Use of a beta-arrestin-1-ser330 site phosphorylation reagent in the preparation of a medicament for treating cardiac injury

Abstract

The present application provides a new finding that beta-arrestin-1-ser330 site phosphorylation is a new inhibitor of sympathetic activation leading to cardiac injury. Specifically, the present application provides a treatment method for promoting beta-arrestin-1-ser330 site phosphorylation by AMPK alpha 2, thereby blocking the activation of the cAMP / PKA pathway, thereby inhibiting the occurrence of sympathetic activation leading to cardiac injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of a β-arrestin-1-ser330 phosphorylation reagent in the preparation of drugs for treating cardiac injury. Background Technology

[0002] Under physiological conditions, low doses of catecholamines secreted by the sympathetic nervous system can activate β-adrenergic receptors (β-AR) in the heart, exerting positive chronotropic, inotropic, and transconductive effects to maintain normal cardiac contraction and pumping function. However, when pathological stimuli such as stress and hypertension occur, the sympathetic nervous system becomes overexcited, leading to the secretion of large amounts of catecholamines and resulting in overactivation of β-AR in the heart. Overactivation of β-AR can, on the one hand, enhance cardiac contractility, mobilize cardiac rate reserve, and improve the body's response to emergency events or compensate for the decline in cardiac function caused by pathological stimuli. On the other hand, overactivation of β-AR can also cause a series of pathological cardiac injuries, such as apoptosis, fibrosis, and inflammation, ultimately leading to a decline in cardiac function. Cardiac inflammation is a central early stage of cardiac injury; effectively inhibiting cardiac inflammation can reduce subsequent myocardial remodeling and decline in cardiac function, and provide a new intervention strategy for acute sympathetic stress-induced cardiac injury. Therefore, clarifying the molecular mechanism of sympathetic stress-induced cardiac inflammation is crucial for inhibiting inflammation-induced cardiac injury.

[0003] β-arrestin is a key protein mediating the functions of β-AR activation, primarily mediating receptor desensitization and non-canonical signaling pathways. There are four isoforms of Arrestin protein, but only two isoforms, arcestin-2 (β-arrestin-1) and arcestin-3 (β-arrestin-2), are expressed in cardiac tissue. Current research on the therapeutic effects of β-arrestin-1 mainly focuses on its application in Alzheimer's disease, Parkinson's disease, intestinal disorders, and liver diseases. In the cardiac field, previous studies have shown that β-arrestin-1 can lead to decreased cardiac function and worsen heart failure after myocardial infarction. The main mechanism is that β-arrestin-1 promotes β1-AR desensitization and downregulation in the heart, thereby inhibiting the positive chronotropic and inotropic effects of catecholamine-activated β1-AR; and promoting cardiac inflammation and cardiomyocyte apoptosis after myocardial infarction. Currently, there are no reports on the therapeutic effects of β-arrestin-1 phosphorylation on cardiac damage. Summary of the Invention

[0004] To fill the gap in the prior art, this invention discloses for the first time the effect of β-arrestin-1 phosphorylation on cardiac damage and specifically provides the following technical solution:

[0005] In a first aspect, the invention provides the use of a β-arrestin-1 phosphorylation agent in the preparation of a treatment for cardiac injury.

[0006] In one implementation, the cardiac injury is caused by acute sympathetic infection.

[0007] In another embodiment, the β-arrestin-1 phosphorylation agent promotes phosphorylation at the β-arrestin-1-ser330 site.

[0008] In another embodiment, the β-arrestin-1 phosphorylation agent can inhibit the activation of the cAMP / PKA pathway.

[0009] In a preferred embodiment, the β-arrestin-1 phosphorylation agent is an AMPKα2 activator.

[0010] A second aspect of the invention provides the use of a β-arrestin-1 phosphorylation agent in the preparation of a formulation for inhibiting cardiomyocyte inflammation and / or oxidative stress.

[0011] In one embodiment, the β-arrestin-1 phosphorylation agent promotes phosphorylation at the β-arrestin-1-ser330 site.

[0012] In another embodiment, the β-arrestin-1 phosphorylation agent can inhibit the activation of the cAMP / PKA pathway.

[0013] In a preferred embodiment, the β-arrestin-1 phosphorylation agent is an AMPKα2 activator.

