Application of transcription factor ZHX2 in diagnosis and treatment of liver ischemia-reperfusion injury

Abstract

The application belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of a transcription factor ZHX2 in diagnosis and treatment of liver ischemia-reperfusion injury. Specifically, it is found through research that the expression of ZHX2 is reduced after ischemia-reperfusion injury, and hepatocyte-specific knockout of Zhx2 significantly promotes HIRI cell necrosis and promotes occurrence and development of HIRI, which is further proved in marginal liver IRI, and ZHX2 has a protective effect on marginal fatty liver ischemia-reperfusion injury. In summary, ZHX2 can be used as a biological marker and a related therapeutic target for liver ischemia-reperfusion injury, especially marginal liver ischemia-reperfusion injury, and therefore has good clinical application value and social benefits.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to the application of transcription factor ZHX2 in the diagnosis and treatment of liver ischemia-reperfusion injury. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] The liver is the largest solid organ in mammals, participating in vital life activities such as immunity, metabolism, and detoxification. Liver transplantation is the most effective treatment for end-stage liver disease, and hepatic ischemia-reperfusion injury (HIRI) is a major cause of poor prognosis in liver transplant patients. Post-transplant liver dysfunction is very common, with 10%–30% of primary transplant liver dysfunction attributed to ischemia-reperfusion injury. HIRI has become an international public health concern. With the increasing global demand for donor livers, the severe shortage of organ supply is a major limiting factor in liver transplantation. Clinically, marginal donor livers, such as those with fatty livers or aged livers, are often used. These marginal transplanted livers, due to their weakened physiological function or the presence of pathological factors, are less tolerant of the ischemia-reperfusion stress during transplantation, leading to post-transplant liver dysfunction and even failure, increasing the probability of post-operative complications and seriously threatening post-operative survival and quality of life. Therefore, elucidating the molecular mechanisms of HIRI and identifying potential intervention targets is of significant clinical importance for improving ischemia-reperfusion injury in marginal donor livers.

[0004] Zinc fingers and homeoboxes 2 (ZHX2) belongs to the zinc finger and homeobox protein family and is a novel transcription factor, first known as Afr1 (Alpha fetal protein regulator 1) in mice. While Afr1 was discovered in mice as early as 1977, its human homolog, ZHX2, was not cloned until 2003. ZHX2 consists of four exons, with the largest exon, exon 3, responsible for protein coding. The ZHX2 protein contains 837 amino acids, including four homeobox domains, two zinc finger domains, and a domain consisting of proline residues at positions 408-448. ZHX2 participates in various physiological and pathological processes, including tumorigenesis and development, cell proliferation, and lipid metabolism. ZHX2 is highly expressed in liver tissue and is highly conserved in mammals. ZHX2 can not only form homodimers but also regulate downstream gene expression and exert transcriptional functions by forming heterodimers with ZHX1 and ZHX3. ZHX2 is a negative regulator of alpha-fetoprotein (AFP), which studies have shown is due to the fact that immature hepatic progenitor cells possess characteristics similar to fetal hepatocytes. Furthermore, ZHX2 expression is dynamically regulated during liver development. Studies have found that ZHX2 participates in regulating multiple genes related to liver development and rapid hepatocyte proliferation, including cytochrome P450 (CYP450), phosphatidylinositol proteoglycan 3 (GPC3), and H19, suggesting an important role for ZHX2 in liver development. In addition, ZHX2 regulates hepatic lipid metabolism by modulating the expression of lipid metabolism-related P450 and lipoprotein lipase (LPL) genes, affecting lipid synthesis and uptake, thereby regulating lipid homeostasis in the liver, suggesting that ZHX2 influences hepatic metabolic remodeling. Previous studies have found that ZHX2 affects liver regeneration. However, whether ZHX2 affects hepatic ischemia-reperfusion injury remains unreported. Summary of the Invention

[0005] Based on the current state of existing research, this invention provides the application of transcription factor ZHX2 in the diagnosis and treatment of hepatic ischemia-reperfusion injury. Specifically, this invention has found that ZHX2 expression decreases after ischemia-reperfusion injury, while hepatocyte-specific knockout of Zhx2 significantly promotes HIRI, and further demonstrates the protective effect of ZHX2 against ischemia-reperfusion injury in marginal fatty liver. Based on these research findings, this invention is thus completed.

