CN114886939A - Application of scutellaria baicalensis extract in resisting haemophilus parasuis disease - Google Patents

Abstract

The invention discloses an application of a scutellaria baicalensis extract in resisting haemophilus parasuis disease, belongs to the technical field of veterinary drugs, and is applied to a medicament containing the scutellaria baicalensis extract in the haemophilus parasuis disease, wherein the scutellaria baicalensis extract contained in the medicament is a scutellaria baicalensis alcohol extract, the content of baicalin in the scutellaria baicalensis extract is more than or equal to 85 percent, in the preparation of a therapeutic medicament for preventing and improving exudative cellulose caused by the haemophilus parasuis disease, the scutellaria baicalensis extract is used as a Wnt/beta-catenin pathway inhibitor and an inflammation inhibiting medicament, the active ingredients of the traditional Chinese medicine scutellaria baicalensis are firstly and separately applied to piglet breeding for clinically preventing haemophilus parasuis disease, the scutellaria baicalensis extract can be found to relieve clinical symptoms caused by infection of haemophilus parasuis, a series of pathological injuries such as thoracic cavity exudative of piglets and the like, the action mechanism of the exudative cellulose resistance of the scutellaria baicalensis extract and the Wnt/beta-plus medicament capable of inhibiting activation by the haemophilus parasuis disease can be found The catenin pathway is related to the inflammation-related pathway.

Description

Application of scutellaria baicalensis extract in resisting haemophilus parasuis disease

Technical Field

The invention relates to application of a scutellaria baicalensis extract, in particular to application of the scutellaria baicalensis extract in resisting haemophilus parasuis disease, and belongs to the technical field of veterinary medicines.

Background

Haemophilus parasuis, also known as glazier's disease, is a conditionally pathogenic infectious disease caused by Haemophilus Parasuis (HPS), mainly causing multiple serositis, arthritis and meningitis in pigs. Haemophilus parasuis is a common bacterium in the upper respiratory tract of pigs and is mainly transmitted through the respiratory tract, and healthy pigs with bacteria can also become a source of infection. Haemophilus parasuis infections are most common in piglets at 4-8 weeks of age, with typical clinical symptoms of high fever, cough, abdominal breathing, joint swelling and lameness, and central nervous symptoms such as side-lying, water-stroke and tremor. The most remarkable characteristic of the haemophilus parasuis systemic infection is that acute exudative cellulose inflammation is caused to be very serious for piglets, a large amount of fibrous exudates appear on various organs of the chest cavity and the abdominal cavity and the surfaces of pleura, pericardium, peritoneum, synovium and cerebrospinal membrane, the physiological functions of the organs and tissues are seriously influenced, and meanwhile, the haemophilus parasuis proliferates in a host body to induce systemic strong inflammatory reaction, so that a large number of piglets are attacked and die, and huge economic loss is brought to the pig industry in China and even the world. Therefore, inhibition of inflammation and formation of a cellulosic film will be the focus of a strategy to prevent this disease.

At present, there are two main ways for preventing and treating the disease at home and abroad: vaccine immunization and antibiotic administration, although multivalent inactivated vaccines have been developed in China, as haemophilus parasuis disease has obvious locality, haemophilus parasuis serotypes are numerous and cross immunity between different serotypes is poor, the protection effect of the commercial vaccine is reflected differently in various pig farms; the incidence and mortality of piglets can be effectively reduced to a certain extent by antibiotic administration, but the wrapping of the cellulose membrane on the surface of organs hinders the exertion of the curative effect of the antibiotic, and meanwhile, hidden dangers such as veterinary drug residues, bacterial drug resistance and the like follow. In conclusion, due to the harmfulness of the disease in the pig industry, with the comprehensive implementation of 'banning resistance order' in livestock breeding, a safe and effective medicine is urgently needed to prevent the disease.

Disclosure of Invention

The invention aims to solve the problems, provides the application of the scutellaria baicalensis extract in resisting haemophilus parasuis, can reduce the permeability of vascular endothelial cells, reduce the cellulose exudation and inflammatory reaction by inhibiting a Wnt/beta-catenin pathway, expands the application range of the scutellaria baicalensis extract, is favorable for further developing health-care medicines applicable to piglets in clinic, has safe components, no side effect, low price, wide action range and excellent effect, and lays a foundation for the research and development of novel preventive and therapeutic medicines for the haemophilus parasuis.

