CN113293190B

CN113293190B - Phycobilin binding peptides and uses thereof

Info

Publication number: CN113293190B
Application number: CN202110847186.4A
Authority: CN
Inventors: 李文军; 秦松; 李亚; 于雁飞; 刘润泽; 张娟娟; 刘正一
Original assignee: Yantai Jiahui Marine Biotechnology Co ltd; Yantai Institute of Coastal Zone Research of CAS
Current assignee: Yantai Jiahui Marine Biotechnology Co ltd; Yantai Institute of Coastal Zone Research of CAS
Priority date: 2021-07-27
Filing date: 2021-07-27
Publication date: 2021-10-29
Anticipated expiration: 2041-07-27
Also published as: CN113293190A

Abstract

The invention discloses a phycobilin binding peptide and application thereof, wherein the preparation process of the phycobilin binding peptide comprises the following steps: enzymolysis, inactivation, centrifugation, plate-and-frame filter pressing, negative pressure concentration and instantaneous spray drying, wherein in the enzymolysis step, phycobiliprotein powder is subjected to enzymolysis by using compound protease MC101 at the enzymolysis temperature of 45-50 ℃ for 4 hours, and in the inactivation step, the inactivation temperature is 70-78 ℃ and is kept for 6-8 min. The invention has the advantages that: the phycobilin binding peptide prepared from the phycobiliprotein is safe to use, has an obvious anti-inflammatory effect, has the effects of resisting oxidation and pulmonary fibrosis, and is small in molecular weight and easy to absorb.

Description

Phycobilin binding peptides and uses thereof

Technical Field

The invention relates to a peptide and application thereof, in particular to a phycobilin binding peptide and application thereof, belonging to the technical field of peptides.

Background

Inflammation, a defensive response to external stimuli, protects the body from damage caused by internal and external related factors. However, when the inflammatory system is disordered, the hyperproliferative and differentiated inflammatory cells and the over-expressed inflammatory factors can damage the body, and various diseases such as pulmonary fibrosis, arthritis, chronic obstructive pulmonary disease, chronic colitis and the like are caused.

Heme oxygenase 1 (HO-1) is an inducing enzyme responsible for the breakdown of heme. HO-1 is a target for the treatment of inflammation due to its anti-inflammatory properties. Traditional HO-1 inducers are mainly metalloporphyrins, which can significantly up-regulate the expression of HO-1, but are not suitable for clinical use due to serious toxicity problems. Recent studies have shown that linear tetrapyrroles such as bilirubin can also activate HO-1, but clinical administration of linear tetrapyrroles such as bilirubin has problems such as toxicity and low bioavailability. Therefore, a linear tetrapyrrole substitute which is safe to use and good in tolerance needs to be found.

Algae are lower plants of photoautotrophic, and the complex external stress endows the algae with unique biological activity function, so that the algae have very wide application prospect, and foods with medicinal value carried by the herbal literature in the past generations of China also relate to the algae, for example: thallus laminariae, thallus Porphyrae, and carrageen. Phycocyanin is light-harvesting chromoprotein existing in blue algae, red algae, cryptophyceae and a few dinoflagellates, and is formed by connecting apoprotein and Phycocyanobilin (PCB) through thioether covalent bond. Phycocyanin is composed of alpha subunit and beta subunit, the alpha subunit is connected with a PCB molecule through Cys84, the beta subunit is connected with two PCB molecules through Cys82 and Cys153, the structure of the PCB molecule and the position of the connection with Cys are shown in figure 1, and the structure can be known from figure 1: the PCB molecules have a linear tetrapyrrole structure. The alpha subunit and the beta subunit are first assembled into a monomer (alpha beta), and the monomer (alpha beta) is further polymerized into a trimer (alpha beta)₃Or hexamer (. alpha. beta.)₆. When phycocyanin forms tripolymer or hexamer, phycocyanobilin is wrapped in a hydrophobic core of phycocyanin and interacts with amino acid residues of Arg, Asp, Tyr and the like to jointly form a special chromophore, so that the phycocyanin has a sharp absorption peak near 620nm and an obvious absorption shoulder near 575 nm.

Disclosure of Invention

The invention aims to prepare a phycobilin binding peptide which is a linear tetrapyrrole substitute with safety and anti-inflammatory effect from phycobiliprotein.

