Methods for detecting the effect of tgfβ1 on neutrophil polarization in hepatolenticular degeneration and uses thereof

Abstract

The application discloses a kind of detection Tgfβ1 to hepatolenticular degeneration neutrophil polarization method and application, including the following steps: step 1, by immunohistochemistry and immunofluorescence co-localization determine hepatolenticular degeneration mouse liver fibrosis formation process, hepatocyte high expression Tgfβ1;Step 2, by inhibiting the high expression of Tgfβ1 of hepatolenticular degeneration mouse liver fibrosis formation process, determine the mitigation of neutrophil polarization;Step 3, collect mouse bone marrow neutrophil, by real-time fluorescence quantitative PCR, protein electrophoresis and methylation reaction determine the mechanism of Tgfβ1 promote neutrophil polarization.The application can detect the effect of Tgfβ1 in hepatolenticular degeneration neutrophil polarization, facilitate to determine hepatolenticular degeneration liver lesion mechanism.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of gene detection, in particular to a method for detecting the effect of Tgf beta 1 on neutrophil polarization in hepatolenticular degeneration and application. BACKGROUND

[0002] Hepatolenticular degeneration is a copper metabolism abnormality disease caused by pathogenic gene ATP7B variation. Copper metabolism disorder in the patient body can cause pathological changes of liver fibrosis, and fibrosis is an important stage of development of liver cirrhosis. About 60% of hepatolenticular degeneration patients eventually develop into liver cirrhosis. Neutrophil polarization can induce liver injury and tumor, therefore, it is very important to determine the factors that affect the neutrophil polarization process in the development process of hepatolenticular degeneration disease, so as to prevent the liver disease from developing into a more serious stage. SUMMARY

[0003] The application aims to provide a method for detecting the effect of Tgf beta 1 on neutrophil polarization in hepatolenticular degeneration and application. The application can detect the effect of Tgf beta 1 on neutrophil polarization in hepatolenticular degeneration, and facilitate the determination of the mechanism of liver lesion in hepatolenticular degeneration.

[0004] The technical scheme of the application is a method for detecting the effect of Tgf beta 1 on neutrophil polarization in hepatolenticular degeneration, which comprises the following steps:

[0005] Step 1, determining that hepatocytes highly express Tgf beta 1 in the process of liver fibrosis formation of hepatolenticular degeneration mice through immunohistochemistry and immunofluorescence co-localization;

[0006] Step 2, determining that neutrophil polarization is reduced by inhibiting the high expression of Tgf beta 1 in the process of liver fibrosis formation of hepatolenticular degeneration mice;

[0007] Step 3, collecting mouse bone marrow neutrophils, and determining the mechanism of Tgf beta 1 promoting neutrophil polarization through real-time fluorescence quantitative PCR, protein electrophoresis and methylation reaction.

[0008] The method for detecting the effect of Tgf beta 1 on neutrophil polarization in hepatolenticular degeneration, the immunohistochemistry is that after 16-week-old ATP7B-KO mice and C57BL6 mice liver paraffin specimens are sliced with a thickness of 4 um, an anti-TGF beta 1 antibody is used, the antibody is diluted at a ratio of 1:200, overnight staining, and DAB color development.

[0009] The aforementioned method for detecting the effect of Tgfβ1 on neutrophil polarization in hepatolenticular degeneration, the immunofluorescence co-localization is that after the liver paraffin specimens of 16-week-old ATP7B-KO mice and C57BL6 mice are sliced at a thickness of 4 μm, anti-TGFβ1 antibody, anti-Albumin antibody and DAPI are used for overnight staining, and a fluorescent secondary antibody is used for color development; wherein the anti-TGFβ1 antibody and the anti-Albumin antibody are diluted at a ratio of 1:500.

[0010] The aforementioned method for detecting the effect of Tgfβ1 on neutrophil polarization in hepatolenticular degeneration, in step 2, by inhibiting the high expression of Tgfβ1 in the process of liver fibrosis formation of the hepatolenticular degeneration mouse, the specific process of determining the reduction of neutrophil polarization is:

[0011] Tgfβ1 receptor inhibitor SM16 is administered to ATP7B-KO mice and C57BL6 mice by gavage, the injection time is from 8 weeks to 16 weeks, and the injection amount is 2 mg / kg, three times a week;

