DNase aggravates intestinal microvascular injury in IBD patients by releasing NET‐related proteins

Neutrophils accumulate in the inflammatory mucosa of patients with inflammatory bowel disease (IBD), and excessive release of NETs (neutrophil extracellular traps may be one of the important factors that cause IBD progression. However, the specific mechanism underlying vascular injury caused by NETs remains unclear. Immunofluorescence, ELISA, and flow cytometry were used in this study to detect the expression of NETs and DNase in the tissue and peripheral blood samples of patients with IBD. DSS mouse model was used to detect colon injury and vascular permeability. We found that NETs and DNase levels increased in the colon of patients with IBD. We found an increase in the activity of NET‐related MPO released by DNase. DNase released NET‐related proteins and damaged vascular endothelial cells in vitro. In DSS mouse model, the synchronous increase of DNase and NETs in the colon leads to an increase in vascular injury markers (CD44, sTM). DNase aggravated colon injury and increased vascular permeability in vivo, which was inhibited by gentamicin sulfate (GS). GS does not reduce the expression of DNase, but rather reduces the release of NET‐related proteins to protect vascular endothelium by inhibiting DNase activity. MPO and histones synergistically damaged the vascular endothelium, and vascular injury can be improved by their active inhibitors. We further found that H2O2 is an important substrate for MPO induced vascular damage. In conclusion, in IBD, DNase, and NET levels increased synchronously in the lesion area and released NET‐related proteins to damage the vascular endothelium. Therefore, targeting DNase may be beneficial for the treatment of IBD.


| INTRODUCTION
The etiology of inflammatory bowel disease (IBD) is a complex process influenced by the external environment, such as high levels of bacteria, and the dysfunction of the immune system, leading to dysregulated immune response. 1The main classes of IBD include ulcerative colitis (UC) and Crohn's disease (CD), which are characterized by recurrence and remission alternately. 2CD has a higher incidence than UC.IBD is a polygenic disease, with family history of disease as one of the main risk factors.The relative risk of IBD in first-degree relatives is five times or higher than other individuals. 3,4ntestinal flora and its products play an important role in the normal development and function of intestinal immune system.Impairment of the integrity of the mucosal barrier results in entry of harmless bacteria through the epithelium, causing immune reactions and inflammation in the intestines. 5The intestinal immune system is well controlled and balanced at all levels in healthy individuals.Inflammatory bowel disease causes epithelial neutrophilic inflammation followed by loss of epithelial integrity, chronic inflammation (usually characterized by secretion of lymphocytes and phagocytes), and epithelial metaplasia. 6However, current studies on immune disorders in IBD mainly focus on the profile and function of lymphocytes, but the role of neutrophils in IBD has not been elucidated.
Neutrophils are the most abundant immune cells in the body, and also the first cells to reach to the infection site when inflammation occurs. 7Neutrophils migrate to the infection site by chemotaxis and kill pathogenic microorganisms through phagocytosis, release of ROS, degranulation and neutrophil extracellular traps (NETs). 8 previous study reported that the intrinsic functions of neutrophils in patients with IBD were not affected. 9owever, significant increase in infiltration and activation of neutrophils is observed in peripheral blood and inflammatory mucosa of IBD patients, 10 and these immune cells are activated in the first stages of IBD attack. 11Calprotectin (S100A8/9, S100A12) is a calcium-and zinc-binding protein secreted by neutrophils and is used as an important indicator to monitor disease progression. 12Studies report that delayed neutrophil apoptosis in IBD aggravates disease progression. 13eutrophils are activated in the colon and migrate to the lung, which may be the main cause of IBD pulmonary disease. 14Myeloperoxidase (MPO) and matrix metalloproteins (MMP) released by neutrophils are implicated in endothelial damage. 15,16revious findings showed that neutrophils actively release DNA and carry proteins which form a network known as NETs, and thus engulfing and killing foreign pathogens. 17Several studies demonstrate that excessive release of NETs is one of the main causes of tissue damage and tumor metastasis. 18,19NETs carry a variety of enzymes, including MPO and NE, which directly damage tissues by destroying cells or intercellular connections. 20,21he findings from our previous research showed that NETs DNA can promote fibroblast differentiation through TLR-9, leading to scar hyperplasia.Inhibiting the excessive release of NETs through use of DNase and PAD4 inhibitors is an effective strategy for treating many diseases. 22,23Studies report that NETs cause intestinal injury and thrombosis in IBD patients. 24However, the mechanism underlying the cause of vascular injury by NETs in IBD is still unclear.Our research demonstrated that the overexpression of DNase in intestinal tissue aggravates the progression of IBD by releasing NET-related proteins, causing microvascular damage.

