阅读说明：本技术 一种全蝎活性肽、药物组合物及其在制备抗肿瘤产品中的应用 (Scorpion active peptide, pharmaceutical composition and application thereof in preparation of anti-tumor products ) 是由 魏永利 王少平 张加余 马传江 代龙 张贺 马睿 于 2021-11-05 设计创作，主要内容包括：本发明涉及一种全蝎活性肽、药物组合物及其在制备抗肿瘤产品中的应用。活性肽是动物类中药的药效物质基础,现有研究证实了全蝎活性肽对肺癌治疗效果明显,本发明目的在于提供一种高效抑制肿瘤的活性单体肽。基于上述目的,本发明从全蝎的肽提取物中筛选得到高活性的肽段,该肽段对肝癌、骨肉瘤等具有高效抑制作用,可通过多种机制抑制肿瘤细胞的生长,有望应用于抗肿瘤相关药物的研发。(The invention relates to scorpion active peptide, a pharmaceutical composition and application thereof in preparing anti-tumor products. The active peptide is the drug effect substance basis of animal traditional Chinese medicines, the existing research proves that the scorpion active peptide has obvious effect on treating the lung cancer, and the invention aims to provide the active monomer peptide for efficiently inhibiting the tumor. Based on the purposes, the invention screens the high-activity peptide segment from the scorpion peptide extract, the peptide segment has high-efficiency inhibition effect on liver cancer, osteosarcoma and the like, can inhibit the growth of tumor cells through various mechanisms, and is expected to be applied to the research and development of anti-tumor related medicines.)

1. The amino acid sequence of the active peptide of scorpion is as follows:

(1)LERTDDPSVA；

(2) is the amino acid sequence with the same physiological activity after adding, reducing and replacing one or more amino acids in the (1).

2. The active peptide of claim 1, wherein the active peptide has the sequence of LERTDDPSVA, and has the structure shown in the following formula 1, the isoelectric point of 11.18, and the hydrophobicity index of +8.21Kcal mol^-1：

3. The scorpion active peptide as claimed in claim 2, further comprising modified forms of the active peptide, wherein the modified forms include but are not limited to salt-forming modification for improving physical properties such as water solubility, active group modification for improving pharmacological activity, or luminescent group and tracer group modification.

4. The scorpion active peptide of claim 2, further comprising a fusion peptide comprising the active peptide;

preferably, a fusion peptide modified from LA-10 is a cell-penetrating peptide or the like, wherein the cell-penetrating peptide is any one of a natural protein, a chimeric peptide and an artificially synthesized peptide;

further preferred are any one of TAT, R9, MPG delta NLS, Stearyl-R8, Transportan, Pep-1;

further preferably, the membrane-penetrating peptide is linked to LA-10 in one of wrapping, electrostatic interaction or covalent bond connection;

preferably, the fusion peptide further comprises a connecting arm, wherein the connecting arm is used for connecting the cell-penetrating peptide and LA-10;

further preferably, the connecting arm is a short peptide with 1-6 amino acids.

5. A pharmaceutical composition comprising the scorpion active peptide of any one of claims 1 to 4.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition comprises other ingredients having anti-tumor activity;

or, in the pharmaceutical composition, further comprising a buffering agent for maintaining the activity of the polypeptide;

or, the pharmaceutical composition also comprises a pharmaceutically necessary carrier.

7. An anti-tumor product, which comprises the scorpion active peptide of any one of claims 1 to 4 or the pharmaceutical composition of claim 5 or 6.

8. The anti-tumor product of claim 7, wherein the anti-tumor product comprises an anti-tumor drug, an anti-tumor health product, and an anti-tumor model drug;

or, the anti-tumor product includes but is not limited to preparations for resisting brain tumor, oral tumor, lung cancer, gastric cancer, liver cancer, intestinal cancer, uterine tumor or osteosarcoma.

9. A product for modulating the intestinal flora, comprising the active peptide of any one of claims 1 to 4 or the pharmaceutical composition of claim 5 or 6.

