Application of Zip7 in prevention and / or treatment of sepsis cardiomyopathy

Abstract

The invention discloses application of Zip7 in prevention and / or treatment of sepsis cardiomyopathy. The research finds that after the Zip7 gene is knocked out, the myocardial function of a sepsis mouse can be remarkably improved. It is prompted that Zip7 is a novel treatment approach for preventing and treating sepsis cardiomyopathy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of Zip7 in preventing and / or treating septic cardiomyopathy. Background Art

[0002] Sepsis-induced cardiomyopathy (SIC) is one of the serious complications of sepsis, with left ventricular dilatation, reduced ejection fraction and decreased myocardial contractility as the main pathophysiological manifestations. The diagnosis of sepsis cardiomyopathy lacks specific markers and is easily confused with other myocardial diseases. At the same time, the treatment of sepsis cardiomyopathy mostly relies on supportive treatment, and there is no direct targeted drug treatment. The circulatory failure caused by sepsis cardiomyopathy is one of the important factors causing the poor prognosis of sepsis patients.

[0003] Zinc is considered an essential trace element in eukaryotes and is critical for maintaining normal cell structure and function. Zinc deficiency can lead to increased oxidative stress and enhanced inflammatory immune responses. The distribution of zinc between the cytoplasm and organelles is regulated by specific zinc transporters. By releasing zinc from proteins and organelles, zinc concentration can be transiently and locally increased, allowing zinc to affect gene expression, enzyme activity and cell signaling. Zinc finger proteins are a special class of proteins that have finger-like domains and play a key role in cell growth, proliferation, apoptosis and intracellular signal transduction. Zinc finger protein Zip7, as a member of the SLC39A family (also known as the Zip family), is a transmembrane protein that is specifically responsible for the transport of zinc ions in cells. The main function of Zip7 is to transport zinc ions from specific organelles in the cell to the cytoplasm at the level of the organelle membrane, thereby ensuring that the zinc ion concentration in different organelles is maintained at a relatively stable level, that is, zinc homeostasis. Summary of the invention

[0004] The present invention provides the use of a reagent for inhibiting Zip7 expression in myocardial cells in preparing a drug for preventing and / or treating septic cardiomyopathy.

[0005] Furthermore, the reagents include reagents used to knock out Zip7 using any of the following technologies: siRNA interference technology, CRISPR technology, TALEN technology, ZFN technology, and Cre-loxP recombination technology.

[0006] Furthermore, the CRISPR technology is accomplished through the CRISPR-cas9 system.

[0007] Furthermore, the sequence of sgRNA in the CRISPR-cas9 system is shown as SEQ ID NO.1 and / or SEQ ID NO.2.

[0008] Furthermore, the reagents also include auxiliary reagents required for the operation of the CRISPR-cas9 system.

[0009] Furthermore, the drug exerts its preventive or therapeutic effects through the following mechanisms: improving cardiac function, alleviating myocardial cell damage, and alleviating myocardial cell mitochondrial function.

[0010] The present invention also provides a medicine for treating septic cardiomyopathy, wherein the effective component of the medicine comprises an agent for inhibiting the expression of Zip7 in myocardial cells.

[0011] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0012] The present invention also provides the use of Zip7 protein or its encoding gene as a target in screening drugs for preventing and / or treating septic cardiomyopathy.

[0013] The present invention also provides a method for screening drugs for preventing and / or treating septic cardiomyopathy, the screening method comprising the following steps:

[0014] The expression level and / or activity of Zip7 protein in cardiomyocytes of a septic cardiomyopathy cell model or an animal model before and after the administration of the test drug, or the change in the transcription level of Zip7 gene, is detected.

[0015] Preferably, the reagents for detecting the content of Zip7 in cells include reagents used in the following methods: hematoxylin-eosin staining (HE staining), safranin O-fast green staining, protein blotting (Western Blot method), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemistry (Immunohistochemistry) staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence resolution technology and protein chip method.

[0016] Preferably, the reagent for detecting the Zip7 content in cells can also be a reagent used in the following methods: PCR-based detection method, Southern hybridization method, Northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, high-throughput sequencing platform method.

[0017] In some embodiments, the Zip7 has a sequence known in the art or is a derivative molecule thereof. Derivative molecules include molecules that hybridize with sequences known in the art under stringent conditions; molecules that have a sequence homology of more than 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more, or any value or range of values ​​therebetween) with sequences known in the art, such as Zip7 molecules obtained by codon optimization.

