CN115804827A - Application of polypeptide in preparation of protective agent for reducing vascular endothelial function damage - Google Patents
Application of polypeptide in preparation of protective agent for reducing vascular endothelial function damage Download PDFInfo
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Abstract
The invention provides an application of polypeptide in preparing a protective agent for reducing vascular endothelial function damage, belonging to the technical field of biomedicine. The effective component of the protective agent provided by the invention is barnacin. The invention discovers that the use of the barnacin for treating the endothelial cells of the human umbilical veins can reverse the cell viability reduction caused by cholesterol, and can reduce the apoptosis-promoting protein BAX up-regulation caused by the cholesterol and the apoptosis-inhibiting protein BCL-2 down-regulation caused by the high cholesterol increase.
Description
The scheme is a divisional application, and the original application name is as follows: a protective agent for reducing vascular endothelial function impairment, as filed on even date herewith: 2022-03-18, the application number of the original application is: CN202210267904.5.
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to application of a polypeptide in preparation of a protective agent for reducing vascular endothelial function damage.
Background
Atherosclerosis is a common vascular disorder in men over 40 years of age and postmenopausal women, and is the major pathological basis of cardiovascular and cerebrovascular disease. Current research has found that pathological progression in atherosclerotic patients often begins with vascular endothelial cell damage and lipid metabolism disorders. The vascular endothelium is an endothelial cell layer that is associated with the inner surface of the lumen of the vessel and, upon debridement, is a natural barrier between blood flow and the vessel wall. Functionally, the vascular endothelium regulates the structure and function of blood vessels, and plays an important role in maintaining various physiological and pathological functions in the body.
The vascular endothelial cells are a layer of mononuclear cells which are covered on the intima of the blood vessel and used for separating blood, vessel walls and various tissues and forming a continuous smooth inner wall of the blood vessel. It is a key physical barrier for many cells, and endothelial cell damage can lead to endothelial dysfunction, leading to the development of atherosclerotic conditions, and thus, effective reduction of endothelial cell damage would be helpful in the treatment of atherosclerotic patients.
Disclosure of Invention
The invention aims to provide a protective agent capable of effectively reducing the functional damage of vascular endothelium.
In order to realize the purpose, the invention provides the following technical scheme:
the invention provides an application of barnacin in preparing a protective agent for reducing vascular endothelial injury, which is characterized in that the barnacin is prepared from barnacin soft body parts, and the preparation method for preparing the barnacin per 100g of the barnacin soft body parts comprises the following steps:
(1) Grinding 100g of barnacle soft body part into powder in a grinder added with liquid nitrogen;
(2) Adding 500ml of distilled water, mixing uniformly, adjusting the pH value to 6-7, adding 10g of papain, and hydrolyzing at 55-65 ℃ for 4-6h;
(3) After hydrolysis, heating in water bath at 90-95 ℃ for 20-30 minutes, centrifuging and collecting supernatant to obtain barnacle enzymatic hydrolysate;
(4) Filtering the barnacle enzymatic hydrolysate by using a 5KDa ultrafiltration membrane, and collecting filtrate to obtain a barnacle peptide extracting solution;
(5) And (3) carrying out freeze drying on the barnacle peptide extracting solution to obtain the barnacle peptide.
Preferably, the protective agent can reduce cholesterol-induced decline in viability of human umbilical vein endothelial cells.
Preferably, the protective agent can reduce cholesterol-induced increase in apoptosis in human umbilical vein endothelial cells.
