CN110787180A - Application of homoplantaginoside and derivatives thereof as Nrf-2 activator - Google Patents
Application of homoplantaginoside and derivatives thereof as Nrf-2 activator Download PDFInfo
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- CN110787180A CN110787180A CN201911318899.0A CN201911318899A CN110787180A CN 110787180 A CN110787180 A CN 110787180A CN 201911318899 A CN201911318899 A CN 201911318899A CN 110787180 A CN110787180 A CN 110787180A
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Abstract
The invention relates to the technical field of medical application of homoplantaginoside and derivatives thereof, and particularly discloses application of homoplantaginoside and derivatives thereof as an Nrf-2 activator. The research of the invention finds that the homoplantaginoside and the derivative thereof can effectively inhibit the apoptosis of endothelial cells caused by oxidative low-density lipoprotein, and play a role in protecting the endothelial cells by oxidation resistance through activating Nrf 2. The invention provides theoretical and experimental basis for researching that the homoplantaginoside and the derivative thereof are used as an activator of Nrf2 to protect endothelial cell injury.
Description
Technical Field
The invention relates to the technical field of medical application of homoplantaginoside and derivatives thereof, in particular to application of homoplantaginoside and derivatives thereof serving as Nrf-2 activators in preparation of medicines for treating diseases related to Nrf2 signal pathways.
Background
The transcription factor Nrf2 belongs to CNC-Bzip (capncollar sub family of basic leucoine-zipper), namely a CNC leucine zipper transcription activator family, is an important transcription factor for regulating and controlling the oxidative stress response of cells and is also a central regulator for maintaining the intracellular redox homeostasis. The Nrf2 can reduce cell damage caused by active oxygen and an electrophile by inducing and regulating the constitutive expression and the inducible expression of a series of antioxidant proteins, so that the cells are in a stable state, and the redox dynamic balance of an organism is maintained, thereby becoming an important path of the cells for resisting oxidative stress. A great deal of research shows that Nrf2 is related to inflammation, chronic kidney disease, diabetes, cardiovascular disease, neurodegenerative disease and tumor.
Proper activation of the Nrf2 signaling pathway can enhance the antioxidant stress capacity of cells, maintain the redox balance in cells, reduce the damage of free radicals and chemical carcinogens to cells, and thus maintain the normal physiological functions of cells. Therefore, the search for safe and effective Nrf2 activators becomes a new direction for drug development.
The hispidoside (homoplantaginin) flavonoid compound mainly exists in common sage herb, and has the following structural formula:
pharmacological research shows that: the hispidoside has effects of treating liver injury, improving endothelial insulin resistance, inhibiting tumor growth, resisting osteoporosis, and inducing cancer cell apoptosis. Another experiment shows that the homoplantaginoside has the functions of reducing blood sugar and regulating blood fat, can obviously improve the generation of NO, and can improve the vascular endothelial insulin resistance caused by free fatty acid. However, no report on the regulation of Nrf2 activity by homoplantaginoside and derivatives thereof is available at present.
Disclosure of Invention
The invention discovers for the first time that the homoplantaginoside and the derivative thereof can be used as an Nrf2 activator, have potential antioxidant stress activity and can be used for preparing health food or medicines for diseases related to Nrf2 signal channels. Based on this, the invention provides the following technical scheme:
in a first aspect of the invention there is provided the use of homoplantaginoside and/or a homoplantaginoside derivative in the preparation of an Nrf2 activator.
In the above application, the homoplantagine derivative is dihydrohomoplantagine, and its structural formula is as follows:
in the above application, the homoplantaginoside and the homoplantaginoside derivative can be obtained commercially; can also be extracted from Salvia plebeia R.Brown. Wherein:
the homoplantaginoside and homoplantaginoside derivatives can be prepared by the following method:
crushing a common sage herb raw material, adding ethanol with the volume fraction of 95% for reflux extraction, and concentrating an extracting solution to obtain a concentrated solution; extracting the concentrated solution with ethyl acetate/n-butanol-water as two-phase solvent system, and concentrating the upper ethyl acetate/n-butanol phase to obtain extract; gradient eluting the extract with macroporous resin, monitoring with thin layer, collecting high plantaginide fraction, concentrating, adjusting the concentrated solution to acidity, filtering to obtain precipitate, washing, and drying to obtain light yellow powder; separating with high performance liquid chromatograph to obtain homocepharaside and homocepharaside derivatives.
