CN111012765A - Application of pseudopterogorgia-hancei alcohol as active substance in preparing medicine for treating cerebral apoplexy - Google Patents
Application of pseudopterogorgia-hancei alcohol as active substance in preparing medicine for treating cerebral apoplexy Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Abstract
The invention provides an application of pseudopterogorgia pseudosciaena active substance (Lemnalol) in preparing a medicine for treating stroke, belonging to the field of pharmacy. The pseudopterogorgia alcohol (Lemnalol) has the obvious effects of protecting nerves and inhibiting inflammatory reaction on tMCAO rats and the like, and accords with the new guidance of the new drug screening in the world at present. Therefore, the application provides the possibility of the pseudopterogorgia pseudosciaenae alcohol (Lemnalol) serving as a novel neuroprotective agent and a potential neuroprotective mechanism, and provides a new experimental basis for clinical treatment application.
Description
Technical Field
The invention belongs to the field of pharmacy, and particularly relates to application of pseudopterogorgia alcohol in preparation of a medicine for reducing brain injury in stroke.
Background
Stroke, commonly known as stroke, is a disorder of cerebral blood circulation which is a sudden onset of disease, and is also the most serious complication of cerebrovascular disease. According to the third national cause of death investigation (2012) in China, the stroke fatality rate is ranked first and is as high as 22.45%, the disability rate is as high as 75%, and serious burden is brought to families and society.
Over the last 30 years, there were nearly 200 stroke drugs entering clinical trials worldwide. However, to date, only tissue-type fibrinolytic agents (tPA) have acquired the us FDA's clinical application approval. However, because tPA can only be used for cerebral thrombosis patients and has the defect of narrow treatment time window (the treatment is effective within 3 hours of the onset of the disease), the medicine has the treatment effect on 3-5 percent of cerebral apoplexy patients. However, the current research on the cerebral apoplexy treatment drugs discovers that the ion channel antagonist, the oxygen radical scavenger, the excitatory transmitter antagonist and the like are effective in basic research, but are really used for clinical discovery and have poor curative effect. For the reasons of clinical trial failure of the cerebral apoplexy medicaments, international pharmacians conduct multi-directional research, summarize possible reasons causing clinical trial failure, and propose to search medicaments with multiple neuroprotective effects such as anti-apoptosis, anti-oxidation and anti-inflammation and the like as research and development objects in the process of developing the cerebral apoplexy medicaments so as to improve the chance of developing the cerebral apoplexy medicaments successfully. Therefore, the active search for new drugs with multiple neuroprotective effects is the focus of drug development for the treatment of ischemic stroke, but no neuroprotective agent with a clear therapeutic effect clinically proven so far has been available.
Ischemic stroke causes a series of early events (from minutes to hours) such as excitotoxicity, oxidative stress, calcium overload and the like due to insufficient blood flow in local areas, so that glial cells are activated to secrete inflammatory mediators such as cytokines, chemokines, matrix metalloproteinases and the like, inflammatory cells derived from blood penetrate into cerebral ischemic areas, infiltrated leukocytes release the cytokines and the chemokines, glial cells are further activated, inflammatory signal cascade amplification reaction is generated, various cytotoxic components are released, blood brain barriers are damaged, and late events (from hours to days) such as immune injury, neuronal death and the like are further aggravated. Since patients often go to hospital for medical treatment hours after the onset of disease, the main pathological feature at this stage is inflammatory injury. Based on the discovery of the pathological mechanism and the failure of research and development of the medicines such as the ion channel antagonist and the like, the inhibition of inflammatory reaction after ischemic stroke has a longer treatment time window, and is considered to be an important direction for research and development of the cerebral stroke medicines.
Microglia is a main innate immune cell of the central nervous system and also a macrophage which plays a role in the nervous system and is responsible for immune monitoring, antigen presentation, phagocytosis, secretion of regulatory immune molecules and the like, CD11b is a marker of microglia and macrophages, and after being activated in inflammatory response after cerebral stroke, microglia/macrophages express Cxcl1 and Cxcl2, IL-1 β, IL-6, TNF- α, iNOS and other marker genes, among which IL-1 β is an important cytokine and plays a key role in neuroinflammation, while under pathological conditions, inflammatory cells, macrophages and glial cells and the like express inducible nitric oxide synthase (indoviborans, iNOS) in large quantities, thereby inducing the production of excessive pro-inflammatory NOS, which causes neurotoxic response in the central nervous system, so that iNOS is considered as an important factor affecting "pathological effect of inflammatory response" after pathological injury, and thus a critical inflammatory response is considered to be an important marker for the inhibition of nociceptive response of nociceptive injury, such as a further research on pathological changes of nociceptive response of nociceptive factors, including Cxcl 44, CD 493, CD44, and a further research on pathological changes of inflammatory response of nociception.