In order to achieve the above object, the present invention adopts the following technical solutions:

the phycobilin binding peptide is characterized in that the preparation process comprises the following steps:

step 1, enzymolysis: pouring the phycobiliprotein powder into an enzymolysis tank filled with pure water, wherein the mass of the phycobiliprotein powder is as follows: the mass of the pure water is =1: 50-80, the pH value is natural, the temperature of water in an enzymolysis tank is increased to 45-50 ℃, the compound protease MC101 with the mass of 1-2% of the phycobiliprotein powder is added, the mixture is uniformly stirred, and the phycobiliprotein binding peptide feed liquid 1 is obtained after enzymolysis is carried out for 4 hours;

step 2, inactivation: heating the phycobilin binding peptide feed liquid 1 to 70-78 ℃, keeping the temperature for 6-8 min, and inactivating to obtain a phycobilin binding peptide feed liquid 2;

step 3, centrifugation: pumping the phycobilin binding peptide feed liquid 2 into a centrifuge for centrifugation at the centrifugation speed of 3000-4000 r/min to obtain a phycobilin binding peptide feed liquid 3;

step 4, plate and frame filter pressing: pouring the phycobilin binding peptide feed liquid 3 into a plate-and-frame filter press for filter pressing, wherein a filter aid is arranged on the plate-and-frame filter press to obtain a phycobilin binding peptide feed liquid 4;

and 5, concentrating under negative pressure: pumping the phycobilin binding peptide feed liquid 4 into a single-effect evaporator for negative pressure concentration, and removing more than 90% of water to obtain phycobilin binding peptide feed liquid 5;

step 6, instantaneous spray drying: rapidly heating the phycobilin binding peptide feed liquid 5 to 90 ℃, and pumping the phycobilin binding peptide feed liquid into a drying tower for instantaneous spray drying to form dry powder, namely phycobilin binding peptide;

the whole preparation process is protected from light.

In the aforementioned phycobilin binding peptide, in step 1, the aforementioned phycobiliprotein powder comprises: phycoerythrin powder, phycocyanin powder and allophycocyanin powder.

The phycobilin binding peptide is characterized in that AG-1000# diatomite is selected as the filter aid in the step 4.

The phycobilin binding peptide is characterized in that in the step 5, the temperature is controlled to be 50 ℃ in the negative pressure concentration process, and the negative pressure is 0.1 MPa.

The phycobilin binding peptide is characterized in that, in the step 6, the drying temperature is 140-155 ℃.

The phycobilin binding peptide in step 6, wherein the phycobilin binding peptide comprises: phycourobilin binding peptide, phycoviolbilin binding peptide, phycoerythrobilin binding peptide and phycocyanobilin binding peptide.

The invention has the advantages that: the phycobilin binding peptide prepared from the phycobiliprotein is safe to use, has an obvious anti-inflammatory effect, has the effects of resisting oxidation and pulmonary fibrosis, and is small in molecular weight and easy to absorb.

Drawings

FIG. 1 is a block diagram of a PCB molecule;

FIG. 2 is a scanning spectrum of a spectrum of phycocyanin raw material and phycocyanobilin-binding peptide;

FIG. 3 is a UV-VIS spectrum of a methanol solution, an acidic methanol solution and phycocyanin of a linear tetrapyrrole phycocyanobilin standard;

FIG. 4 is a graph showing the molecular weight distribution of phycocyanobilin-binding peptides;

FIG. 5 is a graph showing the effect of phycocyanobilin-binding peptides on proliferation of RAW264.7 cells;

FIG. 6 is a graph showing the effect of phycocyanobilin-binding peptides on LPS-induced NO release from mouse RAW264.7 cells;

FIG. 7 is a graph of the effect of phycocyanobilin binding peptides on TNF- α expression levels in cell supernatants;

FIG. 8 is a graph showing the effect of phycocyanobilin-binding peptides on the level of IL-6 expression in cell supernatants;

FIG. 9 is a graph of the effect of phycocyanobilin binding peptides on TGF- β 1-induced A549 cell morphology;

FIG. 10 is a graph showing the effect of phycocyanobilin-binding peptides on the amount of expression of Collagen I in A549 cells induced by TGF- β 1;

FIG. 11 is a graph showing the effect of phycocyanobilin binding peptides on TGF- β 1-induced EMT-associated marker protein expression in A549 cells;

FIG. 12 is a graph showing the results of immunofluorescence staining of TGF-. beta.1-induced HLF-1 cell expression levels of α -SMA.

Detailed Description

The invention is described in detail below with reference to the figures and the embodiments.

First part preparation of phycocyanobilin binding peptides

Phycocyanin is sensitive to light, and phycocyanobilin binding peptide is easily oxidized under light, so the whole preparation process needs to be protected from light.

Step 1: enzymolysis

Pouring phycocyanin powder into an enzymolysis tank filled with pure water (prepared by a reverse osmosis membrane technology), wherein the mass ratio of phycocyanin powder to pure water is =1:65, the pH value is naturally (about 7), raising the temperature of water in the enzymolysis tank to 48 ℃, adding compound protease MC101 (200000 u/g, Takedai Matel Biotech Co., Ltd.) with the mass ratio of phycocyanin powder of 1.5%, uniformly stirring, and carrying out enzymolysis for 4h to obtain phycocyanobilin-binding peptide feed liquid 1.

The solid-liquid ratio is important for the enzymolysis of phycocyanin and directly influences the yield (the mass of phycocyanin peptide freeze-dried powder/the mass of phycocyanin raw materials), and tests show that the yield is higher than 96% when the solid-liquid ratio is the mass of phycocyanin powder and the mass of pure water =1: 50-80.

