CN113244400A

CN113244400A - Application of C5aRA in preparation of product for treating and/or preventing abdominal cavity adhesion

Info

Publication number: CN113244400A
Application number: CN202110556232.5A
Authority: CN
Inventors: 孙红; 陈泽; 蒋丽杰; 陈静; 陈敏
Original assignee: North China University of Science and Technology
Current assignee: North China University of Science and Technology
Priority date: 2021-05-21
Filing date: 2021-05-21
Publication date: 2021-08-13
Anticipated expiration: 2041-05-21
Also published as: CN113244400B

Abstract

The invention discloses an application of C5aRA in preparing a product for treating and/or preventing abdominal cavity adhesion, and relates to the technical field of medical biology; the invention discloses an application of C5aRA in preparing a product for treating and/or preventing abdominal cavity adhesion, namely a new application of C5 aRA; according to the invention, researches show that C5aRA can maintain the forms of RPMCs and human peritoneal mesothelial cells and improve the activities of the forms of the RPMCs and the human peritoneal mesothelial cells; thus, C5aRA may protect damaged mesothelial cells and may be able to inhibit the EMT process of mesothelial cells. Meanwhile, C5aRA can reduce alpha-SMA, TGF-beta 1 and OPN, namely C5aRA can also inhibit fibrosis to improve abdominal cavity adhesion by reducing tissue inflammation.

Description

Application of C5aRA in preparation of product for treating and/or preventing abdominal cavity adhesion

Technical Field

The invention relates to the technical field of medical biology, in particular to application of C5aRA in preparation of a product for treating and/or preventing abdominal adhesion.

Background

The abdominal adhesion is one of the most common complications in surgical operations, and the postoperative incidence rate is extremely high and exceeds 90 percent. The abdominal cavity adhesion causes various complications such as nausea, vomiting, female infertility and the like, and increases the risk of bleeding, infection and even shock caused by postoperative adhesion release operation. In addition, the secondary release of adhesions in the abdominal cavity not only prolongs the time for surgery, anesthesia and recovery, but also brings potential risks to the patient, such as blood loss, infection and even intestinal resection. In order to prevent the occurrence of postoperative abdominal adhesion, a series of measures are taken clinically, such as improving the operation method, adopting a minimally invasive technology to reduce the damage and bleeding of tissues and preventing infection. Shortening the surgical time and reducing abdominal trauma are considered important links in reducing adhesions. The existence of the peritoneal fluid enables some anticoagulant, anti-inflammatory drug, fibrinolytic agent and other drugs to be easily removed when being applied to the peritoneal cavity, so that the ideal anti-adhesion effect is difficult to achieve. The specific mechanism of formation of the abdominal adhesions is very complex, and includes the wide involvement and action of various factors such as inflammatory reaction and cytokines. It is associated with excessive inflammatory reaction caused by tissue ischemia, infection, irritation caused by foreign body, etc. caused by abdominal cavity operation, and also associated with decreased fibrinolytic activity of peritoneum, in short, a series of factors interact together to cause the formation of abdominal cavity adhesion. Excessive inflammatory response is an important factor in the formation of adhesions after abdominal surgery. In the early stage of abdominal cavity operation, inflammation reaction mainly occurs, inflammatory factors are gathered to play a role, and the inflammation reaction is always accompanied with the occurrence and development of the inflammatory reaction in the whole process of postoperative adhesion formation, particularly two factors of the severity degree and the time length of the inflammatory reaction are also key factors for causing the adhesion. Cytokines (pro-inflammatory and anti-inflammatory factors) are usually in dynamic equilibrium under normal physiological conditions to ensure homeostasis in the body. While inflammation occurs, the former often acts more than the latter.

The improvement of the postoperative abdominal adhesion has become a common medical problem, and the postoperative abdominal adhesion is a scientific problem to be solved urgently in clinic due to the universality of the postoperative adhesion of the abdominal cavity and the severity of adhesion consequences. In the whole process of abdominal cavity operation, the peritoneal mesothelial cells of the human body are damaged and even fall off, so that the basement membrane falls off, and the mesothelial cells are fibrillated. Recent studies have shown that mesothelial cells play a very important role in the peritoneal repair process. Under normal physiological conditions, the dissolution of plasminogen relies mainly on the action of peritoneal mesothelial cells and is able to maintain its fibrinolytic action and the release of plasminogen in a stable state. Epithelial-to-mesenchymal transition (EMT) is the process of transformation of Epithelial cells into mesenchymal cells. Epithelial cells are an epithelial tissue that covers both the internal and external surfaces of the body, and these cells polarize and form extensive cell-cell adhesion, including adhesive and tight junctions, and it is known that the most important link for the development of abdominal adhesions is the EMT of HPMCs.

Therefore, the development of a novel EMT agent has important significance and value, and no research report on the effect of C5aRA on the treatment of the abdominal adhesion through the EMT inhibition exists at present.

Disclosure of Invention

The object of the present invention is to provide an application in the preparation of products for treating and/or preventing abdominal adhesions, which solves the above-mentioned problems of the prior art.

In order to achieve the aim, the invention provides application of C5aRA in preparing a product for treating and/or preventing abdominal adhesion.

The invention also provides application of the C5aRA in preparation of products for protecting peritoneal mesothelial cells.

The invention also provides application of the C5aRA in preparation of products for reducing the damage degree of peritoneal mesothelial cells.

Further, the peritoneal mesothelial cells are damaged by lipopolysaccharide.

The invention also provides application of the C5aRA in preparation of products for inhibiting mesenchymal transition between epithelial cells.

The invention also provides application of C5aRA in preparation of products for down-regulating alpha-SMA, TGF-beta 1 or OPN expression.

