Application of succinylation modified protein in preparation of product for diagnosing or treating acute pancreatitis

Abstract

The invention provides an application of a succinylation modified protein in preparation of a product for diagnosing or treating acute pancreatitis. The succinylation modified protein comprises SUCLA2 which is subjected to succinylation modification on 118th lysine and Cyc1 which is subjected to succinylation modification on the 118th lysine. A new thought is provided for diagnosis and / or treatment of acute pancreatitis, and targeted drugs capable of accurately diagnosing and / or treating acute pancreatitis can be developed conveniently.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the use of succinylated modified proteins in the preparation of products for diagnosing or treating acute pancreatitis. Background Art

[0002] Acute pancreatitis is a common digestive system emergency caused by abnormal activation of pancreatic enzymes in the pancreas due to various factors, resulting in inflammatory damage to the pancreas and surrounding tissues. Its incidence rate is increasing year by year. In about 20% of cases, due to the continuous progression of local pancreatic inflammation, systemic inflammatory response syndrome may be triggered, causing damage to multiple organ systems such as circulation, respiratory, kidney, and intestines, and then developing into severe acute pancreatitis. If not treated promptly and effectively, it may rapidly deteriorate into multiple organ dysfunction syndrome and even lead to death.

[0003] The pathogenesis of acute pancreatitis has not been fully elucidated, and there is currently a lack of effective intervention targets and specific therapeutic drugs in clinical practice. Despite the continuous advancement of diagnostic and treatment technologies, the multidisciplinary comprehensive diagnosis and treatment strategy based on organ function support still faces bottlenecks. Therefore, there is an urgent need to deeply explore the key pathophysiological mechanisms of acute pancreatitis, find effective intervention targets and pathways, and develop targeted drugs or methods for precision treatment to provide new strategies for the prevention and treatment of acute pancreatitis. Summary of the invention

[0004] In view of the defects in the prior art, the purpose of the present invention is to provide the use of succinylated modified proteins in the preparation of products for the diagnosis or treatment of acute pancreatitis, providing new ideas for the diagnosis and / or treatment of acute pancreatitis, and providing methods and targeted drugs that can accurately diagnose and / or treat acute pancreatitis.

[0005] The first aspect of the present invention provides the use of a succinylated protein in preparing a product for diagnosing or treating acute pancreatitis. The succinylated protein comprises SUCLA2 in which lysine at position 118 is succinylated and succinylated Cyc1.

[0006] The second aspect of the present invention provides the use of succinylated proteins as targets for treating acute pancreatitis, wherein the succinylated proteins include SUCLA2 with succinylated lysine at position 118 and succinylated Cyc1.

[0007] In an embodiment of the present invention, the target is a drug target.

[0008] The third aspect of the present invention provides a product, the active ingredients of which include SUCLA2 modified by succinylation at lysine 118 and Cyc1 modified by succinylation; the product has at least one of the following functions: (1) predicting the risk of occurrence and progression of pancreatitis; (2) serving as a drug target for the treatment of pancreatitis.

[0009] Compared with the prior art, the embodiments of the present invention have the following beneficial effects: the present invention provides the use of SUCLA2 with succinylated lysine at position 118 and succinylated Cyc1 in the preparation of an acute pancreatitis diagnostic product, and their use as targets for the treatment of acute pancreatitis, providing a new idea for the diagnosis and treatment of acute pancreatitis, so as to provide a targeted drug that can accurately diagnose and / or treat acute pancreatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0011] Figure 1 The PCA and OPLS-DA model construction analysis results of the central carbon metabolomics of AR42J cells after caerulein stimulation in Example 1: Figure 1 A: Changes in amylase activity in the supernatant of AR42J cells at different time points after stimulation with caerulein; Figure 1 B: Changes in lipase activity in the supernatant of AR42J cells at different time points after stimulation with caerulein; Figure 1 C: PCA analysis results of the caerulein-treated group and the control group; Figure 1 D: OPLS-DA score diagram of the caerulein-treated group and the control group; Figure 1 E: Volcano plot of the number of significantly differentially upregulated and downregulated metabolites in AR42J cells after caerulein stimulation; Figure 1 F: VIP_FC score graph of various metabolites in AR42J cells after caerulein stimulation;

[0012] Figure 2 The results of the analysis of the changes in TCA cycle function of AR42J cells and the pancreas of acute pancreatitis rats after caerulein stimulation in Example 1 are as follows: Figure 2 A: KEGG enrichment results of metabolites in different metabolic pathways; Figure 2 B: TCA cycle metabolic pathway diagram of AR42J cells after caerulein stimulation and log2FC and VIP values ​​of key metabolites; Figure 2 C: TCA cycle metabolic pathway diagram and log2FC and VIP values ​​of key metabolites in the rat model of acute pancreatitis; Figure 2D: Venn diagram of differential metabolites of TCA cycle in acute pancreatitis cell and rat models;

[0013] Figure 3 The results of the effect of succinylation modification of SUCLA2 at the K118 site on the SCS activity of pancreatic acinar cells in acute pancreatitis in Example 2 are as follows: Figure 3 A: Results of SCS activity measurement in pancreatic tissue of rats with acute pancreatitis; Figure 3 B: Immunoblotting results of SUCLG1, SUCLA2, and SUCLG2 proteins in AR42J cells after caerulein stimulation; Figure 3 C: A-SCS assay results of AR42J cells after caerulein stimulation; Figure 3 D: G-SCS activity assay results of AR42J cells after caerulein stimulation; Figure 3 E: The results of the determination of SCS activity of wild-type and SUCLA2 point mutant AR42J cells after caerulein stimulation; Figure 3 F: ATP level determination results of wild-type and K118R_SUCLA2-overexpressing AR42J cells after caerulein stimulation;

[0014] Figure 4 The effect of SIRT5-mediated succinylation of SUCLA2 at K118 on the TCA cycle of pancreatic acinar cells in acute pancreatitis in Example 3: wherein, Figure 4 A: Immunoblotting results of SIRT5, CPT1A, KAT2A and SIRT7 proteins in AR42J cells after caerulein stimulation; Figure 4 B: Results of SCS activity measurement in pancreatic tissue of mice with acute pancreatitis; Figure 4 C: Results of ATP level measurement in pancreatic tissue of SIRT5- / - mice with acute pancreatitis; Figure 4 D: Co-immunoprecipitation results of SIRT5 and SUCLA2 in wild-type and SUCLA2_K118R point mutant AR42J cells after caerulein stimulation; Figure 4 E and Figure 4 F: The results of determination of SCS activity and ATP level in AR42J cells overexpressing K118R_SUCLA2 and overexpressing SIRT5 and K118R_SUCLA2 after caerulein stimulation; Figure 4 G: Violin plot of significantly differential metabolites in pancreatic tissue of rats with acute pancreatitis with overexpression of SIRT5; Figure 4 H: Violin plot of significantly differential metabolites in pancreatic tissues of rats with acute pancreatitis with SUCLA2 knockdown; Figure 4 I: Venn diagram of differential metabolites between SUCLA2 knockdown group and SIRT5 overexpression group;

[0015] Figure 5 The results of the effect of exogenous intervention on the level of succinyl-CoA in the pancreas of acute pancreatitis in Example 4 are as follows: Figure 5 A: Immunoblotting results of total protein lysine succinylation levels in AR42J cells after caerulein stimulation; Figure 5 B: Western blot results of exogenously added succinyl-CoA or its inhibitor (SP) to regulate the succinylation level of proteins in AR42J cells after caerulein stimulation; Figure 5 C: Histopathological examination and hematoxylin-eosin staining results of pancreatic tissue after exogenous addition of succinyl-CoA or SP; Figure 5 D: Immunohistochemical analysis showed differential expression of succinylation levels in pancreatic tissue; Figure 5 E: Measurement results of plasma amylase activity in mice with acute pancreatitis treated with exogenous intervention; Figure 5 F: Measurement results of plasma lipase activity in mice with acute pancreatitis treated with exogenous intervention; (Note: CAE stands for caerulein; the magnification of pathological section staining is 20 times, and the magnification of immunohistochemistry is 40 times.)

[0016] Figure 6 The results of the endogenous intervention on succinylation in the pancreas of acute pancreatitis in Example 4. Figure 6 A: Immunoblotting results of protein succinylation levels in AR42J cells induced by SUCLA2 knockdown adenovirus; Figure 6 B: Immunoblotting results of protein succinylation levels in SIRT5-overexpressing AR42J cells after caerulein stimulation; Figure 6 C: Histopathological examination results of acute pancreatitis in SIRT5 overexpression and SUCLA2 knockdown rats and SIRT5- / - mice in vivo; Figure 6 D: Serum amylase activity measurement results of rats with acute pancreatitis with SUCLA2 knockdown or SIRT5 overexpression; Figure 6 E: Serum lipase activity measurement results of rats with acute pancreatitis with SUCLA2 knockdown or SIRT5 overexpression; Figure 6 F: Histopathological examination results of pancreas of SIRT5- / - mice with acute pancreatitis. Figure 6 G and Figure 6 H: Histochemical results of the difference in the expression of myeloperoxidase and protein succinylation in pancreatic tissues of wild-type and SIRT5- / - mice with acute pancreatitis; Figure 6 I: Immunohistochemical results of the difference in SIRT5 expression in pancreatic tissues between wild-type and SIRT5- / - mice with acute pancreatitis; Figure 6 J and Figure 6K: Plasma biochemical assay results of wild-type and SIRT5- / - mice with acute pancreatitis; (Note: CAE stands for caerulein, TAU stands for sodium taurocholate; the magnification of pathological section staining is 20 times, and the magnification of immunohistochemistry is 40 times.)

