CN112516303A

CN112516303A - Application of BAFF antibody in preparation of sepsis drug

Info

Publication number: CN112516303A
Application number: CN202011295535.8A
Authority: CN
Inventors: 付妤
Original assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology; Union Hospital Tongji Medical College Huazhong University of Science and Technology
Priority date: 2020-11-18
Filing date: 2020-11-18
Publication date: 2021-03-19

Abstract

The invention relates to an application of a BAFF antibody in preparing a sepsis medicament; the application of the BAFF antibody in preparing a medicament for preventing sepsis; the application of the BAFF antibody in preparing a medicament for preventing and treating organ damage caused by sepsis; the application of the BAFF antibody in preparing a medicament for controlling inflammatory reaction caused by sepsis; a medicament for treating sepsis, wherein the effective component is a BAFF antibody; the beneficial effects are that: provides a new therapeutic drug for sepsis, which can effectively inhibit the associated diseases such as organ damage, inflammation and the like caused by sepsis; also provides a new application of the BAFF antibody.

Description

Application of BAFF antibody in preparation of sepsis drug

Technical Field

The invention belongs to the technical field of medical treatment, and particularly relates to application of a BAFF (B lymphocyte stimulating factor) antibody in a sepsis medicament.

Background

Sepsis is a systemic infection. That is, the suppurative bacteria invade the bloodstream, multiply in large numbers therein, and diffuse to other tissues or organs of the host through the bloodstream, producing new suppurative foci. Sepsis belongs to one of the more serious general pyogenic infections, and is caused by a local infection, which leads to a general infection, sometimes a local infection, a focus is not clear, but can also cause a general infection, and finally, leads to so-called sepsis. The early stage of the sepsis is the local suppuration focus, and symptoms such as high fever, dysphoria, nausea, vomiting, lethargy, headache, dehydration, acidosis and the like appear after the onset of the sepsis. In the middle and late stages, many subcutaneous abscesses appear, and the abscesses well develop in the trunk and begin to be single. Then, the pus appears frequently, the abscess is in the form of induration at the initial stage, and the fluctuation occurs after about one week, at which time the puncture object is pus.

The current agents for blocking the biological activity of BAFF mainly comprise two main classes of anti-BAFF antibodies and soluble receptors of BAFF, wherein the BAFF antibodies comprise lymphostat-B, EGFP/scFv F8, anti-BAFFscFv, anti-BAFF scFv-Fc and the like.

At present, no BAFF is applied to medicines for treating sepsis and related diseases.

Disclosure of Invention

Aiming at the problems, the invention provides the application of the BAFF antibody in preparing a sepsis medicament, mainly provides a new scheme for treating sepsis and related diseases, and also provides a new application of the BAFF antibody.

In order to solve the problems, the invention adopts the following technical scheme:

use of a BAFF antibody in the manufacture of a medicament for the treatment of sepsis.

Application of BAFF antibody in preparing medicament for preventing sepsis.

Application of the BAFF antibody in preparing a medicament for preventing and treating organ damage caused by sepsis.

Application of BAFF antibody in preparing medicine for controlling inflammatory reaction caused by sepsis.

A medicine for treating sepsis contains BAFF antibody as effective component.

In one aspect, the BAFF antibody is an anti-human BAFF monoclonal antibody.

In one aspect, the organ damage comprises damage to the gut, liver, kidney and myocardium.

In one aspect, the intestinal injury comprises an injury to the barrier function of the intestinal mucosa or an injury to the permeability of the intestinal mucosa.

In one aspect, the inflammatory response comprises an intestinal inflammatory response.

In one aspect, the inflammatory response is caused by secretion of one or more of the proinflammatory cytokines TNF- α, IL-6, IL-1 β.

The use of said antibody to BAFF in medicine as a component for inhibiting the secretion of one or more of the pro-inflammatory cytokines TNF- α, IL-6, IL-1 β.

The term "antibody" is used in its broadest sense and includes single BAFF monoclonal antibodies (including agonists, antagonists, and neutralizing antibodies) as well as anti-BAFF antibody compositions with polyepitopic specificity. As used herein, "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., identical between individual antibodies, except for the possible presence of a minimal number of naturally occurring mutations.

The invention also includes BAFF antibodies that specifically react with BAFF-ligands or their receptors; anti-protein/anti-peptide antisera or monoclonal antibodies can be prepared by standard methods. Mammals, such as mice, hamsters or rabbits, may be immunized with the peptide in immunogenic form. Techniques for conferring immunogenicity on a protein or peptide include coupling to a carrier, or other techniques well known in the art.

The immunogenic portion of a BAFF-ligand or receptor thereof may be administered in the presence of an adjuvant. The progress of the immunization can be monitored by detecting antibody titers in plasma or serum. When an immunogen is used as the antigen, standard ELISA or other immunoassay can be used to assess the level of antibody.