[0018] As used in the present invention, the term "CRISPR technology" may also be referred to as CRISPR / Cas technology, in which CRISPR refers to Clustered Regularly Interspaced Short Palindromic Repeats / Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated proteins; Cas refers to CRISPR-associated proteins, that is, CRISPR-associated proteins, which have the activity of targeting and / or shearing target nucleic acids. There are many types of Cas proteins, the most common of which are Cas9 and Cas12. The guide sequence design principles of different cas proteins are different, but they are all well known to those skilled in the art. Cas proteins reach specific targeting sites through guide sequences to achieve the purpose of precise gene editing (including gene knockout).

[0019] Specifically, the reagents used in the CRISPR technology are well known in the art, specifically including at least Cas protein and guide sequence (can be called sgRNA). For example, the guide sequence used in the present invention is connected to a molecular carrier after annealing two oligos, and the reverse complementary part of the two oligos is the guide sequence.

[0020] Specifically, the present invention designs and synthesizes a specific guide sequence (sgRNA, whose oligo is described in the specific embodiment) for Zip7, and the specific guide sequence is used in combination with the Cas9 protein to achieve the purpose of inhibiting (knocking out) Zip7 expression. That is, in the present invention, the specific guide sequence designed for a specific gene is combined with the Cas protein to achieve the purpose of inhibiting the expression of the specific gene. The reagent including the specific guide sequence and the Cas protein designed for a specific gene is called an inhibitor of the specific gene.

[0021] Preferably, the inhibitor can be introduced into the target cells by any conventional technique. In the specific embodiment of the present invention, a transfection method is specifically used. The transfection reagent can be a commercial product or a self-prepared reagent; exemplary commercial products include Lipofectamine TM2000 (Thermo Fisher Scientific), Lipofectamine TM 3000 (Thermo Fisher Scientific), Lipofectamine TM RNAiMAX (ThermoFisher Scientific), Neon TM Transfection system (Thermo Fisher Scientific), HiPerFect (QIAGEN), X-tremeGENE (Roche), siLentFect TM (Bio-Rad) etc.

[0022] The term "pharmaceutically acceptable carrier" is described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used to help the stability of the formulation or to help improve the bioavailability of the active substance or to produce an acceptable taste or smell when taken orally, and the drug or pharmaceutical composition thus formulated can be administered to a subject in need by any appropriate administration method known to those skilled in the art.

[0023] In some embodiments, the pharmaceutically acceptable carrier and / or excipient include, but are not limited to, a buffer, a surfactant, a diluent, a wetting agent, a binder, a filler, a disintegrant, and an adsorption carrier.

[0024] Further, the buffer includes but is not limited to: phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactate, PIPES, etc.

[0025] Furthermore, the surfactant includes but is not limited to: polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, sodium stearate, calcium stearate, cetyl alcohol and the like.

[0026] Furthermore, the diluent includes, but is not limited to, dextrin, lactose, pregelatinized starch, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, sorbitol, mannitol, glucose, urea, starch, water, and the like.

[0027] Furthermore, the humectant includes but is not limited to: distilled water, ethanol, glycerol, starch, etc.

[0028] Furthermore, the adhesive includes, but is not limited to, starch (e.g., pregelatinized starch), dextrin (e.g., maltodextrin), sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid, alginate, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like.

[0029] Furthermore, the filler includes but is not limited to: mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.

[0030] Furthermore, the disintegrant includes but is not limited to: cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharide and the like.

[0031] Furthermore, the adsorption carrier includes, but is not limited to, liposomes, microcapsules, microspheres, nanocapsules, nanospheres, starch, lactose, bentonite, silica gel, kaolin, bentonite, etc.

[0032] Furthermore, the drug may be in the form of tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules, microcapsules), lozenges, syrups, liquids, emulsions, suspensions, controlled release preparations (e.g., instant release preparations, sustained release preparations, sustained release microcapsules), aerosols, films (e.g., orally disintegrating films, oral mucosa-adhesive films), injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), intravenous drips, transdermal absorption preparations, ointments, lotions, adhesive preparations, suppositories (e.g., rectal suppositories, vaginal suppositories), pellets, nasal preparations, pulmonary preparations (inhalants), eye drops, etc., oral or parenteral preparations (e.g., intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, instillation, intracerebral, intrarectal, etc. administration forms, administration to the vicinity of the tumor and direct administration to the lesion).