The phycocyanin is sensitive to temperature and has a fading phenomenon, the final color of the phycocyanobilin binding peptide is directly influenced, so the enzymolysis temperature is not too high, and experiments show that when the enzymolysis temperature is in the range of 45-50 ℃, the final color of the phycocyanobilin binding peptide is blue-green, and the color change is less compared with the blue color of the phycocyanin.

Phycocyanobilin in phycocyanin is sensitive to pH and temperature, color change is easily caused in the enzymolysis process, in order to reduce the color change of a final product-phycocyanobilin binding peptide as much as possible and ensure the activity of the phycocyanobilin binding peptide, a large number of screening experiments show that when the compound protease MC101 is applied to the enzymolysis process of phycocyanin for the first time, the activity of the compound protease MC101 is high, the yield of the phycocyanin peptide reaches 96%, and the yield of the phycocyanobilin reaches 4.7% (the mass of the phycocyanobilin/the mass of the phycocyanin =4.9%, 4.9% and 96% of the phycocyanin = 4.7%), the spectrum scanning atlas of the phycocyanin raw material and the phycocyanobilin binding peptide obtained by the enzymolysis of the compound protease MC101 is shown in figure 2, and it can be known from figure 2 that: the absorbance of the enzymolysis peptide at 620nm is obviously reduced, the property of the formed absorption spectrum is very close to the property of the absorption spectrum (figure 3) formed by the linear tetrapyrrole phycocyanobilin standard, the phycocyanin is fully decomposed into the linear phycocyanobilin binding peptide, and the good enzymolysis effect of the compound protease MC101 is further verified. Phycocyanobilin binding peptide obtained by enzymolysis of phycocyanin by adopting compound protease MC101 has blue-green color, and has less color change compared with blue color of phycocyanin.

Tests show that when the using amount of the compound protease MC101 is 1% and 2% of the mass of the phycocyanin powder, the enzymolysis time is appropriate, the method is more suitable for industrial mass production, and the yield of the obtained phycocyanin peptide is high; if the dosage is less than 1% of the mass of the phycocyanin powder, the enzymolysis time of the phycocyanin powder can be increased, the color of the final product is influenced, and meanwhile, the phenomenon of incomplete enzymolysis is caused; if the dosage is higher than 2% of the phycocyanin powder, the production cost is increased, and unnecessary waste is caused, so that the dosage of the compound protease MC101 can be within the range of 1-2% of the phycocyanin powder.

Step 2: inactivating

And (3) heating the phycocyanobilin binding peptide feed liquid 1 obtained in the step (1) to 75 ℃, keeping for 7min, and inactivating to obtain a phycocyanobilin binding peptide feed liquid 2.

The inactivation temperature should not be too high, so as to ensure sufficient inactivation and to reduce the damage of temperature to phycocyanobilin in phycocyanobilin binding peptide to the maximum extent. Experiments show that if the inactivation temperature exceeds 80 ℃, the color of the phycocyanobilin binding peptide feed liquid is gradually changed from blue to purple, the original color of the phycocyanobilin binding peptide is lost, and when the inactivation temperature is in the range of 70-78 ℃ and the inactivation time is in the range of 6-8 min, the color of the phycocyanobilin binding peptide feed liquid is blue and is not changed.

And step 3: centrifugation

Pumping the phycocyanobilin binding peptide feed liquid 2 obtained in the step 2 into a horizontal centrifuge for centrifugation, wherein the centrifugation speed is 3500r/min, and reserving the supernatant to obtain phycocyanobilin binding peptide feed liquid 3.

The composite protease MC101 has good enzymolysis effect and small centrifugal precipitation amount, so that the centrifugal rate is not required to be too high and can be within the range of 3000-4000 r/min, and the energy consumption can be reduced.

And 4, step 4: plate frame filter pressing

Pouring the phycocyanobilin binding peptide feed liquid 3 obtained in the step 3 into a plate-and-frame filter press for filter pressing, arranging AG-1000# diatomite (filter aid) on the plate-and-frame filter press, and collecting the feed liquid to obtain phycocyanobilin binding peptide feed liquid 4.

AG-1000# diatomite has a good microporous structure, not only can enable phycocyanobilin binding peptide feed liquid to obtain a good flow rate ratio, but also can filter fine suspended matters, and ensures the clarity of phycocyanobilin binding peptide products. The most important points are: the AG-1000# diatomite has neutral pH value and very low adsorbability to phycocyanobilin color components in the linear phycocyanobilin conjugated peptide, so that the original color of the phycocyanobilin conjugated peptide can be maintained.

And 5: negative pressure concentration

Pumping the phycocyanobilin binding peptide feed liquid 4 obtained in the step 4 into a single-effect evaporator for negative pressure concentration, controlling the temperature at 50 ℃ in the process, controlling the negative pressure at 0.1MPa, discharging evaporation condensate water every 20min, and removing more than 90% of water to obtain the phycocyanobilin binding peptide feed liquid 5.