The marine environment is special and complex, and the survival and metabolic mode of marine organisms is special, so that many marine natural products have specific structures and important pharmacological effects, such as antibacterial, antiviral, antitumor, anti-inflammatory and the like, and are important sources of innovative medicines. Among them, the active substance derived from coralloid is one of the hot spots in the international marine natural product research, and many compounds with novel structures and remarkable biological activity are found in such marine animals. The object of the present invention, Lemnalol (pseudopterogous alcohol), a kind of sesquiterpene (see FIG. 1) of the type of an ylangene, was first extracted from the plant Lagerstroemia speciosa (Lemnalia tenuis Verseveldt), and then also extracted from 2 species of Taiwan Lagerstroemia speciosa (Lemnalia cervicorni and Lemnalia flava) of the genus. The chemical structure of Lemanlol is as follows:
since the inflammatory response traverses the entire pathological process of ischemic brain injury, inhibition of post-stroke inflammatory response provides a longer therapeutic window for the treatment of ischemic stroke, and particularly in clinical practice, inhibition of post-stroke inflammatory response is considered as a potential promising therapeutic approach since the majority of stroke patients are admitted several hours after onset, at which time inflammatory response has become the main pathological mechanism. The study of the invention finds that Lemnalol can effectively reduce infarct volume and neurological function defect on a rat cerebral central artery ischemia (MCAO) reperfusion model, obviously inhibit inflammatory reaction, improve survival rate and have a wider treatment time window (the drug administration is still effective within 24 hours for the first time). Recently, it has been discovered that up-regulation of inflammatory factor gene is one of the characteristics of gene expression change of stroke patients, and effective inhibition of inflammatory reaction is one of the important ways for neuroprotection. Effectively inhibit neuroinflammatory reaction and have obvious protective effect in the treatment of cerebral apoplexy. In addition, in vitro experiments of the invention also show that Lemnalol can effectively inhibit the expression of M1 type marker genes such as BV-2 microglia iNOS and COX2 stimulated by LPS, and can promote the expression of M2 type marker genes such as IL-10, Arg1 and the like, and the conversion of the functional state (M1/M2 type) of microglia/macrophage is a key mechanism influencing the subsequent damage of cerebral apoplexy and is considered as an important way for treating cerebral apoplexy. The invention discovers that Lemnalol has obvious effects of protecting nerves and inhibiting inflammatory reaction on tMCAO rats, and the like, and accords with new guidance of new drug screening in the world at present. Therefore, the research aims to provide the possibility of Lemnalol as a novel neuroprotective agent and a potential neuroprotective mechanism, and provide a new experimental basis for clinical treatment application.
Disclosure of Invention
The invention aims to provide application of pseudopterogorgia pseudosciaenae alcohol in preparation of a medicine for reducing brain injury in stroke, aims to provide the possibility of pseudopterogorgia pseudosciaenae alcohol (Lemnalol) serving as a novel neuroprotective agent and a potential neuroprotective mechanism, and provides a new experimental basis for clinical treatment and application.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of the pseudopterogorgia alcohol (Lemnalol) in preparing the medicine for reducing the brain injury in the stroke is as follows:
the invention has the advantages that:
the invention realizes the improvement of the neuroprotective performance, the pseudopterogorgia pseudosciaenae alcohol (Lemnalol) has obvious neuroprotective effect on cerebral apoplexy, the cerebral infarction volume is up to 30 percent relative to a modeling group, and the administration group is reduced to about 10 percent. The pharmacodynamic characteristics of the traditional Chinese medicine are more in accordance with clinical requirements, and patients can go to hospitals after having a disease for hours, and the main pathological feature at this stage is inflammatory injury. The combined medicine can effectively inhibit inflammatory reaction after cerebral arterial thrombosis, thereby having longer treatment time window. Is expected to be used as a novel neuroprotective agent.
