阅读说明：本技术 多肽vapfpe在改善血脂代谢并调节肠道菌群紊乱中的应用 (Application of polypeptide VAPFPE in improving blood lipid metabolism and regulating intestinal flora disorder ) 是由 郭宇星 孔维梅 王珍 刘明真 姜潇潇 张涛 陶明煊 刘琛 于 2021-09-17 设计创作，主要内容包括：本发明公开了多肽VAPFPE在制备预防或治疗高血脂类疾病的药物和/或调节菌群紊乱的药物和/或肠道胆固醇吸收抑制剂方面的应用,所述多肽VAPFPE的氨基酸序列为：缬氨酸-丙氨酸-脯氨酸-苯丙氨酸-谷氨酸。而本发明提供的六肽在体内具有缓解高脂血症的作用；本发明提供的六肽可在体内通过靶向调节肝脏和肠道血脂代谢相关因子及调节肠道菌群,协同降低血脂,缓解高脂饮食造成的血脂异常。(The invention discloses an application of polypeptide VAPFPE in preparing a medicament for preventing or treating hyperlipidemia diseases and/or a medicament for regulating flora disturbance and/or an intestinal cholesterol absorption inhibitor, wherein the amino acid sequence of the polypeptide VAPFPE is as follows: valine-alanine-proline-phenylalanine-glutamic acid. The hexapeptide provided by the invention has the function of relieving hyperlipemia in vivo; the hexapeptide provided by the invention can be used for synergistically reducing blood fat and relieving dyslipidemia caused by high-fat diet in vivo through targeting regulation of relevant factors of blood fat metabolism of liver and intestinal tract and regulation of intestinal flora.)

1. The application of the polypeptide VAPFPE in the preparation of the medicine for preventing or treating the high blood lipid diseases and/or the medicine for regulating the flora disorder and/or the intestinal cholesterol absorption inhibitor, wherein the amino acid sequence of the polypeptide VAPFPE is as follows: valine-alanine-proline-phenylalanine-glutamic acid.

2. The use according to claim 1, wherein the polypeptide VAPFPE is used for lowering one or more of hyperlipidemic serum cholesterol, hypertriglyceridemia and low density lipoprotein cholesterol.

3. The use of claim 1, wherein the polypeptide VAPFPE is used to modulate the expression of hyperlipidemic liver X receptor alpha, low density lipoprotein receptor, cholesterol 7 alpha hydroxylase protein.

4. The use according to claim 1, wherein the polypeptide VAPFPE is used for the regulation of hepatic lipid metabolism and bile acid synthesis.

5. The use according to claim 1, wherein the polypeptide VAPFPE is used for modulating the expression in a protein associated with lipid metabolism.

6. The use according to claim 1, wherein the polypeptide VAPFPE is used to inhibit the absorption of cholesterol by the small intestine.

7. The use according to claim 1, wherein the polypeptide VAPFPE is used to promote the excretion of cholesterol.

8. The use of claim 1, wherein the polypeptide VAPFPE is used to down-regulate the expression of hepatocyte nuclear factor 4 α, niemann pick C1-type analogous protein 1 in the small intestine; and/or reducing protein expression of acetyl-coa acetyltransferase 2; and/or reducing the formation of cholesterol esters in the intestinal tract; and/or up-regulate the protein expression of the adenosine triphosphate-binding transporter a1, a member of the ATP transporter G family/a member of the ATP transporter G family in the small intestine.

9. Use according to claim 1, wherein the polypeptide VAPFPE is used for decreasing clostridium (f)Clostridium_sensu_stricto_1) Genus Arthrobacter (A), (B), (C)Alistipes) Relative abundance of (a); and/or increasing the amount of rumen coccus: (Ruminococcaceae_UCG−014) Bacillus faecalis (A) and (B)Faecalibaculum) And/or Bifidobacterium (Bifidobacterium) Relative abundance of (a).

Technical Field

The invention relates to the technical field of polypeptides, in particular to application of polypeptide VAPFPE in improving blood lipid metabolism and regulating intestinal flora disturbance.

Background

Hyperlipidemia is a chronic disease caused by abnormal metabolism of blood lipids in the body, and the main clinical manifestations include high serum Total Cholesterol (TC), Triglyceride (TG) and low density lipoprotein cholesterol (LDL-C), and low high density lipoprotein cholesterol (HDL-C). Elevated serum TC levels can also lead to the development of a range of chronic diseases such as cancer, diabetes and obesity. Statins are the first choice drugs for treating hyperlipidemia, and such drugs have many side effects such as liver and kidney function damage, gastrointestinal tract reaction, rhabdomyolysis and the like. Therefore, the development of lipid-lowering active substances which are safe and reliable in food source and accord with dietary habits also becomes a research hotspot at present, and has certain economic and social significance.

