling, and whether or not they were given drugs.

[0043] 1.5.1 The modified Garcia score

[0044] The test items included autonomous movement (5 min), limb movement, forelimb extension movement, grip and climbing ability (the above four items were scored according to 0-3 points), and bilateral body tactile reflex and bilateral mustache touch response (the above two items were scored according to 1-3 points).

[0045] 1.5.2 Rotarod test

[0046] Mice were placed on the fatigue rotarod apparatus and allowed to run, and the time from the start of running until the mouse fell off the rotarod was calculated to assess the motor ability of the mice. The rotarod started at 4 rpm / min and accelerated uniformly to 30 rpm / min within 1 min. The test ended when the mouse fell off, and the time on the rod was recorded. The mouse was considered to have fallen off when it had rotated two full revolutions with the rotarod. All mice were trained for three days before the formal test, and data were recorded on the day before modeling as the baseline, and the rotarod test data were recorded on days 3, 7, and 14 after ICH modeling.

[0047] 1.5.3 Paper removal test

[0048] The paper removal test was used to assess the limb sensory function and motor function of the mice. Before the test, the mice were placed in a glass container for about 1 min to familiarize them with the environment, and then a 3 mm * 4 mm piece of paper was cut as a tactile stimulus and attached to the palm of the mouse's left forelimb. The mouse was then placed back in the glass container, and the timing began. Two times were recorded: contact time, the time when the mouse reacted to the presence of the paper by lifting its forelimb and touching the paper with its mouth and nose; and paper removal time, the time from the start of timing until the mouse tore off the paper. All mice were trained for three days before the formal test, and data were recorded on the day before modeling as the baseline, and the paper removal test data were recorded on days 3, 7, and 14 after ICH modeling.

[0049] 1.5.4 Foot-fault test

[0050] The foot-fault test was used to assess the impairment of the walking function of the mice. The mice were placed on a 25 cm * 35 cm grid woven from metal wires, each with a 1 cm2 gap, to encourage the mice to walk on the metal wires, and the experimenter recorded a video from below the grid to calculate the number of foot-faults and the total number of steps taken by the mice. The number of foot-faults and the total number of steps taken by the mice were calculated for the affected forelimb and hindlimb within 1 min; the ratio of foot-faults / total steps was calculated. All mice were trained for three days before the formal test, and data were recorded on the day before modeling as the baseline, and the foot-fault test data were recorded on days 3, 7, and 14 after ICH modeling.

[0051] 1.6 Collection of mouse brain tissue specimens

[0052] After 3 days of continuous administration, we took the 72-hour postoperative observation node. Anesthesia: After weighing, the mice were anesthetized with isoflurane gas; perfusion and sampling: after complete anesthesia, the mice were fixed on the operating table in a supine position, the skin was cut longitudinally, the chest and abdominal cavity was opened, the ribs on both sides were cut, the xiphoid was clamped and pulled upward, the heart was completely exposed, the right atrial appendage was cut, the syringe needle was fixed in the left ventricle, PBS buffer was slowly injected, the blood flowed out from the right atrial appendage, the blood color gradually faded, until the liver and lung turned white, and the right atrial appendage flowed out colorless and clear liquid; fixation: then continue to perfuse 20 ml of 4% paraformaldehyde, cut off the mouse at the neck, completely peel off the mouse brain tissue, and soak in 4% paraformaldehyde for 24 hours (the fixing solution is sufficient, 20-30 times the volume of the brain tissue); washing: alcohol rinse for several hours; dehydration: the brain tissue was sequentially immersed in 70%, 80%, 90% ethanol solution for 30 min each, and then immersed in 95%, 100% ethanol solution for 20 min each; transparency: the brain tissue was immersed in xylene for 20 min;

[0053] wax and embedding: immersed in paraffin-xylene mixture, then immersed in paraffin for waxing. After waxing, put it in the box and embed it with paraffin; sectioning: sectioning with a sectioning knife, thickness about 4-6 μm; mounting: apply a thin layer of protein glycerol on a clean glass slide, flatten the wax slice in warm water, pick it up and place it on the glass slide, and put the glass slide in a 45°C incubator until it is dry. After the preparation of paraffin sections, store and dry them in a cool and dry place for subsequent Nissl staining, Tunel staining, and Perls staining.