The invention further provides an application of the barnacle peptide in preparing a biological agent for improving the cholesterol-induced decline of the endothelial cell viability of the human umbilical vein, wherein the barnacle peptide is prepared from barnacle soft body parts, and the preparation method for preparing the barnacle peptide per 100g of the barnacle soft body parts comprises the following steps:
(1) Grinding 100g of barnacle soft body part into powder in a grinder added with liquid nitrogen;
(2) Adding 500ml of distilled water, mixing, adjusting pH to 6-7, adding 10g of papain, and hydrolyzing at 55-65 deg.C for 4-6h;
(3) After hydrolysis, heating in water bath at 90-95 ℃ for 20-30 minutes, centrifuging and collecting supernatant to obtain barnacle enzymatic hydrolysate;
(4) Filtering the barnacle enzymatic hydrolysate by using a 5KDa ultrafiltration membrane, and collecting filtrate to obtain a barnacle peptide extracting solution;
(5) And (3) carrying out freeze drying on the barnacle peptide extracting solution to obtain the barnacle peptide.
Secondly, the invention provides an application of barnacle peptide in preparing a biological agent for improving the increase of the apoptosis of human umbilical vein endothelial cells caused by cholesterol, wherein the barnacle peptide is prepared from barnacle soft body parts, and the preparation method for preparing the barnacle peptide per 100g of the barnacle soft body parts comprises the following steps:
(1) Grinding 100g of barnacle soft body part into powder in a grinder added with liquid nitrogen;
(2) Adding 500ml of distilled water, mixing uniformly, adjusting the pH value to 6-7, adding 10g of papain, and hydrolyzing at 55-65 ℃ for 4-6h;
(3) After hydrolysis, heating in water bath at 90-95 ℃ for 20-30 minutes, centrifuging and collecting supernatant to obtain barnacle enzymatic hydrolysate;
(4) Filtering the barnacle enzymatic hydrolysate by using a 5KDa ultrafiltration membrane, and collecting filtrate to obtain a barnacle peptide extracting solution;
(5) And (3) carrying out freeze drying on the barnacle peptide extracting solution to obtain the barnacle peptide.
The invention further provides an application of the barnacle peptide in preparing a biological agent for reducing the increase of the expression of human umbilical vein endothelial cell apoptosis-promoting protein BAX protein caused by cholesterol, which is characterized in that the barnacle peptide is prepared from barnacle soft body parts, and the preparation method for preparing the barnacle peptide from every 100g of the barnacle soft body parts comprises the following steps:
(1) Grinding 100g of barnacle soft body part into powder in a grinder added with liquid nitrogen;
(2) Adding 500ml of distilled water, mixing uniformly, adjusting the pH value to 6-7, adding 10g of papain, and hydrolyzing at 55-65 ℃ for 4-6h;
(3) After hydrolysis, heating in water bath at 90-95 ℃ for 20-30 minutes, centrifuging and collecting supernatant to obtain barnacle enzymatic hydrolysate;
(4) Filtering the barnacle enzymatic hydrolysate by using a 5KDa ultrafiltration membrane, and collecting filtrate to obtain a barnacle peptide extracting solution;
(5) And (3) carrying out freeze drying on the barnacle peptide extracting solution to obtain the barnacle peptide.
Secondly, the invention provides the application of the barnacin in preparing the biological agent for improving the expression reduction of human umbilical vein endothelial cell 5 apoptosis inhibiting protein BCL-2 protein caused by cholesterol, which is characterized in that,
the preparation method of the barnacle peptide from barnacle soft body parts per 100g comprises the following steps:
(1) Grinding 100g of barnacle soft body part into powder in a grinder added with liquid nitrogen;
(2) Adding 500ml of distilled water, mixing, adjusting pH to 6-7, adding 10g of papain 0, and hydrolyzing at 55-65 deg.C for 4-6h;
(3) After the hydrolysis is finished, heating in water bath at 90-95 ℃ for 20-30 minutes, centrifuging and collecting supernatant to obtain barnacle enzymatic hydrolysate;
(4) Filtering the barnacle enzymolysis liquid by using an ultrafiltration membrane of 5KDa, and collecting filtrate to obtain the barnacle enzymolysis liquid
To barnacle peptide extract;
and 5 (5) freeze-drying the barnacle peptide extracting solution to obtain the barnacle peptide.