The BAFF antibodies described herein are intended to include antibody fragments that also specifically react with BAFF-ligands or their receptors; antibody fragments can be fragmented using conventional techniques and screened for use using the same methods as described above for intact antibodies. For example, F (ab') 2 fragments can be produced by treating the antibody with pepsin. The resulting F (ab ') 2 fragments can be treated to reduce disulfide bonds, resulting in Fab' fragments. The antibodies of the invention further include biospecific and chimeric molecules having anti-BAFF-ligand or anti-BAFF-ligand-receptor activity. Thus, monoclonal and polyclonal antibodies (abs) against BAFF-ligands, tumor-ligands and their receptors, as well as antibody fragments such as Fab 'and F (Ab') 2, can be used to block the action of the ligands with their respective receptors.

In addition, recombinant "humanized antibodies" can be synthesized that recognize BAFF-ligands or their receptors. Humanized antibodies are chimeras mainly containing human IgG sequences into which the regions responsible for specific antigen-binding are inserted. The animals are immunized with the desired antigen, the corresponding antibody is isolated, and the portion of the variable region sequence responsible for specific antigen binding is removed. The antigen binding region from the animal is then cloned into the corresponding position in the human antibody gene where the antigen binding region has been deleted.

The invention has the beneficial effects that:

provides a new therapeutic drug for sepsis, which can effectively inhibit the associated diseases such as organ damage, inflammation and the like caused by sepsis; also provides a new application of the BAFF antibody.

Drawings

FIG. 1 is a schematic representation of the expression of BAFF in LPS-induced sepsis mice;

FIG. 2 is a schematic representation of the effect of BAFF antibody intervention on sepsis mouse survival and tissue damage;

FIG. 3 is a graph of the effect of BAFF antibody intervention on the expression of inflammatory factors in the serum of septic mice;

FIG. 4 is a graph showing the effect of BAFF antibody intervention on the damage to intestinal tissue in septic mice;

FIG. 5 is a graph showing the effect of BAFF antibody intervention on gut inflammation and intestinal mucosal permeability in sepsis mice;

FIG. 6 shows that BAFF antibody intervenes in regulating NF-kB/MLCK/MLC signal path up-regulation of ZO-1 and occludin protein expression.

Detailed Description

The invention is further illustrated by the following specific examples:

Application of BAFF antibody in preparing medicament for preventing sepsis.

A medicine for treating sepsis contains BAFF antibody as effective component.

In one aspect, the BAFF antibody is an anti-human BAFF monoclonal antibody.

In one aspect, the organ damage includes damage to the gut, liver, kidney, and heart muscle, among others.

In one aspect, the intestinal injury comprises an injury to the barrier function of the intestinal mucosa, an injury to the permeability of the intestinal mucosa, or the like.

In one aspect, the inflammatory response includes an intestinal inflammatory response, and the like.

Verification method

1. Material

1.1 Experimental animals

Male C57BL/6J mice, 6-8 weeks old, were purchased from the animal technology Co., Ltd, Viton, Beijing and raised in the SPF grade animal testing center.

1.2 Main Equipment, reagent

A cryogenic refrigerator (Panasonic) at low temperature and-80 ℃, a fluorescence photometer (Thermo Fisher Scientific), an optical microscope (Nikon), a Mini-Cycler thermal Cycler (Roche), a multifunctional microplate reader (TECAN), a PCR instrument (Roche), a low temperature centrifuge (Eppendorf), a Western developing instrument (Azure Biosystem), a full-automatic low temperature tissue homogenizer (shanghai yuming instruments ltd), a full-automatic biochemical analyzer (shenzhen ledu life science and technology company), a vortex oscillator (shanghai jing science and technology company), and a shaker (shanghai wisdom honest analyzer manufacturing ltd).

LPS (Sigma-Aldrich); the BAFF neutralizing antibodies (Sandy-2, Adipogen) (BAFF (D7I1U) Rabbit mAb #19944 and Adipogen) (Human BAFF/TNFSF13B (hBAFF) #89628 and Adipogen) all have similar experimental results, the results are slightly different and are approximately the same, and because the experimental results are similar, the Adipogen represents the Sandy-2 and other types of antibody results in order to avoid repeated description and adopt the Sandy-2 in the following results; trizo (ltakara); reverse transcription and PCR kit (bio-technologies ltd, nunjin nuozokenza); protein lysate, primary antibody diluent and secondary antibody diluent (Biyunyan); loading buffer and BCA kit (Thermo Fisher Scientific); PAGE gel Rapid preparation kit (Shanghai Yazyme Biotech Co., Ltd.); tissue fixative (wuhan seiver biotechnology limited); ZO-1 antibody (Thermo Fisher); occludin antibody, p65 antibody, p-p65 antibody, I kappa B alpha antibody, p-I kappa B alpha antibody, rabbit GAPDH antibody (ABClonal); MLC, p-MLC antibody (Cell Signaling Technology); MLCK antibodies (immunological); BSA, donkey anti-rabbit secondary antibody, and ECL kit (wuhan attenti biotechnology limited); mouse BAFF ELISA kit (R & D); mouse IL-1 beta, IL-6, IL-10, TNF-alpha, D-lactate, DAO, MPO ELISA kits (Wuhan Hualianke biotechnology, Inc.).