[0017] Figure 7 The results of the analysis of the involvement of SIRT5-SUCLA2-mediated succinylation level in the regulation of mitochondrial damage in pancreatic acinar cells in acute pancreatitis in Example 5. Figure 7 A: Mass spectrometry analysis results of succinylation modification after 6 hours of caerulein stimulation; Figure 7 B: Cellular subdistribution results of highly succinylated proteins; Figure 7 C: Results of the main enriched categories of highly succinylated proteins in biological processes, cellular components, and molecular functions; Figure 7 D: Enrichment results of highly succinylated proteins in KEGG pathways; Figure 7 E: Immunofluorescence results of mitochondrial membrane potential of AR42J cells after caerulein stimulation treated with succinyl-CoA or SP; Figure 7 F: Immunofluorescence results of ROS levels in AR42J cells after caerulein stimulation treated with succinyl-CoA or SP; Figure 7 G: Immunofluorescence results of succinylation level, mitochondrial membrane potential and ROS level in AR42J cells after caerulein stimulation with SIRT5 and K118R SUCLA2 overexpression (magnification of JC1 and protein succinylation staining is 100 times, and magnification of ROS is 10 times);

[0018] Figure 8 The results of the analysis of SIRT5-SUCLA2-mediated pancreatic acinar cell mtDNA release regulating the inflammatory response of acute pancreatitis in Example 5. Figure 8 A: Immunofluorescence results of mtDNA distribution in AR42J cells overexpressing SIRT5 and K118R_SUCLA2 after caerulein stimulation; Figure 8 B: Quantitative analysis results of AR42J cytoplasmic mtDNA levels; Figure 8 C: RT-PCR results of cGAS mRNA level after AR42J cells were co-cultured with NR8383 cells; Figure 8 D: RT-PCR results of STING mRNA level after AR42J cells were co-cultured with NR8383 cells; Figure 8 E: RT-PCR results of IRF3 mRNA levels after AR42J cells were co-cultured with NR8383 cells; Figure 8 F: RT-PCR results of IFN-β mRNA level after AR42J cells were co-cultured with NR8383 cells; Figure 8G: ELISA results of the effect of exogenous intervention on the IFN-β level of AR42J cells after caerulein stimulation; Figure 8 H: Immunohistochemical results of the expression of cGAS, IRF3, and STING in the pancreas of mice with acute pancreatitis;

[0019] Fig. 9 The results of the analysis in Example 3 show that SIRT5-SUCLA2 may mediate the activity regulation of complex III in the electron transport chain (ETC) by regulating the succinylation level of CYC1. Fig. 9 A: Interaction network diagram of succinylated proteins; Fig. 9 B: MCC method screening results of succinylated proteins; Fig. 9 C: Mass spectrometry identification of CYC1 succinylation sites in AR42J cells after caerulein stimulation; Fig. 9 D: Diagram of the protein network interacting with CYC1; Fig. 9 E: Immunoprecipitation results of succinylation level of CYC1 protein; Fig. 9 F: Results of mitochondrial complex III activity assay in AR42J cells simultaneously overexpressing SIRT5 and K118R_SUCLA2 after stimulation with caerulein. DETAILED DESCRIPTION

[0020] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0021] Succinylation is a post-translational modification that covalently binds succinyl groups to lysine residues in proteins. It can significantly change the charge and three-dimensional structure of proteins by adding negatively charged macromolecular groups, thereby affecting their physicochemical properties and functions. Recent studies have shown that succinylation plays a role in regulating metabolism, gene expression, cell cycle and other processes, and is associated with the occurrence and development of a variety of diseases, including various cancers and metabolic and neurodegenerative diseases. However, the function of succinylation in acute pancreatitis is still unclear.

[0022] SIRT5 is an NAD+-dependent desuccinylase that can remove succinyl groups from proteins and plays an important role in regulating cell metabolism and maintaining homeostasis. By regulating the succinylation modification of proteins, SIRT5 can regulate protein activity, interaction and localization, and affect processes such as cell metabolism, inflammation and apoptosis. At present, the relationship between SIRT5, succinylation modification and metabolic reprogramming in acute pancreatitis has not been fully studied.

[0023] Succinyl-CoA synthase (SCS) consists of a conserved α subunit (SUCLG1) and a β subunit (SUCLA2 or SUCLG2) that determines nucleotide specificity, catalyzing the reaction of succinate with coenzyme A to produce succinyl-CoA. Changes in any subunit of SCS may affect the level of succinyl-CoA-succinate in the TCA cycle. Succinyl-CoA is a substrate for protein succinylation, and succinate plays an important role in inflammation, hypoxia, and metabolic signaling. Cytochrome c1 (Cyc1) is a component of complex III of the electron transport chain and maintains electron transport function.

[0024] In the present invention, the applicant explored the relationship between the imbalance of succinyl-CoA-succinate levels in the TCA cycle and mitochondrial damage in pancreatic acinar cells, as well as its effect on secondary systemic inflammation and multi-organ dysfunction, and then proposed the application of succinylation modification as an intervention and treatment target for acute pancreatitis, and the succinylation modification is the succinylation modification of SUCLA2 and Cyc1. The succinylation modification level of SUCLA2 is upregulated in the experimental model of acute pancreatitis, and the succinylation modification sites upregulated by SUCLA2 include the K118 site. The above-mentioned succinylation is regulated by SIRT5 and succinyl-CoA. The succinylation modification regulated by SIRT5 includes the succinylation modification of SUCLA2, and the succinylation modification regulated by succinyl-CoA includes the succinylation modification of Cyc1. The above-mentioned SUCLA2 succinylation modification participates in the "SIRT5-SUCLA2 succinylation-succinyl-CoA accumulation" positive feedback loop.

[0025] Specifically, this application includes the following:

[0026] The first aspect of the present invention provides the use of SUCLA2 with succinylated lysine at position 118 and succinylated Cyc1 in the preparation of a diagnostic product for acute pancreatitis.

[0027] The second aspect of the present invention provides the use of SUCLA2 with succinylated lysine at position 118 and succinylated Cyc1 as targets for treating acute pancreatitis, wherein the targets are drug targets.

[0028] The third aspect of the present invention provides a product, the active ingredients of which include SUCLA2 modified by succinylation at lysine 118 and Cyc1 modified by succinylation; the product has at least one of the following functions: (1) predicting the risk of occurrence and progression of pancreatitis; (2) serving as a drug target for the treatment of pancreatitis.

[0029] In the present invention, by using these specific succinylation modifications as biomarkers, products for the diagnosis of acute pancreatitis are developed, thereby improving the accuracy and timeliness of disease detection. In addition, these succinylation modifications are also identified as potential targets for the treatment of acute pancreatitis, opening up a new way for targeted intervention. This provides a new solution for precision medicine, and it is expected that targeted drugs can be used to more effectively treat acute pancreatitis, improve the cure rate of patients and reduce the occurrence of complications. In short, the present invention provides innovative strategies and technical support for the diagnosis and treatment of acute pancreatitis.

[0030] The present invention is further described in detail below with reference to specific embodiments.

[0031] The sources of materials used in the following examples are as follows:

[0032] Animal model: C57BL / 6 mice and male Sprague-Dawley rats were obtained from the Department of Animal Experimentation, School of Medicine, Shanghai Jiao Tong University; C57BL / 6 and SIRT5 KO mice were obtained from Shanghai Model Organisms Technology Co., Ltd.

[0033] Antibodies and reagents: Rabbit anti-succinyl lysine polyclonal antibody was purchased from Jingjie Biotechnology Co., Ltd.; rabbit anti-SUCLG1 polyclonal antibody, rabbit anti-SUCLG1 polyclonal antibody, rabbit anti-SUCLG2 polyclonal antibody, mouse anti-CPT1A monoclonal antibody, mouse anti-ACTIN monoclonal antibody, mouse anti-TUBULIN polyclonal antibody, mouse anti-SIRT7 polyclonal antibody, CoraLite488-labeled goat anti-mouse IgG (H+L), and CoraLite594-labeled goat anti-rabbit IgG (H+L) were purchased from Proteintech; mouse anti-SIRT5 monoclonal antibody was purchased from Santa Cruz; HRP-conjugated β-Tubulin mouse monoclonal antibody was purchased from Abcam; mouse anti-dsDNA monoclonal antibody was purchased from Novus; succinylphosphonate (SP), caerulein, and sodium taurocholate were purchased from MedChemExpress; succinyl-CoA sodium salt was purchased from Santa Cruz Company; linear polyethyleneimine MW25000 was purchased from Polysciences Company; HPLC grade acetonitrile, methanol, and formic acid were purchased from Merck Company, Germany; ultrapure water was purchased from Millipore Corporation, USA; SUCLA2 wild-type and mutant plasmids were purchased from VectorBuilder Company; adenovirus expressing SIRT5, SUCLA2 shRNA and corresponding controls were purchased from Heyuan Biotechnology Company; SUCLA2 and K118R_SUCLA3 related lentiviruses were purchased from Shanghai Gene Medical Technology Co., Ltd.