[0033] The "patient", "biological subject", "subject", and "individual" of the present invention are used interchangeably herein to refer to animal subjects, particularly vertebrate subjects, and even more particularly mammalian subjects. Suitable vertebrates falling within the scope of the present invention include, but are not limited to, any member of the subphylum Chordata, including primates, rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), birds (e.g., chickens; ducks; geese; companion birds such as canaries, budgies, etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish. Preferred subjects are primates (e.g., humans, apes, monkeys, chimpanzees).

[0034] The "effective amount" of the present invention refers to an amount that is sufficient to treat or prevent a patient's disease but low enough to avoid serious side effects within the scope of reasonable medical judgment. The therapeutically effective amount will vary depending on the specific compound selected (e.g., considering the potency, effectiveness and half-life of the drug), the selected route of administration, the disease being treated, the severity of the disease being treated, the age, size, weight and physical illness of the patient being treated, the medical history of the patient being treated, the duration of treatment, the nature of concurrent therapy, the desired therapeutic effect, and other factors, but can still be routinely determined by those skilled in the art.

[0035] The term "treatment" generally relates to treatment and therapy of humans or animals (e.g., in veterinary applications) in which some desired therapeutic effect is achieved, such as inhibiting the progression of a disease, and includes reducing the rate of progression, halting the rate of progression, alleviating symptoms of a disease, ameliorating a disease, and curing a disease. Treatment as a preventative measure (i.e., prophylaxis) is also included. For example, the term "treatment" also includes application to patients who have not yet developed a disease but are at risk of developing a disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Shows the experimental flow chart of the present invention;

[0037] Figure 2 The images show the pathological results of myocardial tissues of four groups of mice under light microscope, including HE staining ×40;

[0038] Figure 3 Shows the ultrastructural morphology of mitochondria in four groups of myocardial tissue. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under the conditions recommended by the manufacturer.

[0040] Example

[0041] The experimental flow chart of the present invention is as follows Figure 1 shown.

[0042] 1. Experimental Materials

[0043]

[0044]

[0045] 2. Experimental Methods

[0046] 1) Experimental animals

[0047] 96 wild-type (purchased from Beijing Sibeifu Biotechnology Co., Ltd.) and myocardium-specific knockout Zip7 (cooperated with Nanjing Institute of Biomedical Sciences, Nanjing University) male C57BL / 6 mice, 6-8 weeks old, weighing 20-25g, were selected. Before the experiment, the animals were kept in a specific pathogen-free animal room, maintained at a temperature of 20-22°C, a humidity of 30%-70%, and 12 hours of light and darkness per day, with free access to drinking water and food.

[0048] 2) Construction of myocardium-specific conditional knockout Zip7 mice

[0049] Cardiac-specific Slc39a7 conditional knockout mice (Zip7 cKO) were constructed in collaboration with Nanjing Biomedical Research Institute (NBRI) of Nanjing University using CRISPR / Cas9 technology. The transcript Slc39a7-201 was selected. The start codon ATG of the Slc39a7-201 gene is located in exon 1, and the stop codon TGA is located in exon 7. Cas9 mRNA with endonuclease activity and single-stranded guide RNA (sgRNA) and donor were injected into fertilized eggs at the same time. sgRNA recognizes the target gene DNA and guides Cas9 endonuclease to cleave the DNA, forming double-strand breaks (DSBs). When self-repair is interrupted, the broken double strands will undergo homologous recombination, and the LoxP sites will be inserted into the cut introns respectively. The cre enzyme will excise the DNA fragment between the two loxP sites, and the Slc39a7 gene will successfully undergo frameshift mutation. Tamoxifen (Sigma, USA, T5648, 10 mg / ml) was dissolved in corn oil, mixed and stored at -80°C in the dark. The mice were intraperitoneally injected with tamoxifen solution (75 mg / kg) once a day for 3 consecutive days.

[0050] The sequence information of sgRNA is shown in Table 1.

[0051] sgRNA name sgRNA sequence (5'-3') PAM Slc39a7-5S TGAAGACAACCGGAAAATC(SEQ ID NO.1) TGG Slc39a7-3S CCATCAAAGAACTATACAC(SEQ ID NO.2) AGG

[0052] 3) Identification of cardiomyocyte-specific knockout mice

[0053] Cut the mouse tail tissue and add lysis buffer and proteinase K in a ratio of 50:1 to cover the mouse tail tissue. Lyse in a 55°C water bath for 20 minutes, and then heat in a 100°C metal bath for 5 minutes to inactivate proteinase K. Centrifuge at 12000rpm for 5 minutes, transfer the supernatant to a new centrifuge tube, and you can get the mouse DNA sample. According to the amplification system, add appropriate amounts of PCR MasterMix, upstream and downstream primers, DNA and double distilled water for PCR amplification. Add the PCR amplification product to the prepared 2% agarose gel. Perform electrophoresis at a constant voltage of 130V for 30 minutes and expose. After the PCR amplification product was analyzed by agarose gel electrophoresis, the knockout group mice did not show the expected band at the amplification site of the target gene, indicating that the target gene has been successfully knocked out. The control group mice showed clear expected bands, indicating that the gene had not mutated. The electrophoresis results clearly showed the difference in bands between the knockout group and the control group, confirming the successful knockout of the target gene.

