Use of typhaneoside in preparing medicament for treating ischemic stroke

Abstract

Provided is use of typhaneoside in preparing a medicament for treating ischemic stroke. Experiments have proved that typhaneoside can reduce the cerebral infarction volume, increase the cerebral blood perfusion volume, improve nerve functions and ameliorate motor disorders, reduce ROS levels, and protect neurons, thereby effectively treating ischemic stroke.

Description

Application of typhain in the preparation of drugs for treating ischemic stroke Technical Field

[0001] This invention relates to the field of pharmaceutical biology, and in particular to the application of typhaneoside in the preparation of drugs for treating ischemic stroke. Background Technology

[0002] Data from the Global Burden of Disease Study (GBD; http: / / ghdx.healthdata.org / ) shows that stroke is the leading cause of death and disability among adults in my country. Stroke includes ischemic stroke and cerebral hemorrhage, with ischemic stroke accounting for 69.6%-70.8% of all strokes in my country (Chinese Society of Neurology, Cerebrovascular Disease Group of the Chinese Society of Neurology. Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke in China 2018 [J]. Chinese Journal of Neurology, 2018, 51(9):666-682.). Treatment strategies for ischemic stroke mainly include reperfusion therapy and neuroprotective agents. Reperfusion therapy is currently the main method for treating acute ischemic stroke. However, factors such as a strict time window, widespread treatment delays, and a high risk of hemorrhagic transformation limit the actual clinical benefits of reperfusion therapy. Clinical trials of neuroprotective agents have all failed due to adverse reactions or insufficient efficacy. Therefore, finding effective treatments for ischemic stroke remains a critical issue that urgently needs to be addressed in clinical practice.

[0003] Typhaneoside is an effective component extracted from the traditional Chinese medicine Typha angustifolia L. It is a flavonoid compound with important medicinal value. Modern pharmacological studies have found that typhaneoside can inhibit uterine contraction (Su S, Hua Y, Duan JA, Zhou W, Shang E, Tang Y. Inhibitory effects of active fraction and its main components of Shaofu Zhuyu decoction on uterus contraction. Am J Chin Med. 2010; 38(4): 777-87.), inhibit glutamate release in the cerebral cortex (Chiu KM, Lin TY, Lee MY, Lu CW, Wang MJ, Wang SJ. Typhaneoside Suppresses Glutamate Release Through Inhibition of Voltage-Dependent Calcium Entry in Rat Cerebrocortical Nerve Terminals. Chem Res Toxicol. 2021 May 17; 34(5): 1286-1295.), effectively regulate ventricular remodeling after myocardial infarction (Zhang X, Yang K, Zhang H, Dong W, Peng W, Zhao Y. Effect of typhaneoside on ventricular remodeling and regulation of PI3K / Akt / mTOR pathway. Herz. 2020 Dec; 45(Suppl 1): 113-122.), prevent acute myeloid leukemia progression (Zhu HY, Huang ZX, Chen GQ, Sheng F, Zheng YS. Typhaneoside prevents acute myeloid leukemia (AML) through suppressing proliferation and inducing ferroptosis associated with autophagy. Biochem Biophys Res Commun. 2019 Sep 3; 516(4): 1265-1271.), and has antioxidant activity (Chen P, Cao Y, Bao B, Zhang L, Ding A.Antioxidant capacity of Typha angustifolia extracts and two active flavonoids. Pharm Biol. 2017 Dec; 55(1):1283-1288.), hepatoprotective effect (Zheng Y, Zhao J, Miao D, Xu T, Wang L, Liu C, Gao Y, Yu L, Shen C. Hepatoprotective effect of Typhaneoside on non-alcoholic fatty liver disease via farnesoid X receptor in vivo and in vitro. Biomed Pharmacother. 2023 Aug; 164:114957.) and inhibitory effect on vascular smooth muscle cell proliferation, etc. (Guo Shuzhen, Liu Bin, Wang Wei. Effect of typhaneoside on proliferation of human umbilical artery vascular smooth muscle cells [J]. Journal of Beijing University of Traditional Chinese Medicine, 2006, (05):307-310.).

