Application of Luosan in preventing and treating ischemic stroke

Abstract

The present invention relates to the application field of traditional Chinese medicine, and specifically to the use of Luosan in preventing and treating ischemic stroke. The present invention proposes for the first time the use of Luosan in the preparation of a drug for preventing or treating ischemic stroke, thereby reducing the risk of ischemic stroke and alleviating the symptoms of ischemic stroke. The present invention verifies that Luosan can dose-dependently improve the neurological scores and dose-dependently reduce the cerebral infarction area of ​​rats with cerebral ischemia-reperfusion; after using Luosan, the content of malondialdehyde is significantly reduced and the content of superoxide dismutase is significantly increased, and Luosan reduces cerebral ischemia-reperfusion injury; Luosan inhibits cell apoptosis by activating the PI3K / AKT / mTOR signaling pathway, exerting a neuroprotective effect.

Description

Technical Field

[0001] The present invention relates to the application field of traditional Chinese medicine, and particularly to the application of Luosan in the prevention and treatment of ischemic stroke. Background Art

[0002] Ischemic stroke, also known as cerebral infarction, refers to brain tissue necrosis caused by insufficient blood supply to the brain due to stenosis or occlusion of the brain's blood supply arteries (such as the carotid artery and vertebral artery). The pathogenesis of ischemic stroke is relatively complex, involving multiple pathophysiological processes, such as thrombosis, embolism, and hypoperfusion. Among them, thrombosis is the most common cause, mainly due to atherosclerosis. Embolism is caused by cardiogenic or arterial emboli blocking the brain's blood supply arteries. Hypoperfusion is caused by low blood pressure or severe stenosis of blood vessels, resulting in reduced blood flow to the brain.

[0003] Ischemic stroke falls under the category of "stroke" in Traditional Chinese Medicine. The occurrence of stroke is related to a variety of factors, including emotional imbalance, improper diet, and overwork. Emotional imbalance is a key cause of stroke. Long-term mental stress and emotional instability can lead to liver qi stagnation, which in turn causes qi and blood disorder and can cause stroke.

[0004] In addition, improper diet and excessive fatigue can also lead to poor circulation of qi and blood in the body, thus causing stroke.

[0005] In terms of treatment, Western medicine primarily relies on medications for ischemic stroke, including antiplatelet drugs, anticoagulants, and thrombolytics. Treatment options should be tailored to the patient's condition and etiology, requiring use under a doctor's supervision and potentially associated with side effects. Furthermore, Western medicine may not be effective for some patients, such as those with underlying conditions such as hypertension and diabetes, or those with more severe disease. Thrombolytic therapy is generally considered one of the best treatments for acute ischemic stroke. Tissue plasminogen activator (tPA) is the most commonly used medication, used to dissolve clots and restore blood flow. However, this approach has limitations, including time windows and bleeding risks. Traditional Chinese medicine (TCM) offers potential advantages in expanding treatment options for cerebral hemorrhage, improving limb function, and promoting recovery. The combined effects of TCM medications, acupuncture, massage, fumigation, and qigong can help improve limb function, swallowing, and speech. Acupuncture can achieve therapeutic goals by stimulating acupoints and regulating the flow of qi and blood. In terms of Traditional Chinese Medicine (TCM), commonly used medications include those that promote blood circulation, remove blood stasis, and relax tendons and meridians, such as angelica, Chuanxiong, and safflower. Massage can improve blood circulation in the limbs and alleviate symptoms such as limb paralysis. TCM can tailor treatment plans based on the patient's individual symptoms. Before the onset of symptoms, holistic conditioning can be performed based on the patient's constitution, which can help improve treatment effectiveness.

[0006] Luosan (Lotus) is a tree in the genus Luosan (Araliaceae) with medicinal value. It can be used to treat fever, headache, and cough caused by colds. Luosan also dispels wind and dampness, relaxes muscles and activates blood circulation, and can be used as an adjunct to treat joint pain, swelling, and difficulty flexing and extending caused by rheumatoid arthritis and other conditions. Luosan also relieves cough and reduces phlegm, making it an adjunct to treating cough and phlegm caused by colds in children. Currently, there are no studies investigating the use of Luosan for the prevention and treatment of ischemic stroke. Summary of the Invention

[0007] The present invention provides the application of Luosan in preventing and treating ischemic stroke, provides a new method for treating acute ischemic stroke, and provides reference and inspiration for researching and developing new anti-ischemic stroke Chinese medicines.

[0008] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0009] The present invention provides the use of Luosan in preparing a medicine for preventing or treating ischemic stroke.

[0010] In the above technical solution, further, the effective content of the Luosan is 50-200 mg / kg.

[0011] The invention provides a pharmaceutical composition for preventing or treating ischemic stroke. The pharmaceutical composition uses Lophatherum gracile as an active component.

[0012] In the above technical solution, further, the pharmaceutical composition uses Luosan as the only active ingredient.

