CN114470196B

CN114470196B - Application of CCL3/CCR4 neutralizing antibody and NF- κB inhibitor in preparing NEC therapeutic drug

Info

Publication number: CN114470196B
Application number: CN202210089584.9A
Authority: CN
Inventors: 陈大鹏; 袁溪; 宋志新
Original assignee: Childrens Hospital of Chongqing Medical University
Current assignee: Childrens Hospital of Chongqing Medical University
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2023-06-06
Anticipated expiration: 2042-01-25
Also published as: CN114470196A

Abstract

The invention belongs to the technical field of medical biology, and particularly discloses application of a CCL3/CCR4 neutralizing antibody and an NF- κB inhibitor in preparation of NEC therapeutic drugs. The invention discovers that CCL3 plays a key role in the disease course and progress of the Necrotizing Enterocolitis (NEC) of the newborn for the first time. The invention discovers that CCL3 neutralizing antibodies, CCR4 neutralizing antibodies or NF- κB inhibitors can block relevant links in CCL3-CCR4-NF- κB in a targeted way, thereby blocking CCL3-CCR4 signal axes, effectively reducing apoptosis of intestinal epithelial cells, inhibiting inflammatory macrophage polarization and remarkably improving intestinal damage during NEC. The invention provides a new target point and a new strategy with potential application value for clinical NEC prevention and treatment.

Description

Application of CCL3/CCR4 neutralizing antibody and NF- κB inhibitor in preparing NEC therapeutic drug

Technical Field

The invention relates to the technical field of medical biology, in particular to the technical field of treatment of necrotizing enterocolitis, and particularly relates to application of a CCL3/CCR4 neutralizing antibody and an NF- κB inhibitor in preparation of a medicament for treating neonatal Necrotizing Enterocolitis (NEC).

Background

Necrotizing Enterocolitis (NEC) in newborns is a common and extremely severe acute severe gastrointestinal disorder in neonatal period. Is mainly seen in premature infants and newborns with low birth weight, and is a disease peculiar to newborns. It is characterized in that intestinal tract injury is from mucous membrane injury to injury with different degrees such as full-layer necrosis, perforation, etc. The incidence rate of NEC of infants with pregnancy less than or equal to 33w or birth weight less than or equal to 2500g is as high as 13%, and the mortality rate of NEC is about 20% -30%. The mortality rate of children undergoing surgery is as high as 50%. While surviving NEC infants are susceptible to complications such as short bowel syndrome, cholestasis, significant impaired growth, and neurodevelopment. These infants require long-term care, and the treatment is complex and costly. NEC early treatment is mainly medical conservation treatment, and surgery treatment is needed when combined with intestinal perforation or ineffective medical conservation treatment or disease progression. Because the operation treatment is easy to combine short bowel syndrome, malnutrition and nervous system development disorder, the survival rate and life quality of the children are seriously affected. Thus, a medical conservative treatment of NEC remains the best approach.

Elucidation of the intrinsic pathogenesis of NEC onset is crucial for the discovery of its intrinsic causative factors and the further development of targeted therapeutic drugs. NEC intestinal inflammation is characterized by the production of large amounts of pro-inflammatory factors, infiltration and activation of leukocytes such as neutrophils, macrophages and lymphocytes, and damage to the intestinal mucosal barrier and necrosis of the intestinal wall caused by inflammatory disorders resulting from this. Inflammatory cytokines and chemokines play a push-promoting role therein.

CCL3, also known as macrophage inflammatory protein alpha (MIP-1 alpha), is a pro-inflammatory chemokine that can be produced by a variety of leukocytes and tissue cells and plays an important role in the inflammatory response by binding to the G protein-coupled receptors CCR1, CCR4 and CCR3 on target cells. It has been found that hyperactive CCL3 has a direct and detrimental effect on disease outcome. CCL3 expression has been implicated in the progression of a variety of inflammatory and immune diseases, such as esophageal squamous cell carcinoma, chronic granulocytic leukemia, acute and chronic (fibrotic) liver injury, and the like. However, the role of CCL3 in NEC is still unclear. In addition, CCL3 neutralizing antibodies may neutralize CCL3 and thereby inhibit CCL 3-mediated immune chemotaxis. The CCL3 neutralizing antibodies have been found to have protective effects against a variety of diseases. However, there is no study on whether CCL3 neutralizing antibodies can reduce NEC bowel damage.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide the use of a CCL3/CCR4 neutralizing antibody, NF- κb inhibitor in the preparation of a medicament for treating neonatal Necrotizing Enterocolitis (NEC), and by discussing the effect of CCL3 in NEC, the intervention effect of CCL3 neutralizing antibody, CCR4 neutralizing antibody, NF- κb inhibitor on NEC and the mechanism of action thereof are studied, thereby providing a new target and a new strategy with potential and good application value for clinical prevention, diagnosis and treatment of NEC.

To achieve the above and other related objects, a first aspect of the present invention provides use of a CCL3 neutralizing antibody in the preparation of a medicament for the treatment of necrotizing enterocolitis in newborns.

Further, the administration dose of the CCL3 neutralizing antibody is 0.1-2mg/kg or 2-10mg/kg.

Further, the administration mode of the neonatal necrotizing enterocolitis therapeutic drug is at least one of intraperitoneal injection, intravenous injection, intramuscular injection, subcutaneous injection and the like. In the animal experiments, intraperitoneal injection or tail vein injection is usually adopted, and the intraperitoneal injection operation is simple; for the treatment of human NEC, intravenous injection or other means is typically used.

In a second aspect the invention provides the use of a CCR4 neutralizing antibody for the manufacture of a medicament for the treatment of neonatal necrotizing enterocolitis.

Further, the CCR4 neutralizing antibody is administered at a dose of 0.1-2mg/kg or 2-10mg/kg.

Further, the administration mode of the neonatal necrotizing enterocolitis therapeutic drug is at least one of intraperitoneal injection, intravenous injection, intramuscular injection, subcutaneous injection and the like.

In a third aspect, the invention provides the use of an NF- κb inhibitor for the manufacture of a medicament for the treatment of neonatal necrotizing enterocolitis by blocking the NF- κb pathway to reduce inflammatory damage to intestinal tissue caused by neonatal necrotizing enterocolitis.

Further, the NF- κB inhibitor is selected from PDTC, but is not limited to PDTC, and other NF- κB inhibitors can block NF- κB related signaling pathways to produce similar effects. PDTC, commonly known as Ammonium pyrrolidinedithiocarbamate or Pyrrolidinedithiocarbamic acid, is an NF- κB activation (NF- κBactination) inhibitor that can permeate cell membranes and inhibit NF- κB activation in a variety of cells.

Further, the NF- κB inhibitor may be administered at a dose of 10-30mg/kg, 30-90mg/kg or 90-120mg/kg.

In a fourth aspect, the present invention provides a medicament for the treatment of necrotizing enterocolitis in newborns, comprising a CCL3 neutralizing antibody according to the first aspect and/or a CCR4 neutralizing antibody according to the second aspect and/or an NF- κb inhibitor according to the third aspect.

