CN114031669B - Anti-oxidation active peptide of mytilus coruscus as well as preparation and application thereof - Google Patents
Anti-oxidation active peptide of mytilus coruscus as well as preparation and application thereof Download PDFInfo
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Abstract
The invention provides a thick-shell mussel antioxidant peptide Gln-Glu-Thr-Tyr and Tyr-Glu-Leu-His-Asp for activating Keap1/Nrf2 signal channels, and the polypeptide Gln-Glu-Thr-Tyr and Tyr-Glu-Leu-His-Asp can reduce the generation of intracellular Reactive Oxygen Species (ROS) and form a protective effect. The anti-oxidation agent plays an anti-oxidation role and a protective role on vascular endothelial cells by activating a Keap1/Nrf2 signal pathway, and can provide candidate drugs for preventing and treating cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, atherosclerosis, heart failure and the like.
Description
Technical Field
The invention relates to the technical field of polypeptides, in particular to a mytilus coruscus antioxidant peptide for activating a Keap1/Nrf2 signal channel.
Background
The living body can generate free radicals autonomously, and when the free radicals are generated excessively, the free radicals are harmful to the human body when the free radicals exceed a certain amount. Under normal physiological conditions, living body antioxidant defense systems can use enzymatic and non-enzymatic antioxidants to remove the attack and impact of harmful molecules on the human body. However, once an imbalance between free radicals and endogenous antioxidant defense systems occurs, oxidative stress in cells is induced, causing damage to cells, including deoxyribonucleic acid (DNA), proteins, membrane lipids, and the like. Oxidative damage can cause many chronic diseases such as human diabetes, arthritis, heart disease, alzheimer's disease and cancer. The synthetic antioxidants (tertiary butyl hydroquinone, butyl hydroxy anisole, etc.) can effectively control oxidation. However, it has the disadvantages of high cost and potential toxicity. In addition, we can also reduce the risk of chronic diseases associated with oxidative stress by dietary intake of antioxidants. Therefore, as the demands of high nutrition and functional foods are increasing, people are looking more at natural animals and plants, and the enzymolysis of active antioxidant peptides from marine animals and plants becomes a focus. .
Disclosure of Invention
The inventor takes thick-shell mussels with abundant resources as raw materials to prepare antioxidant active peptides H5 and H1, plays an antioxidant role and protects vascular endothelial cells by activating Keap1/Nrf2 signal paths, and can provide candidate medicaments for preventing and treating cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, atherosclerosis, heart failure and the like.
The invention provides two anti-oxidation active peptides H5 and H1 of mytilus coruscus for activating Keap1/Nrf2 signal paths, wherein the amino acid series of H5 is Gln-Glu-Thr-Tyr, and the molecular weight of the anti-oxidation active peptides is 539.54Da as determined by ESI-MS;
the amino acid sequence of the anti-oxidation active peptide H1 of the mytilus coruscus is Tyr-Glu-Leu-His-Asp, and the molecular weight of the anti-oxidation active peptide H1 is 675.70Da as determined by ESI-MS.
The preparation of the anti-oxidation active peptide of the mytilus coruscus comprises the following steps:
1) Pretreatment of thick-shell mussel samples:
thawing frozen mytilus coruscus with running water at room temperature, oven drying surface water at low temperature, dissecting, mincing, soaking in ethyl acetate for 48 hr, and continuously stirring during degreasing; filtering the degreased sample in vacuum to remove ethyl acetate (rotary evaporation recovery), drying, and pulverizing;
2) Enzymolysis:
weighing a proper amount of pretreated mytilus coruscus body wall powder, adding deionized water according to a feed-liquid ratio of 1:30, adding neutral protease for enzymolysis for 4 hours, and regulating pH to neutral protease (45 ℃ and pH of 7.0) by using 0.5mol/L NaOH and 0.1mol/L HCL solution for enzymolysis;
3) And inactivating the enzymolysis liquid in a water bath at 100 ℃ by adopting a heating enzyme inactivation method. After enzyme deactivation is finished, cooling to room temperature, centrifuging for 10min at 8000r/min, collecting supernatant, and freeze-drying to prepare for detecting ACE inhibition activity of enzymolysis liquid;
4) And (3) separating and purifying active peptides in the enzymolysis peptide: the method comprises the steps of taking the clearance rate of free radicals such as DPPH and the like as evaluation indexes, firstly adopting an ultrafiltration technology to carry out fractionation on polypeptides, then adopting QFF ion exchange chromatography, sephadex G-15 gel filtration chromatography and high performance liquid chromatography to further separate the polypeptides, screening out the separation components with the highest antioxidation capability, and carrying out separation and purification to obtain the mytilus coruscus antioxidation peptide H5 Gln-Glu-Thr-Tyr, wherein the peak time is 6.484min, and the anti-oxidation peptide H1 Tyr-Glu-Leu-His-Asp, and the peak time is 5.970min.