Preferably, the addition amount of the ethanol with the volume fraction of 95 percent is 8 to 12 times of the weight of the litchi grass raw material.
Preferably, the gradient elution is sequentially performed by using 30 percent, 50 percent, 85 percent and 95 percent of ethanol water solution.
In a second aspect of the invention, there is provided the use of homoplantaginoside and/or homoplantaginoside derivatives as Nrf-2 activators in the preparation of a medicament against oxidative stress.
In a third aspect of the invention, there is provided the use of homoplantaginoside and/or homoplantaginoside derivatives as Nrf-2 activators in the preparation of a medicament for inhibiting apoptosis of endothelial cells.
In the above application, the apoptosis is induced by oxidative low density lipoprotein.
In a fourth aspect of the present invention, there is provided an Nrf2 activator, wherein the Nrf2 activator contains homoplantaginin and/or a homoplantaginin derivative as an active ingredient.
The invention has the beneficial effects that:
apoptosis of endothelial cells, disorder of lipid metabolism, and inflammation are major causes of disease development. The homoplantaginoside and the derivative thereof can effectively inhibit the apoptosis of endothelial cells caused by oxidative low-density lipoprotein, play a role in protecting the endothelial cells by oxidation resistance by activating Nrf2, can effectively inhibit the generation and development of arteriosclerosis in ApoE-/-mice, can stabilize plaques, can reduce the ROS expression in the plaques by activating Nrf2, and can improve the HO-1 expression. The homoplantaginoside and the derivative thereof can reduce the action possibly, so the invention provides theoretical and experimental basis for researching that the homoplantaginoside and the derivative thereof are used as activators of Nrf2 and protecting endothelial cell damage, and the homoplantaginoside and the derivative thereof related by the invention can be used as a tool for effectively activating Nrf2 and lay a foundation for researching and developing related diseases.
Description of the drawings:
FIG. 1: homoplantaginoside and its derivatives can activate Nrf 2.
FIG. 2: plantago ovata and derivatives thereof are capable of promoting nuclear entry of Nrf 2.
FIG. 3: the homoplantaginoside and the derivative thereof can promote the expression of protein HO-1 downstream of Nrf 2.
FIG. 4: the homoplantaginoside and the derivative thereof can inhibit the expression of ROS in endothelial cells.
FIG. 5: knockdown of Nrf2 homoplantarin did not protect the endothelial cell inflammatory response.
FIG. 6: mouse body weight injection graph.
FIG. 7: HO-1 expression level in mouse aortic section.
FIG. 8: ROS expression in aortic sections of mice.
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.
As described in the background section, the transcription factor Nrf2 belongs to CNC-Bzip (capncollar subfamily basic leucoine-zipper), namely, the CNC leucine zipper transcription activator family is an important transcription factor for regulating the oxidative stress response of cells and is also a central regulator for maintaining the intracellular redox homeostasis. The Nrf2 can reduce cell damage caused by active oxygen and an electrophile by inducing and regulating the constitutive expression and the inducible expression of a series of antioxidant proteins, so that the cells are in a stable state, and the redox dynamic balance of an organism is maintained, thereby becoming an important path of the cells for resisting oxidative stress. Proper activation of the Nrf2 signaling pathway can enhance the antioxidant stress capacity of cells, maintain the redox balance in cells, reduce the damage of free radicals and chemical carcinogens to cells, and thus maintain the normal physiological functions of cells. There are many reports of Nrf2 activators such as catechin esters, sulforaphane, curcumin, resveratrol, oltipraz, t-butylhydroquinone, and the like. Different Nrf2 activators may activate Nrf2 in different mechanisms and thus may function differently.