[0012] Whole blood specimens are obtained from the orbit of ATP7B-KO mice and C57BL6 mice, and serum is separated from the whole blood by centrifugation at 3500 rpm for 15 minutes; 100 μL of serum sample is extracted, and biochemical indicators of ALT, AST, HDL-C and TChol are detected by a biochemical analyzer;

[0013] The left lobe of the mouse liver is fixed with 4% paraformaldehyde at room temperature for 24 hours, and then the left lobe of the liver is dehydrated with 75%, 80%, 85%, 95% and 100% ethanol solution in turn, and then immersed in xylene solution for 20 minutes to make the tissue transparent; the sample is immersed in wax at 65°C for 3 hours and embedded at 4°C; the tissue is then cut into 4 μm thick sections using a rotary microtome, and after the sections are deparaffinized, hematoxylin-eosin staining and sirius red staining are performed to observe the liver pathology and fibrosis.

[0014] The aforementioned method for detecting the effect of Tgfβ1 on neutrophil polarization in hepatolenticular degeneration, the neutrophil polarization process is that the mouse liver tissue is ground and filtered through a 70 μm filter screen, and then purified and enriched by 35% percoll, and the following antibodies are sequentially incubated: PerCP anti-mouse / human CD11b antibody, APC anti-mouse Ly6G antibody, PE anti-NOS2 antibody, and FITC anti-mouse CD206 antibody, and then analyzed by a flow cytometer; wherein the total aggregation of neutrophils in the liver is represented by CD11b+Ly6G+double positive cells; neutrophil polarization is represented by CD11b+Ly6G+NOS2+triple positive for N1 type and CD11b+Ly6G+CD206+triple positive for N2 type.

[0015] The method for detecting the effect of Tgfβ1 on neutrophil polarization in hepatolenticular degeneration as described above, the collection of mouse bone marrow neutrophils is as follows: after killing 8-12 week old C57BL6 mice, separating their tibial and femoral bones, cutting off both ends of the bone marrow, and exposing the bone marrow cavity; using a 10 mL syringe to fill pre-cooled RPMI 1640 culture medium containing 10% FBS and 1% double antibody to flush, filtering the flushing liquid through a 70 um nylon screen, collecting cells by centrifugation at 3000 rpm for 4 minutes, and resuspending the cells with 3 mL of RPMI medium;

[0016] The percoll gradient liquid is pre-equilibrated to room temperature, 3 mL of 78% and 3 mL of 65% percoll solution are sequentially added to a 15 mL centrifuge tube, the cell suspension is slowly placed on the percoll gradient liquid, and then centrifuged at 800 g, 35 min, 25°C without braking; the solution above the 65% percoll layer is discarded, and the percoll layer between 65% and 78% is collected; 3 mL of 1xPBS is added and mixed, and then centrifuged at 1000 rpm for 3 min; the supernatant is discarded, the precipitate is added with 3 mL of 1xPBS and washed once; 2 mL of red blood cell lysis solution is added, mixed, and reacted for 1 min, and then centrifuged at 1000 rpm for 3 min; the supernatant is discarded, the precipitate is washed twice with 3 mL of 1xPBS; the supernatant is discarded, and the precipitate is resuspended with 500 μL of RPMI 1640 medium containing 10% FBS and 1% double antibody; after being placed in a 37°C carbon dioxide incubator for 2 hours, 10 ng / mL of constitutive human TGFβ1 is added for co-treatment with 1 μM 5-aza for 24 hours or 10 ng / mL of constitutive human TGFβ1 is added for co-treatment with 1 μM STATTIC for 24 hours.

[0017] The method for detecting the effect of Tgfβ1 on neutrophil polarization in hepatolenticular degeneration as described above, the real-time fluorescent quantitative PCR is as follows: the collected mouse bone marrow neutrophils are added with 1 mL of Trizol to extract total RNA, which is reversely transcribed into cDNA by HiScript II first-strand cDNA synthesis kit, and then real-time fluorescent PCR reaction is performed by ChamQ universal SYBR qPCR master mix kit; the detected genes are: CD95, Nos2, Ccl3, Arg2, CD206, Ym1, E-cadherin, Epcam, and Keratin18.

[0018] The aforementioned method for detecting the polarization effect of Tgfβ1 on neutrophils in hepatolenticular degeneration, the protein electrophoresis is to collect mouse bone marrow neutrophils, add RIPA lysis buffer to extract protein, then perform SDS-PAGE electrophoresis, incubate the corresponding primary antibody, and detect the protein expression changes; the antibodies used are: anti-pSTAT3, anti-DNMT3A, and anti-SOCS3.