Preferably, the addition amount of the ethyl acetate in the step 1) is 5 times of the weight of the dissected and minced thick-shell mussel body.
Preferably, in the step 2), the ratio of the thick-shell mussel wall powder to deionized water is 1:30.
Preferably, the enzymolysis temperature in the step 2) is 45 ℃, the enzymolysis time is 4 hours, and the added enzyme amount is 4%.
The antioxidant active peptide H5 and H1 provided by the invention are as follows: the cleaning agent has better cleaning activity on free radicals such as DPPH, HO, O2, ABTS, and the like, and can reduce the generation of Reactive Oxygen Species (ROS) in cells to form a protective effect; the mechanism is that the level of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) and total antioxidant capacity (T-AOC) is improved by promoting the release of Nitric Oxide (NO), and the level of Malondialdehyde (MDA) and Lactate Dehydrogenase (LDH) is reduced. Therefore, H5 and H1 play a role in protecting HUVEC cell oxidative damage induced by hydrogen peroxide; h5 and H1 exert antioxidant effect and protecting vascular endothelial cells by activating Keap1/Nrf2 signal path, and can provide candidate medicine for preventing and treating cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, atherosclerosis, heart failure, etc.
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FIG. 1 is a comparison of DPPH radical scavenging capacity of an enzymatic hydrolysate for each cut-off section of ultrafiltration in accordance with an embodiment of the present invention.
FIG. 2 is a graph showing comparison of the OH radical scavenging capacity of the respective retentate fractions for ultrafiltration in accordance with an embodiment of the present invention.
FIG. 3 is a comparison of the ability of each cut-off segment to scavenge ABTS free radicals from an enzymatic hydrolysate for ultrafiltration in accordance with an embodiment of the present invention.
FIG. 4 is a QFF anion exchange step elution profile of an example of the present invention.
FIG. 5 is a comparison of DPPH radical scavenging ability of components after desalting in accordance with an embodiment of the present invention.
FIG. 6 is a comparison of HO radical scavenging ability of the components after desalting according to an embodiment of the invention.
FIG. 7 is a comparison of the free radical scavenging ability of the components of the present invention after desalting.
FIG. 8 shows the elution profile of Sephadex G-15 gel column chromatography according to the embodiment of the invention.
FIG. 9 is a comparison of DPPH radical scavenging ability of various components of the examples of the present invention.
FIG. 10 is a comparison of HO radical scavenging ability of components of an embodiment of the invention.
FIG. 11 is a comparison of the free radical scavenging ability of the components of the examples of the present invention against ABTS.
FIG. 12 shows the protective effect of different concentrations of H1 and H5 on oxidative damage of HUVEC cells according to the examples of the present invention.
Wherein # P <0.01vs Control group; * P <0.01, P <0.05vs model group.
FIG. 13 shows the effect of different concentrations of H1 and H5 on SOD levels in HUVEC cells according to the examples of the present invention.
Wherein # P <0.01vs Control group; * P <0.01, P <0.05vs model group.
FIG. 14 shows the effect of different concentrations of H1 and H5 on MDA levels in HUVEC cells according to the examples of the present invention.
Wherein # P <0.01vs Control group; * P <0.01, P <0.05vs model group.
FIG. 15 shows the effect of different concentrations of H1 and H5 on GSH-PX levels in HUVEC cells according to the examples of the present invention.
Wherein # P <0.01vs Control group; * P <0.01, P <0.05vs model group.
FIG. 16 shows the effect of different concentrations of H1 and H5 on NO levels in HUVEC cells according to the examples of the present invention.
Wherein # P <0.01vs Control group; * P <0.01, P <0.05vs model group.
FIG. 17 shows the effect of different concentrations of H1 and H5 on the levels of iNOS (A) and eNOS (B) in HUVEC cells according to the examples of the present invention.
Wherein # P <0.01vs Control group; * P <0.01, P <0.05vs model group.
FIG. 18 shows the effect of different concentrations of H1 and H5 on CAT levels in HUVEC cells according to the examples of the present invention.
Wherein # P <0.01vs Control group; * P <0.01, P <0.05vs model group.
FIG. 19 shows the effect of different concentrations of H1 and H5 on LDH levels in HUVEC cells according to examples of the present invention.