[0006] Specifically, the technical solution of this invention is as follows:

[0007] In a first aspect, the invention provides the use of substances for detecting coding genes and their expression products in the preparation of diagnostic products related to liver ischemia-reperfusion injury.

[0008] The liver ischemia-reperfusion injury-related detection products are those with functions of screening, (auxiliary) diagnosis, monitoring and prediction of the progression of liver ischemia-reperfusion injury.

[0009] The expression product of the ZHX2 encoding gene can obviously be the transcription factor ZHX2.

[0010] A second aspect of the present invention provides a product for screening, (aiding) diagnosis, monitoring and predicting the progression of liver ischemia-reperfusion injury, the product comprising a substance for detecting the transcription of the ZHX2-encoding gene based on sequencing methods and / or quantitative PCR methods and / or probe hybridization methods; or a substance for detecting the expression of the transcription factor ZHX2 based on immunoassay methods.

[0011] A third aspect of the invention provides a system for screening, (aided) diagnosis, monitoring, and predicting the progression of liver ischemia-reperfusion injury, the system comprising:

[0012] i) An analysis unit, the analysis unit comprising: a detection substance selected from the expression level of ZHX2 in the test sample of the subject;

[0013] ii) An assessment unit comprising: determining the subject’s disease status based on the ZHX2 expression level determined in i).

[0014] A fourth aspect of the present invention provides the use of the above-mentioned ZHX2 as a target in the preparation and / or screening of drugs for liver ischemia-reperfusion injury.

[0015] A fifth aspect of the invention provides the use of a substance that promotes the expression of the ZHX2 encoding gene and its expression product in any one or more of the following:

[0016] a1) Prepare products that alleviate liver tissue (cell) apoptosis mediated by liver ischemia-reperfusion injury;

[0017] a2) Prepare products to alleviate liver function damage mediated by hepatic ischemia-reperfusion injury;

[0018] a3) Prepare products that inhibit the inflammatory response mediated by hepatic ischemia-reperfusion injury;

[0019] a4) Prepare products for the prevention and / or treatment of hepatic ischemia-reperfusion injury.

[0020] In a1), the relief of liver tissue (cell) apoptosis mediated by liver ischemia-reperfusion injury is specifically manifested by a decrease in the expression of apoptosis-related protein (BAX) in liver tissue, an increase in the expression of apoptosis-inhibiting proteins (BCL2, BCL-XL), and a decrease in the number of TUNEL-positive cells.

[0021] In a2), the reduction of liver function impairment mediated by hepatic ischemia-reperfusion injury is specifically manifested by a decrease in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

[0022] In a3), the inhibition of hepatic ischemia-reperfusion injury-mediated inflammatory response is specifically manifested as a decrease in the expression levels of inflammatory factors (TNF-α, IL-6).

[0023] In this invention, the product can be a drug.

[0024] The liver ischemia-reperfusion injury mentioned above can specifically be classified as marginal liver ischemia-reperfusion injury. The marginal liver can be a donor liver with a high risk of poor transplant function, including fatty liver, aged liver, etc.

[0025] A sixth aspect of the present invention provides the application of substances that inhibit the expression of the ZHX2 encoding gene and its expression products in the construction of an animal model of liver ischemia-reperfusion injury.

[0026] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0027] The aforementioned technical solution discloses for the first time the role of ZHX2 in ischemia-reperfusion injury in both normal and marginal liver. Specifically, the study found that ZHX2 expression decreases after ischemia-reperfusion injury, and hepatocyte-specific knockout of ZHX2 significantly promotes necrosis of hepatic ischemia-reperfusion injury (HIRI), thus promoting the occurrence and development of HIRI. This was further demonstrated in marginal liver HIRI. Furthermore, ZHX2 has a protective effect against ischemia-reperfusion injury in marginal fatty liver. In conclusion, ZHX2 can serve as a biomarker and therapeutic target for hepatic ischemia-reperfusion injury, especially marginal liver ischemia-reperfusion injury, thus possessing significant clinical application value and social benefits. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1This invention presents the expression changes of ZHX2 during the HIRI process. (AB) RT-qPCR and Western blot were used to detect the expression of ZHX2 in liver tissue during ischemia-reperfusion injury, respectively. (C) RT-qPCR and Western blot were used to detect the expression of ZHX2 in hepatocytes during hypoxia-reoxygenation. (D) Changes in ZHX2 expression, data from the GEO database (GSE151648).