The invention discloses the following technical effects: the invention provides a new application of C5 aRA. Research shows that C5aRA can maintain the forms of RPMCs and human peritoneal mesothelial cells and improve the activities of the forms of the RPMCs and the human peritoneal mesothelial cells; thus, C5aRA may protect damaged mesothelial cells and may be able to inhibit the EMT process of mesothelial cells. Meanwhile, C5aRA can down-regulate alpha-SMA, TGF-beta 1 and OPN, namely C5aRA can also improve abdominal cavity adhesion by reducing tissue inflammation and inhibiting fibrosis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

FIG. 1 is a graph showing the effect of LPS on the morphology of RPMCs;

FIG. 2 is a graph showing the effect of LPS on the activity of RPMCs and the effect of C5aRA on the activity of damaged RPMCs; wherein A is the influence of LPS on the activity of RPMCs; b is the effect of C5aRA on the activity of impaired RPMCs;

FIG. 3 shows morphological changes of various groups of cells under an inverted microscope in example 1;

FIG. 4 shows the morphological changes of the cells under a fluorescence microscope after the phalloidin staining in example 1;

FIG. 5 shows morphological changes of various groups of cells under an inverted microscope in example 2;

FIG. 6 shows the morphological changes of the cells under a confocal microscope after the phalloidin staining in example 2;

FIG. 7 shows the immunofluorescence assay for α -SMA and E-cadherin protein expression of example 2;

FIG. 8 shows the results of the neutrophil immunohistochemistry in the sham group, C5aRA group, and model group at 1 day, 3 days, and 5 days after the operation in example 3;

FIG. 9 is a quantitative analysis of neutrophil immunohistochemistry in sham groups, C5aRA groups, model groups at 1 day, 3 days, 5 days after surgery in example 3;

FIG. 10 shows the adhesion of cecum to abdominal wall 1 week after the operation in example 3;

FIG. 11 shows the adhesion of cecum to abdominal wall 2 weeks after surgery in example 3;

FIG. 12 is the post-operative 1-week (1W) and 2-week (2W) adhesion score statistics for each group in example 3;

FIG. 13 shows the results of expression of alpha-SMA and TGF-beta 1 at 1 week and 2 weeks after the operation in example 3;

FIG. 14 shows the results of OPN expression at 1 week and 2 weeks after the operation in example 3;

FIG. 15 shows the histopathological results of each group at 1 week after the operation in example 3, wherein CE is the mucosa of the caecum; AW is abdominal wall; the adhesion is an adhesive tape;

FIG. 16 is the histopathological results of each group at 2 weeks after surgery in example 3, where CE is the mucosa of the caecum; AW is abdominal wall; the adhesion is an adhesive tape;

FIG. 17 shows the results of quantitative analysis of collagen fibril proliferation at 1 week and 2 weeks after the operation in example 3.

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.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.

Example 1

1.1 extraction of RPMCs

Taking 150g-200g of healthy SD rat body weight, injecting 15-25ml of 0.25% trypsin-0.02% EDTA into the abdominal cavity for two hours, putting the rat into an aeroma tank containing isofluorane, closely observing the breathing condition of the rat, and taking out the rat after the breathing stops. Dead rats were sterilized with 75% alcohol for 20 minutes. Sterilized rats were placed on a rat plate under sterile conditions, with abdominal hair first scraped offAfter hair cutting, the abdominal cavity of the rat is opened by using sterile surgical scissors, the abdominal wall is fixed by using sterile curved forceps, the abdominal cavity fluid (15ml) is sucked out by using a suction pipe after the abdominal cavity is fully exposed, and the cell suspension is prepared by blowing, beating and uniformly mixing. If the cell suspension is red, the cell suspension is placed in an ice box and the red cell lysate is used to slightly shake the cell suspension for 2 minutes. And (3) centrifuging the cell suspension by using a centrifuge, wherein the parameter is 1000r/min, keeping the cell mass at the bottom after 5 minutes, discarding the supernatant, and adding 10ml of DMEM blank medium. After the pipette is blown and beaten uniformly, centrifuging the cell suspension again, wherein the parameters are consistent with those of the first time, removing the supernatant, keeping the cell mass at the bottom, and adding 5ml of DMEM complete culture solution containing 10% fetal calf serum. And then evenly blowing and beating the mixture again and inoculating the mixture into a culture bottle. Standing at 37 deg.C for 5% CO₂Culturing in an incubator, and changing the culture solution for the first time after 24 hours. First passage after 72 hours. Mesothelial cells were passaged to the third generation for subsequent experimental studies.

The primary cells were extracted for 0 hour by observation with an inverted microscope, and the cells floated in complete medium and were spherical in shape. After 24 hours of extraction, the primary cells are attached to the wall by non-overlapping monolayers, and the shape of the primary cells is long fusiform or spherical. After extraction for 72 hours, the primary mesothelial cells are tightly attached to the wall of the cell culture flask, the cells are polygonal and star-shaped, are closely arranged, are fused to have a cobblestone-like appearance and are in a typical mesothelial cell shape.

1.2 cell culture

RPMCs were cultured in DMEM complete medium with 10% fetal bovine serum and 1% diabody (penicillin, streptomycin). Standing at 37 deg.C with 5% CO₂Observing the conditions of cell morphology, cell density and the like every day, changing the complete culture medium for 2 to 3 days, and carrying out passage after the cells are completely fused the next day.

1.3CCK-8 assay of RPMCs proliferative Activity

When the state of rat mesothelial cells is good, 0.5ml of trypsin is dropped into the discarded original culture medium at 37 ℃ and 5% CO₂And digesting in an incubator to prepare a cell suspension. After the mixture is blown evenly, 100ml of cell suspension is dripped into each hole of a 96-hole plate according to the plan. At 37 ℃ with 5% CO₂Culturing in an incubator until the cells are completely attached to the wall, and then discarding the original culture medium.

Grouping according to experimental purpose: control group (complete medium), LPS (20 μ g/ml), LPS (50 μ g/ml), LPS (100 μ g/ml), and n is 6. Adding the required culture solution into each group, continuously culturing in an incubator, taking out a 96-well plate after 12h, adding 20 μ l of CCK-8 solution into each well (adding CCK-8 solution and culture medium at a ratio of 1: 20), and shaking uniformly. At 37 ℃ 5% CO₂After incubation in the incubator for 2h, absorbance of the cells at 450nm was measured using a microplate reader. The morphological changes of the cells of each group were observed by an inverted microscope.

As shown in fig. 1, LPS can cause alterations in cell morphology. Under an inverted microscope, the cell density gradually decreased with increasing LPS concentration, the arrangement was no longer tight, and when the LPS concentration reached 100. mu.g/ml, a large number of necrotic cells were visible. After HE staining, it can be seen that the cells in Control group are polygonal, and the cell nucleus is located in the center of the cell. Vacuoles appear in cytoplasm of cells in the LPS group, nuclear fragmentation appears in nucleus and the like, and the LPS is suggested to possibly cause RPMCs to be necrotic.