Wherein # P <0.01vs Control group; * P <0.01, P <0.05vs model group.
FIG. 20 shows the effect of different concentrations of H1 and H5 on T-AOC levels in HUVEC cells according to examples of the present invention.
Wherein # P <0.01vs Control group; * P <0.01, P <0.05vs model group.
FIG. 21 is a chart showing the measurement of intracellular ROS content by DCFH-DA staining method according to an embodiment of the present invention.
Wherein, #P<0.01vs Control group; * P<0.01，*P<0.05vs model group. A is blank group; b is H 2 O 2 A model group; c is NAC+H 2 O 2 A group; d is 50 mu M H1+H 2 O 2 A group; e is 100 mu M H1+H 2 O 2 A group; f is 200 mu M H1+H 2 O 2 A group; g is 50 mu M H5+H 2 O 2 A group; h is 100 mu M H5+H 2 O 2 A group; i is 200 mu M H5+H 2 O 2 A group; j is intracellular ROS content.
FIG. 22 shows the protein protection of the polypeptides of the present invention.
Wherein, #P<0.01vs Control group; * P<0.01，*P<0.05vs model group; a: electropherograms of polypeptide protected BSA lesions; 1: BSA;2: BSA+H 2 O 2 ；3：BSA+NAC+H 2 O 2 ；4：BSA+H1(2.5mM)+H 2 O 2 ；5：BSA+H1(5mM)+H 2 O 2 ；6：BSA+H1(10mM)+H 2 O 2 ；7：BSA+H5(2.5mM)+H 2 O 2 ；8：BSA+H5(5mM)+H 2 O 2 ；9：BSA+H5(10mM)+H 2 O 2 The method comprises the steps of carrying out a first treatment on the surface of the B: analysis of relative intensities of the individual bands.
FIG. 23 shows the effect of polypeptides H1 and H5 of the present invention on the Keap1/Nrf2 pathway-associated proteins of HUVEC cells. Wherein # P <0.01vs Control group; * P <0.01, P <0.05vs model group; a: western blot analysis of Nrf2, HO-1, NQO1, GCLM and Keap 1; b: quantitative analysis of Nrf 2; c: quantitative analysis of HO-1; d: quantitative analysis of NQO 1; e: quantitative analysis of GCLM; f: quantitative analysis of Keap 1.
Detailed Description
The following examples serve to further illustrate the invention, but they do not constitute a limitation or limitation of the scope of the invention.
Examples
1) Pretreatment of thick-shell mussel samples:
thawing frozen mytilus coruscus with running water at room temperature, oven drying surface water at low temperature, dissecting, mincing, soaking in 5 times of ethyl acetate for 48 hr, and continuously stirring during degreasing; filtering the degreased sample in vacuum to remove ethyl acetate (rotary evaporation recovery), drying, and pulverizing;
2) Enzymolysis:
weighing a proper amount of pretreated mytilus coruscus body wall powder, adding deionized water according to a feed-liquid ratio of 1:30, adding neutral protease for enzymolysis for 4 hours, and regulating pH to neutral protease (45 ℃ and pH of 7.0) by using 0.5mol/L NaOH and 0.1mol/L HCL solution for enzymolysis;
3) And inactivating the enzymolysis liquid in a water bath at 100 ℃ by adopting a heating enzyme inactivation method. After enzyme deactivation is finished, cooling to room temperature, centrifuging for 10min at 8000r/min, collecting supernatant, and freeze-drying to prepare for detecting ACE inhibition activity of enzymolysis liquid;
4) And (3) separating and purifying active peptides in the enzymolysis peptide: the method comprises the steps of taking the clearance rate of free radicals such as DPPH and the like as evaluation indexes, firstly adopting an ultrafiltration technology to carry out fractionation on polypeptides, wherein the ultrafiltration steps are as follows: ultrafiltering the mytilus coruscus enzymolysis solution with ultrafiltration membrane with molecular weight cut-off of 1kDa, 3kDa, 5kDa, and 10kDa, M-I (MW < 1 kDa), M-II (MW < 3 kDa), M-III (MW < 5 kDa), M-IV (MW < 10 kDa), and M-V (MW > 10 kDa), concentrating, and lyophilizing. The 5-component freeze-dried sample and the non-ultrafiltered component are respectively prepared into 5mg/mL sample solution, and the free radical scavenging capacity is compared, and the result is shown in figure 1; the HO free radical scavenging ability of the zymolytes of different components is different, as shown in figure 2; the ABTS free radical scavenging ability of the zymolytes of different components is different, and the result is shown in figure 3; the results show that: the M-I component (MW < 1 kDa) has the best antioxidant activity, so that the M-I component is selected for subsequent separation and purification.