[0034] The present invention is further explained below by describing in detail the construction of a cell model in acute pancreatitis, the detection and analysis of metabolomics, the exploration and verification of the "SIRT5-SUCLA2 succinylation-succinyl-CoA accumulation" positive feedback loop and its effect on mitochondrial dysfunction.

[0035] Example 1 Construction of acute pancreatitis model and metabolomics analysis

[0036] This embodiment specifically includes the following steps:

[0037] 1. Construction of Acute Pancreatitis Model

[0038] 1.1 Construction of acute pancreatitis cell model

[0039] 1.1.1 Treatment with caerulein

[0040] AR42J cells were selected and cultured in F12K medium (Gibco, 21127030) containing 20% ​​fetal bovine serum (Gibco, 10099141C) and 1% penicillin-streptomycin mixture at 37°C and 5% CO2. After ensuring that AR42J cells were in good growth state, normal density and morphology, they were stimulated with 100 nM caerulein, and the appropriate treatment time was selected according to the purpose of the experiment. Finally, the cell samples were collected to obtain the acute pancreatitis cell model.

[0041] 1.1.2 Biochemical detection of amylase and lipase

[0042] The supernatants of AR42J cell samples were collected at 3h, 6h, 12h, and 24h after caerulein stimulation, and the amylase and lipase activities of the cell sample supernatants were determined using a colorimetric assay kit (Nanjing Jiancheng, C016-1-1 and A054-1-1). The results are shown in Figure 1 A and Figure 1 As shown in B (AR42J supermatant: AR42J cell supernatant; Amylase: amylase; Lipase: lipase; control: control group; CEA: caerulein.). Figure 1 A and Figure 1 In B, after caerulein stimulation, the amylase and lipase activities of AR42J cells began to increase after 3 hours and reached a peak at 6 hours. The activities decreased after 24 hours, which verified the effectiveness of the acute pancreatitis cell model constructed in step 1.1 and selected 6 hours as the commonly used caerulein treatment time.

[0043] 1.2 Construction of rat model of acute pancreatitis

[0044] 1.2.1 Retrograde injection into the pancreaticobiliary duct

[0045] The acute pancreatitis rat model was established by retrograde injection of 3% sodium taurocholate solution into the pancreaticobiliary duct. 8-week-old SD rats (weight about 300g, half male and half female) were selected and anesthetized with 2.5% tribromoethanol (100μL / 10g body weight), and then the sodium taurocholate solution was slowly and evenly injected into the pancreaticobiliary duct at a dose of 1mL / kg body weight. The entire injection process was completed within 5-10 minutes. The sham operation group rat model was established by retrograde injection of normal saline into the pancreaticobiliary duct.

[0046] 2. Metabolomics analysis of acute pancreatitis model

[0047] 2.1 Sample preparation

[0048] AR42J cell samples stimulated by caerulein for 6 hours and rat pancreatic tissue samples after pancreaticobiliary retrograde injection surgery were obtained by step 1.1, as well as AR42J cell samples not stimulated by caerulein and pancreatic samples of sham-operated rats for experimental control. For cell samples, 100 μL ultrapure water was added to the cell pellet to resuspend it, 50 μL of the resuspended solution was taken, added to 200 μL pre-cooled pure methanol, and then vortexed at 2500 r / min for 2 minutes; for tissue samples, 50 mg ± 2.5 mg of the sample was placed in a 2mL centrifuge tube, 500 μL of pre-cooled 70% methanol aqueous solution was added, and vortexed for 3 minutes. Both samples were centrifuged at 12000 r / min for 10 minutes at 4°C, and 300 μL of supernatant was collected. After standing at 4°C for 30 minutes, they were centrifuged again at 12000 r / min for 10 minutes, and the supernatant was collected to obtain the protein extraction solution. The protein concentration of the supernatant was determined using the BCA kit to ensure that the protein concentration of each experimental group was consistent.

[0049] 2.2 Chromatography-mass spectrometry analysis conditions

[0050] Use the protein extract sample obtained in step 2.1 for chromatography-mass spectrometry analysis.

[0051] 2.2.1UPLC conditions

[0052] Chromatographic column: ACQUITY UPLC BEH Amide column (1.7 μm, 100 mm×2.1 mm inner diameter).

[0053] Mobile phase: Phase A is ultrapure water (containing 10 mM ammonium acetate and 0.3% ammonia water), and phase B is 90% acetonitrile / water (volume ratio). Elution conditions: Flow rate is set at 0.40 mL / min, column temperature is controlled at 40°C, and sample injection volume is 2 μL.

[0054] Gradient elution program: initial A / B was 5:95, linearly changed to 30:70, then changed to 50:50, and finally returned to 5:95.

[0055] 2.2.2MS and mass spectrometry analysis conditions

[0056] Ion source: electrospray ionization source, temperature 550℃.

[0057] Scan mode: positive ion mode voltage is 5500 V, negative ion mode voltage is -4500 V.

[0058] Air curtain pressure: 35psi.

[0059] 2.3 Bioinformatics analysis

[0060] 2.3.1 Principal Component Analysis (PCA)

[0061] Using the PCA method, the original mass spectrometry data obtained in step 2.2 is compressed into n principal components to describe the characteristics of the original data set, and a two-dimensional score graph of PC1 and PC2 is drawn.

[0062] The PCA analysis results of the caerulein-treated group and the control group are shown in Figure 1 As shown in C (CON: control group; CAE: caerulein; PC1: the most prominent feature in the multidimensional data matrix; PC2: the most important feature in the data matrix except for the content captured by PC1). Figure 1 In C, PCA analysis showed significant distribution differences between the control group and the caerulein-treated group on PC1 and PC2, which explained 63.6% and 22.15% of the data variation, respectively.

[0063] 2.3.2 Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA)

[0064] The OPLS-DA analysis was performed using the MrtaboAnalystR package in the R language. The model was evaluated using 7 cross-validation steps and the following three key parameters were calculated:

[0065] R 2 X: the explanation rate of the model for the X variable, which represents the degree of fit of the model to the overall variation of metabolomics data;

[0066] R 2 Y: The model’s explanation rate for the Y variable, reflecting the model’s ability to fit sample classification information;

[0067] Q 2 : The prediction ability index of the model obtained through cross-validation, Q 2 >0.5 indicates that the model has reliable predictive power.

[0068] The OPLS-DA model was subjected to 200 random permutation tests to assess the statistical significance of the model. 2 Y and Q 2The p value was calculated by comparing the values ​​with the corresponding values ​​of the random permutation model. p < 0.05 indicated that there were metabolic differences among the groups.

[0069] The OPLS-DA scores of the caerulein-treated group and the control group are shown in the figure Figure 1 As shown in Figure D (CON: control group; CAE: caerulein; Component1: first predicted component; Component2: first orthogonal component). In Figure D, there are significant differences in metabolic characteristics between the two groups, of which the first predicted component explains 58.7% of the variability and the first orthogonal component explains 30.2% of the variability, which further confirms the significant effect of caerulein treatment on AR42J cell metabolism.

[0070] The S-plot of OPLS-DA integrates the covariance and correlation information between the metabolites and the first predicted component, and intuitively shows the contribution of the metabolites to the discrimination. The metabolites at both ends of the S-plot contribute the most to the discrimination between groups and are potential differential metabolites. The variable projection importance (VIP) value of each metabolite is further calculated, and metabolites with VIP>1 are screened as candidate differential metabolites. A VIP_log2FC value graph of differential metabolites is plotted, where the horizontal axis is the logarithm of the difference factor (FC) of the metabolite with base 2, and the vertical axis is the VIP value.

[0071] Volcano plot of differential metabolites in AR42J cells after caerulein stimulation Figure 1 E (statistics: statistical results; Insignificant: not significant; Down: down-regulated; Up: up-regulated; VIP: variable projection importance; Log2FC: the logarithm of the difference fold of metabolites with base 2.). Figure 1 In E, volcano plot analysis showed that compared with the control group, the levels of 8 differential metabolites were increased and the levels of 27 differential metabolites were decreased in AR42J cells after caerulein stimulation.

[0072] VIP_FC scores of differential metabolites in AR42J cells after caerulein stimulation Figure 1F (Type: Category; Up: Up-regulated; Down: Down-regulated; VIP: Variable projection importance; GTP: Guanosine triphosphate; Isocitrate: Isocitrate; D-Glucose-6-phosphate: D-Glucose-6-phosphate; D-Fructose-1,6-bisphosphate: D-Fructose-1,6-bisphosphate; Succinyl CoA: Succinyl Coenzyme A; L-Glutamate: L-Glutamate; Succinate: Succinate; cAMP: Cyclic adenosine monophosphate; ADP: Adenosine diphosphate; GDP: Guanosine diphosphate; NADP: Nicotinamide adenine dinucleotide phosphate; Pyruvate: Pyruvate; NAD: Oxidized nicotinamide adenine dinucleotide; AMP: Adenosine monophosphate; UDPglucose: Guanosine diphosphate glucose; Log2FC: The logarithm of the difference fold of the metabolite with the base 2.). Figure 1 In F, the VIP_FC score plot shows that among the up-regulated metabolites, GDP, glucose, and succinyl-CoA have higher VIP scores, while among the down-regulated metabolites, ethanolamine phosphate, lactate, L-cysteine, and succinate have higher VIP scores. These metabolites may be potential biomarkers to explore the possible mechanisms of metabolic changes in pancreatic acinar cells in acute pancreatitis.

