CN111110664B

CN111110664B - Application of 6-gingerol in preparing medicine for treating neonatal hypoxic ischemic encephalopathy

Info

Publication number: CN111110664B
Application number: CN202010061480.8A
Authority: CN
Inventors: 郝爱军; 周文娟; 赵曼; 杜静怡
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-01-19
Filing date: 2020-01-19
Publication date: 2021-06-22
Anticipated expiration: 2040-01-19
Also published as: CN111110664A

Abstract

The invention provides application of 6-gingerol in preparing a medicament for treating neonatal hypoxia ischemic encephalopathy, and the invention firstly proves that the 6-gingerol can relieve neonatal mouse HIE postcerebral injury, inhibit death of nerve cells behind neonatal mouse HIE, obviously reduce the level of HIE postlesion lateral cortex Bax protein, increase Bcl-2 protein expression and increase the ratio of Bcl-2/Bax, and simultaneously, the 6-gingerol can relieve the HIE postinflammatory reaction and improve the mouse HIE postshort-term nerve reflex and long-term motor capacity by reducing the expression of proinflammatory factors TNF-alpha and IL-1 beta, so that the 6-gingerol can be considered as an effective candidate medicament for the HIE cerebral injury nerve protective treatment and has important clinical application value.

Description

Application of 6-gingerol in preparing medicine for treating neonatal hypoxic ischemic encephalopathy

Technical Field

The invention belongs to the technical field of crude drug medicines, and particularly relates to application of 6-gingerol in neonatal hypoxic-ischemic encephalopathy.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

In recent years, with the continuous improvement and continuous development of medical level, prenatal and neonatal care is improved, but neonatal hypoxic ischemic encephalopathy (hypoxic ischemic encephalopathy HIE) still remains one of the important causes of morbidity and mortality of newborns all over the world, the morbidity of every thousand newborns in developed countries is 2 to 3, the life health of the newborns is seriously affected, and the social and family burdens are heavy. The research on the pathogenesis of the HIE actively explores a new treatment mode, has important theoretical significance, and is expected to provide a new idea for the prevention and clinical treatment of the HIE.

HIE refers to the hypoxic ischemic injury of the brain of a newborn caused by perinatal asphyxia, and is clinically manifested as a series of encephalopathy symptoms, such as cognitive impairment, myotonia change and abnormal primary reflex, and convulsion and respiratory failure in patients with critical illness. The pathophysiological mechanisms of the nerve cell death include necrosis, apoptosis, autophagy, excitotoxicity, oxidative stress, neuroinflammation and the like. And researches show that low-temperature treatment can reduce cerebral blood flow perfusion and cell metabolism, relieve ischemia reperfusion injury, reduce excessive free radical generation and inhibit immune response and proinflammatory response in HI. Related studies have been conducted to show that neuroprotective therapy as a therapeutic measure for independent or adjunctive hypothermia treatment can ameliorate the adverse consequences of HIE to act synergistically with the neuroprotective effects of hypothermia treatment. Neuroprotective agents include anticonvulsant or sedative agents such as phenobarbital, topiramate, bumetanide, magnesium sulfate, anti-apoptotic agents such as erythropoietin, anti-spasmodic agents such as xenon, anti-inflammatory and anti-oxidant agents such as melatonin, N-acetylcysteine. The clinically approved standard treatment regimen is to provide moderate to severe HI neonates with a therapeutic hypothermia of 33 to 34 ℃ for 72 hours, and the data suggests that the neonates undergoing hypothermic treatment have a low efficacy rate, that only one out of every nine persons on average benefits, and that this treatment method has not been widely popularized clinically. At present, clinical treatment mainly aims at symptomatic support treatment, aims at restoring energy metabolism of nerve cells and promoting repair of damaged nerve cells and regenerative nutrition nerve treatment, commonly used medicaments are phenobarbital to control convulsion, furosemide reduces intracranial pressure, naloxone eliminates brainstem symptoms, neurotrophic factors promote nerve recovery, but the curative effect is not exact, early neonatal death still exists after treatment, survivors also often have a series of nervous system injury sequelae such as cognitive disorder, epileptic seizure and motor function injury, and the occurrence of the nervous system injury sequelae undoubtedly brings heavy burden to society and families, so that the pathogenesis of HIE is fully known to prevent diseases, and a new effective means is explored to reduce disease injury.

