阅读说明：本技术 去氧紫草素在制备宿主防御肽表达促进剂中的用途 (Application of deoxyshikonin in preparation of host defense peptide expression promoter ) 是由 王晶 季海峰 张伟 王四新 王雅民 于 2020-08-06 设计创作，主要内容包括：本发明“去氧紫草素在制备宿主防御肽表达促进剂中的用途”,属于化合物新用途,其特征在于,将去氧紫草素或其衍生物为药效活性成分制成需要的剂型；用于促进宿主防御肽基因上调表达；该化合物对多种宿主防御肽的表达均具有良好的促进作用,且不引起炎性反应,即具有调节宿主内源免疫,提高机体健康水平的能力,可作为新一代抗生素替代物的候选物。(The invention relates to an application of deoxyshikonin in preparing a host defense peptide expression promoter, belonging to a new application of a compound, and being characterized in that deoxyshikonin or a derivative thereof is used as a pharmacodynamic active ingredient to be prepared into a required dosage form; for promoting up-regulated expression of host defense peptide genes; the compound has good promotion effect on the expression of various host defense peptides, does not cause inflammatory reaction, namely has the capability of regulating the endogenous immunity of a host and improving the health level of an organism, and can be used as a candidate of a new generation of antibiotic substitutes.)

1. The application of deoxyshikonin in preparing host defense peptide expression promoter is characterized in that deoxyshikonin shown in formula I or its derivative as drug effect active component is prepared into host defense peptide expression promoter with required dosage form;

2. the use according to claim 1, wherein the host defense peptide expression promoter is an agent promoting up-regulated expression of pBD1, pBD2, pBD3, pBD4, pBD123, pBD125, pBD129, pEP2C, PG1-5, PF1-2, PR-39, PMAP-23, PMAP-36 and PMAP-37 genes.

3. The use according to claim 1, wherein the host defense peptide expression promoter is in the form of an oral preparation or an injection.

4. The use of claim 1, the host defense peptide expression promoter further comprising a pharmaceutically acceptable adjuvant.

5. Use of deoxyshikonin shown as formula I in preparation of feed antibiotic substituteCharacterized in that deoxyshikonin or its derivatives shown in formula I are used as active ingredients to prepare the preparation with required dosage form,

6. the use according to claim 1, in the form of an oral or injectable formulation of the feeding antibiotic substitute.

7. The use according to claim 1, the feed antibiotic substitute further comprising a pharmaceutically acceptable adjuvant.

8. A livestock and poultry feed additive is characterized by containing deoxyshikonin shown in a formula I or a derivative thereof,

9. the feed additive according to claim 8, wherein the livestock is a piglet, preferably a weaned piglet.

10. A weaned piglet breeding method is characterized in that an oral preparation taking deoxyshikonin shown as a formula I or a derivative thereof as an active ingredient is directly provided; or adding deoxyshikonin or its derivative as active ingredient into daily ration,

Technical Field

The invention relates to a new application technology of compounds, in particular to an application of deoxyshikonin in preparing a host defense peptide expression promoter.

Background

Host Defense Peptides (HDPs) are important effector molecules of the body's innate immune system and are important barriers for the body to resist the invasion of foreign pathogenic bacteria. To date, as many as 2385 species of animal-derived HDPs (http:// APs. unmac. edu/AP/main. php). The host defense peptide is an amphoteric small peptide with positive charges, and can directly kill bacteria, fungi and viruses or inhibit the growth of the bacteria, fungi and viruses. The host defense peptides also have powerful immunoregulatory functions, including stimulating immune cell proliferation, modifying expression of relevant genes, promoting wound healing, killing cancer cells, etc.

Induction of synthesis of host endogenous HDPs is beneficial to the body to fight against early infection and inflammatory reaction, so that the expression of endogenous HDPs is regulated and controlled by a nutritional means to enhance the anti-infection capability of the body and improve the innate immunity function of the intestinal tracts of livestock and poultry. Especially under the large background that antibiotics are gradually limited and forbidden in the breeding industry, research and development of new products and strategies for improving animal immunity and disease resistance are urgent.

At present, there are few candidates having the effect of inducing expression of HDPs, and thus, there is a need to research and find more compounds having the effect of inducing expression of HDPs.