Food proteins not only contain abundant nutrients, but also are an important source of bioactive peptides. The food protein can be subjected to enzymolysis or microbial hydrolysis to obtain bioactive peptides with different lengths and special physiological functions. Milk protein is a rich source of bioactive peptides, and the bioactive peptides mainly include Angiotensin-I converting enzyme (ACE) inhibitory peptide, immunoregulatory peptide, casein phosphopeptide, glycomacropeptide and the like, and research on cholesterol-lowering peptides is limited, and is mainly focused on beans, grains and marine products, but the milk-derived cholesterol-lowering reports are few.

The research on milk-derived cholesterol lowering peptides reported at present is still in the primary stage, and mainly focuses on the aspects of polypeptide preparation, in-vitro activity initial detection and the like. Most of the reported milk-derived cholesterol-lowering peptides were obtained by in vitro activity evaluation, and the in vitro activity evaluation indexes included the cholesterol micelle solubility inhibition rate and the effect of inhibiting 3-hydroxymethylglutaryl-CoA reductase (HMG-COAR). However, the entry of polypeptides into the blood circulation is affected by many factors, such as digestibility of polypeptides, hydrolyzability of trypsin and enteroprotease, and permeability of mucous membrane. The active peptide with the function of reducing cholesterol in vitro is easily decomposed by gastrointestinal enzyme systems after being taken orally, and cannot penetrate through an intestinal cell membrane to be absorbed by an intestinal tract. There is a report in the literature that an ACE (angiotensin converting enzyme) inhibitory peptide YAEERYPIL, which is hydrolyzed to YAEER and YP after passing through the brush border membrane of the small intestine. In addition, in vivo blood lipid metabolism involves many factors, such as the body's cholesterol mainly originating from dietary intake and endogenous synthesis, and the liver and intestinal tract are the two major sites of cholesterol metabolism. The cholesterol level in the body depends on the one hand on the dietary cholesterol level and the rate at which it is absorbed by the intestine, and on the synthesis and metabolism of endogenous cholesterol. The polypeptides with the function of reducing cholesterol reported in the prior art are generally polypeptide mixtures, and only the activity of reducing cholesterol in vitro is discussed, and the polypeptides are easy to degrade in the gastrointestinal tract and lose activity, so that the polypeptides cannot play a biological role in vivo, and therefore, the polypeptides with the activity of reducing cholesterol in vitro can not play the roles of reducing cholesterol and regulating blood fat in vivo. The searching for a polypeptide with the function of reducing blood fat in vivo has very important significance.

In recent years, there has been increasing evidence that abnormalities in the intestinal flora are a significant cause of obesity and various metabolic syndromes. The intestinal microbial flora is susceptible to various factors, such as daily diet, use of medicines, individual genotype, immune response or exogenous microbial infection, and the like, about 57% of the composition change of the intestinal flora is related to the change of the daily diet, and in the research on different components of the daily diet, High Fat Diet (HFD) and Dietary Fiber Substances (DFs) can obviously change the composition and functions of the intestinal microbes, so that the balance of the intestinal microbes is influenced, and various diseases can be caused by microbial flora disorder.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide the application of the polypeptide VAPFPE in the preparation of medicines for preventing or treating hyperlipidemic diseases and/or medicines for regulating flora disturbance and/or intestinal cholesterol absorption inhibitors.

The technical scheme is as follows: in order to solve the technical problems, the technical scheme adopted by the invention is as follows: the application of the polypeptide VAPFPE in the preparation of the medicine for preventing or treating the high blood lipid diseases and/or the medicine for regulating the flora disorder and/or the intestinal cholesterol absorption inhibitor, wherein the amino acid sequence of the polypeptide VAPFPE is as follows: valine-alanine-proline-phenylalanine-glutamic acid.

Wherein the polypeptide VAPFPE is used for reducing one or more of serum cholesterol, high triglyceride and low density lipoprotein cholesterol of hyperlipidemia.

The polypeptide VAPFPE is used for regulating the protein expression of a hyperlipidemic liver X receptor alpha (LXR alpha), a low-density lipoprotein receptor (LDL-R) and cholesterol 7 alpha hydroxylase (CYP7A1), regulating the blood fat and bile acid metabolism of the liver, regulating the expression of lipid metabolism related proteins, inhibiting the absorption of cholesterol by the small intestine, promoting the excretion of cholesterol, and regulating the lipid metabolism and the synthesis of bile acid of the liver.

The polypeptide VAPFPE is used for down-regulating the expression of hepatocyte nuclear factor 4 alpha (HNF4 alpha) and Niemannike C1-type analogous protein 1(NPC1L1) in small intestine, reducing the absorption of cholesterol by small intestine epithelial cells, reducing the protein expression of acetyl coenzyme A acetyltransferase 2(ACAT2), reducing the formation of cholesterol ester in intestinal tract, and up-regulating the protein expression of adenosine triphosphate binding transporter A1(ABCA1), ATP transporter G family member (ABCG5)/ATP transporter G family member (ABCG8) in small intestine, thereby promoting the excretion of cholesterol in intestinal tract.