[0014] The present invention achieves the following beneficial technical effects compared to the prior art:

[0015] 1. The inventors of this invention unexpectedly discovered and reported for the first time that promoting phosphorylation of β-arrestin-1-ser330 can significantly inhibit the activation of the cAMP / PKA pathway, inhibit cardiomyocyte apoptosis, inflammation and oxidative stress, thereby inhibiting cardiac damage caused by sympathetic activation;

[0016] 2. This invention is the first to discover that in the process of cardiac inflammation caused by β-AR overactivation, AMPK activation can be used as an intervention to counteract the activation of inflammasomes caused by the β-arrestin-1 signaling pathway. The potential mechanism is that AMPK can phosphorylate the β-arrestin-1-ser330 site, thereby inhibiting the production of ROS and the activation of inflammasomes.

[0017] 3. This invention discloses for the first time that AMPK can play a protective role in cardiac inflammatory damage caused by β-AR overactivation, which can provide theoretical support for the development of new drugs in the future and increase the applicability of the AMPK agonist metformin "old drug". Attached Figure Description

[0018] Figure 1 AMPKα2 binds to β-arrestin-1 and promotes β-arrestin-1-ser330 phosphorylation: a, AMPKα2 binds to β-arrestin-1. In HEK-293, the GFP-β-arrestin-1 plasmid was co-transfected with HA-AMPKα2, and co-IP was used to detect the interaction between β-arrestin-1 and AMPKα2; b, β-arrestin-1 binds to AMPKα2. In HEK-293, the GFP-β-arrestin-1 plasmid was co-transfected with HA-AMPKα2, and co-IP was used to detect the interaction between β-arrestin-1 and AMPKα2; c, Mass spectrum of AMPKα2 phosphorylation at the β-arrestin-1-ser330 site; d, AMPKα2 interacts with β-arrestin-1 at the ser330 site. In HEK-293, p-arrestin-1-wt / S330A / S330D plasmids were co-transfected with HA-AMPKα2, and the interaction between β-arrestin-1 and AMPKα2 was detected by co-IP; e, AMPK activation can promote the interaction between AMPKα2 and β-arrestin-1 in NMCMs. NMCMs were treated with 1 mM AMPK agonist metformin for 1 h, and the interaction between AMPKα2 and β-arrestin-1 was detected by co-IP.

[0019] Figure 2Phosphorylation of β-arrestin-1 at ser330 inhibits ISO-induced cAMP / PKA pathway activation in NMCMs: a) sequence conservation of β-arrestin-1 across different species; b) phosphorylation at ser330 promotes the transition of β-arrestin-1 from a basal state to an activated state. Computer simulations were used to detect the regions of greatest structural change in β-arrestin-1 after phosphorylation at ser330 (amino acid residues 357-383 and 332-341, respectively) and their impact on the overall structure. A schematic diagram of the structural changes of β-arrestin-1 before and after phosphorylation at ser330 is shown; ce simulates the inhibition of ISO-induced cAMP / PKA pathway activation in NMCMs by phosphorylation of β-arrestin-1 at ser330. NMCMs were infected with adenovirus overexpressing β-arrestin-1-wt / S330A / S330D and then treated with ISO (10 μM) for 30 minutes. cAMP concentration (c) and expression levels of p-PKA phosphorylated substrates (d, e) were measured; fh, simulated β-arrestin-1-ser330 phosphorylation did not affect ISO-induced p-SRC and p-ERK expression. NMCMs were infected with adenovirus overexpressing β-arrestin-1-wt / S330A / S330D and then treated with ISO (10 μM) for 30 min. Western blot was used to detect p-ERK and β-SRC expression levels.

[0020] Figure 3 Phosphorylation at β-arrestin-1-ser330 inhibited ISO-induced ROS production and NLRP3 inflammasome activation. NMCM cells were infected with adenovirus overexpressing β-arrestin-1-wt / S330A / S330D and then treated with ISO (10 μM) for 30 min. Phosphorylation at β-arrestin-1-ser330 at site 1 inhibited ISO-induced ROS production; β-arrestin-1-ser330 phosphorylation at site 1 inhibited NLRP3 inflammasome activation. Western blot analysis was performed to detect the expression levels of NLRP3 and P20.