[0073] 2.3.3 Annotation and functional enrichment

[0074] Genes and gene products were annotated using the gene ontology framework, and proteins were classified according to biological processes, cellular components, and molecular functions. Protein domains were annotated using InterProScan, and functional descriptions were obtained using the InterPro domain database. Protein pathways were annotated using KAAS according to the KEGG database, and the results were mapped to the KEGG pathway database using the KEGG mapper. Wolfpsort was used to predict the subcellular localization of eukaryotic sequences. A two-sided Fisher's exact test was used to determine the significant enrichment of the gene ontology framework, protein domains, and KEGG pathways. A heat map was used to perform hierarchical clustering of the functional classification of differentially expressed proteins. Protein-protein interactions were analyzed using the STRING database, and the R package "networkD3" was used to draw a visual interaction network. The KEGG enrichment of metabolites in different metabolic pathways is shown in Figure 2. Figure 2A (P-value: P value; Count: count; Biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid: biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid; Biosynthesis of alkaloids derived from shikimate pathway: biosynthesis of shikimate alkaloids; Phenylalanine metabolism: phenylalanine metabolism; Butanoate metabolism: butanoate metabolism; Citrate cycle (TCA cycle): citric acid cycle (TCA cycle); Carbon fixation pathways in prokaryotes: carbon fixation pathways in prokaryotes; Biosynthesis ofalkaloids derived from terpenoid and polyketide: biosynthesis of alkaloids derived from terpenoid and polyketide; Biosynthesis of phenylpropanoids: biosynthesis of phenylpropanoids; Pyruvate metabolism: pyruvate metabolism; Degradation ofaromatic compounds: degradation of aromatic compounds; Arginine biosynthesis: arginine biosynthesis; Tyrosine metabolism: tyrosine metabolism; proximal tubule bicarbonate reclamation: proximal tubule bicarbonate reabsorption; propanoate metabolism: propionate metabolism; arginine and proline metabolism: arginine and proline metabolism; glyoxylate and dicarboxylate metabolism: glyoxylate and dicarboxylate metabolism; biosynthesis of various antibiotics: biosynthesis of various antibiotics; glutathione metabolism: glutathione metabolism; ferroptosis: iron death; benzoate degradation: benzoate degradation; rich factor: enrichment degree. ). Figure 2 In A, the KEGG enrichment analysis results emphasized that the differential metabolites were involved in TCA cycle-related protein pathways.

[0075] 2.3.4 Analysis of Differential Metabolites in TCA Cycle

[0076] According to the conclusions obtained in step 2.3.3, the metabolomics results involving the TCA cycle obtained in step 2.3.2 were sorted and analyzed. The TCA cycle metabolic pathway diagram and the log2FC and VIP values ​​of key metabolites are shown in the figure below: Figure 2 B and Figure 2 C (Krebs Cycle: tricarboxylic acid cycle; Acetyl CoA: acetyl coenzyme A; Citric acid: citric acid; Isocitric acid: isocitrate; CO2: carbon dioxide; NAD+: oxidized nicotinamide adenine dinucleotide; NADH: reduced nicotinamide adenine dinucleotide; α-Ketoglutaric acid: α-ketoglutaric acid; Succinyl CoA: succinyl coenzyme A; ADP: adenosine diphosphate; GDP: guanosine diphosphate; ATP: adenosine triphosphate; GTP: guanosine triphosphate; Succinate: succinate; FAD: oxidized flavin adenine dinucleotide; FADH2: reduced flavin adenine dinucleotide; Fumarate: fumarate; H2O: water; Malate: malic acid; Oxaloacetate: oxaloacetate; Log2FC: logarithm of the difference fold of metabolites with base 2; VIP: variable projection importance.). Figure 2 B and Figure 2 In C, the levels of various TCA cycle metabolites change in acute pancreatitis, and two numerical values ​​are annotated next to each metabolite, indicating the Log2FC value and VIP value of the experimental group relative to the control group (NA indicates no value).

[0077] Draw a Venn diagram of differential metabolites of TCA cycle in acute pancreatitis cell and rat models, such as Figure 2 D (AR42J with cearulein: AR42J cells treated with cearulein; AP Rats: rats with acute pancreatitis; Succinateacid: succinate; Succinyl-CoA: succinyl-CoA.). Figure 2 In D, succinyl-CoA and succinate are common TCA cycle differential metabolites in cellular and rat pancreas models of acute pancreatitis.

[0078] Among them, the level of succinyl-CoA increased while the level of succinate decreased, which suggested that the abnormal catalytic reaction of succinyl-CoA synthetase may lead to the accumulation of succinyl-CoA, and the imbalance of succinyl-CoA-succinate levels in the TCA cycle may be involved in the pathogenesis of acute pancreatitis.

[0079] Example 2 Effect of SUCLA2 hypersuccinylation on SCS (succinyl-CoA synthase) in acute pancreatitis

[0080] This embodiment specifically includes the following steps:

[0081] 1. To investigate the changes in SCS function in acute pancreatitis

[0082] 1.1 Detection of SCS activity

[0083] To investigate the potential mechanism of TCA cycle dysregulation in acute pancreatitis, we first explored the changes in SCS function. Using the succinyl-CoA synthetase activity colorimetric assay kit (Sigma, MAK217), the absorbance at 450 nm was measured, and the colorimetric product, which was proportional to the enzyme activity present, was used to reflect the total SCS activity. The results of the SCS activity assay in pancreatic tissue of rats with acute pancreatitis are shown in Figure 2. Figure 3 As shown in A (AP Rats pancreas: acute pancreatitis rat pancreas; SCS activity: succinyl-CoA synthase activity; Sham: sham operation group; AP: acute pancreatitis group.). Figure 3 In A, SCS activity in pancreatic tissue of rats with acute pancreatitis was significantly decreased.

[0084] 1.2 Detection of SCS expression

[0085] 1.2.1 Protein sample preparation

[0086] Use 100 μl of RIPA lysis buffer containing protease and phosphatase inhibitors to treat the cell sample stimulated by caerulein for 6 hours obtained in step 1.1.1, vortex thoroughly to mix, and then use an ultrasonic crusher in an ice water bath to break the cells, and then continue cold lysis for 30 minutes, and finally centrifuge at 4°C and 14500r / min for 15 minutes. The supernatant is the protein sample to be tested. Use the BCA kit to detect the protein concentration of the protein sample, which is used to calculate the sample volume required for the immunoblotting loading concentration (100nmol / L).

[0087] 1.2.2 Immunoblotting

[0088] Proteins were separated by 10% SDS-PAGE, blocked with 5% skim milk (Epizyme, PS112L) after transfer, and then incubated with primary antibodies (Anti-SUCLA2, Anti-SUCLG1 or Anti-SUCLG2) and secondary antibodies in sequence. The samples were developed using Tanon ECL detection reagent (Tanon, 180-5001) and quantified using the Tanon 5200 chemiluminescence imaging system.

[0089] The immunoblot results of SUCLG1, SUCLA2 and SUCLG2 proteins in AR42J cells after 6 h of caerulein stimulation are shown in Figure 2. Figure 3As shown in B (TUBULIN: tubulin; SUCLG1: α subunit of succinyl-CoA synthase; SUCLA2: ADP-specific β subunit of succinyl-CoA synthase; SUCLG2: GDP-specific β subunit of succinyl-CoA synthase; CON: control group; CAE: caerulein-treated group; kDa: kilodaltons.). Figure 3 In B, the protein expression levels of SCS subunits (SUCLA2, SUCLG1, and SUCLG2) did not change significantly in the acute pancreatitis cell model.

[0090] 1.3 Detection of the activity of SCS subunits

[0091] In order to distinguish the difference in activity between A-SCS and G-SCS, DTNB (Ellman's reagent) was used to measure the characteristic absorption peak at 412 nm to indirectly determine the catalytic rate of SCS on the substrate.

[0092] The results of the A-SCS and G-SCS activity assays of AR42J cells after 6 h of caerulein stimulation are shown in Figure 3 C. Figure 3 As shown in D (AR42J: AR42J cells; A-SCS Activity: ADP-specific succinyl-CoA synthase activity; G-SCS Activity: GDP-specific succinyl-CoA synthase activity; Units / min / mg protein: units / minute / microgram protein mass (enzyme activity unit); Control: control group; CAE: caerulein-treated group;). Figure 3 C and Figure 3 In D, the activity of A-SCS (composed of SUCLG1 and SUCLA2) decreased, while the activity of G-SCS (composed of SUCLG1 and SUCLG2) remained unchanged. These results suggest that the specific decrease in A-SCS activity may be the main reason for the overall decrease in SCS activity.

[0093] 2. Exploring the molecular mechanism of reduced A-SCS activity

[0094] 2.1 Succinylomic analysis of AR42J cells after caerulein stimulation

[0095] 2.1.1 Protein extraction

[0096] The AR42J cell sample stimulated by caerulein for 6 hours in Example 1 and the control group AR42J cell sample not stimulated by caerulein were taken from a -80°C refrigerator, thawed and homogenized, and a lysis solution containing 1% SDS and 1% protease inhibitor was added to the cells for ultrasonic lysis, and then centrifuged at 4°C and 12000g for 10 minutes. The obtained supernatant was the protein extraction solution. The protein concentration of the sample was determined using a BCA protein quantification kit to ensure that the protein concentration of each experimental group was consistent.

