CN114917237A - Application of rebaudioside A in preparing medicine for preventing and/or treating neuron damage caused by cerebral ischemia - Google Patents
Application of rebaudioside A in preparing medicine for preventing and/or treating neuron damage caused by cerebral ischemia Download PDFInfo
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Abstract
An application of rebaudioside A in preparing a medicament for preventing and/or treating neuron damage caused by cerebral ischemia relates to the field of medicines. Experiments show that the rebaudioside A can inhibit the killing effect of oxygen sugar deprivation on neurons and has a dose-effect relationship, and in vitro studies show that when human SH-SY5Y cells are respectively treated by 5.0mmol/L and 10.0mmol/L of rebaudioside A for 1 hour, the neuron damage and death induced by oxygen sugar deprivation can be remarkably inhibited, and the mechanism of the rebaudioside A is related to the inhibition of endoplasmic reticulum stress reaction, the inhibition of mitochondrial damage and the inhibition of DNA double strand break.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of rebaudioside A in preparing a medicine for preventing and/or treating neuron damage caused by cerebral ischemia.
Background
Rebaudioside a (rebaudioside a) is a small molecular compound of steviol glycoside class extracted from the leaves of stevia rebaudiana Bertoni of Compositae family, and is white crystalline powder with molecular formula: c 44 H 70 O 23 Molecular weight 967.01, CAS number: 58543-16-1. Stevia rebaudiana is widely distributed in 27 provinces such as Hebei, Shanxi, Fujian, Jiangsu, Hunan and Yunnan, and has the characteristics of convenient material acquisition and low price.
Cerebral ischemia refers to cerebral blood supply insufficiency, which can be caused by various diseases, such as cerebral vascular arteriosclerosis, cerebral vasospasm, vertebrobasilar artery compression, and the like. Cerebral ischemia includes two types, transient ischemic attack and cerebral infarction. If cerebral ischemia lasts for a long time, changes such as brain tissue necrosis can be caused, and irreversible damage can be caused. Cerebral ischemia is commonly seen in the elderly, but with the improvement of national living standard, the onset age of cerebral ischemia tends to be younger. Although the brain weighs only 2% of body weight, oxygen consumption is 20% of total body oxygen consumption, and the required blood supply is 15% of cardiac output, so brain tissue is very sensitive to ischemia or hypoxia. Brain tissue is composed primarily of neurons and glial cells, where neurons are the primary performers for neural function. Compared with glial cells, neurons are highly susceptible to ischemic injury, which is the main pathological basis for neurological dysfunction due to cerebral ischemia. Given that cerebral ischemia can lead to an insufficient supply of oxygen and glucose to neurons, Oxygen Glucose Deprivation (OGD) has been widely used to mimic the pathological process of cerebral ischemia in vitro. It has been found that cerebral ischemia can cause neuronal damage or death through various pathways such as proteasome dysfunction, energy metabolism disorder, calcium overload, endoplasmic reticulum stress response, and the like. At present, effective preventive and therapeutic drugs for neurological dysfunction caused by cerebral ischemia are not available. Therefore, prevention and treatment of neuronal damage following cerebral ischemia has become a major strategy to alleviate neurological dysfunction following cerebral ischemia.
Until now, the therapeutic effect of rebaudioside a on nervous system diseases has not been reported.
Disclosure of Invention
The invention aims to provide application of rebaudioside A in preparing a medicine for preventing and/or treating neuronal damage caused by cerebral ischemia, so as to fill up the application blank of rebaudioside A in treating nervous system diseases.
The technical scheme adopted by the invention for solving the technical problem is as follows:
the application of the rebaudioside A in preparing the medicine for preventing and/or treating the neuron damage caused by the cerebral ischemia is disclosed.
