CN112704676B

CN112704676B - Application of oridonin in preparation of medicine for preventing and/or treating cerebral arterial thrombosis

Info

Publication number: CN112704676B
Application number: CN202110188040.3A
Authority: CN
Inventors: 孙秀兰; 李磊; 程淑琪; 李尚泽; 季娟; 孙涛; 胡刚
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2021-02-10
Filing date: 2021-02-10
Publication date: 2022-05-31
Anticipated expiration: 2041-02-10
Also published as: CN112704676A

Abstract

The invention discloses an application of oridonin in preparing a medicament for preventing and/or treating cerebral arterial thrombosis, belonging to the technical field of medicament research and development; oridonin can reduce cerebral oxidative stress level after ischemic stroke, inhibit endothelial cell apoptosis, improve blood brain barrier integrity under ischemia reperfusion state, reduce peripheral inflammatory cell infiltration, and inhibit neuroinflammation.

Description

Application of oridonin in preparation of medicine for preventing and/or treating cerebral arterial thrombosis

Technical Field

The invention belongs to the technical field of drug research and development, and particularly relates to application of oridonin in preparation of a drug for preventing and/or treating cerebral arterial thrombosis.

Background

Stroke (stroke), also known as stroke, is an acute cerebrovascular disease, a group of diseases that cause tissue damage due to sudden rupture of cerebral vessels or due to blood flow failure into the brain caused by vessel occlusion, including ischemic and hemorrhagic stroke. Among them, ischemic stroke accounts for 75% or more of the incidence rate, and is the second leading cause of death in humans. Intravenous tissue plasminogen activator (tPA) is currently the first choice for clinical treatment worldwide, primarily by recanalizing occluded blood vessels with tPA to re-supply oxygen to ischemic brain regions, but often causes a number of complications, of which cerebral ischemia/reperfusion injury (I/R injury) is one of the most serious. Cerebral ischemia/reperfusion injury refers to malignant biochemical cascade reaction caused in ischemic brain tissue after cerebral ischemia receives oxygen supply again, and the cascade reaction can aggravate the injury of the ischemic region and peripheral brain tissue and influence the recovery of brain function. At present, no clear medicament can effectively improve cerebral ischemia/reperfusion injury clinically, so that the search for therapeutic medicaments for limiting stroke progress and promoting recovery after stroke is urgent.

Under the ischemia reperfusion injury state, the active oxygen (ROS) in the brain is rapidly increased abnormally, and the capability of an antioxidant stress system is reduced. The large amount of ROS generated breaks the balance of antioxidants in the body, causing oxidative stress states, leading to cellular toxicity and death, and interacting with multiple events followed by cellular inflammatory responses, cell migration and apoptosis. Endothelial cells are one of the important components of the blood-brain barrier, and their normal cellular function is essential for maintaining the integrity of the blood-brain barrier, and damaged endothelial cells and their mediated dysfunction are closely related to ischemia-reperfusion injury. Research reports that a large number of ROS can activate NF-kB of brain vascular endothelial cells, induce apoptosis of the endothelial cells, cause loss of tight junction proteins including occludin, claudin-5, and ZO-1 and damage integrity and functions of a blood brain barrier. After the blood brain barrier is damaged, a large number of peripheral inflammatory cells infiltrate into the brain, so that the development of neuroinflammation is promoted, and the infarction area is increased.

Oridonin, named as Oridonin, is a bioactive natural substance separated from Rabdosia (Rabdosia) plant of Labiatae, and is a traditional Chinese herbal medicine with main component of ent-kau-reconnectepene natural organic compound. In China, Oridonin is an OTC Chinese herbal medicine which can be used for treating acute and chronic tonsillitis and sphagitis, and research indicates that Oridonin can play an anti-cancer effect by regulating cancer cell cycle, inducing cancer cell apoptosis and inhibiting angiogenesis. A recent study reports that Oridonin can regulate macrophage-related acute oxidative stress and inflammatory response by activating AKT/Nrf-2 pathway, inhibiting activation of NLRP3 and NF-kappa B in LPS-induced macrophage acute inflammation model. In the therapeutic study of AD transgenic mice, researchers found that Oridonin could significantly reduce the a β -induced high ROS levels, improve neuroinflammation, and promote improvement in mouse cognitive behaviours. The previous research shows that Oridonin is a good Chinese herbal medicine for targeting oxidative stress and neuroinflammation and improving neuroinflammatory system diseases, but whether Oridonin can promote the improvement of the integrity and the function of a blood brain barrier by targeting oxidative stress and apoptosis in an ischemia-reperfusion state so as to limit neuroinflammation and reduce infarct size is not reported at present.