| Ethics statement
This study was approved by The Medical Ethical Committee of Nanjing Medical University.For experiments involving human blood samples, signed informed consent was obtained from all patients and healthy volunteers.Blood samples were taken from the cubital veins of patients and healthy donors.All the experimental methods were carried out in accordance with the approved guidelines.All experimental procedures involving mice were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and State Key Laboratory of Pathogens and Biosecurity of the Institute of Microbiology and Epidemiology.Blood samples from patients with IBD were obtained from the Suzhou Municipal Hospital, and blood samples from healthy individuals were obtained from the medical examination center.

| Neutrophil extraction
The Neutrophil Isolation Kit isolates functional, highly purified neutrophils directly from human whole blood by immunomagnetic negative selection.(STEMCELL Technologies, #18103, #19666).Briefly, 50 μL of liquid A and 50 μL of liquid B (magnetic beads) were added to each 1 mL blood sample, mixed and allowed to stand for 5 min.Stem buffer diluted 1:1 was added to the blood sample, which was then placed in a magnetic rack and allowed to stand for 10 min.The supernatant was aspirated, 50 μL of solution B was added to each 1 mL of supernatant, and the mixture was again placed in the magnetic rack for 5 min.The supernatant was aspirated and again placed in the magnetic rack for 5 min.The supernatant was aspirated and centrifuged at 400 g for 5 min to obtain neutrophils.Neutrophils were extracted and stored in RPMI-1640 medium (Gibco, Canada) containing 10% FBS.

| Animals
Male C57BL/6 mice (8 weeks old, Suzhou, China) were maintained at the Animal Experimental Center of Suzhou Municipal Hospital under a 12-h light-night cycle with free access to food and water for at least 1 week before the experiments.
Measurement of Intestinal permeability 7 days after DSS treatment, mice were injected intravenously with 200-mL Evans blue (0.5%, dissolved in phosphate-buffered saline).Thirty minutes later, mice were sacrificed and colons were photographed.Then, Evans blue in the colon was extracted by incubation at 65°C with formamide for 2 h and determined spectrophotometrically at 630 nm against a standard curve.

| NET formation and protein purification from NETs
We inoculated 2× 10 6 neutrophils per well in a 12-well plate with RPMI-1640 medium containing 10% FCS.The cells were allowed to settle at the bottom of the wells in 12well plates for 30 min before stimulation with 100 nM PMA for 4 h.For collection of NET, 2 mL of RPMI per well was added, and NETs (the smear on the wells) were collected in 15 mL tubes by vigorous agitation.After centrifugation at 100 g for 5 min, NETs were collected in the supernatant and subjected to different treatments, including complete digestion by 10 U/mL DNase I (Fermentas, Germany) for 20 min at 37°C or kept undigested.The activity of DNase 1 was blocked with 5 mM ethylenediaminetetraacetic acid (EDTA; Applichem, GmbH, Stockholm, Sweden).
Samples were sequentially centrifuged at 300 g to remove whole cells and at 16 000 g to remove cellular debris. 27 2.5 This protocol used several reagents from the Cell Death Detection ELI-SA kit (Roche).First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at room temperature (RT) with antihuman MPO antibody (RD, DY DY3174). Th plate was washed 2 times with wash buffer (0.05% Tween-20 in PBS) and then blocked with 4% bovine serum albumin (Millipore Sigma) in PBS (with 0.05% Tween-20) for 1 h at RT.The plate was again washed 5 times before incubating for 90 min at room temperature with serum or plasma.The plate was washed 5 times and then incubated for 90 min at room temperature with anti-DNA antibody (HRP conjugated; from the Cell Death kit).After 5 more washes, the plate was developed with TMB followed by a 2 N sulfuric acid stop solution.Absorbance was measured at a wavelength of 450 nm using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek).