[0054] 1.7 Perls staining

[0055] Perls staining, also known as hemosiderin staining, separates trivalent iron ions from proteins in cells or interstitial space by dilute hydrochloric acid, producing insoluble blue compounds that indicate iron deposition in tissues. The experimental operation steps are designed as follows: baking and dewaxing and rehydrating as before; PBS wash the brain tissue sections 3 times, 5 minutes each time, and circle the brain tissue with a histological pen; mix equal amounts of 5% potassium ferrocyanide and 10% hydrochloric acid, add 50 μl of Perls solution to each brain tissue section, and incubate for 1 hour; PBS wash the brain tissue sections 5 times, 5 minutes each time; add 50 μl of DAB to each brain tissue section and incubate for 3 min; add 50 μl of hematoxylin (Sigma-Aldrich) to each brain tissue section for counterstaining for 3 min, and wash with PBS 3 times; sequentially immerse in 70%, 80%, 90%, 95%, and 100% ethanol for 3 min each for dehydration; immerse in xylene twice for 5 min each for transparency; observe the iron deposition in the damaged brain tissue under a light microscope; after taking pictures of the damaged brain tissue around the hematoma under a microscope, take 4 pictures of the 400x field of view of the adjacent damaged brain tissue, and use Image J software to quantify the iron deposition and calculate the ratio of iron deposition area to total area.

[0056] 1.8 Nissl staining

[0057] Nissl staining is to use toluidine blue dye to stain specific basophilic granules (Nissl bodies) in the cytoplasm of neurons. 0.25 g of toluidine blue crystals is dissolved in 25 ml of 70% ethanol, centrifuged at 12,000 rpm / min for 15 minutes, the supernatant is taken and the sediment is discarded. The toluidine blue solution is mixed with 0.9% sodium chloride solution at a volume ratio of 1:9 to prepare a working solution of toluidine blue. Baking: 8 groups of 6 slices from different mice of the same layer are placed in an oven at 70°C for 3 hours to fully melt the paraffin; deparaffinization and rehydration: immerse the slices in two kinds of xylene solution for 10 minutes each, immerse in 95% ethanol and 80% ethanol twice, 5 minutes each time; wash the slices with distilled water for 3 times, 20 seconds each time; staining: use the histology pen to completely circle the brain tissue, add 50 μl of toluidine blue working solution to each brain slice, so that the toluidine blue working solution can fully stain the brain tissue without leaking out of the circle. Place in a 50°C incubator for 40 minutes; washing: wash the slices with distilled water for 3 times, 20 seconds each time; dehydration: immerse in 70%, 80%, 90%, 95%, and 100% ethanol for 3 minutes each; transparency: immerse in xylene twice for 5 minutes each time; mounting: drop neutral balsam on the brain tissue, cover with a cover glass to mount, and observe under an optical microscope to count the number of surviving neurons under each 400x field; select the most effective concentration of roxburghin for subsequent experiments.

[0058] 1.9 Tunel staining

[0059] Recombinant terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) uses recombinant terminal deoxynucleotidyl transferase (rTdT enzyme) to catalyze the incorporation of fluorescein-12-dUTP at the 3'-hydroxyl end of DNA to measure the fragmented DNA of apoptotic cells. The Tunel staining reagent is prepared according to the Tunel kit instructions. The sodium citrate solution is prepared by dissolving 1000 ml of double distilled water with sodium citrate dry powder to obtain a sodium citrate stock solution. The steps are as follows: baking and dewaxing, rehydrating and staining with hematoxylin; antigen repair: take the sodium citrate stock solution, dilute 1:1 (500 ml stock solution diluted with 500 ml double distilled water); take out 500 ml and heat in a microwave oven for 5 min (hot sodium citrate), and take out another 500 ml and place it outside (cold sodium citrate) for standby; place the slices in the hot sodium citrate and boil for 5 min; take out and place in the cold sodium citrate and cool to room temperature. Repeat the boiling and cooling step again; wash: immerse the slices in PBS and shake for 5 min, replace with clean PBS and immerse and shake again for 5 min; perforation: circle the brain tissue with a histology pen, add cell perforation solution dropwise, stand for 15 min, and wash with PBS for 3 times; staining: add 50 μl of Tunel staining solution to each brain slice, and place in a 37°C incubator for 1 hour in the dark (the subsequent steps are all operated in the dark); blocking: wash the staining solution with PBS, add blocking solution, and add 50 μl of blocking solution to each brain slice as above. Place in a 37°C incubator for 1 hour in the dark; add NeuN immunofluorescence primary antibody solution (dilution ratio according to instructions), incubate overnight at 4°C in the dark; wash the slices with PBST for 3 times, 5 min each time. At the same time, add NeuN corresponding species immunofluorescence secondary antibody solution, incubate at 37°C in the dark for 1 hour; wash the slices with PBST for 3 times, 5 min each time. DAPI mounting, stand for 10 min, and observe the brain tissue around the hematoma under a fluorescence microscope.