[0030] Figure 2 In this embodiment of the invention, hepatocyte-specific knockout of Zhx2 significantly promoted HIRI cell necrosis. (A) Western blot detection of BAX, BCL-XL, and BCL-2 expression at specified time points. (B) Representative images and statistical data of TUNEL staining. (C) Serum AST and ALT levels at different time points. (D) RT-qPCR detection of IL-6 and TNF-α expression at specified time points. (E) Flow cytometry detection of ROS levels in liver tissue.

[0031] Figure 3 In this embodiment of the invention, hepatocyte-specific knockout of Zhx2 significantly promotes marginal hepatic inflammatory liver disease (IRI). (AF) Marginal fatty liver mouse model, (A) Representative image of liver Oil Red O staining. (B) Western blot detection of BAX and BCL-2 expression at specified time points. (C) Representative HE images and statistical data. (D) Serum AST and ALT levels at different time points. (E) RT-qPCR detection of IL-6, IL-1β, and TNF-α expression at specified time points. (F) Representative image and statistical data of Sirius Red staining. (GK) Marginal aged liver mouse model, (G) Representative image of liver β-galactose staining. (H) Western blot detection of BAX and BCL-2 expression at specified time points. (I) Representative HE images and statistical data. (J) Serum AST and ALT levels at different time points. (K) RT-qPCR detection of IL-6, IL-1β, and TNF-α expression at specified time points.

[0032] Figure 4 In this embodiment of the invention, ZHX2 improves ischemia-reperfusion injury in fatty liver. A HIRI model was established in 6-week-old C57BL / 6 mice after a 4-month high-fat diet. RT-qPCR and Western blot were used to detect the expression of BAX, BCL-XL, and BCL-2 at specified time points (A), TUNEL representative images and statistical data (B), serum ALT and AST levels (C), and RT-qPCR was used to detect the expression of IL-6 and TNF-α at specified time points (D). Detailed Implementation

[0033] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0035] In a typical embodiment of the present invention, the application of a substance for detecting coding genes and their expression products is provided in the preparation of a detection product related to liver ischemia-reperfusion injury.

[0036] The liver ischemia-reperfusion injury-related detection products are those with functions of screening, (auxiliary) diagnosis, monitoring and prediction of the progression of liver ischemia-reperfusion injury.

[0037] The expression product of the ZHX2 encoding gene can obviously be the transcription factor ZHX2.

[0038] In one or more specific embodiments of the present invention, a product is provided for screening, (aiding) diagnosis, monitoring and predicting the progression of liver ischemia-reperfusion injury, the product comprising a substance for detecting the transcription of the ZHX2-encoding gene based on sequencing methods and / or quantitative PCR methods and / or probe hybridization methods; or a substance for detecting the expression of the transcription factor ZHX2 based on immunoassay methods.

[0039] In one or more specific embodiments of the present invention, methods including but not limited to sequencing, liquid hybridization, Northern blotting, miRNA expression profiling chips, ribozyme protection analysis, RAKE method, and in situ hybridization can be used to detect the transcription of the ZHX2 encoding gene; methods including but not limited to ELISA, colloidal gold test strips, and protein chips can be used to detect the expression of the transcription factor ZHX2 protein.

[0040] The products described can exist in any existing known form, such as primers, probes, nucleic acid membrane strips, (gene or protein) chips, formulations, reagent kits, detection devices, and equipment. Those skilled in the art can realize the above products without inventive effort based on practical experience; therefore, all of the above products fall within the scope of protection of this application.