The homoplantaginoside has effects of reducing blood sugar, regulating blood lipid, improving NO generation, and improving vascular endothelial insulin resistance caused by free fatty acid. However, no report on the regulation of Nrf2 activity by homoplantaginoside and derivatives thereof is available at present.
In order to research the action relationship between the homoplantaginoside (S3) and the homoplantaginoside derivative (S1) and the Nrf2, the invention firstly determines whether the homoplantaginoside and the derivative thereof can activate the Nrf2 by a Luci-hela transfected cell line, and can activate the Nrf2 by measuring the homoplantaginoside (S3) and the derivative thereof (S1) at 540nm by a microplate reader, thereby determining that the homoplantaginoside and the derivative thereof can be used as an activator of the Nrf 2.
The Luci-hela transfected cell line adopted by the invention can stably express firefly luciferase, and is treated by using a firefly luciferase reporter gene kit, wherein a transcription regulation element or a 5 promoter region of Nrf2 is cloned at the upstream of luciferase, or a 3-UTR region is cloned at the downstream of luciferase to form a reporter plasmid, then cells are transfected, the cells are treated by S1 and S3 and then are lysed, the luciferase activity is measured, whether the transcription regulation of Nrf2 can be activated by drug treatment or not is judged according to the activity of the luciferase, and the activation of Nrf2 can be detected by a microplate reader at 540nm by using a homoeosin (S3) and a derivative (S1) thereof. (see FIG. 1).
Under normal physiological conditions, Nrf2 specifically binds to cytoplasm through the N-terminal and Kelch-like ECH associated protein 1(Kelch-like ECH-associated protein 1, Keap1), while the activity of Nrf2 is inhibited, intracellular II-phase enzymes and antioxidants are at basal expression levels, and the cells are in a stable state. When stimulated by Reactive Oxygen Species (ROS) or other electrophiles, Nrf2 uncouples with Keap1 and allows Nrf2 to migrate into the nucleus where it combines with small Maf proteins as a heterodimer, which in turn recognizes and binds Antioxidant Response Elements (AREs) and initiates transcription of downstream antioxidant protective genes and phase ii detoxification enzyme genes. We used endothelial cells to inoculate 2cm dishes and by immunofluorescence staining, we found that homoplantarin and its derivatives were able to activate the nuclear import of Nrf2 (see FIG. 2).
The invention also researches the reaction of the homoplantaginoside and the derivative thereof on HO-1 expression and oxidative stress of downstream protein Nrf 2. The Nrf2 can reduce cell damage caused by active oxygen and an electrophile by inducing and regulating the constitutive expression and the inducible expression of a series of antioxidant proteins, so that the cells are in a stable state and the redox dynamic balance of an organism is maintained. Thus becoming an important pathway for cells to resist oxidative stress. The expression of HO-1 protein of a downstream signal path of the derivative shows that the homoplantaginoside and the derivative thereof can activate Nrf2 to play a role in protecting endothelial cells by antioxidation. The expression of HO-1 protein in the endothelial cells induced by the oxidative low-density lipoprotein is detected by western blotting.
The results show that: compared with a control group ox-LDL group, the compound homoplantain derivative (S10.1 mu M, 1 mu M and 10 mu M) is added, the expression level of HO-1 is obviously higher than that of the control group ox-LDL, and meanwhile, compared with the control group ox-LDL group, the compound homoplantain (S30.1 mu M, 1 mu M and 10 mu M) is added, the expression level of HO-1 is obviously higher than that of the control group ox-LDL, which indicates that the homoplantain and the derivative thereof can activate the antioxidant protein HO-1 of a downstream signal path of Nrf 2. (see FIG. 3), inhibition of oxidative stress by promoting expression of protein HO-1 downstream of Nrf 2.