In addition, the invention provides a protective agent for reducing vascular endothelial function damage, which is characterized in that the effective component of the protective agent is barnacle peptide, the barnacle peptide is prepared from barnacle soft body parts, and the preparation method for preparing the barnacle peptide per 100g of the barnacle soft body parts comprises the following steps:
(1) Grinding 100g of barnacle soft body part into powder in a grinder added with liquid nitrogen; 0 (2) adding 500ml of distilled water, mixing uniformly, adjusting the pH value to 6-7, and adding 10g of pawpaw eggs
Hydrolyzing with white enzyme at 55-65 deg.C for 4-6 hr;
(3) After hydrolysis, heating in water bath at 90-95 ℃ for 20-30 minutes, centrifuging and collecting supernatant to obtain barnacle enzymatic hydrolysate;
(4) Filtering the barnacle enzymatic hydrolysate by using a 5KDa ultrafiltration membrane, and collecting filtrate to obtain a barnacle peptide extracting solution;
(5) And (3) carrying out freeze drying on the barnacle peptide extracting solution to obtain the barnacle peptide.
The invention has the beneficial effects that:
the invention discovers that after the barnacin provided by the invention is used for treating human umbilical vein endothelial cells, the decrease of cell activity caused by cholesterol can be effectively reversed, the increase of apoptosis promoting protein BAX caused by cholesterol can be effectively reduced, and the decrease of apoptosis inhibiting protein BCL-2 caused by high cholesterol can be effectively reduced, so that vascular endothelial injury can be effectively realized, and the barnacin can be used for treating atherosclerosis caused by high-fat diet.
Drawings
FIG. 1 Effect of barnacin on cholesterol-induced decrease in cellular activity of HUVEC;
FIG. 2 Effect of barnacin on cholesterol-induced up-regulation of protein expression of the pro-apoptotic protein BAX and down-regulation of protein expression of the arrestin BCL-2.
Detailed Description
In order to clearly explain the technical features of the present solution, the present solution is explained by the following detailed description.
Example 1
(1) Grinding 100g of barnacle soft body part into powder in a grinder added with liquid nitrogen;
(2) Adding 500ml of distilled water, uniformly mixing, adjusting the pH value to 6.5, adding 10g of papain, and hydrolyzing at 60 ℃ for 4-6h;
(3) After the hydrolysis is finished, heating in water bath at 95 ℃ for 30 minutes, centrifuging and collecting supernatant to obtain barnacle enzymatic hydrolysate;
(4) Filtering the barnacle enzymatic hydrolysate by using a 5KDa ultrafiltration membrane, and collecting filtrate to obtain a barnacle peptide extracting solution;
(5) And (4) carrying out freeze drying on the barnacle peptide extracting solution to obtain the barnacle peptide.
Example 2
(1) The barnacle peptide is prepared into 50mg/ml,100mg/ml and 150mg/ml barnacle peptide reagent by using DMEM culture medium;
(2) 1X 10 of 100ul of logarithmic growth phase 4 HUVEC (human umbilical vein endothelial cells) in a 96-well culture plate in a cell culture box;
(3) After 1d of incubation, the medium was removed and the cells were washed 2 times with PBS, control: serum-free DMEM medium was added and treated for 24h, experimental group a: after the serum-free DMEM medium is treated for 4 hours, adding a serum-free medium containing 10 mu mol/L cholesterol to treat for 20 hours; experimental group B: adding 50mg/ml barnacin for treatment for 4h, and adding serum-free medium containing 10 μmol/L cholesterol for treatment for 20h; experimental group C: adding 100mg/ml barnacin for treatment for 4h, and adding serum-free medium containing 10 μmol/L cholesterol for treatment for 20h; experimental group D: adding 150mg/ml barnacin, treating for 4 hr, adding 10 μmol/L cholesterol-containing serum-free culture medium, treating for 20 hr, setting blank control, and setting 3 repeats per group;
(4) After the treatment is finished, adding an MTT reagent, and placing in a cell culture box for 4h;
(5) Removing supernatant, adding DMSO, and shaking in a shaking table in dark for 20min;
(6) The cells were measured for absorbance at 570nm in a microplate reader and the cell viability was calculated, the experimental results are shown in FIG. 1.