The invention achieves the aim through the following technical scheme, the application of the scutellaria baicalensis extract in resisting the haemophilus parasuis disease is a medicament containing the scutellaria baicalensis extract, the scutellaria baicalensis extract contained in the medicament is a scutellaria baicalensis alcohol extract, the baicalin content in the scutellaria baicalensis extract is not less than 85%, when the medicament is used for preventing the haemophilus parasuis disease, the dosage is 200mg per kg per day according to the weight of an individual, and the scutellaria baicalensis extract is used as a Wnt/beta-catenin pathway inhibitor and an inflammation-inhibiting medicament in the preparation of a therapeutic medicament for preventing and improving exudative cellulose caused by the haemophilus parasuis disease.

Preferably, the medicament comprises at least one of the scutellaria baicalensis extract and a pharmaceutically acceptable modifier synthesized by the scutellaria baicalensis extract as a lead compound.

Preferably, the dosage form of the medicine is one of capsules, pills, tablets, oral liquid and granules.

Preferably, the medicament also contains one or more pharmaceutically acceptable auxiliary materials or carriers, and the auxiliary materials or the carriers are at least one of sustained release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants or lubricants.

The invention has the beneficial effects that: the effective components of the traditional Chinese medicine scutellaria baicalensis, namely the scutellaria baicalensis extract and the baicalin content of which are more than or equal to 85 percent are firstly and independently applied to clinical application of piglet cultivation to prevent haemophilus parasuis diseases, the prevention and administration of the scutellaria baicalensis extract are found to relieve clinical symptoms and pathological injuries caused by haemophilus parasuis infection of piglets, and the cellulose exudation in the pleuroperitoneal cavity of piglets is remarkably reduced, so that the prevention and administration of the scutellaria baicalensis extract has the effect of resisting the exudative cellulose caused by the haemophilus parasuis diseases, and the action mechanism of preventing the exudative cellulose of the administration of the scutellaria baicalensis extract is related to the Wnt/beta-catenin pathway and inflammation related pathway which can be activated by the haemophilus parasuis in vascular endothelial cells.

Moreover, the scutellaria baicalensis is high in yield and low in price in China, and the extract of the scutellaria baicalensis is proved to have a plurality of effects of resisting inflammation, bacteria and viruses, enhancing the immune function and the like by definite research, is safe and effective, can be applied to breeding clinic for a long time, and can effectively solve the more and more serious problem of antibiotic resistance caused by abuse of antibiotics in clinic by replacing antibiotics to prevent various diseases in clinic.

Drawings

FIG. 1 is a photograph showing the cellulose exudation of the pleuroperitoneal cavity and lung of piglets infected by Haemophilus parasuis in example 1 of the present invention.

Fig. 2 is a pathological grading chart of piglets in each experimental group in example 1 of the present invention.

FIG. 3 is a statistical chart of the bacterial load of Haemophilus parasuis in liver, lung, kidney and brain tissues of piglets in example 1 of the present invention.

FIG. 4 shows the result of H & E staining of paraffin sections prepared from lung and kidney tissues of piglets in example 2 of the present invention.

FIG. 5 is a graph showing the activation of inflammation-related pathways in lung and kidney tissues of piglets in example 2 of the present invention.

FIG. 6 is a graph showing the expression levels of inflammatory factors in lung and kidney tissues of piglets in example 2 of the present invention.

FIG. 7 shows the Wnt/β -catenin pathway activation in lung and kidney tissues of piglets in example 3 of the present invention.

FIG. 8 shows the expression of target genes related to cell mobility and permeability in the downstream Wnt/β -catenin pathway in lung and kidney tissues of piglets in example 3 of the present invention.

FIG. 9 is a diagram of immunohistochemical staining of piglet lung and kidney tissues prepared by paraffin sections with anti-beta-catenin antibody in example 3 of the present invention.

FIG. 10 is a graph showing the effect of different concentrations of Scutellaria baicalensis Georgi extract on the activity of Porcine Aortic Endothelial Cells (PAEC) in example 4 of the present invention.

FIG. 11 is a graph showing the activation of inflammatory pathways in PAEC cells in example 5 of the present invention.

FIG. 12 is a graph showing the expression levels of inflammatory factors in PAEC cells in example 5 of the present invention.