Disclosure of Invention

The invention aims to provide the application of oridonin in preparing the medicine for preventing and/or treating cerebral arterial thrombosis, so as to solve the problems in the prior art,

in order to realize the purpose, the invention provides the application of oridonin in preparing the medicine for preventing and/or treating cerebral arterial thrombosis;

the invention also provides an application of oridonin in preparing a medicament for preventing and/or treating cerebral ischemia/reperfusion injury;

preferably, the molecular formula of oridonin is C₂0H₂₈O₆The structural formula is as follows:

preferably, the medicament comprises one or more pharmaceutically acceptable carriers.

Preferably, the oridonin can reduce the permeability of blood brain barrier after cerebral ischemia-reperfusion;

preferably, oridonin can up-regulate the expression of brain tight junction protein in the penumbra region after cerebral ischemia-reperfusion;

preferably, the oridonin can inhibit the proliferation and activation of cerebral glial cells on the affected side after cerebral ischemia-reperfusion;

preferably, after cerebral ischemia-reperfusion, oridonin can reduce the expression of proinflammatory factors and promote the expression of inflammation-inhibiting factors of the affected brain;

preferably, oridonin reduces ROS levels in the affected side brain penumbra region after cerebral ischemia-reperfusion;

preferably, oridonin can up-regulate brain antioxidant stress key enzymes HO-1 and NQO-1 after cerebral ischemia-reperfusion;

preferably, oridonin can down-regulate p-NF-kB of a hemiphrenic region and a pro-apoptotic factor Bax and up-regulate an anti-apoptotic factor Bcl-2 after cerebral ischemia-reperfusion.

The invention discloses the following technical effects:

the invention evaluates the effect of oridonin in the treatment of ischemic stroke, and the oridonin inhibits endothelial cell apoptosis by reducing the brain oxidative stress level after stroke, thereby improving the integrity of blood brain barrier in the ischemia reperfusion state, reducing the infiltration of peripheral inflammatory cells and inhibiting neuroinflammation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

FIG. 1 is a graph showing the results of Oridonin reducing neuromotor dysfunction in mice caused by nerve injury in the acute phase of focal cerebral ischemia reperfusion;

FIG. 2 is a graph showing the results of Oridonin reducing cerebral infarction caused by nerve injury in the acute phase of mouse focal cerebral ischemia reperfusion;

FIG. 3 is a graph of the results of Oridonin improving BBB permeability in example 5;

FIG. 4 is a graph of Western blotting results of up-regulated hemin tight junction protein following Oridonin treatment in example 6;

FIG. 5 is a confocal microscope image showing the results of the section in example 7;

FIG. 6 is a graph of the qPCR results in example 8;

FIG. 7 shows the results of ROS Elisa assays of brain tissue in example 9, with Oridonin reducing reperfusion-induced high levels of ROS in the affected brain;

FIG. 8 shows the protein expression of Oridonin up-regulating the penumbra region key component of antioxidant stress HO-1, NQO-1 in example 9;

FIG. 9 is a graph of inhibition of reperfusion-induced apoptosis by Oridonin and apoptosis of endothelial cells in example 9;

FIG. 10 is a graph showing that Oridonin inhibits NK-kB phosphorylation, decreases expression of pro-apoptotic factor Bax, and increases expression of anti-apoptotic factor Bcl-2 in example 9.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below. Unless otherwise specified, the materials in the examples of the present invention are commercially available.

Example 1 preparation of a focal cerebral ischemia reperfusion animal model

A mouse monofilament intraluminal right Middle Cerebral Artery Occlusion (MCAO) model was prepared using a polylysine-coated nylon wire plug (model: L800, Guangzhou Jialing Biotech, Inc., China) with its tip end referenced to the Longa method. C57/B6L mice are fasted for 8-10 h before operation, 4% chloral hydrate (1ml/100g, prepared by sterile normal saline) is injected into the abdominal cavity to anaesthetize the mice, the mice are fixed in a supine position, a median cervical incision is made after iodophor disinfection, the right common carotid artery, the internal carotid artery and the external carotid artery are separated, a plug thread is inserted from the external carotid artery cut until resistance (about 4-5mm) appears, the plug thread reaches the middle cerebral artery, and the plug thread is ligated. After 45min of ischemia, the thrombus is slowly drawn out, then the external carotid artery is ligated, then the silk thread at the common carotid artery is loosened to recover the blood flow of the right carotid artery, the incision is sutured, then the carotid artery is sterilized by iodophor and placed in a cage, the cage is raised at the room temperature of 25 ℃, and the MCAO is ended to finish the focal cerebral ischemia-reperfusion model. The whole process uses a Laser Doppler flow meter (MoorFLPI Full-field Laser Perfusion Imager, Moor Instruments Let, UK) to monitor local cerebral blood flow to ensure successful model preparation. From the start of surgery to before the animals were awakened, a thermometer was used to ensure that the body temperature of the rats was maintained at 36.5 ℃. + -. 1 ℃. After anesthesia in the Sham (Sham) group of animals, only the carotid internal and external arterial bifurcations were exposed, and no wire plugs were inserted.