2. Method of producing a composite material

2.1 construction of LPS-induced sepsis model in mice and intervention of BAFF neutralizing antibody

1) Grouping experiments: 70 male C57BL/6 mice were randomly divided into a control group, LPS 4h group, LPS 8h group, LPS + anti-BAFF group, wherein LPS 8h group was 10 mice, and the remaining three groups were 20 mice/group. The mice were acclimatized for 7 days in an SPF-grade animal house before subsequent experiments.

2) And (3) experimental intervention: the control group was injected intraperitoneally with 400ul PBS solution; LPS 4h and LPS 8h groups: intraperitoneal injection of LPS (30mg/kg) + control antibody (2 mg/kg); LPS + anti-BAFF group: LPS (30mg/kg) + BAFF antibody (2mg/kg) was intraperitoneally injected.

3) Mouse sacrifice and specimen collection: mice are sacrificed 4 hours after intervention in a control group, an LPS 4h group and an LPS + anti-BAFF group, blood is taken from eyeballs after 0.5 percent pentobarbital sodium abdominal cavity anesthesia of the mice, the mice are kept stand at room temperature for 2 hours, centrifuged at 3000rpm at 4 ℃ for 15min, and then supernatant serum is taken and stored at-80 ℃ for later use. The abdominal cavity and the thoracic cavity were opened along the midline of the abdomen, and the colon, jejunum, ileum, liver, spleen, kidney, and lung were collected. Taking a section of ileum tissue, fixing the ileum tissue in tissue fixing liquid at normal temperature for 24 hours, and then carrying out paraffin section and H & E staining; the remaining tissues were removed and washed and rapidly stored in a-80 ℃ freezer for subsequent ELISA, RT-qPCR and Western Blot assays.

2.2 serum Biochemical marker detection of multiple organ Damage

The serum levels of glutamic pyruvic transaminase (ALT), glutamic oxalacetic transaminase (AST), creatinine (Cr), and Lactate Dehydrogenase (LDH) were measured in each group of mice using a fully automatic biochemical analyzer.

2.3 hematoxylin-eosin staining (H & E)

1) Fixing ileum tissue in tissue fixing solution for 24h, dehydrating with ethanol with gradient concentration, clearing in xylene, embedding with paraffin, and slicing;

2) dewaxing: sequentially placing the glass slide in dimethylbenzene, 100% ethanol, 95% ethanol and 80% ethanol for dewaxing;

3) dyeing: adding hematoxylin staining solution for 10 minutes until the cell nucleus is purple red, washing the cell nucleus with running water for 1 minute until the cell nucleus is bright blue, 0.5% hydrochloric acid alcohol is subjected to color separation for 3-10 seconds, observing under a mirror, washing the cell nucleus with the running water for 5-10 seconds, rewetting the saturated lithium carbonate aqueous solution for 15-20 seconds, washing the cell nucleus with the running water for 1 minute-70% ethanol for 5 minutes, washing the cell nucleus with 80% ethanol for 5 minutes, 1% eosin staining solution for 1-3 minutes-95% ethanol for 5 minutes, 100% ethanol for 5 minutes, xylene and ethanol (1: 1) for 5 minutes, and pure xylene for 5 minutes;

4) sealing: a small drop of neutral resin was added dropwise and mounted with a cover slip and air dried.

2.4 Small intestine histopathology score

Chiu's pathology score evaluates the degree of mouse ileum mucosa damage, which is as follows:

2.5 Effect of BAFF antibody intervention on survival of LPS-induced septic mice

Male C57BL/6 mice 30, divided into control group, LPS + anti-BAFF group, 10/group, were given the same intervention after adaptive feeding for 7 days, given free drinking and eating, and observed and recorded the mortality of each group every 12h, for a total of 72h to all the LPS group mice dead.

2.6 intestinal mucosal Permeability assay

30 male C57BL/6 mice were divided into a control group, an LPS + anti-BAFF group and 10 mice/group, and after the same pretreatment, 200ul FITC-dextran 4kd (25mg/ml) was perfused therein, and after 4 hours, each group of mice was sacrificed, and about 500ul whole blood specimens were collected, left to stand at room temperature for 2 hours, and centrifuged at 3000rpm for 15 minutes at 4 ℃ and then the supernatant was collected, and the fluorescence level in the serum was measured at 490nm and 520nm using a fluorescence spectrophotometer.

2.7 extraction of Primary epithelial cells of the Small intestine

1) A sample of the mouse ileum section of about 5cm is taken, the section of the intestine is cut out longitudinally, and the section of the intestine is fully rinsed in autoclaved ice PBS, so that residual bloodstain and intestinal lumen contents on the surface of the section of the intestine are washed away.

