Application of gentiana macrophylla pall extract in preparation of medicine for relieving and / or treating chronic cerebral hypoperfusion

Abstract

The invention provides application of gentiana straminea extract in preparation of a medicine for relieving and / or treating chronic cerebral hypoperfusion, and belongs to the technical field of medicine. The invention provides a gentiana straminea extract, and particularly relates to related application of a gentiana straminea alcohol extract in chronic cerebral hypoperfusion, the gentiana straminea extract can protect neuron structures, improve cognitive impairment and motor dysfunction caused by chronic cerebral hypoperfusion, reduce inflammatory response of chronic cerebral hypoperfusion, regulate and control STAT1 / STAT6 expression, inhibit M1 polarization, and improve the curative effect of chronic cerebral hypoperfusion. M2 polarization is promoted. The invention further provides a synergistic effect of the gentiana straminea extract and the STAT1 inhibitor in preparation of the medicine for relieving and / or treating the chronic cerebral hypoperfusion, and further provides the medicine for relieving and / or treating the chronic cerebral hypoperfusion.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to the application of Gentiana straminea Maxim. extract in the preparation of a drug for relieving and / or treating chronic cerebral hypoperfusion. Background Art

[0002] The condition of chronic cerebral hypoperfusion (CCH) is caused by insufficient blood flow to the brain due to various reasons. This condition will gradually deteriorate to the point where it cannot meet the physiological needs of normal brain tissue, thereby triggering a series of neurological symptoms. The clinical manifestations of CCH are diverse, including symptoms such as cognitive decline, memory impairment, dementia, depression, anxiety, personality changes, and gait disorders. CCH will produce a series of pathophysiological changes, among which chronic inflammation, oxidative stress, and impaired blood-brain barrier are the key pathophysiological mechanisms for the development and progression of CCH. Currently, the main treatment methods are through intervention of risk factors, treatment targeting the cause, and drugs to improve cerebral circulation, such as butylphthalide (NBP). However, the treatment methods for CCH still need further research.

[0003] Traditional Chinese medicine has good therapeutic effects on diseases due to its multi-component, multi-target, and few side effects. Tibetan medicine is one of the key elements of traditional Chinese medicine. Gentiana straminea Maxim. (abbreviated as GSM) is a Tibetan medicine produced on the Tibetan Plateau. Its unique growth environment endows it with excellent biological characteristics. It is a commonly used medicine in Tibetan medicine and has a history of use of more than 2000 years. GSM is clinically commonly used in the treatment of cardiovascular and cerebrovascular diseases and other diseases. Some studies have shown that GSM provides protective effects on the lungs and brain tissues of hypoxic rats by scavenging oxygen free radicals and regulating energy metabolism. However, the mechanism of action of GSM on CCH is still unclear. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide the application of Gentiana straminea Maxim. extract in the preparation of a drug for relieving and / or treating chronic cerebral hypoperfusion.

[0005] The purpose of the present invention is also to provide the application of Gentiana straminea Maxim. extract combined with a STAT1 inhibitor in the preparation of a drug for relieving and / or treating chronic cerebral hypoperfusion.

[0006] The purpose of the present invention is also to provide a drug for relieving and / or treating chronic cerebral hypoperfusion.

[0007] In order to achieve the above-mentioned invention purposes, the present invention provides the following technical solutions:

[0008] The present invention provides the application of Gentiana straminea Maxim. extract in the preparation of a drug for relieving and / or treating chronic cerebral hypoperfusion.

[0009] Preferably, the Gentiana straminea Maxim. extract is an ethanol extract of Gentiana straminea Maxim.; the preparation method of the ethanol extract of Gentiana straminea Maxim. includes: drying and pulverizing the roots of Gentiana straminea Maxim., mixing with ethanol, soaking, heating for reflux extraction with condensation, collecting the filtrate, and evaporating the filtrate to prepare an extract.

[0010] Preferably, the Gentiana straminea Maxim. extract protects the neuronal structure.

[0011] Preferably, the Gentiana straminea Maxim. extract alleviates cognitive deficits and motor function impairments caused by chronic cerebral hypoperfusion.

[0012] Preferably, the Gentiana straminea Maxim. extract reduces the inflammatory response of chronic cerebral hypoperfusion, regulates the expression of STAT1 / STAT6, inhibits M1 polarization, and promotes M2 polarization.

[0013] Preferably, the Gentiana straminea Maxim. extract improves the inflammatory injury of HMC3 cells induced by OGD and promotes the restoration of HMC3 cells to a normal morphology.

[0014] The present invention also provides the use of the Gentiana straminea Maxim. extract in combination with a STAT1 inhibitor in the preparation of a drug for alleviating and / or treating chronic cerebral hypoperfusion, and the Gentiana straminea Maxim. extract is an ethanol extract of Gentiana straminea Maxim.

[0015] Preferably, the STAT1 inhibitor includes Fludarabine.

[0016] The present invention also provides a drug for alleviating and / or treating chronic cerebral hypoperfusion, and the active ingredient of the drug includes the Gentiana straminea Maxim. extract, and the Gentiana straminea Maxim. extract is an ethanol extract of Gentiana straminea Maxim.

[0017] Preferably, the active ingredient of the drug further includes a STAT1 inhibitor.

[0018] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0019] The present invention has found through research that the GSM extract can protect the neuronal structure, improve the cerebral blood flow condition, and alleviate cognitive dysfunction and motor function impairments caused by chronic cerebral hypoperfusion. The GSM extract can reduce the inflammatory response of chronic cerebral hypoperfusion, regulate the expression of STAT1 / STAT6, improve brain injury, and exert a brain protection effect. The GSM extract can reduce the proportion of the marker CD86 and increase the proportion of the marker CD206, inhibit M1 polarization, and promote M2 polarization, and thus can be used in the preparation of a drug for alleviating and / or treating chronic cerebral hypoperfusion.

[0020] The present invention further discovers that the Gentiana straminea Maxim. extract combined with a STAT1 inhibitor can be used to prepare a drug for alleviating and / or treating chronic cerebral hypoperfusion, and it is verified through cell experiments that the Gentiana straminea Maxim. extract and the STAT1 inhibitor have a synergistic effect. Description of the Drawings

[0021] Figure 1 : Effects of GSM on the neurological function of CCH rats;

[0022] Figure 2 : Effects of GSM on the behavior of CCH rats;

[0023] Figure 3 : Effects of GSM on the cerebral blood flow of CCH rats;

[0024] Figure 4 : Effects of GSM on the pathological damage of the brain tissue of CCH rats;

[0025] Figure 5 : Effects of GSM on the polarization of microglia in the brain tissue of CCH rats;

[0026] Figure 6 : Effects of GSM on the levels of TNF-α, IL-6, IL-4, and IL-10 in the serum of CCH rats;

[0027] Figure 7 : Effects of GSM on the expression of STAT1 / STAT6-related proteins in the brain tissue of CCH rats;

[0028] Figure 8 : Effects of different OGD times on the viability of HMC3 cells;

[0029] Figure 9 : Effects of different concentrations of GSM on the viability of HMC3 cells;

[0030] Figure 10 : Effects of GSM on the viability of OGD-induced HMC3 cells;

[0031] Figure 11 : Effects of GSM on the morphology of HMC3 cells in each group (×100);

[0032] Figure 12 : Effects of GSM on the expression of the microglia marker CD11b in HMC3 cells in each group;

[0033] Figure 13 : Effects of GSM on the expression of the M1 / M2 phenotype markers iNOS / Arg-1 in HMC3 cells in each group;

[0034] Figure 14: Effects of GSM on the levels of TNF-α, IL-6, IL-4, and IL-10 in the supernatant of HMC3 cells;

[0035] Figure 15 : Effects of GSM on the expression of STAT1 / STAT6-related proteins in HMC3 cells of each group. Detailed implementation manners

[0036] The present invention provides the use of Gentiana straminea Maxim. extract in the preparation of a drug for alleviating and / or treating chronic cerebral hypoperfusion.