[0041] In one or more specific embodiments of the present invention, a system for screening, (aided) diagnosis, monitoring and predicting the progression of liver ischemia-reperfusion injury is provided, the system comprising:

[0042] i) An analysis unit, the analysis unit comprising: a detection substance selected from the expression level of ZHX2 in the test sample of the subject;

[0043] ii) An assessment unit comprising: determining the subject’s disease status based on the ZHX2 expression level determined in i).

[0044] The subjects may be humans or non-human mammals (such as mice), and the test samples include, but are not limited to, peripheral blood and liver-related tissues and cells.

[0045] In one or more specific embodiments of the present invention, the use of the above-mentioned ZHX2 as a target in the preparation and / or screening of drugs for liver ischemia-reperfusion injury is provided.

[0046] The method for screening drugs for liver ischemia-reperfusion injury includes:

[0047] 1) Treat the system expressing and / or containing the ZHX2 with the candidate substance; set up parallel controls without the candidate substance treatment;

[0048] 2) After completing step 1), detect the expression level of ZHX2 in the system; if the expression level of ZHX2 in the system treated with the candidate substance is significantly increased compared with the parallel control, the candidate substance can be used as a candidate drug for liver ischemia-reperfusion injury.

[0049] In another specific embodiment of the present invention, the system may be a cell system, a solution system, a tissue system, an organ system, or an animal system.

[0050] In another specific embodiment of the present invention, the cells in the cell system may be hepatocytes;

[0051] In another specific embodiment of the present invention, the tissue in the tissue system may be liver tissue;

[0052] In another specific embodiment of the present invention, the organ in the organ system may be the liver;

[0053] In another specific embodiment of the present invention, the animals in the animal system can be mammals, such as rats, mice, guinea pigs, rabbits, monkeys, humans, etc.

[0054] In one or more specific embodiments of the present invention, the use of a substance that promotes the expression of the ZHX2 encoding gene and its expression product is provided in any one or more of the following:

[0055] a1) Prepare products that alleviate liver tissue (cell) apoptosis mediated by liver ischemia-reperfusion injury;

[0056] a2) Prepare products to alleviate liver function damage mediated by hepatic ischemia-reperfusion injury;

[0057] a3) Prepare products that inhibit the inflammatory response mediated by hepatic ischemia-reperfusion injury;

[0058] a4) Prepare products for the prevention and / or treatment of hepatic ischemia-reperfusion injury.

[0059] The substances that promote the ZHX2 encoding gene and its expression products include, but are not limited to, substances that upregulate ZHX2 expression and / or promote its activity using gene-specific Mimics technology; such as promoters or lentiviruses, adeno-associated viruses, etc. that upregulate ZHX2 expression; and also include compound promoters, etc. In one specific embodiment of the present invention, the upregulation of ZHX2 expression can be achieved by administering an AAV virus containing ZHX2 to the subject.

[0060] In a1), the relief of liver tissue (cell) apoptosis mediated by liver ischemia-reperfusion injury is specifically manifested by a decrease in the expression of apoptosis-related protein (BAX) in liver tissue, an increase in the expression of apoptosis-inhibiting proteins (BCL2, BCL-XL), and a decrease in the number of TUNEL-positive cells.

[0061] In a2), the reduction of liver function impairment mediated by hepatic ischemia-reperfusion injury is specifically manifested by a decrease in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

[0062] In a3), the inhibition of hepatic ischemia-reperfusion injury-mediated inflammatory response is specifically manifested as a decrease in the expression levels of inflammatory factors (TNF-α, IL-6).

[0063] In this invention, the liver ischemia-reperfusion injury specifically refers to marginal liver ischemia-reperfusion injury. The marginal liver can be a donor liver with a high risk of poor transplant function, including fatty liver, aged liver, etc. In this invention, a liver ischemia-reperfusion injury model is constructed using a high-fat diet (HFD) and aging mice to simulate the occurrence of marginal liver ischemia-reperfusion injury.

[0064] In this invention, the product can be a drug.

[0065] According to the present invention, when the product is a drug, the drug further includes at least one inactive pharmaceutical ingredient.

[0066] The inactive components of the drug can be pharmaceutically commonly used carriers, excipients, and diluents. Furthermore, according to conventional methods, it can be formulated into oral, topical, suppository, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0067] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0068] In another specific embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate and mineral oil, etc.