As shown in fig. 2, LPS was able to significantly reduce RPMCs activity and was concentration dependent (fig. 2A); when the C5aRA concentration reached 5. mu.g/ml, the activity of this group of cells was statistically significantly different from that of LPS (100. mu.g/ml) (FIG. 2B). LPS has damage effect on RPMCs, and C5aRA has protection effect on damage and is concentration-dependent.

1.4 changes in morphology of RPMCs

When the fusion of the peritoneal mesothelial cells of the rat reaches 70% -80%, the cells are divided into 3 groups according to the measurement result of the CCK-8 experiment: after grouping treatment, cells of each group are continuously cultured for 6 hours after a Control group, an LPS group (100 mu g/ml LPS) and a C5aRA group (5 mu g/ml C5aRA +100 mu g/ml LPS), and after 12 hours, a light mirror is used, and after the phalloidin is stained, a fluorescence microscope is used for observing morphological changes of each group and taking pictures.

Preparing a working solution 1% BSA working solution by a phalloidin staining method (1): 100mg BSA was added to 10ml phosphate buffer solution at pH 7.4, stirred to dissolve completely to obtain a mother solution, and stored in a refrigerator at 4 ℃ for further use. 0.1% Triton: using a pipette, 5. mu.l of triton stock was added to 5ml of 1% BSA stock solution, pipetted to homogeneity and stored in a freezer at 4 ℃ until use. (2) Preparing phalloidin working solution: using a pipette, 1. mu.l of the stock solution of phalloidin was added to 1ml of 1% BSA solution, and the mixture was whipped to a uniform state and stored in a refrigerator at 4 ℃ for further use. (3) Washing rat peritoneal mesothelial cells with the density of 80% for three times by using a normal-temperature sterile phosphate buffer solution, adding 0.5ml of pancreatin, slightly shaking, digesting the rat peritoneal mesothelial cells in a cell culture box for 1min, then sterilizing the surfaces of cell bottles again, adding 4ml of culture solution to stop the digestion, blowing and beating the cells to uniformly mix the cells, adding 100 mu l of the cells into 5ml of complete culture solution, continuously blowing and beating the cells uniformly, carefully clamping cell crawl sheets by using tweezers and placing the cell crawl sheets into a 24-hole plate, dropwise adding a small amount of physiological saline with the sterility state to enable the cell crawl sheets to be tightly attached to the 24 holes, placing 0.5ml of cell suspension into the hole plate, drawing a character '8' and slightly shaking the cell crawl sheets to enable the cell crawl sheets to be uniformly laid in the hole plate, then placing the cell culture box again for continuous culture, and changing the solution for 1 time every 1 day. And removing the culture solution after the cells are completely attached to the wall and the shape is stretched, washing for three times by using a normal-temperature phosphate buffer solution, and putting 500 mu l of 4% paraformaldehyde into a 24-pore plate for fixing for 20 min. Washing with phosphate buffer solution for 3 times, 5min each time, adding 0.1% triton 200 μ l into 24-well plate, allowing permeation for 20min, and washing with phosphate buffer solution for 3 times, 5min each time. Placing the slide into a wet box in advance, gently clamping cell slide in a 24-pore plate by using sterile forceps, placing the slide at the edge of the slide (convenient clamping), dripping and staining the cell slide with 120 mu l of phalloidin working solution per hole, covering the wet box with a dark cover, incubating at room temperature for 100min, and washing with phosphate buffer solution for 5min 3 times each time. (4) A drop of DAPI was applied to the cell slide, carefully grasped with forceps so that the cell slide faced down, and gently covered on the slide.

RPMCs appeared polygonal, star-shaped under normal conditions under inverted microscope, fluorescent microscope after phalloidin staining, and road stone-like or oval stone-like after fusion, typical mesothelial cell appearance, as shown in fig. 3, 4. After the LPS is given at 100 mu g/ml for treatment for 6 hours, compared with the Control group, most of visual fields of the LPS group show that the cells lose the original forms, are spherical, have reduced cell volumes and cell densities, and have more obvious cell form change after the treatment for 12 hours. It was suggested that LPS may cause damage to RPMCs. After 6h of the cells interfered by LPS and C5aRA together, compared with the LPS group, the damage condition of the cells of the C5aRA group is improved, the cell morphology is restored, and the cell density is increased in a visual field. The cell status further improved after 12h of co-intervention. It was suggested that C5aRA was able to ameliorate the damage caused to cells by LPS, time-dependently.

1.5 conclusion

The abdominal adhesion is a very common complication after abdominal surgery, and the traditional treatment means has poor treatment effect on the abdominal adhesion, so that a novel medicament is clinically needed to improve the adhesion condition. The use of a new type of drug to improve the peritoneal viscosity requires first to determine its use concentration and biosafety. It is therefore essential to examine the effect of C5aRA on peritoneal mesothelial cells before performing in vivo experiments. The research shows that C5aRA is a mild medicament. Compared with the traditional secondary operation adhesion releasing operation, the C5aRA has higher safety and better avoids the potential hidden danger of the secondary operation to the patient.

In the research, different doses of LPS are used for interfering the cell proliferation activity, and the LPS with different doses can inhibit the proliferation activity of rat mesothelial cells, wherein the LPS inhibition effect of 100 mu g/ml is most obvious. The C5aRA concentration of each group did not affect the activity of RPMCs cells. After the cells are induced to be damaged by LPS, C5aRA is used for interference protection, and the result shows that the C5aRA can effectively protect the damaged cells and is concentration-dependent, and the protection effect is optimal when the concentration of the C5aRA is 5 mu g/ml. Morphological observation shows that the mesothelial cells which are not stimulated are polygonal and star-shaped under an inverted microscope and a fluorescence microscope, and are fused to form an oval stone-like or paving stone-like body. After 6h of stimulation by LPS, the cell volume is reduced, the density is reduced, and the cell change is more obvious after 12h, which indicates that the LPS can cause cell damage. After 6h of C5aRA intervention treatment, compared with LPS, the cell density and the cell shape of all cells are more similar to those of a Control group, and the protective effect is improved after 12h of intervention. The C5aRA does not influence the activity of normal rat mesothelial cells, has no toxicity, and is worthy of further researching the specific mechanism of the C5aRA in improving the mesothelial cell injury effect.