Ginger is a rhizome of a plant of the genus Zingiber, and has been used as a traditional food and a Chinese herbal medicine in the past 2000 years in China, and various researches have shown that the ginger has the effects of resisting inflammation, relieving fever and easing pain and reducing blood sugar as a traditional medicine and has certain effects on cold, nausea and vomiting, asthma, cough, bleeding and muscle pain. Rhizoma Zingiberis recens can also be combined with other drugs for protecting nervous system diseases such as ischemic apoplexy. In addition, it has been used clinically in a number of diseases such as tumors, bone diseases, metabolic dysfunctions and cardiovascular diseases with good results. Currently, about 400 ginger components have been identified, including carbohydrates, lipids, terpenes and phenolic compounds. 6-Gingerol (6-Gingerol) is the main pungent compound in fresh ginger, is a phenolic substance, has various biological characteristics and pharmacokinetic studies show that it can be distributed in the whole rat body. And which are associated with a number of chronic diseases in humans and animal models, and which can ameliorate or prevent chronic diseases, have been reported to inhibit the vascular senescence pathway as an important regulator of the senescence process by modulating mammalian rapamycin targets. However, the inventors found that no studies have reported the role of 6-gingerol in HIE.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of 6-gingerol in neonatal hypoxia ischemic encephalopathy, and the invention firstly proves that the 6-gingerol can relieve neonatal mouse HIE hindbrain injury, inhibit death of nerve cells behind neonatal mouse HIE, obviously reduce the level of HIE hind lesion lateral cortex Bax protein, increase Bcl-2 protein expression and increase the ratio of Bcl-2/Bax, and simultaneously, the 6-gingerol can relieve the HIE hind inflammatory reaction and improve the mouse HIE hind short-term nerve reflex and long-term motor capacity by reducing the expression of proinflammatory factors TNF-alpha and IL-1 beta, so that the 6-gingerol can be considered as an effective candidate drug for the HIE brain injury neuroprotection treatment and has important clinical application value.

Specifically, the invention relates to the following technical scheme:

in a first aspect of the invention, the application of 6-gingerol in preparing a medicament for treating neonatal hypoxic-ischemic encephalopathy is provided.

According to the invention, the term "treatment" is intended to mean any relevant measure suitable for the treatment of hypoxic-ischemic encephalopathy in neonates, or the prophylactic treatment of such manifested disease or manifested symptoms, or the avoidance of recurrence of such disease, for example after the end of a treatment period or the treatment of symptoms of already-onset disease.

Specifically, the treatment of the neonatal hypoxic-ischemic encephalopathy is at least shown in any one or more of the following purposes:

a) alleviating hindbrain injury from HIE;

b) reducing apoptosis of nerve cells following HIE;

c) reducing the expression of pro-inflammatory genes after HIE;

d) improving short-term neural reflexes and/or long-term motor ability after HIE.

Wherein, in the a), the reduction of HIE postencephalic injury is specifically represented by any one or more of the following:

a1) reducing area of post-HIE cerebral ischemia;

a2) reducing cerebral edema;

a3) reduce morphological damage of brain nerve cells.

In b), the reduction of apoptosis of nerve cells after HIE is embodied as any one or more of:

b1) reducing the degree of degeneration of nerve cells due to HIE;

b2) reducing the degree of necrosis of nerve cells due to HIE;

b3) the level of Bax protein in a lesion area after HIE is reduced, the expression of Bcl-2 protein is increased, and the ratio of Bcl-2/Bax is increased.