[0069] In another specific embodiment of the invention, the drug of the invention can be administered into the body by known means. For example, it can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Alternatively, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0070] In another specific embodiment of the present invention, the drug can be administered to humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, etc.

[0071] In another specific embodiment of the present invention, the application of a substance that inhibits the expression of the ZHX2 encoding gene and its expression product in constructing an animal model of liver ischemia-reperfusion injury is provided.

[0072] This invention has found through research that mice with Zhx2 knocked out (Zhx2-KO) hepIt can significantly promote the occurrence of liver ischemia-reperfusion injury, specifically manifested as increased expression of the apoptosis-related protein BAX in liver tissue, while significantly decreased expression of apoptosis-inhibiting genes BCL-2 and BCL-XL; after Zhx2 knockout, the number of TUNEL apoptosis-positive cells increased significantly, and the levels of alanine aminotransferase (ALT) and AST were significantly increased; IL-6 and TNF-α were significantly increased in mouse liver tissue, and the level of reactive oxygen species in mouse liver tissue was also significantly increased, thereby aggravating the occurrence of liver ischemia-reperfusion injury.

[0073] The substances that inhibit the ZHX2 encoding gene and its expression products include, but are not limited to, RNA interference molecules or antisense oligonucleotides targeting ZHX2, small molecule inhibitors, shRNA (small hairpin RNA), small interfering RNA (siRNA), substances for lentiviral infection or gene knockout, and specific antibodies against the transcription factor ZHX2 protein itself or its upstream and downstream molecules, such as anti-ZHX2 antibodies. In one specific embodiment of the invention, this is achieved by applying hepatocyte-specific knockout of Zhx2.

[0074] The animals mentioned can be non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, etc.

[0075] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0076] Example

[0077] I. Materials and Methods

[0078] 1. Laboratory animals

[0079] The C57BL / 6 mice used in this study were purchased from the Experimental Animal Center of Shandong University and Beijing Vital River Laboratory Animal Technology Co., Ltd. Zhx2 flox / flox The mice were generously donated by Professor Brett T. Spear of the University of Kentucky, the Alb-Cre mice by Professor Gong Yaoqin of the Department of Genetics at Shandong University, and the hepatocyte-specific knockout Zhx2 mice (Zhx2-KO) hep The animals were constructed in our laboratory (PMID: 37980429). All experiments were conducted using male rats approximately 8 weeks old from the same litter or raised together for more than one week. They were housed in an SPF-grade environment in the animal facility of the School of Basic Medical Sciences, Shandong University. All animal experiments were approved by the Shandong University Animal Ethics Committee and followed the guidelines of the Shandong University Animal Ethics Committee.

[0080] 2.2 / 3 Hepatectomy Model Preparation

[0081] According to the literature, 8- to 12-week-old mice were selected and fasted for approximately 12 hours before surgery. Before surgery, the mice were weighed and anesthetized with an intraperitoneal injection of 0.5% sodium pentobarbital at a dose of 10 μL / g. After successful anesthesia (approximately 5 minutes), the mice were placed on a mouse operating table, their limbs were secured with tape, and after alcohol disinfection, a midline abdominal incision was made. Significant reperfusion injury was induced in the middle and left lobes of the liver by blocking and then re-opening blood flow to these lobes. The details to be noted during the construction of the mouse liver ischemia-reperfusion model are as follows: A 1 cm midline abdominal incision was made, and the skin and peritoneum were dissected layer by layer; abdominal organs were exposed using a retractor; sterile gauze moistened with physiological saline was prepared; the middle and left lobes of the liver were everted; the branches of the portal vein and hepatic artery in the middle and left lobes were located; the portal vein and hepatic artery in the middle and left lobes were clamped with non-invasive vascular clamps, causing ischemia in the middle and left lobes of the liver. The ischemic liver lobes would then clearly turn white. The vessels were released after the predetermined ischemia time was reached. The collected liver tissue was then fixed with 4% paraformaldehyde, and the remaining liver was rapidly frozen in liquid nitrogen and stored at -80°C.