Example 2

2.1 cell culture

HPMCs were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% diabase (penicillin, streptomycin) in 25ml cell culture flasks at 37 ℃ with 5% CO₂The complete medium is replaced every 2 to 3 days, and the cells are subjected to grouping passage after being completely fused.

Freezing and recovering cells, freezing: when HPMCs are fused to 80%, the complete culture medium is replaced by half or all of the day before operation, on the day, the original culture medium is firstly discarded, the cells are slightly washed for 1 to 2 times by using phosphate buffer solution, the excessive phosphate buffer solution remained in the cell culture bottle is absorbed by a pipette, then 0.5ml of trypsin is absorbed by using a sterile pipette and added into the culture bottle, the temperature is controlled to be 37 ℃, and the concentration of 5% CO is controlled to be lower than that of the trypsin₂The incubation chamber (a) was digested for 1 minute to prepare a cell suspension. After being taken out from the incubator, 5ml of complete culture medium is quickly added into the culture flask to stop digestion, so that damage caused by excessive digestion of cell trypsin is prevented. The adherent mesothelial cells are repeatedly blown and beaten by a liquid transfer gun for about 5 minutes to prepare the cell suspension. After transferring the cell suspension to a 15ml centrifuge tube, the centrifugation parameters were: 800r/min and 5 min. Discarding the supernatant, retaining the cell mass at the bottom of the centrifuge tube, adding the freezing solution, transferring into a freezing tube, performing gradient freezing, retaining in a refrigerator at 4 ℃ for 40 minutes, retaining in a refrigerator at-20 ℃ for 2 hours, and storing in a refrigerator at-80 ℃ or an ultra-low temperature refrigerator for a long time. And (3) resuscitation: taking out the frozen mesothelial cells from a refrigerator at minus 80 ℃, quickly placing the cells into a preheated water bath kettle at 37 ℃, and observing and continuously shaking until the cell mass in the frozen tube is completely melted into a liquid state. Placing the freezing tube in a sterile table, sucking out cell suspension in the freezing tube, placing the cell suspension in a culture bottle containing 5ml of complete culture medium, uniformly blowing to make the cells flatly spread in the culture bottle, and changing the liquid the next day.

2.2 morphological changes of HPMCs after TGF-. beta.1 and C5aRA treatment

When the fusion of HPMCs reaches around 80%, cell suspensions were prepared by trypsinizing the cells: and (4) uniformly blowing and beating. Preparing cell climbing sheet, placing the climbing sheet in 24-pore plate, and washing with phosphate buffer solution to make the pore plate have humid environment or fix the climbing sheet in the pore plate. The cell suspension prepared in advance was dropped into a 24-well plate at about 500. mu.l per well. The experiment groups comprise a Control group, a TGF-beta 1 group (10ng/ml TGF-beta 1) and a C5aRA group (10ng/ml TGF-beta 1+5 mu g/ml C5 aRA). After grouping treatment, each group of cells are continuously cultured for 6 hours, after 12 hours, a light mirror is used, after the phalloidin is dyed, each group of morphological changes are observed under a confocal microscope, and then a picture is taken.

As shown in FIG. 5, under an inverted microscope, HPMCs were polygonal under normal conditions and appeared as cobblestone after fusion. Typical mesothelial cell appearance. After 10ng/ml of TGF-beta 1 is given for treatment for 6h and 12h, compared with the Control group, most of cells in the TGF-beta 1 group are seen to lose the original shape and are in a long fusiform shape, the cells are not arranged tightly, and the TGF-beta 1 is suggested to possibly cause the process of mesenchymal transformation of HPMCs. After treatment with TGF-beta 1 and C5aRA for 6 and 12h, compared with the TGF-beta 1 group, the cell morphology of the C5aRA group is more similar to that of the Control group and is more closely arranged, and the suggestion that C5aRA can inhibit the process of TGF-beta 1 inducing the mesenchymal transformation of HPMCs is carried out. The result of the Coprinus cinereus cyclopeptide staining under a confocal microscope is shown in FIG. 6, the cells in the Control group are well spread and are polygonal, actin is clearly visible, and cytoskeleton is neat and in a bundle shape. Compared with the Control group, the TGF-beta 1 group has the advantages of reduced cell volume, poor spreading, flocculent rolling of cell internal frameworks and damaged F-action structure. Compared with the TGF-beta 1 group, the cell volume of the C5aRA group is increased, floccules basically disappear, and the cell skeleton is similar to the cell shape of the Control group.

2.3 immunofluorescence detection of EMT-related protein expression

When the fusion of HPMCs reaches 70% -80%, cell climbing tablets are prepared according to the method described above. Grouping experiments: the cells of the two groups are continuously cultured for 6 hours and 12 hours after the group treatment of a Control group, a TGF-beta 1 group (10ng/ml TGF-beta 1) and a C5aRA group (10ng/ml TGF-beta 1+5 mu g/ml C5 aRA).

Cellular immunofluorescence staining operation steps: (1) blocking with 10% donkey serum: cells were washed with phosphate buffer, 3 min x 3 times, and blocked in 10% donkey serum for 30 min at room temperature. (2) Antigen-antibody reaction: 10% donkey serum is not discarded, E-cadherin, alpha-SMA antibody is diluted according to the proportion and is dripped on the marked cells to completely cover the whole slide and is incubated overnight at 4 ℃ in a refrigerator. Taking out the cell slide the next day, keeping the experiment environment strictly dark after taking out, washing the slide 10 minutes times by 3 times in phosphate buffer, dripping 488 and 594 fluorescent secondary antibodies to completely cover the whole cell slide, and incubating the cell slide for 1.5 hours in an incubator at 7 ℃. (3) DAPI staining: in phosphate buffer, wash 10 min × 3 times, and drop DAPI color developing solution on cell-slide (DAPI is toxic and careful to handle, never touch the skin). (4) Sealing the plate with an anti-fluorescence quencher and storing in dark.

As shown in fig. 7, the EMT-related factor expression was detected using immunofluorescence techniques. The E-cadherin protein has positive expression on the cell membrane of normal HPMCs, and the alpha-SMA protein is not expressed. After 10 ng/mLTGF-beta 1 acts for 6 hours, the expression of E-cadherin begins to be reduced, and the reduction is more obvious after 12 hours. After 6h of treatment with C5aRA, E-cadherin expression gradually recovered, and after 12h, it was essentially normal. HPMCs begin to express a-SMA after 6 hours and 12 hours of 10ng/ml of GF-beta 1, and this process is inhibited by C5 aRA. Suggesting that C5aRA is able to modulate alterations in EMT-related protein expression.