[0037] The Gentiana straminea Maxim. extract of the present invention is preferably an ethanol extract of Gentiana straminea Maxim.; the preparation method of the ethanol extract of Gentiana straminea Maxim. includes: drying and pulverizing the roots of Gentiana straminea Maxim., mixing with ethanol, soaking, heating for condenser reflux extraction, collecting the filtrate, and evaporating the filtrate to prepare an extract. Preferably, the dried roots of Gentiana straminea Maxim. are pulverized through a 40-mesh sieve to obtain the dried root powder of Gentiana straminea Maxim.; preferably, the dried root powder of Gentiana straminea Maxim. is mixed with ethanol at a mass-to-volume ratio of 1 g:(4 - 6) mL, and the mass-to-volume ratio is more preferably 1 g:5 mL; preferably, the ethanol is 95% ethanol; preferably, the soaking is static soaking at room temperature, and the soaking time is 20 - 26 h, more preferably 21 h, 22 h, 23 h, 24 h, or 25 h; preferably, the heating temperature for the heating condenser reflux extraction is 80 - 85 °C, and the extraction times are preferably repeated extraction 3 times, each lasting 1 h; preferably, the solutions (filtrates) after each filtration are combined and collected, and an extract is obtained by evaporation. The evaporation is preferably carried out using a rotary evaporator. The extract obtained in the present invention is the ethanol extract of Gentiana straminea Maxim. As an alternative implementation manner, the extract obtained in the present invention is prepared into a solution form of the ethanol extract of Gentiana straminea Maxim. by using distilled aqueous solution and ultrasonic mixing; as another alternative implementation manner, the extract obtained in the present invention is freeze-dried to obtain a freeze-dried powder; the freeze-drying treatment includes pre-freezing at -80 °C and freeze-drying at -40 °C.

[0038] Permanent bilateral common carotid artery occlusion (2-VO) in rats is considered a suitable animal model for simulating the CCH state; oxygen-glucose deprivation (OGD) is an important cell model for simulating ischemic diseases. It is verified by animal experiments and cell experiments of the present invention that the Gentiana straminea Maxim. extract can protect the neuronal structure and improve the cerebral blood flow condition; it can alleviate the cognitive defects and motor function deficits (loss of spontaneous motor ability or motor function) caused by chronic cerebral hypoperfusion; it can reduce the inflammatory response of chronic cerebral hypoperfusion, regulate the expression of STAT1 / STAT6, inhibit M1 polarization, and promote M2 polarization, thereby playing a role in improving brain injury. The Gentiana straminea Maxim. extract improves the inflammatory injury of OGD-induced HMC3 cells and promotes the restoration of HMC3 cells to a normal morphology.

[0039] The present invention also provides the use of Gentiana straminea Maxim. extract combined with a STAT1 inhibitor in the preparation of a drug for alleviating and / or treating chronic cerebral hypoperfusion. The Gentiana straminea Maxim. extract is an ethanol extract of Gentiana straminea Maxim., and the STAT1 inhibitor includes Fludarabine.

[0040] When the Gentiana straminea Maxim. extract combined with the STAT1 inhibitor is used in the present invention, the preparation method of the ethanol extract of Gentiana straminea Maxim. preferably includes: drying and pulverizing the roots of Gentiana straminea Maxim., mixing with ethanol, soaking, heating for condenser reflux extraction, collecting the filtrate, and evaporating the filtrate to prepare an extract. Preferably, the dried roots of Gentiana straminea Maxim. are pulverized through a 40-mesh sieve to obtain the dried root powder of Gentiana straminea Maxim.; preferably, the dried root powder of Gentiana straminea Maxim. is mixed with ethanol at a mass-to-volume ratio of 1 g:(4 - 6) mL, and the mass-to-volume ratio is more preferably 1 g:5 mL; preferably, the ethanol is 95% ethanol; preferably, the soaking is static soaking at room temperature, and the soaking time is 20 - 26 h, more preferably 21 h, 22 h, 23 h, 24 h or 25 h; preferably, the heating temperature for condenser reflux extraction is 80 - 85°C, and the extraction times are preferably repeated 3 times, each lasting 1 h; preferably, the solutions (filtrates) after each filtration are combined and collected, and the extract is obtained by evaporation. The evaporation is preferably carried out using a rotary evaporator. The extract obtained in the present invention is the ethanol extract of Gentiana straminea Maxim. As an alternative embodiment, the extract obtained in the present invention is mixed with distilled aqueous solution by ultrasonic to prepare a solution form of the ethanol extract of Gentiana straminea Maxim.; as another alternative embodiment, the extract obtained in the present invention is freeze-dried to obtain a freeze-dried powder; the freeze-drying treatment includes pre-freezing at -80°C and freeze-drying at -40°C.

[0041] The present invention discovers that the Gentiana straminea Maxim. extract and Fludarabine can synergistically regulate the levels of inflammatory factors TNF-α, IL-6, IL-4 and IL-10, as well as the expression of STAT1 / STAT6-related proteins in HMC3 cells. The synergistic effect between the two is obvious, and they can be effectively used for the development of drugs for chronic cerebral hypoperfusion.

[0042] The present invention also provides a drug for alleviating and / or treating chronic cerebral hypoperfusion. The active ingredient of the drug includes the Gentiana straminea Maxim. extract, and the Gentiana straminea Maxim. extract is the ethanol extract of Gentiana straminea Maxim.

[0043] The preparation method of the ethanol extract of Gentiana straminea Maxim. as the active ingredient of the drug in the present invention is preferably as follows: the dried roots of Gentiana straminea Maxim. are crushed and then mixed with ethanol, soaked, heated for condensation and reflux extraction, the filtrate is collected, and the filtrate is evaporated to prepare an extract. Preferably, the dried roots of Gentiana straminea Maxim. are crushed and passed through a 40-mesh sieve to obtain the dried root powder of Gentiana straminea Maxim.; preferably, the dried root powder of Gentiana straminea Maxim. is mixed with ethanol at a mass-to-volume ratio of 1 g:(4-6) mL, and the mass-to-volume ratio is more preferably 1 g:5 mL; preferably, the ethanol is 95% ethanol; preferably, the soaking is static soaking at room temperature, and the soaking time is 20-26 h, more preferably 21 h, 22 h, 23 h, 24 h or 25 h; preferably, the heating temperature for the heating condensation and reflux extraction is 80-85 °C, and the extraction times are preferably repeated 3 times, each lasting 1 h; preferably, the solutions (filtrates) after each filtration are combined and collected, and the extract is obtained by evaporation. The evaporation is preferably carried out using a rotary evaporator. The extract obtained in the present invention is the ethanol extract of Gentiana straminea Maxim. As an alternative embodiment, the extract obtained in the present invention is mixed with distilled aqueous solution by ultrasonic to prepare a solution form of the ethanol extract of Gentiana straminea Maxim.; as another alternative embodiment, the extract obtained in the present invention is freeze-dried to obtain a freeze-dried powder; the freeze-drying treatment includes pre-freezing at -80 °C and freeze-drying at -40 °C.