Wherein the polypeptide VAPFPE is used for reducing the relative abundance of Clostridium (Clostridium _ sensu _ stricoto _1) and other bacilli (Alisipes); and/or increasing the relative abundance of ruminococcus (ruminococcus _ UCG-014), coprinus (faecalibaccum) and/or Bifidobacterium (Bifidobacterium).

Has the advantages that: compared with the existing polypeptide, the hexapeptide has the advantages that by establishing a hyperlipidemia model and applying to VAPFPE intervention, the hexapeptide can reduce the contents of serum cholesterol, triglyceride and low-density cholesterol lipoprotein in a hyperlipidemia mouse; the hexapeptide regulates the liver lipid metabolism and bile acid synthesis by regulating the expression of liver lipid metabolism related protein in a hyperlipidemic mouse, reduces the accumulation of lipid in the liver, and has no toxic or side effect; the hexapeptide can regulate and control the expression of intestinal cholesterol absorption related protein in a hyperlipidemic mouse, inhibit the absorption of cholesterol by small intestine and promote the excretion of cholesterol; the hexapeptide can regulate intestinal flora disorder caused by high fat diet, and VAPFPE increases the composition of Bifidobacterium, presumably related to bile acid metabolism regulation. The polypeptide with the function of reducing cholesterol reported in the prior art is generally a polypeptide mixture, and only the activity of reducing cholesterol in vitro is discussed, the polypeptide is easy to degrade in the gastrointestinal tract and loses activity, and cannot play a biological effect in vivo, but the hexapeptide provided by the invention has the function of relieving hyperlipemia in vivo; the cholesterol lowering function reported in the prior art is mainly determined by inhibiting the solubility of cholesterol micelles and the activity of HMG-CoAR, and only the in vitro function can be evaluated.

Drawings

FIG. 1 lipid concentration in serum 6 weeks after mice were fed high fat diet; note: different letters indicate that there is a significant difference between Normal and Model groups between the same assay group (P < 0.05);

FIG. 2 Effect of different peptide treatment groups on TC in mouse serum (a), the Effect of different peptide treatment groups on TG in mouse serum (b), the Effect of different peptide treatment groups on HDL-C in mouse serum (C) and the Effect of different peptide treatment groups on LDL-C in mouse serum (d); note: different letters indicate significant differences between groups (P < 0.05);

FIG. 3 effect of different peptide treatment groups on TC in mouse liver (a), effect of different peptide treatment groups on TG in mouse liver (b), effect of different peptide treatment groups on HDL-C in mouse liver (C) and effect of different peptide treatment groups on LDL-C in mouse liver (d); note: different letters indicate significant differences between groups (P < 0.05);

FIG. 4 HE staining of mouse liver (400X);

FIG. 5 classification heat map of gut microbiota at the phylum level for different peptide treated groups;

FIG. 6 categorical heat map of gut microbiota at genus level for different peptide treated groups;

FIG. 7 Linear discriminant analysis of LEfSe (Linear differential analysis Effect size) among different peptide-treated groups of intestinal microorganisms; VAPFPE (a), LQPE (b) and VLPVPQ (c);

FIG. 8 effect of VAPFPE on cholesterol metabolism-related protein expression in mouse liver; note: significant differences P < 0.05, P < 0.01 compared to normal group; significant differences compared with the model group^#P＜0.05，^##P＜0.01

FIG. 9 Effect of VAPFPE on the expression of cholesterol metabolism-related proteins in mouse small intestine; note: significant differences P < 0.05, P < 0.01 compared to normal group; significant differences compared with the model group^#P＜0.05，^##P＜0.01。

Detailed Description

The present invention is further illustrated by the following specific examples, it should be noted that, for those skilled in the art, variations and modifications can be made without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention.

Both ND and HFD feeds were purchased from indolojia feeds ltd, tokyo. The ND comprises the following components: 51.8% corn starch (w/w), 24.7% casein (w/w), 12.1% sucrose (w/w), 5.1% lard (w/w), 4.1% mineral blend (w/w), 2.1% vitamin blend (w/w), 0.1% DL-methionine (w/w). The composition of the HFD comprises: 90% basal diet, 2% cholesterol (w/w), 0.2% propylthiouracil (w/w), 0.3% sodium cholate (w/w), 7.5% lard (w/w).

EXAMPLE 1 Regulation of lipid levels in hyperlipidemic mice by three Polypeptides

1. Laboratory animal

All procedures are carried out according to the ethical examination method for experimental animals of traditional Chinese medicine institute of Jiangsu province, and the ethical approval certificate of the animals is numbered 2019-DWNL-002. 135 male mice, 4-6 weeks old, C57BL/6J, were purchased from sbeful (beijing) biotechnology limited.