[0097] 2.1.2 Protein enzymatic hydrolysis

[0098] An equal amount of protein sample was taken from each sample in each group and adjusted to the same volume with the lysis buffer used in step 2.2.1. Subsequently, an equal volume of cold acetone was added to each sample, and after thorough vortex mixing, four times the volume of cold acetone was added and placed at -20°C for precipitation for 2 hours. After precipitation, the supernatant was removed by centrifugation at 4500g for 5 minutes, and the precipitate was washed twice with cold acetone. After the precipitate was dried, it was redissolved with 200mM tetraethylammonium bromide (TEAB) and dispersed by ultrasonic treatment. Subsequently, trypsin was added for overnight enzymatic digestion at a ratio of 1:50. Dithiothreitol was added to the enzymatic digestion solution to a final concentration of 5mM, and the reduction reaction was carried out at 56°C for 30 minutes. Finally, iodoacetamide was added to the sample to a final concentration of 11mM and incubated at room temperature in the dark for 15 minutes to achieve acetylenic etherification of the peptide segment.

[0099] 2.1.3 TMT mass spectrometry labeling

[0100] Dissolve the peptides obtained in step 2.1.2 in 0.5M TEAB and label each sample with the corresponding TMT reagent according to the experimental protocol of ThermoFisher Scientific. Incubate at room temperature for 2 hours. Take 5μl of each sample for desalting and then perform mass spectrometry analysis to check the TMT labeling efficiency. After confirming that the labeling is correct, use 5% hydroxylamine to terminate the reaction, use Strata X C18 SPE column for desalting, and then vacuum centrifuge and dry to obtain the required TMT mass spectrometry labeled sample.

[0101] 2.1.4 Enrichment of succinylation modification

[0102] The peptide sample obtained in step 2.1.3 was dissolved in a special immunoprecipitation buffer (containing 100nM NaCl, 1nM EDTA, 50mM Tris-HCl, 0.5% NP-40, and the pH was adjusted to 8.0), and the peptide was bound to the succinylation-specific antibody resin. Then, an eluent containing 0.1% trifluoroacetic acid was used, and the eluent was collected and vacuum freeze-dried. After desalting, the desired succinylation-modified enriched peptide sample was obtained.

[0103] 2.1.5 High performance liquid chromatography-mass spectrometry analysis

[0104] The succinylated peptide obtained in step 2.1.5 was dissolved in an aqueous solution containing 0.1% formic acid and 2% acetonitrile (mobile phase A) and finely separated using a NanoElute ultra-high performance liquid chromatography system. The mobile phase B used for separation was a solution containing 0.1% formic acid and 100% acetonitrile. The liquid phase gradient was set as follows: from 0 to 40 minutes, phase B increased from 6% to 22%; from 40 to 52 minutes, phase B increased from 22% to 30%; from 52 to 56 minutes, phase B increased from 30% to 80%; from 56 to 60 minutes, phase B was maintained at 80%, and the flow rate was controlled at 450nL / min. The peptides separated by the ultra-high performance liquid chromatography system were then ionized in the capillary ion source and subsequently transported to the timsTOF Pro2 mass spectrometer for in-depth analysis. The ion source voltage was set to 1.65 kV, and at the same time, the original ions and fragment ions of the peptides were detected by high-resolution time-of-flight mass spectrometry. The scanning range of the secondary mass spectrometer was 100 to 1700 m / z. The data acquisition strategy adopted the parallel accumulation serial fragmentation PASEF mode. After a full scan, 10 rounds of PASEF scans were performed on the parent ions with charge states between 0 and 5 to obtain their secondary mass spectrum information. To prevent repeated acquisition of parent ions, the dynamic exclusion time of the tandem mass spectrometer scan was set to 24 seconds.

[0105] 2.1.6 Data search and analysis of mass spectrometry data

[0106] The "Rattus_norvegicus_10116_PR_20210721.fasta" database (containing 29934 sequences) was used to perform a matching search analysis on the mass spectrometry analysis results, and a reverse database was used to estimate the false positive rate, and a common pollutant database was used to exclude pollutants in the identification results. The enzyme cutting rule is Trypsin / P, allowing up to 4 missed cutting sites, the minimum length of the peptide segment is set to 7 amino acids, and the maximum number of modifications is limited to 5. The mass tolerance of the primary parent ion and the secondary fragment ion is 20ppm, and the succinylation modification of lysine is specially marked. The false positive rate of protein and PSM identification is controlled within 1%.

[0107] 2.1.7 Quantitative analysis

[0108] According to the search results, the signal intensity of each peptide in different samples is used to calculate the relative quantitative value of the modification site. The relative quantitative value of the modified peptide is obtained by centralizing the signal intensity in different samples. The calculation formula is: Rij = Iij / Mean(Ij), where i represents the sample, represents the peptide, I represents the signal intensity value, and R represents the relative quantitative value. Further, the relative quantitative value of the modification site is divided by the relative quantitative value of the corresponding protein to eliminate the effect of protein expression changes on the modification expression. The comparison samples are selected, and the ratio of the relative quantitative values ​​of the modification sites between samples is used as the difference change FC. For example, the calculation formula for the difference multiple of the modification sites between sample A and sample B is: FCA / B, k = RAk / RBk, where R represents the relative quantitative value of the modification site, and k represents the modification site. The changes in the modification regulation level of the A-SCS subunit SUCLA2 were analyzed by mass spectrometry. In the acute pancreatitis cell model, 6 lysine residues on the SUCLA2 protein underwent high levels of succinylation modification, and the results are shown in Table 1.

[0109] Table 1 Changes in SUCLA2 succinylation level in AR42J cells after 6 hours of caerulein stimulation

[0110] Location Amino Acids AR42J_T / AR42J_CRatio Changes in succinylation levels 118 Lysine 6.904 Increase 110 Lysine 3.95 Increase 226 Lysine 2.923 Increase 90 Lysine 2.867 Increase 370 Lysine 1.699 Increase 217 Lysine 1.523 Increase

[0111] 2.2 Detection of SCS activity and ATP synthesis level in K118R_SUCLA2-overexpressing cells

[0112] 2.2.1 Plasmid transfection

[0113] In order to study the correlation between succinylation modification of the above sites and SCS activity, SUCLA2 point mutation plasmids were first constructed, and the six lysine residues in Table 1 were mutated to arginine to simulate the state without succinylation modification. The rat plasmids were constructed by Guangzhou Yunzhou Biotechnology and include:

[0114] (1)Prp[Exp]-EGFP / Puro-CMV-SUCLA2-HA

[0115] (2)Prp[Exp]-EGFP / Puro-CMV-SUCLA2(K90R)-HA

[0116] (3)Prp[Exp]-EGFP / Puro-CMV-SUCLA2(K110R)-HA

[0117] (4)Prp[Exp]-EGFP / Puro-CMV-SUCLA2(K118R)-HA

[0118] (5)Prp[Exp]-EGFP / Puro-CMV-SUCLA2(K217R)-HA

[0119] (6)Prp[Exp]-EGFP / Puro-CMV-SUCLA2(K226R)-HA

[0120] (7)Prp[Exp]-EGFP / Puro-CMV-SUCLA2(K370R)-HA

[0121] One day before transfection, AR42J cells were seeded onto culture plates and transfected using polyethylenimine (PEI) plasmid transfection reagent with a PEI:DNA mass ratio of 3:1 or higher.

[0122] 2.2.2 The same operation as in "1.1 Detection of SCS activity in Example 2" was performed. The results were as follows: Figure 3 E (AR42J: AR42J cells; A-SCS Activity: ADP-specific succinyl-CoA synthase activity; Control: control group; CAE: caerulein-treated group; SUCLA2 OE CAE: AR42J cells overexpressing wild-type SUCLA2 after caerulein stimulation; K90R SUCLA2 OE CAE: AR42J cells overexpressing SUCLA2_K90R after caerulein stimulation; K110R SUCLA2 OE CAE: AR42J cells overexpressing SUCLA2_K110R after caerulein stimulation; K118R SUCLA2 OE CAE: AR42J cells overexpressing SUCLA2_K118R after caerulein stimulation; K217R SUCLA2 OE CAE: AR42J cells overexpressing SUCLA2_K217R after caerulein stimulation; K226R SUCLA2 OE CAE: AR42J cells overexpressing SUCLA2_K226R mutant after stimulation with caerulein; K370R SUCLA2 OE CAE: AR42J cells overexpressing SUCLA2_K370R mutant after stimulation with caerulein;). Figure 3 In E, SCS activity of AR42J cells overexpressing K118R_SUCLA2 was higher after caerulein stimulation compared with the wild type and other mutants.

[0123] 2.2.3 After transfection of SUCLA2 point mutation plasmid, ATP level was detected using ATP assay kit (Beyotime, S0026). Figure 3As shown in F (AR42J: AR42J cells; ATP Luminescence RLU: ATP relative luminescence unit; Control: control group; CAE: caerulein treatment group; SUCLA2-OE CAE: AR42J cells overexpressing wild-type SUCLA2 after caerulein stimulation; K118R-SUCLA2-OE CAE: AR42J cells overexpressing mutant SUCLA2_K118R after caerulein stimulation;). Figure 3 In F, in AR42J cells after caerulein stimulation, ATP levels were increased in cells overexpressing K118R_SUCLA2 compared with those in cells overexpressing wild type.