2) The sections were cut rapidly on ice and placed in 40ml autoclaved ice PBS containing 1mmol/L DTT, 1mmol/L EDTA and incubated in a 37 ℃ water bath for 20min with constant gentle shaking.

3) Filtering the tissue suspension treated in step 2) with 200 mesh filter cloth, removing undigested intestinal tissue, collecting cell filtrate containing intestinal epithelial cells, and repeating the step 2 times.

4) The cell filtrate was centrifuged at 800g at 4 ℃ for 3min, the supernatant carefully discarded, 10ml of 0.1% BSA/PBS solution resuspended the cell pellet, and gently inverted upside down to wash the cells.

5) The cell suspension was centrifuged at 800g at 4 ℃ for 5min and then steps 4) and 5) were repeated 2 times.

6) The cell pellet was resuspended in sterile ice PBS to make a cell suspension, and the cell was counted on a cell counting plate for use.

2.8 preparation of tissue homogenate

1) Approximately 50mg of ileum, colon, spleen, liver, kidney, and lung tissue were excised from each group of mice, washed with PBS (0.01M, pH 7.4), and the remaining blood or impurities on the tissue surface were washed away.

2) The tissue blocks are cut into pieces, and the pieces are as small as possible, so that the homogenate is more sufficient.

3) Tissues were added to pre-chilled 450ul PBS (tissue weight: PBS volume 1:9, protease inhibitor added just before use), homogenized at low temperature in a fully automatic grinder.

4) And (3) sucking the homogenate liquid into a centrifuge tube, centrifuging for 5-10 min at 4 ℃ and 10000rpm, taking the supernatant, and storing at-80 ℃ for subsequent ELISA detection of BAFF.

2.9 enzyme-linked immunosorbent assay (ELISA)

Detecting the levels of BAFF, MPO (myeloperoxoxidase) of ileum tissues and IL-1 beta, IL-6, IL-10, TNF-alpha, D-lactate and DAO (diamine oxidase) in serum and each tissue, operating according to the kit instruction, taking the BAFF kit as an example, and operating steps are briefly described as follows:

1) rewarming: taking out the kit and the sample, standing at room temperature for 20min, and shaking up the reagent;

2) preparation of reagent solution and sample:

BAFF capture antibody: the BAFF capture antibody is prepared by 0.5ml PBS, gently and uniformly blown by a pipette, placed for 15min to be fully dissolved, and diluted to the working concentration by Reagent.

BAFF detection antibody: preparing BAFF detection antibody with 1ml of reagent diluent, gently blowing and uniformly mixing by using a pipette, standing for 15min to fully dissolve the BAFF detection antibody, and then diluting the BAFF detection antibody to a working concentration by using the reagent diluent.

③ standard substance: preparing BAFF standard substance with 0.5ml of reagent diluent, gently blowing and uniformly mixing by using a pipette, and sequentially carrying out multiple dilution according to the instruction.

(iv) Streptavidin-HRP: it was diluted to working concentration with reagent diluent.

3) Taking out the required lath from the aluminum foil bag, sealing the rest, and storing at 4 ℃;

4) adding 100ul BAFF CaptureAntibody per well to coat, sealing the plate with a sealing plate film, and incubating overnight;

5) washing the plate: discarding liquid in the batten, adding 400ul of plate washing liquid into each hole, standing for 1min, repeatedly washing for 3 times, and then patting the batten on clean filter paper;

6) 300ul Reagent solution was added to each well and incubated for 1 hour with a coversheet membrane.

7) Repeating the step 5);

8) adding a standard substance and a sample to be detected, sealing by a sealing plate film at 100 ul/hole, and incubating for 2 hours;

9) repeating the step 5);

10) adding BAFF detectiontfoods, 100 ul/well; sealing the plate films, and incubating for 2 hours at room temperature;

11) repeating the step 5);

12) adding Streptavidin-HRP with working concentration, 100 ul/hole; sealing the plate films, and incubating for 20 minutes at room temperature in a dark place;

13) repeating the step 5);

14) adding substrate solution, 100 ul/well; sealing the plate films, and incubating for 20 minutes at room temperature in a dark place;

15) adding stop solution at 50 ul/hole, and keeping out of the sun;

16) measuring OD values at 450nm and 570nm wavelength by using an enzyme-labeling instrument, and correcting the OD value at 570nm wavelength;

17) and drawing a 4-parameter logistic regression curve by using the OD value of the standard substance, calculating the concentration of the sample to be detected according to the standard curve, and multiplying the concentration by the sample dilution times.