Example 2 Experimental randomized Subdivision and administration

The mice were grouped as: the sham surgery group (Con-Vehicle), the tMCAO model group (tMCAO-Vehicle and tMCAO-Oridonin). Randomly dividing male C57/B6L mice weighing 22-25g into two batches, one batch is a sham group, and the other batch is a tMCAO model group; after molding in the manner of example 1, the molding groups were randomly divided into two groups, one group was intraperitoneally administered with Oridonin (15mg/kg), and the other group was intraperitoneally administered with the same dose of solvent.

Example 3 neuromotor function scoring

Grading and scoring the nerve function defect of the mouse according to a Bederson method after 72 hours of operation, removing the mouse with the nerve function score of 0 and 5 after 72 hours, and calculating a 3d scoring mean value. The criteria are as follows:

0 minute: normal, no neurological symptoms were observed;

1 minute: when the tail is lifted and suspended, the contralateral forelimb of the animal can not be fully extended, and the symptoms are that the wrist and the elbow are flexed, the shoulder is rotated inwards, the elbow is expanded outwards and is tightly attached to the chest wall;

and 2, dividing: placing the animal on a smooth plane, and rotating or circling towards the opposite side of the operation when the animal freely walks;

and 3, dividing: when the animal walks freely, the animal is toppled towards the opposite side of the operation;

and 4, dividing: animals were placed on smooth surfaces without spontaneous walking with a concomitant decrease in the level of consciousness;

and 5, dividing: and death.

In animal experiments, excessive bleeding occurs in the operation process; abnormal breathing, premature death and subarachnoid hemorrhage found at sacrifice after operation are all discarded, and animals removed are randomly supplemented in later experiments. The results of the experiment are shown in FIG. 1.

Example 4: determination of cerebral infarction volume

Cutting head of mouse 72h after ischemia reperfusion to obtain brain, rapidly taking out brain tissue, removing olfactory bulb, cerebellum and lower brain stem, and freezing at-20 deg.C for 8 min; (2) 2 mm/slice thickness coronal sections of brain were cut continuously from the anterior pole (electrode pole), incubated in 1% 2,3, 5-triphenyltetrakis (2,3,5-triphenyltetrazolium chloride, TTC, Sigma, America) physiological saline solution at 37 ℃ in the dark for 5min, and then fixed in 4% paraformaldehyde solution (pH7.4) at 4 ℃ overnight; (3) after taking the picture, the cerebral infarct volume was analyzed and calculated using Image J Image analysis software.

As shown in fig. 2, the percentage of the volume of infarct in the Oridonin-treated mice was reduced by 24.2% compared to the model group.

Example 5 detection of blood brain Barrier permeability by mouse Evans blue

(1) Treated animals (mice as an example) were treated and were treated with Evans blue staining solution (0.5%) by intravenous injection, which resulted in blue color on the eyes and skin of the mice. The mice are sacrificed after 0.5-1 h, and the target brain tissue is taken out. (2) The affected brain tissue was placed in a 1.5ml centrifuge tube, 1ml PBS was added, the brain tissue was rapidly homogenized with a tissue homogenizer, and centrifuged at 4 ℃. (3) The supernatant was taken, and the same amount of trichloroacetic acid was added thereto, followed by incubation at 4 ℃. The steps may also be performed as follows: taking the supernatant, and pressing the supernatant: acetone was added at a ratio of 3:7 and incubated at room temperature for 24 h. (3) Centrifuge at 4 ℃ for 15 min. (4) The solution was taken and measured for OD at 620nm with a spectrophotometer. And simultaneously measuring the OD values of the standard Evans blue with different known gradients, and drawing a standard curve. And calculating the Evans blue content of the sample to be detected according to the standard curve.