Step 6: instantaneous spray drying

And (3) quickly heating the phycocyanobilin binding peptide feed liquid 5 obtained in the step (5) to 90 ℃, pumping the phycocyanobilin binding peptide feed liquid into a drying tower for instantaneous spray drying, wherein the drying temperature is 140 ℃ (the drying temperature can be properly raised to 155 ℃) to form dry powder, namely the phycocyanobilin binding peptide.

The phycocyanobilin binding peptide prepared by the method is blue-green powder, the yield of phycocyanin peptide is about 96%, and the yield of phycocyanobilin is about 4.7%.

The molecular weight distribution of the phycocyanin peptide prepared by the method is shown in figure 4, and a table corresponding to figure 4 is as follows:

as shown in FIG. 4 and the above table, the average molecular weight of the phycocyanin peptide prepared by the method is 583Da, and the component of <1000Da reaches 89.66%.

In addition, according to the simulated enzymolysis, the following steps are calculated: the average molecular weight of the phycocyanobilin is about 586Da and accounts for about 4.9% of the total peptide content, and the molecular weight of the phycocyanobilin binding peptide is 700-1000 Da and accounts for about 4.7% of the total peptide content.

At present, bilirubin, a commonly used HO-1 activator, is fat-soluble, because two carboxyl groups of bilirubin respectively form hydrogen bonds with carbonyl and N in a pyridine ring. In the phycocyanobilin binding peptide, the hydrogen bond in the phycocyanobilin binding peptide can be broken by the existence of the peptide, so that the water solubility of the phycocyanobilin binding peptide is better, which means that: the phycocyanobilin binding peptide has good solubility. It was found by experiment that a 2 wt% solution of the phycocyanobilin binding peptide was clear and transparent.

Phycobiliproteins are light-harvesting chromoproteins existing in blue-green algae, red algae, cryptophyceae and a few dinoflagellates, and can be divided into three types, namely: phycoerythrin, phycocyanin, and allophycocyanin. Each phycobiliprotein subunit consists of an apoprotein and 1-5 linear tetrapyrroles, and the linear tetrapyrroles can be connected with a cysteine residue (Cys) on the apoprotein of the phycobiliprotein in a thioether bond mode. Among phycobiliproteins, four common linear tetrapyrroles are Phycocyanobilin (PCB), Phycoerythrobilin (PEB), Phycourobilin (PUB) and phycoviologen (PXB or PVB), which have absorption maxima at 620-650 nm (PCB), 540-565 nm (PEB), 568nm (PXB) and 490nm (PUB), respectively. In cryptophyceae, linear tetrapyrroles, specifically 15, 16-dihydrobiliverdin (Cryptoviolin 15, 16-dihydrobiliverdin, DBV) and Mesobiliverdin (MBV) are also present. These linear tetrapyrroles are also known as phycobilins in phycobiliproteins. The nuclear magnetic resonance spectrum proves that the carbon skeletons of the phycobilin molecules are basically the same, the molecular weight is about 586kDa, the phycobilin molecules all contain 2 ketone groups (C = O), seven carbon-carbon double bonds (C = C) and the like, and the difference is shown in the difference of the positions and the number of the double bonds.

Therefore, in addition to phycocyanobilin binding peptides prepared from phycocyanin powder, phycoerythrobilin binding peptides, phycourobilin binding peptides, phycoviolbilin binding peptides, and the like having a linear tetrapyrrole structure can be prepared from phycoerythrin powder and allophycocyanin powder.

The second part is to study the anti-inflammatory effect of phycocyanobilin binding peptides prepared by the invention

RAW264.7 mouse macrophage is selected as a cell model, Lipopolysaccharide (LPS) is adopted to induce RAW264.7 cells to enable the cells to express related inflammatory factors, the phycocyanobilin binding peptide prepared by the method is used to treat cell supernatant, the content of the inflammatory factors is measured by a Griess method and an Elisa method, and the scavenging capacity of the phycocyanobilin binding peptide on the inflammatory factors is researched.

1. Test method

(1) RAW264.7 cell viability assay

Diluting the sample to 50 mug/ml, 100 mug/ml and 200 mug/ml by adopting a DMEM culture medium; taking logarithmic growth RAW264.7 cells, adjusting the cell concentration to be about 5X 10⁴One/ml of the cells were inoculated into a number of 96-well plates, each 100. mu.l, and the culture was continued for 24 hours at 37 ℃ with 5% CO₂Culturing; after 24 hours, replacing the culture medium containing each group of medicines and continuing to culture for 24 hours; adding medicine for 24h, changing the solution, adding 100 μ L of culture medium reaction solution containing 10 μ L of CCK-8 into each hole, taking the reaction solution without cells as a zero hole, and incubating for a proper time; absorbance at 450nm was measured with a microplate reader. Cell viability was calculated using the following formula:

(2) determination of NO content in supernatant of RAW264.7 cells

Adding 50 mu L of 100ng/mL LPS solution into the administration group and the LPS group, adding DMEM culture solution with the same volume into the control group, culturing for 24h, sucking 100 mu L of supernatant, and determining the NO content by adopting a Biyuntian NO detection kit according to a method shown by the kit.