QFF ion exchange chromatography: the M-I component is subjected to QFF ion exchange chromatography for further separation and purification. The M-I component was prepared as a 50mg/mL sample solution, and after loading, the sample was eluted with a Tris-HCl gradient, and the results are shown in FIG. 4. As can be seen from FIG. 4, the samples were eluted stepwise with 5 peaks designated M-I-1, M-I-2, M-I-3, M-I-4, M-I-5, respectively, in Tris-HCl buffer, tris-HCl (containing 0.05M NaCl), tris-HCl (containing 0.1M NaCl) buffer, tris-HCl (containing 0.25M NaCl) buffer, tris-HCl (containing 0.5M NaCl) buffer. The obtained unimodal component is subjected to rotary evaporation concentration, and is subjected to rotary evaporation freeze-drying after dialysis for desalting. Samples were prepared as 1mg/mL of sample solution, and DPPH free radical, HO free radical and ABTS free radical scavenging activities of 5 components were compared to conduct the next experiment.
Comparing the DPPH free radical scavenging ability of the polypeptides of each component after dialysis and desalination, and the results are shown in figure 5; comparing the scavenging ability of HO free radical, the result is shown in figure 6; the ability of ABTS radicals to scavenge was compared and the results are shown in fig. 7. The results show that: the M-I-5 component has the best antioxidant activity, so that the M-I-5 component is selected for the next experiment.
Sephadex G-15 gel filtration chromatography: subjecting the M-I-5 component obtained by QFF chromatographic column to Sephadex G-15 gel filtration chromatography, detecting peak at 214nm wavelength to obtain components M-I-5-1 and M-I-5-2, and measuring activity of the separated components as shown in figure 8.
After the two components are freeze-dried, preparing a sample solution with the concentration of 1mg/mL, and comparing the DPPH free radical scavenging capacity of the sample solution, wherein the result is shown in figure 9; comparing the scavenging ability of HO free radical, the result is shown in figure 10; the ability of ABTS radicals to scavenge was compared and the results are shown in fig. 11. The results show that: the M-I-5-1 component has the best antioxidant activity, so that the M-I-5-1 component is selected for the next experiment.
Purifying and sequencing by high performance liquid chromatography: the component with the best antioxidant activity after passing through the Sephadex G-15 chromatographic column in the last step is further separated and purified by HPLC, and the obtained component is entrusted to related scientific research detection institutions for amino acid sequencing. The component M-I-5-1 is subjected to high performance liquid phase analysis and sequencing to obtain 2 polypeptides, and the peak time, the amino acid sequence and the molecular weight of the polypeptides are shown in table 1.
TABLE 1
Radical scavenging Activity assay: by DPPH free radical, OH free radical, ABTS free radical, O 2 - Free radical scavenging Rate evaluation of pure peptide free radical scavenging Activity of Mytilus coruscus and determination of EC 50 Values, results are shown in table 2.
TABLE 2
Four radical scavenging Activity assays, EC, were performed on 2 polypeptides 50 The lower the value, the greater the antioxidant capacity of the polypeptide.
Polypeptide pairs H with different concentrations 2 O 2 Protection of induced injury HUVEC cells: 500 mu M H is selected for use 2 O 2 HUVEC cell damage was induced and model groups were established. Acetylcysteine (NAC, final concentration 1 mM) was used as a positive control sample group. The sample group was pre-added with polypeptides (H1, H5) at concentrations of (50. Mu.M, 100. Mu.M, 200. Mu.M) to act on HUVEC cells, followed by H 2 O 2 Cell damage is induced. The cell viability of each experimental group was determined by MTT assay, whereby the protective effect of different concentrations of the polypeptide on Human Umbilical Vein Endothelial Cell (HUVEC) injury was determined. The results are shown in FIG. 12, in which the cell viability of the model group was significantly different from that of the blank group (P<0.01 48.58.+ -. 1.16%, indicating that the constructed oxidative damage model is available. Compared with the model group, the cell viability of the polypeptide sample group (H1, H5) increases with the concentration, and the cell viability increases with the concentration of H 2 O 2 The protective effect of injured HUVEC cells gradually increased. Wherein H1, H5 are fine to HUVEC at a concentration of 50. Mu.M, 100. Mu.MThe protective effect of cell injury is less obvious than that of the model group (P<0.05). But they can significantly increase the H-channel at 200. Mu.M concentration 2 O 2 Damaged HUVEC cell survival (P<0.01 Cell viability was increased from 48.58.+ -. 1.16% to 60.34.+ -. 1.87% and 58.47.+ -. 1.37%, respectively. From a general trend, the protective effect of polypeptides H1, H5 on oxidative damage HUVEC cells is dose-dependent.