[0124] Taken together, the high succinylation modification of SUCLA2 at the K118 site significantly inhibited the SCS activity and ATP synthesis function of pancreatic acinar cells in acute pancreatitis, suggesting that SUCLA2 succinylation may regulate mitochondrial metabolism in acute pancreatitis.

[0125] Example 3 Effect of SIRT5 on SUCLA2 hypersuccinylation and succinyl-CoA in acute pancreatitis This example specifically includes the following steps:

[0126] 1. Study the role of succinylation-modifying enzymes in SUCLA2 succinylation

[0127] The changes in protein expression levels of several succinylation-modifying enzymes in AR42J cells after 6 hours of caerulein stimulation were studied. The same immunoblotting operation as in "1.2 Detection of SCS expression in Example 2" was performed. The primary antibody selected was Anti-CPT1A, Anti-SIRT5, Anti-KAT2A, Anti-TUBULIN or Anti-SIRT7. The results are as follows: Figure 4 As shown in A (CPT1A: carnitine palmitoyltransferase 1A; SIRT5: sirtuin 5; KAT2A: lysine acetyltransferase 2A; TUBULIN: tubulin; SIRT7: sirtuin 7; CON: control group; CAE: caerulein-treated group; kDa: kilodaltons). Figure 4 In A, the SIRT5 protein level of AR42J cells decreased after caerulein stimulation, while the CPT1A, KAT2A, and SIRT7 protein levels showed no significant changes, suggesting that SIRT5 may play a role in the occurrence and development of acute pancreatitis.

[0128] 2. Study the role of SIRT5 in SCS activity in acute pancreatitis

[0129] 2.1 Construction of SIRT5- / - acute pancreatitis mouse model

[0130] Using CRISPR / Cas9 technology, a frameshift mutation was introduced into the mouse SIRT5 gene through a non-homologous recombination repair pathway (as shown in Table 2). The mutation was a 1724 bp deletion, resulting in the production of a truncated non-functional protein, and the mutant transcribed mRNA may undergo nonsense-mediated degradation, ultimately leading to the loss of SIRT5 gene function.

[0131] Table 2 Sequencing sequences of SIRT5 gene in wild-type and SIRT5 knockout mice

[0132]

[0133] Eight-week-old SIRT5 knockout mice (weighing approximately 20 g) were selected and intraperitoneally injected with caerulein (50 μg / kg) once every 1 hour for 7 consecutive times to simulate the action of cholecystokinin to construct an animal model of acute pancreatitis.

[0134] 2.2 Detection of SCS activity and ATP levels in pancreatic tissue of SIRT5- / - acute pancreatitis mice

[0135] The same operation as in "Example 21.1 Detection of SCS activity" and "Example 22.2.3" was performed. The results were as follows Figure 4 As shown in B and 4C (APMouse pancreas: pancreatic tissue of acute pancreatitis mouse model; SCS Activity: succinyl-CoA synthase activity; ATP Luminescence RLU: ATP luminescence relative unit; Control: control group; CAE: caerulein-treated group; SIRT5KO: SIRT5 gene knockout mice; SIRT5 KO CAE: SIRT5 gene knockout mice treated with intraperitoneal injection of caerulein;). Figure 4 B and Figure 4 In C, compared with wild-type mice, the SCS activity and ATP levels in the pancreatic tissue of SIRT5- / - mice were lower; under the acute pancreatitis modeling conditions, the decrease in SCS activity and ATP levels in SIRT5- / - was more obvious compared with wild-type mice.

[0136] 3. Study the interaction between SIRT5 and SUCLA2 succinylation at K118

[0137] 3.1 Construction of acute pancreatitis models in AR42J cells overexpressing wild-type SUCLA2 and K118R_SUCLA2

[0138] Two AR42J cell stable strains overexpressing wild-type SUCLA2 and K118R_SUCLA2 were constructed by lentiviral transfection. Rat lentivirus was constructed by Shanghai GeneCare Biotechnology Co., Ltd., including:

[0139] (1)Ubi-MCS-3FLAG-SUCLA2-SV40-puromycin

[0140] (2)Ubi-MCS-3FLAG-SUCLA2(NC)-SV40-puromyci

[0141] (3)Ubi-MCS-3FLAG-SUCLA2(K118R)-SV40-puromycin

[0142] The same operation as in “Example 11.1 Construction of acute pancreatitis cell model” was performed to construct two AR42J cell acute pancreatitis models overexpressing wild-type SUCLA2 and K118R_SUCLA2.

[0143] 3.2 Co-immunoprecipitation

[0144] Collect the two cell samples obtained in step 3.1, use protease / phosphatase inhibitors (Roche, Switzerland) and IP lysis / wash buffer (Biolinkedin, L-7101) to lyse the cells, and collect the supernatant, which is the cell lysate. Mix 2-10μg IP antibody with cell lysate containing 500-1500μg total protein to form an "antibody-antigen" mixture, dilute 300-500μL with IP lysis / wash buffer, and incubate at room temperature for 1-2h or 4℃ for 2-4h. Then add 25μL anti-HA magnetic beads (L-1004) to the "antibody-antigen" mixture and incubate at room temperature for 1-2h or 4℃ for 2-4h. Subsequently, the magnetic beads were separated by a magnetic stand, the supernatant was discarded, the magnetic beads were retained, and the beads were washed with IP lysis / wash buffer to remove non-specific binding. The magnetic beads were then placed in 80-100 μL 1×SDS loading buffer and boiled to denature and elute the target protein. The supernatant was retained for immunoblot analysis, which was the same as the immunoblot operation in “Detection of SCS expression in 1.2 Example 2”. The primary antibody was Anti-SIRT5, Anti-Flag, Anti-TUBULIN or Anti-succinyllysine.

[0145] The results of immunoprecipitation of SIRT5 and SUCLA2 in wild-type and SUCLA2_K118R point mutant AR42J cells after 6 h of caerulein stimulation are shown in Figure 2. Figure 4D (WT-SUCLA2_OE: wild-type overexpressing SUCLA2; K118R-SUCLA2_OE: K118R overexpressing SUCLA2 mutant; CAE: caerulein; INPUT: input; IP: immunoprecipitation; SIRT5: sirtuin 5; Flag: tag protein; TUBULIN: tubulin; SIRT7: sirtuin 7; kDa: kilodaltons.). Figure 4 In D, in AR42J cells after caerulein stimulation, SIRT5 was able to interact with SUCLA2, while the K118R mutation of SUCLA2 weakened its binding ability with SIRT5, suggesting that K118 is the key site for SIRT5 to bind to SUCLA2.

[0146] 3.3 Construction of AR42J cell acute pancreatitis model with overexpression of K118R_SUCLA2 and simultaneous overexpression of SIRT5 and K118R_SUCLA2

[0147] The same operation as in Example 33.1 Construction of AR42J cell acute pancreatitis model overexpressing wild-type SUCLA2 and overexpressing K118R_SUCLA2 was performed, and adenovirus transfection was also used. The adenovirus in this study was completed by Shanghai Heyuan Biotechnology Co., Ltd., including:

[0148] (1)pADV-U6-shRNA(SUCLA2)-CMV-EGFR

[0149] (2) pADV-U6-shRNA(NC)-CMV-EGFR

[0150] (3)pcADV-EF1-mScarlet-CMV-MCS-SIRT5-3Xflag

[0151] (4)pcADV-EF1-mScarlet-CMV-MCS-3xFLAG

[0152] 3.4 Detection of SCS activity and ATP levels

[0153] The same operation as “1.1. Detecting SCS activity in Example 2” and “2.2.3 in Example 2”.

[0154] The results of SCS activity and ATP level determination in AR42J cells overexpressing K118R_SUCLA2 and overexpressing SIRT5 and K118R_SUCLA2 after caerulein stimulation are shown in Figure 4 E and Figure 4As shown in F (AR42J: AR42J cells; SCS Activity: succinyl-CoA synthase activity; ATP Luminescence RLU: ATP relative unit; Control: control group; CAE: caerulein treatment group; SIRT5 OE CAE: mutant AR42J cells overexpressing SIRT5 after caerulein stimulation; K118RSUCLA2 OE CAE: mutant AR42J cells overexpressing SUCLA2_K118R after caerulein stimulation; SIRT5 OEK118R SUCLA2 OE CAE: mutant AR42J cells overexpressing both SIRT5 and SUCLA2_K118R after caerulein stimulation.). Figure 4 E and Figure 4 In F, in AR42J cells stimulated by caerulein, compared with the overexpressed wild type, the SCS activity of cells overexpressing K118R_SUCLA2 was enhanced and the ATP level was increased; and compared with the overexpression of a single gene, the SCS activity of cells co-overexpressing SIRT5 and K118R_SUCLA2 was further enhanced and the ATP level was further increased.

[0155] 4. Metabolomics analysis of the acute pancreatitis rat model with SIRT5 overexpression and SUCLA2 knockdown 4.1 Construction of the acute pancreatitis rat model with SIRT5 overexpression and SUCLA2 knockdown

[0156] Rat adenovirus transfection was performed by intraperitoneal injection using the adenovirus described in step 3.3.