[0021] Figure 4Phosphorylation at β-arrestin-1-ser330 inhibits ISO-induced cardiac inflammation. Male 10-week-old C57BL / 6J wild-type mice (WT) or mice with a β-arrestin-1-ser330-specific mutation (S330A KI, S330DKI) were subcutaneously injected with a single dose of ISO (5 mg / kg). Samples were taken on day 1 [ad] or day 3 [ef]. ab, β-arrestin-1-ser330 phosphorylation inhibits ISO-induced NLRP3 inflammasome activation; cd, β-arrestin-1-ser330 phosphorylation inhibits ISO-induced cardiac inflammatory factor production; ef, β-arrestin-1-ser330 phosphorylation inhibits ISO-induced cardiac macrophage infiltration.

[0022] Figure 5 Plasmid map of the mutated β-arrestin-1-ser330 site. Detailed Implementation

[0023] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, were performed in accordance with conventional practices in the art or under conditions recommended by the manufacturer.

[0024] Example: Phosphorylation mechanism of β-arrestin-1 at site 330

[0025] 1. Isolation and culture of neonatal mouse cardiomyocytes:

[0026] Cardiac cells were extracted from C57BL / 6 neonatal mice aged 1-3 days for subsequent experiments. The simplified procedure is as follows: After disinfecting the chest and abdomen of the mice twice with 75% alcohol (approximately 15 seconds each time), the heart was immediately removed via thoracotomy and placed in Hank's balanced salt buffer (NaCl 138 mmol / L; KCl 5.33 mmol / L; NaHCO3 4.17 mmol / L; KH2PO4 0.441 mmol / L; NaH2PO4 0.338 mmol / L; D-glucose 5.56 mmol / L) pre-cooled to 4°C and washed twice. The atria and major blood vessels at the base of the heart were then removed using ophthalmic scissors. The ventricles were then cut into small pieces and digested with an enzyme solution containing 0.07% trypsin and 0.04% type II collagenase prepared in Hank's balanced salt solution. The entire digestion process was carried out under constant temperature and stirring conditions at 37°C (140 rpm). After every 4 minutes of digestion, the supernatant was collected, and an equal volume of Dulbecco's Modified Eagle Medium (DMEM) high-glucose medium containing 15% fetal bovine serum (FBS) was added and mixed thoroughly. This step was repeated 8-10 times until the myocardial tissue was completely digested. The resulting myocardial cell suspensions were combined, centrifuged at 1000 rpm for 6 minutes at room temperature, the supernatant was discarded, and the cells were washed once more with DMEM medium containing 15% FBS. The cells were then resuspended in DMEM medium containing 15% FBS. Cells were seeded into 10 cm diameter cell culture plates and cultured at 37°C with 5% CO2 for 2.5 hours to allow non-cardiac cells (mainly fibroblasts) to adhere. The supernatant culture medium (containing cardiac cells) was collected, and bromodeoxyuridine (BrdU, final concentration 0.1 mmol / L) was added to inhibit fibroblast growth. Finally, the cardiac cells were seeded into appropriate cell culture plates and cultured in DMEM medium containing 15% FBS for 36 hours. The medium was then replaced with DMEM medium without serum for 4 hours for subsequent experiments.

[0027] 2. Co-IP experiment:

[0028] (1) Protein extraction: HKE-293A cells overexpressing hemagglutinin (HA)-labeled β-arrestin-1 (HA-β-arrestin-1) or HA-β-arrestin-2 were collected using IP lysis buffer. Protein quantification was performed using the BCA method. The protein concentration was adjusted to 1 μg / μl, and half of the sample was used as the Input group and the other half as the IP group, with a total system volume of 100 μl.

[0029] (2) Removal of non-specific binding proteins: Add 5 μl of protein A / G agarose beads to each sample and incubate on a shaker at 4°C for 2 hours. Then centrifuge at 1000 rpm / min at 4°C for 1 minute, remove the precipitate, and collect the supernatant.

[0030] (3) Antibody co-incubation: Add 2 μg of HA antibody or rabbit negative IgG to the lysate of HA-β-arrestin-1 or HA-β-arrestin-2 protein overexpression, and incubate overnight at 4°C on a rotating shaker.

[0031] (4) Co-incubation with agarose beads: Add 15 μl of βrotein A / G agarose beads to each of the lysis buffers in step (3) and incubate overnight on a shaker at 4°C.