[0004] Although there have been many reports on the application of typhain in existing technologies, its application in the preparation of drugs for treating ischemic stroke has not yet been reported.

[0005] Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of typhaein in the preparation of drugs for treating ischemic stroke.

[0007] The technical solution of this invention is summarized as follows:

[0008] Application of typhain in the preparation of drugs for treating ischemic stroke.

[0009] Advantages of this invention:

[0010] Experiments have shown that typhain can reduce the volume of cerebral infarction, increase cerebral blood flow perfusion, improve neurological function and motor disorders, reduce ROS levels, and protect neurons, thus effectively treating ischemic stroke. Attached Figure Description

[0011] Figure 1 shows the protective effect of typhain on OGD / R-damaged HT-22 hippocampal neurons.

[0012] Figure 2 shows the effect of typhaein on ROS levels in OGD / R-damaged HT-22 cells.

[0013] Figure 3 shows the effects of typhain on the neurological function of tMCAO mice.

[0014] Figure 4 is the effect of typhaneoside on the movement disorder of tMCAO mice.

[0015] Figure 5 is the effect of typhaneoside on the cerebral blood perfusion (A) and cerebral infarction volume (B) of tMCAO mice. DETAILED DESCRIPTION

[0016] The typhaneoside involved in the present application is a flavonoid compound, whose molecular formula is C 34 H 42 O 20 , and the CAS number is 104472-68-6.

[0017] The reagent materials, methods and results used in the examples are as follows:

[0018] 1. Drugs and reagents

[0019] Typhaneoside (B21367) was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. CCK-8 kit (CK04, DOJINDO), serum-free cell freezing solution (C40100, New Cell Bioscience), trypsin digestion solution 0.25% (C100C1, New Cell Bioscience), active oxygen detection kit (S0033S, Biyun Tian).

[0020] 2. Experimental consumables

[0021] Cell counting plate (JSB01); sealing film (imported from the United States) (0105021);

[0022] 3. Main experimental instruments

[0023] Full-wavelength multifunctional enzyme label instrument (Varioskan Flash, Thermo scientific); three-gas incubator (Germany Memmert); oven (51028150 / OMS180, Thermo scientific); carbon dioxide incubator purchased from Japan SANYO (MCO-20AIC); clean bench (HCB-1300V, Haier).

[0024] 4. In vitro cell experiment method and experimental results

[0025] 4.1 Cell source

[0026] The cell line used in the experiment is SV40T-transformed mouse hippocampal neuron cells (HT-22), which is purchased from the National Biomedical Experimental Cell Resource Library, item number: 1101MOU-PUMC001005. The morphological characteristics of HT-22 cells are fibroblast-like cells, which grow adherently, are passaged at a ratio of 1:3-1:4, and are used once every 1-2 weeks. The normal culture conditions of HT-22 cells are DMEM high-sugar medium (12100, Solabio) + 10% FBS.

[0027] 4.2 Preparation and grouping of OGD / R cell model

[0028] An oxygen and glucose deprivation / restoration (OGD / R) model was prepared. HT-22 cells were seeded in a 96-well plate at a density of 1×10 5 cells / ml, 100 μL / well, and cultured in a CO2 incubator at 37°C for 24 h. Then, the culture medium was replaced with serum-free DMEM without glucose (D6540, Solabio), and the cells were cultured in a three-gas incubator (1% O2) for 6 h. Then, the medium was replaced with DMEM high-sugar medium for continued incubation for 24 h. The process is referred to as OGD 6h / R 24h below. The normal group was cultured normally, and the drug group was cultured in a three-gas incubator (1% O2) for 6 h. Then, 1 μM, 10 μM, and 100 μM of typhonii rhizoma and radix paeoniae rubra glycoside (DMEM high-sugar medium) were added, respectively, and incubated for 24 h.