[0082] 3. Real-time quantitative PCR

[0083] 1. RNA extraction

[0084] (1) Collect the cells into a 1.5 mL EP tube without RNase, add 1 mL TRIzol, and disperse the cells by blowing. For tissue RNA extraction: Take a small piece of mouse liver tissue and place it in a 1.5 mL EP tube without RNase, add 200 μL TRIzol, grind it thoroughly with a tissue homogenizer, then add 800 μL TRIzol, and repeatedly blow to lyse the cells.

[0085] (2) Add 200 μL of chloroform, mix thoroughly using a vortex mixer, and let stand on ice for 15 min until the layers separate.

[0086] (3) Centrifuge at 4℃ and 12000rpm for 15min, carefully aspirate the upper aqueous phase and transfer it to a 1.5mL EP tube that does not contain RNase.

[0087] (4) Add an equal volume of isopropanol, gently shake up and down, and let stand on ice for 10 minutes.

[0088] (5) Centrifuge at 4℃ and 12000rpm for 15min, discard the supernatant, add 1mL of pre-cooled 75% ethanol, gently invert to mix, and wash the RNA.

[0089] (6) Centrifuge at 4℃ and 12000rpm for 15min, remove the supernatant, add an appropriate amount of DEPC water, detect the RNA concentration and purity, and then perform reverse transcription or store at -80℃.

[0090] 2. Reverse transcription

[0091] (1) Add 1 μL of Random Hexamer Primer and 1 μg of RNA solution to a new EP tube on ice, and make up to 12 μL with DEPC water. Mix well.

[0092] (2) Incubate in a 65°C water bath for 5 minutes, then immediately remove the EP tube and place it on ice. Add each component to the EP tube according to the following system, for a total of 20 μL.

[0093]

[0094] (3) After mixing, incubate at 42℃ for 60 min. (4) Terminate the reaction by incubating at 70℃ for 5 min. The obtained cDNA can be used for the next step of PCR or stored at -20℃.

[0095] 3. RT-qPCR

[0096] (1) Using the cDNA obtained above as a template, add each component according to the following system, totaling 20 μL.

[0097]

[0098]

[0099] (2) RT-qPCR amplification

[0100]

[0101] (3) RT-qPCR results based on 2 -ΔΔCT Conversion.

[0102]

[0103] 4. Western Blot

[0104] (1) Cell collection and protein extraction: After removing the culture medium, wash the cells twice with pre-cooled PBS, collect the cells in a 1.5 mL EP tube, centrifuge at 2000 rpm at 4℃ for 5 min, and collect the cell pellet. Add 50 μL of WB / IP lysis buffer (containing 1× protease inhibitor PMSF), lyse on ice for 30 min, inverting and mixing once every 10 min. After lysis, centrifuge at 12000 rpm at 4℃ for 10 min, and collect the supernatant in a new EP tube.

[0105] (2) BCA method for protein concentration determination: Prepare protein standards with concentrations of 2000, 1000, 750, 500, 250, 125, and 25 μg / mL. Add 20 μL of each protein standard and the sample to be tested to a 96-well plate, repeating 3 times. Prepare BCA working solution according to the ratio of reagent A: reagent B = 50:1, add 200 μL to each well, mix carefully, and incubate at 37℃ for 30 min. Measure the OD value at a single wavelength of 562 nm using a microplate reader. Plot a standard curve based on the protein concentration and OD value of the standards, and then calculate the concentration of the sample protein to be tested based on the standard curve. Add 5×SDS protein loading buffer according to the protein volume, and heat at 100℃ for 6 min to denature the protein. Add the sample to the PAGE gel according to the sample protein concentration, ensuring that the amount of protein loaded for each sample is consistent.

[0106] (3) Electrophoresis: Add about 30 μg of protein sample to each well. Perform electrophoresis on the upper gel at 80 V for 30 min and on the lower gel at 120 V for about 90 min.

[0107] (4) Transfer: Activate the PVDF membrane in advance, soak it in methanol for 2 minutes, and transfer the membrane under a constant current of 400mA. The order of placement of each part is: white clamping plate - sponge - two layers of filter paper - PVDF membrane - protein glue - two layers of filter paper - sponge - black clamping plate.