2.4 conclusion

In order to more visually observe the change of the morphology of each group of cells, the invention uses phalloidin staining and a confocal microscope to observe the cell morphology. The Control group cells are polygonal and star-shaped, and are fused to form oval stone-like or paving stone-like cells; after 10ng/mL TGF-. beta.1 stimulation, cells lost the typical mesothelial appearance, transitioning from polygonal to long spindle; after treatment with 5 μ g/mLC5aRA, the cells restored a stone-like appearance, resembling the Control group cell morphology.

Further detecting the expression change of E-cadherin and alpha-SMA by immunofluorescence technique. The expression of E-cadherin starts to be reduced after TGF-beta 1 acts for 6 hours, and the reduction is more obvious after 12 hours; after treatment with C5aRA, E-cadherin expression gradually recovers; TGF-beta 1 at 10ng/mL for 6-12 hours can induce HPMCs to express alpha-SMA, and the process can be inhibited by 5 mu g/mLC5 aR. The research results show that C5aRA can effectively inhibit the EMT process.

Example 3C5aRA adhesion prevention experiment and evaluation of Effect thereof

3.1C 5aRA adhesion prevention experiment operation

Clean-grade Kunming male mice 45 weighing 22g + -2 g were randomly divided into 3 groups, which were a sham group A, a model group B, and an C C5aRA group, respectively.

Group A: after the abdominal cavity of the mouse is opened by a sterile scalpel, the wound is flushed by normal saline and then sutured; group B: after the abdominal wall cecum injury is finished, the surgical wound surface is respectively cleaned by 1ml of sterile normal saline, and then the surgical wound surface is sutured; group C: before operation, C5aRA (1mg/kg) is injected into rat tail vein, abdominal wall cecal injury is completed, and the operation wound is cleaned by 1ml of sterile physiological saline.

The abdominal wall cecum injury operation steps of the group B are as follows: normally raising the mice before the model building for 1 week, injecting 10 wt% chloral hydrate into the abdominal cavity to anaesthetize the mice, giving the mice according to the injection amount of 3ml per kilogram of the weight of the mice, fixing the mice on a mouse board for experiment after full anaesthesia, disinfecting an operation area by using 75% alcohol under a shadowless lamp, preparing the lower abdomen of the mice, flatly paving an operation hole towel on the surface of the abdomen after iodine Erkang disinfection to just expose the lower abdomen, cutting an incision with the length of about 1cm from bottom to top along the central part of the skin of the lower abdomen by using an aseptic surgical scissors, exposing a leucorrhea line, and then cutting the abdominal wall along the leucorrhea line. The right abdominal wall was fixed and fully exposed using sterile curved forceps, and a wound surface of about 0.5mm in depth, about 0.5cm in length, and 1cm in width was first gently scribed with a small scalpel blade at about 0.5cm from the edge of the incision, preferably with a bleeding surface. Gently rub the surface of the cecum (avoiding mesentery to avoid bleeding) with medical gauze to punctate bleeding. And then, when the abdominal cavity is closed, the cecum wound surface is required to be butted with the abdominal wall wound surface, so that the cecum and the abdominal wall wound surface can be fully jointed, the success of the model creation is facilitated, and the operation wound surface is sterilized by using ioerkang after the abdominal wall muscular layer skin layer is sutured by adopting a continuous suturing method. All manipulations were performed under sterile conditions. Mice were observed for bleeding and conscious status after surgery, and attention was paid to warmth.

After 45 mice are normally fed in a single cage for 1 day, 3 days and 5 days after operation, each 5 mice are placed in a gas anesthetic tube containing isofluorane for about 1 minute to be killed, abdominal cavity material drawing is opened, histochemical staining is applied to detect the expression level of neutrophils, and the effect of inhibiting inflammation by C5aRA is evaluated. Neutrophils, as the first response of the immune system, can respond rapidly to bacterial infections, thereby minimizing the inflammatory response, infiltration in tissues being characteristic of the inflammatory response. The experimental results are shown in fig. 8 and 9, wherein fig. 8 shows the neutrophil immunohistochemistry results of the sham surgery group, the C5aRA group and the model group at 1 day, 3 days and 5 days after the operation; FIG. 9 shows the neutrophil immunohistochemical quantitative analysis of the sham group, C5aRA group and model group at 1 day, 3 days and 5 days after the operation. Compared with the sham operation group, the nucleus and cytoplasm of the neutrophil granulocytes in the model group are strongly positively expressed and are more in number. Compared with the model group, the quantity of the neutrophil positive expression in the C5aRA group is obviously reduced, and the inflammatory response is proved to be weakened. The intervention of C5aRA is further shown to inhibit the transitional activation of complement and reduce the exudation of neutrophils in tissues, thereby relieving the inflammatory reaction after the abdominal wall and the caecum are injured.

After 1 and 2 weeks of normal single-cage rearing of 30 postoperative animals, 5 mice were sacrificed in each experimental group for about 1 minute in a gas anesthetic tube containing isofudane.

3.2 general post-operative observations

Open the abdominal cavity and observe the adhesion status of the injured part of the animal:

all mice did not die after surgery, and vital signs, food intake, water intake, stool, weight and activity were observed and recorded daily after surgery. In the model group, 3 mice had reduced motility, and abdominal swelling was evident and was suspected to be accompanied by intestinal gas. In the C5aRA group, 1 mouse had a slightly less mental and mild abdominal swelling. After normal feeding for 1 week, 5 experimental mice of each experimental group were sacrificed by using a decapitation, and the abdominal cavity of each group was observed by opening the abdominal cavity with a postoperative knife. As shown in fig. 10, no adhesion occurred between the intestinal wall and the abdominal wall of all mice in the sham group 1 week after the operation. In the model group, different amounts of ascites formation were observed after the abdominal opening of most mice, and all the tissues of the cecum and abdominal wall of the mice were seriously adhered. The adhesion area is wide, and the intestinal flatulence occurs in individual mice. In the C5aRA group, mice also developed similar adhesions, but compared to the injured group, the adhesions between the cecum and abdominal wall were reduced to some extent, mostly to a lesser extent, and were not firm, and could be separated by blunt instruments.