[0044] The drug in the present invention may take the ethanol extract of Gentiana straminea Maxim. as the sole active ingredient, or may also be used in combination with the ethanol extract of Gentiana straminea Maxim. + STAT1 inhibitor. The STAT1 inhibitor includes Fludarabine. The ethanol extract of Gentiana straminea Maxim. in the present invention is preferably the ethanol extract of Gentiana straminea Maxim. The drug for relieving and / or treating chronic cerebral hypoperfusion in the present invention also includes pharmaceutically acceptable excipients to facilitate the preparation of the drug dosage form and the convenience of clinical application. The present invention does not limit the specific drug dosage form and excipient selection.

[0045] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0046] In the embodiments of the present invention, the dried root of GSM is the dried root of Gentiana straminea Maxim. of the Gentianaceae family. The experimental animals, SPF-grade male SD rats, were obtained from Chengdu Dashuo Experimental Animal Co., Ltd., with the license number SCXK(Sichuan)2020-0030. All experimental animal operations complied with the regulations of the Experimental Animal Ethics Committee of Shaanxi University of Chinese Medicine (Ethical batch number: SUCMDL20241208002). 3-N-butylphthalide (NBP), batch number: 1182212113, was purchased from Shijiazhuang Pharmaceutical Group Enppi Pharmaceutical Co., Ltd. Human microglial cells (HMC3) were purchased from Wuhan Punosai Life Science Co., Ltd., product number: CL-0620.

[0047] In the embodiments of the present invention, all data were processed and analyzed using SPSS 26.0 software and GraphPad Prism 8 software. The experimental data were expressed as mean±SD. The Kolmogorov-Smirnov test and Shapiro-Wilk test were used to verify the normal distribution of the data. One-way ANOVA test (for normal distribution) or non-parametric test (for non-normal distribution) was used to determine the significance of differences between two groups. A P value less than 0.05 was considered statistically significant.

[0048] In the following examples, unless otherwise specified, all are conventional methods.

[0049] In the following examples, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0050] Example 1

[0051] Investigate the pharmacological effects and mechanisms of GSM against chronic cerebral hypoperfusion:

[0052] 1. Preparation of the drug and establishment and grouping of the rat CCH model

[0053] Preparation of the ethanol extract of GSM: Take the dried root of GSM, crush it through a 40-mesh sieve, take 200 g of the powder, add 1000 mL of 95% ethanol according to the mass-volume ratio of 1 (g): 5 (mL), seal it with plastic wrap, soak it at room temperature for 24 h, heat (85 °C) for condensation reflux extraction, repeat the extraction 3 times, each time for 1 h, combine and collect the filtered solution, and obtain the extract (30.9146 g) after completely evaporating the ethanol with a rotary evaporator. The extract was fixed to 200 mL with preheated (45 °C) distilled water, and after ultrasonic mixing (frequency 40 kHz, temperature 25 °C, time 10 min), the ethanol extract of GSM (1 g of crude drug·mL -1 ) was obtained and stored refrigerated for later use.

[0054] Establishment and grouping of the rat CCH model: The rat CCH model was prepared by permanent ligation of the bilateral common carotid arteries (2-VO). After anesthesia, the rats were fixed on the rat board, the neck was shaved, and disinfected with iodophor. An incision about 1.5 cm long was made along the middle of the neck, and the bilateral common carotid arteries were exposed by blunt dissection and ligated. Forty rats with successful surgery were grouped. According to the random number table method, they were divided into 4 groups: the model group (Model group), the positive drug group (NBP, 80 mg·kg -1 ), the low-dose GSM group (GSM-L, 3.33 g crude drug·kg -1 , administered with the ethanol extract of GSM), the high-dose GSM group (GSM-H, 6.66 g crude drug·kg -1 , administered with the ethanol extract of GSM), with 10 rats in each group. At the same time, a sham operation group (Sham group, 10 rats) was established. The rats in the sham operation group were treated in the same way without ligation. Gastric gavage of the corresponding drugs started on the third day after surgery and continued for 28 days.

[0055] (1) Evaluation of the neurological function of rats in each group by Zea Longa score

[0056] The neurological injury was evaluated according to the Zea Longa rating system. The definitions of this rating system are as follows: 0 points, rats with no neurological deficit; 1 point, the contralateral forepaw flexes and cannot fully extend when the tail is suspended; 2 points, cannot walk in a straight line and shows a following phenomenon when walking; 3 points, topples to the contralateral side when walking; 4 points, no spontaneous activity or loss of consciousness.

[0057] The neurological function of rats was evaluated by the Zea Longa scoring method. The results are as Figure 1 shown in the figure. In the figure, compared with the Sham group, ** P < 0.01; compared with the Model group, ## P < 0.01; n = 6. The results showed that compared with the Sham group, the neurological function score of the Model group increased significantly (P < 0.01). Compared with the Model group, the neurological function scores of GSM and NBP decreased significantly after treatment (P < 0.01). It indicates that GSM can effectively repair the neurological injury caused by CCH.

[0058] (2) Behavioral experiments of rats in each group

[0059] Morris water maze experiment: From the 23rd to the 27th day of drug administration, the rats underwent continuous 5-day place navigation training. During this process, the time taken by the rats to find and reach the designated platform within 90 s (i.e., the escape latency) was observed. On the second day after the end of the place navigation training, the platform was removed to perform the spatial probe test. At this stage, the number of times the rats crossed the position where the platform was previously placed within 90 s and the duration of staying in the quadrant where the platform was located were counted.

[0060] Open field test: Each rat in each group was placed into the open field exercise box one by one to allow it to move freely for 10 min. The first 5 min was for getting familiar with the environment, and the last 5 min was for the formal experiment. The movement trajectory was recorded by the upper camera, and the ANY-maze software was used to analyze the stored video to obtain data such as the average speed, total movement distance, and time in the central area of each group of rats within 5 min. Before each rat's experiment, the open field test box was wiped with water and alcohol to exclude external factors such as the smell of rats, so as not to let the remaining information of the previous rat affect the experimental results of the subsequent rats.

[0061] Rotarod test: Before the formal experiment, the rats were given 3 times of rotarod adaptability training. The initial rotation speed was 4 r / min, the final rotation speed was 40 r / min, and the acceleration time was set to 5 min. The staying time of the rats on the rotarod was recorded, and the average value of the 3 tests was taken as the experimental result of the rat. After each experiment, 75% alcohol was sprayed to eliminate the smell, and ultrapure water was sprayed to eliminate the alcohol smell. During the experiment, the environment was kept quiet.

[0062] The results of the effects of GSM on the behavior of CCH rats are as Figure 2 shown in the figure. In the figure, a: Representative trajectory diagrams of the water maze spatial probe test of each group of rats; b: Statistical results of the escape latency of each group of rats; c: Statistical results of the number of times each group of rats passed through the position where the platform was placed before; d: Statistical results of the duration that each group of rats stayed in the quadrant where the platform was located; e: Representative movement trajectory diagrams of each group of rats in the open field test; f: Statistical results of the total movement distance of each group of rats in the open field; g: Statistical results of the average speed of each group of rats in the open field; h: Statistical results of the time of each group of rats in the central area of the open field; i: Statistical results of the staying time of each group of rats on the rotarod. Compared with the Sham group, ** P < 0.01; compared with the Model group, # P < 0.05, ## P < 0.01.