As a preferred embodiment, the functions of rebaudioside a include:
a) inhibiting neuronal cell damage and death caused by oxygen sugar deprivation;
b) inhibiting stress on the endoplasmic reticulum of a neuron cell caused by oxygen sugar deprivation;
c) inhibiting upregulation of expression of GRP78 and CHOP due to oxygen sugar deprivation;
d) inhibiting nuclear translocation of CHOP due to oxygen sugar deprivation;
e) inhibiting mitochondrial damage to neuronal cells caused by oxygen sugar deprivation;
f) inhibiting the increase of mitochondrial superoxide compounds in neuronal cells following oxygen sugar deprivation;
g) inhibiting in a concentration-dependent manner a decrease in AIF in a mitochondrial component of neuronal cells caused by oxygen sugar deprivation;
h) inhibiting the increase of AIF in neuronal nuclear components caused by oxygen sugar deprivation;
I) inhibiting neuronal cell protein aggregation caused by oxygen sugar deprivation;
J) inhibiting the up-regulation of the amount of Ubiquitin-tagged protein by oxygen sugar deprivation in a concentration-dependent manner;
k) inhibiting DNA double-strand damage of neuron cells caused by oxygen sugar deprivation;
l) inhibiting the up-regulation of the expression of the marker γ -H2AX of DNA double strand breaks due to oxygen sugar deprivation in a concentration-dependent manner.
In a preferred embodiment, the concentration of rebaudioside A is 5.0 to 10.0 mmol/L.
In a preferred embodiment, the concentration of rebaudioside A is 10.0 mmol/L.
As a preferred embodiment, the dosage form of the drug is an injection or a tablet.
The medicine for preventing and/or treating neuron damage caused by cerebral ischemia has the effective component of rebaudioside A.
In a preferred embodiment, the concentration of rebaudioside A is 5.0 to 10.0 mmol/L.
In a preferred embodiment, the concentration of rebaudioside A is 10.0 mmol/L.
The preparation containing the medicament for preventing and/or treating the neuron damage caused by the cerebral ischemia is disclosed.
The beneficial effects of the invention are:
the present invention provides a novel use of rebaudioside A, and specifically, rebaudioside A is capable of inhibiting neuronal cell damage and death caused by oxygen sugar deprivation. Experiments show that the rebaudioside A can inhibit the killing effect of oxygen sugar deprivation on neurons and has dose-effect relationship; in vitro studies showed that treatment of human SH-SY5Y cells with 5.0mmol/L and 10.0mmol/L rebaudioside A for 1 hour significantly inhibited oxygen deprivation induced neuronal damage and death by mechanisms associated with inhibition of endoplasmic reticulum stress responses, inhibition of mitochondrial damage, and inhibition of DNA double strand breaks. The invention develops the new application of the rebaudioside A, and provides a new diagnosis and treatment method and a new treatment scheme for preventing and treating common diseases of the nervous system which affect the life quality of the old, namely cerebral ischemia.
Drawings
FIG. 1 shows the OD release values of LDHs of each group in example 1.
FIG. 2 is a graph showing the effect of rebaudioside A on the changes in expression of ER stress-associated proteins GRP78 and CHOP induced by oxygen deprivation in example 2.
FIG. 3 is a graph of the effect of rebaudioside A on the increase in superoxide in mitochondria induced by oxygen deprivation as measured using Mitosox red in example 3.
FIG. 4 is a graph showing the effect of rebaudioside A on AIF nuclear translocation induced by oxygen deprivation as measured by Western blot analysis in example 3.
FIG. 5 shows the effect of rebaudioside A on the expression of Ubiquitin protein as determined by Western blotting in example 4.
FIG. 6 is a graph showing the effect of rebaudioside A on DNA double strand breaks induced by oxygen deprivation as detected by Western blotting in example 5.
FIG. 7 is a graph showing the effect of rebaudioside A in inhibiting DNA double strand damage in human SH-SY5Y cells caused by oxygen deprivation in example 5.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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.
Example 1 use of rebaudioside A to inhibit oxygen deprivation (OGD) killing of human SH-SY5Y cells
Rebaudioside a was purchased from Shanghai leaf Biotech, and when used, it was diluted with DMSO to give a mother liquor, which was administered. Human SH-SY5Y cells were purchased from Gykey Biotechnology, China. A cell model of "neuronal damage after cerebral ischemia" was prepared by Oxygen Glucose Depletion (OGD), and damage and death of cells were detected by LDH (lactate dehydrogenase) release.