2.10 real-time quantitative polymerase chain reaction (RT-qPCR)

2.10.1 extraction of tissue RNA

1) Using an autoclaved ophthalmic scissors and forceps to cut about 20mg of ileum tissue of the mouse, quickly placing the tissue into an autoclaved 2ml EP tube containing 1ml of Trizol, adding 3 small magnetic beads, and homogenizing for 2 times at a low temperature of 65Hz 120sec in a grinding instrument;

2) to the tissue homogenate was added 200ul chloroform, the EP tube was shaken vigorously upside down for 15s, allowed to stand at room temperature for 5min, and centrifuged at low temperature for 15min (12000 rpm). After centrifugation, the liquid is divided into three layers, wherein the upper colorless solution is an RNA phase;

3) sucking 300ul of the colorless upper layer solution by a pipette, adding 300ul of isopropanol, slightly inverting and mixing, standing at room temperature for 5min, and centrifuging at low temperature for 15min (12000 rpm);

4) carefully removing all the solution in the EP tube by using a pipettor, wherein the sediment at the bottom of the EP tube is the extracted RNA sediment, adding 1ml of precooled 75% ethanol into the EP tube, gently blowing and cleaning the RNA sediment by using the pipettor, and centrifuging at low temperature for 5min (12000 rpm);

5) discarding the supernatant, inverting the EP tube on clean filter paper, and drying for 1min to volatilize the residual ethanol.

6) And adding a proper amount of DEPC water to dissolve the precipitate by using a pipette according to the RNA precipitation amount, and gently blowing and beating to fully dissolve the precipitate.

7) And (3) RNA concentration detection: the RNA concentration was determined spectrophotometrically by first washing the cuvette at high pressure and then zeroizing it by adding 100ul ddH 2O. 2ul of the extracted RNA solution and 98ul of ddH2O were added to the cuvette and the RNA concentration was determined at an absorbance of 280nm and the purity and concentration of the RNA was recorded.

2.10.2 reverse transcription

1) cDNA synthesis was performed according to the reverse transcription kit instructions, and the amount of RNA required was calculated based on the amount of cDNA to be synthesized and the concentration of RNA extracted.

2) Preparing a reaction system: take 10ul reaction system as an example

3) Reverse transcription: reverse transcription is carried out in a mini PCR instrument, the program is 15min to 85 ℃ at 37 ℃ and 15sec to 4 ℃, and then the corresponding cDNA can be obtained.

2.10.3 real-time quantitative PCR

1) Preparing a PCR reaction system: 10ul of amplification system: TB Green 5ul, primer 1ul, cDNA 1ul, ddH2O ul;

2) sample adding: and adding the prepared reaction system into a 96-well PCR plate, sealing the 96-well plate by using a sealing plate membrane after the sample is added, and centrifuging.

3) And (3) amplification procedure: 10min at 95 ℃ to 30s at 60 ℃ to 1min at 72 ℃, 40 cycles of amplification-dissolution curve, 0s at 95 ℃, 15s at 65 ℃ and 0s at 95 ℃.

4) ResultsAnd (3) analysis: beta-actin is used as an internal reference, and 2 is adopted^-△△CTThe method performs calculations and analysis.

2.11 Western immunoblotting (Western Blot, WB)

2.11.1 extraction of Total protein from tissues

1) And (3) total protein extraction: approximately 50mg of ileal tissue was cut with sterile ophthalmic scissors and forceps and quickly placed into a 2ml sterile EP tube, 250ul of protein lysate (protein lysate: protease inhibitor: 100: 1) and 2 magnetic beads were added. Homogenizing at low temperature in a tissue homogenizer, shaking on a vortex shaking instrument at intervals of 5min, repeatedly shaking for 6 times, centrifuging at low temperature for 15min (12000rpm), and sucking supernatant to obtain the extracted tissue protein.

2) Determination of protein concentration: according to the BCA kit instructions: 1) diluting the standard substance in multiple proportion; 2) adding samples (24ul PBS and 1ul protein sample to be detected obtained in the step 1) and 25ul diluted standard substance with each concentration into a 96-well plate; 3) preparing working solution, namely uniformly mixing 50 parts of reagent A and 1 part of reagent B, adding 200 mu l of working solution into each hole, and putting a 96-hole plate into a thermostat at 37 ℃ for incubation for 30 min; 4) measuring the OD value of each hole by using a 570nm wavelength in an enzyme labeling instrument, making a standard curve according to the OD value and the concentration of a standard substance, and substituting the OD value of a sample to be measured into a standard curve equation to calculate the concentration of the protein to be measured;

3) protein denaturation: adding the sample buffer solution into the extracted protein supernatant at a ratio of 4:1, placing in an instrument, reacting at 100 deg.C for 10min, and storing the sample in an ultra-low temperature refrigerator at-80 deg.C.

2.11.2WB Experimental procedure

1) Washing the glass plate and assembling the glue maker.

2) Preparing a separation gel: preparing a proper amount of 10% separation gel according to the specification, adding about 7ml of separation gel into each glass plate, slowly adding distilled water into the separation gel for water sealing, and standing at room temperature for 30min to wait for the solidification of the separation gel. The supernatant water was discarded and the water was blotted dry with filter paper.

3) Preparing concentrated glue: preparing 5% concentrated glue according to the specification, adding the concentrated glue into the glass plate, quickly inserting the comb into the concentrated glue, and standing at room temperature for 30min until the concentrated glue is solidified.