[0063] The spatial learning and memory deficits of CCH rats were evaluated using the Morris water maze test. In the place navigation test, spatial learning ability was evaluated by escape latency. In the first 5 days of continuous training, as training increased, the escape latency of rats in each group shortened, indicating that rats performed spatial learning and memory through daily training. On the 5th day, the escape latency of the Model group was longer than that of the Sham group (P < 0.01), indicating that CCH indeed caused a decline in the spatial learning and memory abilities of rats, leading to cognitive impairment. The escape latency of the GSM-L and GSM-H groups was shorter than that of the Model group (P < 0.01). GSM administration significantly shortened the escape latency of rats on the 5th day and significantly increased the frequency of passing through the removed platform area and the time spent in the platform quadrant during spatial exploration on the 6th day. Compared with the Sham group, the frequency of passing through the removed platform area of the Model group was lower (P < 0.01), and the time spent in the platform quadrant decreased (P < 0.01). The frequency of passing through the removed platform area of the GSM-L and GSM-H groups was higher than that of the Model group (P < 0.01), and the time spent in the platform quadrant was also significantly prolonged (P < 0.01). This indicates that GSM alleviated the cognitive deficits of CCH rats to a certain extent.

[0064] The spontaneous locomotor ability of CCH rats was evaluated using the open field test. The results showed that after modeling, the spontaneous locomotor ability and motor function of rats were significantly weakened. The total distance traveled by rats in the Model group shortened, the average speed slowed down, and the time spent in the central area decreased (P < 0.01). Compared with the Model group, the spontaneous locomotor ability of rats improved after GSM and NBP treatment. The total distance traveled, average speed, and time spent in the central area of rats increased significantly (P < 0.05 or P < 0.01). In this regard, GSM-H was more effective than GSM-L.

[0065] The motor function of CCH rats was evaluated using the rotarod test. The results showed that GSM treatment could improve the motor dysfunction of CCH model rats. The time spent on the rod by rats in the Model group was shorter than that in the Sham group (P < 0.01), while the time spent on the rod by rats in the GSM-H and NBP groups was longer than that in the Model group (P < 0.01).

[0066] The open field test and rotarod test showed that GSM administration improved the behavior of rats in the open field and rotarod tests, revealing that GSM treatment had a certain degree of improvement effect on the motor ability of CCH rats.

[0067] (3) Laser Doppler flow imaging perfusion instrument was used to detect the cerebral blood flow of rats in each group

[0068] Anesthetize the rats, prepare the skin on the posterior part of the head, and disinfect it with iodophor. Make an incision about 2 cm along the midline of the brain, remove the subcutaneous fascia to fully expose the skull. Hold a micro-cranial grinder to thin the skull, place the rat's head under the detector and stabilize it for 2 minutes to record the whole-brain blood perfusion. Set the detection distance of the laser Doppler blood flow imaging perfusion instrument to 13 cm, the parameter area to 3 cm × 3 cm, and set the sensing region ROI (Region of Interest), and detect 3 times in parallel and take the average value.

[0069] The effect of GSM on the cerebral blood flow of CCH rats is as Figure 3 shown in the figure. In the figure, a: whole-brain blood flow heat map; b: statistical chart of the whole-brain blood perfusion volume of rats in each group. Compared with the Sham group, ** P < 0.01; compared with the Model group, ## P < 0.01; n = 3. Detection of the whole-brain blood perfusion by the laser Doppler blood flow imaging perfusion instrument found that the whole-brain blood perfusion volume of rats in the Model group decreased significantly (P < 0.01), indicating that CCH can reduce the cerebral blood flow of the cortex; compared with the Model group, the GSM-L and GSM-H groups can increase the whole-brain blood perfusion volume (P < 0.01). The blood flow recovered significantly after GSM intervention, suggesting that GSM plays a beneficial role in the brain.

[0070] (4) Detection of the pathological conditions of the brain tissues of rats in each group

[0071] After anesthetizing the rats, collect blood from the abdominal aorta, decapitate and take the brain. The brain tissues of 3 rats in each group are placed in 4% tissue fixative and fixed for 48 hours, and then undergo routine dehydration, wax infiltration, embedding, and sectioning, and finally operate according to the HE and Nissl staining steps and observe under the microscope.

[0072] The effect of GSM on the pathological damage of the brain tissues of CCH rats is as Figure 4 shown in the figure. In the figure, a: brain tissue stained with HE (×400); b: brain tissue stained with Nissl (×400); c: statistical chart of the number of Nissl + neurons in rats in each group; n = 3; compared with the Sham group, ** P < 0.01; compared with the Model group, ## P < 0.01.

[0073] The results of HE staining of the cerebral cortex of rats in each group showed that the cells in the cerebral tissue of the Sham group were closely arranged, with intact cell morphology and clear boundary lines. At the same time, the cell nuclei were large and full in shape, and the nucleoli were relatively obvious. However, the cells in the Model group showed obvious changes, including a decrease in the number of cells, scattered cell arrangement, cell degeneration, disappearance of cytoplasm, irregular and shrunken nuclei with deep staining, indicating that the cell structure in the Model group was damaged after 2-VO modeling. Compared with the Model group, GSM could significantly improve this damage, significantly reduce the number of damaged cells, relatively increase the number of cells, the cell arrangement was relatively neat, and the number of degenerated cells with irregular and shrunken nuclei and deep staining decreased, indicating that GSM could protect the neuronal structure to a certain extent.

[0074] The results of Nissl staining of the cerebral cortex of rats in each group showed that the cell morphology and structure of the Sham group were normal, the number of neurons did not change significantly, and the nucleoli were clearly centered. In the Model group, the cell arrangement was disordered, the cell structure was unclear, the cell nuclei were shrunken and deeply stained, and cell degeneration and necrosis were visible, and the number of neurons decreased significantly (P<0.01). Compared with the Model group, after GSM intervention, the number of neurons increased significantly (P<0.01), and some still had degeneration and necrosis, indicating that GSM could reduce the neuronal damage caused by CCH and protect the neuronal structure to a certain extent.

[0075] (5) Detection of microglial markers and M1 / M2 phenotype markers in the brain tissues of rats in each group

[0076] Detection of the number of IBA-1 positive cells, a microglial marker, in the brain tissues of rats by immunohistochemical staining: Paraffin sections were processed, incubated with relevant antibodies, and observed under a microscope after DAB and hematoxylin staining. The positive expression of IBA-1 was brownish-yellow, and the number of positive cells in each image was counted using Image pro plus.

[0077] Detection of the proportion of positive areas of CD86 / CD206, M1 / M2 phenotype markers, in the brain tissues of rats by immunofluorescence staining: Paraffin sections were dewaxed to water, incubated with relevant antibodies, and the cell nuclei were counterstained with DAPI. The sections were imaged using a scanning imaging system, and the proportion of positive areas in each image was calculated using the Halo data image analysis system.