[0032] (5) Obtaining purified protein: Centrifuge at 1000 r / min, 4℃ for 1 minute, and discard the supernatant; add 500 μl of IP lysis buffer to resuspend the precipitate, centrifuge at 1000 r / min, 4℃ for 1 minute, repeat twice, discard the supernatant, and take the precipitate.

[0033] (6) Dissociation of precipitated protein: Add 50 μl of 1× loading buffer to each sample and resuspend the precipitate; then boil in a 100°C water bath for 10 minutes, centrifuge at 1000 rpm for 1 minute, and take the supernatant, which is the protein purified by immunoprecipitation.

[0034] The loading buffer containing the aforementioned interdependent proteins can be used to detect whether the loading buffer contains the interdependent protein (HA-β-arrestin-1) by the following Western blot assay.

[0035] 3. Protein imprinting experiment:

[0036] 293T or neonatal mouse cardiomyocytes (NMCMs) were washed three times with cold PBS, then lysed with cell lysis buffer (20 mmol / L Tris-HCl pH 7.4, 150 mmol / L NaCl, 2.5 mmol / L EDTA, 50 mmol / L NaF, 0.1 mmol / L Na₄P₂O₇, 1 mmol / L Na₃VO₄, 1% Triton X-100, 10% glycerol, 0.1% SDS, 1% deoxycholic acid, 1 mmol / L PMSF, and 1 mg / ml aβrotinin). After sonication, the cells were centrifuged at 12000g for 15 minutes at 4°C. The supernatant was collected. 5 μL of the supernatant was used for protein quantification, and the remaining supernatant was added to 5X gel loading buffer and incubated at 100°C for 5 minutes to ensure protein denaturation. After 10% SDS-PAGE gel electrophoresis, the nitrocellulose membrane was transferred and blocked with 5% skim milk at room temperature for 1 hour. The membrane was then incubated overnight at 4°C with the corresponding primary antibody. After washing three times with the appropriate species-specific secondary antibody, the membrane was incubated at room temperature for 1 hour, washed again with TBST, and developed. The membrane was then placed in developing solution (Millipore Corporation), drained, and exposed in a luminescence detection machine. Band intensity was quantified using NIH ImageJ software.

[0037] In HEK-293, the GFP-β-arrestin-1 plasmid was co-transfected with HA-AMPKα2. Co-IP analysis of the interaction between β-arrestin-1 and AMPKα2 revealed that β-arrestin-1 can interact with AMPKα2. Figure 1 (ab). AMPK acts as a protein kinase, transmitting signals by phosphorylating substrate proteins. Therefore, we used purified human AMPKα2 and β-arrestin-1 proteins to detect whether AMPK phosphorylates β-arrestin-1 and the relevant phosphorylation sites using a combination of kinase assays and mass spectrometry. After the purified AMPKα2 and β-arrestin-1 proteins (purchased from Abcam) underwent a kinase reaction, we separated the phosphorylated β-arrestin-1 protein by SDS-polyacrylamide gel electrophoresis. After Coomassie brilliant blue staining, a strip of approximately 55 kDa was excised and analyzed by time-of-flight mass spectrometry. Mass spectrometry revealed that AMPKα2 phosphorylates nine sites on β-arrestin-1: Ser13, Tyr21, Thr19, Thr98, Ser163, Ser202, Ser234, Ser320, and Ser330. Figure 1c). The Ser330 site received the highest mass spectrometry score and the strongest confidence level, therefore we conducted further research on this site. To investigate the effects of phosphorylation at the β-arrestin-1-ser330 site on downstream signaling pathways and cardiac inflammation induced by ISO, we first constructed two plasmids with mutations at the β-arrestin-1-ser330 site ( Figure 5 ), along with adenovirus, were used in subsequent experiments. β-arrestin-1-S330A mutated the ser330 site to the inert amino acid alanine (A), preventing phosphorylation at this site; β-arrestin-1-S330D mutated the ser330 site to aspartic acid (D), mimicking phosphorylation at this site. In HEK-293 cells, we overexpressed plasmids co-transfected with HA-AMPKα2+β-arrestin-1-wt / S330A / S330D, and used co-IP assays to detect whether the interaction between AMPKα2 and β-arrestin-1 changed after the ser330 site mutation. The results showed that regardless of whether the ser330 site was mutated to A or D, the amount of AMPKα2 protein binding to β-arrestin-1 in HEK293 cells was significantly reduced. Figure 1 d) indicates that the mutation of the β-arrestin-1-ser330 site can inhibit the interaction between AMPKα2 and β-arrestin-1, that is, the β-arrestin-ser330 site is one of the binding sites of AMPKα2 and β-arrestin-1.