[0013] In the above technical solution, further, the pharmaceutical composition includes pharmaceutically acceptable excipients.

[0014] In the above technical solution, further, the pharmaceutical composition is an oral preparation or an injectable preparation.

[0015] Compared with existing technologies, the present invention offers the following benefits: It is the first to propose the use of Luosan for the prevention and treatment of ischemic stroke, reducing the risk of ischemic stroke and alleviating its symptoms. Luosan dose-dependently improved neurological scores and reduced cerebral infarction area in rats with cerebral ischemia-reperfusion. Luosan significantly reduced malondialdehyde levels and increased superoxide dismutase levels after use, demonstrating that Luosan reduced cerebral ischemia-reperfusion. Luosan also exerted a neuroprotective effect by inhibiting apoptosis through activation of the PI3K / AKT / mTOR signaling pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Neurological function score results: (A) Longa score; (B) mNSS score;

[0017] Note: ####P<0.0001vs Sham group.

[0018] Figure 2 Example 3 Test results: (A) TTC staining of cerebral infarction area; (B) Determination of cerebral infarction area; The cerebral infarction area of ​​rats in the Model group increased, while the cerebral edema and cerebral infarction area of ​​rats in the low-, medium-, and high-dose Luosan treatment groups decreased; (C) Determination of cerebral edema content; The brain water content of rats in the Model group increased, while the cerebral edema content of rats in the low-, medium-, and high-dose Luosan treatment groups decreased; Note: ####P<0.0001vs Sham group, ****P<0.0001vs Model group.

[0019] Figure 3 Example 4 Test results: (A) Changes in reactive oxygen species (ROS) levels, ####P<0.0001 vs Sham group; *P<0.05, **P<0.01, ****P<0.0001 vs Model group; (B) Changes in malondialdehyde (MDA) levels, ####P<0.0001 vs Sham group; *P<0.05, **P<0.01, ****P<0.0001 vs Model group.

[0020] Figure 4 Example 5 Test Results: (A) Effects of Luosan on the expression of PI3K, AKT, mTOR, and their phosphorylated proteins in rat brain tissue; (B) Changes in p-PI3K / PI3K ratio in rat brain tissue; (C) Changes in p-AKT / AKT ratio in rat brain tissue; (D) Changes in p-mTOR / mTOR ratio in rat brain tissue. ###P<0.001, ####P<0.0001 vs. Sham group; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. Model group. DETAILED DESCRIPTION

[0021] The present invention is further described below with reference to specific examples, but is not intended to limit the present invention in any way.

[0022] Example 1

[0023] Establishment of a rat model of focal cerebral ischemia: A rat model of focal cerebral ischemia was established by incomplete ligation of the unilateral common carotid artery. Rats were weighed and anesthetized with 3 ml / kg of 10% chloral hydrate intraperitoneally at room temperature. Ischemia-reperfusion injury was induced by making a 2-2.5 cm incision along the midline of the neck. The neck mucosa and muscle were carefully dissected, and the vagus nerve and capillaries were carefully isolated to expose the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). A suture plug (0.43 ± 0.02 mm in diameter) was inserted through the ECA into the ICA to occlude the middle cerebral artery. The CCA and ECA were simultaneously ligated. After 2 hours of occlusion, the suture plug was removed to restore cerebral blood supply, and reperfusion was performed for 24 hours.

[0024] Sixty male SPF SD rats weighing 220-250 g were selected. Before the experiment, the SD rats were adaptively fed in a well-ventilated room for one week and randomly divided into 6 groups, with 10 rats in each group; (I) sham operation group (Sham group), (II) model group (Model group), (III) positive control nimodipine group (Nimodipine group, 15 mg / kg), (IV) low-dose group (L group, 50 mg / kg); (V) medium-dose group (M group, 100 mg / kg); (VI) high-dose group (H group, 200 mg / kg).

[0025] The sham-operated group did not receive a suture inserted into the middle cerebral artery. Two weeks prior to model establishment, the model group, positive control group, and low-, medium-, and high-dose Losan groups were administered normal saline, nimodipine, low-, medium-, and high-dose Losan via oral gavage. The saline and high-dose Losan dosages were identical. Neurobehavioral differences among the three groups were assessed using the Longa Neurological Function Scale (mNSS) and modified Neurological Severity Score (mNSS) scores, as well as TCC staining.

[0026] Example 2

[0027] Effects of Luosan on neurological evaluation in rats with cerebral ischemia-reperfusion

[0028] According to the method of Example 1, the middle cerebral artery occlusion method was used to establish a rat cerebral ischemia-reperfusion model. The suture was removed 2 hours after cerebral ischemia in the model rats. After 24 hours of reperfusion, the researchers used the Longa score and mNSS to evaluate the rat behavior.