Disclosure of Invention

Based on the needs in the field, the inventor of the invention obtains a natural compound Deoxyshikonin (Deoxyshikonin) by using cell model screening, and carries out a verification test of host defense peptide expression of pig in vitro cell lines and intestinal tissues, and the result shows that the compound has good promotion effect on the expression of various host defense peptides and can promote the antibacterial activity of immune cells. Cell level experiments show that the polypeptide does not cause inflammatory reaction within the effective concentration range for inducing host defense peptide expression, and can be used as a candidate of a new generation antibiotic substitute, so the invention claims the following technical scheme:

the application of deoxyshikonin in preparing host defense peptide expression promoter is characterized in that deoxyshikonin shown in formula I or its derivative as drug effect active component is prepared into host defense peptide expression promoter with required dosage form;

the host defense peptide expression promoter is a preparation for promoting the up-regulation expression of pBD1, pBD2, pBD3, pBD4, pBD123, pBD125, pBD129, pEP2C, PG1-5, PF1-2, PR-39, PMAP-23, PMAP-36 and PMAP-37 genes.

The dosage form of the host defense peptide expression promoter is oral preparation or injection.

The host defense peptide expression promoter also comprises pharmaceutically acceptable auxiliary materials.

According to another aspect of the invention, the application of the deoxyshikonin shown in the formula I or the derivative thereof in preparing a feed antibiotic substitute is provided, and the deoxyshikonin shown in the formula I or the derivative thereof is used as an active ingredient to prepare a preparation in a required dosage form.

The dosage form of the feed antibiotic substitute is oral preparation or injection.

The feed antibiotic substitute also comprises pharmaceutically acceptable auxiliary materials.

In another aspect of the present invention, a livestock and poultry feed additive is provided, which is characterized by comprising deoxyshikonin represented by formula I or a derivative thereof.

The livestock and poultry refer to piglets, preferably weaned piglets.

Based on the above results, the present invention provides a weaned piglet feeding method, which is characterized in that it is directly provided with an oral preparation having deoxyshikonin represented by formula I or its derivative as an active ingredient; or adding deoxyshikonin or its derivative as active ingredient into daily ration.

The invention discloses a natural compound Deoxyshikonin (Deoxyshikonin) with the function of promoting the up-regulation expression of host defense peptide, which is screened by constructed high-throughput screening, and the structural formula of the Deoxyshikonin is as follows: c₁₆H₁₆O₄The molecular weight is: 272.3, CAS No. is: 43043-74-9, the chemical structural formula is shown as the following formula:is one of the pigment components of lithospermum.

As shown by the exemplary experimental data in the detailed description section, deoxyshikonin was able to induce expression of the porcine IPEC-J2 cell host defense peptide pBD3 gene. Can also induce the expression of host defense peptides pBD2, pEP2C and PG1-5 genes in IPEC-J2 cells. The inventors further verified the effect of Deoxyshikonin in inducing expression of host defense peptides in swine on in vitro culture of porcine jejunal tissue.

In addition, the inventor carries out bacteriostatic tests and confirms that the 3D4/31 bacteriostatic activity of the porcine alveolar macrophage is improved after the stimulation by Deoxyshikonin. Finally, the inventors investigated the effect of Deoxyshikonin on the expression of proinflammatory cytokines and showed that Deoxyshikonin was not observed to elicit an inflammatory response in the concentration range that increased host defense peptide expression.

The natural compound Deoxyshikonin (Deoxyshikonin) capable of effectively inducing the expression of host defense peptides in pig intestinal tracts is one of the pigment components of the common Chinese medicinal material lithospermum, is provided for livestock and poultry as a substitute of feed antibiotics without worrying about residue and safety, and has an antibacterial mechanism which does not directly act on pathogenic bacteria but induces the expression of host endogenous defense peptides so as to improve the antibacterial activity and the immune function, so that the possibility of causing bacterial drug resistance is very low. In conclusion, Deoxyshikonin (Deoxyshikonin) is expected to be developed into a novel feed additive to replace antibiotics, and meets the requirements of modern animal husbandry.