[0038] To clarify the potential mechanism by which β-arrestin-1 participates in AMPK inhibition of ISO-induced cardiac NLRP3 inflammasome activation and cardiac inflammation, we first examined the interaction between β-arrestin-1 and AMPKα2 in NMCMs before and after treatment with the AMPK agonist metformin. The results showed that, using the co-IP method, compared with the untreated group, 1 hour of metformin treatment (1 mmol / L) significantly enhanced the interaction between endogenous AMPKα2 and β-arrestin-1 protein in NMCMs (metformin(-) vs. metformin(+): 0.60±0.18 vs. 1.52±0.21, P<0.001), indicating that AMPK activation may exert its anti-inflammatory effect in ISO-induced cardiac inflammation by binding to β-arrestin-1. Figure 1 e).

[0039] Example 2: Phosphorylation of β-arrestin-1-ser330 inhibits ISO-induced cAMP production and PKA activation. live

[0040] To clarify the impact of phosphorylation at β-arrestin-1-ser330 on the structure of β-arrestin-1 and its downstream signaling pathways after β-AR activation, we first used molecular dynamics simulations to predict the effect of phosphorylation at this site on the structure of β-arrestin-1 based on the resolved crystal structure of β-arrestin-1 before and after activation (PDB: 1G4M). The results showed that after phosphorylation of β-arrestin-1-ser330, the root mean partial deviation (RMSD) of amino acid residues at positions 357-383 and 332-341 of the bovine β-arrestin-1 protein changed the most over time, and the overall RMSD of the protein also changed. Figure 2 ab). RMSD values ​​reflect the degree of change in protein structure; therefore, phosphorylation at ser330 has the greatest impact on the structure of amino acid sites 357-383 and 332-341 of β-arrestin-1, and to some extent affects the overall structure of the protein. Compared with the resolved basal / activated state crystal structures of β-arrestin-1, phosphorylation at ser330 promotes the structural transition of β-arrestin-1 from the basal state to the activated state. Figure 2 b) indicates that phosphorylation at ser330 may promote the activation of β-arrestin-1.

[0041] To clarify the impact of β-arrestin-1-ser330 phosphorylation on the downstream canonical cAMP / PKA signaling pathway and the non-canonical ERK and SRC signaling pathways induced by ISO, we used adenoviruses that simulated or blocked β-arrestin-1-ser330 phosphorylation to examine the effect of ISO on the activation levels of related proteins in these two signaling pathways in NMCMs. The results showed that, using cAMP accumulation and Western blotting experiments, ISO (10 μmol / L) treatment of NMCMs for 30 minutes resulted in less cAMP accumulation and lower PKA substrate phosphorylation levels compared to β-arrestin-1-S330A / wt virus; while compared to wt virus, ISO infection with S330A virus resulted in greater cAMP accumulation and higher PKA substrate phosphorylation levels. Figure 2 However, regardless of whether phosphorylation at this site was simulated or inhibited, after 30 minutes of ISO treatment, there was no significant difference in protein levels between p-ERK and p-SRC in NMCMs. Figure 2The above results indicate that simulated phosphorylation of β-arrestin-1-ser330 can inhibit the classical cAMP / PKA signaling pathway after β-AR activation, but does not affect the non-classical ERK and SRC signaling pathways.