[0019] The aforementioned method for detecting the polarization effect of Tgfβ1 on neutrophils in hepatolenticular degeneration, the methylation reaction collects mouse bone marrow neutrophils, uses a blood / cell / tissue genomic DNA extraction kit for genomic extraction; takes 500 ng of genomic DNA and performs bisulfite treatment using a DNA bisulfite conversion kit; takes 50 ng of bisulfite DNA as a template, adds methylation or non-methylation primers for amplification reaction; takes 10 μL of amplification product for agarose gel electrophoresis, and the band is compared by relative gray scale using Image J software;

[0020] The methylation primers used are as follows:

[0021] F 5'-TTTTTTGTTGTTTGTTGTAAAATTC-3'

[0022] R 5'-TTTAAAATATCTAAACTTTCCCGAT-3'

[0023] The non-methylation primers used are as follows:

[0024] F 5'-TTTTTTGTTGTTTGTTGTAAAATTTG-3'

[0025] R 5'-TTTAAAATATCTAAACTTTCCCAAT-3'.

[0026] The aforementioned method for detecting the polarization effect of Tgfβ1 on neutrophils in hepatolenticular degeneration, the methylation primers or non-methylation primers used are applied in the detection of Tgfβ1 level for non-diagnostic purposes.

[0027] Compared with the prior art, the present application determines that hepatocytes highly express Tgfβ1 in the process of liver fibrosis of a hepatolenticular degeneration mouse by immunohistochemistry and immunofluorescence co-localization, and determines that the polarization of neutrophils is reduced by inhibiting the high expression of Tgfβ1 in the process of liver fibrosis of a hepatolenticular degeneration mouse. Finally, the mechanism and factors of Tgfβ1 promoting the polarization of neutrophils are determined by collecting mouse bone marrow neutrophils, using real-time fluorescence quantitative PCR, protein electrophoresis and methylation reaction. Thus, the present application further clarifies the mechanism of liver lesions of hepatolenticular degeneration by detecting the expression of Tgfβ1 in the development process of hepatolenticular degeneration and the effect on the polarization of neutrophils, and provides an alternative for improving liver diseases of hepatolenticular degeneration. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a Tgfβ1 expression site; (A) immunohistochemistry of liver specimens; (B) immunofluorescence co-localization of liver specimens;

[0029] Figure 2A indicates a flow cytometry analysis diagram of neutrophils in the liver of a wild-type mouse after SM16 treatment;

[0030] Figure 2B indicates a flow cytometry analysis diagram of neutrophils in the liver of a hepatolenticular degeneration mouse after SM16 treatment;

[0031] Figure 3 B of the figure is the change in the number of neutrophils in the liver of a wild-type mouse and a hepatolenticular degeneration mouse after SM16 treatment; Figure 3 C of the figure is the change in the polarization of neutrophils in the liver of a wild-type mouse and a hepatolenticular degeneration mouse after SM16 treatment; Figure 3 D of the figure is the change in ALT; Figure 3 E of the figure is the change in AST;

[0032] Figure 4 F of the figure is the change in HDL-C; Figure 4 G of the figure is the change in TChol; Figure 4 H of the figure is the change in liver index; Figure 4 I of the figure is the change in liver copper level;

[0033] Figure 5 is H&E staining of the liver of a wild-type mouse and a hepatolenticular degeneration mouse after SM16 treatment;

[0034] Figure 6 is Sirius red staining of the liver of a wild-type mouse and a hepatolenticular degeneration mouse after SM16 treatment;

[0035] Figure 7 A of the figure indicates the change in the expression of neutrophil polarization-related genes in bone marrow detected by real-time fluorescence quantitative PCR;Figure 7 Figure B in the figure shows the changes in the expression of genes related to epithelial-mesenchymal transition in bone marrow neutrophils as detected by real-time quantitative PCR.