As shown in fig. 3, Evans blue content in the homogenate of the affected lateral brains was significantly reduced in the Oridonin-treated mice compared to the model group, suggesting that permeability of the blood-brain barrier was improved.

Example 6 Western immunoblotting (Western blotting)

Mice were anesthetized by intraperitoneal injection with 4% chloral hydrate solution, sacrificed by decapitation, and brains were rapidly removed on ice, dividing cerebral hemispheres into injured side and non-injured side. Taking fresh brain tissue in a penumbra area, fully homogenizing the brain tissue and lysate according to the mass-volume ratio of 1:10, putting the homogenate in an ice box, placing the ice box on a shaking table, shaking for 30min, centrifuging at 4 ℃ and 12000rpm for 15min, sucking supernatant, adding 5 times of protein loading buffer solution according to the volume ratio, boiling in a metal bath kettle at 100 ℃ for denaturation for 10min, and storing at-80 ℃ after subpackaging. Protein samples at-80 ℃ were taken out of solution, centrifuged, added to the lanes with a microsyrin, subjected to SDS-polyacrylamide constant pressure gel electrophoresis separation, and transferred to PVDF membrane (Millipore, USA) using an electric transfer system (minirotor-III wet transfer unit, BioRad Hercules, California, USA). Blocking the mixture for 1-2 h at room temperature by 5% BSA-TBST. Primary antibody (antibody concentration see Table 1) was added and incubated overnight in a shaker at 4 ℃. The next day, the primary antibody was discarded and the membrane washed with TBST for 10min × 3 times, and a secondary antibody labeled with horseradish peroxidase (antibody concentration shown in Table 2) was added thereto, incubated at room temperature for 1h on a shaker, rinsed with TBST (10min × 3), and added with ECL (Pierce, Thermo scientific, USA) as a luminescent substrate to develop the color. Tanon5200 full-automatic chemiluminescence imaging analysis system development analysis. And carrying out semi-quantitative analysis on the ratio of the gray value of the target protein to the gray value of the internal reference beta-actin.

TABLE 1 Primary antibody for immunoblotting

TABLE 2 Secondary antibodies for immunoblotting

As shown in figure 4, the mouse penumbra tight junction protein expression was significantly reduced after stroke, and brain tight junction protein expression was significantly upregulated after Oridonin treatment.

Example 7 frozen tissue immunofluorescence

(1) Heart perfusion fixation, material selection: anesthetizing a mouse by using 4% chloral hydrate, and sequentially perfusing ice PBS and 4% paraformaldehyde through the left ventricle of the mouse after anesthesia until the liver becomes white and the texture becomes hard; taking a complete brain tissue of a mouse, and soaking the complete brain tissue in a 4% paraformaldehyde solution for 48 hours for fixation treatment; (2) tissue dehydration and slicing: placing the brain tissue which is subjected to external fixation into a 30% sucrose solution for dehydration until the brain tissue sinks to the bottom; after dehydration, OCT gel is embedded and frozen in a refrigerator at minus 80 ℃ for 6 hours; after freezing, slicing with a freezing microtome (Leica, Germany) to a thickness of 15um, taking 1 every 10, and storing in a refrigerator at-20 deg.C; (3) penetrating membrane rupture, sealing: taking out the storage slices, washing with PBS (phosphate buffer solution), and washing for 10min by 3X; after cleaning, placing the slices on a glass slide treated by lysine, and drying; circling the section by using a grouping pen, adding 200 mu L of 0.2% Triton PBST solution into the circle for membrane rupture treatment for 10 minutes, and after the membrane rupture treatment is finished, sealing the section by using 5% BSA PBST solution for 2 hours; (4) primary and secondary antibodies were incubated, stained with DAPI, and observed: after blocking, the antibody was diluted with blocking solution (see fig. 3 and table 4 for antibody information and dilution ratio), incubated overnight at 4 ℃ for the first time, and after blocking, the sections were washed with PBST for 10min, 3X; after the washing is finished, the diluted secondary antibody is incubated in a dark room and sliced for two hours; after the secondary antibody incubation is finished, washing the section by using PBST for 10min by 3X; after drying, sealing by using a sealing liquid containing DAPI; the observation was performed under a confocal microscope.