[0060] 1.10 Brain water content analysis

[0061] The wet weight and dry weight of the left brain, right brain and cerebellum of each drug-treated mouse after modeling and drug administration for 3 days were weighed, and the water content of different parts of the mouse brain was calculated. The higher the water content, the more severe the brain edema, and because brain edema is often caused by inflammation, it is also a side proof that the higher the water content, the more severe the inflammation.

[0062] The water content of the left brain, right brain and cerebellum of each group of mice was calculated according to the formula for calculating brain water content

(wet weight-dry weight) / wet weight

[0063] 1.11 Evans blue

[0064] The blood-brain barrier damage caused by ICH hematoma compression can cause hemoglobin, hematin, iron ions and other substances in the brain blood vessels to enter the brain tissue, exacerbate the inflammation of the brain tissue around the hematoma, and inflammation can exacerbate BBB damage, entering a vicious cycle. Only when the BBB is damaged can Evans blue injected into the circulatory system enter the brain tissue, and because of the efflux function of living cells, only dead cells will be stained blue. We use the penetration characteristics of Evans blue dye to reflect the BBB damage and surrounding brain cell necrosis around the hematoma. The deeper the blue penetration, the more severe the BBB damage and the more brain cells around the hematoma die. Reference: WANG D, LI S, CHEN J, et al. The Effects of Astilbin on Cognitive Impairments in a Transgenic Mouse Model of Alzheimer's Disease [J]. Cellular and molecular neurobiology, 2017, 37(4): 695-706.

[0065] The ratio of absorbance of the affected cerebral hemisphere to that of the contralateral cerebral hemisphere was used for statistical comparison.

[0066] 1.12 ELISA

[0067] At 72 hours after ICH model establishment, we collected blood samples from the anterior orbital plexus of each mouse. The blood samples were centrifuged at 3000 rpm / min at 4°C for 10 minutes. Then the supernatant was collected. According to the manufacturer's instructions, the levels of TGF-β1, IL-35, IL-10, CCL-1 and CCL-20 were detected using specific ELISA kits (purchased from Wuhan Fine Biotechnology Co., Ltd.).

[0068] 1.13 Immunofluorescence staining

[0069] The steps are as follows: antigen repair: use sodium citrate boiling-cooling method for antigen repair, the specific method is the same as before; washing: immerse the section in PBS for 5 minutes, replace PBS and immerse again for 5 minutes; perforation: use a histological pen to circle the brain tissue, add 0.2% Triton X-100 dropwise, stand for 15 min, wash with PBS for 3 times; blocking: wash with PBS for 3 times, add 5% BSA blocking solution, add 50 μl blocking solution to each brain section as above. Place in a 37℃ incubator for 1 hour; primary antibody incubation: dilute Foxp3 antibody according to the instructions 1:50, add 50 μl to each brain section, incubate at 4℃ overnight; wash the section with PBST for 3 times, 5 min each time. At the same time, add the corresponding species of immunofluorescence secondary antibody solution (dilution ratio according to the instructions), incubate at 37℃ in the dark for 1 hour; wash the section with PBST for 3 times as before, 5 min each time; DAPI mounting, stand for 10 min, then observe the brain tissue around the hematoma under a fluorescence microscope.

[0070] 1.14 Statistical analysis

[0071] All data were expressed as means + SD, and analyzed by one-way ANOVA using SPSS 22.0 statistical analysis software. P<0.05 was considered statistically significant.

[0072] 2. Results

[0073] 2.1 Survival rate of mice

[0074] 8 groups of mice were modeled and administered, a total of 410 mice were used, of which 45 mice in the sham group all survived, and 365 mice in the ICH model group finally survived 310 mice, with an overall survival rate of 84.9%. Among the 355 surviving mice, 42 mice were randomly selected from each group for subsequent experiments.