As described above, the use of the CCL3/CCR4 neutralizing antibody, NF- κb inhibitor, of the invention in the preparation of NEC therapeutic drugs has the following beneficial effects:

the invention discovers that CCL3 plays a promoting role in the progress of NEC. Based on the above, the invention researches and discusses the intervention effect of CCL3 neutralizing antibody, CCR4 neutralizing antibody and NF- κB inhibitor on NEC and the action mechanism thereof, and discovers that the CCL3 neutralizing antibody, the CCR4 neutralizing antibody or the NF- κB inhibitor can target and block the relevant links in CCL3-CCR4-NF- κB, thereby blocking the CCL3-CCR4 signal axis, effectively reducing apoptosis of intestinal epithelial cells, inhibiting inflammatory macrophage polarization and obviously improving NEC intestinal injury. The research of the invention provides the basis for developing and preparing the innovative medicines for treating NEC, provides a new strategy for clinical internal medicine treatment of NEC, and has important significance for NEC treatment.

Drawings

FIG. 1-1 shows a bar graph of the expression levels of CCL3 from surgically resected necrotic intestinal tissue and nearby non-necrotic sites in NEC patients in example 1 of the present invention.

FIGS. 1-2 are bar graphs showing the expression levels of CCL3 in NEC model mouse intestinal tissue and control intestinal tissue in example 1 of the present invention.

FIGS. 1-3 are immunohistochemical graphs showing CCL3 expression levels in NEC model mouse intestinal tissue versus control intestinal tissue in example 1 of the present invention.

FIG. 2-1 shows body weight graphs of NEC model mice, NEC model mice injected intraperitoneally with recombinant CCL3 protein (rCCL 3), NEC model mice injected intraperitoneally with CCL3 neutralizing antibody (anti-CCL 3) in example 2 of the present invention.

FIG. 2-2 shows the survival rate of NEC model mice in example 2 of the present invention, NEC model mice injected intraperitoneally with rCCL3, and NEC model mice injected intraperitoneally with anti-CCL 3.

FIGS. 2-3 show the intestinal appearance and HE staining patterns of NEC model mice, NEC model mice injected intraperitoneally with rCCL3, NEC model mice injected intraperitoneally with anti-CCL3, and control groups in example 2 of the present invention.

FIGS. 2 to 4 are graphs showing the intestinal injury scores of NEC model mice, NEC model mice injected intraperitoneally with rCCL3, NEC model mice injected intraperitoneally with anti-CCL3, and control groups in example 2 of the present invention.

FIGS. 2-5 are bar graphs showing the expression levels of IL-6, TNF-a, IFN-gamma, IL-1β in intestinal tissue of NEC model mice, in-vitro rCCL3 NEC model mice, in-vitro anti-CCL3 NEC model mice, and control groups in example 2 of the present invention.

FIG. 3-1 shows TUNEL fluorescence maps of NEC model mice, NEC model mice injected intraperitoneally with rCCL3, NEC model mice injected intraperitoneally with anti-CCL3, and intestinal tissue sections of control group in example 3 of the present invention.

FIG. 3-2 shows immunoblot analysis of expression levels of the intestinal tissue apoptosis protein BAX/BCL-2 of NEC model mice, NEC model mice injected with rCCL3 intraperitoneally, NEC model mice injected with anti-CCL3 intraperitoneally, and control groups in example 3 of the present invention.

FIGS. 3-3 are graphs showing apoptosis of intestinal epithelial cells of mice intestinal epithelial cell strain IEC-6 of example 3 of the invention 4h after LPS stimulation of rCCL3, anti-CCL3 pretreatment.

FIG. 4-1 is a bar graph showing the evaluation of NEC mouse CCR1, CCR3, CCR4 mRNA and NEC infant human CCR4 mRNA expression levels by qRT-PCR in example 4 of the present invention.

FIG. 4-2 shows immunofluorescence of CCR4 (pink) expression levels of NEC model mouse intestinal tissue versus control intestinal tissue in example 4 of the present invention.

FIGS. 4-3 are immunoblot analysis of expression levels of CCR4 in NEC model mice, in NEC model mice injected intraperitoneally with rCCL3, in NEC model mice injected intraperitoneally with anti-CCL3, and in control intestinal tissues in example 4 of the present invention.

FIGS. 4-4 show immunofluorescence graphs showing co-localization of CCR4 (green) and CCL3 (red) in intestinal tissue of NEC model mice in example 4 of the present invention.

FIG. 5-1 shows HE staining patterns of NEC model mice, CCR4 neutralizing antibody (anti-CCL 4) intraperitoneally injected NEC model mice, PDTC intraperitoneally injected NEC model mice, and control intestinal tissues in example 5 of the present invention.

FIG. 5-2 shows the intestinal injury score of NEC model mice, anti-CCR4 intraperitoneally injected NEC model mice, PDTC intraperitoneally injected NEC model mice, and control intestinal tissue in example 5 of the present invention.

FIGS. 5-3 are TUNEL fluorescence maps of NEC model mice, anti-CCR4 intraperitoneally injected NEC model mice, PDTC intraperitoneally injected NEC model mice, and control intestinal tissue sections in example 5 of the present invention.

FIGS. 5-4 are graphs showing apoptosis of intestinal epithelial cells of mice in example 5 of the present invention, IEC-6, stimulated by LPS for 4h after anti-CCR4 pretreatment.

FIG. 6-1 shows immunoblot analysis of expression levels of p-ERK1/2 and p-NF- κB of mouse intestinal epithelial cells IEC-6 of example 6 of the invention after pretreatment of rCCL3, anti-CCL3 and LPS treatment at different times (0, 1, 3, 6 h).

FIG. 6-2 shows immunoblot analysis of expression levels of p-ERK1/2 and p-NF- κB after LPS treatment at different times (0, 1, 3, 6 h) after anti-CCR4 pretreatment of mouse intestinal epithelial cells IEC-6 in example 6 of the invention.

FIGS. 6-3 are graphs showing apoptosis of intestinal epithelial cells of mice in example 6 of the present invention, IEC-6, stimulated with LPS for 4h after rCCL3+PDTC pretreatment.

FIGS. 6-4 are Western blot analysis of expression levels of the intestinal tissue apoptosis protein BAX/BCL-2 of NEC model mice, PDTC-injected NEC model mice intraperitoneally, and control groups according to example 6 of the present invention.

FIG. 7-1 is a flow chart showing the preparation of single cell suspensions for detecting F4/80+ cell infiltration after digestion of intestinal tissues of NEC model mice, NEC model mice injected with rCCL3 intraperitoneally, NEC model mice injected with anti-CCL3 intraperitoneally, in example 7 of the present invention.

FIG. 7-2 is a bar graph showing the evaluation of mRNA expression levels of human iNOS/human CD86 gene M1 associated with NEC infant by qRT-PCR in example 7 of the present invention.