10. The product for modulating the intestinal flora of claim 9, wherein the medicament for modulating the intestinal flora comprises but is not limited to the medicament for modulating the intestinal flora of firmicutes, bacteroidetes, tenericutes; further, the intestinal flora regulating medicament is used for up-regulating firmicutes or down-regulating bacteroidetes and tenebrio.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to scorpion bioactive peptide, a pharmaceutical composition containing the scorpion bioactive peptide, and application of the scorpion bioactive peptide in preparation of anti-tumor products.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The active peptide is the material basis of the drug effect of animal traditional Chinese medicines, can enter a human body to play the drug effect by active absorption in a complete molecular form through a peptide transport channel of intestinal epithelial cells, and has the advantages of rapid absorption, complete absorption and the like. In recent years, researchers at home and abroad have studied active peptides derived from animal traditional Chinese medicines. Active peptides obtained by enzymolysis of earthworm by pepsin, such as Chenping, can obviously reduce the content of total cholesterol and triglyceride in plasma of mice. Researches on Xuchengqin and the like find that the leech active peptide can regulate and control the activity of lipid synthesis related enzyme to play a role in reducing blood fat by inhibiting related factors such as NKK3 and P38 at the downstream of a P38AMPK signal channel. The research reports fully confirm that the active peptide is the basis of the drug effect substances of the animal traditional Chinese medicines.

Proved by verification, the scorpion is continuously enzymolyzed by pepsin and trypsin, and is assisted by an ultrafiltration membrane treatment technology to obtain the scorpion bioactive peptide, and the scorpion bioactive peptide has an obvious treatment effect on lung cancer.

Research shows that the number of conditioned pathogens is increased to different degrees and the number of probiotics is greatly reduced in models of lung cancer patients, rats, mice and the like, for example, the content of harmful bacteria such as lacospirillum and coprococcus is increased rapidly, and the content of beneficial bacteria such as bifidobacterium and lactobacillus is greatly reduced. The diversity of intestinal flora can ensure the normal absorption and metabolism of lipid, and the adjustment of intestinal flora can reduce the blood lipid index in the blood serum of lung cancer patients. This also indicates a close relationship between the intestinal flora and lung cancer.

Disclosure of Invention

Based on the above technical background, the present invention aims to provide an active monomeric peptide for efficiently inhibiting tumors. The active peptide for inhibiting the tumor in vitro with high efficiency is obtained by screening the scorpion protein extract, and further research proves that the active peptide can induce the tumor cell apoptosis and the tumor cell scorching, and is expected to be applied to the development of anti-tumor related products.

Based on the technical effects, the invention provides the following technical scheme:

in a first aspect of the present invention, a scorpion active peptide is provided, wherein the amino acid sequence of the active peptide is as follows:

(1)LERTDDPSVA；

(2) is the amino acid sequence with the same physiological activity after one or more amino acids are added, reduced and replaced in the step (1).

In a preferred scheme, the sequence of the scorpion active peptide is LERTDDPSVA (LA-10), the structure of the scorpion active peptide is shown as the following formula 1, the isoelectric point is 11.18, and the hydrophobic index is +8.21Kcal & mol^-1：

It is known in the art that the polypeptide extract in scorpion has good anti-tumor activity, but the scorpion peptide extract provided in the prior art is usually a whole peptide part, is a mixture with high molecular weight, and may face the defects of inactivation and low bioavailability in the practical application process. The present invention relates to a method for screening high-activity portion from scorpion peptide. The LA-10 has proper molecular weight, and can meet the requirements of various pharmaceutical dosage forms on the molecular weight of active ingredients. Moreover, the active peptide has good inhibition effect on liver cancer and sarcoma through verification, and the inhibition effect is obviously better than that of other parts in scorpion peptide.

In addition, the invention also verifies that the polypeptide can influence the change of intestinal flora in an animal model after being used for a lung cancer model, and shows that the polypeptide component can realize the tumor inhibition effect through an intestinal flora-anti-tumor mechanism. The existing research proves that the anti-tumor mechanism of the intestinal flora comprises various ways of activating the release of anti-tumor factors of organisms, preventing the formation of carcinogens in the intestines, preventing gene mutation, influencing the activity of tumor telomerase and the like to realize the tumor inhibition effect, the intestinal regulation effect of the polypeptide is a treatment effect realized by enhancing the immune function of the organisms, and the treatment idea of strengthening the body resistance and consolidating the foundation by traditional Chinese medicines is well embodied.

In a preferred embodiment, the active peptide also includes a modified form of the active peptide, and the modified form includes, but is not limited to, salt-forming modification for improving physical properties such as water solubility, active group modification for improving pharmacological activity, or luminescent group, tracer group modification, and the like.