The invention also researches the influence of the homoplantaginoside and the derivative thereof on the expression of ROS, and Nrf2 belongs to a CNC regulatory protein family, is a transcription factor with an alkaline leucine zipper structure, widely exists in each organ of an organism, and is a main regulatory factor of cell redox reaction. Under normal physiological conditions, Nrf2 binds primarily to the inhibitor Keap1, in the hope that the inactive state exists in the cytoplasm and degrades rapidly under the ubiquitin proteasome pathway to maintain low transcriptional activity of Nrf2 under physiological conditions. When the cells ARE stimulated by active oxygen (ROS) or other nucleophilic agents, the Nrf2 and the Keap1 ARE uncoupled, the activated Nrf2 is transported into cell nucleus, combined with Maf protein to form heterodimer to be combined with ARE, the expression of target genes is activated, and the transcriptional activity of II-phase metabolic enzyme, antioxidant enzyme or drug transporter is regulated and controlled, so that the effect of resisting oxidative damage is exerted. The expression amount of ROS in endothelial cells is detected by a DCFH-DA probe method, which shows that the adaptoglycoside and the derivatives thereof can reduce the expression level of ROS by activating Nrf 2. (see FIG. 4)
The results show that: compared with a control group ox-LDL group, the compound homoplantain derivative (S10.1 mu M, 1 mu M and 10 mu M) is added, the expression level of ROS is obviously lower than that of the control group ox-LDL, and the compound homoplantain (S30.1 mu M, 1 mu M and 10 mu M) is added and the expression level of ROS is obviously higher than that of the control group ox-LDL compared with the control group ox-LDL group, so that the homoplantain and the derivative thereof can activate Nrf2 to reduce the expression of ROS in cells.
The invention also researches the influence of the knockout of the post-Nrf 2 homoeoside on the endothelial cell injury protection effect, and Nrf2 drives a free radical cation channel to be an important endogenous steady-state mechanism, thereby playing an important role in the aspects of inflammation resistance, apoptosis resistance, tumor resistance, atherosclerosis resistance, neuroprotection and the like. The nuclear factor kB (NF-kB) system is mainly involved in the information transfer of the processes of body defense reaction, tissue damage and stress, cell differentiation and apoptosis, and tumor growth inhibition. In most cell types, NF-kB binds to inhibitory proteins in the cytoplasm to form an inactive complex. When tumor necrosis factor, etc. acts on the corresponding receptor, the system can be activated by a second messenger, Cer, etc., while virus infection, lipopolysaccharide, active oxygen intermediates, phorbol esters, double-stranded RNA, RKC, PkA, etc. activated in the aforementioned message transmission pathway can directly activate NF-kB. The activated NF-kB enters the nucleus, contacts DNA, and initiates or inhibits transcription of the gene of interest. Following knockdown of Nrf2, homoplantagins do not protect endothelial cells under stimulation with ox-LDL. (see FIG. 5).
The experimental results are as follows: compared with a control group random interfering RNA group, the Sinrf2 interfering group added with the compound homoplantarin can not inhibit the nuclear entry of NF-kB, and the homoplantarin is used for protecting endothelial cells from being damaged by activating Nrf 2.
The invention also researches the influence of the homoplantaginoside and the derivative thereof on antioxidant protein HO-1 downstream of Nrf2 in the heart slice of the ApoE-/-mouse. A classical animal model ApoE-/-mouse for evaluating the drug effect of the anti-atherosclerosis drug is taken as a model. ApoE-/-mice were randomly grouped, including Control group (Control), high plantaginin derivative low dose group (S15mg/kg), high plantaginin derivative high dose group (S110 mg/kg) and high plantaginin low dose group (S35 mg/kg), high plantaginin high dose group (S310 mg/kg), Control group injected with the corresponding solvent amount of PBS. The specific injection mode is shown in figure 6.
Intraperitoneal injection is adopted, the injection is performed once every 24h for 6 weeks, and the weight change of the mice is tracked at any time during the injection. HO-1 was observed in aortic sections. Statistical analysis of the data summarized the expression of hispidoside and its derivatives on Nrf2 downstream signals in aortic plaques.
The results show that: there was no significant difference in body weight between groups during injection, indicating that the S1 and S3 injected doses were within the normal tolerance range of the mice, with no toxic effect on the mice. The HO-1 protein content of aortic plaque was significantly increased in the S1 high dose group (10mg/kg) compared to the S1 low dose group (5mg/kg) or the control group, and the HO-1 protein content was significantly increased in the S3 high dose group (10mg/kg) compared to the S3 low dose group (5mg/kg) or the control group (see FIG. 7).