TABLE 3 Primary antibody for immunochemical staining of frozen tissues

TABLE 4 Secondary antibodies for immunochemical staining of frozen tissues

As shown in fig. 5, after stroke, the mice had a large infiltration of Ly6G + neutrophils and CD45+ leukocytes on the affected side, Oridonin significantly reduced infiltration of peripheral inflammatory cells; in addition, the IBA1+ microglia and GFAP + astrocyte of the stroke affected side are obviously proliferated and activated, and after the Oridonin is injected into the abdominal cavity, the proliferation and activation of the glial cells are obviously inhibited.

Example 8 tissue qPCR (Quantitative Real-time PCR) assay

Extracting brain tissue RNA using an RNA extraction kit Trizon (TAKARA, japan), and reverse-transcribing the RNA into cDNA using an RNA reverse transcription kit (Vazyme, china); and detecting the RNA expression quantity of related target genes in brain tissues by using 2xCHAMQ SYBR qPCR Master Mix (Vazyme, China), wherein the sequences of related target gene primers are shown in a table 5.

TABLE 5 Gene primer sequences

As shown in figure 6, after stroke, the mouse affected tissue highly expresses proinflammatory factors such as IL-1 beta, IL-6, TNF-alpha, MCP-1, IFN-gamma and the like, and lowly expresses inflammation-inhibiting factors such as IL-10, Arg-1 and the like, and Oridonin can remarkably reduce the expression of the proinflammatory factors and promote the expression of the inflammation-inhibiting factors. As indicated above, Oridonin inhibits neuroinflammation caused by ischemia reperfusion.

Example 9 brain tissue ROS Elisa assay

Injecting 4% chloral hydrate into abdominal cavity of mouse for anesthesia, taking the brain in penumbra area after anesthesia, weighing, placing in a nuclease-free tube, adding 100ul PBS solution with pH7.4 into each 0.01g tissue, and ultrasonically homogenizing; after homogenizing, centrifuging at 3000rpm for 20min by a 4-degree centrifuge; after centrifugation, the cell phone supernatant is carefully selected, and detection is carried out according to the operation steps of a mouse ROS Elisa kit (winged flying blood, YFXEH01863, Nanjing, China);

as shown in figure 7, ischemia reperfusion injury resulted in a massive increase in mouse brain ROS, which significantly decreased its levels in the brain following Oridonin injection.

As shown in fig. 8, the key enzymes of antioxidant stress HO-1 and NQO-1 were significantly down-regulated in the ischemia-reperfusion state, while the expression of the mouse affected brain after Oridonin treatment was significantly increased. The above results suggest that stroke mice treated with Oridonin have improved brain oxidative stress.

As shown in fig. 9: in the ischemia-reperfusion state, the number of apoptotic brain cells including endothelial cells is obviously increased, and the apoptosis of the endothelial cells is obviously reduced after the Oridonin treatment.

As shown in fig. 10: under the ischemia reperfusion state, the up-regulation of p-NF-kB and apoptosis promoting factor Bax in the penumbra region is obvious, the down-regulation of anti-apoptosis factor Bcl-2 is obvious, and the trend is obviously reversed after Oridonin treatment is given.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. An application of oridonin as the only active component in preparing the medicines for preventing and/or treating cerebral arterial thrombosis is disclosed.

2. An application of oridonin as the only active component in preparing the medicines for preventing and/or treating cerebral ischemia/reperfusion injury is disclosed.

3. The use according to claim 1 or 2, wherein the medicament comprises one or more pharmaceutically acceptable carriers.

4. The use according to claim 1 or 2, wherein the oridonin reduces blood brain barrier permeability after cerebral ischemia-reperfusion.

5. The use according to claim 1 or 2, wherein the oridonin up-regulates the expression of brain claudin following cerebral ischemia-reperfusion.

6. The use according to claim 1 or 2, wherein the oridonin inhibits the proliferation and activation of glial cells following cerebral ischemia-reperfusion.

7. The use according to claim 1 or 2, wherein the oridonin reduces the expression of pro-inflammatory factors and promotes the expression of anti-inflammatory factors after cerebral ischemia-reperfusion.

8. Use according to claim 1 or 2, characterized in that oridonin reduces ROS levels after cerebral ischemia-reperfusion.

9. The use according to claim 1 or 2, wherein the oridonin up-regulates brain antioxidant stress critical enzymes HO-1 and NQO-1 after cerebral ischemia-reperfusion.

10. The use according to claim 1 or 2, wherein, after cerebral ischemia-reperfusion, oridonin down-regulates the hemiphrenic region p-NF- κ B and the pro-apoptotic factor Bax, up-regulates the anti-apoptotic factor Bcl-2.