4) Sample adding: and (3) preparing an electrophoresis solution according to the formula, filling the electrophoresis solution into the electrophoresis tank, calculating the sample amount according to the protein concentration of the sample, and then loading the sample.

5) Electrophoresis: electrophoresis was terminated after 150V until bromophenol blue reached the bottom of the glass plate.

6) Cutting the glue and transferring the film: the PVDF membrane was activated for 15s in methanol. Cutting glue at corresponding positions by using a small blade according to the molecular weight of a protein marker and a protein to be detected, placing a PVDF membrane on a black surface of a rotating membrane clamp, sticking the corresponding glue on the corresponding PVDF membrane, paying attention to the fact that bubbles cannot be generated, and injecting glue and keeping the PVDF membrane wet in the whole operation process; film transfer: the membrane transferring clamp is tightly bound by a rubber band and then placed in a membrane transferring groove, the membrane transferring groove is placed in an ice-water mixture to reduce the temperature, 350mA constant flow membrane transferring is carried out, different target proteins are different in membrane transferring time, and the specific membrane transferring time is determined according to the molecular weight.

7) And (3) sealing: the PVDF membrane after membrane transfer was placed in blocking solution (5% BSA) and blocked at room temperature on a shaker for at least 1h with slow shaking.

8) Incubating the primary antibody: the primary antibody was diluted with the primary antibody diluent at the appropriate ratio according to the antibody specification, and the bands were placed in an antibody incubation cassette and incubated overnight on a shaker in a refrigerator at 4 ℃.

9) Washing a primary antibody: the strip was taken out the next day, washed with TBST solution on a room temperature shaker for 3 times with 10 min/time shaking, and replaced with new TBST solution after each washing.

10) Hatching a secondary antibody: according to the antibody specification at 1: 2000 of the total amount of the antibody, the rabbit secondary antibody was diluted with a secondary antibody dilution and the bands were incubated in an antibody incubation box for 1h at room temperature on a shaker with slow shaking.

11) Washing a secondary antibody: and taking out the strips, placing the strips on a shaking table at room temperature by using the TBST solution, quickly shaking and washing the strips for 10 min/time and 3 times in total, wherein the TBST solution needs to be replaced after each washing.

12) Chemiluminescence: and (3) fully and uniformly mixing the solution A and the solution B in the ECL kit in equal volume to prepare an exposure solution, uniformly incubating the exposure solution in a strip, paving the strip on a developing plate, and placing the strip in a gel imager for exposure.

13) Image analysis: grey value analysis was performed with aic. alphaview software.

2.12 statistical analysis

Statistical analysis and plotting of experimental data was performed using GraphPad Prism 7.0, with statistical results expressed as mean ± standard deviation. The survival rate curve of each group of mice is statistically analyzed by adopting a Kaplan-Meier method. Analysis of differences between groups was performed using One-way analysis of variance (One-way anova) or Mann-Whitney test, depending on the type of experimental data. Differences between groups were considered statistically significant when P < 0.05.

Results

Increased expression of BAFF in LPS-induced sepsis mice

Expression of BAFF in serum and organs of LPS-induced septic mice was examined by ELISA. As shown in FIG. 1, the expression of BAFF in serum, small intestine, colon, liver, spleen, kidney and lung of septic mice was significantly increased (P <0.05) compared to the control group. The BAFF concentration in the kidney and the liver is higher than that in the LPS intervention 8h group at 4h of LPS intervention, but the difference is not statistically significant, while the BAFF expression in the serum and other organ tissues of the LPS intervention 4h group mice is obviously higher than that in the LPS intervention 8h group (P <0.05), and the BAFF content in the spleen and the lung of the LPS 8h group mice is not obviously different from that in the control group, which indicates that the BAFF expression in the blood circulation and local organ tissues starts to rise and reach the peak at the early stage of sepsis and then gradually declines.

FIG. 1BAFF is expressed in serum and organs of tissues of mice with sepsis. Control: a control group; LPS 4 h: injecting LPS into the abdominal cavity to mold for 4 h; LPS 8 h: injecting LPS into the abdominal cavity to mold for 8 h; LPS + anti-BAFF 4 h: the 4h group was intervened by intraperitoneal injection of LPS and BAFF antibodies. P <0.05, P <0.01 compared to Control group; in comparison to LPS 4h group, # # P < 0.01; ns indicates that P >0.05 compared to either the Control or LPS 4h group.

BAFF antibody intervention increases sepsis mouse survival and reduces organ tissue damage

Figure 2A graphs of survival rates for groups of mice, P <0.01 compared to model groups; figure 2B serum ALT, AST, Cr, LDH of mice in each group, P <0.01 compared to Control group; compared to the LPS group, # P <0.05, # P < 0.01.