Drawings

FIG. 1 Effect of Deoxyshikonin on the inducible expression of pBD3 Gene

Wherein the control is a blank group without adding Deoxyshikonin, and the rest are relative expression levels of pBD3 gene caused by Deoxyshikonin with final stimulation concentration of 0.3125. mu.M, 0.625. mu.M, 1.25. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M and 20. mu.M relative to the control;

FIG. 2 Effect of Deoxyshikonin on inducible expression of other host defense peptides

Wherein the control is a blank group without adding Deoxyshikonin, and the rest are relative expression amounts of pBD2, pEP2C and PG1-5 genes caused by Deoxyshikonin with final stimulation concentrations of 0.3125. mu.M, 0.625. mu.M, 1.25. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M and 20. mu.M relative to the control;

FIG. 3 induced expression effects of Deoxyshikonin on host defense peptides in porcine intestinal tissue

Wherein the control is a blank group without adding Deoxyshikonin, and the rest are relative expression levels of pBD3, pBD2, pEP2C and PG1-5 genes caused by the stimulation final concentration of Deoxyshikonin (Deoxyshikonin) of 2.5. mu.M, 5. mu.M and 10. mu.M relative to the control.

FIG. 4 shows the bacteriostatic activity of porcine alveolar macrophage 3D4/31 after Deoxyshikonin treatment

The upper panel shows enterotoxigenic Escherichia coli carrying F4 fimbriae (F4)⁺Enterotoxigenic Escherichia coli，F4⁺ETEC), and the lower graph is the result of the antibacterial activity experiment on staphylococcus aureus. The control is a blank without addition of Deoxyshikonin.

FIG. 5 Effect of Deoxyshikonin on proinflammatory cytokine expression

Wherein white bars represent the control as a blank without adding Deoxyshikonin, and black bars represent the relative expression levels of the proinflammatory cytokines IL-1 beta, IL-8 and TNF alpha genes after Deoxyshikonin treatment of cells.

Detailed Description

Example 1 screening for Deoxyshikonin

1261 kinds of known pure natural products and their derivatives separated from plant, animal and microbe are screened, which belong to more than 30 kinds of alkaloid, lipid, terpene, diterpene, pentacyclic triterpene and sterol.

48 compounds are obtained which have induction activity on the pBD3 gene in IPEC-J2/pBD3-luc stable reporter cells; it was found by a dose-response test that 21 compounds, including Deoxyshikonin, increased luciferase activity by more than 2-fold at least one of the three concentrations tested (5. mu.M, 20. mu.M, 80. mu.M).

Experimental reagent:

IPEC-J2/pBD3-luc luciferase reporter cells containing the Porcine pBD3 gene promoter (described in the inventors' paper: Deng, Z., et al.2018.development of a Cell-Based High-Throughput Screening Assay to Identify Port Host feed Peptide-Inducing company. journal of Immunology Research, 2018, 5492941.).

DMEM/F12 medium: purchased from Gibco^TM，Thermo Fisher Scientific，Waltham，MA，USA。

Purchased from Gibco^TM，Thermo Fisher Scientific，Waltham，MA，USA。

Candidate compounds: purchased from TargetMol, Boston, MA, USA.

The screening process is as follows:

IPEC-J2/pBD3-luc luciferase reporter cells containing porcine pBD3 gene promoter and constructed in the early stage are adopted to carry out 2X 10 reaction⁴One/well was inoculated and high throughput screening assays were performed in 96-well plates. The adopted complete culture solution comprises the following components: DMEM/F12 medium + 10% Fetal Bovine Serum (FBS) + 1% diabody (penicillin/streptomycin) + 1. mu.g/mL puromycin. The culture conditions were 37 ℃ and 5% CO₂。

After cells were grown overnight for adherence, they were stimulated with the candidate compound at a final concentration of 20. mu.M, and luciferase activity was measured using L-Max II Luminescence Microplate Reader after 24h of culture. Added 4h before luciferase activity detectionTo detect cellular activity.

Z-score values of 48 compounds obtained by adopting a Z-score calculation method are larger than 2, which indicates that the 48 compounds are possible to activate a promoter of a pBD3 gene so as to trigger transcriptional expression of the compounds, and can be used for follow-up research verification, including Deoxyshikonin.