In a preferred embodiment, the active peptide further includes a fusion peptide comprising the active peptide, such as a fusion peptide obtained by modifying LA-10 with a cell-penetrating peptide, and the like, wherein the cell-penetrating peptide is any one of a natural protein, a chimeric peptide and an artificially synthesized peptide; further preferred are any of TAT, R9, MPG Δ NLS, Stearyl-R8, Transportan, Pep-1, but not limited thereto.

Further preferably, the attachment of the cell-penetrating peptide to LA-10 is one of encapsulation, electrostatic interaction or covalent bond attachment.

Preferably, the fusion peptide further comprises a connecting arm for connecting the cell-penetrating peptide and LA-10.

Further preferably, the connecting arm is a short peptide with 1-6 amino acids.

In a second aspect of the present invention, a pharmaceutical composition is provided, which comprises the scorpion active peptide of the first aspect.

Preferably, the pharmaceutical composition comprises other components with antitumor activity.

Preferably, the pharmaceutical composition further comprises a buffer agent for maintaining the activity of the polypeptide and the like.

Preferably, the pharmaceutical composition further comprises a pharmaceutically necessary carrier.

In a third aspect of the present invention, an anti-tumor product is provided, wherein the anti-tumor product comprises the scorpion active peptide of the first aspect or the pharmaceutical composition of the second aspect.

Preferably, the anti-tumor product comprises an anti-tumor drug, an anti-tumor health product and an anti-tumor model drug.

Preferably, the anti-tumor product includes but is not limited to preparations for resisting brain tumor, oral tumor, lung cancer, gastric cancer, liver cancer, intestinal cancer, uterine tumor or osteosarcoma.

In a fourth aspect of the present invention, there is provided a product for modulating the intestinal flora, said product comprising the active peptide of the first aspect or the pharmaceutical composition of the second aspect.

Preferably, the medicament for modulating the intestinal flora includes, but is not limited to, the medicament for modulating the intestinal flora of firmicutes, bacteroidetes, tenericutes; further, the intestinal flora regulating medicament is used for up-regulating firmicutes or down-regulating bacteroidetes and tenebrio.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a relative abundance analysis of the gates described in example 1;

wherein FIG. 1A is the mean gate relative abundance; FIG. 1B is the relative abundance of all sample gates; figure 1C is a heat map of gate relative abundance.

FIG. 2 is a graph showing the relative abundance analysis of the classes described in example 1;

wherein FIG. 2A is the mean relative abundance of the classes; FIG. 2B is the relative abundance of all classes of samples; FIG. 2C is a heat map of class relative abundance.

FIG. 3 is the relative abundance of the targets described in example 1;

wherein, FIG. 3A is the mean value of the relative abundance of the eye; FIG. 3B is the relative abundance of all sample mesh; figure 3C is a heat map of relative abundance of the eye.

FIG. 4 is a microbiota differential species analysis described in example 1;

wherein FIG. 4A is the average relative abundance of different species; FIG. 4B is the relative abundance of all sample differential species; figure 4C is a thermal map of relative abundance of different species.

FIG. 5 is a graph showing the results of LA-10 in example 1 reducing the serum IL-6 level in mice of the orthotopic lung cancer model.

FIG. 6 is a graph showing the results of LA-10 in example 1 reducing the serum IL-8 level in mice of the orthotopic lung cancer model.

FIG. 7 is a graph showing the results of LA-10 in example 1 reducing the serum TNF-. alpha.content in mice of the orthotopic lung cancer model.

FIG. 8 is a morphological observation of LA-10 on S180 sarcoma cell apoptosis;

wherein, the left figure is a cell morphology figure before administration, and the right figure is a cell morphology figure after administration.

FIG. 9 is a morphological observation of scorching of liver cancer cells HepG2 by LA-10;

wherein, the left figure is a cell morphology figure before administration, and the right figure is a cell morphology figure after administration.

FIG. 10 shows the results of flow cytometry for LA-10 pro-cell apoptosis;

wherein, the left picture is the result of LA-10 scorching of HepG2 human liver cancer cells; the right panel shows the results of apoptosis of S180 sarcoma cells by LA-10.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the technical solution of the present invention will be described in detail below with reference to specific examples and comparative examples.