(3) Content determination of TNF-alpha and IL-6 in supernatant of RAW264.7 cells

And adding 50 mu L of 100ng/mL LPS solution into the administration group and the LPS group, culturing the control group in DMEM culture solution with the same volume for 24h, sucking 100 mu L of supernatant, detecting TNF-alpha and IL-6 by adopting a Wallace Elisa detection kit, and carrying out the steps indicated by the kit.

2. Test results

(1) Effect on the viability of RAW264.7 cells

The CCK-8 method is adopted to observe the influence of the phycocyanobilin binding peptide prepared by the invention on the proliferation of RAW264.7 cells, and further to observe whether the phycocyanobilin binding peptide has toxicity to the cells.

The effect of the phycocyanobilin binding peptide on the proliferation of RAW264.7 cells is shown in FIG. 5, based on the relative cell survival rate of 90% or more as the standard of no cytotoxicity.

As can be seen from FIG. 5, the phycocyanobilin binding peptides with different concentrations (50. mu.g/ml, 100. mu.g/ml and 200. mu.g/ml) have no obvious difference in the effect on the growth of RAW264.7 cells, and the phycocyanobilin binding peptides with high concentration (200. mu.g/ml) have weak promotion effect on the proliferation of RAW264.7 cells, that is, the phycocyanobilin binding peptides prepared by the invention have no obvious cytotoxicity in the concentration range of 0-200. mu.g/ml.

(2) Inhibition of NO in RAW264.7 cells

NO is an active nitrogen radical in the body, which mediates many biological functions. Macrophage-derived NO plays an important role in physiology and pathology, and a proper amount of NO can promote the immune response of the organism, and the over-expression of NO can cause inflammatory diseases such as rheumatoid arthritis, atherosclerosis, tissue injury and the like. The expression level of NO thus determines the induction or inhibition of inflammation by the sample.

Experiment the influence of the phycocyanobilin binding peptide prepared by the invention on the NO release amount of mouse RAW264.7 cells induced by LPS is detected by adopting a Griess method, and the detection result is shown in figure 6.

As can be seen from fig. 6, the amount of NO released in the supernatant of RAW264.7 cells induced by LPS was significantly increased compared to the control group, that is, the expression level of NO in the supernatant of RAW264.7 cells could be inhibited after the administration of the phycocyanobilin-binding peptide prepared in the present invention for dry prognosis, indicating that the phycocyanobilin-binding peptide prepared in the present invention has a certain anti-inflammatory activity.

(3) Inhibition of TNF-alpha and IL-6 in RAW264.7 cells

TNF-alpha and IL-6 are important inflammatory factors produced by macrophages, and over-expression of TNF-alpha and IL-6 often leads to the production of various diseases. Thus, by detecting changes in TNF- α and IL-6 levels, the anti-inflammatory ability of the sample can be studied.

In the experiment, on the basis of an NO inhibition test, an Elisa method is adopted to determine the influence of the phycocyanobilin binding peptide prepared by the invention on the expression levels of TNF-alpha and IL-6 in cell supernatant, the anti-inflammatory activity of the phycocyanobilin binding peptide is further verified, and the detection result is shown in figure 7 and figure 8.

As can be seen from FIGS. 7 and 8, the phycocyanobilin-binding peptides prepared in the present invention can effectively inhibit the expression levels of TNF- α and IL-6 in the supernatant, and the secretion amounts of TNF- α and IL-6 were significantly reduced compared to the model group.

In conclusion, the phycocyanobilin binding peptide prepared by the invention has obvious anti-inflammatory effect. That is, the phycocyanobilin binding peptide prepared by the invention can be applied to the preparation of anti-inflammatory drugs.

The carbon skeletons of other three phycobilin molecules, phycourobilin, phycoerythrobilin and phycoerythrobilin, are the same as the carbon skeletons of phycocyanobilin, and the difference is only expressed in the difference of positions and numbers of double bonds, so the phycourobilin binding peptide, the phycoerythrobilin binding peptide and the phycoerythrobilin binding peptide are also the same as the phycocyanobilin binding peptide, have obvious anti-inflammatory effect and can be applied to the preparation of anti-inflammatory drugs.