2. Establishing a high fat model

All mice are kept in 12/12h light/dark cycle at room temperature (22 +/-2 ℃), water is freely taken, after adaptive feeding is carried out for 5 days, all mice are divided into two groups, a normal group (15) and a model group (120) are placed in the same room, the temperature is 22 +/-2 ℃, the relative humidity is 40% -60%, the mice are fed in cages, 5 mice are fed in each cage, food and water are freely eaten, fresh food is replaced every 2 days, padding is replaced regularly, mouse cages are cleaned, and the like, and the weight of the mice is measured once a week. After 6 weeks, 3 mice were randomly selected from the model group and the normal group, respectively, and fasted for 12 hours before blood collection, and whether a hyperlipidemia model was established was determined by measuring the contents of TC, TG, HDL-C and LDL-C in the mouse serum.

3. Animal experiment grouping

The three polypeptides LQPE (Leu-Gln-Pro-Glu), VLPVPQ (Val-Leu-Pro-Val-Pro-Gln) and VAPFPE (Val-Ala-Pro-Phe-Pro-Glu) are derived from milk casein, have a purity of more than 95 percent and are synthesized by Shanghai Baotai Biotech limited. From the normal group and the model group, 10 and 110 well-grown mice were selected, respectively. Model group mice were randomly divided into 11 groups (n ═ 10): high fat diet group (HFD) + saline (model group, model); HFD +10mg/kg body weight lovastatin; HFD +20mg/kg bw LQPE (LQPE low dose group, L-LQPE); HFD +40mg/kg bw LQPE (dose group in LQPE, M-LQPE); HFD +80mg/kg bw LQPE (LQPE high dose group, H-LQPE); HFD +20mg/kg bw VLPVPQ (VLPVPQ low dose group, L-VLPVPQ); HFD +40mg/kg bw VLPVPQ (VLPVPQ middle dose group, M-VLPVPQ); HFD +80mg/kg bw VLPVPQ (VLPVPQ high dose group, H-VLPVPQ); HFD +20mg/kg bw VAPFPE (VAPFPE low dose group, L-VAPFPE); HFD +40mg/kg bw VAPFPE (VAPFPE middle dose group, M-VAPFPE); HFD +80mg/kg bw VAPFPE (VAPFPE high dose group, H-VAPFPE).

4. Intragastric administration scheme

The normal control group was given Normal Diet (ND), the other experimental groups were given HFD, and the ND and HFD feeds were purchased from inojia feeds ltd, tokyo, south. The treatment group was given the corresponding test polypeptides LQPE, VLPVPQ, VAPFPE and lovastatin, and the normal group and the model group were given the same dose of saline, and were separately gavaged daily at the same time in the morning for 8 weeks. Body weights were recorded once a week and drug doses were adjusted according to body weight.

5. Animal treatment and detection

5.1 organ indices

The mouse feces were collected 3 days before the end of the experiment and stored rapidly at-80 ℃. After all mice are fasted for 12 hours before the experiment is finished, blood is collected from the orbit, the liver, the kidney, the heart, perirenal fat, the testis and epididymal fat are immediately taken out, the organs are cleaned by ice-cold normal saline, water is sucked by filter paper, the organ indexes are weighed and calculated, and the liver and small intestine tissues are quickly placed at the temperature of minus 80 ℃ for storage, so that the subsequent detection is convenient.

Organ index (%) ═ weight of organ (g)/weight of animal (g) × 100 (1)

5.2 detection of blood lipid indicators

Blood samples were collected in EP tubes at 3000 Xg, 4 ℃ for 15min and serum was collected. And (3) measuring the contents of TG, TC and LDL-C, HDL-C in serum by using a full-automatic biochemical analyzer.

5.3 detection of indexes related to hepatic lipid metabolism

Frozen mouse liver was homogenized in physiological saline, centrifuged at 3000 Xg for 15min at 4 ℃ and the supernatant was collected. The contents of TG, TC, LDL-C and HDL-C were determined by a full-automatic biochemical analyzer.

5.4 liver microsome preparation

Weighing 0.5g of mouse liver tissue, adding 1mL of precooled Tris-HCl-sucrose buffer (with the concentration of 0.1M and the pH value of 7.4), homogenizing by using a tissue homogenizer, centrifuging the tissue homogenate at 10000 Xg and 4 ℃ for 10min, sucking froth on the surface of a sample, transferring the rest supernatant into an ice-bath ultra-high-speed centrifuge tube, adding precooled Tris-HCl-sucrose buffer to dilute to 8.333mL, centrifuging at 100000 Xg and 4 ℃ for 60min, taking out the sample after the centrifugation is finished, immediately putting the sample into ice, discarding the supernatant, slightly adding 30% Tris-HCl-sucrose buffer along the wall to clean the surface and suck the sample, adding 0.1mL of 30% Tris-HCl-glycerol precooled buffer (with the pH value of 7.4 and 20% glycerol) to resuspend the sample to obtain a liver microparticle sample, and subpackaging and storing the sample at-80 ℃.