The remaining postoperative mice of the sham, model, and C5aRA groups were all sacrificed at postoperative week 2 as shown in fig. 11. After 2 weeks of surgery, all 5 mice in the sham group did not develop adhesions. Compared with one week after operation, the caecum and abdominal wall tissues of all Kunming mice in the model group have larger and wider adhesion area which is obviously larger than the wound area of the initially manufactured model, and more serious vascularized adhesion is generated. Although most of animals in the C5aRA group have adhesion between cecum and abdominal wall, the adhesion degree is lighter than that of a sham operation group, a small part of adhesion tissues have slight vascularization reaction, the adhesion is not tight, and bleeding can occur when blunt instruments are used for separating the tissues at the adhesion parts.

3.3 postoperative adhesion Scoring

To quantify the different degrees of adhesion in each experimental group, we scored tissue adhesion using international scoring criteria. From high to low are: 5, forming vascularized adhesion in a relatively large area; 4, forming more than one part for close adhesion; 3, forming relatively tight adhesion; 2, more than one adhesion occurs; 1, a thinner adhesion occurs; 0 is no blocking. The scoring personnel are three-position classmates which do not participate in model building operation, and the objectivity of the experiment is ensured.

As shown in table 1, fig. 12, 5 mice in the Control group did not develop adhesion at 1 week after the operation, and the adhesion score was 0; the degree of adhesion was not changed after 2 weeks. Mice developed 4-dominated adhesions in the model group at 1 week post-surgery, and the degree of adhesions at 2 weeks post-surgery was mostly rated 5. In the C5aRA group, 1 mouse has an adhesion score of 1 after 1 week of operation, and the other mice have an adhesion score of 2; adhesion scores were 3 for 2 mice 2 weeks after surgery and 2 for the remaining mice.

Table 1 adhesion scores for each group at 1 and 2 weeks post-surgery (n ═ 5)

3.4 detection of alpha-SMA, TGF-beta 1 and OPN expression at adhesion sites 1 week and 2 weeks after surgery by immunofluorescence staining

The development of EMT and intraperitoneal inflammatory responses by HPMCs is considered to be two important links in adhesion formation. TGF-. beta.1 is a major inducer of mature fibrosis. TGF-beta 1 activity and total concentration are reported to be more than twice that of unaffected peritoneum, and blocking TGF-beta 1 and TGF-beta 2 using neutralizing antibodies prevents abdominal adhesion formation. These reports indicate the importance of TGF-. beta.1 for adhesion formation. Thus, application of immunostaining showed the formation of an adhesive bond between the intestinal wall and abdominal wall within 1 week, 2 weeks after surgery, and TGF- β 1 positive expression was present in the damaged serosa and adjacent submucosa. At the same time, α -SMA + myofibroblasts, which may be mesothelial derived, produced similar levels of TGF- β 1. Taken together, these results indicate that TGF- β 1 expression is upregulated in the adhesive zone between the intestinal wall and abdominal wall, thereby inducing mesothelial cell-derived myofibroblasts to produce collagen and promoting adhesion formation. As shown in FIG. 13, positive expression of α -SMA and TGF- β 1 was up-regulated in the model group compared to the sham group, suggesting that EMT may be involved and inflammatory factors exuded during the development of abdominal adhesions. Compared with the model group, the positive expression of alpha-SMA and TGF-beta 1 in the C5aRA group is reduced, and the fact that the C5aRA can inhibit the generation of EMT and the exudation of inflammatory factors is suggested. After 2 weeks, compared with 1 week, the positive expression of the alpha-SMA and the TGF-beta 1 is not obviously changed, but the area of the adhesive band is larger.

The expression of OPN factor detected by immunofluorescence technique after 1 week is shown in FIG. 14, compared with the sham operation group, the OPN positive expression in the model group is up-regulated, which indicates that the expression of OPN factor is generated in the process of abdominal adhesion. The expression of OPN was down-regulated in the C5aRA group compared to the model group, suggesting that C5aRA is able to down-regulate the expression of OPN. No significant change was seen in OPN positive expression compared to 1 week after 2 weeks, but the area of the adhesive band was larger.

3.5 histological observations

The cecum and abdominal wall of the injured part are cut off 7 days and 14 days after the operation, the adhered part is cut off from the part which is already adhered, but the tissue which is not adhered is selected, the tissue which is injured in the molding process is selected, the tissue which is taken off is placed in an embedding box and numbered, and then the tissue is fixed in 4% paraformaldehyde for 30 minutes and is kept running overnight. Then, each group of tissues fixed by 4% paraformaldehyde is embedded in paraffin and then is continuously sliced, and the tissues are stained by using an HE staining method, a Masson special staining method and a picric acid sirius red staining method (hereinafter, the two are abbreviated as sirius red staining).

HE staining method is a traditional contrast staining method, is widely applied in histology, and is a commonly used staining method for clinical pathological diagnosis. In addition to HE Staining, Special Staining (SS) is also commonly used. Masson staining belongs to a common special staining method, is commonly used for staining fibrous connective tissues, can clearly observe the distribution of the fibrous connective tissues in tissues, and is not easy to distinguish similar to a normal structure because collagen fibers in the tissues stained by HE are light pink. However, the muscle fibers stained by Masson appear red and the collagen fibers appear blue, so that the muscle fibers and the collagen fibers in the connective tissue can be distinguished. Because the existing amount of the collagen fibers directly reflects the degree of adhesion and is a key link for forming the adhesion, the content of the collagen fibers in the damaged tissues can be directly observed in a special dyeing mode for the damaged tissues, and the adhesion degree of the tissues of each experimental group can be further explained, so that the effect of preventing the abdominal cavity postoperative adhesion by the C5aRA is evaluated.

1) HE dyeing step

(1) Dewaxing to water: the slices were harvested and baked in an oven at 60 ℃ for 30 minutes, the tissue surface paraffin was removed using xylene i15 minutes and xylene II15 minutes, then the tissue surface xylene was removed using absolute ethanol, 95% ethanol, 85% ethanol, 70% ethanol for 5 minutes each, and finally the tissue slices were hydrated in tap water for 1 minute.