The third part is to research the anti-fibrosis effect of the phycocyanobilin binding peptide prepared by the invention on A549 cells and HLF-1 cells

Idiopathic Pulmonary Fibrosis (IPF) is considered to be a chronic, interstitial pneumonia. The major manifestations of histopathology are alveolar epithelial cell damage, inflammatory cell infiltration at the site of fibrosis, ECM accumulation, EMT overexpression and myofibroblast transformation, ultimately leading to impaired gas exchange and respiratory function. Although the cause of IPF is complex, current studies have demonstrated that IPF is often associated with an inflammatory response during its development. Under the induction of LPS, macrophages can secrete inflammatory factors such as TGF-beta 1 and the like, and induce the fibroblast to be transformed into myofibroblast to aggravate IPF. Therefore, in the experiment, human lung epithelial cells A549 cells and human lung fibroblast HLF-1 cells are selected, TGF-beta 1 is used for induction, fibrosis formation conditions are simulated in vitro, cells are treated by the phycocyanobilin binding peptide prepared by the method, the changes of EMT related markers and the synthetic amount of Collagen Collagen I of the cells are detected, and the anti-pulmonary fibrosis effect of the phycocyanobilin binding peptide prepared by the method is observed.

1. Test method

(1) Effect on A549 cell morphology

A549 cells were selected as the cells, and the cell culture conditions and groups are shown in the following table.

Epocyte culture conditions and grouping

	Control group	Model set	Low dose group	High dose group
					Culture medium	1640 medium	1640 medium	1640 medium	1640 medium
FBS
		10%	10%	10%	10%
Temperature of						37℃	37℃	37℃	37℃
	CO₂	5%	5%	5%	5%
TGF-β1						-	10ng/mL	10ng/mL	10ng/mL
	Phycocyanobilin binding peptides	-	-	10µg/mL	30µg/mL
Sample size						100µL	100µL	100µL	100µL
	Incubation time	72h	72h	72h	72h

After the culture is finished, the morphology of the A549 cells is observed by an optical microscope and is subjected to comparative analysis.

(2) Effect on A549 cell Collagen I expression

A549 cells are selected as the cells, and the cell culture conditions and the groups are the same as the table above. Continuously culturing for 24 h; after removing the supernatant, the cells were washed twice with PBS; cells were fixed with 4% paraformaldehyde overnight, washed with PBS for 5min for 3 times in total; incubating 1% TritonX-100 prepared by PBS for 5min, and washing with PBS for 5 min; 3% H₂O₂Incubating for 10min, and washing for 5min by PBS; washing with PBS for 5min for 3 times, preparing a second antibody diluent according to a volume ratio of 1:100, and incubating for 2h at 37 ℃; and (4) washing with PBS for 5min for 3 times, dyeing the DAPI nucleus for 5-10 min, and taking a picture with a fluorescence microscope.

(3) Effect on EMT-related protein expression of A549 cells

Selecting A549 cells as cells, culturing the cells under the same conditions and grouping as those in the table above, administering the phycocyanobilin binding peptide prepared by the invention when the cell density reaches 60%, taking a cultured cell plate after 72 hours, removing a culture medium, washing with 100 mu l of PBS for 3 times, and washing at 3min intervals; fixing the cells by adopting 4% formaldehyde at room temperature for 20 min; washing 3 times with 100 mul PBS, wherein the time interval of each washing is 5 min; adding 100 mul of confining liquid, and incubating for 1h at room temperature; discarding the confining liquid, not washing, adding 100 microliter of diluted primary antibody, and standing overnight at 4 ℃; primary antibody is recovered, and 100 mul PBS is washed for 3 times, wherein the time interval of each washing is 5 min; adding diluted fluorescent secondary antibody in a dark place, immediately placing in a box, placing in a dark place, and incubating at room temperature for 1 h; discarding the secondary antibody, washing 3 times by 100 mul PBS, and washing each time at an interval of 5 min; and adding 50 mul of DAPI, incubating for 5min in a dark place, carrying out nuclear staining on the specimen, and observing by a fluorescence microscope after recovering the DAPI.

(4) Effect on expression of HLF-1 cell alpha-SMA

HLF-1 cells are selected as the cells, and the cell culture conditions and the groups are the same as those in the table above; continuously culturing for 24 h; after removing the supernatant, the cells were washed twice with PBS; cells were fixed with 4% paraformaldehyde overnight, washed with PBS for 5min for 3 times in total; incubating 1% TritonX-100 prepared by PBS for 5min, and washing with PBS for 5 min; 3% H₂O₂Incubating for 10min, and washing for 5min by PBS; washing with PBS for 5min for 3 times, preparing a second antibody diluent according to a volume ratio of 1:100, and incubating for 2h at 37 ℃; and (4) washing with PBS for 5min for 3 times, dyeing the DAPI nucleus for 5-10 min, and taking a picture with a fluorescence microscope.

2. Results of the experiment

(1) Effect of phycocyanobilin-binding peptides on A549 cell morphology

Under the induction of TGF-beta 1, human lung epithelial cells (A549) are polarized, the cells present a slender spindle shape, and an EMT process is carried out, and the fibrosis activity is shown. The effect of phycocyanobilin binding peptides on TGF- β 1-induced a549 cell morphology is shown in fig. 9, in which (a) is a control group, (b) is a model group, (c) is a low dose group, (d) is a high dose group, the low dose is 10 μ g/mL, and the high dose is 30 μ g/mL.