5.5 liver HE staining

Cleaning and drying liver, collecting liver right leaf three tissue specimens, and cutting with scalpel to obtain 1cm³It was quickly placed in a glass vial containing formaldehyde fixative and cooled to 4 ℃ until ready for use. Taking out the liver tissue block, fixing, embedding with conventional paraffin, and slicing at 4 μm; dewaxing by xylene after slicing, washing by xylene for 5min, then washing by new xylene for 5min, washing by 100% ethanol for 2min, washing by 95% ethanol for 1min, washing by 80% ethanol for 1min and washing by 75% ethanol for 1min, and finally washing by distilled water for 2 min; then staining with hematoxylin for 5min, and washing with tap water until no residue is left on the surface; differentiating with hydrochloric acid ethanol for 30s, and soaking in tap water for 15 min; taking out and placing in eosin solution for dyeing for 2 min; and finally, carrying out conventional dehydration and sealing, wherein the dehydration steps sequentially comprise 95% ethanol decolorization for 1min, new 95% ethanol decolorization for 1min, 100% ethanol decolorization for 1min, new 100% ethanol (II) decolorization for 1min, xylene carbonic acid decolorization for 1min, xylene (I) decolorization for 1min and xylene (II) decolorization for 1min, and finally sealing with neutral resin. Liver histopathological examination was performed.

6. Analysis of results

6.1 hyperlipidemia model establishment analysis

As can be seen from FIG. 1, after the mice were fed with the high-fat diet for 6 weeks, the serum TC, TG and LDL-C of the model group mice were significantly increased (P < 0.05) compared to the normal group, which proves that the high-fat diet causes the lipid metabolism disorder of the mice, and the model of hypercholesterolemia was successfully established.

6.2 organ index of mice in each group

The results are shown in table 1, and compared with the normal group, the fat coefficients of the liver, the testis and the epididymis are obviously increased (P is less than 0.05); compared with a model group, the fat coefficients of testis and epididymis are reduced by the gastric lavage lovastatin and the three polypeptides, wherein the fat coefficients of testis in a VAPFPE high dose group and a LQPE medium dose group are remarkably reduced (P is less than 0.05) compared with the model group, except for a VLPVPQ high dose group, the fat coefficients of epididymis in other treatment groups are remarkably reduced (P is less than 0.05), and the three polypeptides can reduce the deposition of lipid in the organs of mice to a certain extent.

TABLE 1 organ index of each group of mice

6.3 serum and liver lipid levels

As can be seen from FIGS. 2 and 3, the serum TG, TC, LDL-C, HDL-C and TC and LDL-C in the liver were significantly increased in the model group mice as compared with the normal group. Comparing the results in fig. 1, it can be seen that the HDL-C of the model mice after 6 weeks of high fat diet has no significant difference from the HDL-C of the normal group, while after 14 weeks of experiment, the model mice after continuous high fat diet are dyslipidemic disorder and HDL-C may increase in inflammatory tendency to form a mixed type hypercholesterolemia model.

FIG. 2 shows that after 8 weeks of gastric lavage, both the VAPFPE medium and high dose groups significantly reduced serum TC levels (P < 0.05), but not LQPE and VLPVPQ, compared to the model group; the three polypeptides can reduce the content of TG in serum (P is less than 0.05), but the VAPFPE has the best reduction effect; except for the LQPE low dose group, each of the other treatment groups had the best effect (P < 0.05) on reducing LDL-C in serum, but VAPFPE. FIG. 3 shows that the VAPFPE high dose group significantly reduced TC and LDL-C levels in the liver of hyperlipidemic mice, while the other two polypeptides were not effective; there was no significant difference in hepatic TG content (P > 0.05) in each experimental group.

In conclusion, VAPFPE has better effect of reducing the TC, TG and LDL-C levels of the serum and the liver of a hyperlipemia model mouse compared with LQPE and VLPVPQ.

6.4 HE staining results for groups of mice

As can be seen from FIG. 4, after HE staining of mouse liver, the liver tissue section of the normal control group has clear structure, obvious boundary between cells, liver cell cords in peripheral divergent arrangement, clear outline, centered nucleus and more cytoplasm. Compared with the normal group, the model group has obvious damage to the structure of the liver cells, cell swelling, a large amount of white fat droplets, cell nucleus shift and no complete liver cell cord. Compared with a high-fat model group, lipid droplets in the VLPVPQ high-dose group are obviously reduced, partial cells of the VAPFPE medium-dose group and the VAPFPE high-dose group are recovered to be normal, the cell boundary is clear, the lipid droplets are obviously reduced, the overall cell degeneration is obviously improved, and compared with lovastatin, the VAPFPE has more obvious improvement on the liver tissue structure and the liver cells. Lovastatin is a common statin lipid-lowering drug in the market, and has side effects of liver and kidney function damage and the like after long-term administration, while the milk-derived peptide VAPFPE used in the experiment has a good improvement effect on liver tissue pathological changes caused by high-fat diet and has no obvious toxic or side effect.