Example 1

1. Preparation of active peptide LA-10

(1) Preparing the scorpion bionic enzymatic hydrolysate: pulverizing Scorpio, sieving with 50 mesh sieve, adding 10 times of deionized water, stirring, heating to boil, standing to room temperature, adjusting pH to 1.5-2.5 with dilute hydrochloric acid solution, adding pepsin 1% of substrate amount, stirring, standing at 37 deg.C, oscillating for 2 hr, adjusting pH to 7.5-8.5 with 10% sodium hydroxide, adding trypsin 2% of substrate amount, stirring, standing at 50 deg.C, oscillating for 4 hr, adjusting pH constantly to 8.0, boiling to inactivate enzyme, standing to room temperature, 5000 r.min^-1Centrifuging for 15min, and separating supernatant to obtain Scorpio active peptide.

(2) The mass spectrometry complete sequence analysis of the scorpion active peptide:

preparing a sample: the sample was dissolved in 200. mu.l of 25mM ammonium bicarbonate solution, added with 2. mu.L of 1M DTT and placed in 60 ℃ water bath for >40min, then added with 8. mu.L of freshly prepared 1M iodoacetamide and placed in the dark at room temperature for 30 min. Desalting the sample by using a trace desalting column, adding 10 mu L of loading buffer solution into the dried sample, and oscillating and dissolving the sample to be detected.

The testing method comprises the following steps: the nanoliter liquid chromatography column is a C18 reversed phase analytical column (50mm × 15cm × 3 μm); the mobile phase A is a mixed solution of 99.9 percent of water and 0.1 percent of formic acid, and the mobile phase B is a mixed solution of 80 percent of acetonitrile and 0.1 percent of formic acid. The liquid phase gradient is: 0-3min, 2-8% B; 3-42min, 8-20% B; 42-48min, 20% -35% B; 48-49min, 35-100% B; 49-60min, 100% B. The flow rate of the mobile phase was 300 nL/min. ESI + mode, using a data-dependent scan mode, full scan acquisition (m/z 200-. The separated first 20 peptide signal (charge state ≥ 1) parent ions were fragmented by high energy collision (HCD), with a Normalized Collision Energy (NCE) of 28.0. The temperature of the capillary was 275 ℃ and the spray voltage was 1800V. The daughter ions were measured on an orbit with a resolution of 17500(AGC 1e 5). The maximum filling times for the full scan and the MS-MS scan were set to 50MS and 45MS, respectively, and the dynamic exclusion time was set to 30 s.

Analysis of data: the data of the test sample are subjected to sequence analysis by using a Peaks studio10.0 software search engine. The analysis parameters were: mass tolerance of parent ion: 10ppm, mass tolerance of secondary spectrum: 0.020u, modified by deamidation (NQ), oxidation (M), or non-enzymatic digestion. Selecting the polypeptide sequence with peptide FDR less than or equal to 1 percent as the identified polypeptide sequence. The mass spectrum data of the test article is analyzed by a Peaks studio10.0 software search engine, and the polypeptide sequences in the first ten relative contents obtained by searching the library are shown in Table 1, wherein the peptide sequence with the highest relative content is AVFPSIVGR.

TABLE 1 Scorpio active peptide Mass Spectrometry sequence analysis results

(3) Solid phase synthesis of LA-10 with the other 9 active peptides:

1) resin treatment

Resin swelling: Fmoc-Leu-Wang Resin (molar substitution coefficient is 0.6mmol/g) is selected as starting Resin to be added into a 500mL reaction column, DCM is added for soaking, and the Resin is drained to complete the swelling of the Resin.

Deprotection: adding 20% piperidine in DMF, introducing N₂Stirring for 30min, and draining the solvent; the resin was washed with DMF 6 times and dried by suction to complete the deprotection of the resin.

2) Amino acid coupling reaction

The detection method comprises the following steps: the reaction progress was monitored by the indetrione method.

The specific operation process is as follows: weighing corresponding amount of TBTU and protected amino acid in a beaker, and adding DMF for dissolving; then adding the reaction solution into resin, adding DIEA, and introducing N₂Blowing for about 90min, and detecting through ninhydrin reaction. After the reaction was complete, the solvent was removed and the resin was washed 3 times with DMF. Then 20% piperidine in DMF was added to the resin and N was added₂Blowing was continued for 30 minutes, then the solvent was removed and the resin was washed 6 times with DMF, i.e. the coupling of the amino acid was completed. The above reaction procedure is repeated until the condensation reaction of all protected amino acids is completed. After completion, the polypeptide was contracted, sequentially washed 3 times with DMF/DCM/methanol, drained and weighed.