FIG. 7-3 is a bar graph showing the mRNA expression levels of M1-related genes (iNOS, CD86, IRF 5) and M2-related genes (FIZZ 1, YM1, arg-1) of NEC model mice, NEC model mice injected with rCCL3 intraperitoneally, NEC model mice injected with anti-CCL3, and control groups in example 7 of the present invention by qRT-PCR.

FIGS. 7-4 show preparation of single cell suspensions for detection of F4/80 after digestion of intestinal tissue of NEC model mice, CCL3 recombinant protein-injected NEC model mice, CCL3 neutralizing antibody-injected NEC model mice and control group in example 7 of the present invention ⁺ CD86 ⁺ /F4/80 ⁺ CD206 ⁺ Flow chart of cell infiltration.

FIGS. 7-5 show mouse peritoneal macrophages in example 7 of the present invention

Flow cytometry detection of F4/80 after rCCL3, anti-CCL3 pretreatment ⁺ CD86 ⁺ /F4/80 ⁺ CD206 ⁺ Flow chart of cell infiltration.

FIGS. 7-6 show flow cytometry detection of F4/80 after rCCL3, anti-CCL3 pretreatment for mouse Bone Marrow Derived Macrophages (BMDM) in example 7 of the present invention ⁺ CD86 ⁺ /F4/80 ⁺ CD206 ⁺ Flow chart of cell ratio.

Detailed Description

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

The invention aims at providing a brand new strategy for clinical treatment of NEC, discovers that CCL3 is a key harmful internal cause in NEC occurrence and development through animal experiments, and definitely blocks CCL3 and a receptor CCR4 thereof and an important role of downstream pathway NF- κB in NEC treatment.

Based on this, the invention proposes the use of CCL3 neutralizing antibodies, CCR4 neutralizing antibodies, NF- κb inhibitors in the preparation of NEC therapeutic drugs.

The nucleotide/amino acid sequence related to the invention is as follows:

information on genes and proteins of human CCL3 in the NCBI database (Gene ID: 6348) is found in https:// www.ncbi.nlm.nih.gov/Gene/6348. Wherein the nucleotide sequence of the human CCL3 is shown as SEQ ID NO.1, and the amino acid sequence of the human CCL3 is shown as SEQ ID NO. 2.

Gene and protein related information on human CCL3 in the NCBI database (Gene ID: 1233) is found in https:// www.ncbi.nlm.nih.gov/Gene/1233. Wherein the nucleotide sequence of human CCR4 is shown as SEQ ID NO.3, and the amino acid sequence of human CCR4 is shown as SEQ ID NO. 4.

In the following examples, murine rCCL3, murine anti-CCL3 were purchased from R & D Systems, inc., U.S.A., anti-CCR4 was purchased from SANTA CRUZ biotechnology, inc., and NF-. Kappa.B inhibitor PDTC was purchased from Selleck, inc., U.S.A..

The following specific exemplary examples illustrate the invention in detail. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the invention, as many insubstantial modifications and variations are within the scope of the invention as would be apparent to those skilled in the art in light of the foregoing disclosure. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a suitable selection from the description herein and are not intended to be limited to the specific values described below.

Example 1

Expression of CCL3 in NEC infants and model mice

1. Materials and methods

1.1, patient intestinal tissue specimens

Approved by the ethics committee of the affiliated children hospital of Chongqing medical university and signed informed consent. The specimens required for this experiment were all from discarded intestinal tissue specimens excised during the patient surgery and were used only for scientific research for non-commercial use, and were disposed of in a prescribed manner after use.

1.2, laboratory animals

The experiment is approved by the animal ethics committee of Chongqing medical university. Newborn C57BL/6 mice pups (male and female) 7-10 days old were purchased from the university animal center of Zoose medical science. SPF environment rearing, weighing, random grouping, control group 10, NEC group 10.

1.3 construction of mouse NEC model

The NEC mice are filled with 20-30 ul/g hypertonic formula milk every 4 hours for 5 times. The formula contains Similac 60/40 (Abbott Laboratories, saint-Laurent, canada) and Esbilac (PetAg, hampshire, illinois). In addition, pups were exposed twice daily to cold stimulation (4 ℃,10 min) and low oxygen (100% N2, 90 seconds) and fed lipopolysaccharide (LPS, sigma-Aldrich, st. Louis) 5ug/g/d. The pups of the control group were breast fed. Mice that were inappropriately fed or died within 24 hours after NEC induction were excluded. All surviving pups were euthanized 96 hours after NEC induction.

1.4 detection of CCL3 in mouse intestinal tissue

Taking small intestine tissues of a mouse, homogenizing, centrifuging and collecting supernatant, detecting the level of CCL3 by adopting an enzyme-linked immunosorbent assay (ELISA) method, operating according to the instruction of a kit, and detecting by using an enzyme-labeled instrument.

1.5 immunohistochemistry

Antigen retrieval was performed with reference to the first antibody instructions, a suitable amount of endogenous peroxidase blocker was added to the sample, incubated at room temperature for 10min, and then rinsed with Phosphate Buffered Saline (PBS). Goat anti-mouse CCL3 antibody (1:40, AF-450-SP) was dropped onto the specimen and incubated at 37℃for 60 minutes. Tissues were stained using Anti-coat HRP-DAB cells and tissue staining kit (Brown; R & D, CTS 008) and counterstained with hematoxylin (blue).

1.6 statistical method

GraphPad Prism 7 (GraphPad Software, la Jolla, calif.) was used for all statistical analyses and data. Consecutive data are expressed as mean ± standard deviation, ordinal data are expressed using median and quarter bit spacing (IQR). A two-tailed unpaired t-test or one-way anova was performed in the comparison between two or more groups. p <0.05 is considered statistically significant.

2. Results

2.1, as shown in fig. 1-1, NEC patients showed significantly higher expression levels of surgically resected necrotic intestinal CCL3 than nearby non-necrotic sites.

2.2 as shown in FIGS. 1-2, the expression level of CCL3 in the intestinal tissue of NEC model mice was significantly increased compared to that of the intestinal tissue of the control group.

2.3 as shown in FIGS. 1-3, CCL3 (buffy) expression levels in NEC model mice intestinal tissue sections were significantly higher than in control intestinal tissue sections.

3. Conclusion(s)

The level of CCL3 expression in mouse intestinal tissue was detected by ELISA, and the results showed that: the expression of CCL3 was significantly increased in both NEC-affected infants and model mice, suggesting that CCL3 may be involved in the development of NEC.

Example 2

CCL3 is a key harmful endogenous factor in NEC occurrence and development, and blocking CCL3 by anti-CCL3 can remarkably reduce intestinal damage of NEC model mice.

1. Materials and methods

1.1, laboratory animals and medicaments

The experiment is approved by the animal ethics committee of Chongqing medical university. Newborn C57BL/6 mice pups (male and female) 7-10 days old were purchased from the university animal center of Zoose medical science. SPF environment feeding, weighing, random grouping, control group 10, NEC group 10, NEC+rCCL3 group 10, NEC+anti-CCL3 group 10.