| Immunohistochemistry
Tissue was fixed in 4% formaldehyde, paraffin-embedded, and sectioned at 5 μm.The sections were exposed to 3% hydrogen peroxide for 10 min to inhibit endogenous peroxidase activity, followed by blocking with 3% bovine serum albumin for 30 min.The tissue sections were stained with hematoxylin and eosin (H&E) and then observed by light microscopy.
Tissue sections were prepared as described above and incubated with Ly6G antibody (Abcam, ab238132; 1:2000) or DNase I antibody (HUABIO, ER1907-64; 1:400) at 4°C overnight.The sections were then washed and incubated with anti-rabbit IgG secondary antibody for 30 min at RT.Then, the sections were restained with hematoxylin and developed using a diaminobenzidine (DAB) kit.The results were obtained by examination under an IX73 microscope (Olympus).

| ELISA
The procedure was carried out based on a kit protocol: MPO (RD, DY9167-05, DY3174), sTM (Elabscience, E-EL-H0166c), DNase I (Uscn, SEB127Hu), CD44 (Abcam, ab49512), MPO activity (Sigma, MAK068), and DNase I activity (Abcam, ab234056).Then, 50-100 μL volumes of standards or samples were added to a 96-well plate and incubated at RT.Samples were added to three wells each.Biotinylated antibody, streptavidin-HRP reagent, and TMB substrate were added sequentially according to the protocol.The color was developed by incubation for 5-30 min at RT while the sample was protected from light.Stop solution was added to each well.The absorbance at the protocol required was measured within 5 min to measure protein levels, and the data were recorded.

| Endothelial cell permeability assay
Human Colonic Microvascular Endothelial Cells (HCMEC) (Sciencell, Catalog #2930) were cultured in endothelial cell culture medium (ScienCell, USA) supplemented with FBS, ECGS, and P/S.HCMEC cells were seeded in 6.5 mm Transwell plates with 0.4 μm pore polycarbonate membrane inserts (Corning, USA).Fibronectin (Sigma-Aldrich, USA) was added during culture to achieve complete fusion.BSA-HRP (Solarbio, China) and recombinant HBP (Novoprotein, China) were added to the upper compartment of the Transwell chamber.After 30 min, a fixed volume of medium was collected from the lower chamber, and the OD value was measured at 450 nM by adding TMB substrate.The concentration of BSA was calculated with the BSA-HRP standard curve.Native cow aprotinin protein (Abcam, UK) was used.

| Statistical analyses
All statistical analyses were performed, and graphs were prepared with GraphPad Prism 8.0 software and Adobe Illustrator.The Shapiro-Wilk test was used to test the normality of continuous variables.The results are expressed as the mean ± standard deviation (SD).For group comparisons, one-way ANOVA was used to compare continuous variables with a normal distribution.The Kruskal-Wallis test was used to compare continuous variables with a skewed distribution.Tukey's post hoc test or Dunn's post hoc test was used for multiple comparisons.Student's t test and the Wilcoxon paired signed rank test were used to compare differences between two groups.The Pearson correlation coefficient was used for correlation analysis.Significant differences are noted by asterisks (*p < .05,**p < .01,***p < .001,****p < .0001).

| The release of NETs in IBD patients increased
We collected peripheral blood neutrophils from patients with IBD and healthy volunteers respectively and tested their ability to generate NETs.We found a significant increase in the number and proportion of neutrophils in the peripheral blood of IBD patients (Figure 1A).We found that the expression of citrullinated histones in the nucleus of neutrophils in peripheral blood of IBD was significantly increased (Figure 1B,C).We continued to detect NETs (MPO-DNA) in the peripheral blood plasma of the two groups and found that NETs in the circulation of IBD patients increased (Figure 1D).In vitro, neutrophils from IBD patients release more NETs (Figure 1E,F).