[0075] 2.2 ICH modeling and verification in mice

[0076] Autologous blood was injected into the striatum through the skull, and a stable round hematoma was formed about 24 hours later. Mice in the sham operation group ( Figure 1 a) After PBS perfusion, the brain was taken out, the brain was complete, no blood filaments were left, and there was a needle hole on the right side of the cerebral cortex above the striatum, which was the top of the puncture channel, and a micro-injection device entered the striatum from there, and the coronal brain section had no hematoma formation except the bloody needle channel; ICH group mice ( Figure 1 b) After PBS perfusion, the whole body was similar to the sham operation group, the brain was complete, no blood filaments were left, excluding the interference caused by insufficient perfusion, and the coronal brain section had hematoma at 4 levels, especially at the 2nd and 3rd levels, round hematoma formed compression to the surrounding brain tissue.

[0077] 2.3 Improvement of ICH-induced neurological dysfunction in mice by crocin

[0078] The results of the foot-fault test are shown in Figure 2 Figure 6. The most significant difference was observed on the third day after modeling. Overall, the foot-fault rate of the contralateral forelimb and hindlimb of the mice was significantly higher than that of the sham group after ICH modeling. The administration of 100 mg / kg of rhein significantly reduced the foot-fault rate of the forelimb (P < 0.01) and hindlimb (P < 0.05). The effect on the hindlimb was slightly worse than that on the forelimb.

[0079] The results of the paper-removal test are shown in Figure 3 Figure 7. After ICH modeling, the mice showed a significant decrease in responsiveness, and the time taken to remove the paper from the palm of the contralateral forelimb was significantly longer than that of the sham group (P < 0.0001). The effect of rhein was very significant in this test, and the administration of 3 concentrations of rhein significantly shortened the reaction time and removal time compared with the DMSO control group (P < 0.0001).

[0080] The results of the rotarod test are shown in Figure 4 Figure 8. The neurological dysfunction caused by ICH modeling made it difficult for the mice to coordinate their limbs, and the mice with ICH modeling fell off the rotarod much more easily than the sham group, with a significant statistical difference (P < 0.0001). The administration of 3 concentrations of rhein and CD28SA significantly improved the effect, and the mice ran on the rotarod for a longer time, with a significant statistical difference compared with the respective solvent control groups (P < 0.0001).

[0081] The results of the modified Garcia score test are shown in Figure 5 Figure 9. The mice showed significant neurological deficits after modeling, especially on the third day after modeling. Overall, the administration of 3 concentrations of rhein and CD28SA significantly improved the neurological function, with a significant statistical difference compared with the respective solvent control groups (P < 0.0001).

[0082] 2.4 Rhein improves iron deposition in brain tissue after ICH

[0083] After ICH, iron ions enter the brain tissue around the hematoma, are phagocytosed by immune cells, or diffuse between tissues, all of which induce an inflammatory response in the brain tissue around the hematoma. As shown in Figure 6 a, the dark brown particles stained with potassium ferrocyanide dye were dispersed in the brain tissue around the hematoma. Microscopic observation showed that the iron deposition in the rhein administration group and the CD28SA administration group was more sparse and more localized around the hematoma. Figure 6b. For the statistical analysis of the area of Perls staining positive brain tissue, we found that the iron deposition of the brain tissue around the hematoma in the drug group was significantly reduced compared with the solvent control group after 4 adjacent 400x fields around the integrated hematoma.

[0084] 2.5 Astilbin improves ICH-induced neuronal death

[0085] As shown in Figure 7 a, the simple ICH modeling group, DMSO solvent control group, and PBS solvent control group all showed loose structure of brain tissue around the hematoma, more round cavities left after cell necrosis dissociation, and fewer neurons with relatively complete tissue morphology (for example, the cells pointed to by the red arrows). Under 400x field, we counted the number of surviving neurons in the brain tissue around the hematoma in each group, as shown in Figure 7 b: On average, the number of surviving neurons in each group after ICH modeling was reduced compared with the sham operation group. After statistical analysis, we found that the number of surviving neurons in the astilbin drug group of three concentrations was significantly higher than that in the DMSO solvent control group (P<0.0001).

[0086] Nissl staining to evaluate the number of surviving neurons: a. Nissl staining of brain tissue around the hematoma in each group under a microscope, the left side is the overall situation around the hematoma, and the scale bar is 200 μm; the right side is an enlarged view of the black square area, and the arrow points to the surviving neurons in the damaged brain tissue around the hematoma, and the scale bar is 50 μm. As can be seen, the proportion of Nissl staining positive neurons in the astilbin drug group was significantly higher than that in the solvent control group, and the higher the drug concentration, the more surviving neurons.