To further verify the induction activity of the above 48 compounds on the pBD3 gene in IPEC-J2/pBD3-luc stability reporter cells, the inventors performed a dose-response assay. Of these, 21 compounds increased luciferase activity by more than 2-fold at least one of the three concentrations tested (5. mu.M, 2. mu.M and 80. mu.M). Including Deoxyshikonin.

TABLE 1 screening information for natural compounds Deoxyshikonin

Derived from: medchem express(https://www.medchemexpress.cn/)

Example 2 verification of Effect of Deoxyshikonin on the Induction of expression of the pBD3 Gene on IPEC-J2 cells

Experimental reagent:

primary cell line IPEC-J2: this cell line was also available from the gulong Zhang heyday, oklahoma state university, usa, for healthy piglet small intestinal epithelial cells, through other commercial routes.

Deoxyshikonin stock solution: the concentration was 20mM and the solvent was dimethyl sulfoxide (DMSO), purchased from TargetMol, Boston, MA, USA.

The experimental steps are as follows:

culture of IPEC-J2 cells

The IPEC-J2 cell culture adopts DMEM/F12 culture medium, and the complete culture solution comprises: DMEM/F12 medium + 10% Fetal Bovine Serum (FBS) + 1% diabody (penicillin/streptomycin).

Taking out the frozen cells from the liquid nitrogen tank, and quickly putting the cells into a 37 ℃ water bath for thawing. In a biological safety cabinet, sucking the cell suspension, completely transferring into a centrifugal tube, adding 4mL of complete culture solution for dilution, centrifuging at 1000 Xg for 7min, discarding the supernatant, adding 10mL of complete culture solution, gently blowing and mixing the cell suspension, transferring into a 10cm cell culture dish, placing at 37 ℃ and 5% CO₂Culturing in a cell culture box.

When the cells grow to 70-80% of fusion in an adherent manner, digesting for 3-5 min by using 0.25% pancreatin, adding 5mL of complete culture solution to terminate digestion, centrifuging for 7min by 1000g, and removing supernatant. Resuspend with complete medium for subsequent stimulation or culture.

Stimulation treatment and detection of IPEC-J2 cells

When the cultured cells are fused to 70-80%, digesting and counting by 1.25 × 10⁵The amount per well was seeded in 12-well cell culture plates. Stimulation experiments were performed after overnight growth adherent.

Cell stimulation test: the stock Deoxyshikonin solution was diluted to 2mM with DMSO as a stock solution, and the stock solution was added to the complete culture medium in 12-well cell culture plates at different ratios to give final concentrations of 0.375. mu.M, 0.75. mu.M, 1.25. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M and 20. mu.M, respectively.

Cell culture: after the treatment, 5% CO is added at 37 DEG C₂Culturing in an incubator for 24 h.

After the culture is finished, the supernatant is discarded, the mixture is washed for 3 times by sterile PBS, 0.5mL of RNAzol reagent is added into each hole, the mixture is cracked for 5min at normal temperature, and the mixture is transferred into a 1.5mL RNA-free enzyme centrifugal tube after being completely blown and stored at the temperature of minus 80 ℃.

Extraction of total RNA of cells: following the procedures of the kit (RNAzol RT, Molecular Research Center, Cincinnati, OH, USA) instructions. RNA concentration was determined using a NanoDrop spectrophotometer and RNA quality was determined as the ratio of A260: A280 to A260: A230.

Using iScript^TMcDNA Synthesis kit 1. mu.g of RNA was reverse transcribed into cDNA.

Using iTaqTM UniversalGreen Supermix the qPCR assay was performed on a QuantStaudio 3 Real-Time PCR system.

Relative expression amount 2^-ΔΔctAnd (4) calculating.

The primer sequences used were as follows:

GAPDH，F：5′-GCTACACTGAGGACCAGGTTG(SEQ ID No.1)，

R：5′-CCTGTTGCTGTAGCCAAATTC(SEQ ID No.2)；

pBD3，F：5′-CCTTCTCTTTGCCTTGCTCTT(SEQ ID No.3)，

R：5′-GCCACTCACAGAACAGCTACC(SEQ ID No.4)。

3. the result of the detection

Deoxyshikonin was verified to induce pBD3 gene expression using the primary cell line IPEC-J2.

The results show that Deoxyshikonin can induce the expression of pBD3 gene of IPEC-J2 cells of the original cell line, and has obvious concentration gradient effect.