[0042] Example 3: Phosphorylation of β-arrestin-1-ser330 inhibits ISO-induced ROS production in cardiomyocytes and NLRP3 inflammasome activation

[0043] ROS assay: Lactating mouse cardiomyocytes were routinely seeded into 12-well or 96-well plates. Twenty-four hours later, cells were infected with adenovirus overexpressing β-arrestin-1-wt / S330A / S330D protein (10 mol / 10 oz). 6 Cells were starved for 48 hours; or pretreated with different concentrations of glibenclamide or metformin for 30 minutes. Cells were then serum-free starved for 4 hours, treated with 10 μmol / L ISO for 30 minutes or 2 hours, and washed three times with PBS at 3°C. Cells were then incubated with 5 μmol / L total ROS probe Dihydroethidium (DHE) at 37°C for 30 minutes, and washed three times with pre-warmed PBS. Nuclei were then stained with Hoechst (1:1000) for 5 minutes. Intracellular fluorescence intensity was detected using the Array Scan system of a ThermoScientific tm HCS microscope (Hoechst excitation wavelength 389 nm; DHE excitation wavelength 570 nm; exposure time 0.15 seconds, magnification 20×, 10 fields of view per well, 3 replicates per group). After all the photos were taken, the Disk Scan module of the high-content microscope was used to quantify the cell number based on Hoechst-labeled cell nuclei and to quantify the ROS fluorescence intensity of cardiomyocytes based on the DHE emission intensity. The average value of 10 fields of view was taken as the average ROS fluorescence intensity of each well.

[0044] Protein overexpression: Plasmid transfection was performed according to the lipo2000 transfection reagent instructions, briefly described below: When HKE-293A cells in a six-well plate grew to approximately 50% confluency (approximately 0.5 × 10⁻⁶ cells), 6For each cell / well, mix 1.5 μg / well of HA-β-arrestin-1 or HA-β-arrestin-2 plasmid, or 1 μg / well of GFP-AMPKα2+HA-β-arrestin-1-wt / S330A / S330D plasmid, with 100 μl of OPTI-MEM medium and incubate at room temperature for 5 minutes. Mix 3 μl / well of lipo2000 with 100 μl of OPTI-MEM medium and incubate at room temperature for 5 minutes. Mix the plasmid with the lipo2000 transfection reagent and incubate at room temperature for 15 minutes to allow the transfection reagent to fully coat the plasmid. Then, add this mixture to HEK-293A cells and transfect in serum-free OPTI-MEM for 8 hours. Discard the medium, wash once with pre-warmed PBS, and then add complete medium containing 10% FBS. Harvest cells for subsequent experiments after 48 hours. Adenovirus encapsulating the HA-β-arrestin-1-wt / S330A / S330D plasmid was synthesized by Hanheng Biotechnology (vector: PEGFP-N1). Approximately 1 × 10⁻⁶ NMCMs were to be isolated into 6-well plates. 6 After culturing (cells / well) for 24 hours, the medium was changed to complete medium containing 10% FBS. Then, 10-30 moles of virus were added to each well. After 48 hours, the infection efficiency of the virus was observed under a fluorescence microscope.

[0045] To clarify the role of β-arrestin-1-ser330 phosphorylation in ISO-induced cardiac inflammation, we first examined the effect of this phosphorylation on ROS production and NLRP3 inflammasome activation in cardiomyocytes. In non-myocardial micromolecular cells (NMCMs), patients were first infected with either adenovirus overexpressing β-arrestin-1-S330D (which mimics phosphorylation at this site) or β-arrestin-1-S330A (which inhibits phosphorylation at this site) for 48 hours, followed by treatment with 10 μM ISO for 1 hour. ROS production was then detected by DHE staining. The results showed that ROS production in ISO-induced NMCMs was lower after β-arrestin-1-S330D adenovirus infection compared to S330A infection; however, ROS production was lower after wild-type (wt) virus infection than after β-arrestin-1-S330A virus infection, but higher than after β-arrestin-1-S330D virus infection. This indicates that simulated phosphorylation at the β-arrestin-1-ser330 site can inhibit ISO-induced ROS generation on NMCMs. Figure 3ab). We further investigated the effect of simulated phosphorylation at this site on ISO-induced NLRP3 inflammasome activation. We infected NMCMs with adenovirus overexpressing β-arrestin-1-wt / S330A / S330D, followed by serum-free starvation for 48 hours, and then treatment with 10 μM ISO for 1 hour. NLRP3 inflammasome activation was then assessed. The results showed that compared to NMCMs infected with β-arrestin-1-S330A virus, NMCMs infected with β-arrestin-1-S330D virus exhibited significantly reduced NLRP3 and Caspase-1(p20) protein expression after 1 hour of ISO treatment; compared to wild-type (wt) virus, NMCMs infected with β-arrestin-1-S330A virus showed higher NLRP3 inflammasome activation levels after 1 hour of ISO (10 μmol / L), indicating that simulated β-arrestin-1-ser330 site phosphorylation can inhibit ISO-induced NLRP3 inflammasome activation on NMCMs. Figure 3 ce).