| Both NETs and DNase in the lesion area of IBD were increased and followed with vascular endothelial damage
Excessive release of NETs has been proved tissue damage.In the process of DSS mice model, we found that the expression of DNase and NETs in the colon of DSS mice increased at fourth day and the increase was synchronous (Figure 2A,B).Three rounds of DSS experiment were conducted and the number of neutrophils in the peripheral blood of DSS-treated mice was displayed, and DSS-treated mice also display increased blood NETs (Figure 2C,D).We also found a significant NETs release increase in colon tissue of IBD patients (Figure 2E).Interestingly, we also found that the DNase expression increased in colon tissue of IBD patients (Figure 2F).We further detected the peripheral blood of IBD patients and found that the expression of DNase in the circulation of patients increased significantly (Figure 2G).In addition, the expression of circulating NET-related proteins, including citH3 and MPO, increased significantly (Figure 2H).The expression of markers of vascular endothelial injury such as sTM and CD44 increased in peripheral blood of IBD patients (Figure 2I,J).

| DNase releases NET-related proteins to damage vascular endothelium
To distinguish whether NETs or its related proteins cause vascular damage, we use DNase to degrade DNA to release NET-related proteins in vitro.When NETs or related proteins co-cultured with intestinal microvascular endothelial cells for 24 h, NET-related proteins promoted more apoptosis of vascular endothelial cells (Figure 3A,B).Under microscope, NET-related proteins also lead to more apoptosis than NETs (Figure 3C).We further detected CD44 and sTM (soluble thrombokinin) in the culture supernatant and found that NET-related proteins promote more release of vascular injury markers (Figure 3D,E).We tested the activity of MPO carried or released by NETs, and found that the activity of MPO released was higher (Figure 3F).NETs or its related proteins co-cultured with platelets to detect the content of PF4 (platelet factor 4) in the culture supernatant and platelets.It was found NETrelated proteins were more capable of activating platelets (Figure 3G).We found that after co-culturing with vascular endothelial cells for 24 h, a large number of NETs adhered to the surface of vascular endothelial cells by immunofluorescence (Figure 3H).

F I G U R E 1
The release of NETs in IBD patients increased.The peripheral blood of healthy volunteers and IBD patients were collected respectively.(A) neutrophil numbers and proportion were significant increased than healthy.(B) Neutrophils were purified, and cell membrane (laminB1, red) and citrullinated histone (citH3, green) were stained with immunofluorescence respectively.The citH3 in the nucleus of patients was significantly higher than that of normal people.(C) Flow cytometry showed that the intracellular citH3 in patients was significantly higher than that in normal people.

| DNase aggravates colon injury
To verify whether DNase can aggravate the damage of vascular endothelium due to the release of NET-related proteins.We increased DNase in mice via the tail vein of DSS mice.It was found that on the seventh day after the DSS mouse model was established, the colon damage of the mice was aggravated (Figure 4A,B).We also set the cytochalasin group and AT791 (TLR-9 inhibitor)group as controls to prove that the colon damage is not aggravated by NETs itself or DNA (Figure 4A,B).Similarly, on the seventh day of modeling, the colonic permeability of mice was tested by measuring Evans blue exudation evaluation, and it was found that DNase could significantly aggravate the colonic exudation (Figure 4C).We injected DNase around the inferior abdominal vessels of DSS mice and found that compared with the control group, the local increase of DNase can significantly increase the vascular permeability (Figure 4D).Reduced neutrophils could also explain why pathology is increased during DNase treatment during DSS, but neutrophil numbers are not altered during administration of DNase into mice (Figure 4E).We continue to use the DNase activity inhibitor gentamicin sulfate (GS) to inhibit the DNase activity of DSS mice.We found that GS could decrease the weight loss, mortality, and disease activity index induced by DSS or DSS+DNase (Figure 4F-H).The mesenteric venous exudation of mice is significantly reduced after GS used (Figure 4I).At the same time, GS reduced the colon injury and exudation of DSS mice (Figure 4J).We also found that the amount and activity of MPO in peripheral blood of DSS mice decreased after GS inhibited DNase activity (Figure 4K).