[0029] 4.3 Detection of viable cell activity by CCK-8 method

[0030] The OD value of each group of HT-22 cells was measured at 450 nm. The results showed that typhonii rhizoma and radix paeoniae rubra glycoside had a protective effect on OGD / R-injured HT-22 hippocampal neuron cells. Specifically, after OGD 6h / R 24h, the OD value of the cells decreased significantly (P<0.01), indicating a decrease in cell activity and cell injury. After 6 hours of hypoxia, 10 μM and 100 μM of typhonii rhizoma and radix paeoniae rubra glycoside were added to the cells for continued incubation for 24 hours, and the OD value of the cells increased significantly (P<0.05, P<0.01), indicating a recovery of cell activity and a reversal of cell injury (see Figure 1).

[0031] Figure 1 Protective effect of typhonii rhizoma and radix paeoniae rubra glycoside on OGD / R-injured HT-22 hippocampal neuron cells. Compared with the normal group, ##P<0.01; compared with the OGD / R group, *P<0.05, **P<0.01.

[0032] 4.4 Detection of reactive oxygen species

[0033] The reactive oxygen species (ROS) of each group of cells was detected using a reactive oxygen species detection kit (S0033S, Biyun Tian). The specific steps were performed according to the kit instructions. A laser confocal microscope was used to observe the strength of cell fluorescence using an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0034] The results showed that typhonioside could reduce the ROS level of OGD / R damaged HT-22 cells. Specifically, after OGD 6h / R 24h, the ROS level of the cells was significantly increased (P<0.01), indicating oxidative stress damage. After 6 hours of hypoxia, 10 μM typhonioside was added and incubated for 24 hours, and the ROS level of the cells was significantly reduced (P<0.01), indicating that typhonioside could reverse the oxidative stress damage of the cells (see Figures 2A and B).

[0035] Figure 2 Effect of typhonioside on ROS level of OGD / R damaged HT-22 cells. Compared with the normal group, ##P<0.01; compared with the OGD / R group, **P<0.01.

[0036] 5 In vivo animal experiment method and experimental results

[0037] 5.1 Experimental animals

[0038] SPF male C57BL / 6 mice weighing 23±2g were purchased from Beijing Huafukang Biotechnology Co., Ltd. [SCXK(Jing)2019-0008]. They were raised in the barrier environment animal room of Dongzhimen Hospital of Beijing University of Chinese Medicine [SCYK(Jing)2015-0001]. The room temperature was 22-25℃, the humidity was 60-65%, the daily light period was 12h, and the animals had free access to water and food. The experiment was reviewed by the Medical and Experimental Animal Ethics Committee of Dongzhimen Hospital of Beijing University of Chinese Medicine and met the relevant ethical requirements. The ethical review number was 20-40.

[0039] 5.2 Preparation and grouping of mouse tMCAO model

[0040] A mouse transient Middle Cerebral Artery Occlusion (tMCAO) model was constructed using a thread plug method. The mice were anesthetized by inhaling 1.5% isoflurane, and when the mice were completely anesthetized, they were placed in a supine position on the operation table. The skin was cut in the midline of the neck with ophthalmic scissors, and the thyroid gland was bluntly separated. Under a microscope, the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully separated. A small opening was made in the external carotid artery with a Venus scissors, and the thread plug was inserted from the small opening into the ECA. The thread plug was gently turned into the internal carotid artery (ICA) when a significant resistance was felt. The thread plug was removed after 1 hour of ischemia.

[0041] The left common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) of the sham operation group mice were isolated, but no subsequent operation was performed. The degree of neurological impairment of the mice after operation was evaluated by Longa score (LONGA E Z, WEINSTEIN P R, CARLSON S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats [J / OL]. Stroke, 1989, 20(1): 84-91.), and the mice with scores of 0 and 4 were excluded. The mice successfully replicating the model were divided into a model group, a typhaneoside 10 mg / kg group, a typhaneoside 20 mg / kg group and a typhaneoside 40 mg / kg group (solvent: 0.5% Na-CMC aqueous solution), a total of 4 groups, 6 mice in each group. There were 6 mice in the sham operation group. Two hours after the operation, the mice in the typhaneoside 10 mg / kg group, the typhaneoside 20 mg / kg group and the typhaneoside 40 mg / kg group were administered with the corresponding dose of typhaneoside prepared with Na-CMC. The mice in the sham operation group and the model group were administered with the same volume of 0.5% Na-CMC aqueous solution, and the gavage was administered for 3 days.