EXAMPLE 2 modulation of intestinal flora in hyperlipidemic mice by three Polypeptides

1. DNA extraction/quality control

Extracting DNA from mouse feces by using DNA extraction kitdsDNA HS Assay Kit to detect DNA concentration.

2. PCR amplification and library construction

Prokaryotes were amplified using a series of PCR primers designed with Kingchi using 20-30ng of DNA as template. The V3 and V4 regions were amplified using a forward primer comprising the sequence "CCTACGGRRBGCASCAGKVRVGAAT" and a reverse primer comprising the sequence "GGACTACNVGGGTWTCTAATCC". In addition, linkers with Index were added to the ends of the PCR products of 16SrDNA by PCR for NGS sequencing.

TABLE 2 PCR amplification System

PCR reaction parameters: the pre-denaturation parameters 94 ℃ and 3min, the denaturation parameters 94 ℃ and 5s, the annealing parameters 57 ℃ and 90s, the elongation 72 ℃ and 10s, and the final elongation parameters 72v and 5min, for 24 cycles.

And (3) identifying a PCR amplification result: the PCR product was detected by electrophoresis on a 1.5% agarose gel.

3. Sequencing on machine

The library concentration was detected by a microplate reader. The library was quantitated to 10nM, paired-end sequencing was performed according to the Illumina MiSeq/NovaSeq sequencer instructions, and sequence information was read by the associated software onboard the instrument.

4. Data analysis

And firstly splicing the forward and reverse reads obtained by double-end sequencing in pairs, filtering sequences containing N in the splicing result, and reserving the sequences with the sequence length being more than 200 bD. The chimera sequence was removed by mass filtering, the final sequence was used for OTU clustering, VSEARCH was used for sequence clustering (sequence similarity set to 97%), and the 16S rRNA reference database for alignment was silvera 132. Representative sequences of OTUs were then analyzed for taxonomy of species using RDP classifier (ribosol Database Program) bayesian algorithm and the community composition of each sample was counted at different taxonomic levels of species.

5. Experimental data processing and analysis

The experimental data were statistically analyzed using SPSS 19.0 statistical software, and the one-way analysis of variance examined the differences in significance between groups, with data expressed as mean + -SD and different letters indicating significant differences at levels P < 0.05.

6. Results and analysis

6.1 Regulation of the groups at the Classification level of the mouse intestinal flora

FIG. 5 shows the variation of species abundance at phylum taxonomic level for the intestinal flora of mice in each experimental group. It can be seen from the figure that intestinal microorganisms belong mainly to the phylum Firmicutes, Bacteroidetes, Proteobacteria and actinomycetes. Compared with a normal group, the high-fat diet causes the relative abundance of Firmicutes and Proteobacteria in the intestinal tracts of mice to be increased, the relative abundance of Bacteroidetes is reduced, Firmicutes are increased, bacteroides are reduced, lipid metabolism abnormality and obesity are closely related, pathogenic bacteria such as escherichia coli, salmonella, vibrio cholerae, helicobacter pylori and the like are included in Proteobacteria, the Proteobacteria is one of the markers of intestinal homeostasis disorder, and the increase of the abundance is generally related to the metabolic disorder and inflammatory response of a host. After three polypeptides of VAPFPE, VLPVPQ and LQPE are respectively given for prediction, the abundance of intestinal microorganisms changes to different degrees, and compared with a model group, the relative abundance of firmicutes and proteobacteria in a VAPFPE high-dose group is reduced, and the relative abundance of bacteroidetes is increased; the relative abundance of firmicutes in the dose group in LQPE was decreased and the relative abundance of bacteroidetes was increased; relative abundance of proteobacteria in the dose group in the vlppq was reduced.

As can be seen from fig. 6, the relative abundance of Lactobacillus (Lactobacillus), allobacter (AlistiDes), Clostridium (Clostridium _ sensu _ stricoto _1), and lachnospira (Lachnospiraceae _ NK4a136_ group) in the intestinal tract of the model group mice increased compared to the normal group.