From the figure, it can be seen that the cell viability of the experimental group a is 62.84 ± 2.84, the cell viability of the experimental group B is 73.12 ± 3.61, the cell viability of the experimental group C is 84.09 ± 3.10, and the cell viability of the experimental group D is 84.99 ± 3.12, and from the results, after the barnacin pretreatment, the HUVEC cell viability decrease caused by cholesterol can be effectively improved, and the difference has statistical significance.
Example 2
(1) 1 × 10 of logarithmic growth phase 5 Putting HUVEC (human umbilical vein endothelial cells) in a 96-well culture plate for culturing in a cell culture box;
(2) When the cell density reached 90%, the medium was removed and the cells were washed 2 times with PBS, control: serum-free DMEM medium was added and treated for 24h, experimental group 1: after the serum-free DMEM medium is treated for 4 hours, adding a serum-free medium containing 10 mu mol/L cholesterol to treat for 20 hours; experimental group 2: adding 100mg/ml barnacin for treatment for 4h, and adding serum-free medium containing 10 μmol/L cholesterol for treatment for 20h;
(3) After the treatment, the culture medium was removed, the cells in the 6-well plate were washed 3 times with PBS, and then 100ul of lysate was added to each well;
(4) Scraping the cells with a cell scraper, mixing the lysate and the cells thoroughly, and performing lysis on ice for 30min;
(5) After lysis for 30min, the mixture of cell debris and lysate was pipetted into a 2ml centrifuge tube;
(6) 12000g, centrifuging for 10min at 4 ℃, removing supernatant, placing in a new centrifuge tube, measuring protein concentration, adding protein sample buffer solution, boiling protein for 10min at 100 ℃ to obtain a protein sample;
(7) Installing an electrophoresis tank, carrying out protein sample loading, and adding a protein standard Marker into the first hole;
(8) Performing constant voltage electrophoresis by using 80V voltage, adjusting the voltage to 130V when the blue sample buffer solution reaches the separation gel, and stopping electrophoresis when the sample buffer solution reaches the bottom edge;
(9) Soaking a PVDF membrane in methanol for 1min, sequentially placing a sponge pad, filter paper, separation glue, a membrane, filter paper and the sponge pad from a cathode to an anode, clamping a rotating membrane clamp, placing the rotating membrane clamp into a membrane rotating groove, adding an ice box and a membrane rotating buffer solution, and then, performing 250mA constant current membrane rotation for 90min;
(10) After the membrane conversion is finished, taking out the PVDF membrane, washing for 3 times by using TBSR, preparing 5% skimmed milk powder by using TBST, putting the membrane into a confining liquid, and slightly shaking and confining for 1h at room temperature;
(11) Rinsing the sealed membrane with TBST for 10min for 3 times, removing TBST, adding BAX, BCL-2 and beta-actin, and incubating at 4 deg.C overnight;
(12) After absorbing the primary antibody, rinsing with TBST for 3 times, 10min each time, adding the secondary antibody, and shaking gently for 1h at room temperature;
(13) The TBST rinse was 3 times followed by chemiluminescence and development, and the results obtained are shown in fig. 2.
From the figure, it can be seen that in experimental group 1, the protein expression of the pro-apoptotic protein BAX was up-regulated, while the protein expression of the anti-apoptotic protein BCL-2 was down-regulated; in the experimental group 2, after the barnacin treatment, the up-regulation of protein expression of pro-apoptotic protein BAX and down-regulation of apoptosis-inhibiting protein BCL-2 caused by cholesterol are obviously reversed, which indicates that barnacin can effectively reverse the increase of HUVEC apoptosis caused by cholesterol.