[0157] 4.2 Metabolomics analysis

[0158] The same operation as "2. Metabolomics analysis of acute pancreatitis model in Example 1". The violin plot of significantly different metabolites in pancreatic tissue of rats with acute pancreatitis with overexpression of SIRT5 and knockdown of SUCLA2 is shown in Figure 4 G and Figure 4 As shown in H (TAU_NC: control group of sodium taurocholate modeling; TAU_SIRT5OE: SIRT5 overexpression group of sodium taurocholate modeling; TAU_SUCLA2DE: SUCLA2 knockdown group of sodium taurocholate modeling; Raw Intensity: raw data intensity; 3-phenyllactic-acid: 3-phenyllactic acid; Succinyl-CoA: succinyl coenzyme A; Tyrosine: tyrosine; BPG: diphosphoglycerate; Oxaloacetate: oxaloacetate; Fructose-1,6-bisphosphate: fructose-1,6-bisphosphate.). Figure 4In G, the levels of 3-phenyllactate, succinyl-CoA, and tyrosine in the SIRT5 overexpression group were significantly decreased compared with the control group. Figure 4 In H, compared with the control group, the levels of oxaloacetate and succinyl-CoA were increased, while the levels of BPG and fructose-1,6-bisphosphate were decreased in the SUCLA2 knockdown group.

[0159] The Venn diagram of differential metabolites between SUCLA2 knockdown group and SIRT5 overexpression group is shown in Figure 2. Figure 4 I (TAU_SUCLA2-DE vs TAU_NC: comparison between SUCLA2 knockdown group and control group in sodium taurocholate modeling; TAU_SIRT5-OE vs TAU_NC: comparison between SIRT5 overexpression group and control group in sodium taurocholate modeling; Succinyl-CoA: succinyl coenzyme A). Figure 4 In I, succinyl-CoA was a common differentially altered metabolite between the SUCLA2 knockdown group and the SIRT5 overexpression group.

[0160] The results of Example 2 and Example 3 suggest that SIRT5-mediated desuccinylation of SUCLA2 plays a key role in regulating TCA cycle metabolism in pancreatic acinar cells in acute pancreatitis. SIRT5 levels in pancreatic acinar cells in acute pancreatitis are downregulated, which promotes high succinylation of SUCLA2, reduces SCS activity, and leads to TCA cycle obstruction. This process may form a SIRT5-SUCLA2 succinylation-succinyl CoA accumulation positive feedback loop, leading to the continuous deterioration of metabolic disorders in pancreatic acinar cells in acute pancreatitis.

[0161] Example 4 Relationship between protein hypersuccinylation and severity of acute pancreatitis

[0162] This embodiment specifically includes the following steps:

[0163] 1. Detection of total protein succinylation level in AR42J cells after 6h stimulation with caerulein

[0164] The immunoblotting operation was the same as that in "1.2 Detection of SCS expression in Example 2", with Anti-ACTIN or Anti-succinyllysine selected as the primary antibody, and 10 μM succinyl-CoA or 5 mM succinyl-CoA inhibitor (succinylphosphonate, SP) used as exogenous intervention.

[0165] The results of total protein succinylation level are as follows Figure 5 A and Figure 5 As shown in B (PAN-KSUC: protein succinylation; ACTIN: actin; CON: control group; CAE: caerulein treatment; SUC-COA: exogenously added succinyl coenzyme A; SP: succinylphosphonate.). Figure 5A and Figure 5 In B, the level of total protein lysine succinylation in AR42J pancreatic acinar cells increased after 3 and 6 hours of caerulein stimulation, while exogenous addition of succinyl-CoA or its inhibitor (SP) could significantly regulate the level of protein succinylation in AR42J cells after caerulein stimulation, suggesting that succinyl-CoA plays a key role in regulating protein succinylation.

[0166] 2. Effects of succinylation-related exogenous interventions on acute pancreatitis

[0167] 2.1 Construction of a mouse model of acute pancreatitis with succinylation-related exogenous intervention

[0168] Succinyl-CoA or SP was added to the aqueous solution of caerulein (50 μg / kg) to construct a C57BL / 6 mouse model of acute pancreatitis, and the operation was the same as that in “2.1 Construction of SIRT5- / - acute pancreatitis mouse model in Example 3”.

[0169] 2.2 Histopathological examination

[0170] Pancreatic tissue was obtained 12 hours after modeling, fixed with 4% paraformaldehyde, and then gradually dehydrated with 75%-100% ethanol. It was then transparentized with a mixed solution of ethanol and a transparent agent, then immersed in wax at 62-65°C, and embedded in an ice box. The embedded tissue was sliced ​​using a microtome, and pathological staining was performed using hematoxylin-eosin (HE) staining. The magnification of histopathological examination was 20 times.

[0171] Histopathological examination results Figure 5 C (SHAM: sham operation group; CAE: caerulein treatment group; CAE+SUC-COA: caerulein + succinyl-CoA treatment group; CAE+SP: caerulein + succinylphosphonate treatment group; PANCREAS: pancreatic tissue.). Figure 5 As shown in C, exogenous addition of succinyl-CoA aggravated pancreatic tissue damage, while SP alleviated pancreatic tissue damage. Figure 6 In C, SIRT5 overexpression adenovirus intervention in vivo can alleviate pancreatic damage in rats with acute pancreatitis, while SUCLA2 knockdown in vivo aggravated pancreatic damage in rats with acute pancreatitis.

[0172] 2.3 Immunohistochemistry

[0173] The tissue sections were obtained in the same manner as in "Example 42.2 Histopathological Examination", dewaxed with xylene, dexylene with gradient ethanol, and finally rinsed with distilled water. Antigens were repaired using citric acid buffer (PH 6.0) in a pressure cooker. Blocked with 5% BSA at room temperature for 30 minutes, and then succinyl lysine antibody was used as the primary antibody and incubated overnight at 4°C. After washing with PBS, a secondary antibody labeled with horseradish peroxidase was added and incubated at room temperature for 1 hour. DAB was used for color development, hematoxylin counterstaining, gradient ethanol dehydration, xylene transparent, neutral resin sealing, microscopic examination and photography, and the magnification was 40 times.

[0174] Immunohistochemical results of differential expression of succinylation levels in pancreatic tissues Figure 5 D (SHAM: sham operation group; CAE: caerulein treatment group; CAE+SUC-COA: caerulein + succinyl-CoA treatment group; CAE+SP: caerulein + succinylphosphonate treatment group; KSUC: succinylation.). Figure 5 As can be seen in D, there are differences in the levels of succinylated proteins in pancreatic tissues of each group.

[0175] 2.4 Serum amylase and lipase activity determination

[0176] The same operation as "1.1.2 Biochemical detection of amylase and lipase in Example 1" was performed. The results were as follows Figure 5 E and Figure 5 As shown in F (AP Mouse serum: acute pancreatitis mouse serum; Amylase: amylase; Lipase: lipase; control: control group; CEA: caerulein; CAE SUC-COA: caerulein + succinyl coenzyme A treatment group; CAE SP: caerulein + succinylphosphonate treatment group.). Figure 5 E and Figure 5 In F, exogenous addition of succinyl-CoA or SP could significantly regulate the levels of plasma amylase and lipase in mice with acute pancreatitis.

[0177] The above results suggest that the level of succinylated protein in the pancreas is closely related to the severity of acute pancreatitis.

[0178] 3. Effects of succinylation-related endogenous interventions on acute pancreatitis

[0179] 3.1 In vitro experiments on succinylation-related endogenous interventions

[0180] 3.1.1 Construction of an acute pancreatitis cell model with SIRT5 overexpression and SUCLA2 knockdown

[0181] The operation is the same as that of “Example 33.3 Construction of AR42J cell acute pancreatitis model overexpressing K118R_SUCLA2 and simultaneously overexpressing SIRT5 and K118R_SUCLA2”.

[0182] 3.1.2 Immunoblotting of succinylation-related endogenous interventions

[0183] The same immunoblotting operation as in “1.2 Detection of SCS expression in Example 2” was performed to detect the protein succinylation level in AR42J cells with SUCLA2 knockdown or SIRT5 overexpression after caerulein stimulation.

[0184] Immunoblotting results Figure 6 A and Figure 6 As shown in B (PAN-KSUC: protein succinylation; SUCLA2: ADP-specific β subunit of succinyl-CoA synthase; TUBULIN: tubulin; SIRT5: sirtuin 5; ACTIN: actin; CAE: caerulein treatment; sh-SUCLA2: adenovirus treatment carrying SUCLA2 knockdown shRNA; sh-NC: adenovirus treatment of shRNA blank control; SIRT5 OE: SIRT5 overexpression group; kDa: kilodaltons.). Figure 6 In A, SUCLA2 knockdown adenovirus resulted in increased protein succinylation in AR42J cells, which exacerbated the caerulein-induced increase in protein succinylation. Figure 6 In B, adenovirus-mediated overexpression of SIRT5 effectively reduced the succinylation level at the cellular level and reduced the level of caerulein-induced protein succinylation in AR42J cells. These results suggest that SIRT5 and SUCLA2 are involved in regulating protein succinylation in pancreatic acinar cells in acute pancreatitis.

[0185] 3.2 In vivo experiments on succinylation-related endogenous interventions

[0186] 3.2.1 Histopathological examination

[0187] The rats obtained in "Example 34.1 Construction of acute pancreatitis rat model with SIRT5 overexpression and SUCLA2 knockdown" were used to perform the same operation as "2.2 Histopathological examination in Example 4".