As shown in FIG. 1, the expression level of pBD3 gene reached the highest level at the final Deoxyshikonin stimulation concentration of 5. mu.M, and was increased by about 23-fold compared to the blank control.

The results demonstrate the effect of Deoxyshikonin, a compound screened by IPEC-J2/pBD3-luc luciferase reporter cells, on the inducible expression of the pBD3 gene of the original IPEC-J2 cell line.

Example 3 Effect of Deoxyshikonin on Induction of expression of other host defense peptides

Cell culture and stimulation, total RNA extraction, and mRNA expression amount measurement As in example 2, the following primer sequences were used:

GAPDH，F：5′-GCTACACTGAGGACCAGGTTG-3′(SEQ ID No.1)，

R：5′-CCTGTTGCTGTAGCCAAATT-3′C(SEQ ID No.2)；

pBD2，F：5′-TGTCTGCCTCCTCTCTTCC-3′(SEQ ID No.5)，

R：5′-AACAGGTCCCTTCAATCCTG-3′(SEQ ID No.6)；

PG1-5，F：5′-ACGGTGAAGGAGACTGTG-3′(SEQ ID No.7)，

R：5′-CGCAGAACCTACGCCTACAA-3′(SEQ ID No.8)；

pEP2C，F：5′-ACTGCTTGTTCTCCAGAGCC-3′(SEQ ID No.9)，

R：5′-TGGCACAGATGACAAAGCCT-3′(SEQ ID No.10)。

the detection results are as follows: the compound Deoxyshikonin has an induction effect on the expression of a host defense peptide pBD3 gene of a pig IPEC-J2 cell, and also has an obvious induction effect on the expression of a host defense peptide pBD2 gene, a pEP2C gene and a PG1-5 gene.

As shown in FIG. 2, the expression levels of PG1-5 gene were increased 100-fold or more at the concentrations of Deoxyshikonin of 2.5. mu.M, 5. mu.M and 10. mu.M, respectively, as compared with the blank control, and the expression level was the highest at the concentration of 5. mu.M and increased nearly 150-fold. Deoxyshikonin has relatively low capacity of inducing expression of pBD2 and pEP2C genes, reaches the highest expression level at the concentration of 5 mu M and 1.25 mu M respectively, and is improved by 5.0 times and 6.6 times compared with a blank control respectively.

The results show that the compound Deoxyshikonin not only has an induction effect on the expression of the pBD3 gene, but also has a remarkable induction expression effect on other HDPs.

Example 4 Effect of Deoxyshikonin on induced expression of host defense peptides on porcine intestinal tissue

Experimental reagent:

RPMI 1640: purchase Source Gibco^TM，Thermo Fisher Scientific，Waltham，MA，USA。

HEPES (high efficiency particulate air): the source Solarbio Life Sciences, Beijing, China was purchased.

The experimental steps are as follows:

1. obtaining porcine intestinal tissue

The jejunum tissue of the piglets is taken from weaned piglets (growing white/large white; 7.85 plus or minus 0.23kg) with the age of 5 weeks, and the weaning day age is 28 days. After the piglets are slaughtered, the middle segment of the jejunum is taken, the external muscular layer is removed, and the jejunum tissue is cut to 0.5 multiplied by 0.5cm by a pair of scissors²Size, and fully washed with pre-cooled PBS for later use.

PBS contained 100. mu.g/mL gentamicin, 100U/mL penicillin and 100. mu.g/mL streptomycin.

The sheared tissue was placed in a 6-well cell culture plate, and 4mL of RPMI1640 complete medium was added for culture.

Wherein the composition of the complete culture solution of RPMI1640 is RPMI1640 + 10% FBS +20mM HEPES + 100. mu.g/mL gentamicin +100U/mL penicillin + 100. mu.g/mL streptomycin.

2. Stimulation treatment and detection of porcine intestinal tissue

Porcine jejunal tissue was stimulated with Deoxyshikonin at final concentrations of 2.5. mu.M, 5. mu.M, 10. mu.M, 3 replicates per concentration.

The tissue culture conditions of pig intestine are 37 ℃ and 95 percent of 0₂+5％C0₂The culture time is 24 h.