[0046] Example 4: β-arrestin-1 ser330 phosphorylation inhibits ISO-induced cardiac inflammation in mice.

[0047] Heart Acquisition and Weighing: After weighing the mice (accurate to 0.1g), they were anesthetized by intraperitoneal injection of sodium pentobarbital at 200mg / kg. After complete anesthesia, the eyeballs were enucleated to collect blood. The thorax was then quickly cut open with scissors, the heart was dissected, and the right atrial appendage was cut open with ophthalmic scissors. A perfusion needle was inserted, and the heart and all organs were perfused with pre-cooled physiological saline on ice. The heart was then cut off and placed in phosphate buffer solution (PBS) at 4°C. Attached blood vessels and adipose tissue were removed from the heart. The moisture was blotted with filter paper, and the whole heart was weighed on a precision balance (accurate to 0.01g). The tibia was then separated, and its length was measured with calipers. The whole heart weight / body weight (HW / BW) and whole heart weight / tibia length (HW / TL) were calculated respectively. The middle portion of the heart tissue was fixed in 4% paraformaldehyde (pH=7.4) for 24 hours and used as a tissue section. The remaining myocardial tissue was placed in cryovials, flash-frozen in liquid nitrogen, and stored at -80℃ for protein extraction and detection in the heart tissue.

[0048] To clarify the effect of β-arrestin1-ser330 phosphorylation on ISO-induced cardiac inflammation in mice, β-arrestin1-330A and β-arrestin1-330D site-directed mutant mice and WT wild-type mice were administered a single subcutaneous injection of ISO (5 mg / kg body weight). Compared with WT wild-type mice, β30A KI mice showed a significant increase in NLRP3 and P20 expression after a single subcutaneous injection of ISO (5 mg / kg body weight), while β30D KI attenuated NLRP3 and P20 expression. Figure 4 ab). We then examined the effect of the 330D mutation on ISO-induced cardiac cytokine expression in mice. The results showed that, compared to wild-type mice, 330D KI mice showed significantly reduced expression of cardiac macrophage chemokines MCP-1 and MCP-5, as well as pro-inflammatory cytokines TNF-α and IL-6, on day 1 after subcutaneous ISO injection, indicating that 330D can inhibit ISO-induced cardiac inflammation in mice. Figure 4 Furthermore, we found that on day 3, the Mac3+ area in the heart tissue of 330DKI mice was significantly reduced, indicating that 330DKI can inhibit ISO-induced macrophage infiltration in mouse hearts (cd). Figure 4 ef).

[0049] The preferred embodiments of the present invention have been described above, but are not intended to limit the invention. Those skilled in the art can make modifications and variations to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

1. The use of a β-arrestin-1 phosphorylation agent in the preparation of a drug for treating cardiac injury, wherein the β-arrestin-1 phosphorylation agent is an adenovirus overexpressing β-arrestin-1-S330D or a plasmid overexpressing β-arrestin-1-S330D, and the cardiac injury is caused by acute sympathetic infection.

2. The application as described in claim 1, characterized in that, The β-arrestin-1 phosphorylation reagent can promote phosphorylation of β-arrestin-1 at the ser330 site.

3. The application as described in claim 1, characterized in that, The β-arrestin-1 phosphorylation reagent can inhibit the activation of the cAMP / PKA pathway.

4. The use of a β-arrestin-1 phosphorylation agent in the preparation of a formulation for inhibiting inflammation and / or oxidative stress in cardiomyocytes, wherein the β-arrestin-1 phosphorylation agent is an adenovirus overexpressing β-arrestin-1-S330D or a plasmid overexpressing β-arrestin-1-S330D.

5. The application as described in claim 4, characterized in that, The β-arrestin-1 phosphorylation reagent can promote phosphorylation of β-arrestin-1 at the ser330 site.

6. The application as described in claim 4, characterized in that, The β-arrestin-1 phosphorylation reagent can inhibit the activation of the cAMP / PKA pathway.

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