DNase activity
After using GS, we detected NETs (MPO+citH3) and DNase in colon tissue of DSS mice and found that GS did not reduce the expression of NETs and DNase, But GS can significantly reduce colon damage (Figure 5A,B).However, the detection of colon tissue homogenate showed that GS the activity of MPO (Figure 5B), and this may be due to MPO not being released by NETs.We found that the inflammatory factors in the plasma of DSS mice and patients were significantly increased (Figure 5C), and GS could reduce the expression of inflammatory factors in DSS mice (Figure 5C).We also found that DNase activity in patients and DSS mice did not increased (Figure 5D), which is similar to the results of other studies, but the DNase activity decreased even lower after the use of GS (Figure 5D).

| MPO and histone damage intestinal microvasculature synergistically
Neutrophil granule protein and histone are reported to be the most abundant proteins carried by NETs, and granule proteins mainly include MPO, NE, and MMP9.In previous experiments, we have proved that local DNase releases a large number of NET-related proteins.We used histone inhibitor (activated protein C, APC), MPO inhibitor (AZD5904), NE inhibitor (Sivelestat), and MMP9 inhibitor (Llomastat) to treat NETs or NET-related proteins in vitro.Then co-cultured with intestinal microvascular endothelial cells, it was found that only MPO and histone could cause epithelial damage.In addition, histones cause more serious damage to endothelial cells (Figure 6A).We further tested the damage of MPO and histone to cell junction, and found that histone can damage cell junction at the same time, but MPO has poor effect (Figure 6B).We found that MPO did not increase the permeability of vascular endothelium.However, MPO together with hydrogen peroxide can increase vascular endothelial permeability (Figure 6C).Histone proteins increase the permeability of vascular to some extent.Moreover, histone and MPO have synergistic effects, which can increase vascular permeability and damage (Figure 6C,D).We injected MPO and Histone into wild mice and found that MPO and Histone jointly damaged mesenteric vessels and increased permeability (Figure 6E).At the same time, the vascular endothelium of the mice femoral vein was observed under the transmission electron microscope, and it was found that MPO and Histone damaged the vascular endothelial glycocalyx synergistically (Figure 6F).