Superoxide dismutase (SOD) content determination: 500 mu M H is selected for use 2 O 2 HUVEC cell damage was induced and model groups were established. Acetylcysteine (NAC, final concentration 1 mM) was used as a positive control sample group. The sample group was pre-added with polypeptides (H1, H5) at concentrations of (50. Mu.M, 100. Mu.M, 200. Mu.M) to act on HUVEC cells, followed by H 2 O 2 Cell damage is induced. The protective effect of polypeptides at different concentrations on Human Umbilical Vein Endothelial Cell (HUVEC) injury was evaluated by SOD levels in the cells. As shown in FIG. 13, the SOD content of the model group was significantly reduced as compared with that of the blank group (P<0.01 Indicating that the oxidative damage model was successfully established. Compared with the model group, the SOD content in the cells of the polypeptide sample group (H1, H5) increases with the increase of the polypeptide concentration, which indicates that the SOD content is relative to H 2 O 2 The protective effect of injured HUVEC cells gradually increased. Wherein H1 and H5 slightly increased the SOD content in HUVEC cells at 50 μm concentration compared with model group, but no significant difference. The polypeptides H1, H5 significantly increased SOD content (P) in oxidatively damaged cells at a concentration of 100. Mu.M, 200. Mu.M<0.01). From a general trend, polypeptides H1, H5 can increase SOD content in oxidatively damaged HUVEC cells and are dose dependent.
Malondialdehyde (MDA) content determination: 500 mu M H is selected for use 2 O 2 HUVEC cell damage was induced and model groups were established. Acetylcysteine (NAC, final concentration 1 mM) was used as a positive control sample group. The sample group was pre-added with polypeptides (H1, H5) at concentrations of (50. Mu.M, 100. Mu.M, 200. Mu.M) to act on HUVEC cells, followed by H 2 O 2 Cell damage is induced. The protection of Human Umbilical Vein Endothelial Cells (HUVEC) against damage was assessed by varying concentrations of the polypeptide with respect to MDA content in the cells. The results are shown in FIG. 14, where the MDA content of the model group was significantly increased as compared with that of the blank group (P<0.01) Indicating that the oxidative damage model is successfully established. Compared with the model group, MDA content in the cells of the polypeptide sample group (H1, H5) is reduced along with the increase of the concentration of the polypeptide, which indicates that the MDA content is reduced on H 2 O 2 The protective effect of injured HUVEC cells gradually increased. Wherein H1 and H5 at 50 mu M concentration slightly reduced MDA content in HUVEC cells compared with model group, but no significant difference. Polypeptides H1, H5 significantly reduced MDA content in oxidatively damaged cells at a concentration of 100 μΜ,200 μΜ (P<0.01). From a general trend, polypeptides H1, H5 can reduce MDA levels in oxidative damaged HUVEC cells and are dose dependent.
Glutathione peroxidase (GSH-PX) content determination: 500 mu M H is selected for use 2 O 2 HUVEC cell damage was induced and model groups were established. Acetylcysteine (NAC, final concentration 1 mM) was used as a positive control sample group. The sample group was pre-added with polypeptides (H1, H5) at concentrations of (50. Mu.M, 100. Mu.M, 200. Mu.M) to act on HUVEC cells, followed by H 2 O 2 Cell damage is induced. The protection effect of different concentrations of polypeptide on Human Umbilical Vein Endothelial Cell (HUVEC) injury was evaluated by GSH-PX content in the cells. The results are shown in FIG. 15, in which the GSH-PX content of the model group was significantly reduced as compared with that of the blank group (P<0.01 Indicating that the oxidative damage model was successfully established. Compared with the model group, the GSH-PX content in the cells of the polypeptide sample group (H1, H5) increases with the increase of the polypeptide concentration, which indicates that the GSH-PX content is relative to H 2 O 2 The protective effect of injured HUVEC cells gradually increased. Wherein H1, H5 at 50. Mu.M concentration resulted in slightly increased GSH-PX content in HUVEC cells compared to model group, but no significant difference. Polypeptides H1, H5 significantly increase GSH-PX content (P) in oxidatively damaged cells at a concentration of 100. Mu.M, 200. Mu.M<0.01). From a general trend, polypeptides H1, H5 can increase GSH-PX content in oxidatively damaged HUVEC cells, and are dose dependent.