The invention also investigated the effect of hispidoside and its derivatives on ROS expression in the aorta of ApoE-/-mice. Oxidative stress is a stress reaction generated in the body due to the disruption of the oxidative/antioxidant homeostasis in the body by the external environmental stimuli. Under normal conditions, the systems involved in the production and clearance of ROS in the body are in a state of dynamic equilibrium. Due to the stimulation of the external environment or the change of the organism, and the like, the ROS in the organism is increased or the scavenging capacity of the organism to the ROS is reduced, and the organism can generate oxidative stress. The Nrf2-Keap1-ARE signal pathway system plays an important role in the mechanism of resisting endogenous or exogenous oxidative stress of cells and is an important member of a cell defense system. A reduction in oxidative stress can also indicate activation of Nrf 2. (see FIG. 8)
The results show that: compared with the control group, the ROS expression level of the S1 low-dose group (5mg/kg), the S1 high-dose group (10mg/kg), the S3 low-dose group (5mg/kg) and the S3 high-dose group (10mg/kg) is obviously reduced, which indicates that the homoplantaginin and the derivatives thereof can effectively inhibit the generation of ROS in aorta and activate Nrf 2.
From the above experiments and the results thereof, the following conclusions can be drawn:
endothelial cell apoptosis, disorder of lipid metabolism, and inflammation are the main causes of disease development. The homoplantaginoside and the derivative thereof can effectively inhibit the apoptosis of endothelial cells caused by oxidative low-density lipoprotein, play a role in protecting the endothelial cells by oxidation resistance by activating Nrf2, can effectively inhibit the generation and development of arteriosclerosis in ApoE-/-mice, can stabilize plaques, can reduce the ROS expression in the plaques by activating Nrf2, and can improve the HO-1 expression. Therefore, the invention provides theoretical and experimental basis for researching the homoplantaginoside and the derivative thereof as the activator of the Nrf2 and protecting endothelial cell injury, and the homoplantaginoside and the derivative thereof can be used as a tool for effectively activating the Nrf2 and lay a foundation for inventing and researching related diseases.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
Example 1: preparation of homoplantaginoside and its derivatives
The specific operation is as follows:
1) pulverizing herba Salviae Plebeiae, adding 10 times of 95% ethanol solution, heating and reflux-extracting for 2 times (4 hr each time), mixing extractive solutions, and recovering ethanol under reduced pressure to obtain concentrated solution;
2) extraction and column separation: taking ethyl acetate/n-butanol-water as a two-phase solvent system, taking the upper layer as an acetic acid acetyl/n-butanol phase, taking the lower layer as a water phase, taking the upper layer of acetic acid acetyl/n-butanol phase, concentrating into an extract, diluting and filtering the concentrated solution with proper amount of water, loading onto a macroporous resin column, eluting with 30%, 50%, 85% and 95% ethanol water solution, monitoring by a thin layer, collecting the high plantaginin fraction, and concentrating the eluent;
3) removing impurities: adjusting the concentrated solution to acidity, filtering out precipitate, washing with acetone, and vacuum drying to obtain light yellow powder;
4) high performance liquid phase separation and purification: separating with 60% volume fraction of chromatographic methanol-water by high performance liquid chromatograph to obtain pure homocephem derivatives S1 and homocephem S3.
Example 2: higher plantaginide activates Nrf2
1. Experimental methods
Endothelial cells were seeded in 96-well culture dishes, and after the cell plating area reached 80%, a control group was set: culturing under normal conditions without serum; experimental groups: adding 0.1 mu M, 1 mu M and 10 mu M of adaptoside S3 and a derivative S1 thereof under the condition of serum removal, culturing for 24 hours, fixing for one hour by 10% of TCA, washing for 5 times by deionized water, naturally drying, adding 50 mu L of SRB dye into each hole, uniformly mixing for 10 minutes by a shaking table, washing for 5 times by the deionized water, naturally drying, adding 100 mu L of Trisbase into each hole, dissolving, and measuring the light absorption value by a microplate reader at 540 nm. Luci-hela cells (purchased and ATCC) were seeded in 96-well plates, and after the cell plating area reached 80%, controls were set: culturing under normal conditions without serum; experimental groups: adding 0.1. mu.M, 1. mu.M and 10. mu.M of hispidoside S3 and its derivative S1 under serum-free condition, culturing for 24h, removing supernatant, adding 100. mu.L of lysate, transferring to ep tube, centrifuging at 4 deg.C and 15000g for 5min, adding 80. mu.L of luciferase, and measuring data with microplate reader. Nrf2 (fluorescence value-value per well-value for blank lysis, then per group averaged/con, SRB-value per well-value for blank well, then per group averaged/con).