FIG. 13 shows the activation of Wnt/β -catenin pathway in PAEC cells of example 6.

FIG. 14 shows the expression of target genes related to cell mobility and permeability downstream of the Wnt/β -catenin pathway in PAEC cells of example 6.

FIG. 15 shows the activation of Wnt/β -catenin pathway in PAEC cells of example 6.

FIG. 16 is a graph showing the change in cell permeability in PAEC cells in example 7 of the present invention.

FIG. 17 shows the change in the migration ability of PAEC cells in example 8 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Observing the influence of the scutellaria baicalensis extract on piglets infected with haemophilus parasuis.

Experimental Material

Experimental animals:

15 weaned piglets with the age of 20 days are all provided with male and female piglets with the weight of 10-15kg, and the temperature of a feeding room is 26 +/-3 ℃ during the test period.

Primary reagents and drugs:

scutellariae radix extract (EESB) with baicalin content of more than or equal to 85%, storing at room temperature in dark place, weighing 2g and adding 14mL sterilized pure water to obtain suspension, and feeding to one piglet once a day.

Tryptone Soy Agar (TSA): weighing 40g of TSA powder, dissolving in 800mL of distilled water, diluting to a constant volume of 1L, subpackaging, sterilizing at 121 ℃ for 15min by high-temperature high-pressure steam, and storing at room temperature. 5% (v/v) newborn bovine serum and NAD at a final concentration of 20. mu.g/mL were added before use.

The Haemophilus parasuis HPS-SH0165 strain is preserved in the room.

Main apparatus and equipment:

electronic balance, ultra clean bench, Bioprep-24 biological sample homogenizer.

Experimental methods

Animal grouping: 15 weaned piglets with age of 20 days are provided, the weight of the piglets is 10-15kg, and the weaned piglets are randomly divided into three groups, namely a blank control group, an HPS infection group and a scutellaria baicalensis extract prevention administration group, and 5 piglets in each group.

The administration scheme is as follows: all experimental piglets are raised for two days after being transported to an animal room, the scutellaria baicalensis extract (200 mg/(kg. d)) is grouted to the scutellaria baicalensis extract preventive administration group piglets from the third day, each piglet is fed once a day, and the HPS infection group and the scutellaria baicalensis extract preventive administration group piglets are inoculated with HPS-SH0165(10^9 CFU/piglet) in the abdominal cavity after being continuously administered for 7 days. Animal status manifestations are observed and recorded 48h after challenge, pathological dissection is carried out immediately if piglets are in poor status or die, and all piglets are pathologically scored according to typical symptoms and clinical manifestations of haemophilus parasuis.

And (3) determining the bacterial load of haemophilus parasuis in the diseased tissue of the piglet: taking 0.2g of tissue samples of liver, lung, kidney and brain tissues of experimental piglets in a 2mL grinding tube under aseptic condition, adding 1mL of normal saline for homogenization, taking 100 mu L of TSA coated flat plate after dilution by multiple times, and culturing at 37 ℃ for 48 h. The results were observed and haemophilus parasuis counts were performed, the number of single colonies was effectively counted within 30-300, and the number of CFU per g of homogeneous tissue was determined by statistics and calculation.

Results of the experiment

The pathological injury of experimental piglets infected with haemophilus parasuis of the administration group of the scutellaria baicalensis extract is obviously reduced compared with that of the piglets infected with HPS, and specifically, the symptoms of dyspnea, nervous disorder and lameness caused by haemophilus parasuis disease of the piglets of the administration group of the scutellaria baicalensis extract after challenge are greatly reduced, and the pathological anatomical results show that the symptoms of chest abdominal cavity cellulose exudation, pericardial effusion and joint effusion of the piglets fed with the scutellaria baicalensis extract after haemophilus parasuis infection are obviously reduced.

The bacterial load of haemophilus parasuis in liver, lung, kidney and brain tissues of experimental piglets of the radix scutellariae extract prevention and administration group is obviously reduced compared with that of the experimental piglets infected by HPS.

Animal experiments prove that the scutellaria baicalensis extract can obviously relieve related pathological changes and clinical symptoms of exudative cellulose inflammation and the like caused by HPS infection of piglets by preventing administration, and reduce the proliferation of haemophilus parasuis in animal tissues.