[0078] The effect of GSM on microglial polarization in the brain tissues of CCH rats was as Figure 5 shown. In the figure, a: Immunohistochemical staining of IBA-1 in the brain tissue (×400); b: Statistical analysis of the number of IBA-1 positive cells in each group of rats; c: Immunofluorescent staining of CD86 and CD206 in the brain tissue (×200); d: Proportion of positive areas of CD86 in each group of rats; e: Proportion of positive areas of CD206 in each group of rats. Compared with the Sham group, **P < 0.01; compared with the Model group, # P < 0.05, ## P < 0.01.

[0079] The activity of microglia was detected by immunohistochemical staining of IBA-1. The results showed that the number of IBA-1 positive cells in the Model group was significantly increased (P < 0.01), while compared with the Model group, GSM treatment could reduce the number of IBA-1 positive cells (P < 0.05 or P < 0.01).

[0080] The expressions of M1 / M2 phenotype markers CD86 and CD206 in the brain tissues of CCH rats were detected by immunofluorescence staining. The results showed that the proportion of CD86 positive area in the Model group was significantly increased (P < 0.01), and the proportion of CD206 positive area was significantly decreased (P < 0.01). After GSM intervention, the proportion of CD86 positive area was significantly decreased (P < 0.01), and the proportion of CD206 positive area was significantly increased (P < 0.01).

[0081] (6) Detection of the levels of inflammatory factors in the sera of rats in each group

[0082] The levels of pro-inflammatory factors and anti-inflammatory factors were detected by ELISA: After the rats were anesthetized, blood was collected from the abdominal aorta, left standing at room temperature for 30 min, centrifuged at 3000 rpm / min for 15 min, the upper-layer serum was aliquoted into EP tubes and stored at -80 °C for later use. According to the requirements of the ELISA kit, the levels of pro-inflammatory factors TNF-α and IL-6 and anti-inflammatory factors IL-4 and IL-10 in the sera of rats in each group were detected. The results are shown in the figure Figure 6 as shown. In the figure, compared with the Sham group, ** P < 0.01; compared with the Model group, ## P < 0.01; n = 6.

[0083] The results showed that compared with the Sham group, the contents of TNF-α and IL-6 in the Model group were significantly increased (P < 0.01), and at the same time, the amounts of IL-4 and IL-10 were also significantly decreased (P < 0.01). Comparing with the Model group again, it was found that GSM-L and GSM-H could effectively slow down the increase of TNF-α and IL-6 (P < 0.01), and increase the levels of IL-4 and IL-10 (P < 0.01), and the effect of GSM-H was better than that of GSM-L. It is suggested that GSM plays a neuroprotective role by inhibiting M1 polarization and enhancing M2 polarization, thereby reducing the inflammatory response in CCH rats.

[0084] (7) Detection of the expressions of STAT1 / STAT6-related proteins in the brain tissues of rats in each group by Western Blot

[0085] Total protein extraction from tissues: Wash the tissue blocks with pre-cooled PBS 1-2 times, cut them into small pieces and place them in a grinding tube. Add 3 grinding beads of 3 mm, add lysis buffer at 10 times the tissue volume, and set the grinding program to grind the tissues. Take out the completed grinding tube, place it on ice or in the lysis buffer at 4°C for 30 min. Centrifuge at 12,000 rpm at 4°C for 10 min, and collect the supernatant, which is the total protein solution.

[0086] Protein concentration determination: Take the undenatured protein solution and measure the protein concentration with reference to the instructions of the BCA protein concentration determination kit.

[0087] Protein denaturation: Add 5× reducing protein loading buffer to the protein solution at a ratio of 4:1, denature at 95°C in a metal bath for 10 min, and store at -80°C in the refrigerator for later use.

[0088] SDS-PAGE electrophoresis: Clean the glass plates and prepare the gel. Place the gel in the electrophoresis tank, fill the inner side with electrophoresis buffer, carefully remove the comb, and load the sample. Stop the electrophoresis until the bromophenol blue is about 1 cm from the bottom, and prepare for the membrane transfer operation.

[0089] Membrane transfer: Prepare the transfer filter paper and PVDF membrane. The PVDF membrane needs to be activated with methanol before use. Place the transfer cassette, two sponges, filter paper, and the activated PVDF membrane in a container with transfer buffer. Carefully peel off the separating gel and place it on the filter paper, rinse the bubbles on the gel with transfer buffer, slowly attach the PVDF membrane to the gel without bubbles, and then attach the transfer filter paper and transfer sponge in sequence.

[0090] Immune reaction: Place the transferred membrane in an incubation box containing 5% skim milk, place it on a shaker, and block it at room temperature. Dilute the primary antibody according to the antibody instructions. After preparation, pour out the blocking solution in the incubation box, add the prepared primary antibody, and incubate at 4°C overnight. Recover the primary antibody and wash the membrane three times with TBST. Dilute the secondary antibody with TBST, then add it to the incubation box, place it on a shaker, and incubate at room temperature. Wash the membrane three times with TBST.

[0091] Chemiluminescence: Mix ECLA and solution B at a ratio of 1:1 and set aside. Take out the eluted PVDF membrane and place it on absorbent paper to slightly dry the liquid on the membrane. Place the membrane in the mixed ECL luminescent solution to completely immerse the membrane in the liquid. After reacting for 1 min, take out the membrane and place it on the tray of the chemiluminescence instrument to start exposure.

[0092] Result analysis: Save the original image in TIFF format after exposure. Analyze the data of the saved TIFF format original image with ImageJ analysis software.

[0093] The effect of GSM on the expression of STAT1 / STAT6-related proteins in the brain tissue of CCH rats is asFigure 7 As shown in the figure, compared with the Sham group, * P < 0.05, ** P < 0.01; compared with the Model group, ## P < 0.01. STAT 1 increases M1 polarization, while STAT 6 plays a role in M2 polarization. The results show that microglia in the Sham group are in a resting state, with low expression of STAT1 / STAT6-related proteins and in a micro-equilibrium state. In the Model group, the expression of p-STAT1 / STAT1 and p-STAT6 / STAT6 increased (P < 0.01 or P < 0.05). After CCH in rats, activated microglia not only produce pro-inflammatory factors to exacerbate brain injury, but also secrete some anti-inflammatory factors to initiate the repair process of brain injury, so the expression of STAT1 / STAT6-related proteins is up-regulated. Compared with the Model group, after GSM intervention, the expression of p-STAT1 / STAT1 can be significantly reduced (P < 0.01), and the expression of p-STAT6 / STAT6 can be significantly increased (P < 0.01). The GSM-H group has a better effect. It shows that GSM intervention improves brain injury and plays a brain-protective role, with up-regulated expression of STAT6-related proteins and down-regulated expression of STAT1-related proteins. In summary, it is suggested that GSM may play a therapeutic role by regulating the expression of STAT1 / STAT6, inhibiting M1 polarization and promoting M2 polarization.

[0094] Example 2

[0095] Study on the protective effect and mechanism of GSM on oxygen-glucose deprivation HMC3 cells:

[0096] 1. Preparation of GSM test solution

[0097] GSM extraction: Take the dried roots of GSM, crush them through a 40-mesh sieve, take 200 g of the powder, add 1000 mL of 95% ethanol according to the mass-volume ratio of 1 (g): 5 (mL), seal it with plastic wrap, soak it at room temperature for 24 h, heat (85 °C) for reflux extraction with condensation, repeat the extraction 3 times, each time for 1 h, combine and collect the filtered solution, and completely evaporate the ethanol with a rotary evaporator to obtain an extract (30.9146 g). Put the medicinal extract into an evaporating dish and leave it at -80 °C overnight. The next day, place it in a freeze dryer for treatment (-40 °C, 24 h), and finally obtain 18.3829 g of GSM extract powder.