| DISCUSSION
The imbalance of intestinal mucosal immune system is considered to be the main factor leading to the progress of IBD. 30 As an important part of innate immunity, neutrophils are activated during the active phase of IBD. 31 Neutrophils participate in the progress of IBD through trans epithelial migration, degranulation, release of cytokines, etc. 32 As NETs is reported to be the cause of many diseases, it is also found to cause tissue damage in IBD. 33n our study, we found that the production of NETs in peripheral blood of patients with IBD was increased, and intact neutrophils were more likely to generate NETs.We also found a large number of NETs in the intestinal tissues of active patients, which is not found in the intestinal tissues of healthy people, which is consistent with the results of many studies. 34,35NETs can activate platelets through their surface proteins and capture blood cells through DNA networks, and both of them can synergistically promote thrombosis. 36The excessive release of NETs during sepsis, mainly including MPO, NE, and other NET-related proteins, directly damages cells or cell junctions. 19,37In the process of scar or pneumonia fibrosis, the DNA structure of NETs promotes fibroblast differentiation through TLR9. 38Nase can eliminate the structure of NETs by degrading DNA.The application of DNase in tumor, 39  thrombosis, 41 and other animal effectively inhibits the progress of the disease.Interestingly, in our study, we found that DNase and NETs increased synchronously on the fourth day during the process of DSS mice modeling.We also found that the expression of DNase in the colon tissue of IBD patients was significantly increased, and this increase did not alleviate the damage of colon microvessels.After DNA is degraded, the released protein aggravates the damage of vascular endothelium.It has been reported that the DNA structure of NETs reduces the activity of NET-related enzymes, but also protects NET-related enzymes from being degraded by endogenous proteases. 42,43n another report, it was also mentioned that after DNA was degraded by DNase, NET-related proteins remained in the body for a long time and maintained enzyme activity. 44herefore, we speculate that locally increased DNase releases NET-related enzymes and aggravates endothelial damage.We further used DNase to degrade DNA and release NETrelated proteins in vitro.After co-culture with microvascular endothelial cells, it was found that NET-related proteins caused more serious vascular endothelial cell damage than NETs.We further proved that the MPO activity after release was higher than that under combined condition.Our results do not conflict with other reports on the improvement of tumor, lupus erythematosus, and thrombosis by DNase, because these diseases progress mainly through DNA aggregation cells or the production of DNA antibodies.However, the role of NET-related proteins released after DNA degradation is rarely discussed.Especially in IBD, the intestinal mucosal immune function is disturbed, and the activity of NET-related enzymes cannot be degraded in time.In de Bruyn M's report, IBD patients could not produce enough TIMPs to antagonize MMPs. 45e found that the intestinal injury caused by injecting DNase into the tail vein during the modeling of DSS mice was more serious than that in the group without DNase injection.The use of Cytb to directly inhibit the formation of NETs can reduce intestinal injury.This further indicates that the release of NET-related proteins aggravates intestinal injury.We further used the mesenteric vein and abdominal wall vein of mice for modeling.It was found that local infiltration of DNase could cause more leakage of mesenteric vein in DSS mice.After the use of DNase activity inhibitor (GS), this situation was significantly improved, and the expression of inflammatory factors and the amount and activity of MPO in DSS mice were reduced.In our previous study, we found that MPO can accelerate vascular leakage by destroying the glycocalyx of vascular endothelium. 41However, there are many NETrelated enzymes, which will be further discussed later. of (H 2 O 2 which consistent with our previous research on burn leakage.MPO mainly damages the glycocalyx of vascular endothelium.Histone damages the connection of vascular endothelium, and it has been reported that histone has cytotoxic effect.Our study also found that MPO and histone can synergistically increase the permeability of mesenteric vessels in mice.

| CONCLUSION
In conclusion, our study reported for the first time that the local DNase and NETs increased simultaneously in IBD, and the release of a large number of NET-related proteins was the main cause of vascular endothelial damage (Figure 7).It is also discussed that NETs may be one of the causes of IBD related diseases.It provides a new therapeutic target for clinical treatment of IBD.

AUTHOR CONTRIBUTIONS
Yiming Shao, Yunxi Yang, and Bingwei Sun designed the study and wrote the paper.Yulan Ye, Xi Gao, Zaiwen Guo, Jiamin Huang, and Linbin Li performed the experiments.Zaiwen Guo, Lu Liu, Yulan Ye, and Yi Chen performed collection of clinical data.Yiming Shao and Bingwei Sun performed the statistical analysis.All authors read and approved the final manuscript.

F I G U R E 2
Both NETs and DNase in the lesion area of IBD were increased and accompanied with vascular endothelial damage.(A, B) The pathological colon of DSS mice was sectioned.DNase and NETs (citH3 red+MPO green) in colon increased synchronously on the fourth day.(C, D) Three rounds of DSS experiment were conducted and the number of neutrophils in the peripheral blood of DSS-treated mice was increased, and DSS-treated mice also display increased blood NETs.(E) The NETs (citH3 red+MPO green) in the tissues of the patients were significantly increased after the tissues were sliced and stained with polychrome immunofluorescence.(F) Immunohistochemical staining showed that DNase in the tissues of patients increased significantly.(G-J) Collect peripheral blood from healthy volunteers and patients.ELISA showed that DNase, citH3, sTM, and CD44 in patients were significantly increased.*p < .05,**p < .01,***p < .001,****p < .0001.