Example 2

And observing the influence of the scutellaria baicalensis extract on the in-vivo inflammatory reaction of the piglets infected with the haemophilus parasuis disease.

Experimental Material

Experimental animals:

15 weaned piglets with the age of 20 days are all provided with male and female piglets with the weight of 10-15kg, and the temperature of a feeding room is 26 +/-3 ℃ during the test period.

Primary reagents and drugs:

scutellariae radix extract (EESB) with baicalin content of more than or equal to 85%, storing at room temperature in dark place, weighing 2g and adding 14mL sterilized pure water to obtain suspension, and feeding to one piglet once a day.

The Haemophilus parasuis HPS-SH0165 strain is preserved in the room.

Tissue lysate (2 × LBA lysate containing 10% protease inhibitor (PMSF)).

Reverse transcription kit, SYBR Green Real-time PCR Mix and PAGE gel Rapid preparation kit (10%).

Main apparatus and equipment:

PDVF membrane, an electronic balance, a LightCycler480 fluorescence quantitative PCR instrument, a NanoDrop spectrophotometer, an ice maker, a temperature control shaking table, an ultra-clean bench, an ultra-speed refrigerated centrifuge and a Bioprep-24 biological sample homogenizer.

Experimental methods

Animal grouping: 15 weaned piglets with the age of 20 days are provided with male and female piglets and the weight of the piglets is 10-15 kg. The piglets were randomly divided into three groups, namely a blank control group, an HPS infected group and a Scutellariae radix extract prophylactic administration group, and 5 piglets per group.

The administration scheme is as follows: all experimental piglets are raised for two days after being transported to an animal room, the scutellaria baicalensis extract (200 mg/(kg. d)) is grouted to the scutellaria baicalensis extract preventive administration group piglets from the third day, each piglet is fed once a day, and the HPS infection group and the scutellaria baicalensis extract preventive administration group piglets are inoculated with HPS-SH0165(10^9 CFU/piglet) in the abdominal cavity after being continuously administered for 7 days. And observing and recording the animal state performance 48h after the challenge, and immediately performing pathological dissection to collect animal tissue samples if piglets are in poor states or die.

And (3) preparing paraffin sections from lung and kidney tissues of piglets of each experimental group, carrying out H & E staining, and observing inflammatory cells and cellulose exudation conditions in the tissue sections.

0.5g of lung and kidney tissues are added with 1mL of tissue lysate for grinding and lysis, tissue samples are prepared for Western blot experiment, and the activation conditions of inflammation-related pathways such as NF-kB pathway, p38 and JNK MAPK are detected.

Taking 0.5g of lung and kidney tissues to extract tissue RNA, carrying out reverse transcription, then carrying out fluorescence quantitative PCR, and detecting the expression level of inflammatory factors.

Results of the experiment

H & E staining results show that lung tissues of the piglets in the HPS-infected group are edematous and are accompanied by the phenomena of cellulose exudation and hemorrhage, a large amount of inflammatory cells infiltrate and diffuse, the number of the inflammatory cells and the cellulose exudation in the administration-preventing group of the scutellaria baicalensis extract are obviously reduced, and only slight pathological changes are detected; tissue edema, inflammatory cell accumulation and cellulose exudation were observed in kidney tissue sections of the HPS-infected piglets, while the kidney tissue structure of the piglets of the group to which the scutellaria extract was administered for prevention tended to be normal.

In order to analyze the effect of the scutellaria baicalensis extract on the in-vivo inflammatory response of the piglets infected with the haemophilus parasuis during the preventive administration, the activation condition of inflammation-related pathways in lung and kidney tissues of the piglets of each experimental group is detected, and the result shows that the phosphorylation levels of p65, p38 and JNK in the lung and kidney tissues of the piglets of the scutellaria baicalensis extract preventive administration group are obviously reduced, which indicates that the activation of the in-vivo inflammation-related pathways is inhibited; the fluorescent quantitative RT-PCR detection result shows that after the scutellaria baicalensis extract is prevented from being administrated, the transcriptional levels of inflammatory factors IL-8, CCL5, IL-1 beta and TNF alpha in a piglet infected by haemophilus parasuis are obviously or extremely obviously inhibited. Therefore, the scutellaria baicalensis extract can inhibit the inflammatory reaction of the haemophilus parasuis infected piglets in vivo and relieve tissue damage by preventing administration.