Nitric Oxide (NO) content determination: 500 mu M H is selected for use 2 O 2 HUVEC cell damage was induced and model groups were established. Acetylcysteine (NAC, final concentration 1 mM) was used as a positive control sample group. The sample group was pre-added with polypeptides (H1, H5) at concentrations of (50. Mu.M, 100. Mu.M, 200. Mu.M) to act on HUVEC cells, followed by H 2 O 2 Cell damage is induced. The protective effect of different concentrations of the polypeptide on Human Umbilical Vein Endothelial Cell (HUVEC) injury was evaluated by the NO content in the cells. The results are shown in FIG. 16, where the model group has a significant decrease in NO content over the blank group (P<0.01 Indicating that the oxidative damage model was successfully established. Compared with the model group, the NO content in the cells of the polypeptide sample group (H1, H5) increases with the increase of the concentration of the polypeptide, which indicates that the polypeptide sample group has NO activity on H 2 O 2 The protective effect of injured HUVEC cells gradually increased. Wherein H1 and H5 slightly increased the NO content of HUVEC cells at 50. Mu.M concentration, but without significant difference. The polypeptides H1, H5 significantly increased the NO content (P) in oxidatively damaged cells at a concentration of 100. Mu.M, 200. Mu.M<0.01). From a general trend, polypeptides H1, H5 increased NO content in oxidatively damaged HUVEC cells and were dose dependent.
Nitric Oxide Synthase (NOS) content assay: 500 mu M H is selected for use 2 O 2 HUVEC cell damage was induced and model groups were established. Acetylcysteine (NAC, final concentration 1 mM) was used as a positive control sample group. The sample group was pre-added with polypeptides (H1, H5) at concentrations of (50. Mu.M, 100. Mu.M, 200. Mu.M) to act on HUVEC cells, followed by H 2 O 2 Cell damage is induced. The protection of Human Umbilical Vein Endothelial Cells (HUVEC) against injury was assessed by varying concentrations of the polypeptide in terms of iNOS and eNOS levels in the cells. As can be seen from FIG. 17 (A), the model group had a significant increase in iNOS content over the blank group (P<0.01 Indicating that the oxidative damage model was successfully established. Compared with the model group, the iNOS content in the cells of the polypeptide sample group (H1, H5) was decreased with the increase of the polypeptide concentration, which indicates that the cell was specific for H 2 O 2 The protective effect of injured HUVEC cells gradually increased. Wherein H1, H5 at 50. Mu.M concentration resulted in slightly lower iNOS content in HUVEC cells than in the model group, but without significant differences. The polypeptides H1, H5 significantly reduced iNOS content in oxidatively damaged cells at a concentration of 100 μm,200 μm (P<0.01). From a general trend, polypeptides H1, H5 can reduce iNOS content in oxidatively damaged HUVEC cells and are dose dependent. As can be seen from FIG. 17 (B), the model group had a significant decrease in eNOS content as compared with the blank group (P<0.01 Indicating that the oxidative damage model was successfully established. And mouldCompared with the type group, the eNOS content in the cells of the polypeptide sample group (H1, H5) increases with the increase of the concentration of the polypeptide, which indicates that the eNOS content increases with the increase of the concentration of the polypeptide 2 O 2 The protective effect of injured HUVEC cells gradually increased. Wherein H1, H5 at a concentration of 50. Mu.M resulted in a slight increase in eNOS content in HUVEC cells over the model group, but without significant differences. The polypeptides H1, H5 significantly increased eNOS content in oxidatively damaged cells at a concentration of 100 μm,200 μm (P<0.01). From a general trend, polypeptides H1, H5 increased eNOS content in oxidatively damaged HUVEC cells and were dose dependent.
Catalase (CAT) assay: 500 mu M H H is selected for use 2 O 2 HUVEC cell damage was induced and model groups were established. Acetylcysteine (NAC, final concentration 1 mM) was used as a positive control sample group. The sample group was pre-added with polypeptides (H1, H5) at concentrations of (50. Mu.M, 100. Mu.M, 200. Mu.M) to act on HUVEC cells, followed by H 2 O 2 Cell damage is induced. The protective effect of different concentrations of the polypeptide on Human Umbilical Vein Endothelial Cell (HUVEC) injury was evaluated by CAT content in cells. The results are shown in FIG. 18, where the CAT content of the model group was significantly reduced as compared with that of the blank group (P<0.01 Indicating that the oxidative damage model was successfully established. Compared with the model group, the CAT content in the cells of the polypeptide sample group (H1, H5) increases with the increase of the concentration of the polypeptide, which indicates that the CAT content is higher than that of the cell of the polypeptide sample group for H 2 O 2 The protective effect of injured HUVEC cells gradually increased. Wherein H1 and H5 increased CAT content in HUVEC cells slightly at 50. Mu.M concentration, but were not significantly different from that in the model group. Polypeptides H1, H5 significantly increase CAT content (P) in oxidatively damaged cells at a concentration of 100. Mu.M, 200. Mu.M<0.01). From a general trend, polypeptides H1, H5 increased CAT content in oxidatively damaged HUVEC cells and were dose dependent.