[0042] 5.3 mNSS evaluation of neurological impairment

[0043] The Modified Neurological Severity Score (mNSS) scale was used to evaluate the degree of neurological impairment of tMCAO mice. The score was 0-14, 0 indicating normal and healthy mice, and 14 indicating the most severe functional impairment. The higher the total score, the more severe the neurological impairment. The mNSS neurological function score was performed at 1 day and 3 days after modeling.

[0044] The results are shown in Figure 3. Typhaneoside improved the neurological function of tMCAO mice. Specifically, the neurological function of the mice in the sham operation group was normal, and the mNSS score of the mice in the tMCAO group (model group) was significantly higher than that of the mice in the sham operation group, indicating that there was obvious neurological impairment. Compared with the model group, typhaneoside (20 mg / kg and 40 mg / kg) administered for 1 day could significantly reduce the neurological impairment (P<0.01), and typhaneoside (10 mg / kg, 20 mg / kg and 40 mg / kg) administered for 3 days could significantly reduce the neurological impairment (P<0.05 or P<0.01).

[0045] Figure 3 Effect of typhaneoside on the neurological function of tMCAO mice.

[0046] 5.4 Rota-Rod rotarod test to evaluate motor impairment

[0047] The mice were subjected to the rotarod test at 1 d, 3 d after tMCAO operation, and the mode was set to uniform acceleration mode with speed of 4-40 rpm and time of 300 s. The test was performed for 3 times per day, and the average fall time of 3 times was taken as the latency to fall.

[0048] The results are shown in Figure 4. Typhonii Rhizoma Extract improved the motor impairment of tMCAO mice. Specifically, compared with the sham operation, the latency to fall of the model mice was significantly shortened at 1 d, 3 d after modeling (P<0.01), and the latency to fall of the mice was significantly prolonged after administration of Typhonii Rhizoma Extract (10 mg / kg, 20 mg / kg and 40 mg / kg) for 3 days (P<0.01).

[0049] Figure 4 Effect of Typhonii Rhizoma Extract on motor impairment of tMCAO mice

[0050] 5.5 Laser speckle blood flow imaging instrument to observe the changes of cerebral blood perfusion of mice

[0051] The laser speckle blood flow imaging instrument was used to observe the changes of regional cerebral blood flow of mice at 3 days after tMCAO operation.

[0052] The results are shown in Figure 5A. Typhonii Rhizoma Extract improved the cerebral blood perfusion of tMCAO mice. Specifically, the cerebral blood perfusion of the left brain of the mice was significantly reduced after 3 days of left tMCAO operation, indicating that the left brain of the mice had obvious infarction, and the cerebral blood perfusion of the tMCAO mice was significantly improved after administration of Typhonii Rhizoma Extract (10 mg / kg, 20 mg / kg and 40 mg / kg) for 3 days.

[0053] 5.6 TTC to detect cerebral infarction volume

[0054] The TTC staining method was used to determine the cerebral infarction volume of mice, and the specific staining steps were performed according to the instructions of TTC staining solution (G3005, Solabio).

[0055] The results are shown in Figure 5B. Typhonii Rhizoma Extract can reduce the cerebral infarction volume of tMCAO mice. Specifically, no infarction occurred in the sham operation group, and the left cerebral cortex of the tMCAO model mice was obviously infarcted at 3 days after operation, and the cerebral infarction volume of the mice was significantly reduced after administration of 20 mg / kg Typhonii Rhizoma Extract for 3 days (P<0.01).

Claims

1. The use of typhaneoside in the preparation of a drug for treating ischemic stroke.

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