[0098] GSM in vitro experimental drug preparation: Weigh 0.2 g of GSM extract powder precisely and directly add it to MEM (containing NEAA) basal medium to make up 20 mL. Sonicate it (40 kHz, 25 °C, 30 min) to dissolve it completely. Aliquot the dissolved GSM and store it sealed at -20 °C for later use. This solution is the stock solution with a concentration of 10 mg / mL. In subsequent experiments, dilute it to 1 mg / mL before use, filter it through a microporous membrane to sterilize, and then dilute it to the required concentration for use.

[0099] 2. HMC3 cell culture

[0100] HMC3 cells are cultured in MEM medium containing 10% FBS and 1% P / S and placed in a cell incubator at a constant temperature of 37 °C and 5% CO2. Observe the growth state of the cells, and change the medium 2 - 3 times a week. When the cell confluence in the culture flask is greater than 80%, cell passage is carried out.

[0101] Cell passage: Aspirate the original culture medium; add 2 mL of PBS, gently shake the culture flask to wash the cells, and aspirate and discard the PBS; add 1 mL of 0.25% trypsin solution (containing EDTA), gently shake the culture flask to make it soak all the cells; place it in the incubator for digestion. When most cells are detached from the wall under the microscope, digestion can be terminated. The digestion time is 2 min; add 3 mL of serum-containing medium to terminate digestion, pipette to detach the cells from the wall and pipette repeatedly in the liquid to make the cells as single-cell suspension as much as possible; collect the cell suspension and centrifuge at 1200 rpm / min for 3 min. After centrifugation, aspirate and discard the supernatant; add fresh medium, pipette a few times to mix the cells evenly, inoculate them into a new culture flask according to the ratio, the passage ratio is 1:5, supplement the medium, and culture with a breathable bottle cap.

[0102] Cell cryopreservation, the cryopreservation steps are as follows: HMC3 cells are adherent cells. When it is observed under the microscope that the cells cover the bottom of the culture flask, digest with trypsin and centrifuge, discard the supernatant, quickly add a specific cell cryopreservation solution (70% basal medium + 20% fetal bovine serum + 10% DMSO), pipette to resuspend the cells, aliquot them into cryotubes, about 2 mL per tube, and seal. To ensure the safe storage of cells, first place the cryotubes in a 4 °C refrigerator and let them stand for 20 min. Subsequently, transfer the cryotubes to a -20 °C refrigerator and let them stand for 2 h. Then, place the cryotubes in an -80 °C refrigerator, and the cells can be stored for 3 - 6 months. If longer-term storage is needed, finally place the cell cryotubes in a liquid nitrogen environment to ensure the long-term stability of the cells.

[0103] Cell resuscitation: Take out the cryopreserved HMC3 cells from the liquid nitrogen tank. After the liquid nitrogen evaporates, place them in a 37°C water bath. During this period, hold the cryopreservation tube and shake it up and down continuously to dissolve it quickly. After dissolution, take out the cryopreservation tube, dry the moisture with sterile paper, spray 75% alcohol, and quickly transfer it to the operating table. Add the dissolved HMC3 cells to a 15 mL centrifuge tube containing 3 mL of MEM medium, centrifuge at 1200 rpm for 3 min, discard the supernatant, resuspend the cells with 1 mL of complete MEM medium, mix well, and inoculate them into a T-25 culture flask pre-added with 4 mL of medium. Gently shake the culture flask to disperse the cells evenly, and place it in an incubator at 37°C and 5% CO2 for culture. After 24 h, observe the cell status, perform medium replacement, and continue culturing.

[0104] Cell counting: Drop the cell suspension along the edge of the coverslip onto the counting plate (before use, both the coverslip and the counting plate should be cleaned with alcohol). Let it stand for 3 min until the cells are fixed, and then count the cells under the microscope (following the principle of counting cells on the upper and left sides but not on the lower and right sides. Aggregated clumped cells are counted as 1). The calculation formula is: Number of cells / mL = Total number of cells in four large grids / 4 × 10 4 cells / mL.

[0105] 3. Establishment of oxygen-glucose deprivation (OGD) model

[0106] Take HMC3 cells in the logarithmic growth phase, digest the cells, and suspend them with MEM to make the density of the cell suspension 1×10 5 cells / mL. Inoculate them into a 96-well plate, 100 μL per well. After inoculation, place them in a CO2 incubator for normal culture. The next day, replace the MEM in the HMC3 cells with sugar-free MEM, keep other conditions unchanged, and place them in a triple-gas incubator (37°C, 94% N2, 5% CO2, 1% O2) for culture to establish an HMC3 oxygen-glucose deprivation (OGD) model.

[0107] 4. Effects of different OGD times on HMC3 cells

[0108] Take HMC3 cells in the logarithmic growth phase, resuspend the cells with complete medium, adjust the density to 1×10 5 cells / mL, divide them into 6 groups, namely normal culture for 1, 2, 3, 4, 5, 6 h (Control group), and OGD for 1, 2, 3, 4, 5, 6 h (experimental group). After the corresponding treatment time ends, detect the cell viability with CCK-8.

[0109] An in vitro CCH model was established by OGD, and the effects of different OGD times on the viability of HMC3 cells were detected by CCK-8. The results are as Figure 8 shown. In the figure, compared with the Control group, **P < 0.01, n = 6. The results showed that after OGD, the cell viability of HMC3 cells decreased significantly with the prolongation of time. Compared with the Control group, the cell viability of HMC3 cells was significantly reduced under the condition of OGD for 4 h (P < 0.01). To reduce cell viability without causing massive cell death, OGD for 4 h was finally determined as the cell modeling time, and OGD for 4 h was used as the cell modeling time for subsequent experimental contents.

[0110] 5. Effects of different concentrations of GSM on HMC3 cells

[0111] Closely monitor the growth of HMC3 cells. When they grow to a good state and show logarithmic growth, suspend them with MEM. Subsequently, the density of HMC3 cells can be adjusted to approximately 1×10 5 cells / mL, and inoculate them into a 96-well culture plate, adding 100 μL in each well. After plating and stabilizing for 24 h, add medium containing different concentrations of GSM (800, 400, 200, 100, 50, 25, 12.5, 6.25 μg / mL) to the cells in the drug-adding wells. Set 6 replicates for each concentration. At the same time, set the corresponding control wells and blank wells respectively. After 24 h, 48 h, and 72 h, replace the fresh culture medium and add 10 μL of CCK-8 solution and incubate for 1.5 h. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value (OD) at 450 nm, calculate the relative cell survival rate at different concentrations and different time points, and select the appropriate drug concentration and time.

[0112] The effects of different concentrations of GSM on the viability of HMC3 cells are as Figure 9 shown in the figure. In the figure, compared with the Control group, ** P < 0.01, n = 6. The results showed that GSM at 6.25 - 200 μg / mL had no obvious effect on the viability of normally cultured HMC3 cells (P > 0.05), while when the concentration of GSM was greater than 200 μg / mL, it could significantly inhibit the viability of HMC3 cells (P < 0.01); the effect of GSM acting for 24 h on the viability of normally cultured HMC3 cells was the lowest. Therefore, it was finally determined that the concentration of GSM in subsequent studies should be in the range of 6.25 - 200 μg / mL, and the action time of GSM should be 24 h.