[0108] (5) Blocking: After the transfer is completed, the membrane is blocked in 5% skim milk solution at room temperature on a shaker for 1-2 hours.

[0109] (6) Incubation with primary antibody: The PVDF membrane was placed in a dilution solution containing primary antibody and incubated overnight at 4°C. The membrane was then washed with 1×TBST three times, for 8-10 minutes each time.

[0110] (7) Incubation with secondary antibody: Place the PVDF membrane in the secondary antibody and incubate at room temperature for 1-2 hours. Wash the membrane with 1×TBST 3 times, 8-10 minutes each time.

[0111] (8) Exposure and development: Prepare developer A and developer B in advance and use them in a 1:1 ratio on a chemiluminescence imaging system.

[0112] 5. Detection of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST)

[0113] (1) Blood was collected from the inner canthus of the mouse, centrifuged at 2000 rpm for 20 min, and the serum was collected for testing.

[0114] (2) Preheat the matrix solution in the kit in a 37°C incubator beforehand. Use a 96-well plate. Add 20 μL of matrix solution to each well for testing and control. Add 5 μL of serum to each well for testing. Gently shake the plate and incubate at 37°C for 30 min.

[0115] (3) Add 20 μL of 2,4-dinitrophenylhydrazine to each test well and control well, and add 5 μL of the test sample to the control well. Gently shake the plate and incubate at 37°C for 20 min.

[0116] (4) Add 200 μL of 0.4 M sodium hydroxide to all test wells and control wells, gently shake the plate, and let it stand at room temperature for 15 min.

[0117] (5) The wavelength is 510nm. The OD value is measured by an enzyme-linked immunosorbent assay (ELISA) reader. The absolute OD value (OD value of the test well minus the OD value of the control well) is used to calculate the corresponding ALT and AST activity units according to the standard curve.

[0118] 6. Immunofluorescence: The above liver tissue was prepared into paraffin sections, and the expression of the above proteins was detected through steps such as dewaxing, antigen retrieval, antibody incubation, and fluorescence development.

[0119] II. Test Results

[0120] 1. Dynamic changes in ZHX2 expression during the HIRI process

[0121] A HIRI model was established in WT mice to detect ZHX2 mRNA and protein expression. Significant dynamic changes were observed in ZHX2 expression. Figure 1 AB). Primary hepatocytes were extracted from C57BL / 6 mice to construct a hypoxia-reoxygenation model. ZHX2 expression was decreased after HIRI (High-Intensity Respiratory Intake). Figure 1 C), further analysis of clinical ischemia-reperfusion injury data in the GEO database (GSE151648) revealed decreased ZHX2 expression after ischemia-reperfusion injury. Figure 1 D).

[0122] 2. Hepatocyte-specific knockout of Zhx2 significantly promotes HIRI

[0123] To clarify the role of ZHX2 in HIRI, liver-specific knockout Zhx2 mice (Zhx2-KO) were used. hep Ischemia-reperfusion injury models were established using mice (Zhx2-WT) and control mice (Zhx2-WT). Subsequently, the expression of apoptosis genes and liver function in mouse hepatocytes were dynamically monitored. hep The expression of the apoptosis-related protein BAX in mouse liver tissue was significantly higher than that in the control group at different time points, while the expression of apoptosis-inhibiting genes BCL-2 and BCL-XL was significantly decreased. Figure 2A); After Zhx2 knockout, the number of TUNEL apoptotic positive cells increased significantly, especially after 6 hours of HIRI ( Figure 2 B); Collect serum from mice after injury, Zhx2-KO hep Compared with Zhx2-WT mice, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly increased. Figure 2 C). Dynamic monitoring of inflammatory factor markers in mouse hepatocytes, Zhx2-KO hep The levels of IL-6 and TNF-α in mouse liver tissue were significantly higher than those in the Zhx2-WT group. Figure 2 D); In addition, Zhx2-KO hep The level of reactive oxygen species in mouse liver tissue was significantly higher than that in the control group. Figure 2 E).

[0124] 3. Hepatocyte-specific knockout of Zhx2 significantly promotes borderline liver IRI.