3) TFA cleavage

The resin was added to a previously prepared lysate (86% TFA/5% EDT/5% thioanisole/3% phenol/2% pure water) and stirred for 150 min. Then the resin and the lysate are extracted, and the polypeptide is fully precipitated by adding ether. The polypeptides were filtered using a buchner funnel and washed thoroughly 6 times with ether to obtain 10 peptides including LA-10 after purification.

Effect of LA-10 on IL-6, IL-8 and TNF- α in serum of orthotopic Lung cancer mice

(1) LLC cell culture

Recovering Lewis lung cancer cell strain (LLC), and culturing with DMEM medium and 10% Fetal Bovine Serum (FBS); the culture conditions are as follows: 37 ℃ and 5% CO₂The cell fusion degree is 70% -80%, and the cell is digested by pancreatin and passaged.

(2) Construction of LLC-GFP stable transfer cell line by lentivirus transfection

And (2) transfecting an LLC cell in a logarithmic growth phase by taking a Green Fluorescent Protein (GFP) overexpression lentivirus suspension, observing the quantity and the intensity of fluorescence under an inverted fluorescence microscope after 24 hours, replacing a new culture medium, screening positive clone cells by using G418 after 48 hours, observing the cell morphology under an inverted phase contrast microscope, observing the fluorescence under the inverted fluorescence microscope, digesting by trypsin, culturing, and screening to obtain an LLC-GFP stable cell line.

(3) Lung in situ inoculation of LLC-GFP cells in C57BL/6 mice

Amplifying LLC-GFP cells, taking log-phase cells, digesting with pancreatin, centrifuging, resuspending with PBS, counting with a cell counter, adjusting the cell order to 1 × 10⁵Mix well with equal volume of Matrigel gel per mL for use. After 8-week-old male C57BL/6 mice were anesthetized, the upper edge of the left thoracic costal arch was surgically excised from the muscle, 100. mu.L of the mixed cell matrix glue mixture was extracted with a microsyringe, the mixture was slowly injected into the left lung lobe with a slow needle, and the incision was closed. After the operation, the respiratory rate is observed, the body temperature of the mouse is evaluated after the mouse naturally revives, the general state of the mouse and the glossiness of hair are observed, and the weight of the mouse is weighed every day.

(4) Evaluation of mouse LLC-GFP Lung cancer in situ model

And (3) after 10 days of inoculation, scanning the chest of the mouse by using a small animal radiation instrument for tomographic CT imaging, observing whether a tumor body exists in the lung, observing the biological activity of the tumor body in the lung by using a small animal living body imaging system (conditions are that the excitation wavelength is 483nm, the emission wavelength is 535nm, and the exposure time is 1.2s), and further observing whether the left lung has tumor tissue characteristics by using an HE (high intensity intrinsic growth) staining method, thereby identifying whether the lung in-situ tumor model is successful.

(5) Grouping and administration of drugs

After the model building is successful, the mice are divided into a model group, a positive drug group (pentafluorouracil), a LA-10 high dose group and a LA-10 low dose group. The feeding mode of each group of mice is kept unchanged, and the administration by gastric lavage is adopted. The administration dosage of the positive medicine is 20.00 mg/kg^-1The dosage of LA-10 high dose group is 50.00 mg/kg^-1The administration dosage of the LA-10 low-dosage group is 25.00 mg/kg^-1The concentration of each drug dissolved in normal saline is 2 mg/mL^-1，5mg·mL^-1，2.5mg·mL^-1The intragastric volume of0.2mL, blank and model groups were given daily equal volumes of saline for 3 consecutive weeks.

(6) Taking materials and measuring

Firstly, material taking: mice were fasted for 12h before drawing materials, and water was not prohibited. After anesthetizing each group of mice with 10% chloral hydrate, blood was taken from abdominal aorta and packed in heparin sodium anticoagulant test tube at 3500 r.min^-1Centrifuging for 10min, and storing the upper layer plasma at-20 deg.C. Fresh feces were collected and stored in a refrigerator at-80 ℃.

Measuring biochemical indexes: and (3) measuring the values of inflammatory factors such as IL-6, IL-8, TNF-alpha and the like by adopting an ELISA kit. The experiment results show that LA-10 can obviously reduce the contents of IL-6, IL-8 and TNF-alpha in the serum of the orthotopic lung cancer model mouse, and the results show that LA-10 has obvious anti-tumor activity and are shown in Table 2.