In said c), the proinflammatory factors include, but are not limited to TNF-alpha and IL-1 beta, and the test proves that the mRNA level of TNF-alpha and IL-1 beta in mouse lesion cortex is increased after HIE injury, and the mRNA level of TNF-alpha and IL-1 beta in lesion cortex is obviously reduced after 6-gingerol treatment.

In the d), the improvement of short-term neural reflex after HIE is specifically shown in the improvement of HIE retroversion positive reflex disorder and/or the reduction of the prolongation of geotaxic reflex time caused by HIE;

the improvement of long-term exercise ability is specifically reflected in the improvement of exercise coordination.

According to the present invention, not only is the use of 6-gingerol in the preparation of a medicament for the treatment of hypoxic-ischemic encephalopathy in neonates disclosed, but it is also disclosed that this effect can be enhanced by administering 6-gingerol in combination with at least one other pharmaceutically active ingredient. As an alternative or in addition to other pharmaceutically active ingredients, 6-gingerol may also be used in combination with other non-pharmaceutically active ingredients.

In view of the above, the second aspect of the present invention provides a pharmaceutical composition for treating neonatal hypoxic-ischemic encephalopathy, said pharmaceutical composition comprising 6-gingerol and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.

In the sense of the invention, the pharmaceutical composition provided by the invention represents a substance, and the contained 6-gingerol has an obvious treatment effect on the hypoxic ischemic encephalopathy of the newborn.

In a third aspect of the invention, there is provided the use of 6-gingerol in the manufacture of any one or more of:

1) an inhibitor of a proinflammatory factor;

2) inhibitors of Bax protein;

3) a Bcl-2 protein promoter;

among these, proinflammatory factors include, but are not limited to TNF- α and IL-1 β.

The invention has the beneficial technical effects that:

the invention discovers the effect of 6-gingerol in neonatal hypoxic ischemic encephalopathy for the first time, and the specific expression is that the 6-gingerol can relieve the hindbrain injury of neonatal mouse HIE, relieve the apoptosis of nerve cells behind the neonatal mouse HIE, obviously reduce the level of HIE lesion lateral cortex Bax protein, increase the expression of Bcl-2 protein and increase the ratio of Bcl-2/Bax. The 6-gingerol reduces the expression of proinflammatory factors TNF-alpha and IL-1 beta so as to relieve the inflammatory reaction after HIE. Meanwhile, the 6-gingerol can improve short-term nerve reflex and long-term motor ability of mice after HIE. The results show that the 6-gingerol can be used as an effective candidate drug for neuroprotective treatment of HIE brain injury.

In conclusion, the invention opens up a new medicinal application for 6-gingerol, lays an experimental foundation for developing high-efficiency medicaments related to the neonatal hypoxic ischemic encephalopathy and provides a new visual field.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing that 6-gingerol in the examples of the present invention reduces the post-HIE brain damage in neonatal mice; wherein (a) representative TTC stained coronal sections of brain were selected from different groups 72 hours after HIE injury. (B) The ischemic area was measured in each group by TTC staining. Ischemic area (contralateral area-ipsilateral non-ischemic area)/contralateral area. The quantitative analysis of the lesion area for each group represents the mean ± standard deviation. (C) Representative photographs of cortex and hippocampus in coronal sections stained with hematoxylin-eosin (HE) 72 hours after HIE injury, N ═ 3,^＊＊P＜0.01。

FIG. 2 is a graph showing the effect of 6-gingerol on HIE-induced neuronal cell death in an example of the present invention. (A) Cortex and hippocampal representative Nissl staining patterns of coronal sections of brains of different groups of mice 72 hours after HIE injury. (B) Western Blot representation of Bcl-2, Bax and cleared-PARP in the cortex of different groups of rats 72 hours after HIE injury, with β -actin as the internal control. (C) Western Blot quantification of Bcl-2/Bax and cleaned-PARP, data expressed as mean. + -. standard deviation. N-5^＊P＜0.05,^＊＊P＜0.01，^＊＊＊P＜0.001。