After culturing, removing supernatant, adding a proper amount of RNAzol reagent to fully crack pig jejunum tissues, transferring the pig jejunum tissues into a 1.5mL RNA-free enzyme centrifugal tube after complete air-assisted beating, and extracting the following total RNA in the same way as the extraction of the total RNA of the cells.

The procedure for detecting the target gene was the same as in example 2.

3. The result of the detection

As shown in FIG. 3, the addition of Deoxyshikonin also induced the expression of HDPs in porcine intestinal tissue. Deoxyshikonin has the best effect on the induction expression of the pBD3 gene in the porcine jejunum tissue at the concentration of 2.5 mu M, and has obvious difference with a blank control.

Deoxyshikonin with the concentration of 2.5 mu M, 5 mu M and 10 mu M can remarkably induce the expression of PG1-5 gene of porcine jejunum tissue, and the relative expression quantity is improved by more than 30 times.

Deoxyshikonin did not induce the expression of pBD2 and pPE2C genes in porcine jejunum tissues to a significant level, but showed an increasing trend.

The results show that the compound Deoxyshikonin obtained by in vitro cell screening also has the effect of inducing expression on host defense peptides of porcine intestinal tissues simulating in vivo tissues.

Example 5 bacteriostatic Activity of porcine alveolar macrophages after Deoxyshikonin treatment

Experimental reagent:

F4⁺ETEC: obtained from China Veterinary Culture Collection Center (China Veterinary Culture Collection Center), and the strain number is CVCC 225.

Staphylococcus aureus: from China Veterinary Culture Collection Center (China Veterinary Culture Collection Center), strain number CVCC 546.

TSA/TSB Medium:and (7) discharging.

3D4/31 cells: porcine alveolar macrophages were offered by the university of oklahoma state, gulong zhang, usa, which is also a conventional cell line and is commercially available.

Hank's Balanced Salt Solution (HBSS): purchased from Gibco^TM，Thermo Fisher Scientific，Waltham，MA。

The experimental steps are as follows:

1. preparation of bacteria

The pathogenic bacteria used in the bacteriostasis test are enterotoxigenic escherichia coli carrying F4 fimbriae (F4)⁺Enterotoxigenic Escherichia coli，F4⁺ETEC, strain number CVCC225) and Staphylococcus aureus (strain number CVCC546), representing gram-negative and gram-positive pathogenic bacteria, respectively.

The culture of pathogenic bacteria uses TSA/TSB culture medium. The original colonies streaked out from the TSA plate were picked and placed in 3mL of a medium containing 25mM NaHCO₃And 1mM NaH₂PO₄The culture was performed in 20% TSB medium at 250rpm and 37 ℃ for about 2.5 hours with shaking. Centrifuging at 1000 Xg for 10min, discarding the supernatant, resuspending in the above medium at OD₆₀₀The absorbance values were measured by diluting the bacteria to 2.5X 10 using the above medium according to the curve equation (curve fitting absorbance value and actual number of bacteria)⁵CFU/mL, spare.

2.3 culture, stimulation of D4/31 cells and preparation of lysates

The culture of the pig 3D4/31 cells adopts an RPMI1640 culture medium, and the complete culture solution comprises the following components: RPMI1640 medium + 10% Fetal Bovine Serum (FBS) + 1% diabody (penicillin/streptomycin) +1mM sodium pyruvate.

The 3D4/31 cell thawing and culturing steps were the same as IPEC-J2 cells. The culture conditions were 37 ℃ and 5% C0₂. When the 3D4/31 cells are cultured to 70-80% fusion, digestion count is 4 multiplied by 10⁵The amount per well was seeded in 12-well cell culture plates.

After overnight growth the anchorage wall was stimulated with Deoxyshikonin at a final concentration of 5. mu.M, at which time the medium was changed to antibiotic-free medium. After 24h of culture, all cells were collected, washed 2 times with Hank's balanced salt solution containing no calcium and magnesium ions, and then resuspended to 100. mu.L with purified water.

Freezing the collected cells in a refrigerator at-80 deg.C for 20min, thawing on ice, ultrasonic lysing for 30s, centrifuging at 12,000 Xg 4 deg.C for 10min, and collecting the supernatant.