Drawings
FIG. 1 is a graph of the cerebral infarct volume and volumetric ratio statistics for Lemnalol (LN) 2 hours after MCAO reperfusion, after 6 days of continuous intravenous injection (15 mg/kg/day); wherein (A) is the cerebral infarction volume; (B) the volume ratio of cerebral infarction is counted.
FIG. 2 is an analysis of the expression level of inflammatory factors.
FIG. 3 shows the effect of Lemnalol (LN) on the mRNA and protein expression levels of IL-6 and IL-1 β, where a is the IL-6 mRNA expression level, b is the IL-1 β mRNA expression level, c is the IL-6 protein expression level, and d is the IL-1 β protein expression level.
FIG. 4 is a graph of the effect of Lemnalol (LN) on the expression levels of iNOS and COX-2 mRNA; wherein a is the iNOS mRNA expression level; b is COX-2 mRNA expression level.
FIG. 5 shows the effect of Lemnalol (LN) on the expression of M2-related genes, such as IL-10 and Arg 1.
Detailed Description
Example 1
1 Material
1.1 healthy adults of laboratory animals
SPF grade SD rats 60, body mass (280 ± 20) g, purchased from shanghai slyke laboratory animals ltd, certification No.: 2007000509960, license number: SCXK 2007 and 0005, which is bred in SPF animal laboratory of the university of Fujian Chinese medicine laboratory animal center. Keeping the temperature and humidity constant for 24h, ensuring sufficient food and drinking water, simulating natural day and night change, giving illumination at 6:00-18:00, keeping the day at 18: 00-next day and keeping the day at 6:00 in a dark state, and carrying out adaptive feeding for one week.
1.2 Total RNA extraction Kit RNeasy Mini Kit (QIAGEN, Cat. No.: 74106) of main reagent and tested drug, RT Kit (Fermentas, Cat. No.: K1622) of reverse transcription Kit, fluorescent quantitative PCR Master Mix (Applied Biosystems, Cat. No.: R11022), PCR primers were synthesized by Shanghai Jieli bioengineering, Inc.
TNF- α gene primers:
the upstream sequence 5 '-GCCACCACGCTCTTC-TGTC-3';
the downstream sequence 5'-GCTACGGGCTTGTCACTCG-3' is a sequence of sequences,
the length of the PCR amplified fragment was 149 bp.
IL-6 gene primers:
upstream sequence 5'-AGACTTCACAGAGGATACCACCCAC-3';
the downstream sequence 5'-CAATCAGAATTGCCATTGCACAA-3' is a sequence of sequences,
the length of the PCR amplified fragment is 129 bp.
IL-1 β gene primer:
an upstream sequence 5'-ACAAGGAGAGACAAGCAACGACAA-3';
the downstream sequence of the flow line 5'-TTTCCATCTTCTTCTTTGGGTATTG-3',
the length of the PCR amplified fragment was 149 bp.
iNOS gene primers:
an upstream sequence 5'-CAGATCCCGAAACGCTACACTT-3';
the downstream sequence 5'-TGCGGCTGGACTTCTCACTC-3' is a sequence of sequences,
the PCR amplified fragment was 175bp in length.
Cxcl1 gene primer:
an upstream sequence 5'-CCACTCACCTGCTGCTACTCATT-3';
the downstream sequence 5'-GTATGTCTGGACCCATTCCTTCTTG-3' is a sequence of sequences,
the length of the PCR amplified fragment is 113 bp.
Cxcl2 gene primer:
an upstream sequence 5'-GCTCCCAGCCAGGTGTCATTTT-3';
the downstream sequence 5'-AAGACTCTCAGGCATTCAGTTCCAG-3' is a sequence of sequences,
the length of the PCR amplified fragment was 105 bp.
Arg1 gene primer:
an upstream sequence 5'-CGTTGACCTTGTCTTGTTTTGG-3';
the downstream sequence 5'-CTGGTTCTGTTCGGTTTGCTG-3' is a sequence of sequences,
the PCR amplified fragment was 138bp in length.
GAPDH primer upstream sequence 5'-CAACGGGAAACCCATCACCA-3';
the downstream sequence 5 '-ACGCCAGTAGACTC-CACGACAT-3',
the length of the PCR amplified fragment is 96 bp.
iNOS, Cox2 and Agr1 antibody (Santa Cruz); β -actin antibody was purchased from Biyun Biotech research institute.