FIG. 3 shows that 6-gingerol reduces the expression of pro-inflammatory genes following HIE in mice in accordance with an embodiment of the present invention. TNF-. alpha.IL-1. beta. mRNA levels in ipsilateral cortex were detected by qPCR 72 hours after HIE injury. N is 5.^＊P＜0.05,^＊＊P＜0.01。

FIG. 4 shows the effect of 6-gingerol on the short-term neuroreflex and long-term motor function of mice in the present invention. (A) Mice were tested for righting reflex response time 24 hours after HIE injury. (B) Mice were tested for negative geotaxic reflex time 24 hours after HIE injury. (C) 4 weeks after HIE injury, mice were tested for motor coordination by rotarod testing. The value represents the mean value. + -. standard deviation, N is 3-6,^＊P＜0.05,^＊＊p <0.01, ns, no statistical difference.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.

As described in the background, no study has been reported in the prior art of the role of 6-gingerol in HIE.

In view of the above, in one embodiment of the present invention, there is provided an application of 6-gingerol in preparing a medicament for treating neonatal hypoxic-ischemic encephalopathy.

In yet another embodiment of the invention, the treatment of hypoxic-ischemic encephalopathy in neonates is indicated at least by any one or more of the following:

a) alleviating hindbrain injury from HIE;

b) reducing apoptosis of nerve cells following HIE;

c) reducing the expression of pro-inflammatory genes after HIE;

In still another embodiment of the present invention, the reducing of HIE postbrain injury in a) is embodied in any one or more of:

a1) reducing area of post-HIE cerebral ischemia;

a2) reducing cerebral edema;

a3) reduce morphological damage of brain nerve cells.

In still another embodiment of the present invention, in b), the reduction of apoptosis of nerve cells after HIE is embodied as any one or more of:

b1) reducing the degree of degeneration of nerve cells due to HIE;

b2) reducing the degree of necrosis of nerve cells due to HIE;

In yet another embodiment of the present invention, the pro-inflammatory factors in c) include, but are not limited to, TNF- α and IL-1 β, and the present invention has been tested to demonstrate that mRNA levels of TNF- α and IL-1 β are increased in the lesion cortex of mice after HIE injury, while mRNA levels of TNF- α and IL-1 β are significantly decreased in the lesion cortex after treatment with 6-gingerol.

In still another embodiment of the present invention, in said d), the improvement of short-term neuroreflex after HIE is specifically characterized by improving disorder of retroversion and positive reflex of HIE and/or reducing prolongation of geotaxic reflex time by HIE;

In another embodiment of the present invention, a pharmaceutical composition for treating hypoxic-ischemic encephalopathy in neonates is provided, said pharmaceutical composition comprising 6-gingerol and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.

In another embodiment of the present invention, the pharmaceutical composition of the present invention represents a substance containing 6-gingerol having a significant therapeutic effect on hypoxic ischemic encephalopathy of newborn infants.

Wherein, the treatment of the neonatal hypoxic ischemic encephalopathy is specifically shown as follows:

a) alleviating hindbrain injury from HIE;

b) reducing apoptosis of nerve cells following HIE;

c) reducing the expression of pro-inflammatory genes after HIE;

a1) reducing area of post-HIE cerebral ischemia;

a2) reducing cerebral edema;

a3) reduce morphological damage of brain nerve cells.

b1) reducing the degree of degeneration of nerve cells due to HIE;

b2) reducing the degree of neuronal necrosis caused by HIE;

In the d), the improvement of short-term neuroreflex after HIE is specifically manifested in improving HIE retroversion positive reflex disorder and/or reducing HIE-induced prolongation of geotaxic reflex time.