Endothelial cells were seeded in 2ml culture dishes, and after the cell plating area reached 80%, a control group was set: culturing under normal conditions without serum; experimental groups: the homoplantaginoside and its derivatives (S3 and S1) were added at concentrations of 0.1. mu.M, 1. mu.M and 10. mu.M, respectively, under serum-free conditions for 24h of culture followed by drug-addition treatment. 37 ℃ CO2After culturing for 6 hours in an incubator, removing the old culture solution, and gently washing the cells for 2 times by using 0.1M PBS; 0.1M PBS was discarded, and cells were fixed with 4% paraformaldehyde for 15min at room temperature; discarding 4% paraformaldehyde, washing with 0.1M PBS for 3 times, each for 5 min; discarding PBS, adding prepared 30% normal serum sealing solution, sealing at room temperature for 20-40 min; removing the blocking solution, adding Nrf2 primary antibody (diluted by 1:100 antibody diluent), and incubating overnight (more than 18 h) in a wet box at 4 ℃; returning to room temperature, discarding primary antibody, washing with 0.1M PBS for 3 times, each for 5 min; discarding PBS, adding fluorescent group labeled rabbit-488 secondary antibody (antibody diluent 1:200 dilution), and incubating at 37 deg.C for 50 min; returning to room temperature, discarding the secondary antibody, washing with 0.1M PBS buffer for 3 times, each time for 5 min; and observing by using a laser scanning confocal microscope, and taking a picture. Immunofluorescence detects the nuclear entry of Nrf2 in endothelial cells.
2. The experimental results are as follows:
as shown in fig. 1 and fig. 2, respectively, it can be seen from fig. 1 that homoplantaginin (S3) and its derivative (S1) can activate Nrf 2; as can be seen from fig. 2, homoplantaginoside (S3) and its derivatives (S1) were able to activate the internuclear of Nrf 2.
Example 3: the homoplantaginoside and its derivatives can activate downstream antioxidant protein HO-1 of Nrf2
1. Experimental methods
Endothelial cells were seeded in 6cm diameter culture dishes, normal group: adding Low Density Lipoprotein (LDL) to culture under normal condition without serum; setting a control group: adding oxidized low density lipoprotein (ox-LDL) to culture under normal condition of removing serum; experimental groups: the culture was performed by adding S1 and S3 at concentrations of 0.1. mu.M, 1. mu.M and 10. mu.M, respectively, under serum-free conditions. 37 ℃ CO2And (3) after culturing for 6 hours in an incubator, lysing cells, collecting cell lysate, centrifuging for 15min at 4 ℃ at 12000g, taking supernatant, and detecting the expression level of HO-1 in endothelial cells by western blot.
2. Results of the experiment
The experimental results are shown in fig. 3, and the results show that: compared with the control group ox-LDL group, the compound homoplantain derivative (S10.1. mu.M, 1. mu.M and 10. mu.M) is added and ox-LDL is added simultaneously, the expression level of HO-1 is obviously higher than that of the control group ox-LDL, meanwhile, compared with the control group ox-LDL group, the compound homoplantain (S30.1. mu.M, 1. mu.M and 10. mu.M) is added and ox-LDL is added simultaneously, the expression level of HO-1 is obviously higher than that of the control group ox-LDL, and the fact that the homoplantain and the derivative thereof can activate the antioxidant protein HO-1 of a downstream signal channel of Nrf2 is shown.