[0036] Figure 8 This indicates that protein electrophoresis showed changes in pSTAT3 expression;

[0037] Figure 9 This indicates that protein electrophoresis showed changes in DNMT3A expression;

[0038] Figure 10 This indicates that protein electrophoresis showed changes in SCOS3 expression; Figure 11 This indicates that the methylation reaction shows changes in SOCS3 methylation. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0040] Example: A method for detecting the effect of Tgfβ1 on the polarization of Wilson's disease neutrophils, comprising the following steps:

[0041] Step 1: By co-localization of immunohistochemistry and immunofluorescence, it was determined that hepatocytes highly express Tgfβ1 (transforming growth factor β1) during the formation of liver fibrosis in Wilson's disease mice;

[0042] In this step, immunohistochemistry was performed as follows: paraffin-embedded liver specimens from 16-week-old ATP7B-KO and C57BL6 mice were sectioned to a thickness of 4 μm, stained overnight with anti-TGFβ1 antibody diluted at a ratio of 1:200, and then developed with DAB.

[0043] Immunofluorescence co-localization was performed as follows: paraffin-embedded liver specimens from 16-week-old ATP7B-KO and C57BL6 mice were sectioned to a thickness of 4 μm and stained overnight with anti-TGFβ1 antibody (1:500), anti-Albumin antibody (1:500), and DAPI, followed by fluorescence secondary antibody development.

[0044] Step 2: By inhibiting the high expression of Tgfβ1 in the process of liver fibrosis formation in Wilson's disease mice, the reduction of neutrophil polarization was determined;

[0045] This step includes drug injection: SM16 was administered to ATP7B-KO mice and C57BL6 mice by gavage at a dose of 2 mg / kg, three times a week, from 8 to 16 weeks of age.

[0046] Mouse liver enzyme metabolism detection: Whole blood samples were obtained from the orbital blood of ATP7B-KO mice and C57BL6 mice, and serum was separated from whole blood by centrifugation at 3500 rpm for 15 minutes. 100 μL of serum samples were drawn and biochemical indicators such as ALT, AST, HDL-C, TChol, etc. were detected by a biochemical analyzer.

[0047] Liver pathology: The left lobe of the mouse liver was fixed with 4% paraformaldehyde at room temperature for 24 hours, and then the left lobe of the liver was dehydrated with 75%, 80%, 85%, 95%, and 100% ethanol solutions. Then the tissue was transparentized by immersing in xylene solution for 20 minutes. The sample was immersed in wax at 65°C for 3 hours and embedded at 4°C. The tissue was cut into 4 μm thick sections using a rotary microtome. After deparaffinization, the sections were stained with hematoxylin and eosin (H&E) and Sirius red to observe liver pathology and fibrosis.

[0048] Mouse liver neutrophil polarization: Mouse liver tissue was ground and filtered through a 70 μm filter, then purified and enriched by 35% percoll, and then incubated with the following antibodies: PerCP anti-mouse / human CD11b antibody, APC anti-mouse Ly6G antibody, PE anti-NOS2 (iNOS) antibody, FITC anti-mouse CD206 (MMR) antibody, and analyzed by flow cytometry. The total aggregation of liver neutrophils was represented by CD11b+Ly6G+double positive cells; neutrophil polarization was represented by CD11b+Ly6G+NOS2+triple positive for N1 type and CD11b+Ly6G+CD206+triple positive for N2 type.

[0049] Step 3, collect mouse bone marrow neutrophils, and determine the mechanism of Tgfβ1 promoting neutrophil polarization by real-time fluorescent quantitative PCR, protein electrophoresis and methylation reaction.

[0050] In this step, the collection of mouse bone marrow neutrophils is as follows: after the C57BL6 mice of 8-12 weeks old are sacrificed, their tibial and femoral bones are separated, the bone marrow at both ends is cut off, and the bone marrow cavity is exposed. A 10 mL syringe is filled with pre-cooled RPMI1640 culture medium containing 10% FBS and 1% double antibody for flushing. The flushing liquid is filtered through a 70 um nylon screen and the cells are collected by centrifugation at 3000 rpm for 4 minutes, and the cells are resuspended with 3 mL of RPMI culture medium. The percoll gradient liquid is pre-equilibrated to room temperature, and 3 mL of 78% and 3 mL of 65% percoll solution are sequentially added to a 15 mL centrifuge tube. The cell suspension is slowly placed on the percoll gradient liquid, and then centrifuged at 800g, 35min, 25℃ without braking. The solution above the 65% percoll layer is discarded, and the percoll layer between 65%-78% is collected. After adding 3 mL of 1xPBS and mixing, centrifugation at 1000 rpm for 3 min is performed. The supernatant is discarded, and the precipitate is added with 3 mL of 1xPBS and washed once. 2 mL of red blood cell lysis solution is added, mixed and reacted for 1 min, and then centrifuged at 1000 rpm for 3 min. The supernatant is discarded, and the precipitate is washed twice with 3 mL of 1xPBS. The supernatant is discarded, and the precipitate is resuspended with 500 μL of RPMI 1640 culture medium containing 10% FBS and 1% double antibody. After being placed in a 37℃ carbon dioxide incubator for 2 hours, 10 ng / mL of constitutive human TGFβ1 is added for co-treatment with 1 μM 5-aza for 24 hours or 10 ng / mL of constitutive human TGFβ1 is added for co-treatment with 1 μM STATTIC for 24 hours.