Lactate Dehydrogenase (LDH) assay: 500 mu M H is selected for use 2 O 2 HUVEC cell damage was induced and model groups were established. Acetylcysteine (NAC, final concentration 1 mM) was used as a positive control sample group. The sample group was pre-added with polypeptides (H1, H5) at concentrations of (50. Mu.M, 100. Mu.M, 200. Mu.M) to act on HUVEC cells, followed by H 2 O 2 Cell damage is induced. Assessment of different concentration of polypeptide by LDH content in cellsProtection against Human Umbilical Vein Endothelial Cell (HUVEC) injury. The results are shown in FIG. 19, in which the LDH content of the model group was significantly increased as compared with that of the blank group (P<0.01 Indicating that the oxidative damage model was successfully established. Compared with the model group, the LDH content in the cells of the polypeptide sample group (H1, H5) is reduced along with the increase of the concentration of the polypeptide, which indicates that the LDH content is reduced on H 2 O 2 The protective effect of injured HUVEC cells gradually increased. Wherein H1 and H5 at 50 mu M concentration slightly reduced the LDH content in HUVEC cells compared with model group, but no significant difference. Polypeptides H1, H5 significantly reduced LDH content in oxidatively damaged cells at a concentration of 100 μm,200 μm (P<0.01). From a general trend, polypeptides H1, H5 can reduce LDH content in oxidatively damaged HUVEC cells and are dose dependent.
Total antioxidant capacity (T-AOC) determination: 500 mu M H is selected for use 2 O 2 HUVEC cell damage was induced and model groups were established. Acetylcysteine (NAC, final concentration 1 mM) was used as a positive control sample group. The sample group was pre-added with polypeptides (H1, H5) at concentrations of (50. Mu.M, 100. Mu.M, 200. Mu.M) to act on HUVEC cells, followed by H 2 O 2 Cell damage is induced. The protective effect of different concentrations of the polypeptide on Human Umbilical Vein Endothelial Cell (HUVEC) injury was evaluated at the level of T-AOC in the cells. The results are shown in FIG. 20, where the model group had a significant decrease in T-AOC levels compared to the blank group (P<0.01 Indicating that the oxidative damage model was successfully established. The increase in T-AOC levels in the cells of the polypeptide sample group (H1, H5) with increasing polypeptide concentration compared to the model group, indicates that it was specific for H 2 O 2 The protective effect of injured HUVEC cells gradually increased. Wherein H1, H5 at a concentration of 50. Mu.M resulted in a slight increase in T-AOC levels in HUVEC cells over the model group, but without significant differences. Polypeptides H1, H5 significantly increased T-AOC levels in oxidatively damaged cells at a concentration of 100. Mu.M, 200. Mu.M (P<0.01). From a general trend, polypeptides H1, H5 can increase T-AOC levels in oxidatively damaged HUVEC cells and are dose dependent.
DCFH-DA staining: at 500 mu M H 2 O 2 HUVEC cell injury is induced, and a HUVEC cell oxidative injury model is established. Acetylcysteine (NAC, final concentration 1 mM) was used as positive control sample group. The HUVEC cells were acted on by 50. Mu.M, 100. Mu.M, 200. Mu.M polypeptides (H1, H5) followed by H 2 O 2 Cell damage is induced. Whether the polypeptide has the effect of reducing active oxygen in oxidative damage cells or not is observed through a fluorescence microscope. The results are shown in FIG. 21. After DCFH-DA staining, the intracellular ROS content of HUVEC was significantly increased compared with the normal group in the model group (P<0.01 Indicating that the constructed oxidative damage model is available. The polypeptide sample group (H1, H5) increased with the concentration of the polypeptide sample compared to the model group such that H 2 O 2 The ROS content in injured HUVEC cells gradually decreased. From the results, the mytilus coruscus polypeptides H1, H5 were able to reduce the increase of intracellular ROS caused by oxidative damage, and were concentration-dependent.