F I G U R E 3
DNase releases NET-related proteins to damage vascular endothelium.In vitro, DNase is used to digest the DNA of NETs and release NET-related proteins.NETs and NET-related proteins were cultured with intestinal microvascular epithelium for 24 h.(A, B) Flow cytometry showed that NET-related proteins led to more apoptosis.(C) Microscopy showed that NET-related proteins caused more cell death.(D, E) ELISA detected the culture medium and found that NET-related proteins caused more cell damage markers (CD44, sTM, VE cadherin) to increase.(F) The activity of MPO also increased.(G) The detection of PF4 (platelet factor 4) in the culture medium by ELISA and the detection of intracellular PF4 by WB showed that NET-related proteins promoted the increase of more PF4.(H) Immunofluorescence showed that NETs (green) gathered around the cells when co-cultured with intestinal microvascular endothelium.**p < .01,***p < .001,****p < .0001.
lupus F I G U R E 4 DNase aggravates colon injury.DSS mice model, (A-C) At the same time of making DSS model, cytochalasin (cytB), DNase, and TLR-9 receptor inhibitor (AT791) were injected into the tail vein of mice, respectively.It was found that DNase caused more severe colon damage and increased permeability.(D) DNase was injected around the abdominal wall vessels of DSS mice, and DNase caused more vascular leakage.(E) DNase treatment did not reduce the number of neutrophils in DSS mice.(F-H) GS significantly reduced the weight loss, motility, and disease activity index of DSS or DSS+DNase treated mice.(I-K) Gentamicin sulfate (GS) inhibits DNase activity, effectively reduces colon injury and exudation, and reduces the amount and activity of MPO in peripheral blood.*p < .05,**p < .01,***p < .001,****p < .0001.

F I G U R E 5
Gentamycin sulfate inhibits DNase activity and alleviates colon injury.(A, B) Fluorescent staining of NETs and immunohistochemical analysis of DNase were performed on colon tissue of DSS mice.GS reduced colon damage and MPO activity without reducing DNase.(C, D) The activity of inflammatory factors and DNase in mouse plasma and patient plasma was tested.The expression of peripheral blood inflammatory factors in DSS mice and patients increased, but the activity of DNase was lower than that in the control group.GS can effectively reduce the expression of inflammatory factors and the activity of DNase.*p < .05,**p < .01,***p < .001,****p < .0001.NET-related proteins mainly and neutrophil granule proteins.We used a variety enzyme inhibitors to treat NET-related proteins, and found that MPO and histones are the main components that cause vascular endothelial damage.The damage of MPO to vascular endothelium requires the presence F I G U R E 6 MPO and histone damage intestinal microvasculature synergistically.(A) In vitro, NET-related proteins were co-cultured with intestinal microvascular endothelium, and different enzyme inhibitors were used at the same time.CD44 in the culture supernatant was detected by ELISA.Only histone inhibitors and MPO inhibitors could reduce the expression of CD44.(B) MPO and histone were used to co-culture with intestinal microvascular endothelium, and VE cadherin in the culture supernatant was detected by ELISA.Histone can cause more cell connection damage.(C) The intestinal microvascular endothelial cells were laid on the upper layer of transwell and cocultured with MPO/MPO+H 2 O 2 /histone for 24 h.At the same time, BSA-HRP is added, BSA-HRP is detected at the lower layer, and CD44 is detected at the upper layer.In the presence of substrate, MPO cooperates with histone to damage cells.(D) Under the microscope, MPO and histone damage cells synergistically in the presence of substrate.(E) MPO/histone was injected into the tail of mice, and MPO cooperated with histone to cause more obvious mesenteric vascular leakage.(F) MPO/histone was injected into the tail of mice, and the vascular endothelium of mouse femoral vein was observed under transmission electron microscope.MPO and histone cooperated to cause the loss of sugar calyx on the surface of vascular endothelium more obviously.**p < .01,***p < .001,****p < .0001.F I G U R E 7 DNase releases NET-related protein to damage vascular endothelium during IBD.Neutrophils in the lesion colon were activated to release NETs.Local DNase and NETs increased synchronously, releasing NET-related proteins.MPO and histone cooperate to damage vascular endothelial cells.