[0087] According to the overall improvement of behavior and the improvement of cell level shown by Nissl staining test results, we selected the 100 mg / kg concentration of astilbin intraperitoneal drug group as the representative of the drug group for subsequent tests, and referred to the 100 mg / kg concentration of astilbin intraperitoneal drug group as "Astilbin" to reduce the loss of biology, chemistry, and materials. As shown in Figure 8 a: Under 400x field, the density of red-stained 3'-OH exposed after DNA fragmentation by Tunel staining was significantly increased in the simple ICH modeling group, DMSO solvent control group, and PBS solvent control group; the density of apoptotic cells in these groups after fusion was also higher. As shown in Figure 8 b, the statistical analysis results suggest that the number of Tunel staining positive apoptotic neurons in the astilbin drug group is significantly less than that in the DMSO solvent control group (P<0.0001).

[0088] 2.6 Astilbin improves ICH-induced brain edema

[0089] As shown in Figure 9 , the brain water content of the quercetin 80 mg / kg group (P=0.0018) and the quercetin 100 mg / kg group (P=0.0002) were significantly lower than that of the DMSO solvent control group.

[0090] 2.7 Quercetin reverses ICH-induced BBB damage

[0091] As shown in Figure 10 a, the BBB damage caused by edema, mechanical compression, inflammatory factors, etc. after ICH was visually presented. After quercetin administration, although there was still BBB damage around the hematoma, the diffuse edema was greatly improved, and the blue-stained area was significantly controlled. As shown in Figure 10 b, the three groups of quercetin administration showed obvious improvement, and compared with the DMSO solvent control group, the Evans blue penetration was significantly controlled (P<0.0001).

[0092] 2.8 Quercetin promotes the secretion of chemotactic factors and anti-inflammatory factors after ICH

[0093] In the ELISA experiment, as shown in Figure 11 , the promoting effect of high concentration quercetin was very significant, and it significantly up-regulated the expression of CCL-1 and CCL-20 two chemotactic factors in peripheral blood (P<0.0001 vs ICH+DMSO). Similar effects were also observed in the CD28SA administration group.

[0094] As shown in Figure 12 , the anti-inflammatory factors IL-10, IL-35, and TGF-β1 in the peripheral blood were significantly up-regulated under the action of quercetin compared with the DMSO solvent control group, and had a significant promoting effect on the three anti-inflammatory factors.

[0095] 2.9 Quercetin promotes the aggregation of Treg cells around the hematoma in the brain tissue after ICH

[0096] In the immunofluorescence staining experiment, as shown in Figure 13 a, the immunofluorescence intensity of the brain tissue around the hematoma in the quercetin administration group and the CD28SA administration group was significantly higher than that of the DMSO solvent control group and the PBS solvent control group, which indicated that Foxp3 protein was significantly up-regulated in the quercetin administration group and the CD28SA administration group, and side-proved the accumulation of Treg cells around the hematoma in the brain tissue of the two groups. As shown in Figure 13 b, compared with the DMSO solvent control group, intraperitoneal injection of quercetin significantly promoted the enrichment of Foxp3+Treg cells around the hematoma in the brain tissue (P<0.0001).

[0097] Treg cells in brain tissues around ICH, secreted anti-inflammatory factors, created an environment to suppress inflammation, improved the prognosis of SBI in mice after ICH, such as edema, iron deposition, neuronal necrosis and apoptosis, and reduced neurological damage caused by ICH in mice. This study provides an experimental basis and theoretical basis for treating SBI after ICH with astilbin.

[0098] This study focuses on inflammation, although the autologous blood injection model also has the defect of not simulating small vessel rupture, but it may be closer to the true face of ICH disease in terms of inflammation. This study shows that astilbin reduces a series of secondary injuries such as neuronal necrosis and apoptosis, and ferric iron deposition in mice after ICH, and improves neurological damage in mice after ICH. It proves that astilbin has a significant effect in the mouse ICH model, significantly improves SBI after ICH in mice, and accelerates the recovery of neurological function in mice. This indicates that astilbin has the potential to be applied in the treatment of ICH. According to the experimental results of this study, we recommend a dosage of 100 mg / kg of astilbin in mice to achieve better efficacy. In this study, we found that astilbin produced similar results to CD28SA in reducing secondary inflammatory damage after ICH, which may suggest that astilbin and CD28SA have similar anti-inflammatory mechanisms.