2 method of experiment
2.1 MCAO animal model rat preoperative fasting 12 h, free drinking water, intraperitoneal injection of 10% chloral hydrate (300 mg. kg-1) anesthesia, supine position fixation, nylon monofilament line plug from the left common carotid artery to the middle cerebral artery initiation end. 2 h after embolism, the wire plug is pulled out and then is perfused for 24 h. Sham operated animals did not embolize middle cerebral arteries. During the procedure, the temperature of the animals was continuously monitored and maintained at 37 ℃. And (4) evaluating the nerve function damage degree according to a Longa evaluation method, wherein 3-4 grades are successful model making and can be used for experiments.
2.2 Experimental groups and dosing methods the experimental rats were divided into sham (sham), Model Control (MCAO), and Lemnalol dosing (MCAO + LN) (15 mg/kg/d) drugs, 6 per group. The Lemnalol administration group is subjected to reperfusion after 2 h of embolism, immediately subjected to intraperitoneal injection after nerve function injury evaluation, and is subjected to intraperitoneal injection of Lemnalol (15 mg/kg/d) or normal saline for 24h, wherein 3 animals in each group are randomly selected, and brain tissues are taken for inflammation index analysis. In addition, the drug is given every 24h, and after 6d, the head of each group of experimental animals is cut off and the brain is taken for TTC analysis.
2.3 statistics of rat neural function impairment score and cerebral infarction volume determination
Evaluation of nerve function impairment: according to Longa evaluation method: grade 0, no defect; grade 1, inability to extend contralateral forelimb; grade 2, contralateral forelimb flexion; level 3, slightly turning to the opposite side; grade 4, severe revolutions; grade 5, contralateral paralysis.
After 6 days of administration, rat brain tissue was collected, and coronal sections were prepared at the visual cross and 2mm before and after the visual cross, and incubated in TTC for 30min at 37 ℃ in the dark. Normal tissue was stained rose-red, while infarcted tissue was white. After staining, the tissue was photographed and infarct volume was calculated using image analysis.
2.4 fluorescent real-time quantitative PCR (RT-qPCR) for detecting gene expression of Cxcl1, Cxcl2, TNF- α, IL-6, IL-1 β, iNOS, etc
Extracting total RNA according to a QIAGEN RNeasy Mini Kit, carrying out reverse transcription by using a RT reverse transcription Kit of Fermentas to obtain cDNA, and establishing a qPCR system by using the cDNA as a template, wherein the system comprises 10 mu l of SYBR Select Master mix, 2 mu MF-primer 2 mu l, 2 mu M R-primer 2 mu l, 2 mu l of cDNA and 20 mu l of ddH2O 4 mu l; the reaction conditions are 50 ℃ for 2min, 95 ℃ for 10 min, 95 ℃ for 15 s, annealing at 58 ℃ for 15 s and polymerization at 72 ℃ for 40 cycles. The expression level of mRNA among the groups was calculated by the 2-. DELTA.Ct method using GAPDH as an internal reference.
2.5 cell culture
Mouse microglia BV2 was cultured in RPMI1640 medium containing 10% final fetal bovine serum and 1% Penicillin-Streptomycin diabody, at 37 ℃ in an incubator with 5% CO 2. When the cells exceeded 80% confluence, cell subcultures were performed.
2.6 ELISA kits (R & D Systems)
After the 96-well plate is coated overnight, the target inflammatory factor is detected by an antibody sandwich method. And (3) measuring the absorbance (A value) at the wavelength of 450 nm by a chromogenic reaction and a microplate reader, calculating the concentration of the sample, and quantitatively detecting the inflammatory factor.
2.7 statistical analysis was performed using SPSS 18.0 statistical software, data are expressed in x. + -.s, and differences between groups of data were analyzed using ANOVA method. 3 results
3.1 after reperfusion of MCAO rat for 2 hours, Lemnalol (15 mg/kg/d) or normal saline is injected into abdominal cavity for 6 days, and then the material is obtained for analysis, so that Lemnalol has neuroprotective effect with wider treatment time window.