[0051] Real-time fluorescent quantitative PCR: The mouse bone marrow neutrophils are collected, 1 mL of Trizol is added to extract total RNA, which is reversely transcribed into cDNA by HiScript II first-strand cDNA synthesis kit, and then real-time fluorescent PCR reaction is performed by ChamQ universal SYBR qPCR master mix kit. The detection genes are: CD95, Nos2, Ccl3, Arg2, CD206, Ym1, E-cadherin, Epcam, Keratin18.

[0052] Protein electrophoresis: The mouse bone marrow neutrophils are collected, RIPA lysis buffer is added to extract protein, and then SDS-PAGE electrophoresis is performed, the corresponding primary antibody is incubated, and the protein expression change is detected. The antibodies used are: anti-pSTAT3, anti-DNMT3A, anti-SOCS3.

[0053] Methylation reaction: collect mouse bone marrow neutrophils, use blood / cell / tissue genomic DNA extraction kit for genome extraction. Take 500 ng genomic DNA for DNA bisulfite conversion kit for bisulfite treatment. Take 50 ng bisulfite DNA as template, add methylation or non-methylation primer for amplification reaction. Take 10 μL amplification product for agarose gel electrophoresis, and the band is compared by Image J software.

[0054] The methylation primers used are as follows:

[0055] F 5'-TTTTTTGTTGTTTGTTGTAAAATTC-3'

[0056] R 5'-TTTAAAATATCTAAACTTTCCCGAT-3'

[0057] The non-methylation primers used are as follows:

[0058] F 5'-TTTTTTGTTGTTTGTTGTAAAATTTG-3'

[0059] R 5'-TTTAAAATATCTAAACTTTCCCAAT-3'.

[0060] Based on the above steps, the corresponding results are compared and analyzed after each step. As shown in Figure 1 Figure 1 is the expression site of Tgfβ1. (A) Immunohistochemistry of liver specimens. (B) Immunofluorescence co-localization of liver specimens. Albumin is used to indicate hepatocytes, and DAPI is used to indicate cell nuclei. It can be seen that by using the immunohistochemical method of mouse liver specimens, it is observed that Tgfβ1 is highly expressed in the process of liver fibrosis formation in hepatolenticular degeneration mice Figure 1 A). Further, in order to clarify the cell type expressing Tgfβ1, by using the method of immunofluorescence co-localization, it is observed that Tgfβ1 is co-localized with the hepatocyte-specific marker antibody anti-Albumin. And the Tgfβ1 receptor inhibitor SM16 is administered by gavage to ATP7B-KO mice and wild-type mice, which can eliminate the expression of Tgfβ1 in hepatocytes Figure 1 B). The above data show that in the process of liver fibrosis formation in hepatolenticular degeneration mice, hepatocytes highly express Tgfβ1.

[0061] Further, Figure 2A Figure shows the flow cytometry analysis chart of neutrophils in the liver of wild-type mice after SM16 treatment; Figure 2B Figure shows the flow cytometry analysis chart of neutrophils in the liver of hepatolenticular degeneration mice after SM16 treatment; Figure 3 ​Figure B in the figure shows the changes in the number of neutrophils in the livers of wild-type mice and Wilson's disease mice after SM16 treatment; Figure 3 Figure C in the figure shows the changes in liver neutrophil polarity in wild-type mice and Wilson's disease mice after SM16 treatment; Figure 3 The D-plot in the diagram represents the changes in ALT. Figure 3 The E-plot in the figure represents the AST variation; Figure 4 The F-plot in the diagram represents the HDL-C variation; Figure 4 The G-plot shows the TChol variation; Figure 4 Changes in liver index in the H-plot; Figure 4 Figure I shows changes in liver copper levels; Figure 5 H&E staining of livers from wild-type mice and Wilson's disease mice treated with SM16; Figure 6 Sirius red staining of the livers of wild-type mice and Wilson's disease mice treated with SM16.