3. Bacteriostatic detection

50 mul of the prepared cell lysate is taken, 50 mul of the bacterial suspension prepared in the step 1 is added, and the bacterial suspension is placed into a 96-well plate for culture at 37 ℃. At OD 3h, 6h and 12h, respectively₆₀₀Bacteria turbidity was measured under the conditions.

4. The result of the detection

The results show that the Deoxyshikonin can improve the resistance of 3D4/31 cells to gram-negative pathogenic bacteria (F4)⁺ETEC) and gram-positive pathogenic bacteria (staphylococcus aureus).

As shown in FIG. 4, the cell lysate treated with Deoxyshikonin at a final concentration of 5 μ M significantly reduced F4 at 3h and 6h, respectively⁺The number of ETECs, at 3h, 6h and 12h significantly reduced the number of staphylococcus aureus.

Presumably, Deoxyshikonin could reduce the number of pathogenic bacteria by inducing 3D4/31 cells to produce antibacterial substance host defense peptides.

Example 6 Effect of Deoxyshikonin on proinflammatory cytokine expression

The culture and stimulation of cells, extraction of total RNA, and measurement of mRNA expression were performed as in example 2.

The primer sequences used were as follows:

GAPDH，F：5′-GCTACACTGAGGACCAGGTTG-3′(SEQ ID No.1)，

R：5′-CCTGTTGCTGTAGCCAAATTC-3′(SEQ ID No.2)；

IL-8，F：5′-TTCGATGCCAGTGCATAAATA-3′(SEQ ID No.11)，

R：5′-CTGTACAACCTTCTGCACCCA-3′(SEQ ID No.12)；

TNF-α，F：5′-CCCCTCTGAAAAAGACACCA-3′(SEQ ID No.13)，

R：5′-TCGAAGTGCAGTAGGCAGAA-3′(SEQ ID No.14)；

IL1β，F：5′-GCCCTGTACCCCAACTGGTA-3′(SEQ ID No.15)，

R：5′-CCAGGAAGACGGGCTTTTG-3′(SEQ ID No.16)。

and (3) displaying a detection result: deoxyshikonin has no induction promoting effect on the expression of proinflammatory cytokines IL-1 beta, IL-8 and TNF alpha of pig IPEC-J2 cells;

FIG. 5 illustrates the relative expression levels of Deoxyshikonin on porcine IPEC-J2 cell pro-inflammatory cytokines IL-1 β, IL-8 and TNF α at a concentration of 5 μ M.

Therefore, it is presumed that Deoxyshikonin is a preferable antibiotic substitute candidate, is added as a feed additive, and is safe because it hardly causes inflammatory reaction in the intestinal tract under a concentration condition capable of effectively inducing expression of host defense peptide.

SEQUENCE LISTING

<110> agriculture and forestry academy of sciences of Beijing City

Application of deoxyshikonin in preparation of host defense peptide expression promoter

<130> P190848-NLK

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> GAPDH forward primer

<400> 1

gctacactga ggaccaggtt g 21

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> GAPDH reverse primer

<400> 2

cctgttgctg tagccaaatt c 21

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> pBD3 Forward primer

<400> 3

ccttctcttt gccttgctct t 21

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> pBD3 reverse primer

<400> 4

gccactcaca gaacagctac c 21

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> pBD2 Forward primer

<400> 5

tgtctgcctc ctctcttcc 19

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> pBD2 reverse primer

<400> 6

aacaggtccc ttcaatcctg 20

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> PG1-5 forward primer

<400> 7

acggtgaagg agactgtg 18

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> PG1-5 reverse primer

<400> 8

cgcagaacct acgcctacaa 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> pEP2C Forward primer

<400> 9

actgcttgtt ctccagagcc 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> pEP2C reverse primer

<400> 10

tggcacagat gacaaagcct 20

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> IL-8 Forward primer

<400> 11

ttcgatgcca gtgcataaat a 21

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> IL-8 reverse primer

<400> 12

ctgtacaacc ttctgcaccc a 21

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> TNF-alpha Forward primer

<400> 13

cccctctgaa aaagacacca 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> TNF-alpha reverse primer

<400> 14

tcgaagtgca gtaggcagaa 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> IL1 beta Forward primer

<400> 15

gccctgtacc ccaactggta 20

<210> 16

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> IL1 beta reverse primer

<400> 16

ccaggaagac gggcttttg 19