Protein protection: in order to study the protective effect of the polypeptide on oxidative damage of Bovine Serum Albumin (BSA), the Bovine Serum Albumin (BSA) is damaged by hydrogen peroxide and hydroxyl radicals generated by iron (II) ions (Fenton reaction). The polypeptide was added to the reaction solution and incubated at 37℃for 60min. After the incubation, the extent of oxidative damage to BSA was measured by Coomassie Brilliant blue R-250 staining. FIG. 22 (A) is a SDS-PAGE graph of Bovine Serum Albumin (BSA) treated under different conditions, from which it can be seen that the bands of the model group damaged by Fenton reaction were significantly shallower than the normal group, the bands were deeper than the model group after protection with 2.5mM,5mM,10mM polypeptides (H1, H5), and shallower than the normal group, and the more significant changes with increasing concentration, indicating that the model was more successful. It can also be seen from the relative intensity analysis of the bands in fig. 22 (B) that the model set has significantly reduced band intensities (P < 0.01) compared to the normal set, indicating that the model set is available. The band intensities of the polypeptide sample groups (H1, H5) increased gradually with increasing concentration of the polypeptide samples compared to the model group. From the results, the mytilus coruscus polypeptides H1, H5 are capable of protecting against the action of hydroxyl radical induced damage to Bovine Serum Albumin (BSA) and are concentration dependent.
Western blot detection Nrf2 and related protein expression:
(1) Polyacrylamide gel electrophoresis (SDS-PAGE): the glass plate is cleaned, dried and aligned and then clamped. Preparing 10% of separation gel: ultrapure water, 30% acrylamide, 4 XSDS-PAGE gel buffer, 10% ammonium persulfate and TEMED were added in this order according to the formulation. And (3) immediately pouring the glue into the glass plate interlayer along the glass plate wall after uniformly mixing, adding a layer of ultrapure water liquid seal into the glue, separating the glue, solidifying and removing the ultrapure water. Preparing 5% concentrated glue: ultrapure water, 30% acrylamide, 4 XSDS-PAGE concentrated gel buffer, 10% ammonium persulfate and TEMED were added in this order according to the formulation. After being uniformly mixed, the glass plate wall is poured above the separating glue, a comb is inserted, and bubbles cannot be generated in the glue making process. Loading, namely, running the concentrated glue layer at the voltage of 80V and running the separated glue layer at the voltage of 120V.
(2) Transferring: the PVDF film is soaked in the anhydrous methanol, and the sponge, the filter paper, the gel, the PVDF film, the filter paper and the sponge are sequentially clamped according to instructions, so that bubbles are continuously removed in the operation process. The clips were placed in a transfer tank to transfer the protein.
(3) Closing: the PVDF membrane after completion of the transfer was washed 3 times with TBST for 15 min/time. The PVDF membrane was blocked with a 5% skimmed milk powder shaker for 2h.
(4) Incubating primary antibodies: TBST was used for 3 times, 15 min/time, and primary antibody was added for incubation at 4℃overnight.
(5) Incubating a secondary antibody: TBST is used for washing the membrane for 3 times, 15 min/time, secondary antibody is added, and the membrane is incubated for 2h at room temperature.
(6) ECL chemiluminescent development: TBST was washed 3 times for 15 min/time, and a developing solution was dropped onto the PVDF film, and developed and photographed by a gel imager.
As shown in FIG. 23, compared with the blank group, the Keap1 expression level in the model group is increased, the Nrf2 protein expression level is reduced, the content of downstream antioxidant proteins regulated by Nrf2 is further reduced, and the downstream proteins HO-1, NQO1 and GCLM expression are obviously reduced (P is less than 0.01), which indicates that hydrogen peroxide causes damage to HUVEC cells and inhibits the expression of an Nrf2 channel. After adding the polypeptides H1 and H5, compared with a model group, the Keap1 expression level is reduced, the Nrf2 protein expression level is obviously increased, the expression of the downstream proteins HO-1, NQO1 and GCLM is obviously increased (P is less than 0.01), and the concentration dependence is shown, so that the polypeptides H1 and H5 play a role in protecting through a Keap1/Nrf2 pathway.
Finally, it should be noted that the above list is only one embodiment of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (3)
1. The anti-oxidation active peptide H5 of the mytilus coruscus is characterized in that the amino acid series of the anti-oxidation active peptide H5 is Gln-Glu-Thr-Tyr, and the molecular weight of the anti-oxidation active peptide H5 is 539.54Da as measured by ESI-MS.
2. The use of the thick-shell mussel antioxidant active peptide H5 as claimed in claim 1 in the preparation of medicaments for the prevention and treatment of cardiovascular and cerebrovascular diseases.
3. The use according to claim 2, wherein said cardiovascular and cerebrovascular diseases comprise hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, atherosclerosis and heart failure.
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