TABLE 2 Effect of LA-10 on serum IL-6, IL-8 and TNF- α content in orthotopic Lung cancer model mice

Regulating effect of LA-10 on intestinal flora of mice with lung cancer in situ

(1) Preparing an in-situ lung cancer model: as above.

(2) Grouping and administration: the feeding mode of each group of mice is kept unchanged, and the administration by gastric lavage is adopted. The administration dosage of the positive medicine is 20.00 mg/kg^-1The dosage of LA-10 high dose group is 50.00 mg/kg^-1The administration dosage of the LA-10 low-dosage group is 25.00 mg/kg^-1The concentration of each drug dissolved in normal saline is 2 mg/mL^-1，5mg·mL^-1，2.5mg·mL^-1The gavage volume of the solution (2) was 0.2mL, and the blank group and the model group were administered with an equal volume of physiological saline daily for 3 weeks.

(3) Taking materials and measuring

Firstly, material taking: mice were fasted for 12h before drawing materials, and water was not prohibited. After anesthetizing each group of mice with 10% chloral hydrate, blood was taken from abdominal aorta and packed in heparin sodium anticoagulant test tube at 3500 r.min^-1Centrifuging for 10min, and storing supernatant plasma at-20 deg.CThe application is as follows. Fresh feces were collected and stored in a refrigerator at-80 ℃.

Measuring the flora of the mice: taking fresh excrement of blank group, model group and LA-10 high-dose group mice, extracting intestinal microorganism DNA, designing corresponding primers according to conserved regions in the sequences, adding sample-specific Barcode sequences, and carrying out PCR amplification on rRNA gene variable regions (single or continuous multiple) or specific gene segments by adopting Q5 high-fidelity DNA polymerase of NEB company. The PCR amplification products were detected by 2% agarose gel electrophoresis, followed by preparation of a sequencing Library using TruSeqnano DNA LT Library Prep Kit from lllumina, and finally paired-end sequencing of 2X 300bp using a MiSeq sequencer.

(4) Data processing method

And performing OTUs clustering analysis based on effective data by using SMCAP 15.1 software (USA; 2016) by adopting an omics data processing method, performing Alpha diversity and Beta diversity calculation on the obtained OTUs, and obtaining species annotation, species information and species-based abundance distribution of a representative sequence by taking P < 0.05 as a threshold value in combination with t test.

(5) Results of bacterial colony assay

See fig. 1 gate relative abundance analysis: compared with the blank group, the relative abundance of firmicutes in intestinal tracts of the model group mice is obviously reduced, and the relative abundance of bacteroidetes and tenebrio is obviously increased. After LA-10 dry prediction, the relative abundance of firmicutes in intestinal tracts of mice in the administration group is increased, and the relative abundance of bacteroidetes and tenebrio molitorum is obviously reduced.

See FIG. 2 for relative abundance analysis: compared with the blank group, the relative abundance of Bacteroides in intestinal tracts of the model group mice is obviously increased, and the relative abundance of Clostridium is obviously reduced. After LA-10 intervention, the abundance of bifidobacteria and clostridia in intestinal tracts of mice in the administration group is obviously increased, and the abundance of bacteroides is obviously reduced.

See figure 3 for relative abundance analysis: compared with the blank group of mice, the relative abundance of bacteroides and clostridiales in intestinal tracts of the model group of mice is obviously increased, and the relative abundance of lactobacillales is obviously reduced. After LA-10 stem prognosis, the abundance of lactobacillales and bacillales in intestinal tracts of mice in an administration group is obviously increased. And the abundance of clostridiales and bacteroides is obviously reduced.

See fig. 4 flora differential species analysis: compared with the blank group, the composition of the intestinal flora of the mice in the model group is obviously changed, the bacillus, the lactobacillus, the parabacteroides dieldii and the bifidobacteria are obviously reduced, the content of the lactobacillus bacteriophage of the Marasmius bacteria and the number of the trichinella spiralis obviously increased, after LA-10 dryout, the content of the lactobacillus, the bacillus and the bifidobacteria in the intestinal tract of the mice in the administration group is obviously increased, and the content of the acid-producing bacteria of the bacteroides and the Marasmius bacteria is obviously reduced.