[0054] 4) Experimental Grouping

[0055] The two types of mice were randomly divided into two groups (n=48) using a random number table method, namely: 1) Sham group: wild-type C57BL / 6 mice underwent sham surgery with only abdominal opening and slight intestinal traction; 2) cKO+Sham group: Zip7 cKO mice underwent sham surgery; 3) CLP group: wild-type C57BL / 6 mice underwent cecal ligation and puncture surgery; 4) cKO+CLP: Zip7 cKO mice underwent cecal ligation and puncture surgery. The mice in the four groups were raised normally after surgery, and according to the results of the previous study, subsequent experimental studies were performed 18 hours later.

[0056] 5) Cecal ligation and puncture (CLP) model of sepsis in mice

[0057] The mice were anesthetized by inhalation of 2% sevoflurane. Fixation, skin preparation, and disinfection were performed in sequence. An incision of about 1 cm long was made along the midline of the abdomen of the mouse, and the abdominal cavity was carefully opened along the linea alba for exploration. Sutures were used to make a ligation at a position about 1 / 3 away from the end of the cecum, and a needle was used to penetrate the cecum between the ligation point and the end of the cecum, and a small amount of intestinal contents was gently squeezed out. The abdominal cavity was sutured layer by layer. After the operation, 1 ml of normal saline was injected subcutaneously into the mouse to replenish its fluid loss. For mice undergoing sham surgery, only their abdominal cavity was opened and the intestines were slightly pulled, but the cecum was not ligated or perforated.

[0058] 6) Determination of mouse survival rate

[0059] After surgical modeling, the four groups of mice were housed in separate cages, and the survival status of the four groups of mice was observed and recorded every day. After 10 days of observation, the survival status of the mice in each group was measured (n=20).

[0060] 7) Small animal cardiac ultrasound measurement

[0061] The mouse was placed in a supine position on the ultrasound table, a catheter was connected to the mouth and nose, 1% isoflurane was inhaled for anesthesia, the limbs were fixed, the skin was prepared to expose the body surface, and coupling agent was applied. The Vevo770 ultrasound instrument was used, and the M400 probe was selected for M ultrasound monitoring. After the outflow tract was detected with the probe, the probe was placed on the long and short axes of the mouse's left ventricle, and the left ventricular ejection fraction (LVEF) and shortening fraction (LVFS) were detected by LV Trace, and at least 5 cardiac cycles were continuously monitored (n=6).

[0062] 8) Myocardial histopathological observation

[0063] 18 hours after modeling or sham operation, mice were anesthetized with 2% sevoflurane, the chest was opened, the right atrial appendage was carefully cut off, and the heart was taken out by perfusing pre-cooled PBS and paraformaldehyde through the left apex. The heart was fixed, dehydrated, transparent, dipped in wax, and embedded in paraffin in sequence. The embedded paraffin block was trimmed into a trapezoid, dewaxed after sectioning, and HE and Masson staining were performed and observed under an optical microscope.

[0064] 9) Determination of cardiac function indexes and inflammatory factor levels

[0065] 18 hours after the sham operation, 6 mice were randomly selected from each group. After anesthesia with 2% sevoflurane, 1 ml of blood was collected from the apex of the heart. After standing at room temperature for 2 hours to precipitate serum, the blood was centrifuged at 1000 g for 10 minutes, and the supernatant was taken as a sample. According to the instructions of the corresponding kits (purchased from Beijing Solebow Technology Co., Ltd. and Shanghai Biyuntian Biotechnology Co., Ltd.), the levels of mouse cardiac troponin (cTNT) and serum inflammatory factors (IL-6, TNF-α) were determined by ELISA (n=5).