[0113] 6. Effects of GSM on the viability of OGD-induced HMC3 cells

[0114] Take cells in the logarithmic growth phase, resuspend them with MEM, and adjust the cell density to approximately 1×10 5Cells were inoculated into a 96-well plate at a density of 1×10 cells / mL, 100 μL per well, and divided into a Control group, an OGD group, and a GSM group. After inoculation, the cells were cultured in a CO2 incubator. After the cells adhered and grew, the medium of the drug treatment group was replaced with a medium containing the corresponding concentration of GSM. An OGD model was constructed according to the method in step 3 (OGD for 4 h was used as the cell modeling time). After OGD, 10 μL of CCK-8 solution was added to each well and incubated for 1.5 h. The absorbance value at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader to calculate the effect of different concentrations of GSM on the viability of OGD-induced HMC3 cells, and the optimal drug dosage was selected for subsequent experiments.

[0115] After treating HMC3 cells with 6.25 - 200 μg / mL of GSM for 24 h, OGD was performed for 4 h, and CCK-8 was used to detect cell viability to investigate the protective effect of GSM on OGD-induced HMC3 cells. The effect of GSM on the viability of OGD-induced HMC3 cells is shown as Figure 10 follows. In the figure, compared with the Control group, ** P < 0.01; compared with the OGD group, # P < 0.05, ## P < 0.01, n = 6. The results showed that compared with the Control group, the viability of HMC3 cells in the OGD group was significantly decreased (P < 0.01); compared with the OGD group, 12.5 - 200 μg / mL of GSM improved cell viability to varying degrees (P < 0.05 or P < 0.01), and the effects of 50 μg / mL and 100 μg / mL of GSM were the best. Therefore, in this study, 50 μg / mL and 100 μg / mL of GSM were finally selected as the low and high doses for subsequent drug administration experiments.

[0116] 7. Drug administration experiment on HMC3 cells

[0117] Cells in the logarithmic growth phase were suspended with MEM to make the cell density approximately 1×10 5Cells were inoculated at a density of cells / mL into six-well plates, 2 mL per well. This experiment was divided into 8 groups: Control group, OGD group, AS1517499 (STAT6 inhibitor, 8 μM) group, Fludarabine (STAT1 inhibitor, 50 μM) group, GSM (50 μg / mL and 100 μg / mL) group, GSM + AS1517499 (100 μg / mL GSM + 8 μM AS1517499, denoted as AS + 100 μg / mL) group, and GSM + Fludarabine (100 μg / mL GSM + 50 μM Fludarabine, denoted as Flu + 100 μg / mL) group. After inoculation, the cells were cultured in a CO2 incubator. After adherent growth, the medium in each drug administration group was replaced with MEM medium containing the corresponding drug concentration, and an OGD model was constructed according to the method in step 3 (OGD for 4 h was used as the cell modeling time). After the treatment, the cells and cell supernatants were collected for subsequent operations.

[0118] (1) Observation of the morphology of HMC3 cells in each group

[0119] After the treatment of HMC3 cells in the Control group, OGD group, AS1517499 (STAT6 inhibitor, 8 μM) group, Fludarabine (STAT1 inhibitor, 50 μM) group, GSM (50 μg / mL) group, GSM (100 μg / mL) group, AS + 100 μg / mL group, and Flu + 100 μg / mL group, the liquid in the six-well plates was replaced with PBS, and the cell density and cell morphology of each group were carefully observed under an inverted microscope. Pictures were taken and saved. The results are as Figure 11 shown. The results showed that the HMC3 cells in the Control group grew adherently, and the cell morphology was epithelial-like; compared with the Control group, the cell density in the OGD group was significantly decreased, the boundary was blurred, and the cell morphology was amoeba-like, with pseudopodia and ciliated protrusions; compared with the OGD group, the cells in the AS1517499 group showed more severe changes. The cell density in the 100 μg / mL group, Fludarabine group, and Flu + 100 μg / mL group increased, the boundary was clear, and the cell morphology tended to be that of the Control group cells. The improvement effect of the Fludarabine group was better than that of the 100 μg / mL group, and the Flu + 100 μg / mL group had the best improvement effect. It was indicated that the HMC3 cells were activated after OGD, GSM was beneficial for the activated cells to return to the normal morphology, and Fludarabine had a synergistic effect with GSM.

[0120] (2) Detection of microglial markers and M1 / M2 phenotype markers in HMC3 cells in each group

[0121] Place the disinfected round cell culture slides at the bottom of a six-well plate. Uniformly inoculate the cell suspension into the six-well plate containing the slides. After 24 hours of inoculation, perform drug treatment. After the treatment is completed, fix the cell slides with 4% paraformaldehyde. After the fixation is completed, collect the cell slides of each group and perform immunofluorescence staining. The specific operation steps are as follows: Steps for single staining of microglial marker CD11b: After gently shaking off the liquid on the slides, use a histochemical pen to draw a circle at the position where the cells are evenly distributed in the middle of the coverslip, and add 50 - 100 μL of permeabilization working solution and incubate at room temperature for 20 minutes. Drop 3% bovine serum albumin (BSA) within the circle, evenly cover it, and incubate at room temperature for 30 minutes for blocking. Gently shake off the blocking solution, add the prepared rabbit anti-CD11b, and incubate overnight at 4°C. Add the corresponding secondary antibody, CY3-labeled goat anti-rabbit, and incubate at room temperature for 50 minutes. Drop DAPI staining solution within the circle and incubate at room temperature for 10 minutes in the dark. Finally, mount the slides with an anti-fluorescence quenching mounting medium. After image acquisition using a scanner, process and analyze the obtained images with ImageJ software.

[0122] Steps for double staining of M1 / M2 phenotype markers iNOS / Arg-1: Add the permeabilization working solution and incubate at room temperature for 20 minutes. Add the first prepared primary antibody, rabbit anti-iNOS, and incubate overnight at 4°C. Add the corresponding secondary antibody, HRP-labeled goat anti-rabbit, and incubate at room temperature for 50 minutes. Drop TSA and incubate at room temperature for 10 minutes in the dark. Add the second prepared primary antibody, rabbit anti-Arg-1, and incubate overnight at 4°C. Drop the corresponding secondary antibody, CY3-labeled goat anti-rabbit, to cover the cells, and incubate at room temperature for 50 minutes. Drop DAPI staining solution and incubate at room temperature for 10 minutes in the dark. Finally, mount the slides with an anti-fluorescence quenching mounting medium. After image acquisition using a scanner, process and analyze the obtained images with ImageJ software.

[0123] The effects of GSM on the polarization of HMC3 cells in each group are as Figures 12 - 13 shown, Figure 12 For the results of immunofluorescence detection of the expression of microglial marker CD11b in HMC3 cells of each group, Figure 13 For the results of immunofluorescence detection of the expression of M1 / M2 phenotype markers iNOS / Arg-1 in each group. In the figure, compared with the Control group, ** P < 0.01; compared with the OGD group, ## P < 0.01; compared with the 100 μg / mL group, @@P<0.01, n=3. The results showed that the expression of CD11b and iNOS in the OGD group was significantly increased compared with the Control group (P<0.01), and the expression of Arg-1 was significantly decreased (P<0.01). Compared with the OGD group, GSM downregulated the expression of CD11b and iNOS (P<0.01) and upregulated the expression of Arg-1 (P<0.01). Compared with the 100μg / mL group, the AS+100μg / mL group significantly increased the expression of CD11b and iNOS (P<0.01) and significantly decreased the expression of Arg-1 (P<0.01); the Flu+100μg / mL group significantly decreased the expression of CD11b and iNOS (P<0.01) and significantly increased the expression of Arg-1 (P<0.01). These results suggest that GSM can inhibit M1 polarization and promote M2 polarization after OGD, thereby improving the inflammatory damage of HMC3 cells. AS1517499 can antagonize the effect of GSM, and Fludarabine has a synergistic effect with GSM.