(2) And (3) carrying out hematoxylin nuclear staining: the tissue sections were placed in hematoxylin stain for 2 to 3 minutes and washed slightly with water.

(3) Differentiation: quickly differentiate in 1% alcohol hydrochloride differentiation solution for 4 seconds.

(4) Anti-blue: the blue color is reversed for 10 minutes using running water.

(5) Eosin staining: placing the mixture in eosin dye liquor for 1 minute, taking out the mixture and washing the mixture with water slightly.

(6) And (3) dehydrating: placing in 70% ethanol, 85% ethanol, 95% ethanol, and anhydrous ethanol gradient ethanol for 5min respectively to dehydrate.

(7) And (3) transparency: the mixture was placed in xylene I for 15 minutes and xylene II for 15 minutes to be transparent.

(8) Sealing: a neutral gum mounting was used.

2) Masson staining procedure

(2) And (3) staining nuclei with weigert iron hematoxylin: mixing the solution A and the solution B in equal proportion, shaking up, dripping to completely cover the tissue for dyeing for 12 minutes, and slightly washing with running water (taking care that the dye solution is prepared as before use).

(3) The tissue was differentiated in 1% hydrochloric acid alcohol for 3-4 seconds and then washed slightly with running water.

(4) Ponceau acid fuchsin is stained by dripping to completely cover tissues for staining for 8 minutes, and the staining solution is washed clean by running water.

(5) The tissue surface was covered completely with 1% phosphomolybdic acid solution and differentiated for 4 minutes.

(6) The tissue was covered with aniline blue dye by drop-staining for 5 minutes and washed with water.

(7) Coating 1% glacial acetic acid on tissue surface, differentiating for 1min, dehydrating with 95% ethanol for several times, and soaking tissue in anhydrous ethanol for dehydration.

(8) And (3) transparency: the mixture was placed in xylene I and xylene II for 15 minutes to be transparent.

(9) Sealing: the tissues were mounted using neutral gum.

3) Dyeing step of sirius red

(2) The tissue was rinsed with distilled water for 2 minutes.

(3) The tissue is completely covered by the sirius red staining solution and placed for 30 minutes, and the water around the tissue is wiped off after the tissue is washed by running water.

(4) The tissue is completely covered by Mayer hematoxylin staining solution and placed for 3 minutes, and water around the tissue is wiped off after the tissue is washed by running water.

(5) Differentiation: differentiation was performed for several seconds using 1% hydrochloric acid alcohol differentiation solution.

(6) Anti-blue: slightly washed with running water.

(7) Dehydrating a transparent sealing sheet: placing the tissue slices in 95% alcohol and absolute ethyl alcohol in sequence for 5 minutes respectively to dehydrate; placing in xylene I and xylene II for 10 min, respectively, and sealing with neutral gum.

4) Immunohistochemical staining procedure:

(1) dewaxing to water, baking the slices in an oven at 60 ℃ for 1 hour, placing the tissue slices in xylene I for 10 minutes, xylene II for 10 minutes, absolute ethyl alcohol I for 5 minutes, absolute ethyl alcohol II for 5 minutes, 95% alcohol, 85% alcohol and 70% alcohol for 3 minutes respectively, and hydrating for 20 minutes.

(2) Repairing: the method comprises the steps of filling a proper amount of citrate repairing liquid with the pH value of 8.0 into a pressure cooker, firstly adjusting an electromagnetic oven to be in a big fire state to preheat the liquid in the pressure cooker until the liquid is boiled, then putting prepared slices into the pressure cooker, heating the pressure cooker in a microwave oven until water is boiled again, lifting a boiling valve to start air bleeding, changing to be in a middle-low fire state, and timing for 2 minutes and 30 seconds. And (4) directly placing the pressure cooker in cold water prepared in advance for natural cooling after heating is finished, and taking out the slices when the liquid in the pressure cooker is cooled to room temperature.

(3)3％H₂O₂And (3) sealing: sections were placed in PBS 3 min X3 times 3% H₂O₂And sealing for 15 minutes at the middle room temperature.

(4) Antigen-antibody reaction: in PBS, wash the film 3 minutes times by 3 times, dilute the antibody proportionally and drop-wise onto the labeled tissue and allow it to completely cover the entire tissue, and incubate overnight at 4 ℃ in a refrigerator. Taking out the slices the next day, recovering the slices at room temperature for half an hour, washing the slices in PBS for 3 minutes by 3 times, dripping secondary antibody to completely cover the whole tissue, and incubating in an incubator at 37 ℃ for half an hour.

(5) Color development: washing the film in PBS for 3 min × 3 times, diluting DAB color developing solution proportionally, dripping on tissue, controlling color development time through tissue color, and washing with tap water to stop color development (DAB should be prepared and toxic before use, and should be carefully handled, and should not contact skin).

(6) Counterdyeing: staining with hematoxylin for 1min, soaking in water, differentiating with 1% hydrochloric acid alcohol differentiation solution for 3 s, and bluing in running water for 20 min.

(7) Dehydrating, and sealing.

5) Fluorescent staining procedure for immune tissue

(1) Dewaxing to water: the tissue is more tightly attached to the glass slide by baking the baked sheet in an oven at 60 ℃ for 1 hour. The paraffin sections were immersed in xylene i for 10 minutes, xylene II for 10 minutes, absolute ethanol i for 5 minutes, absolute ethanol II for 5 minutes, 95% ethanol, 85% ethanol, and 70% ethanol each for 2 minutes, and hydrated for 20 minutes.

(2) Repairing: pouring prepared citrate repairing solution with pH of 8.0 into the pressure cooker until all slices are submerged. And preheating the liquid in the pressure cooker by using an electromagnetic oven until the liquid is boiled, putting the prepared slices into the pressure cooker, heating the pressure cooker in a microwave oven until the water is boiled again, lifting a boiling valve, and starting to deflate, changing to medium and low fire, and timing for 3 minutes and 30 seconds. And (4) directly placing the pressure cooker in cold water prepared in advance for natural cooling after heating is finished, and taking out the slices when the liquid in the pressure cooker is cooled to room temperature.

(3) Blocking with 10% donkey serum: sections were washed 3 times in phosphate buffer for 3 minutes each, with 10% donkey serum completely covering the tissue and blocked for 30 minutes at room temperature.