Human SH-SY5Y cells were inoculated in DMEM medium in a 96-well plate, incubated overnight with carbon dioxide, incubated with different concentrations of rebaudioside A (5.0mmol/L, 10.0mmol/L) 1 hour earlier, and the extent of LDH (lactate dehydrogenase) release was measured 24 hours after oxygen-sugar deprivation.
As a result, as shown in FIG. 1, LDH release OD values (490nm) of Control group, 5.0mmol/L rebaudioside A group and 10.0mmol/L rebaudioside A group which were not treated with oxygen deprivation were 0.328, 0.330 and 0.329, respectively, and there was no significant difference among the groups (Table 1). After the oxygen sugar deprivation treatment, the LDH release OD value of the Control group is obviously increased to 0.576; however, pretreatment of rebaudioside A group (5.0mmol/L, 10.0mmol/L) for 1 hour in advance inhibited release of LDH by oxygen deprivation, with OD values falling to 0.544(p <0.01) and 0.522(p <0.01), respectively. Among them, 10.0mmol/L of rebaudioside A group showed more significant inhibitory effect (Table 2).
In conclusion, rebaudioside A was effective in inhibiting the injury and death of human SH-SY5Y cells induced by oxygen sugar deprivation.
TABLE 1 LDH Release levels for groups not treated with oxygen sugar deprivation
TABLE 2 LDH Release levels for groups treated with oxygen sugar deprivation
Note: p <0.01, rebaudioside a + OGD group compared to OGD alone.
Example 2 use of rebaudioside A to inhibit human SH-SY5Y endoplasmic reticulum stress caused by oxygen deprivation (OGD)
Endoplasmic reticulum stress (ER stress) has been shown to be a major factor in oxygen deprivation-induced neuronal death. Studies have shown that endoplasmic reticulum stress leads to neuronal death by up-regulating CHOP expression and facilitating CHOP translocation into the nucleus. In order to clarify the mechanism of the damage and death of neurons induced by oxygen deprivation by rebaudioside A, human SH-SY5Y cells were inoculated into DMEM medium in a culture dish, incubated overnight with carbon dioxide for 1 hour in advance, incubated with rebaudioside A (5.0mmol/L, 10.0mmol/L) for 24 hours after oxygen deprivation, the cells were collected, lysed, the cytoplasm fraction and the nucleus fraction were separated by differential centrifugation, and then the effect of rebaudioside A on the changes in expression of endoplasmic reticulum stress-associated proteins GRP78 and CHOP induced by oxygen deprivation was examined by Western blot analysis.
As a result, as shown in fig. 2, oxygen deprivation significantly up-regulated the expression levels of GRP78 and CHOP, and also promoted the translocation of CHOP into the nucleus. However, with 5mmol/L rebaudioside A, up-regulation of GRP78 and CHOP induced by oxygen deprivation and nuclear translocation of CHOP induced by oxygen deprivation could be significantly inhibited; moreover, when the concentration of rebaudioside A is increased to 10mmol/L, the inhibitory effect is more pronounced.
In conclusion, the protective effect of rebaudioside a on neurons is associated with the inhibition of endoplasmic reticulum stress induced by oxygen sugar deprivation.
Example 3 use of rebaudioside A to inhibit mitochondrial damage to human SH-SY5Y cells due to oxygen deprivation (OGD)
1. Mitochondrial damage is an important factor in oxygen deprivation-induced neuronal damage and death. Mitochondrial damage is mainly manifested by: increased intramitochondrial superoxide, and the release and translocation of AIF into the nucleus within mitochondria. Thus, this experiment used Mitosox red to examine the effect of rebaudioside a on the increase in superoxide in mitochondria induced by oxygen deprivation.
Human SH-SY5Y cells are inoculated in DMEM medium in a 96-well plate, incubated overnight in a carbon dioxide incubator with rebaudioside A (5.0mmol/L, 10.0mmol/L) 1 hour earlier, added with Mitosox red after 24 hours of oxygen sugar deprivation, and the fluorescence intensity of the Mitosox red is detected after half an hour.