1.2 modeling and intervention

The modeling method is the same as 1.3 in example 1. Nec+rccl3 group: during NEC modeling, 31mg/kg of intraperitoneal injection rCCL was administered daily for 4 consecutive days; NEC+anti-CCL3 group: during NEC modeling, anti-CCL31mg/kg was given daily for 4 consecutive days. Control group: the mice were kept in the same cage as the female mice, and the mice were fed with milk and fed freely. Mice that were inappropriately fed or died within 24 hours after NEC induction were excluded. All surviving pups were euthanized 96 hours after NEC induction.

1.3, NEC intestinal tissue injury score

Rat small intestine tissues were fixed by soaking them in 4% paraformaldehyde, dehydrated with alcohol having a gradient concentration of 70% to 100%, and after dehydration, tissue transparence was performed with 100% xylene. And (5) after the transparency is finished, immersing the tissues after the transparency is finished, and carrying out paraffin slicing. Following H & E staining of the sections, pathological changes were observed by microscopy and intestinal injury scoring was performed. And judging that the score of the intestinal injury is more than or equal to 2 points is NEC.

1.4, detection of inflammatory factors of intestinal tissue of mice

Taking small intestine tissue of a mouse, homogenizing, centrifuging and collecting supernatant, detecting inflammatory factors interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) by an enzyme-linked immunosorbent assay (ELISA) method, operating according to a kit instruction, and detecting by an enzyme-labeled instrument.

1.5 statistical methods

2. Results

This example experiment found that NEC mice injected with rCCL3 had significantly reduced body weight (FIG. 2-1) and survival (FIG. 2-2) and macroscopic changes in intestinal flatulence, hemorrhage, necrosis, etc., and HE staining showed increased intestinal injury (FIG. 2-3). However, intestinal tissue damage was significantly reduced in NEC mice injected with anti-CCL3 (fig. 2-4).

Uncontrolled inflammation is another important feature of NEC, one of the causes of intestinal damage during NEC. Thus, the present experiment evaluates intestinal inflammation by measuring intestinal inflammatory factor expression. The levels of inflammatory factors in the intestinal tract, IL-6, TNF-a, IFN-gamma, IL-1β, etc., were significantly elevated after NEC induction, while anti-CCL3 significantly inhibited NEC-mediated expression and release of inflammatory factors, IL-6, TNF-a, IFN-gamma, IL-1β, etc. (FIGS. 2-5).

3. Conclusion(s)

By detecting and analyzing the survival rate curve, the weight change curve, the HE staining and the inflammatory factors, the results show that: the release of CCL3 plays a key role in the pathogenesis of NEC, CCL3 being a key detrimental internal cause in the development of NEC, rCCL3 being able to exacerbate intestinal damage. And anti-CCL3 plays a role in protecting NEC, and can relieve intestinal damage.

Example 3

rCCL3 aggravates apoptosis of intestinal epithelial cells, while blocking CCL3 can effectively alleviate apoptosis of intestinal epithelial cells.

1. Materials and methods

1.1, laboratory animals

The experiment is approved by the animal ethics committee of Chongqing medical university. Newborn C57BL/6 mice pups (male and female) 7-10 days old were purchased from the university animal center of Zoose medical science. SPF environment feeding, weighing, random grouping, control group 10, NEC group 10, NEC+rCCL3 group 10, NEC+anti-CCL3 intervention group 10.

1.2 modeling and intervention

The modeling method is the same as 1.3 in example 1. Nec+rcccl3 group: during NEC modeling, 31mg/kg of intraperitoneal injection rCCL was administered daily for 4 consecutive days; NEC+anti-CCL3 group: during NEC modeling, anti-CCL31mg/kg was given daily for 4 consecutive days. Control group: the mice were kept in the same cage as the female mice, and the mice were fed with milk and fed freely. Mice that were inappropriately fed or died within 24 hours after NEC induction were excluded. All surviving pups were euthanized 96 hours after NEC induction.

1.3 TUNEL staining

Apoptosis IN ileum sample sections was assessed by a commercially available TUNEL staining kit (Roche Diagnostics, indianapolis, IN) according to the manufacturer's instructions.

1.4 Western blot analysis

Adding RIPA buffer solution into mouse ileocecum tissue, grinding by a homogenizer, centrifuging for 10min at 12,000r/min, taking supernatant as tissue lysate, preparing SDS-polyacrylamide gel electrophoresis to separate protein, and transferring the protein into PVDF membrane. After sealing for 2 hours at room temperature with 5% skim milk, washing 3 times with TBST, adding BAX (1:1000), BCL-2 (1:1000), GAPDH (1:1000) and then incubating the primary antibody-containing membrane overnight at 4℃and then adding horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:8000) and goat anti-mouse IgG antibody (1:10000), incubating at room temperature for 1 hour, developing PVDF membrane using ultrasensitive ECL chemiluminescent kit after exposure, and quantifying using Image J (National Institutes of Health, bethesda, MD). The following antibodies were used: anti-BAX (ab 32503) and anti-BCL-2 (ab 182858) from Abcam.

1.5 flow cytometry detection for apoptosis

IEC-6 cells were pre-incubated overnight at a density of 1X 10≡5 cells/mL. After 24 hours, the cells were pretreated with rCCL3/anti-CCL3 for 24 hours and then stimulated with 100. Mu.g/ml LPS for 4 hours. Then, the cells were resuspended at 1X 10≡6 cells/mL, then 5. Mu.l annexin V-FITC and propidium iodide (Becton Dick-inson and Company, lake Franklin, USA) were added. Apoptosis was detected by flow cytometry (Becton Dickinson and Company, lake Franklin, USA).

1.6 statistical method

2. Results

2.1 As shown in FIG. 3-1, the proportion of apoptotic cells in the NEC group was significantly greater than that in the control group, rCCL3 treatment aggravated NEC-induced apoptosis, while anti-CCL3 treatment almost completely abrogated NEC-induced apoptosis.

2.2 as shown in fig. 3-2, the changes in apoptosis factor reflect the severity of NEC intestinal apoptosis. Thus, BAX and BCL-2 expression in the intestinal tract was also assessed in ileal tissue samples collected after NEC. Expression of the NEC group pro-apoptotic factor BAX was up-regulated and expression of the anti-apoptotic factor BCL-2 was down-regulated compared to the control group. Up-and down-regulation of both proteins was more evident after CCL3 recombinant protein treatment. While CCL3 neutralizing antibodies significantly reversed the up-and down-regulation of both proteins to levels similar to those of the control and NEC groups.