The results show that the radix scutellariae extract can reduce the inflammation of HPS infected piglets in vivo by preventing administration, obviously inhibit the expression of inflammatory factors and reduce pathological injury.

Example 3

And observing the influence of the scutellaria baicalensis extract on the Wnt/beta-catenin pathway in the bodies of the piglets infected with the haemophilus parasuis disease.

Experimental Material

Experimental animals:

15 weaned piglets with age of 30 days are provided with male and female piglets with weight of 10-15 kg. Experimental animal experiment ethical number: HZAUSW-2020-: 202012110006. the temperature of the rearing room during the test was 26. + -. 3 ℃.

Primary reagents and drugs:

scutellariae radix extract (EESB) with baicalin content of more than or equal to 85%, storing at room temperature in dark place, weighing 2g and adding 14mL sterilized pure water to obtain suspension, and feeding to one piglet once a day.

Beta-catenin antibody, beta-actin, horseradish peroxidase (HRP) labeled goat anti-rabbit IgG, tissue lysate (2 × LBA lysate containing 10% protease inhibitor (PMSF)), reverse transcription kit, SYBR Green Real-time PCR Mix, and PAGE gel rapid preparation kit (10%).

Main apparatus and equipment:

PDVF membrane, an electronic balance, a LightCycler480 fluorescence quantitative PCR instrument, a NanoDrop spectrophotometer, an ice maker, an ultra clean bench, an ultra-speed refrigerated centrifuge, and a Bioprep-24 biological sample homogenizer.

Experimental methods

Animal grouping: 15 weaned piglets with the age of 20 days are provided with male and female piglets and the weight of the piglets is 10-15 kg. The piglets were randomly divided into three groups, namely a blank control group, an HPS infected group and a Scutellariae radix extract prophylactic administration group, and 5 piglets per group.

The administration scheme is as follows: all experimental piglets are raised for two days after being transported to an animal room, the scutellaria baicalensis extract (200 mg/(kg. d)) is grouted to the scutellaria baicalensis extract preventive administration group piglets from the third day, each piglet is fed once a day, and the HPS infection group and the scutellaria baicalensis extract preventive administration group piglets are inoculated with HPS-SH0165(10^9 CFU/piglet) in the abdominal cavity after being continuously administered for 7 days. And observing and recording the animal state performance 48h after the challenge, and immediately performing pathological dissection to collect animal tissue samples if piglets are in poor states or die.

And (3) preparing paraffin sections from lung tissues and kidney tissues of piglets of each experimental group, performing immunohistochemical staining, and observing the expression condition of the beta-catenin in the tissues.

0.5g of lung and kidney tissues are taken and added with 1mL of tissue lysate (2 x LBA lysate containing 10% PMSF) for grinding and lysis, a tissue sample is prepared for Western Blot experiment, and the activation condition of the Wnt/beta-catenin channel is detected.

Taking 0.5g of lung and kidney tissues to extract tissue RNA, carrying out reverse transcription, and then carrying out fluorescent quantitative PCR (polymerase chain reaction) to detect the expression level of target genes related to cell mobility and permeability at the downstream of the Wnt/beta-catenin channel.

Results of the experiment

The preliminary study of the subject group shows that the Wnt/beta-catenin pathway plays an important role in regulating the integrity and permeability of cells in the haemophilus parasuis infection process, and the activation condition of the Wnt/beta-catenin pathway in the lung and kidney tissues of piglets is detected in order to explore the influence of a Scutellaria baicalensis extract on the structural integrity of the tissue cells of the piglets. Compared with the HPS infected group, the expression levels of beta-catenin and GSK-3 beta in the piglet tissues of the scutellaria baicalensis extract prevention and administration group are reduced, and the phosphorylation of the GSK-3 beta is obviously inhibited, which indicates that the Wnt/beta-catenin pathway activated by the haemophilus parasuis infection in the piglet is inhibited.

The transcription levels of target genes COX-2, MMP7 and PAI-1 related to cell mobility and permeability at the downstream of a Wnt/beta-catenin pathway in lung and kidney tissues of piglets of a drug administration group are obviously reduced and the transcription level of an epithelial phenotype gene E-cadherin is obviously increased compared with that of an HPS infection group by using a fluorescence quantitative RT-PCR technology.