The results of fig. 1 show that the BAFF antibody has a significantly reduced content of BAFF in mouse serum and tissues of various organs after the antibody is dried, which indicates that the effect of the BAFF neutralizing antibody is significant. The results show (fig. 2A), that blocking BAFF significantly increases the 72h survival rate in LPS-induced sepsis mice. Because the death of sepsis is closely related to multiple organ dysfunction, biochemical index detection of organ injuries such as ALT, AST, Cr, LDH and the like in serum of each group of mice is carried out, and the serum markers respectively reflect the degree of the injury of liver, kidney and cardiac muscle. The results are shown in fig. 2B, compared with the control group, ALT, AST, Cr and LDH in the serum of sepsis mice induced by LPS are all significantly increased, and BAFF antibody intervention can significantly reduce the expression levels of the four serum markers, which indicates that blocking BAFF has a protective effect on multi-organ injury.

FIG. 2BAFF antibody intervention can improve the survival rate of sepsis mice and reduce the degree of multi-organ injury.

Interference of BAFF antibody in inhibition of inflammatory factor expression in serum of sepsis mice

Excessive inflammatory response is one of the important mechanisms leading to sepsis multiple organ failure and poor prognosis. The production of inflammatory mediators such as IL-1 beta, IL-6, TNF-alpha and the like in serum is obviously increased during sepsis. The results show (figure 3), the level of the inflammatory factors in the serum of the sepsis mice is obviously higher than that of the control group, the BAFF antibody intervention can reverse the effect, the expression level of IL-1 beta, IL-6 and TNF-alpha in the serum of the BAFF antibody intervention group mice is obviously reduced compared with that of the LPS group, but the blocking of BAFF has no obvious influence on the level of IL-10 in the serum, and the blocking of BAFF inhibits the systemic inflammatory factor expression of the sepsis mice.

Figure 3BAFF antibody intervention reduces systemic inflammatory factor expression in septic mice. P <0.01 compared to Control group; compared to the LPS group, # P <0.05, ns indicates P > 0.05.

Intervention of BAFF antibody in reducing damage to intestinal tissue of sepsis mouse

The damage of the intestinal mucosa barrier function in the sepsis is an important reason for multi-organ failure and death, and the influence of BAFF antibody intervention on the inflammation and damage degree of intestinal tissues of sepsis mice is evaluated. The ileum partial segment is subjected to H & E staining (figure 4A), and Chiu's pathological damage scoring (figure 4B) is carried out on the ileum partial segment, and the results show that the control group mice have complete small intestinal mucosa and orderly arranged small intestinal villi, the LPS group small intestinal mucosa is seriously damaged and is expressed by massive infiltration of inflammatory cells, short or broken small intestinal villi, exposure of mucosa lamina propria, telangiectasia and the like, and the damage degree of the LPS + anti-BAFF group small intestinal mucosa is obviously reduced compared with the LPS group. The local neutrophil granulocytes of the small intestinal mucosa are heavily infiltrated in the early stage of the sepsis, and the MPO activity of the small intestinal tissue is an important inflammation marker of the small intestinal tissue damage in the sepsis. As shown in fig. 4C, LPS intervention significantly increased the MPO concentration in small intestine tissue, while BAFF antibody intervention decreased the MPO concentration in septic mouse small intestine tissue. The results show that the intervention of the BAFF antibody can relieve the inflammation of intestinal tissues and the damage degree of mucous membranes of the septic mice.

Figure 4BAFF antibody intervention reduced septic mouse ileal tissue damage. (A) Mouse ileum histopathological section H & E staining, 100X; (B) ileum tissue Chiu's pathology score; (C) ileal tissue MPO. P <0.01 compared to Control group; compared to LPS group, # P < 0.05.

Interference of BAFF antibody reduces inflammation of intestinal tract of sepsis mice and improves permeability of intestinal mucosa

Blocking BAFF can reduce the level of inflammatory factors such as IL-1 beta, IL-6, TNF-alpha and the like in the serum of a sepsis mouse, improve the systemic inflammatory reaction, and find that the damage of the small intestinal mucosa of the sepsis mouse of a BAFF antibody intervention group is also obviously reduced through H & E staining.

The mRNA level expression of IL-1 beta, IL-6, TNF-alpha and IL-10 in mouse intestinal tissues is detected, and the result is shown in figure 5A, compared with the control group, LPS intervention obviously increases the local inflammatory factor expression of small intestine, while BAFF antibody intervention obviously decreases the mRNA expression of IL-1 beta, IL-6, TNF-alpha and IL-10. Intestinal mucosa epithelial cells play an important role in intestinal injury inflammatory reaction and are one of important sources of intestinal local inflammatory factors, primary intestinal epithelial cells of mice of each group are extracted, the expression levels of mRNA (messenger ribonucleic acid) of IL-1 beta, IL-6, TNF-alpha and IL-10 in the primary intestinal epithelial cells are detected (figure 5B), and the result shows that the expression levels of the mRNA of the four inflammatory mediators in the primary intestinal epithelial cells of the LPS group are all increased, and the effect can be reversed by BAFF antibody intervention. The results show that the intervention of the BAFF antibody can reduce intestinal inflammatory reaction by regulating the expression level of local IL-1 beta, IL-6, TNF-alpha and IL-10mRNA of small intestine.