2.3 As shown in FIGS. 3-3, to further investigate the effect of CCL3 on intestinal epithelial apoptosis, the experiment of this example established a model of LPS-stimulated IEC-6 cell inflammation, and flow cytometry showed that LPS stimulation increased apoptosis of IEC-6 cells compared to the control group, whereas CCL3 recombinant protein pretreatment significantly increased apoptosis, whereas pretreatment with CCL3 neutralizing antibodies significantly decreased apoptosis.

3. Conclusion(s)

Detection by TUNEL fluorescence, immunoblotting, flow cytometry, results showed: CCL3 plays a key role in the pathogenesis of NEC by affecting intestinal epithelial apoptosis, and CCL3 recombinant proteins are able to exacerbate intestinal epithelial apoptosis. And CCL3 neutralizing antibody plays a role in protecting NEC and can reduce intestinal epithelial apoptosis.

Example 4

CCL3 exacerbates intestinal tissue damage in experimental NEC through CCL3-CCR4 axis

1. Materials and methods

1.1, Q-PCR analysis

Intestinal tissue specimens were obtained from NEC patients and NEC mice as in example 1. Total RNA was extracted from whole intestinal tissue using Trizol reagent (Invitrogen) and then reverse transcribed into cDNA using PrimeScript RT kit (RR 037A, takara, japan) according to the manufacturer's instructions. Real-Time PCR was performed on the cDNA using the SYBR Premix Ex Taq II (Tli RNaseH Plus) Kit (RR 820A, takara) and Applied Biosystems 7500Fast Real-Time PCR System (ABI, torrance, calif.).

1.2 immunofluorescence

Murine intestinal tissue was treated with anti-CCL3 from R & D (1:40, AF-450-SP), anti-CCR4 from SANTA CRUZ (1:200, sc-101375). Three consecutive histological sections were examined per mouse.

1.3 Western blot analysis

Adding RIPA buffer solution into mouse ileocecum tissue, grinding by a homogenizer, centrifuging for 10min at 12,000r/min, taking supernatant as tissue lysate, preparing SDS-polyacrylamide gel electrophoresis to separate protein, and transferring the protein into PVDF membrane. After sealing for 2 hours at room temperature with 5% skim milk, washing 3 times with TBST, adding anti-CCR4 (1:1000), then incubating the membrane containing the primary antibody overnight at 4℃and then adding horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:8000), incubating at room temperature for 1h, developing the PVDF membrane after exposure using ultrasensitive ECL chemiluminescent kit, and quantifying using Image J (National Institutes of Health, bethesda, MD).

1.4 statistical methods

2. Results

2.1, experiments in this example found that CCR4 expression was increased in both human and mouse NEC, as shown by quantitative reverse transcriptase PCR (FIG. 4-1) and immunofluorescent staining of tissue sections (FIG. 4-2).

2.2, as shown in FIGS. 4-3, NEC induction enhanced intestinal CCR4 expression. After treatment with rCCL3, the expression of CCR4 was significantly increased, whereas treatment with anti-CCL3 significantly attenuated NEC-induced protein expression of CCR 4.

2.3 immunofluorescent co-localization of tissue sections in NEC showed direct binding of CCL3 to CCR4, as shown in fig. 4-4.

3. Conclusion(s)

Through Q-PCR, immunoblotting and immunofluorescence co-localization detection, the results show that: CCL3 functions in NEC through the CCL3-CCR4 axis.

Example 5

CCR4 neutralizing antibodies can block CCL3-CCR4 axes, and can protect NEC intestinal tracts.

1. Materials and methods

1.1 NEC mice intestinal tissue injury score

The specific procedure was as in 1.3 of example 2.

1.2, TUNEL staining

1.3 flow cytometry detection for apoptosis

IEC-6 cells (rat intestinal crypt epithelial cells) were pre-incubated overnight at a density of 1X 10≡5 cells/mL. After 24 hours, the cells were pretreated with CCL3/anti-CCL3 for 24 hours and then stimulated with 100. Mu.g/ml LPS for 4 hours. Then, the cells were resuspended at 1X 10≡6 cells/mL, then 5. Mu.l annexin V-FITC and propidium iodide (Becton Dick-inson and Company, lake Franklin, USA) were added. Apoptosis was detected by flow cytometry (Becton Dickinson and Company, lake Franklin, USA).

1.4 statistical methods

2. Results:

2.1, as shown in FIGS. 5-1 and 5-2, inhibition of CCR4 in vivo by injection of CCR4 neutralizing antibodies resulted in reduced tissue damage and reduced NEC severity scores.

2.2, as shown in FIGS. 5-3, anti-CCR4 injection significantly reduced NEC-induced apoptosis in intestinal tissue.

2.3 As shown in FIGS. 5-4, flow cytometry showed that pretreatment with anti-CCR4 significantly reduced LPS-induced IEC-6 apoptosis.

3. Conclusion(s)

Detection by TUNEL fluorescence, HE staining, flow cytometry, results showed: NEC causes apoptosis of intestinal epithelial cells through CCL3-CCR4 signaling axis, exacerbating intestinal injury. And CCR4 neutralizing antibodies can block CCL3-CCR4 signal axes, reduce apoptosis of intestinal epithelial cells and play a role in protecting intestinal tracts.

Example 6

CCL3 aggravates intestinal injury through the ERK/NF- κB signaling pathway, and the NF- κB inhibitor PDTC reduces intestinal epithelial cell apoptosis by blocking the CCL3/CCR4/ERK/NF- κB signaling pathway.

1. Materials and methods

1.1 Western blot analysis

Adding RIPA buffer solution into mouse ileocecum tissue, grinding by a homogenizer, centrifuging for 10min at 12,000r/min, taking supernatant as tissue lysate, preparing SDS-polyacrylamide gel electrophoresis to separate protein, and transferring the protein into PVDF membrane. After sealing 5% skim milk for 2 hours at room temperature, washing 3 times with TBST, adding p-ERK1/2 (1:1000), T-ERK1/2 (1:1000), p-NF- κB (1:1000), T-NF- κB (1:1000), BAX (1:1000), BCL-2 (1:1000), GAPDH (1:1000), then incubating the primary antibody-containing membrane overnight at 4℃and then adding horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:8000), incubating at room temperature for 1 hour, developing PVDF membrane using ultrasensitive ECL chemiluminescent kit after exposure, and quantifying using Image J (National Institutes of Health, bethesda, MD).

1.2 flow cytometry detection for apoptosis

IEC-6 cells were pre-incubated overnight at a density of 1X 10≡5 cells/mL. After 24 hours, the cells were pretreated with CCL3/anti-CCL3 for 24 hours and then stimulated with 100. Mu.g/ml LPS for 4 hours. Then, the cells were resuspended at 1X 10≡6 cells/mL, then 5. Mu.l annexin V-FITC and propidium iodide (Becton Dick-inson and Company, lake Franklin, USA) were added. Apoptosis was detected by flow cytometry (Becton Dickinson and Company, lake Franklin, USA).