The lung and kidney tissues of piglets in each experimental group are used for preparing paraffin sections and immunohistochemical staining is carried out by using an anti-beta-catenin antibody, so that the significant reduction of the expression of the beta-catenin in the tissues of the HPS-infected piglets after the prevention administration of the scutellaria baicalensis extract can be observed, and the prevention administration of the scutellaria baicalensis extract can be prompted to inhibit the expression of the beta-catenin in the HPS-infected piglets.

The results show that the radix scutellariae extract can inhibit the activation of the Wnt/beta-catenin pathway in the HPS infected piglet body by preventing administration, and obviously reduce the expression of target genes related to cell mobility and permeability at the downstream of the pathway.

Example 4

Observing the influence of the baicalin content of the scutellaria baicalensis extracts with different concentrations, wherein the baicalin content is more than or equal to 85 percent, on the activity of Porcine Aortic Endothelial Cells (PAEC).

Experimental Material

Main reagents and drugs:

the baicalin content of Scutellariae radix extract (EESB) is not less than 85%, storing at room temperature in dark place, sterilizing in sealed sterile glass bottle at 121 deg.C for 15min, adding PAEC cell culture solution to obtain suspension, and making into suspension.

Preparing a PAEC cell culture solution: adding 20% fetal calf serum, 1% endothelial cell growth factor, 1% double antibody and 1% glutamine into M199 culture medium according to mass volume ratio, and storing at 4 ℃ for later use.

Porcine Aortic Endothelial (PAEC) cells were primary cells isolated from 30 day old piglets; the Haemophilus parasuis HPS-SH0165 strain is preserved in the room.

M199 medium, Fetal Bovine Serum (FBS).

MTT detection kit, LDH detection kit.

Main apparatus and equipment:

electronic balance, enzyme-labeling instrument, inverted microscope, and pipette.

Experimental method

Preparing the scutellaria baicalensis extract into suspensions with the concentrations of 0, 12.5, 25, 50, 100, 250, 500 and 1000 mu g/mL according to the mass-volume ratio, placing the suspensions in a constant temperature biological incubator at 37 ℃, culturing PAEC cells in an environment of 5% CO2 to form a compact monolayer, adding the scutellaria baicalensis extract suspension, treating the suspension by using an MTT (methyl thiazolyl tetrazolium) and LDH (LDH) detection kit after culturing for 12 hours, and detecting by using an enzyme-labeling instrument.

Results of the experiment

The optimum concentration of the scutellaria baicalensis extract used in PAEC was determined to be 100 μ g/mL, and the survival rate of cells was affected by the scutellaria baicalensis extract exceeding this concentration, so the cell experiment was subsequently performed using 100 μ g/mL of the scutellaria baicalensis extract.

Example 5

Observation of the Effect of Scutellaria Baicalensis extract on the level of inflammation of PAEC cells infected with Haemophilus parasuis

Experimental Material

Primary reagents and drugs:

scutellariae radix extract (EESB) with baicalin content of not less than 85% is stored at room temperature in dark place. Placing the mixture into a sterile glass bottle for sterilization at 121 ℃ for 15 minutes, adding PAEC cell culture solution to prepare suspension with the concentration of 100 mu g/mL according to the mass-volume ratio, and preparing the suspension at present.

PAEC was isolated from 30 day old piglets; the haemophilus parasuis HPS-SH0165 strain is stored in the room, and an M199 culture medium, fetal calf serum, a protease inhibitor PMSF, a reverse transcription kit, a SYBR Green Real-time PCR Mix and a PAGE gel rapid preparation kit (10 percent).

Main apparatus and equipment:

electronic balance, inverted microscope, pipette.

Experimental method

Culturing PAEC to a compact monolayer, treating PAEC cells for 1h by using 100 mu g/mL scutellaria baicalensis extract suspension, then adding HPS-SH0165(10^8CFU) for stimulation for 12h, and detecting the activation conditions of inflammation-related pathways such as NF-kappa B pathway, p38 and JNK MAPK and the like in the PAEC by using Western blot; the expression level of inflammatory factors in cells is detected by using a fluorescent quantitative RT-PCR technology.