[0062] As can be seen, when ATP7B-KO mice and wild-type mice were administered the Tgfβ1 receptor inhibitor SM16 by gavage, it was observed that SM16 treatment reduced the number of aggregated neutrophils in the liver of Wilson's disease mice, and also reduced the number of N2 neutrophils (Figure 2 and...). Figure 3 (BC diagram in the image) shows that the pre-existing abnormal liver enzymes in the diseased mice were improved. Figure 3 DE and Figure 4 (FG diagram in the image). SM6 treatment reduced the liver index in diseased mice ( Figure 4 (H-plot in the image), but liver copper levels did not improve ( Figure 4 Figure I in the table). H&E and Sirius red staining of liver specimens showed that SM16 treatment reduced liver fibrosis in mice with pre-existing disease. Figure 5 and Figure 6 The above data indicate that, during the progression of liver disease in Wilson's disease mice, inhibiting the high expression of Tgfβ1 can alleviate neutrophil polarization.

[0063] Furthermore, Figure 7 Figure A in the figure shows the changes in the expression of polarity-related genes in bone marrow neutrophils detected by real-time quantitative PCR. Figure 7 Figure B in the figure shows the changes in the expression of genes related to epithelial-mesenchymal transition in bone marrow neutrophils as detected by real-time quantitative PCR. Figure 8 This indicates that protein electrophoresis showed changes in pSTAT3 expression; Figure 9 This indicates that protein electrophoresis showed changes in DNMT3A expression; Figure 10 This indicates that protein electrophoresis showed changes in SCOS3 expression; Figure 11 This indicates that the methylation reaction shows changes in SOCS3 methylation.

[0064] It can be seen that the application uses the real-time fluorescent quantitative PCR method to observe that Tgfβ1 promotes the polarization of neutrophil by adding human Tgfβ1 to the neutrophil separated from the bone marrow of wild-type mice in vitro, and the effect is dependent on methylation (A-B figure in the middle). Figure 7 It is further observed by protein electrophoresis that Tgfβ1 can promote STAT3 phosphorylation by methylation SOCS3. Figures 8-11 The above data show that Tgfβ1 can promote STAT3 phosphorylation by methylation SOCS3, and finally lead to the polarization of neutrophil.

[0065] In summary, the application detects the expression of Tgfβ1 in the development process of hepatolenticular degeneration and the effect on the polarization of neutrophil by a series of methods such as immunohistochemistry, immunofluorescence co-localization, mouse drug gavage, extraction of mouse bone marrow neutrophil, real-time fluorescent quantitative PCR, protein electrophoresis, methylation reaction, and further clarifies the mechanism of liver lesion of hepatolenticular degeneration, and provides an optional solution for improving the liver disease of hepatolenticular degeneration.

Claims

1. The application of Tgfβ1 inhibitors in the preparation of drugs to alleviate neutrophil polarization in liver fibrosis, characterized in that: The Tgfβ1 inhibitor is the Tgfβ1 receptor inhibitor SM16; the liver fibrosis is Wilson's disease-associated liver fibrosis.

2. The application according to claim 1, characterized in that: The drug is administered by gavage at a dose of 2 mg / kg, three times a week.

3. The application according to claim 1, characterized in that: The neutrophil polarization includes N1 polarization and N2 polarization, wherein N1 neutrophils are represented by CD11b+Ly6G+NOS2+ triple positivity, and N2 neutrophils are represented by CD11b+Ly6G+CD206+ triple positivity.

4. The application according to claim 1, characterized in that: The drug reduces the aggregation of CD11b+Ly6G+ double-positive neutrophils in liver tissue by inhibiting the high expression of Tgfβ1, thereby alleviating neutrophil polarization.

5. The application according to claim 1, characterized in that: The drug reduces neutrophil polarization by regulating the STAT3 phosphorylation pathway through SOCS3 methylation.

6. The application according to claim 5, characterized in that: The detection of methylated SOCS3 employs specific primer pairs, which include methylating primers and non-methylating primers. The methylating primers are as follows: F 5'-TTTTTTGTTGTTTGTTGTAAAATTC-3' R 5'-TTTAAAATATCTAAACTTTCCCGAT-3' The unmethylated primers are as follows: F 5'-TTTTTTGTTGTTTGTTGTAAAATTTG-3' R 5'-TTTAAAATATCTAAACTTTCCCAAT-3'.

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