The pharmaceutically inactive ingredients may be carriers, excipients, diluents and the like which are generally used in pharmacy. Further, the composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

Such pharmaceutically inactive ingredients, which may include carriers, excipients and diluents, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.

In still another embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In yet another embodiment of the present invention, the medicament of the present invention may be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In still another embodiment of the present invention, the subject to which the pharmaceutical composition is administered may be human and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, and the like.

In a further embodiment of the invention, there is provided the use of 6-gingerol in the preparation of any one or more of the following products:

1) an inhibitor of a proinflammatory factor;

2) inhibitors of Bax protein;

3) bcl-2 protein promoter.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1

Materials and methods

HIE model

Animal experiments were conducted according to the International guidelines for animal research, supplied by the International organization for medical science (CIOMS), and procedures were approved by the animal ethics and welfare Committee, university of Shandong. The Rice-Vannucci model was used in this study with minor modifications. Briefly, day 7 postnatal (P7) mouse pups were anesthetized by 2-3% isoflurane inhalation, the right common carotid artery of the mouse was exposed, double ligated with 4-0 surgical suture, and after surgery, the mouse was allowed to recover for 1 hour, then placed in an anoxic incubator at 37 ℃ containing 5% carbon dioxide, 9% oxygen, and 86% nitrogen for 90 minutes under hypoxia, and the sham group was anesthetized and the right carotid artery exposed without ligation and anoxic treatment of the common carotid artery.

2. Intraperitoneal injection and drug treatment of mice

A 6-gingerol (desserts, chinese achievements) working solution and a negative control solution were prepared according to the manufacturer's instructions. (6-gingerol dissolved in physiological saline containing 1% DMSO).

Mice were randomized into three groups: the sham operation group, the HIE + 6-gingerol group, and the HIE + physiological saline group. Briefly, 6-gingerol (2mg/kg) was intraperitoneally injected 24 hours prior to mouse HIE treatment once a day for 4 days. Mice in the HIE + saline group were injected intraperitoneally with saline (2mg/kg) containing 1% DMSO.

3. Infarct size measurement

Cerebral infarct size was measured by 2,3, 5-triphenyltetrazolium chloride (TTC) staining. Briefly, 72 hours after HIE injury, mice were anesthetized, and mouse brain tissue was removed, washed with physiological saline, frozen at-20 ℃ for 20 minutes, cut into 2 mm-thick coronal sections, and the cut brain sections were stained in 2% TTC (Solarbio, Beijing, China) solution in dark at 37 ℃ for 30 minutes, and photographed after the staining was finished. The cerebral infarct size was analyzed using ImageJ software (national institute of health, beuserda, maryland, usa).

4. Histological staining

3 days after HIE injury, mice were euthanized by deep anesthesia with isoflurane and heart perfused with 10mL of normal saline, then perfused with 10mL of 4% PFA, and mouse brains were fixed in 4% PFA for 24 hours. Gradient dehydration was performed using xylene and ethanol, and the brains were embedded in paraffin and cut into 4 μm thick coronal sections for subsequent staining. Hematoxylin-eosin (HE) and Nissl staining (KeyGEN BioTECH, jiangsu, china) staining was performed according to the manufacturer's instructions to observe histopathological changes. Images were obtained using an optical microscope (OLYMPUS-BX 51).

RNA isolation and real-time quantitative PCR

72 hours after HIE, mouse brain tissue was collected and homogenized in TRIZOL solution (Invitrogen). Total RNA was extracted and its concentration was then measured by spectrophotometer. Using RevertAid^TMFirst Strand cDNA Synthesis Kit (Thermo Fisher Scientific) converts RNA to cDNA and performs real-time PCR by SYBR Green real-time PCR Master Mix (TOYOBO). Beta actin gene as internal reference, use 2^-ΔΔCTThe method calculates the relative expression level of the gene. The gene primer sequences are shown in Table 1.