Results of the experiment

HPS infection can activate inflammation-related pathways in PAEC cells, and scutellaria baicalensis extract pretreatment can remarkably reduce phosphorylation levels of p65, p38 and JNK which are up-regulated by HPS infection in cells and inhibit the activation of inflammation-related pathways in the PAEC cells infected by HPS; the detection result of the fluorescent quantitative RT-PCR technology shows that the scutellaria baicalensis extract pretreatment can remarkably inhibit the up-regulation of the transcription levels of inflammatory factors CCL5 and TNF alpha in HPS infected PAEC (p is less than 0.01), and obviously inhibit the transcription of IL-8 and IL-1 beta.

The results show that the scutellaria baicalensis extract pretreatment can inhibit the inflammatory reaction in the PAEC cells infected by HPS, and obviously inhibit the expression of inflammatory factors, which is consistent with the results in animal tissues.

Example 6

Observing the influence of the scutellaria baicalensis extract on the Wnt/beta-catenin pathway in PAEC cells infected with haemophilus parasuis.

Experimental Material

Primary reagents and drugs:

scutellariae radix extract (EESB) with baicalin content of not less than 85% is stored at room temperature in dark place. Placing the mixture into a sterile glass bottle, sterilizing the mixture for 15 minutes at 121 ℃, adding a PAEC cell culture solution into the mixture to prepare suspension with the concentration of 100 mu g/mL according to the mass-volume ratio, and preparing the suspension on site.

PAEC was isolated from 30 day old piglets; the haemophilus parasuis HPS-SH0165 strain is stored in a room, and comprises an M199 culture medium, Fetal Bovine Serum (FBS), a Lipofection kit Lipofectin 2000 and a serum-free culture medium OPTI-MEM, a DNA rapid recovery kit, a plasmid small-amount extraction kit, an endotoxin-free plasmid extraction kit, a dual-luciferase activity detection kit, a beta-catenin antibody, beta-actin, horseradish peroxidase (HRP) -labeled goat anti-rabbit IgG, a protease inhibitor PMSF, a reverse transcription kit, a SYBR Green Real-time PCR Mix and a PAGE gel rapid preparation kit (10%).

Main apparatus and equipment:

electronic balance, inverted microscope, pipette.

The experimental method comprises the following steps:

culturing PAEC to a compact monolayer, pretreating PAEC cells for 1h by using 100 mu g/mL scutellaria baicalensis extract suspension, then adding HPS-SH0165(10^8CFU) for stimulation for 12h, and detecting the activation condition of the Wnt/beta-catenin channel in the PAEC by using Western blot; the expression conditions of target genes VEGF, PAI-1 and COX-2 related to cell mobility and permeability at the downstream of the Wnt/beta-catenin pathway in cells are detected by using a fluorescent quantitative RT-PCR technology.

The method comprises the steps of pretreating PAEC cells transfected with TOP flash or FOP flash reporter gene plasmids by using scutellaria baicalensis extracts for 1h, then inoculating HPS-SH0165(10^8CFU) for stimulating for 12h, and judging the activation degree of a Wnt/beta-catenin channel according to the ratio of TOP flash to FOP flash.

The experimental results are as follows:

the dual-luciferase detection and Western blot results show that a Wnt/beta-catenin channel in a PAEC cell can be remarkably activated by HPS infection, and the pretreatment of a scutellaria baicalensis extract remarkably inhibits the protein expression level of the beta-catenin and the phosphorylation of GSK-3 beta in the PAEC cell infected by HPS, and inhibits the activation of the channel in the PAEC cell; the fluorescent quantitative RT-PCR detection result shows that after the scutellaria baicalensis extract is pretreated, the transcription of VEGF and COX-2 genes in HPS infected PAEC cells is remarkably inhibited (p is less than 0.05), and the transcription of PAI-1 genes is extremely remarkably inhibited (p is less than 0.01).

The results show that the scutellaria baicalensis extract pretreatment can inhibit the Wnt/beta-catenin pathway in PAEC cells activated by HPS infection, and remarkably reduce the expression of target genes related to cell mobility and permeability at the downstream of the pathway, which is consistent with the results in animal tissues.

Example 7

The effect of scutellaria baicalensis extract on PAEC cell permeability of haemophilus parasuis infection was observed.