[0124] (3) Detection of inflammatory factor levels in HMC3 cells in each group

[0125] After the cell administration treatment, the cell supernatant was collected and centrifuged at 3000rpm / min for 10min to obtain the supernatant. The levels of TNF-α, IL-6, IL-4 and IL-10 in the supernatant of each group of HMC3 cells were detected according to the requirements of the TNF-α, IL-6, IL-4 and IL-10 ELISA kits. The specific steps are as follows: Strictly follow the operating steps in the instructions to prepare the working solution. Set up standard wells, blank wells and sample wells, add 50μL of standards of different concentrations to each standard well, no blank wells, add 10μL of the sample to be tested to the sample well, and then add 40μL of sample diluent. Except for the blank wells, add 100μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard well and sample well, seal the reaction wells with a sealing film, and incubate in a 37℃ water bath or incubator for 1h. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times. Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes. In the Excel worksheet, use the concentration of the standard as the ordinate and the corresponding OD value as the abscissa to draw the linear regression curve of the standard. Substitute the OD value into the formula according to the curve equation to calculate the concentration value of each sample.

[0126] Effects of GSM on the levels of inflammatory factors TNF-α, IL-6, IL-4 and IL-10 in HMC3 cells of each group Figure 14As shown, the results showed that compared with the Control group, the contents of TNF-α and IL-6 in the cell supernatant of the OGD group increased significantly (P < 0.01), while the contents of IL-4 and IL-10 decreased significantly (P < 0.05). Compared with the OGD group, GSM inhibited the levels of TNF-α and IL-6 factors (P < 0.01) and increased the levels of IL-4 and IL-10 factors (P < 0.05 or P < 0.01). Compared with the 100 μg / mL group, the AS + 100 μg / mL group significantly increased the levels of TNF-α and IL-6 (P < 0.01) and significantly decreased the levels of IL-4 and IL-10 (P < 0.01); the Flu + 100 μg / mL group significantly inhibited the levels of TNF-α and IL-6 (P < 0.01) and significantly increased the levels of IL-4 and IL-10 (P < 0.05 or P < 0.01). It is suggested that GSM can improve the inflammatory injury of HMC3 cells induced by OGD, AS1517499 can antagonize the effect of GSM, and Fludarabine has a synergistic effect with GSM.

[0127] (4) Detection of the expression of STAT1 / STAT6-related proteins in HMC3 cells of each group by Western Blot

[0128] After the cell drug treatment was completed, the original medium in the six-well plate was discarded, and the cells were rinsed 3 times with PBS. The PBS was discarded, and the residual liquid was aspirated as dry as possible with a pipette. An appropriate volume of RIPA lysis buffer was added to the culture plate for 4 min. During this period, the culture plate was shaken repeatedly to ensure that the reagent was in full contact with the cells. The cells were scraped off with a cell scraper and transferred to a 1.5 mL centrifuge tube. The cells were lysed on ice for 30 min, and during this period, the cells were repeatedly pipetted to ensure complete lysis of the cells. Centrifuge at 12,000 rpm at 4 °C for 10 min, and collect the supernatant, which is the total protein solution. The remaining steps were carried out according to the method in step (7) of the example to detect the expression of STAT1 / STAT6-related proteins in HMC3 cells of each group.

[0129] The effect of GSM on the expression of STAT1 / STAT6-related proteins in HMC3 cells of each group was as Figure 15 shown. In the figure, compared with the Control group, * P < 0.05, ** P < 0.01; compared with the OGD group, # P < 0.05, ## P < 0.01; compared with the 100 μg / mL group, @@P < 0.01. STAT1 increases M1 polarization, while STAT6 plays a role in M2 polarization. The results of Western blot detection of STAT1 / STAT6-related protein expression showed that compared with the Control group, the expression of p-STAT1 / STAT1 in the OGD group was significantly increased (P < 0.01), and the expression of p-STAT6 / STAT6 was significantly decreased (P < 0.05). Compared with the OGD group, the expression of p-STAT1 / STAT1 could be significantly reduced after intervention with 100 μg / mL GSM (P < 0.01); compared with the OGD group, GSM significantly increased the expression of p-STAT6 / STAT6 (P < 0.01). Compared with the 100 μg / mL group, the expression of p-STAT1 / STAT1 in the AS + 100 μg / mL group was significantly increased (P < 0.01), and the expression of p-STAT6 / STAT6 was significantly decreased (P < 0.01); the expression of p-STAT1 / STAT1 in the Flu + 100 μg / mL group was significantly reduced (P < 0.01), and the expression of p-STAT6 / STAT6 was significantly increased (P < 0.01). It is suggested that AS1517499 can antagonize the effect of GSM, and Fludarabine has a synergistic effect with GSM.

[0130] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of Gentiana straminea Maxim. extract in the preparation of a drug for relieving and / or treating chronic cerebral hypoperfusion.

2. The application according to claim 1, characterized in that, The Gentiana straminea Maxim. extract is an ethanol extract of Gentiana straminea Maxim.; the preparation method of the ethanol extract of Gentiana straminea Maxim. includes: drying and pulverizing the roots of Gentiana straminea Maxim., mixing with ethanol, soaking, heating and refluxing for extraction, collecting the filtrate, and evaporating the filtrate to prepare an extract.

3. The application according to claim 1, characterized in that The Gentiana straminea Maxim. extract protects the neuronal structure.

4. The application according to claim 1, characterized in that, The Gentiana straminea Maxim. extract relieves cognitive deficits and motor function impairments caused by chronic cerebral hypoperfusion.

5. The application according to claim 1, characterized in that, The Gentiana straminea Maxim. extract reduces the inflammatory response of chronic cerebral hypoperfusion, regulates the expression of STAT1 / STAT6, inhibits M1 polarization, and promotes M2 polarization.

6. The application according to claim 1, characterized in that, The Gentiana straminea Maxim. extract improves the inflammatory damage of OGD-induced HMC3 cells and promotes the restoration of HMC3 cells to a normal morphology.

7. Use of Gentiana straminea Maxim. extract combined with STAT1 inhibitor in the preparation of a medicament for relieving and / or treating chronic cerebral hypoperfusion, characterized in that, The Gentiana straminea Maxim. extract is an ethanol extract of Gentiana straminea Maxim.

8. The application according to claim 7, wherein The STAT1 inhibitor includes Fludarabine.

9. A drug for alleviating and / or treating chronic cerebral hypoperfusion, characterized in that, The active ingredient of the drug includes the Gentiana straminea Maxim. extract, and the Gentiana straminea Maxim. extract is an ethanol extract of Gentiana straminea Maxim.

10. The drug according to claim 9, characterized in that, The active ingredient of the drug also includes a STAT1 inhibitor.

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