Example 4: the homoplantaginoside and the derivative thereof can inhibit ROS by activating Nrf2
1. Experimental methods
Inoculating endothelial cells into a culture dish with the diameter of 2cm, and adding LDL to culture under the normal condition of serum removal; setting a control group: adding ox-LDL to culture under the normal condition of serum removal; experimental groups: the culture was performed by adding S1 and S3 at concentrations of 0.1. mu.M, 1. mu.M and 10. mu.M, respectively, under serum-free conditions. 37 ℃ CO2After 6 hours incubation in the incubator, the cells were washed twice with PBS, added with DCFH dye, given 30min at 37 ℃ and washed three times with stock solution and photographed under an inverted fluorescence microscope.
2. Results of the experiment
The experimental results are shown in fig. 4, and the results show that: compared with a control group ox-LDL group, the compound homoplantain derivative (S10.1 mu M, 1 mu M and 10 mu M) is added, the expression level of ROS is obviously lower than that of the control group ox-LDL, and the compound homoplantain (S30.1 mu M, 1 mu M and 10 mu M) is added and the expression level of ROS is obviously higher than that of the control group ox-LDL compared with the control group ox-LDL group, so that the homoplantain and the derivative thereof can activate nrf2 to reduce the expression of ROS in cells.
Example 5: knockout of Nrf2 post-hispidoside does not inhibit the internuclear of NFKB
1. Experimental methods
The design of siRNA for Nrf2 was done by jema bio, shanghai, lipofectamine2000 transfection technique to transfect cells. Inoculating endothelial cells into a culture dish with the diameter of 2cm, performing serum-free culture for 6h after cell transfection, replacing a complete culture medium, continuously culturing for 24h, and adding drugs for treatment. Setting a control group: culturing under a normal condition of negative interference and serum removal; the negative interference experiment components are LDL group, ox-LDL group and S1 group of ox-LDL +10 mu M; experimental groups: nrf2 siRNA interference in serum-free culture divided into LDL group, ox-LDL + 10. mu.M S1 group, 37 ℃, CO2After culturing for 6 hours in an incubator, removing the old culture solution, and gently washing the cells for 2 times by using 0.1M PBS; 0.1M PBS was discarded, and cells were fixed with 4% paraformaldehyde for 15min at room temperature; discarding 4% paraformaldehyde, washing with 0.1M PBS for 3 times, each for 5 min; discarding PBS, adding prepared 30% normal serum sealing solution, sealing at room temperature for 20-40 min; removing blocking solution, adding P65 primary antibody (diluted by 1:100 antibody diluent), and incubating overnight (more than 18 h) in a wet box at 4 deg.C; returning to room temperature, discarding primary antibody, washing with 0.1M PBS for 3 times, each for 5 min; discarding PBS, adding fluorescent group labeled rabbit-488 secondary antibody (antibody diluent 1:200 dilution), and incubating at 37 deg.C for 50 min; returning to room temperature, discarding the secondary antibody, washing with 0.1M PBS buffer for 3 times, each time for 5 min; and observing by using a laser scanning confocal microscope, and taking a picture. Immunofluorescence detects the nuclear entry of NFKB in endothelial cells.
2. Results of the experiment
The experimental results are shown in fig. 5, and the results show that: compared with the negative interference group, the group with Nrf2 was interfered by adding the compound homoplantaginide derivative (S110. mu.M) and ox-LDL, and NFKB entered the nucleus, which indicates that the homoplantaginide and its derivatives can not inhibit endothelial cell apoptosis after interfering with Nrf2 by activating Nrf 2.
Example 6: effect of homoplantaginoside and derivatives thereof on antioxidant protein HO-1 downstream of Nrf2 in ApoE-/-mouse heart sections.