[0125] To clarify the role of ZHX2 in marginal hepatic ischemia-reperfusion injury (IRI), ischemia-reperfusion injury models were established using high-fat diet (HFD) and aging mice, respectively. Subsequently, the expression of apoptosis genes and liver function in mouse hepatocytes were dynamically monitored. Zhx2-KO hep The expression of the apoptosis-related protein BAX in mouse liver tissue was significantly higher than that in the control group at different time points, while the expression of apoptosis-inhibiting genes BCL-2 and BCL-XL was significantly decreased. Figure 3 AB); After Zhx2 knockout, the area of ​​liver necrosis significantly increased, especially after 6 hours of HIRI ( ). Figure 3 C); Collect serum from mice after injury, Zhx2-KO hep Compared with Zhx2-WT mice, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly increased. Figure 3 D). Dynamic monitoring of inflammatory factor markers in mouse hepatocytes, Zhx2-KO hep The levels of IL-6 and TNF-α in mouse liver tissue were significantly higher than those in the Zhx2-WT group. Figure 3 E); In addition, aging mice Zhx2-KO hep The degree of liver tissue damage in mice was higher than that in the control group. Figure 3 FJ).

[0126] 4. Protective effect of ZHX2 against ischemia-reperfusion injury in borderline fatty liver

[0127] C57BL / 6 mice were fed a high-fat diet for 6 weeks for 4 months, and then injected via tail vein with AAV-Vehicle or AAV-ZHX2 virus, respectively. An ischemia-reperfusion injury model was subsequently established. Mice were sacrificed at different time points, and liver tissue and serum samples were collected to detect apoptosis and inflammation-related markers in the liver tissue. Compared with AAV-Vehicle mice, the expression of apoptosis-related protein (BAX) in the liver tissue of AAV-ZHX2 mice was significantly decreased, while the expression of apoptosis-inhibiting proteins (BCL2, BCL-XL) was significantly increased. Figure 4 A), the number of TUNEL-positive cells was significantly reduced ( Figure 3 B), serum ALT and AST levels decreased significantly. Figure 4 C) The expression levels of inflammatory factors (TNF-α, IL-6) were significantly decreased. Figure 4 D).

[0128] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. Application of reagents for detecting the ZHX2 encoding gene in the preparation of kits for the auxiliary diagnosis of liver ischemia-reperfusion injury.

2. A system for assisting in the diagnosis of liver ischemia-reperfusion injury, characterized in that, The system includes: i) An analysis unit, the analysis unit comprising: a detection reagent for determining the expression level of ZHX2 in a test sample of a subject; ii) An assessment unit comprising: determining the subject’s disease status based on the ZHX2 expression level determined in i).

3. The system as described in claim 2, characterized in that, The subjects are human or non-human mammals, and the test samples include liver tissue and hepatocytes.

4. Application of ZHX2 as a target in the preparation of drugs for marginal hepatic ischemia-reperfusion injury.

5. Application of adeno-associated virus vectors expressing ZHX2 in any one or more of the following: a1) To prepare drugs that alleviate hepatocyte apoptosis mediated by marginal hepatic ischemia-reperfusion injury; a2) To prepare drugs that can alleviate liver function impairment mediated by marginal hepatic ischemia-reperfusion injury; a3) Prepare drugs to inhibit the inflammatory response mediated by marginal hepatic ischemia-reperfusion injury; a4) Prepare drugs for the prevention and / or treatment of marginal hepatic ischemia-reperfusion injury.

6. The application as described in claim 5, characterized in that, In a1), the relief of hepatocyte apoptosis mediated by liver ischemia-reperfusion injury is specifically manifested by a decrease in the expression of apoptosis-related protein BAX in liver tissue, an increase in the expression of apoptosis-inhibiting proteins BCL2 and BCL-XL, and a decrease in the number of TUNEL-positive cells. In a2), the reduction of liver function damage mediated by hepatic ischemia-reperfusion injury is specifically manifested by a decrease in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. In a3), the inhibition of hepatic ischemia-reperfusion injury-mediated inflammatory response is specifically manifested in the decreased expression levels of inflammatory factors TNF-α and IL-6.

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