The detection of TTC and the like on the affected side brain tissue shows that Lemnalol reduces the infarct volume of MCAO rats and has a wider treatment time window: lemnalol (LN) 6 days of continuous intravenous injection (15 mg/kg/day) was effective in reducing cerebral infarct volume 2 hours after MCAO reperfusion (FIG. 1A). TTC staining showed that the cerebral infarction volume ratio statistics showed that the administration after 2 hours after reperfusion was up to 30% of cerebral infarction volume relative to the model group, and the administration group was also reduced to around 10% (fig. 1B).
3.2 Lemnalol reduces the expression of a series of inflammatory factors in the affected side of the semi-brain tissue of MCAO reperfusion model rats.
After MCAO rats are perfused for 2 hours again, Lemnalol (15 mg/kg/d) or normal saline is injected into the abdominal cavity for 24 hours, then the materials are taken out, brain tissues are taken for subsequent analysis, and the reanalalol is found to be capable of inhibiting the expression levels of a series of inflammatory factors such as Cxcl1, Cxcl2, TNF- α -6, IL-1 β and the like through reanaltime PCR analysis, and the results are shown in figure 2.
3.3 BV-2 microglia inflammation model (100 ng LPS stimulation), and analyzed 24h after administration, Lemnalol effectively inhibits M1 type-related marker gene expression and has dose effect.
(1) Lemnalol effectively inhibited IL-6 and IL-1 β expression (FIG. 3);
(2) lemnalol was effective in inhibiting iNOS and COX-2 expression (FIG. 4);
3.4 BV-2 microglia inflammation model (100 ng LPS stimulation), analyzed 24h after administration, Lemnalol promoted the expression of M2-related genes like IL-10 and Arg1 with dose effect (FIG. 5).
4 conclusion
In conclusion, the study of Lemnalol on the neuroprotective and anti-inflammatory effects of stroke is based on in vivo and in vitro experiments. The study is established that Lemnalol has a remarkable neuroprotective effect on MCAO rats, and when an administration group of the MCAO rats after 2 hours of reperfusion injury is analyzed, Lemnalol shows a remarkable inflammation inhibition effect in the protection effect on cerebral ischemia injury, and directly participates in the regulation and control of inflammatory response in the treatment of cerebral apoplexy, and clinical investigation and research find that the up-regulation of inflammatory factor genes is one of the characteristics of gene expression change of patients with cerebral apoplexy, and the effective inhibition of inflammatory response is one of important ways for neuroprotection. Therefore, the anti-inflammatory action of Lemnalol after stroke is one of the neuroprotective action mechanisms. Based on the fact that the functional states of microglia/macrophages (M1 and M2) directly affect secondary injury after cerebral apoplexy, and the functional states are reported to have an important role in regulating neuroprotection in the literature, the change of the activity states of the macrophages in M1 and M2 can be an important mechanism of neuroprotection and anti-inflammatory action of Lemnalol.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian university of traditional Chinese medicine
<120> application of pseudopterogorgia lancifolium active substance sciadol in preparing medicine for treating cerebral apoplexy
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Claims (2)
1. An application of pseudopterogorgen alcohol in preparing the medicines for decreasing the cerebral damage caused by apoplexy is disclosed.
2. The use of claim 1, wherein the chemical formula of the pseudopterogorgia alcohol is as follows:
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101164540A (en) * | 2006-08-03 | 2008-04-23 | 中山大学 | Application of marine steroids compound in preparing medicine for treating neuronal damage |
| TW200841875A (en) * | 2007-04-24 | 2008-11-01 | Univ Nat Sun Yat Sen | Lemnalol applying for treating inflammation and pain |
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2019
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101164540A (en) * | 2006-08-03 | 2008-04-23 | 中山大学 | Application of marine steroids compound in preparing medicine for treating neuronal damage |
| TW200841875A (en) * | 2007-04-24 | 2008-11-01 | Univ Nat Sun Yat Sen | Lemnalol applying for treating inflammation and pain |
Non-Patent Citations (2)
| Title |
|---|
| EUGENIA GENTILE等: "Marine pharmacology: therapeutic targeting of matrix metalloproteinases in neuroinflammation", 《DRUG DISCOVERY TODAY》 * |
| 丁伟等: "《血性脑血管并研究新进展》", 31 July 2009, 中国海洋大学出版社 * |
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