[0066] 10) Determination of reactive oxygen species in myocardial tissue

[0067] 18h after modeling or sham operation, mice were anesthetized with 2% sevoflurane, the chest was opened, the right atrial appendage was carefully cut off, and pre-cooled PBS was perfused through the left apex. An appropriate amount of myocardial tissue was taken, 1 ml of extract was added, homogenized under ice bath, and the supernatant was taken as the sample to be tested after centrifugation. According to the instructions of the hydroxyl radical (OH-) content kit (Suzhou Grace Biotechnology Co., Ltd.) and the peroxynitrite anion (ONOO-) detection kit (Shanghai Lianzu Biotechnology Co., Ltd.), the active oxygen content of the sample was determined by colorimetry (n=5).

[0068] 11) Observation of myocardial mitochondrial morphology under transmission electron microscopy

[0069] 18 hours after modeling or sham operation, mice were anesthetized with 2% sevoflurane, and the chest was opened. Fresh heart tissue was cut into small pieces of about 1 mm3, fixed, rinsed, fixed, dehydrated, and embedded in sequence. The embedded blocks were semi-thinly positioned and ultra-thinly sliced ​​with an ultrathin slicer, and the sections were double-stained with uranyl acetate and lead citrate, and the ultrastructure of myocardial mitochondria was observed with a transmission electron microscope.

[0070] 12) Extraction of mitochondrial proteins from myocardial tissue

[0071] Fresh mouse heart tissue was used to extract myocardial tissue mitochondria using a mitochondrial extraction kit (purchased from Beijing Solebow Technology Co., Ltd.) according to the instructions.

[0072] 13) Protein content determination by western blotting

[0073] 18h after modeling or sham surgery, mice were anesthetized with 2% sevoflurane, the chest was opened, the right atrial appendage was carefully cut off, and pre-cooled PBS was perfused through the left apex. An appropriate amount of tissue was weighed, and 10 times its weight of lysis solution (RIPA: PMSF = 100: 1) was added, fully ground, and centrifuged at 15000rpm for 20min. The supernatant was the heart tissue protein sample. SDS protein loading buffer was added and the protein was denatured at 100℃. 4% to 12% SDS polyacrylamide gel electrophoresis, fast wet transfer instrument transfer for 16min, 5% skim milk blocking for 2h, PVDF membrane was placed in the corresponding prepared antibody (antibody concentration 1: 1000, internal reference concentration 1: 5000), and incubated overnight on a shaker at 4℃. The corresponding secondary antibody was incubated at room temperature for 90min. ECL luminescence method was used for development and exposure. After adjusting the exposure parameters, the grayscale value of the bands was statistically analyzed using Image J, and the ratio of the target protein to the internal reference protein (GAPDH) was used to reflect the expression of the target protein.

[0074] 14) Data statistics and analysis

[0075] All data were statistically analyzed using GraphPad Prism 9.5 software. Before comparing the groups, the Shapiro-Wilk test was used to confirm whether the data conformed to normal distribution. The measurement data that conformed to normal distribution were expressed as mean ± standard deviation. The independent samples t test was used to compare two groups of independent samples, and one-way ANOVA was used to compare multiple groups of independent samples. The differences between the groups were further compared using Tukey's HSD test. All statistical tests were two-sided, and P < 0.05 was considered statistically significant.

[0076] 3. Experimental Results

[0077] The 10-day survival rates of mice in the Sham, cKO, CLP, and cKO+CLP groups were 100%, 90%, 0%, and 50%, respectively. Compared with the Sham group, the survival rates of the CLP and cKO+CLP groups were significantly decreased (P<0.01); compared with the CLP group, the survival rate of the cKO+CLP group was significantly increased (P<0.01).

[0078] Compared with the Sham group, the cardiac ultrasound of the CLP group mice showed a significant decrease in LVEF and LVFS (P<0.05). The LVEF and LVFS of the cKO+CLP group mice were significantly improved compared with the CLP group (P<0.05). See Table 2.

[0079] Table 2 Comparison of cardiac ultrasound cardiac function indices in four groups of mice

[0080]

[0081] Note: LVEF is left ventricular ejection fraction, LVFS is left ventricular shortening fraction

[0082] Under light microscopy, the myocardial cells in the Sham and cKO groups were arranged in a regular pattern, with no obvious damage to myocardial fibers and no obvious inflammatory cell infiltration. In the CLP group, myocardial tissue was severely damaged and cells were arranged in a disordered pattern. Under light microscopy, myocardial cell degeneration, interstitial edema, and increased inflammatory cell infiltration were observed. At the same time, the number of fibrotic myocardial cells in the CLP group increased significantly. At the same time, myocardial tissue damage in the cKO+CLP group was alleviated, inflammatory cell infiltration was relatively reduced, and interstitial edema was alleviated. Figure 2 .