In sepsis, the permeability of the intestinal mucosa is impaired, so that intestinal bacteria and endotoxin are displaced into the blood, and systemic inflammatory reaction and multiple organ dysfunction are aggravated. In a healthy physiological state, FITC-dextran is unable to penetrate the intestinal mucosal barrier into the blood, whereas FITC-dextran is detectable in the blood when the integrity of the intestinal mucosa is compromised. DAO is expressed in small intestinal villus epithelial cells, and when the intestinal mucosa is damaged, the mucosal epithelial cells are exfoliated and DAO is released into the blood circulation. D-lactate is a metabolite of intestinal bacteria, and can permeate through the intestinal mucosa to enter blood when the barrier function of the intestinal mucosa is damaged. FITC-dextran, DAO, D-lactate levels in the mouse sera were examined to reflect impaired small intestinal mucosal permeability and barrier function. The result is shown in figure 5C, compared with the control group, the levels of FITC-dextran, DAO and D-lactate in the serum of the LPS-induced sepsis mice are obviously increased, and the BAFF antibody intervention can reduce the three indexes in the serum, which shows that the BAFF blocking can improve the permeability of the small intestine mucous membrane and reduce the damage of the small intestine mucous membrane barrier function.

FIG. 5BAFF antibody intervention can relieve sepsis mouse ileum tissue inflammation and improve intestinal mucosal permeability.

(A) Mouse ileum tissue IL-1, IL-6, TNF-alpha, IL-10mRNA expression; (B) mouse primary intestinal epithelial cells IL-1, IL-6, TNF-alpha and IL-10mRNA expression; (C) mouse serum FITC-dextran, DAO, D-lactate. P <0.01 compared to Control group; compared to the LPS group, # P <0.05, # P < 0.01.

BAFF antibody intervention upregulated ZO-1 and occludin protein expression by modulating NF- κ B/MLCK/MLC signaling pathway

The integrity of intestinal mucosal permeability and barrier function is closely related to intestinal tight junction proteins. Expression levels of ZO-1 and occludin tight junction proteins were examined using western blot. The results showed that LPS intervention reduced ZO-1 and occludin protein expression, and BAFF antibody intervention increased ZO-1 and occludin protein expression back (FIGS. 6A and 6B). Results as shown in fig. 6C and 6D, while MLCK/MLC pathway activation was significantly activated in sepsis, intervention with the BAFF antibody inhibited LPS-induced MLCK/MLC pathway activation.

The western blot is used for detecting the expression of p65 and I kappa B alpha phosphorylated protein in small intestinal tissues of a mouse with sepsis, and whether an NF-kappa B passage participates in the regulation effect of BAFF on the barrier function of intestinal mucosa is researched. The results show that p-p65 and p-I κ B α are significantly elevated in LPS mice, while BAFF antibody intervention down-regulates the expression levels of p-p65 and p-I κ B α, suggesting that BAFF antibody inhibits activation of NF- κ B signaling pathway (FIGS. 6E and 6F). The results show that BAFF antibody intervention can up-regulate ZO-1 and occludin expression by inhibiting NF-kB/MLCK/MLC signal path, thereby improving the barrier function of intestinal mucosa.

FIG. 6BAFF antibody intervention upregulates ZO-1 and occludin expression in small intestinal tissues. (A) ZO-1; (B) occludin; (C) p-MLC/MLC; (D) an MLCK; (E) p-p65/p 65; (F) p-I kappa B alpha/I kappa B alpha. P <0.05, P <0.01 compared to Control group; compared to the LPS group, # P <0.05, # P < 0.01.

It will be apparent to those skilled in the art that various modifications may be made to the above embodiments without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.

Claims

The application of the BAFF antibody in preparing a medicament for treating sepsis.
Application of the BAFF antibody in preparing a medicament for preventing sepsis.
Application of the BAFF antibody in preparing a medicament for preventing and treating organ damage caused by sepsis.
Application of the BAFF antibody in preparing a medicament for controlling inflammatory response caused by sepsis.
5. A medicine for treating sepsis contains BAFF antibody as effective component.
6. The use of any one of claims 1-5, wherein said BAFF antibody is an anti-human BAFF monoclonal antibody.
7. The use of claim 3, wherein the organ damage comprises damage to the gut, liver, kidney and heart muscle.
8. The use of claim 7, wherein the intestinal injury comprises an injury to intestinal mucosal barrier function, an injury to intestinal mucosal permeability.
9. The use of claim 4, wherein the inflammatory response comprises an intestinal inflammatory response.
10. Use according to any one of claims 1 to 5, wherein the BAFF antibody is used in a medicament as a component for inhibiting the secretion of one or more of the pro-inflammatory cytokines TNF- α, IL-6, IL-1 β.