As can be seen from the data in FIG. 3, Table 3 and Table 4, the superoxide content in the mitochondria of human SH-SY5Y cells increased by 27.9% under the effect of oxygen-deprivation; in the 5mmol/L rebaudioside A group, the increase of superoxide in mitochondria was only 22.0%; when the concentration of rebaudioside A was increased to 10mmol/L, the increase in mitochondrial superoxide was only 16.6%.
In conclusion, rebaudioside A can significantly inhibit the increase of mitochondrial superoxide compounds in human SH-SY5Y cells after oxygen sugar deprivation.
TABLE 3 superoxide content in mitochondria of human SH-SY5Y cells of groups not treated with oxygen deprivation
| Item | Control group | Rebaudioside A (5mmol/L) | Rebaudioside A (10mmol/L) |
| 1 | 176 | 172 | 166 |
| 2 | 172 | 167 | 175 |
| 3 | 168 | 177 | 173 |
| 4 | 173 | 174 | 171 |
| 5 | 177 | 169 | 165 |
| 6 | 166 | 168 | 179 |
| Mean value of | 172 | 171.1666667 | 171.5 |
| SD | 4.335896678 | 3.868677638 | 5.357238094 |
| Ratio of | 1 | 0.995155039 | 0.997093023 |
TABLE 4 superoxide content in mitochondria of groups of human SH-SY5Y cells treated with oxygen deprivation
Note: p <0.05, rebaudioside a (5mmol/L) + OGD compared to OGD alone; p <0.01, rebaudioside a (10mmol/L) + OGD compared to the OGD only group.
2. The release of aif (apoptosis inducing factor) from mitochondria and into the nucleus is also a major cause of neuronal death. In order to detect the inhibition effect of rebaudioside A on human SH-SY5Y cell death induced by oxygen deprivation, human SH-SY5Y cells are inoculated in a DMEM culture medium in a culture dish in the experiment, a carbon dioxide incubator is used for overnight incubation for 1 hour in advance with rebaudioside A (5.0mmol/L and 10.0mmol/L), after 24 hours of oxygen deprivation, the cells are collected and lysed, a differential centrifugation method is adopted to separate a mitochondrion component and a cell nucleus component, and then a western blot analysis method is adopted to detect the influence of rebaudioside A on AIF nuclear translocation induced by oxygen deprivation.
As a result, as shown in FIG. 4, rebaudioside A inhibited the decrease in AIF in mitochondrial fractions of human SH-SY5Y cells induced by oxygen sugar deprivation in a concentration-dependent manner, while also inhibiting the increase in AIF in nuclear fractions of human SH-SY5Y cells, indicating that rebaudioside A significantly inhibited AIF nuclear translocation induced by oxygen sugar deprivation. Wherein TOMM20 is a mitochondrial marker protein, and H2A is a nuclear marker protein.
Example 4 application of rebaudioside A to inhibition of aggregation of human SH-SY5Y cellular proteins due to oxygen deprivation (OGD)
"protein aggregation" has been shown to be one of the leading causes of neuronal death. In order to elucidate the effect of rebaudioside a on protein aggregation induced by oxygen deprivation, the effect of rebaudioside a on Ubiquitin protein expression was examined by western blotting.
As a result, as shown in FIG. 5, although oxygen sugar deprivation could significantly up-regulate the amount of Ubiquitin-tagged protein, rebaudioside A inhibited the up-regulation of the amount of Ubiquitin-tagged protein induced by oxygen sugar deprivation in a concentration-dependent manner, indicating that rebaudioside A inhibited human SH-SY5Y cell protein aggregation induced by oxygen sugar deprivation.
Example 5 application of rebaudioside A to inhibition of double-stranded DNA damage in human SH-SY5Y cells caused by oxygen deprivation (OGD)
DNA is a carrier of genetic information, and double strand breaks in DNA are the basis for causing DNA lysis and cell death. To illustrate the protective effect of rebaudioside a on neurons, the effect of rebaudioside a on DNA double strand breaks induced by oxygen deprivation was examined by western blotting.