Example 2 in vitro verification of antitumor Activity

Evaluation of LA-10 antitumor Activity in vitro based on HepG2 cell and S180 cell

(1) Cell processing

1) Cell resuscitation

Taking out cells from a low-temperature refrigerator at minus 80 ℃ and quickly placing the cells in a metal bath at 37 ℃ to completely melt cell freezing solution;

placing the cell frozen stock solution on a super clean workbench, slightly blowing and beating the cell frozen stock solution, transferring the cell frozen stock solution into a 1.5mL EP tube, and centrifuging the cell frozen stock solution for 5min at 1000 rpm;

after centrifugation is finished, abandoning the supernatant, adding 1mL of PBS buffer solution, gently mixing, and centrifuging again;

fourthly, centrifuging, removing the supernatant, adding 1mL of DMEM medium to resuspend the cells, transferring the cells to a 6-well plate, supplementing 1mL of DMEM medium, mixing uniformly, and placing at 37 ℃ and 5% CO₂In the incubator, the growth of the cells was observed the next day.

2) Cell passage

Firstly, taking out cells from an incubator, observing the cell morphology under an inverted microscope, and passaging when the cell morphology is normal and the cell fusion degree reaches 80%;

removing the original culture medium, slightly rinsing the cells for 3 times by PBS (phosphate buffer solution), adding 300 mu L of 0.25% pancreatin into each hole, slightly shaking the culture plate to ensure that the pancreatin is fully contacted with the cells, then removing the pancreatin, putting the cells into a culture box for digestion for 3min, and observing the form of the cells to be round under an inverted microscope to prove that the digestion is finished;

adding 1mL of DMEM into each hole, gently blowing and beating the DMEM to enable the cells to fall off from the bottoms of the holes and be blown into single cell suspension, continuously adding a proper amount of DMEM to adjust the cell concentration, and performing the steps of 1: 2 or 1: 3, cell passage is carried out.

3) Cell counting

Taking cells which are transferred to the second generation and grow vigorously, washing the cells for 3 times by PBS, digesting 0.25 percent of pancreatin, and then resuspending 1mL of DMEM to prepare single cell suspension;

secondly, 10uL of cell suspension is taken, and is lightly loaded from one side of the cover glass to ensure that the cell suspension just fills the gap between the counting plate and the cover glass, so that excessive overflow of the cover glass or insufficient vacuole is avoided;

counting under an inverted microscope, counting the total number of cells in four squares of the counting plate, and calculating the cell density according to the following formula:

cell number (/ mL) ═ 4 large lattice cell number sum/4) × 10⁴。

The same cell suspension was counted 3 times in succession and the average was taken.

(2) CCK-8 measures LA-10 toxicity to tumor cells: the experiment was performed using cells in the logarithmic growth phase. Each well of the 96-well plate was pre-cultured by adding 100. mu.L of cell suspension at a concentration of 70000 cells/mL. After the cells had grown to a logarithmic growth phase, the stock culture was discarded, 100. mu.L of pre-prepared LA-10 and 9 other synthetic peptide solutions were added at concentrations of 0. mu.M (with DMSO solvent only), 0.5. mu.M, 2. mu.M, and 8. mu.M, respectively, and control wells (without addition of drugs) and background-zeroed wells (with culture medium only) were set. In order to prevent the liquid in the test well from evaporating and affecting the OD value of the test well, PBS with the same volume is added to one circle of the periphery of the test well. When the predetermined action time of the medicine is reached, CCK-8 reagent with the volume of 10 percent of the solution is added into each hole, and the operation is carried out by taking care of avoiding light. And (4) slightly shaking the 96-well plate left and right, putting the 96-well plate into an incubator for incubation for 1h, and detecting the absorbance at 450nm by using an enzyme-labeling instrument. The cytostatic rate was calculated according to the instructions. (3) And (3) observing cell morphology: the experiment was performed using cells in the logarithmic growth phase. After the cells in the 6-well plate are grown to a level that can be tested, the cells are washed twice with PBS, 2.5mL of fresh culture solution is added to each well of the control group, 2. mu.M LA-10 solution is added to each well of the test group, and the cells are transferred into an incubator to be cultured for 24 h. The 6-well plate is taken out when the preset time is reached, and the morphological change of the cells is observed under a 3-mesh inverted microscope and photographed.