Experimental Material

Primary reagents and drugs:

scutellariae radix extract (EESB) with baicalin content of not less than 85% is stored at room temperature in dark place. Placing the mixture into a sterile glass bottle, sterilizing the mixture for 15 minutes at 121 ℃, adding a PAEC cell culture solution into the mixture to prepare suspension with the concentration of 100 mu g/mL according to the mass-volume ratio, and preparing the suspension on site.

PAEC was isolated from 30 day old piglets; the haemophilus parasuis HPS-SH0165 strain is stored in the room, and is cultured in M199 culture medium and Fetal Bovine Serum (FBS).

Main apparatus and equipment:

electronic balance, inverted microscope, Transwell cell co-culture plate, pipette gun, transepithelial resistance meter EVOM.

Experimental method

PAEC were cultured in a Transwell chamber to a dense monolayer, PAEC cells were treated with 100. mu.g/mL of a suspension of Scutellaria baicalensis extract for 1h, HPS-SH0165(10^8CFU) was added, and TEER values of PAEC were measured at 6h, 12h, 24h, 48h, and 72h after stimulation with HPS-SH 0165.

Results of the experiment

The cell permeability of PAEC monolayer is obviously increased by HPS infection, and the cell permeability of PAEC cell up-regulated by HPS infection is obviously reduced (p is less than 0.01) after the cell is pretreated by the scutellaria baicalensis extract, which indicates that the pretreatment of the scutellaria baicalensis extract can obviously recover the cell permeability of PAEC infected by HPS.

Experiments prove that the scutellaria baicalensis extract can remarkably recover the permeability of HPS infected PAEC cells.

Example 8

The effect of the scutellaria baicalensis extract on the migration ability of PAEC cells infected with haemophilus parasuis was observed.

Experimental Material

Primary reagents and drugs:

scutellariae radix extract (EESB) with baicalin content of not less than 85% is stored at room temperature in dark place. Placing the mixture into a sterile glass bottle, sterilizing the mixture for 15 minutes at 121 ℃, adding a PAEC cell culture solution into the mixture to prepare suspension with the concentration of 100 mu g/mL according to the mass-volume ratio, and preparing the suspension on site.

PAEC cells were isolated from 30 day old piglets; the haemophilus parasuis HPS-SH0165 strain is stored in the room, and is cultured in M199 culture medium and Fetal Bovine Serum (FBS).

Main apparatus and equipment:

electronic balance, inverted microscope, pipette.

The experimental method comprises the following steps:

culturing PAEC in a six-hole cell culture plate until the PAEC is fused to 90 percent, using 100 mu g/mL scutellaria baicalensis extract suspension to treat PAEC cells for 1h after scratching by using a tip of a sterile pipette, continuously adding HPS-SH0165(10^8CFU) for stimulation for 12h, and observing and collecting images by using an inverted microscope.

The experimental results are as follows:

PAEC cell motility increased after HPS infection, while scutellaria baicalensis extract pretreatment could significantly alleviate PAEC cell migration caused by HPS infection.

Experiments prove that the scutellaria baicalensis extract can inhibit PAEC cell migration caused by HPS infection.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (4)

1. The application of the scutellaria baicalensis extract in resisting haemophilus parasuis is characterized in that: the scutellaria baicalensis extract is a scutellaria baicalensis alcohol extract, the baicalin content in the scutellaria baicalensis extract is more than or equal to 85%, when the anti-haemophilus parasuis disease is used, the dosage is 200mg per kg per day according to the weight of an individual, and the scutellaria baicalensis extract is used as a Wnt/beta-catenin pathway inhibitor and an inflammation inhibiting medicine in the preparation of a therapeutic medicine for preventing and improving exudative cellulose caused by haemophilus parasuis disease.

2. The use of a scutellaria baicalensis extract according to claim 1 for resisting haemophilus parasuis disease, characterized in that: the medicine contains at least one of the scutellaria baicalensis extract and a pharmaceutically acceptable modifier synthesized by the scutellaria baicalensis extract as a lead compound.

3. The use of a scutellaria baicalensis extract according to claim 1 for resisting haemophilus parasuis disease, characterized in that: the dosage form of the medicine is one of capsules, pills, tablets, oral liquid and granules.

4. The use of a scutellaria baicalensis extract according to claim 1 for resisting haemophilus parasuis disease, characterized in that: the medicine also contains one or more pharmaceutically acceptable auxiliary materials or carriers.

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