As a result, as shown in fig. 6, oxygen deprivation was able to significantly up-regulate the protein level of γ -H2AX, a marker of DNA double strand break, but rebaudioside a inhibited the expression up-regulation of γ -H2AX induced by oxygen deprivation in a concentration-dependent manner, indicating that rebaudioside a inhibited DNA double strand damage induced by oxygen deprivation. Wherein, gamma-H2 AX is a marker protein of DNA double strand break, H2A is a nuclear marker protein.
To further illustrate the effect of rebaudioside A in inhibiting DNA double strand damage of human SH-SY5Y cells caused by oxygen deprivation, in the experiment, human SH-SY5Y cells are inoculated in a DMEM culture medium of a culture dish with the diameter of 3cm, a carbon dioxide incubator is used for overnight incubation with rebaudioside A (10.0mmol/L) 1 hour in advance, and the cells are fixed by ethanol after 24 hours of oxygen deprivation for immunocytochemical staining, wherein the primary antibody adopted is a marker protein for DNA double strand breakage: γ -H2AX (red), nuclei were labeled with Hochest33258 (blue) and observed with a confocal laser microscope.
As a result, as shown in fig. 7, under the effect of oxygen sugar deprivation, red spots in the nucleus of human SH-SY5Y increased significantly, suggesting that γ -H2AX expression was upregulated; however, rebaudioside a significantly inhibited the increase in red spots, indicating that rebaudioside a inhibited DNA double strand damage induced by oxygen deprivation.
The invention discloses an application of rebaudioside A in preparing a medicament for preventing and/or treating neuron damage caused by cerebral ischemia, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the products described herein without departing from the spirit and scope of the invention.
Claims (9)
1. Application of rebaudioside A in preparing medicine for preventing and/or treating neuron damage caused by cerebral ischemia is provided.
2. The use of claim 1, wherein the functions of rebaudioside a comprise:
a) inhibiting neuronal cell damage and death caused by oxygen sugar deprivation;
b) inhibiting stress on the endoplasmic reticulum of a neuron cell caused by oxygen sugar deprivation;
c) inhibiting upregulation of expression of GRP78 and CHOP due to oxygen sugar deprivation;
d) inhibiting nuclear translocation of CHOP due to oxygen sugar deprivation;
e) inhibiting mitochondrial damage to neuronal cells caused by oxygen sugar deprivation;
f) inhibiting the increase of mitochondrial superoxide compounds in neuronal cells following oxygen sugar deprivation;
g) inhibiting in a concentration-dependent manner a decrease in AIF in a mitochondrial component of neuronal cells caused by oxygen sugar deprivation;
h) inhibiting the increase of AIF in neuronal nuclear components caused by oxygen sugar deprivation;
I) inhibiting neuronal cell protein aggregation caused by oxygen sugar deprivation;
J) inhibiting the up-regulation of the amount of Ubiquitin-tagged protein by oxygen sugar deprivation in a concentration-dependent manner;
k) inhibiting DNA double-chain damage of neuron cells caused by oxygen sugar deprivation;
l) inhibiting the up-regulation of the expression of the marker γ -H2AX of DNA double strand breaks due to oxygen sugar deprivation in a concentration-dependent manner.
3. The use of claim 1, wherein the rebaudioside a is at a concentration of 5.0 to 10.0 mmol/L.
4. The use of claim 3, wherein the concentration of rebaudioside A is 10.0 mmol/L.
5. The use according to claim 3, wherein the medicament is in the form of an injection or tablet.
6. A medicament for preventing and/or treating neuronal damage caused by cerebral ischemia, characterized in that the medicament contains rebaudioside A as an active ingredient.
7. The agent of claim 6, wherein the concentration of rebaudioside A is 5.0 to 10.0 mmol/L.
8. The medicament of claim 7, wherein the concentration of rebaudioside A is 10.0 mmol/L.
9. A preparation containing the agent for preventing and/or treating neuronal damage caused by cerebral ischemia according to any one of claims 6 to 8.
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