TABLE 1

6. Western blot

Briefly, 72 hours after HIE, brain tissue was collected and prepared as a homogenate on ice after addition of protease inhibitors and RIRA lysate. The homogenate was centrifuged at 12,000rpm for 20 minutes at 4 ℃. The supernatants were collected and quantified using bicinchoninic acid (BCA) assay (Pierce Biotechnology). The sample was mixed with loading buffer and boiled for 5 minutes. Equal amounts of protein were loaded into each well and separated by SDS-PAGE. Primary antibody was used at the following dilutions: murine anti-Bcl-2 (1: 500, Santa), Rabbit anti-Bax (1:1,000, CST), Rabbit anti-cleaned PARP1(1:1000, Abcam), murine anti-beta-actin (1: 2,000, CST).

7. And (5) righting the reflection.

The mice were fixed in the supine position and the time it took for the mice to roll over to the prone position was recorded. (in seconds). Each mouse was tested 3 times, and the average of 3 tests was calculated.

8. Negative geotaxis test

The mice were placed on a 30 cm long inclined plate (45 degrees) with the head facing down. In a 60 second test, the time (in seconds) required for the animal to turn 180 ° was recorded. Each mouse was tested 3 times, and the average of 3 tests was calculated.

9. Rotating rod testing

To evaluate the post-HIE motor coordination in mice, a rotarod test was performed 4 weeks after HIE injury using a rotarod fatigue apparatus (seons, jiangsu, china). Each mouse was acclimated to training at a fixed speed of 10rpm for 10s before the start of the official experiment. After training, the speed of the rod was gradually accelerated from 10rpm to 40rpm for 120s and the mouse movement time was recorded by the device. The time recording was stopped until the animal fell off the rotating rod. This time period is referred to as the mouse-in-stick time. If the animal continues to run for more than 120s, the test is terminated and the mouse is considered 120s at the time of the wand.

10. And (5) carrying out statistical analysis.

Statistical analysis was performed using GraphPad Prism 5.0 software. Values are expressed as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by multiple comparisons of the means using Tukey's test with significance set at P < 0.05.

Results

1.6-gingerol reduced hindbrain injury of neonatal mice HIE.

Fig. 1A is a representative TTC-stained mouse coronal brain section with normal brain tissue appearing uniformly red and ischemic areas white. The area of brain injury is expressed as the ratio of total infarct area (left white area) to total brain area. The mice in the sham operation group had no obvious ischemic area, the percentage of ischemic area in the HIE group was 27.2% (FIG. 1B), and the treatment in the 6-gingerol group significantly reduced the percentage of ischemic area in the brain to 17.8% (P <0.01), indicating that 6-gingerol significantly reduced the ischemic area in the hindbrain of HIE.

The mice were examined for brain damage by hematoxylin-eosin (HE) staining, as shown in fig. 1c, with clear structural layers in the cerebral cortex and normal neuronal morphology in the sham group. In the HIE group, cells were sparsely arranged, cell contours were blurred, neurons were contracted, and significant edema was observed in the cerebral cortex. After the treatment by using the 6-gingerol, the cerebral edema and the cellular morphological injury are obviously reduced.

2.6-gingerol reduction of apoptosis of neuronal cells after HIE in neonatal mice

To evaluate neuronal apoptosis Nissl staining was performed, as shown in fig. 2A, in the sham group, the neurons of the cortex and hippocampus were morphologically intact, clearly stained, and densely arranged. The HIE group exhibited substantial tissue loss, loose neuronal alignment and cell atrophy, swelling and even lack of normal cellular structure. The degeneration and necrosis degree of the 6-gingerol group nerve cells are obviously reduced, and the number of intact neurons is obviously higher than that of the HIE group.