Research reports that increased xenobacter (Alistipes) disrupts the balance of the serotonin-activated system in the gut and may increase the incidence of gut inflammation; clostridium _ sensu _ stricoto _1 belongs to the family Clostridiaceae (Clostridiaceae), which contains many pathogenic bacteria. Studies have shown that increased relative abundance of Lachnospiraceae (Lachnospiraceae) in the intestinal tract compared to normal populations may cause disturbances in carbohydrate metabolism in vivo. Compared with the model group, the relative abundance of other bacilli (Alistipes) in the intestinal tract is reduced by each of the LQPE low dose group, the VLPVPQ high dose group and the vappq, the relative abundance of Clostridium sense strain _1 is reduced by each of the LQPE medium dose group, the vappe high dose group and the LQPE high dose group, the relative abundance of ruminococcus (ruminococcus _ UCG-014) in the intestinal tract of mice is increased by each of the LQPE medium dose group, the LQPE high dose group, the VLPVPQ low dose group and the VLPVPQ high dose group, the relative abundance of ruminococcus _ UCG-014 in the intestinal tract is significantly increased by each of the ruminococcus _ UCG-013 in the intestinal tract, and the relative abundance of ruminococcus _ UCG-lansised in the intestinal tract is increased by each of the LQPE low dose group and the high dose group. More bacteria in the rumen coccaceae family (Ruminococcaceae) are capable of converting primary bile acids into secondary bile acids, and are closely related to the metabolism of cholesterol.

As can be seen from fig. 7, the dose groups in VAPFPE, LQPE and vlppq significantly increased the relative abundance of erysipelomyces (Erysipelotrichia), erysipelomyces (Erysipelotrichales), erysipelomyces (erysipelotrichaee) and zurich (turibacter) in the mouse intestine (P < 0.05), and it was reported that the relative abundance of Erysipelotrichales and Turicibacter was significantly decreased and the probability of colitis in mice was increased. The VAPFPE medium dose group and LQPE low dose group significantly increased the relative abundance (P < 0.05) of faecalibacillus, a class of Short Chain Fatty Acid (SCFAs) producing bacteria, maintaining the integrity of the intestinal wall barrier and preventing intestinal inflammation. In addition, the VAPFPE high-dose group enables the relative abundance of bifidobacteria (bifidobacteria), Bifidobacteriaceae and bifidobacteria (Bifidobacterium) in intestinal tracts of mice to be remarkably increased (P is less than 0.05), the bifidobacteria are dominant bacteria in healthy intestinal tracts, have bile salt hydrolase activity, can effectively remove cholesterol in the intestinal tracts through various mechanisms such as assimilation absorption, thallus adsorption and the like, and reduce serum cholesterol. In conclusion, VAPFPE can regulate intestinal flora of hyperlipidemic mice and promote the increase of flora which is beneficial to reducing the cholesterol content in vivo.

Example 3 Effect of VAPFPE on expression level of genes involved in Cholesterol metabolism in mice

Through experiments of example 1 and example 2, it can be found that among the three polypeptides, VAPFPE can significantly reduce the content of TC, TG and LDL-C in a hyperlipidemic mouse, significantly reduce the liver fat vacuole area of the hyperlipidemic mouse, reduce the accumulation of liver fat, and regulate the intestinal flora disorder of the hyperlipidemic mouse, promote the increase of the flora beneficial to reducing the cholesterol content in vivo, such as bifidobacterium, while the other two polypeptides, LQPE and VLPVPQ, have insignificant effect of improving the lipid level of the hyperlipidemic mouse, and do not increase the number of the intestinal flora bifidobacterium. Therefore, the temperature of the molten metal is controlled,

example 4 study of the regulatory mechanism of VAPFPE on mouse lipid metabolism by Western blot

1. Total tissue protein extraction

Taking out tissues (liver and intestinal tract) stored at minus 80 ℃, rapidly shearing 0.2g of each of the liver and intestinal tract tissue samples, dividing the tissues into homogenizers arranged on ice, adding 0.4mL of RIPA lysate containing 1% PMSF into each tissue, homogenizing on ice until no obvious tissue residues exist, then cracking on ice for 1.5h, 13000 Xg, centrifuging for 30min at 4 ℃, sucking supernatant, measuring protein content in Nano-100, unifying the protein into the same concentration by the RIPA lysate, adding SDS protein loading buffer, denaturing at 100 ℃ for 8min, rapidly placing on ice for cooling, and centrifuging before Western blot is used.