Claims (3)
1. The application of the barnacle peptide in preparing a protective agent for reducing vascular endothelial injury caused by cholesterol is characterized in that the barnacle peptide is prepared from barnacle soft body parts, and the preparation method for preparing the barnacle peptide per 100g of the barnacle soft body parts comprises the following steps:
(1) Grinding 100g of barnacle soft body part into powder in a grinder added with liquid nitrogen;
(2) Adding 500ml of distilled water, mixing uniformly, adjusting the pH value to 6-7, adding 10g of papain, and hydrolyzing at 55-65 ℃ for 4-6h;
(3) After the hydrolysis is finished, heating in water bath at 90-95 ℃ for 20-30 minutes, centrifuging and collecting supernatant to obtain barnacle enzymatic hydrolysate;
(4) Filtering the barnacle enzymatic hydrolysate by using an ultrafiltration membrane of 5KDa, and collecting filtrate to obtain a barnacle peptide extracting solution;
(5) Freeze-drying the barnacle peptide extracting solution to obtain barnacle peptide;
the protective agent can simultaneously reduce the activity of human umbilical vein endothelial cells caused by cholesterol and reduce the apoptosis in the human umbilical vein endothelial cells caused by cholesterol.
2. The application of the barnacle peptide in preparing a biological agent for reducing the increase of the expression of human umbilical vein endothelial cell apoptosis-promoting protein BAX protein caused by cholesterol is characterized in that the barnacle peptide is prepared from barnacle soft body parts, and the preparation method for preparing the barnacle peptide per 100g of the barnacle soft body parts comprises the following steps:
(1) Grinding 100g of barnacle soft body part into powder in a grinder added with liquid nitrogen;
(2) Adding 500ml of distilled water, mixing, adjusting pH to 6-7, adding 10g of papain, and hydrolyzing at 55-65 deg.C for 4-6h;
(3) After hydrolysis, heating in water bath at 90-95 ℃ for 20-30 minutes, centrifuging and collecting supernatant to obtain barnacle enzymatic hydrolysate;
(4) Filtering the barnacle enzymatic hydrolysate by using a 5KDa ultrafiltration membrane, and collecting filtrate to obtain a barnacle peptide extracting solution;
(5) And (3) carrying out freeze drying on the barnacle peptide extracting solution to obtain the barnacle peptide.
3. The application of the barnacle peptide in preparing a biological agent for improving the reduction of the expression of human umbilical vein endothelial cell apoptosis inhibiting protein BCL-2 protein caused by cholesterol is characterized in that the barnacle peptide is prepared from a barnacle soft body part, and the preparation method for preparing the barnacle peptide from each 100g of the barnacle soft body part comprises the following steps:
(1) Grinding 100g of barnacle soft body part into powder in a grinder added with liquid nitrogen;
(2) Adding 500ml of distilled water, mixing, adjusting pH to 6-7, adding 10g of papain, and hydrolyzing at 55-65 deg.C for 4-6h;
(3) After hydrolysis, heating in water bath at 90-95 ℃ for 20-30 minutes, centrifuging and collecting supernatant to obtain barnacle enzymatic hydrolysate;
(4) Filtering the barnacle enzymatic hydrolysate by using a 5KDa ultrafiltration membrane, and collecting filtrate to obtain a barnacle peptide extracting solution;
(5) And (3) carrying out freeze drying on the barnacle peptide extracting solution to obtain the barnacle peptide.
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| CN202210267904.5A CN114404561B (en) | 2022-03-18 | 2022-03-18 | Protective agent for reducing vascular endothelial function damage |
| CN202211614887.4A CN115804827B (en) | 2022-03-18 | 2022-03-18 | Application of polypeptide in preparation of protective agent for reducing vascular endothelial function injury |
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