1.3 statistical methods

2. Results

2.1, as shown in FIG. 6-1, activation of IEC-6 apoptosis-related signaling pathways was observed after LPS stimulation for various time periods (0-6 h), and rCCL3 pretreatment was found to significantly enhance LPS-mediated increase in the levels of p-ERK1/2 and p-NF- κB, while anti-CCL3 blocked CCL 3-mediated phosphorylation of p-ERK1/2 and p-NF- κB.

2.2, as shown in FIG. 6-2, the activation of IEC-6 apoptosis-related signal pathways was observed at different time periods (0-6 h) after LPS stimulation, and it was found that the phosphorylation levels of p-ERK and p-NF- κB increased after LPS stimulation for 1-3h, and then anti-CCR4 significantly inhibited the phosphorylation of the pathways.

2.3 As shown in FIGS. 6-3, treatment with the NF- κB inhibitor PDTC significantly reduced LPS-induced IEC-6 apoptosis.

2.4 As shown in FIG. 5-1, treatment with PDTC significantly alleviates intestinal damage in NEC model mice.

2.5 As shown in FIGS. 6-4, treatment of NEC model mice with PDTC reduced pro-apoptotic protein BAX and increased expression of apoptosis-inhibiting protein BCL-2.

3. Conclusion(s)

Detection by immunoblotting, HE staining, flow cytometry, results showed: CCL3 activates a signal path ERK1/2/NF- κB through a CCL3-CCR4 signal shaft and regulates the expression level of an apoptosis related gene BAX/BCL-2, so that intestinal epithelial cells are apoptotic and serious intestinal damage is caused, and blocking the ERK1/2/NF- κB signal path through an NF- κB inhibitor PDTC can obviously relieve the intestinal epithelial cell apoptosis and relieve intestinal tissue damage caused by NEC.

Example 7

CCL3 promotes NEC intestinal inflammation imbalance by chemotactic macrophages to intestinal tissues and inducing inflammatory M1 type polarization, damages intestinal mucosa barrier, causes intestinal mucosa injury, and CCL3 neutralizing antibodies can reverse macrophage chemotaxis in NEC, reduce inflammation and relieve intestinal tissue injury.

1. Materials and methods

1.1, Q-PCR analysis

The procedure is as in example 4.

1.2 isolation of leukocytes from mouse intestinal tissue

After day 4 of NEC mouse modeling, all surviving pups were euthanized and intestinal tissue samples were collected. The intestinal tract was dissected longitudinally and washed in PBS until the PBS cleared to remove faeces. The intestinal tissue was then cut into 0.5x0.5 cm pieces and suspended in DMEM medium containing 5mM ethylenediamine tetraacetic acid (EDTA; sigma-Aldrich), 2mM 1, 4-dithiothreitol (DTT; sigma-Aldrich) and 1% FBS, followed by shaking and incubation (37 ℃) in a shaker (180 RCF) for 2x 20 minutes to isolate the epithelial layer. Intestinal tissue without epithelial layer was minced and supplemented with 1mg/ml (0.15U/mg) collagenase D (Roche), 1% fetal bovine serum (FBS; biological Industries) and 1000U/ml Dnase I (Worthington Biochemical Corporation's DMEM) incubated for 30min on a shaker at 37 degrees (180 RCF), and finally the cell-containing supernatant was filtered through a 70mm filter (Falcon, corning) to obtain a single cell suspension.

1.3 isolation and culture of mouse Bone Marrow Derived Macrophages (BMDM)

Bone marrow cells were isolated from the femur and tibia of 16-18g male C57BL/6J mice and induced to differentiate into macrophages in DMEM complete medium supplemented with 10ng/ml M-CSF (Peprotech, USA), 10% fetal bovine serum. Ausbian, australia), 1% penicillin/streptomycin (Gibco, usa). The medium was refreshed on

days

3 and 5, and then on day 7, rCCL3/anti-CCL3 treated cells were collected from the dishes with a cell scraper. The cells were centrifuged at 500RCF for 5 minutes to form a pellet, and then suspended in complete DMEM medium for further use.

1.4, mouse peritoneal macrophages

Is isolated and cultured

Mice were euthanized by intraperitoneal injection of 1ml of sterile liquid paraffin for 3-5 days. Mice were sterilized with 70% alcohol and the limbs were fixed on the control plate. The abdomen of the mice was massaged for several minutes. The skin on the left or right side of the midline of the peritoneal base was then cut longitudinally with surgical scissors to expose the clear peritoneal membrane. 10mL of pre-chilled PBS containing EDTA was injected into the abdominal cavity of the mice and collected in a 15mL centrifuge tube after multiple lavages. Cells were washed 3 times with PBS and then incubated in cell culture plates for 45-60 minutes. Cells were then washed 2 times with PBS to discard non-adherent cells and adherent cells were cultured in complete DMEM medium for use.

1.5 Flow Cytometry (FCM)

The cell suspension was incubated with different fluorescent-labeled surface molecule antibodies and isotype control antibodies for 30min, after which the cells were washed and detected by flow cytometry and analyzed according to the product instructions.

1.6 statistical method

2. Results

2.1, as shown in FIG. 7-1, CD11b+F4/80+ cells were significantly increased in the intestinal tract of NEC mice injected with rCCL3, while anti-CCL3 reversed macrophage chemotaxis in NEC.

2.2 As shown in FIG. 7-2, expression of the M1 macrophage associated genes iNOS and CD86 was higher in necrotic intestinal tissue of NEC patients compared to control group.

2.3 As shown in FIGS. 7-3, expression of M1 macrophage associated genes iNOS, CD86 and IRF5 was increased in rCCL3 treated mice compared to control group in NEC mouse model. Whereas the expression of M2 macrophage-related genes Arg1, FIZZ1 and YM1 was decreased. Whereas anti-CCL3 group showed the opposite result, and M2-related gene expression was increased and M1-related gene expression was decreased.

2.4, as shown in FIGS. 7-4, F4/80 in the intestinal tract of rCCL 3-treated NEC mice ⁺ CD86 ⁺ The cells (M1) were significantly increased, whereas F4/80 was observed in the anti-CCL3 group ⁺ CD206 ⁺ Cells (M2) increased.

2.5, as shown in FIGS. 7-5, CCL3 recombinant protein treatment

The proportion of F4/80+CD86+ cells (M1) in the cells increases, F4/80 ⁺ CD206 ⁺ The proportion of cells (M2) decreases. Whereas anti-CCl3 treated +.>

F4/80 in cells ⁺ CD86 ⁺ The proportion of cells (M1) decreased and the proportion of F4/80+CD206+ cells (M2) increased.

2.6, as shown in FIGS. 7-6, BMDMs were induced to form M2 by GS-CSF and IL-4 and then treated with rCCL 3. The proportion of F4/80+CD86+ cells in the CCL3 treated group was significantly increased.

3. Conclusion(s)

CCL3 acts as a detrimental endogenous factor to NEC, exacerbating NEC-mediated inflammatory bowel injury by modulating macrophage chemotaxis and polarization of inflammatory phenotypes. Blocking CCL3 by anti-CCL3 can effectively inhibit macrophage chemotaxis and polarization, and reduce NEC intestinal tissue inflammation injury, thus providing a new idea for NEC clinical treatment.