[0083] Compared with the Sham group, there were no significant differences in the levels of cTNT, TNF-α, and IL-6 in the cKO group (P>0.05), and the levels of cTNT, TNF-α, and IL-6 in the CLP group were significantly increased (P<0.05); compared with the CLP group, the levels of cTNT, TNF-α, and IL-6 in the cKO+CLP group were significantly decreased (P<0.05). See Table 3.

[0084] Table 3 Comparison of serum cTNT, TNF-α and IL-6 concentrations in four groups of mice

[0085]

[0086]

[0087] Note: cTNT is troponin, TNF-α is tumor necrosis factor, IL-6 is interleukin 6

[0088] There was no significant change in the OH- and ONOO- levels of mice in the Sham group and the cKO group (P>0.05). Compared with the Sham group, the levels of serum reactive oxygen molecules OH- and ONOO- in the CLP group were significantly increased (P<0.05); compared with the CLP group, the reactive oxygen species in the cKO+CLP group were significantly reduced (P<0.05), as shown in Table 4.

[0089] Table 4 Comparison of serum OH- and ONOO- concentrations in four groups of mice

[0090]

[0091] Note: OH- is hydroxyl radical, ONOO- is peroxynitrite anion

[0092] There was no obvious swelling of vacuoles in the mitochondria of the cardiomyocytes of the mice in the Sham and cKO groups, and the mitochondrial cristae were intact. Compared with the Sham group, the ultrastructural destruction of mitochondria was obvious in the CLP group, the oval structure of mitochondria changed and swollen, and the mitochondrial cristae were obviously broken. Compared with the CLP group, the degree of mitochondrial swelling was significantly alleviated after knocking out Zip7, and the dissolution and breakage of mitochondrial cristae were improved (see Figure 3 ).

[0093] Compared with the Sham group, the expression of mitochondrial protein Drp1 in the cKO+Sham group was not significantly different (P>0.05), and the expression of mitochondrial protein Drp1 in the CLP group was significantly increased (P<0.05); compared with the CLP group, the expression of Drp1 in the cKO+CLP group was significantly decreased (P<0.05). Compared with the Sham group, the expression of Mfn2 and Opa1 in the myocardial tissue of mice in the CLP group was significantly decreased (P<0.05), and compared with the CLP group, the expression of Mfn2 and Opa1 in the cKO+CLP group was significantly increased (P<0.05), as shown in Table 5.

[0094] Table 5 Comparison of the expression of Drp1, Mfn2 and Opa1 in myocardial tissue of four groups of mice

[0095]

[0096] Note: Drp1 is dynamin-related protein 1, Mfn2 is mitochondrial fusion protein 2, Opa1 is optic atrophy protein 1

[0097] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. Use of an agent for inhibiting ZIP7 expression in cardiomyocytes in the preparation of a drug for preventing and / or treating septic cardiomyopathy.

2. The use according to claim 1, characterized in that: The reagents include reagents used to knock out ZIP7 using any of the following technologies: siRNA interference technology, CRISPR technology, TALEN technology, ZFN technology, and Cre-loxP recombination technology.

3. The use according to claim 2, characterized in that: The CRISPR technology is accomplished through the CRISPR-cas9 system.

4. The use according to claim 3, characterized in that: The sequence of sgRNA in the CRISPR-cas9 system is shown as SEQ ID NO.1 and / or SEQ ID NO.

2.

5. The use according to claim 4, characterized in that: The reagents also include auxiliary reagents required for the operation of the CRISPR-cas9 system.

6. The use according to claim 1, characterized in that: The drug exerts its preventive or therapeutic effects through the following mechanisms: improving cardiac function, alleviating myocardial cell damage, and alleviating myocardial cell mitochondrial function.

7. A drug for treating septic cardiomyopathy, characterized in that: The active ingredient of the drug includes an agent that inhibits the expression of ZIP7 in cardiomyocytes.

8. The use according to claim 7, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

9. Use of ZIP7 protein or its encoding gene as a target in screening drugs for preventing and / or treating septic cardiomyopathy.

10. A method for screening drugs for preventing and / or treating septic cardiomyopathy, characterized in that: The screening method comprises the following steps: The expression amount and / or activity of ZIP7 protein in cardiomyocytes of a cell model or an animal model of septic cardiomyopathy before and after the administration of the test drug, or the change in the transcription level of ZIP7 gene, is detected.

Images