[0099] SBI caused by ICH, including inflammation, seriously interferes with the patient's outcome process. In order to determine the efficacy of the anti-inflammatory drug astilbin in the ICH disease model, we established an ICH model by injecting autologous blood into the striatum to form a hematoma, and established a gradient of astilbin dosages to explore the ICH efficacy of astilbin from multiple perspectives. We studied the effect of intraperitoneal injection of astilbin on brain tissue edema, BBB damage, neuronal damage, brain tissue apoptosis, anti-inflammatory factors in peripheral blood, and improvement of neurological function recovery in mice after ICH. In addition, we explored whether astilbin can enrich regulatory T cells in the brain tissue around the hematoma to exert immunosuppressive effects. We obtained the following conclusions:

[0100] 1. The success rate of the ICH model by injecting autologous blood can reach 84.9%. In the behavioral experiment to evaluate the recovery of neurological function for up to 2 weeks after modeling, we found that high concentrations of astilbin administered intraperitoneally significantly improved the recovery of neurological function after ICH in all aspects, and significantly accelerated the rehabilitation process of ICH mice.

[0101] 2、Mice brain tissues were made into paraffin coronal brain tissue sections after modeling, and a series of section staining experiments were carried out. Nissl staining and Tunel staining experiments verified that quercetin effectively reduced the death of neurons after ICH; Perls staining experiments confirmed that quercetin significantly reduced the iron deposition of the damaged brain tissue around ICH. Quercetin reduces brain tissue damage and improves ICH-induced neurological dysfunction. The above can reduce the inflammatory response induced by ferric iron entering the brain tissue after necrosis and apoptosis of neuronal cell fragments.

[0102] 3、Dry-wet method for measuring brain water content confirmed that quercetin reduced secondary brain edema after ICH; Evans blue penetration method showed that quercetin improved BBB damage after ICH; ELISA experiments confirmed that quercetin promoted the chemotaxis of immunosuppressive cells and promoted the high expression of anti-inflammatory factors in the internal environment, thereby playing an anti-inflammatory role.

[0103] 4、By immunofluorescence staining of Foxp3 protein, we found that quercetin can effectively promote the accumulation of Foxp3+ Treg cells in the damaged brain tissue around the hematoma, and play the immunosuppressive role of Treg cells. As shown in Figure 14 , we preliminarily revealed the partial mechanism of quercetin in playing an anti-inflammatory role after ICH. When ICH causes inflammation and causes SBI in brain tissue, quercetin promotes the expression of CCL-1 and CCL-20 chemokines, and chemotaxis of Treg cells into the damaged brain tissue, while expressing anti-inflammatory factors IL-10, IL-35 and TGF-β1, thereby inhibiting the inflammatory response of the damaged brain tissue and reducing SBI.

[0104] The above is only a preferred embodiment of the present application, and the protection scope of the present application is not limited to the above-mentioned embodiments. Any technical solutions falling within the scope of the present application are within the protection scope of the present application. It should be noted that for ordinary skilled persons in the art, some improvements and refinements without departing from the principles of the present application are also considered to be within the protection scope of the present application.

Claims

1. Use of quercetin in the preparation of a medicament for improving secondary brain injury after cerebral hemorrhage, characterized in that, The preparation method of the medicine is as follows: completely dissolving the powder of astilbin in dimethyl sulfoxide, diluting with 37℃ physiological saline to the concentration of dimethyl sulfoxide not higher than 0.4%, and adopting intraperitoneal injection for administration.

2. Use according to claim 1, characterized in that, Astilbin reduces the apoptosis of brain cells after cerebral hemorrhage, reduces the necrosis of neurons after cerebral hemorrhage, reduces the iron deposition of brain tissue after cerebral hemorrhage, and improves the prognosis of neurological function induced by cerebral hemorrhage.

3. Use according to claim 1, characterized in that, Astilbin reduces the secondary brain injury after cerebral hemorrhage by regulating the penetration of Treg cells through the blood-brain barrier.

4. Use according to claim 3, characterized in that, Astilbin promotes the accumulation of Treg cells of characteristic transcription factor Foxp3 in the damaged brain tissue around the cerebral hematoma, and plays the immunosuppressive role of Treg cells.

5. Use according to claim 4, characterized in that, Astilbin promotes the expression of CCL-1 and CCL-20 chemokines, and at the same time expresses anti-inflammatory factors IL-10, IL-35 and TGF-β1, which promotes the formation of an anti-inflammatory microenvironment in the brain tissue around the cerebral hematoma, thereby inhibiting the inflammatory response of the damaged brain tissue and reducing the secondary brain injury.

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