By detecting CCL3 in the intestinal tissues of a clinical NEC patient and the intestinal tissues of a model mouse, the invention discovers that CCL3 is remarkably and highly expressed in the intestinal tissues of NEC lesions. Through further animal experiments, the invention discovers that blocking CCL3 by anti-CCL3 can obviously reduce intestinal tissue damage caused by NEC, and administration of rCCL3 obviously aggravates intestinal tissue damage of NEC mice. The above results indicate that CCL3 may be an important detrimental cause of NEC development and blocking CCL3 may be a new strategy for treating NEC. Through further research, the invention discovers that CCL3 aggravates intestinal tissue injury in different ways during NEC, on one hand, CCL3 directly induces a large amount of apoptosis of intestinal epithelial cells by combining with CCR4 and activating intracellular signaling pathways such as Erk1/2, NF- κB and the like to regulate the expression of apoptosis related genes BAX/BCL-2, thereby causing serious intestinal injury; CCL3, on the other hand, chemotaxis macrophages to the lesion during NEC and strongly induces polarization of macrophages towards the pro-inflammatory phenotype (M1 type) and causes an unbalanced excessive inflammatory response, exacerbating inflammatory lesions of the intestinal tissue. Based on this, the present invention found that blocking CCL3 and CCR4 by neutralizing antibodies and blocking NF- κb pathway with inhibitors are both effective in reducing intestinal tissue damage and improving survival in NEC mice.

In conclusion, the invention discovers that blocking the CCL3-CCR4 related loop by an antibody or inhibitor can significantly and effectively reduce NEC intestinal tissue damage. Based on the above, the invention provides a novel therapy for treating the necrotizing enterocolitis of the newborn by using the CCL3 neutralizing antibody, the CCR4 neutralizing antibody and the NF- κB inhibitor PDTC, and provides a novel strategy with good application value for medical treatment of clinical NEC.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

SEQUENCE LISTING

<110> Chongqing medical university affiliated children Hospital

<120> use of CCL3/CCR4 neutralizing antibodies, NF- κB inhibitors in the preparation of NEC therapeutic drugs

<130> PYZYK2216023

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 780

<212> DNA

<213> Artificial

<220>

<223> human CCL3 nucleotide sequence

<400> 1

agaaggacac gggcagcaga cagtggtcag tcctttcttg gctctgctga cactcgagcc 60

cacattccgt cacctgctca gaatcatgca ggtctccact gctgcccttg ctgtcctcct 120

ctgcaccatg gctctctgca accagttctc tgcatcactt gctgctgaca cgccgaccgc 180

ctgctgcttc agctacacct cccggcagat tccacagaat ttcatagctg actactttga 240

gacgagcagc cagtgctcca agcccggtgt catcttccta accaagcgaa gccggcaggt 300

ctgtgctgac cccagtgagg agtgggtcca gaaatatgtc agcgacctgg agctgagtgc 360

ctgaggggtc cagaagcttc gaggcccagc gacctcggtg ggcccagtgg ggaggagcag 420

gagcctgagc cttgggaaca tgcgtgtgac ctccacagct acctcttcta tggactggtt 480

gttgccaaac agccacactg tgggactctt cttaacttaa attttaattt atttatacta 540

tttagttttt gtaatttatt ttcgatttca cagtgtgttt gtgattgttt gctctgagag 600

ttcccctgtc ccctccccct tccctcacac cgcgtctggt gacaaccgag tggctgtcat 660

cagcctgtgt aggcagtcat ggcaccaaag ccaccagact gacaaatgtg tatcggatgc 720

ttttgttcag ggctgtgatc ggcctgggga aataataaag atgctctttt aaaaggtaaa 780

<210> 2

<211> 92

<212> PRT

<213> Artificial

<220>

<223> human CCL3 amino acid sequence

<400> 2

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Leu Cys Asn Gln Phe Ser Ala Ser Leu Ala Ala Asp Thr Pro Thr Ala

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Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile Ala

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Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Gly Val Ile Phe

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Leu Thr Lys Arg Ser Arg Gln Val Cys Ala Asp Pro Ser Glu Glu Trp

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Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala

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<210> 3

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<212> DNA

<213> Artificial

<220>

<223> human CCR4 nucleotide sequence

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ggcattgcct cacagacctt cctcagagcc gctttcagaa aagcaagctg cttctggttg 60