Based on the above results, we further identified proteins that may be associated with this effect, and analyzed the expression of Bcl-2, Bax and cleared PARP by western blot (FIG. 2B). As shown in FIG. 2C, the expression of Bax protein was significantly increased in the HIE group, the expression of Bcl-2 protein was lower, and the ratio of Bcl-2/Bax protein was decreased, as compared to the sham group. In contrast, 6-gingerol treatment significantly reduced the level of Bax protein in the lesion lateral cortex after HIE, increased Bcl-2 protein expression, and increased the Bcl-2/Bax ratio.

3.6-gingerol reduced the expression of pro-inflammatory genes following HIE in mice.

The levels of TNF-alpha and IL-1 beta mRNA were increased in the mouse lesion cortex after HIE injury compared to the sham group, while the levels of TNF-alpha and IL-1 beta mRNA were significantly reduced in the lesion cortex after treatment with 6-gingerol (FIG. 3).

4.6-Effect of gingerol on short-term neural reflexes and long-term motor ability after HIE in mice.

Mice were tested for righting reflex 24 hours after HIE injury. The results show that the HIE group has longer flip reflex time (P is less than 0.05) compared with the sham operation group, the mice have shorter flip reflex time (P is less than 0.05) after the treatment by 6-gingerol, and the 6-gingerol can improve the flip reflex disorder after the hypoxic-ischemic brain injury of the newborn mice (figure 4A); the mice were subjected to a geotaxic reflex test 24 hours after HIE injury, and the results showed that the geotaxic reflex time was prolonged (p < 0.05) in mice of HIE group compared to the sham group, and decreased after treatment with 6-gingerol, but there was no statistical difference. (FIG. 4B).

4 weeks after HIE injury, mice were subjected to a rotarod test. The results show that mice in the post-HIE group fell rapidly off the rotarod during the trial period and exhibited poor motor coordination compared to the sham group, whereas the 6-gingerol treated group of mice exhibited increased residence time on the rotarod as exhibited strong motor coordination (fig. 4C).

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

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Application of <120> 6-gingerol in neonatal hypoxic ischemic encephalopathy

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Claims

1.6-gingerol is used in preparing medicine for treating neonatal hypoxic ischemic encephalopathy.

2. The use according to claim 1, wherein the treatment of hypoxic-ischemic encephalopathy in neonates is indicated by any one or more of the following:

a) alleviating brain damage after hypoxic-ischemic encephalopathy of newborn;

b) reducing apoptosis of nerve cells after hypoxic-ischemic encephalopathy of the newborn;

c) reducing the expression of proinflammatory factors after the hypoxic ischemic encephalopathy of the newborn;

d) improving short-term nerve reflex and/or long-term motor ability of newborn infants after hypoxic-ischemic encephalopathy.

3. The use according to claim 2, wherein in a) the reduction of brain damage following hypoxic-ischemic encephalopathy in a neonate is manifested by any one or more of:

a1) reducing the cerebral ischemic area after the hypoxic ischemic encephalopathy of the newborn;

a2) reducing cerebral edema;

a3) reduce morphological damage of brain nerve cells.

4. The use according to claim 2, wherein in b) the reduction of neuronal apoptosis following hypoxic-ischemic encephalopathy in the neonate is manifested by any one or more of:

b1) reducing the degree of degeneration of nerve cells caused by hypoxic-ischemic encephalopathy of newborn infants;

b2) reducing the degree of nerve cell necrosis caused by neonatal hypoxic-ischemic encephalopathy;

b3) reducing the Bax protein level of the pathological change area after the hypoxic ischemic encephalopathy of the newborn, increasing the Bcl-2 protein expression and increasing the Bcl-2/Bax ratio.

5. The use of claim 2, wherein in c) the proinflammatory factors comprise TNF- α and IL-1 β.

6. The use according to claim 2, wherein in d) the improvement of short-term neuroreflex after hypoxic-ischemic encephalopathy in neonates is manifested in an improvement of flip-up reflex disorder after hypoxic-ischemic encephalopathy and/or a reduction of prolongation of time to geotaxis reflex caused by hypoxic-ischemic encephalopathy in neonates; or the like, or, alternatively,