(4) Antigen-antibody reaction: washing the film for 3 min × 3 times in phosphate buffer, dropping the diluted primary antibody onto the marked tissue to cover the whole tissue completely, and preserving in a wet box at 4 deg.C for overnight incubation in a refrigerator. The next day, the slices were removed and returned to room temperature for half an hour, washed 3 times in phosphate buffer, 5 minutes each time. Fluorescent secondary antibody was added dropwise to completely cover the entire tissue, and incubated at 37 ℃ for 1.5 hours.

(5) Color development: in phosphate buffer, wash 10 min × 3 times, and drop DAPI color for 10 min onto the tissue (DAPI is toxic, handling is cautious, and does not touch the skin).

(6) And sealing the anti-fluorescence quencher.

As a result: the histology of the adhesion tissues and the wound surface parts of the original abdominal wall and the caecum of the sham operation group, the model group and the C5aRA group are respectively checked. Fig. 15 shows the histopathological results of each experimental group after 1 week of surgery, all the experimental mice in the sham surgery group had no adhesion to the cecum and abdominal wall, and the experimental mice in the model group had adhesion to the cecum and abdominal wall, and we stained collagen fibers specifically by Masson staining and sirius red staining, and collagen fibers were stained dark blue or sky blue by Masson staining and red collagen fibers by sirius red. The C5aRA group formed loose connective tissue with a smaller number of collagen fibrils than the Control group.

As shown in FIG. 16, the histopathological results of each experimental group at 2 weeks after the operation were that the connective tissue became denser and the thickness of the adhesion tissue was significantly increased in the dotted line region at 1 week after the operation in the sham group, accompanied by a larger amount of collagen fibers. In the C5aRA group, although the connective tissue was dense compared with the postoperation period 1 week, the thickness of the adhesion area was not increased but significantly decreased (shown by a dotted line), and the collagen fiber content and the infiltration amount of inflammatory cells such as neutrophils were small. The C5aRA group has a very significant effect on preventing adhesion after abdominal cavity surgery. The results of quantitative analysis of collagen fibril proliferation at 1 week and 2 weeks after surgery are shown in fig. 17, and collagen fibril proliferation in the model group is higher than that in the C5aRA group. Suggesting that C5aRA can inhibit the proliferation of collagen fibers at the adhesion part.

3.6 conclusion

During abdominal surgery, various inflammatory factors of acute inflammation are generated due to abdominal trauma, and are widely involved in the formation process of abdominal adhesions. The postoperative abdominal adhesion refers to adhesion between the surface mucosa of visceral organs in the abdominal cavity and adhesion between the abdominal wall mucosa and the abdominal wall surface, which often causes a series of complications such as infertility and abdominal pain, and possibly causes more serious complications of intestinal obstruction and intestinal necrosis, and threatens the life of patients. The adhesion is mainly caused by that fibrin at the wound surfaces of the abdominal wall and the viscera can not be completely dissolved, so that the fibrin is continuously deposited until the adhesion occurs. The peritoneal mesothelium is damaged and is accompanied by an inflammatory response, the release of prostaglandins and histamine results in increased permeability of capillaries in the wound tissue, and a serum exudate rich in inflammatory cells is distributed in large quantities around the capillaries, which can solidify within 3 hours. Under the condition of no interference of other factors, the fibrinolytic enzyme system plays a role in accelerating the degradation of substances generated by fibrin aggregation and preventing adhesion. However, as other factors interfere with fibrin deposition at a rate greater than the dissolution rate, and for more than 3 days, proliferation of fibroblasts occurs, eventually leading to adhesion. Therefore, the pathogenesis and preventive measures thereof have been the most important subjects of researchers, and the researches have been conducted in many ways. Since the postoperative abdominal adhesion is a very complicated process, the discovery of each link may become a potential target for improving the abdominal adhesion. In order to reduce the occurrence of postoperative abdominal adhesion in clinic, a series of measures are taken, such as improving an operation mode, reducing bleeding by adopting a minimally invasive technology and preventing infection. However, the effect is general. Covering the postoperative wound with the material is a mature treatment means in the current research stage, but the long-term existence of the material in the body can cause foreign body reaction.

The formation of the abdominal cavity adhesion is a complex process, the accumulation of inflammatory factors is an early pathological characteristic after the abdominal cavity operation, the inflammatory reaction always exists in the whole process of the development of the postoperative adhesion, and particularly, two factors, namely the severity of the inflammatory reaction and the time of the inflammatory reaction, are also key factors for the adhesion. After the body is infected, a series of anti-infection defense reactions occur, wherein complement is widely involved in and plays roles of opsonophagocytosis, cell lysis mediation and immunoadhesion, and in addition, the complement also participates in inflammatory reactions to cause body immune injury and the like. In pathological tissue damage, since immune damage mediated by complement activation plays a great role therein, reducing abnormal complement activation becomes an effective way to reduce inflammatory response.

The stable establishment of the model is explained through general observation, adhesion scoring and histopathology analysis, and a foundation is laid for the smooth implementation of later-stage experiments. The majority of animals in the C5aRA group did not adhere, only individual animals had slight adhesion rated as 1, the content of connective tissue collagen fibers observed in the adhesion part was less, and the exudation of inflammatory cells such as neutrophils was less. As can be seen by observing the expression conditions of TGF-beta 1 and OPN in tissues of the postoperative injury part of the abdominal cavity, the positive expression of the TGF-beta 1 expression C5aRA group is obviously weakened compared with the positive expression of the model group in 1 week and 2 weeks after operation. The wound surface of the injury part of the C5aRA group is well repaired, compared with a model group, the repair is greatly improved, and the expression of TGF-beta 1 and OPN is weakened. The TGF-beta 1 and OPN expression results of each experimental group are consistent with general observation and pathological histological evaluation.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. Application of C5aRA in preparation of products for treating and/or preventing abdominal cavity adhesion.

2. Application of C5aRA in preparation of products for protecting peritoneal mesothelial cells.

3. Application of C5aRA in preparation of products for reducing damage degree of peritoneal mesothelial cells.

4. Use according to claim 3, wherein the peritoneal mesothelial cell damage is caused by lipopolysaccharide.

5. Application of C5aRA in preparation of products for inhibiting mesenchymal transition of epithelial cells.

6. Application of C5aRA in preparation of products for down-regulating expression of alpha-SMA, TGF-beta 1 or OPN.