1. Experimental methods
ApoE-/-mice were randomly grouped, including Control group (Control), high plantaginin derivative low dose group (S15mg/kg), high plantaginin derivative high dose group (S110 mg/kg) and high plantaginin low dose group (S35 mg/kg), high plantaginin high dose group (S310 mg/kg), Control group injected with the corresponding solvent amount of PBS. The change in body weight of the mice was followed over the injection period and dissected 6 weeks after administration. And (3) placing the frozen section of the aortic root in cold acetone for fixing at 4 ℃ for 10min, carrying out immunofluorescence staining on the HO-1 of the blood vessel section of the ApoE-/-mouse, and observing the expression quantity of the HO-1 in the plaque. The staining was observed using a Leica DMI8 fluorescence microscope and photographed.
2. Results of the experiment
The results of the experiment are shown in fig. 7, and the results show that: the HO-1 protein content of the aortic plaque is obviously increased in the S1 high dose group (10mg/kg) compared with the S1 low dose group (5mg/kg) or the control group, and the HO-1 protein content is obviously increased in the S3 high dose group (10mg/kg) compared with the S3 low dose group (5mg/kg) or the control group.
Example 7: the homoplantaginoside and the derivative thereof can activate Nrf2 to reduce the expression of ROS in the aorta of ApoE-/-mice
1. Experimental methods
The aortic root is frozen and embedded and sliced for standby, after being washed by PBS, DCFH-DA is added for staining for 30 minutes, then the aortic root is washed by PBS for three times, and the aortic root is observed and photographed under an inverted microscope.
2. Results of the experiment
The experimental results are shown in fig. 8, and the results show that: compared with the control group, the ROS expression level of the S1 low-dose group (5mg/kg), the S1 high-dose group (10mg/kg), the S3 low-dose group (5mg/kg) and the S3 high-dose group (10mg/kg) is obviously reduced, which indicates that the homoplantaginin and the derivatives thereof can effectively inhibit the generation of ROS in aorta and activate Nrf 2.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (9)
1. Use of homoplantaginoside and/or a homoplantaginoside derivative in the preparation of an Nrf2 activator.
2. Use according to claim 1, wherein the homoplantagine derivative is dihydrohomoplantagine.
3. The use according to claim 1, wherein the homoplantagine, homoplantagine derivative is prepared by a process comprising:
crushing a common sage herb raw material, adding ethanol with the volume fraction of 95% for reflux extraction, and concentrating an extracting solution to obtain a concentrated solution; extracting the concentrated solution with ethyl acetate/n-butanol-water as two-phase solvent system, and concentrating the upper ethyl acetate/n-butanol phase to obtain extract; gradient eluting the extract with macroporous resin, monitoring with thin layer, collecting high plantaginide fraction, concentrating, adjusting the concentrated solution to acidity, filtering to obtain precipitate, washing, and drying to obtain light yellow powder; separating with high performance liquid chromatograph to obtain homocepharaside and homocepharaside derivatives.
4. The use as claimed in claim 3, wherein the 95% by volume of ethanol is added in an amount of 8-12 times the weight of the raw material of litchi chinensis grass.
5. Use according to claim 3, characterized in that the gradient elution is carried out sequentially with 30%, 50%, 85% and 95% aqueous ethanol by volume fraction.
6. Application of homoplantagine and/or homoplantagine derivatives as Nrf-2 activators in preparation of anti-oxidative stress drugs.
7. Application of homoplantaginoside and/or homoplantaginoside derivatives as Nrf-2 activators in preparation of medicines for inhibiting apoptosis of endothelial cells.
8. Use according to claim 7, wherein apoptosis is induced by oxidative low density lipoprotein.
9. An Nrf2 activator characterized in that the Nrf2 activator contains homoplantaginin and/or a homoplantaginin derivative as an active ingredient.
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| CN109810959A (en) * | 2019-01-15 | 2019-05-28 | 中国人民解放军第二军医大学 | It is a kind of in conjunction with KEAP1 and to adjust the polypeptide of NRF2 protein stability |
| CN109810959B (en) * | 2019-01-15 | 2022-03-15 | 中国人民解放军第二军医大学 | Protein polypeptide combined with KEAP1 and used for regulating stability of NRF2 protein |
| TWI813440B (en) * | 2022-09-13 | 2023-08-21 | 國立臺灣師範大學 | Use of hispidulin derivatives for preparing medicament to treat or defer nmda receptor overactivation |
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