As can be seen from FIG. 9, the morphology of the A549 cells after TGF-beta 1 is changed obviously, from the original cobblestone-shaped cells to the very obvious spindle-shaped cells, and the cells are slender. After the phycocyanobilin binding peptide prepared by the invention is treated, the recovery of the A549 cell morphology is not obvious under low dosage, most cells still present fusiform, and the recovery of the A549 cell morphology is obvious under high dosage, and the cells present cobblestone-shaped cells similar to a control group.

Therefore, the phycocyanobilin binding peptide prepared by the invention can effectively relieve the change of the A549 cell morphology induced by TGF-beta 1, so that the A549 cell maintains a relatively normal cell morphology, the recovery effect is better under a high dose (30 mu g/mL), and the A549 cell morphology is basically recovered. That is, phycocyanin and phycocyanobilin binding peptide prepared by the present invention can restore lung tissue fibrosis symptoms caused by lung epithelial cell polarization to some extent.

(2) Effect of phycocyanobilin-binding peptides on the expression of A549 cells Collagen I

One very significant condition of IPF is the excessive deposition of extracellular matrix between tissues, and the excessive deposition of ECM exacerbates the development of IPF. Collagen i plays an important role in ECM excessive deposition as a main component of ECM, and Collagen is not easily degraded, its conversion rate is very slow, and has strong resistance to general proteases, so that once Collagen is produced, it is difficult to be removed by the human body, thereby continuously aggravating IPF. Therefore, Collagen I is an important index for IPF, and the inhibition effect of Collagen I on IPF can be researched by detecting the difference of the expression quantity of A549 cell Collagen I before and after the action of phycocyanin and phycocyanobilin binding peptide prepared by the invention.

Since the collagen production rate was slower than that of other EMT-labeled proteins, the present experiment conducted an investigation on the construction of an a549 collagen expression model, and the results showed that the collagen expression level was good 72 hours after 10ng/mL TGF- β 1 induction, and the collagen expression level was able to be used as a pulmonary fibrosis cell model.

The influence of the phycocyanobilin binding peptide prepared by the invention on the expression level of A549 cell Collagen I is shown in figure 10, wherein, (a) is a control group, (b) is a model group, (c) is a low-dose group, (d) is a high-dose group, the low dose is 10 mu g/mL, and the high dose is 30 mu g/mL.

As can be seen from FIG. 10, the cells in the control group do not substantially express Collagen I, and the expression level of Collagen I in A549 cells is remarkably increased after TGF-beta 1 is induced for 72 h. Although the phycocyanobilin binding peptide with low dose (10 mu g/mL) has a certain relieving effect on the expression of the Collagen I of the A549 cells, the influence is not obvious, and under high dose (30 mu g/mL), the phycocyanobilin binding peptide has the best inhibition effect on the expression of the Collagen I, and the fading amplitude of green fluorescence is larger.

(3) Effect of phycocyanobilin-binding peptides on EMT-associated protein expression of A549 cells

EMT refers to the process of transformation of epithelial cells into mesenchymal cells, and plays a very important role in tissue inflammation and tissue fibrosis. TGF-beta can induce A549 cells to generate an EMT process through an NF-kB pathway, express marker proteins such as alpha-SMA, E-cadherin, N-cadherin and Vimentin and the effect of a sample on pulmonary fibrosis can be researched by measuring the change of the expression content of several marker proteins in cell supernatant. The expression of the alpha-SMA, the N-cadherin and the Vimentin can promote the development of IPF, and plays a very important role in the occurrence process of pulmonary fibrosis. And the E-cadherin can play a role in maintaining epithelial cell connection, when the expression level of the E-cadherin is reduced, the connection between epithelial cells is damaged, the adhesion is lost, the migration capacity of the cells is greatly increased, the morphology is changed, and finally fibrosis is caused.

The effect of phycocyanobilin binding peptide on TGF-beta 1-induced EMT-associated marker protein expression of A549 cells is shown in FIG. 11, wherein the low dose is 10. mu.g/mL, and the high dose is 30. mu.g/mL.

As can be seen from FIG. 11, the expression levels of alpha-SMA, N-cadherin and Vimentin of A549 cells induced by TGF-beta 1 are remarkably increased, while the expression level of E-cadherin is reduced, which proves that the A549 cells have the EMT process. That is, after the treatment with the phycocyanobilin-binding peptide, the EMT of the A549 cells is weakened, and the effect of inhibiting Vimentin is better particularly under high dosage (30 mu g/mL).