[0029] The Longa score ranges from 0 to 4, with 0 indicating normal gait and no neurological deficits; 1 indicating inability to fully extend the forepaw on the paralyzed side; 2 indicating inability to walk in a straight line and rotation toward the paralyzed side; 3 indicating falling toward the paralyzed side while walking; and 4 indicating inability to walk independently, lack of free gait, and loss of consciousness.

[0030] The mNSS score ranges from 0 to 18 points, with severity levels as follows: 13-18 points, severe damage; 7-12 points, moderate damage; 1-6 points, mild damage. The higher the score, the more severe the neurological impairment.

[0031] The results are as follows Figure 1 The lower the neurological score, the better the recovery. Figure 1 It can be seen that with the increase of the dose of Luosan, the injury recovery is getting better and better, and is getting closer and closer to the neurological function score of normal mice. Luosan can improve the neurological score of rats with cerebral ischemia-reperfusion in a dose-dependent manner.

[0032] Example 3

[0033] Effects of Luosan on cerebral infarction and brain edema volume evaluation in rats with cerebral ischemia-reperfusion

[0034] Following the method of Example 1, a rat cerebral ischemia-reperfusion model was established using middle cerebral artery occlusion. The suture was removed 2 hours after ischemia in the model mice. Twenty-four hours later, brain samples were collected and subjected to TTC staining to assess the infarct size in each group of rats. During the experiment, the room's light and temperature were kept constant to maintain consistent environmental conditions throughout the experiment.

[0035] Depend on Figure 2 The results showed that the cerebral infarction area of ​​the model group rats increased, while the cerebral edema and cerebral infarction area of ​​the low, medium and high doses of Luosan treatment groups decreased. Luosan can dose-dependently reduce the cerebral infarction area of ​​rats with cerebral ischemia-reperfusion.

[0036] Example 4

[0037] Luosan improves oxidative stress level in brain tissue of MCAO / R rats

[0038] According to the method of Example 1, the middle cerebral artery occlusion method was used to establish a rat cerebral ischemia-reperfusion model in the experiment. The suture was removed after 2 hours of cerebral ischemia in the model mice. 24 hours later, the rats were immediately decapitated and killed. The injured hemibrain of the rats was quickly removed on ice and then homogenized with pre-cooled PBS. The homogenate was then centrifuged at 4°C, and the supernatant was used as the test sample for detection of SOD and MDA.

[0039] Depend on Figure 3The results showed that after using Luosan, the content of malondialdehyde in rats with cerebral ischemia-reperfusion was significantly lower than that in rats without cerebral ischemia-reperfusion, and the content of superoxide dismutase was significantly higher than that in rats without cerebral ischemia-reperfusion, indicating that Luosan can reduce cerebral ischemia-reperfusion.

[0040] Example 5

[0041] Effects of different doses of Luosan on the PI3K / AKT / mTOR signaling pathway

[0042] Following the method of Example 1, a rat cerebral ischemia-reperfusion model was established using middle cerebral artery occlusion. After 2 hours of cerebral ischemia, the sutures were removed. 24 hours later, the rats were immediately decapitated. The brain tissue was rapidly dissected on ice, the olfactory bulb removed, and the intact injured hemisphere was removed. To every 10 mL of RIPA protein lysis buffer, 100 μL of 100 mM PMSF was added, along with phosphatase and protease inhibitors. Mix thoroughly and place on ice for 20 minutes until ready for use. The ischemic brain tissue of the mouse was thoroughly minced on ice, placed in the lysis buffer, and thoroughly homogenized on ice using a grinding rod. The extract was centrifuged at 12,000 rpm and 4°C for 15 minutes. The supernatant obtained was the protein extract. Protein concentration was determined by adding equal amounts of protein to each well, performing SDS-PAGE gel electrophoresis, transferring to a PVDF membrane, blocking with fetal bovine serum for 1 hour, and incubating with the primary antibody at 4°C overnight. The next day, the secondary antibody was incubated for 1 hour at room temperature, followed by ECL development. The expression of proteins related to the PI3K / AKT / mTOR signaling pathway was detected.

[0043] Depend on Figure 4 The results showed that the expression levels of p-PI3K, p-AKT, and p-mTOR in the Luosan and Nimodipine groups were significantly increased in a dose-dependent manner. There were no significant differences in the expression levels of total PI3K, AKT, and mTOR proteins among the six groups. These results suggest that Luosan may exert its neuroprotective effects by inhibiting cell apoptosis through activation of the PI3K / AKT / mTOR signaling pathway.

[0044] Anyone skilled in the art will be able to utilize the above-disclosed technical content to make many possible changes and modifications to the technical solution of the present invention, or to modify it into equivalent embodiments with equivalent changes, without departing from the scope of the technical solution of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention that do not depart from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. Use of Luosan in preparing a drug for preventing or treating cerebral ischemia-reperfusion injury caused by ischemic stroke, wherein the only active ingredient of the drug is Luosan.

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