(4) Detecting cell apoptosis by flow cytometry: cell digestion in logarithmic growth phase is counted, diluted to 1X 10⁵one/mL. 2mL of cell suspension was added to each 6-well plate, and three groups of three parallel control wells were provided for each group. Transferring the 6-hole plate into a cell culture box for culture. When the cell density was appropriate, the plate was removed and the stock culture was completely removed. Adding 2mL of fresh culture solution into each well of the control group, adding fresh culture solution of 10 μ M and 20 μ M LA-10 into the administration group, and placing into a cell culture box for further culture for 24 h. When the preset administration time is reached, the 6-well plate is taken out. The culture solution is sucked out and transferred into a centrifuge tube for standby. PBS was washed once and pancreatin digest was added. Care should be taken to prevent excessive digestion of pancreatin, damage to cells, and influence on the assay results. The collected culture medium from each well was added back to the original well to terminate the trypsinization. The cell suspension was centrifuged and the cell pellet was retained, resuspended in PBS and counted. 10 ten thousand of the resuspended cells were removed, centrifuged at 1000g for 5min and the bottom cell pellet retained. Gradually adding fluorescent dye for dyeing according to the instruction. And wrapping the mixture with aluminum foil paper, incubating the mixture for 20min at room temperature in a dark place, and transferring the mixture to ice for storage. And completing the flow type on-machine detection within 1 h.

(5) Results of the experiment

1) Effect of LA-10 on tumor cell inhibition Rate

After 10 active peptides with different concentrations are respectively acted on the HepG2 human liver cancer cell and the S180 sarcoma cell for 24h, 48h and 72h, absorbance OD values of each group at 450nm are obtained, and the cell inhibition rate is calculated. The drug action time is fixed, and the cell inhibition rate of LA-10 with different concentrations is greatly and obviously increased (P is less than 0.01) compared with that of a control group, as shown in Table 3. At the same administration concentration, the inhibitory effect was very significant in the groups of 2 μ M and 8 μ M for 48h and 72h compared to 24h in culture (P <0.01), as shown in table 4. Therefore, the results of cytotoxicity tests indicate that the inhibition rate of LA-10 on HepG2 human liver cancer cells and S180 sarcoma cells is increased along with the increase of concentration and action time and is in time-concentration dependence.

TABLE 3 LA-10 results on the inhibition rate of HepG2 human hepatoma cells

TABLE 4 LA-10 results on the suppression rate of S180 sarcoma cells

In this example, the anti-tumor effect of other 9 active peptides was also examined, and comparing the inhibition rates of the active peptides in tables 3 and 5 to the hepatoma cells and the inhibition rates of the active peptides in tables 4 and 6 to the sarcoma S180 cells, it can be seen that LA-10 has better anti-tumor activity than other active peptides. Considering that LA-10 has higher mass ratio in the scorpion active peptide, the suggestion that LA-10 is probably an important site for playing an anti-tumor effect in the scorpion active peptide and has important guiding significance for the development of anti-tumor drugs related to the scorpion active peptide.

TABLE 5 72h inhibition rate results of other 9 active peptides on HepG2 human hepatoma cells

TABLE 6 72h inhibition results of other 9 active peptides on S180 sarcoma cells

2) Cell morphology observation

After LA-10(2 mu M) acts on HepG2 human liver cancer cells and S180 sarcoma cells for 24h, the result of light microscopy shows that the cells in the control group are in irregular squamous epithelial morphology, and the cell morphology of the administration group is obviously changed. Some of the cells are reduced in volume and become round, are disconnected with surrounding cells, are remarkably increased in cytoplasm density, and present typical apoptotic cell characteristics, which indicates that LA-10 can inhibit the growth of HepG2 human liver cancer cells and S180 sarcoma cells by promoting apoptosis; some cells swell and the cell membrane has many protrusions, suggesting that LA-10 may also inhibit cell growth by inducing apoptosis.

3) Detection of cell apoptosis

After HepG2 human liver cancer cells and S180 sarcoma cells are treated by LA-10(2 mu M) for 24 hours, the results of flow cytometry detection show that the LA-10 can lead HepG2 human liver cancer cells and S180 sarcoma cells to be scorched. The scorch rate of the HepG cells treated by LA-10(2 mu M) is (7.67 +/-0.45)%, and the scorch rate of the S180 sarcoma cells treated by LA-10(2 mu M) is (15.4 +/-1.2)%.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.