(4) Effect of phycocyanobilin-binding peptides on expression of HLF-1 cell alpha-SMA

In the development process of IPF, fibroblasts are induced to be transformed into myofibroblasts, and myofibroblasts have the characteristics of fibroblasts and smooth muscle cells, participate in the processes of tissue injury repair and fibrosis, and finally, inflammatory injury is caused by pulmonary fibrosis by activating myofibroblasts. The alpha-SMA is used as an important index for activating myofibroblasts, and the increase of the expression level of the alpha-SMA is often used for indicating the activation of the myofibroblasts and the development of pulmonary fibrosis. Therefore, the inhibition effect of the sample on pulmonary fibrosis can be demonstrated only by measuring the change of alpha-SMA expression level of the fibroblast under the induction of TGF-beta 1. In the experiment, TGF-beta 1 is adopted to induce human lung fibroblasts (HLF-1), then phycocyanobilin binding peptide with different concentrations is added for processing, the condition of HLF-1 cell alpha-SMA expression after 72h processing is measured by an immunofluorescence staining method, and the anti-pulmonary fibrosis capability of the phycocyanobilin binding peptide prepared by the invention is researched by comparing with a control group and a model group.

The results of immunofluorescence staining for the expression level of alpha-SMA in HLF-1 cells induced by TGF-beta 1 are shown in FIG. 12, wherein (a) is a control group, (b) is a model group, (c) is a low dose group, (d) is a high dose group, the low dose is 10. mu.g/mL, and the high dose is 30. mu.g/mL.

As can be seen in FIG. 12, the model group expressed a large amount of α -SMA after 72h of TGF- β 1 induction, and the cells gradually transformed from fibroblasts to myofibroblasts. While the green fluorescence of HLF-1 cells treated by the phycocyanobilin binding peptide is obviously reduced.

In conclusion, the phycocyanobilin binding peptide prepared by the invention shows better anti-pulmonary fibrosis activity in both A549 cells and HLF-1 cell experiments. That is, the phycocyanobilin binding peptide prepared by the invention can be applied to the preparation of anti-pulmonary fibrosis drugs.

The carbon skeletons of other three phycobilin molecules, phycourobilin, phycoerythrobilin and phycocyanobilin are the same as the carbon skeletons of phycocyanobilin, and the difference is only expressed in the difference of the positions and the number of double bonds, so the phycourobilin binding peptide, the phycoerythrobilin binding peptide and the phycoerythrobilin binding peptide are the same as the phycocyanobilin binding peptide, have better anti-pulmonary fibrosis activity and can be applied to the preparation of anti-pulmonary fibrosis drugs.

Fourth, other uses of phycobilin binding peptides

The phycourobilin binding peptide, the phycoerythrobilin binding peptide and the phycocyanobilin binding peptide have a large number of conjugated double bonds, so the phycobilin binding peptides also have good application prospects in the aspect of oxidation resistance.

In addition, since phycourobilin binding peptide, phycoerythrobilin binding peptide, and phycocyanobilin binding peptide have good color and are more stable than that of phycocyanin, these phycobilin binding peptides can be used as novel pigments.

It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.

Claims

1. The phycobilin binding peptide is characterized in that the preparation process comprises the following steps:

step 1, performing enzymolysis, namely pouring phycobiliprotein powder into an enzymolysis tank filled with pure water, wherein the mass ratio of phycobiliprotein powder to pure water is =1: 50-80, the pH value is natural, raising the temperature of water in the enzymolysis tank to 45-50 ℃, adding compound protease MC101 with the mass ratio of phycobiliprotein powder of 1-2%, uniformly stirring, and performing enzymolysis for 4 hours to obtain phycobiliprotein binding peptide feed liquid 1;

step 2, inactivating, namely heating the phycobilin binding peptide feed liquid 1 to 70-78 ℃, keeping for 6-8 min, and inactivating to obtain a phycobilin binding peptide feed liquid 2;

step 3, centrifuging, namely pumping the phycobilin binding peptide feed liquid 2 into a centrifuge for centrifuging at the centrifuging speed of 3000-4000 r/min to obtain phycobilin binding peptide feed liquid 3;

step 4, plate-and-frame filter pressing, namely pouring the phycobilin binding peptide feed liquid 3 into a plate-and-frame filter press for filter pressing, wherein a filter aid is arranged on the plate-and-frame filter press to obtain a phycobilin binding peptide feed liquid 4;

the whole preparation process is protected from light;

in step 1, the phycobiliprotein powder is phycocyanin powder;

in step 4, AG-1000# diatomite is selected as the filter aid;

in step 6, the phycobilin binding peptide is a phycocyanobilin binding peptide.

2. The phycobilin binding peptide of claim 1, wherein in step 5, the temperature of the negative pressure concentration process is controlled to be 50 ℃ and the negative pressure is 0.1 MPa.

3. The phycobilin binding peptide of claim 1, wherein the drying temperature in step 6 is 140-155 ℃.

4. Use of a phycobilin binding peptide according to any one of claims 1 to 3 for the preparation of an anti-inflammatory medicament.

5. Use of the phycobilin binding peptide of any one of claims 1 to 3 for the preparation of a medicament for the treatment of pulmonary fibrosis.