2. Western blot method for detecting protein expression

SDS gels with different concentrations are prepared according to the molecular weight of target protein, the loading amount of each porin is 120 mug, the electrophoresis conditions are that the constant pressure of concentrated gel is 75V, the constant pressure of separation gel is 135V, and the electrophoresis time is about 2 h. After electrophoresis is finished, the glass plate is peeled off, the concentrated gel is removed, a proper separation gel block is cut according to the required protein molecular weight, and the membrane is transferred by using a semi-dry transfer membrane method. The method comprises the following specific steps: cutting out proper separation gel, paving a conversion membrane sandwich structure in the sequence of filter paper-gel-membrane-filter paper from the negative electrode to the positive electrode, carefully removing bubbles, calculating current according to the multiplication of the membrane area (cm2) by a coefficient of 1.2(mA), performing constant current conversion on the membrane for 60-90 min, and transferring the protein to the PVDF membrane. Full wet transfer membrane method: cutting out proper separation glue, paving a film transfer sandwich structure in the sequence from the cathode to the anode filter paper, glue, film and filter paper, clamping a frame, placing the frame into a film transfer groove filled with a film transfer liquid, placing the film transfer groove in an ice bath, and transferring the film at a constant current of 300mA for 2.5-4 h; after the membrane transfer is finished, sealing the PVDF membrane for 2 hours on a shaking table at 4 ℃ by using BSA with the concentration of 3%, discarding the sealing liquid, and cleaning the PVDF membrane for 10min for 3 times by using PBST; adding primary antibody with appropriate concentration, incubating overnight at 4 deg.C in a shaking table, recovering primary antibody every other day, and washing with PBST for 10min for 3 times; adding a secondary antibody diluted by PBST, incubating for 2h at a constant temperature of 4 ℃ by using a shaking table, removing the secondary antibody after the incubation is finished, and cleaning the strip by using PBST for 3 times, wherein each time lasts for 10 min; preparing fresh ECL luminescent liquid according to a certain proportion, dripping the ECL luminescent liquid on the PVDF membrane to be detected, scanning the membrane in a gel imaging analysis system for imaging, and analyzing the Western Blot result.

3. Quantitative and statistical analysis

And quantitatively calculating a gray value by using imageJ software, wherein the protein expression quantity of the target gene is the ratio of the gray value of the target protein to the gray value of the internal reference.

4. Results and analysis

4.1 Effect of VAPFPE on expression levels of proteins involved in the metabolic pathways of cholesterol in mouse liver

The results are shown in fig. 8, the expression level of liver X receptor alpha (LXR alpha) in the hyperlipidemia model group is increased, LXR alpha plays an important role in fat metabolism, and target genes thereof include related enzymes of bile acid synthesis, fatty acid synthesis and cholesterol excretion, which indicates that high-fat diet affects lipid metabolism of mice, and VAPFPE can restore LXR alpha to normal level and improve blood lipid metabolism; the cholesterol 7 alpha hydroxylase (CYP7A1) expression of the hyperlipemia model group is increased, the CYP7A1 expression quantity of the VAPFPE group can be restored to a normal level relative to the model group, and the VAPFPE is also proved to be capable of regulating the bile acid metabolism of the liver; the protein expression of liver low-density lipoprotein receptor (LDL-R) is reduced in the hyperlipidemia model group, LDL-C in blood is accumulated, and the VAPFPE group can promote the transportation of LDL-C from blood to liver to participate in metabolism again, so that the LDL-C level in blood is reduced.

4.2 Effect of VAPFPE on expression levels of related proteins in metabolic pathways of cholesterol in mouse Small intestine

The results are shown in fig. 9, compared with the model group, the VAPFPE treatment group can significantly improve the protein expression of LXR α, down-regulate the protein expression of hepatocyte nuclear factor 4 α (HNF4 α) and niemann-pick C1-type analogous protein 1(NPC1L1) in small intestine, and reduce the absorption of cholesterol by small intestine epithelial cells; compared with the model group, the VAPFPE treatment group reduces the protein expression level of acetyl coenzyme A acetyltransferase 2(ACAT2) and reduces the formation of cholesterol ester in intestinal tract; compared with the model group, the VAPFPE treatment group can remarkably up-regulate the protein expression amount of an adenosine triphosphate binding transporter A1(ABCA1), an ATP transporter G family member 5(ABCG5) and an ATP transporter G family member (ABCG8) in the small intestine, thereby promoting the excretion of cholesterol in the intestinal tract.

Therefore, the VAPFPE can reduce the absorption of cholesterol and the formation of cholesteryl ester by the intestinal tract, promote the discharge of the cholesterol in the intestinal tract, regulate the reverse transport of the hepatic LDL-C and the metabolism of bile acid, reduce the contents of TC, TL and LDL-C in blood and further relieve the hyperlipidemia by regulating the expression of the intestinal tract and liver lipid metabolism related target genes.

Sequence listing

<110> university of Nanjing university

Application of polypeptide VAPFPE in improving blood lipid metabolism and regulating intestinal flora disorder

<160> 3

<170> SIPOSequenceListing 1.0

<210> 2

<211> 4

<212> PRT

<213> LQPE(Artificial Sequence)

<400> 2

Leu Gln Pro Glu

1

<210> 1

<211> 6

<212> PRT

<213> VLPVPQ(Artificial Sequence)

<400> 1

Val Leu Pro Val Pro Gln

1 5

<210> 3

<211> 6

<212> PRT

<213> VAPFPE(Artificial Sequence)

<400> 3

Val Ala Pro Phe Pro Glu

1 5