ggcccagacc tgccttgagg agcctgtaga gttaaaaaat gaaccccacg gatatagcag 120

acaccaccct cgatgaaagc atatacagca attactatct gtatgaaagt atccccaagc 180

cttgcaccaa agaaggcatc aaggcatttg gggagctctt cctgccccca ctgtattcct 240

tggtttttgt atttggtctg cttggaaatt ctgtggtggt tctggtcctg ttcaaataca 300

agcggctcag gtccatgact gatgtgtacc tgctcaacct tgccatctcg gatctgctct 360

tcgtgttttc cctccctttt tggggctact atgcagcaga ccagtgggtt tttgggctag 420

gtctgtgcaa gatgatttcc tggatgtact tggtgggctt ttacagtggc atattctttg 480

tcatgctcat gagcattgat agatacctgg caattgtgca cgcggtgttt tccttgaggg 540

caaggacctt gacttatggg gtcatcacca gtttggctac atggtcagtg gctgtgttcg 600

cctcccttcc tggctttctg ttcagcactt gttatactga gcgcaaccat acctactgca 660

aaaccaagta ctctctcaac tccacgacgt ggaaggttct cagctccctg gaaatcaaca 720

ttctcggatt ggtgatcccc ttagggatca tgctgttttg ctactccatg atcatcagga 780

ccttgcagca ttgtaaaaat gagaagaaga acaaggcggt gaagatgatc tttgccgtgg 840

tggtcctctt ccttgggttc tggacacctt acaacatagt gctcttccta gagaccctgg 900

tggagctaga agtccttcag gactgcacct ttgaaagata cttggactat gccatccagg 960

ccacagaaac tctggctttt gttcactgct gccttaatcc catcatctac ttttttctgg 1020

gggagaaatt tcgcaagtac atcctacagc tcttcaaaac ctgcaggggc ctttttgtgc 1080

tctgccaata ctgtgggctc ctccaaattt actctgctga cacccccagc tcatcttaca 1140

cgcagtccac catggatcat gatctccatg atgctctgta gaaaaatgaa atggtgaaat 1200

gcagagtcaa tgaactttcc acattcagag cttacttaaa attgtatttt agtaagagat 1260

tcctgagcca gtgtcaggag gaaggcttac acccacagtg gaaagacagc ttctcatcct 1320

gcaggcagct ttttctctcc cactagacaa gtccagcctg gcaagggttc acctgggctg 1380

aggcatcctt cctcacacca ggcttgcctg caggcatgag tcagtctgat gagaactctg 1440

agcagtgctt gaatgaagtt gtaggtaata ttgcaaggca aagactattc ccttctaacc 1500

tgaactgatg ggtttctcca gagggaattg cagagtactg gctgatggag taaatcgcta 1560

ccttttgctg tggcaaatgg gccctctaat taatttcttg cttttgcgga acaatataga 1620

taactgtttt tctaataaca tatctcaggc aaagtatatt ccattgagcc agatgtatga 1680

agaaacaatt agcgaagtga tgaaaccaga tctcaattat ttattgtaaa ggattatctg 1740

ttaattgaaa ccaaactttt tatactgata taagggtaag gatatgaaga cattagccaa 1800

ggtctgcttt ccaaacgtga actacaaggc attcaaaatc caaacatatt tatgaaaatt 1860

caaacacagt ttctcacttg tttgtggaca tgttttgttc taattttaac agaggaatat 1920

taaaaaattt taaataggct gggcacggtg gcctgtaatc ccagcactgt gggaggccaa 1980

ggtgggcgga tcacctgagg tcaggagttc gagaccagcc tggccaacat ggagaaaccc 2040

tgtctctact aaaaaataca aaattagcca ggtgtggtgg cgcatgcctg taatcccagc 2100

tactcaggag gctgaggctg gagaataact tgaatccggg aggtggaggt tgcggtgagc 2160

cgagatcgcg ccattgtact ccaacctggg caaaaagagc gaaactctgt ctcaaaaaaa 2220

aaaaaaaaaa attaaataat acataggcca agaatacatt tatttgaggt catttacttg 2280

tttttttttt tttttttttt tttgagatgg aatcttgctc tgtcacccag tctggagtgc 2340

agtggcgcga tctcggctca ctgcaagctc tgcctcacgg gttcgcacca ttctcctgcc 2400

tcagcctccc aagtaggtgg gactacaggc acctgccccc atgcctggct aattttttgt 2460

attttcagta gagatggggt ttcaccatgt tagcaaggat ggttttaatc tcctgacctc 2520

gtgatccacc cgcctcggcc tcccaaagtg ctgggattac aggtgtgagc catcacgccc 2580

ggccacttgt ttatttttta ttttatttta ttttattttt gagatggagt ctcactctgt 2640

cacccaagct ggagtgcagt ggcactcggt tcactgcaaa gtctgcctcc caggttcaag 2700

cgattctcct gcctcagctt ctcaagcagc tgggattaga ggtgtgcacc actacgccag 2760

gctaattttt gtatttttag tagagatggg gtttcaccat attggccagg ctagtcttga 2820

actcctgacc tcaggtgatc tgcctgcttc agcctcccaa agtgctggga ttacaggcgt 2880

gagccacctc gcccagccaa ggtcttttac ttgtttataa acagtctctt cataattaaa 2940

attaaggatt aataaagtat gacaatacct ccttaatcat tttgaagtgc ctgctatcaa 3000

ttgaaataaa aacaatcaac taaaa 3025

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<213> Artificial

<220>

<223> human CCR4 amino acid sequence

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Ser Asn Tyr Tyr Leu Tyr Glu Ser Ile Pro Lys Pro Cys Thr Lys Glu

20 25 30

Gly Ile Lys Ala Phe Gly Glu Leu Phe Leu Pro Pro Leu Tyr Ser Leu

35 40 45

Val Phe Val Phe Gly Leu Leu Gly Asn Ser Val Val Val Leu Val Leu

50 55 60

Phe Lys Tyr Lys Arg Leu Arg Ser Met Thr Asp Val Tyr Leu Leu Asn

65 70 75 80

Leu Ala Ile Ser Asp Leu Leu Phe Val Phe Ser Leu Pro Phe Trp Gly

85 90 95

Tyr Tyr Ala Ala Asp Gln Trp Val Phe Gly Leu Gly Leu Cys Lys Met

100 105 110

Ile Ser Trp Met Tyr Leu Val Gly Phe Tyr Ser Gly Ile Phe Phe Val

115 120 125

Met Leu Met Ser Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val Phe

130 135 140

Ser Leu Arg Ala Arg Thr Leu Thr Tyr Gly Val Ile Thr Ser Leu Ala

145 150 155 160

Thr Trp Ser Val Ala Val Phe Ala Ser Leu Pro Gly Phe Leu Phe Ser

165 170 175

Thr Cys Tyr Thr Glu Arg Asn His Thr Tyr Cys Lys Thr Lys Tyr Ser

180 185 190

Leu Asn Ser Thr Thr Trp Lys Val Leu Ser Ser Leu Glu Ile Asn Ile

195 200 205

Leu Gly Leu Val Ile Pro Leu Gly Ile Met Leu Phe Cys Tyr Ser Met

210 215 220

Ile Ile Arg Thr Leu Gln His Cys Lys Asn Glu Lys Lys Asn Lys Ala

225 230 235 240

Val Lys Met Ile Phe Ala Val Val Val Leu Phe Leu Gly Phe Trp Thr

245 250 255

Pro Tyr Asn Ile Val Leu Phe Leu Glu Thr Leu Val Glu Leu Glu Val

260 265 270

Leu Gln Asp Cys Thr Phe Glu Arg Tyr Leu Asp Tyr Ala Ile Gln Ala

275 280 285

Thr Glu Thr Leu Ala Phe Val His Cys Cys Leu Asn Pro Ile Ile Tyr

290 295 300

Phe Phe Leu Gly Glu Lys Phe Arg Lys Tyr Ile Leu Gln Leu Phe Lys

305 310 315 320

Thr Cys Arg Gly Leu Phe Val Leu Cys Gln Tyr Cys Gly Leu Leu Gln

325 330 335

Ile Tyr Ser Ala Asp Thr Pro Ser Ser Ser Tyr Thr Gln Ser Thr Met

340 345 350

Asp His Asp Leu His Asp Ala Leu

355 360

Claims

Use of a ccl3 neutralizing antibody in the manufacture of a medicament for the treatment of neonatal necrotizing enterocolitis.
2. Use according to claim 1, characterized in that: the administration dosage of the CCL3 neutralizing antibody is 0.1-2mg/kg or 2-10mg/kg.
3. Use according to claim 1, characterized in that: the administration mode of the neonatal necrotizing enterocolitis therapeutic drug is at least one of intraperitoneal injection, intravenous injection, intramuscular injection and subcutaneous injection.
Use of a ccr4 neutralizing antibody in the preparation of a medicament for the treatment of neonatal necrotizing enterocolitis.
5. Use according to claim 4, characterized in that: the administration dosage of the CCR4 neutralizing antibody is 0.1-2mg/kg or 2-10mg/kg.
6. Use according to claim 4, characterized in that: the administration mode of the neonatal necrotizing enterocolitis therapeutic drug is at least one of intraperitoneal injection, intravenous injection, intramuscular injection and subcutaneous injection.