[0188] Histopathological findings of acute pancreatitis in rats with SIRT5 overexpression and SUCLA2 knockdown Figure 6As shown in C (SHAM: sham operation group; TAU: sodium taurocholate model group; TAU SIRT5OE: SIRT5 overexpression group of sodium taurocholate model; TAU SUCLA2DE: SUCLA2 knockdown group of sodium taurocholate model; PANCREAS: pancreatic tissue.). Figure 6 In C, SIRT5 overexpression adenovirus intervention in vivo can alleviate pancreatic damage in rats with acute pancreatitis, while SUCLA2 knockdown in vivo aggravated pancreatic damage in rats with acute pancreatitis.

[0189] 3.2.2 Serum amylase and lipase activity determination

[0190] The same operation as "1.1.2 Biochemical detection of amylase and lipase in Example 1" was performed. The results were as follows Figure 6 D and Figure 6 As shown in E (AP Rat serum: serum of rats with acute pancreatitis; Amylase: amylase; Lipase: lipase; control: control group; TAU: sodium taurocholate modeling group; SIRT5 OE TAU: SIRT5 overexpression group of sodium taurocholate modeling; SUCLA2 KDTAU: SUCLA2 knockdown group of sodium taurocholate modeling.). Figure 6 D and Figure 6 In E, SIRT5 overexpression reduced the levels of plasma digestive enzymes in acute pancreatitis compared with the control group, whereas SUCLA2 knockdown increased the levels of digestive enzymes.

[0191] 3.3 Study on the relationship between SIRT5-mediated protein succinylation and acute pancreatitis

[0192] 3.3.1 Histopathological examination

[0193] The mice obtained in "Example 32.1 Construction of SIRT5- / - acute pancreatitis mouse model" were used, and the same procedures as "2.2 Histopathological examination in Example 4" were performed.

[0194] Histopathological findings of SIRT5- / - acute pancreatitis mice Figure 6 As shown in F (SHAM: sham operation group; CAE: caerulein model group; SIRT5 KO SHAM: sham operation group with SIRT5 knockout; SIRT5 KO CAE: caerulein model group with SIRT5 knockout; PANCREAS: pancreatic tissue.). Figure 6 In F, pancreatic structural destruction and inflammatory cell infiltration were more severe in SIRT5- / - acute pancreatitis mice than in wild-type acute pancreatitis mice.

[0195] 3.3.2 Immunohistochemistry

[0196] The same operation as in "2.3 Immunohistochemistry in Example 4" was performed, and myeloperoxidase antibody, succinyl lysine antibody and SIRT5 antibody were selected for staining.

[0197] Histochemical results of the differences in myeloperoxidase, protein succinylation, and SIRT5 expression in pancreatic tissues of wild-type and SIRT5- / - mice with acute pancreatitis Figure 6 G Figure 6 H and Figure 6 As shown in I (SHAM: sham operation group; CAE: caerulein model group; SIRT5 KO SHAM: sham operation group with SIRT5 knockout; SIRT5 KO CAE: caerulein model group with SIRT5 knockout; MPO: myeloperoxidase; KSUC: succinylated protein; SIRT5: sirtuin 5.). Figure 6 G and Figure 6 In H, compared with wild-type acute pancreatitis mice, SIRT5- / - acute pancreatitis mice had increased myeloperoxidase expression and increased protein succinylation levels in the pancreas. Figure 6 In Figure 1, SIRT5 expression was reduced in the pancreas of SIRT5- / - mice compared with wild-type mice.

[0198] 3.3.3 Serum amylase and lipase activity determination

[0199] The same operation as "1.1.2 Biochemical detection of amylase and lipase in Example 1" was performed. The results were as follows Figure 6 J and Figure 6 K shows (AP Mouse pancreas: acute pancreatitis mouse pancreas; Amylase: amylase; Lipase: lipase; control: control group; CAE: caerulein model group; SIRT5 KO: SIRT5 knockout group; SIRT5 KO CAE: caerulein model SIRT5 knockout group.). Figure 6 J and Figure 6 In K, the levels of amylase and lipase in the plasma of SIRT5- / - acute pancreatitis mice were increased compared with those of wild-type acute pancreatitis mice.

[0200] The above results suggest that the level of protein succinylation in pancreatic tissue is closely related to the pathological process of acute pancreatitis. More specifically, the increase in succinylation level is positively correlated with the severity of acute pancreatitis. Among them, SIRT5 and SUCLA2 play an important role in the pathological process of acute pancreatitis by regulating the succinylation level.

[0201] Example 5 Relationship between hypersuccinylated proteins and mitochondrial dysfunction

[0202] This embodiment specifically includes the following steps:

[0203] 1. Succinylomics Analysis

[0204] The succinylated proteins identified in “2.1 Succinylomics analysis of AR42J cells after caerulein stimulation in Example 2” were subjected to the same bioinformatics analysis of succinylated proteins as in “2.3.3 Annotation and functional enrichment in Example 1”.

[0205] The results of mass spectrometry analysis of succinylation after 6 hours of caerulein stimulation are as follows Figure 7 As shown in A (Number: quantity; Type: category; Down-Protein: down-regulated protein; Down-Site: down-regulated site; Up-Protein: up-regulated protein; Up-Site: up-regulated site.). Figure 7 In A, succinylation levels of 564 lysine sites in 230 proteins increased after 6 hours of cerulein stimulation.

[0206] The cellular sub-distribution results of highly succinylated proteins are as follows Figure 7 As shown in B (cytoplasm: cytoplasm; mitochondria: mitochondria; nucleus: cell nucleus; others: others.). Figure 7 In B, highly succinylated proteins are mainly enriched in mitochondria.

[0207] The main enriched categories of highly succinylated proteins in biological processes, cellular components, and molecular functions are shown in the following table. Figure 7C (Fisher's exact test p value: Fisher's exact test p value; cellular component: cellular component; Molecular Function: Molecular Function; Biological Process: Biological Process; nucleoid: ribosome; mitochondrial nucleoid: mitochondrial ribosome; tricarboxylic acid cycle enzyme complex: tricarboxylic acid cycle enzyme complex; carbon-carbon lyase activity: carbon-carbon lyase activity; lyase activity: lyase activity; pyridoxal phosphate binding: nicotinamide phosphate binding; vitamin B6 binding: vitamin B6 binding; carboxy-lyase activity: decarboxylase activity; aldehyde dehydrogenase (NAD+) activity: acetyltransferase activity: acetyltransferase activity; NADPH binding: NADPH binding; aerobic respiration: aerobic respiration; tricarboxylic acid metabolic process: tricarboxylic acid metabolic process; lipid modification: lipid oxidation: lipid oxidation; cellular lipid catabolic process: cellular lipid decomposition metabolic process; dicarboxylic acidmetabolic process: dicarboxylic acid metabolic process; learning or memory: learning or memory; anterogradetrans-synaptic signaling: anterograde trans-synaptic signaling pathway; trans-synaptic signaling: trans-synaptic signaling pathway. ). Figure 7 In C, in terms of biological processes, highly succinylated proteins are mainly enriched in processes closely related to energy metabolism, including "aerobic respiration", "tricarboxylic acid metabolic process" and "lipid modification"; in terms of cellular composition, highly succinylated proteins are mainly enriched in organelles and complexes, such as "ribosomes", "mitochondrial ribosomes" and "tricarboxylic acid cycle enzyme complexes"; in terms of molecular functions, highly succinylated proteins are mainly enriched in functions related to metabolic enzyme activity, such as "carbon-carbon lyase activity", "lyase activity" and "nicotinamide phosphate binding".

[0208] The enrichment results of highly succinylated proteins in KEGG pathways are as follows Figure 7 D (Protein number: protein number; P value: P value; beta-Alanine metabolism: β-alanine metabolism; Lysine degradation: lysine degradation; Tryptophan metabolism: tryptophan metabolism; Propanoate metabolism: propionate metabolism; Citrate cycle (TCA cycle): citric acid cycle (TCA cycle); Arginine and proline metabolism: arginine and proline metabolism; Pyruvate metabolism: pyruvate metabolism; Valine, leucine and isoleucine degradation: valine, leucine and isoleucine degradation; Log2 Fold enrichment: the logarithm of the enrichment factor with base 2.). Figure 7 In D, KEGG pathway enrichment analysis showed that highly succinylated proteins were highly enriched in the “TCA cycle” pathway.

[0209] 2. Immunofluorescence

[0210] 2.1 Assessment of mitochondrial function

[0211] AR42J cells stimulated with caerulein for 6 h obtained in “Example 11.1.1 Caerulein treatment”, AR42J cells overexpressing K118R_SUCLA2 obtained in “Example 33.1 Construction of an acute pancreatitis model of AR42J cells overexpressing wild-type SUCLA2 and overexpressing K118R_SUCLA2”, and AR42J cells overexpressing SITR5 and K118R_SUCLA2 simultaneously obtained in “Example 43.1.1 Construction of an acute pancreatitis cell model with SIRT5 overexpression and SUCLA2 knockdown” were selected, and exogenously intervened with 10 μM succinyl-CoA or 5 mM SP. DAPI (Beyotime C1002) was used for nuclear staining, and JC-1 fluorescent dye (Beyotime C2006) and ROS detection kit (Beyotime, S0033S) were used to detect mitochondrial membrane potential and reactive oxygen species (ROS) levels, respectively. Images were acquired under a ZEISS inverted fluorescence microscope.

[0212] Immunofluorescence results of mitochondrial membrane potential and ROS level in AR42J cells after caerulein stimulation Figure 7 E. Figure 7 F. Figure 7 As shown in G (JC-1: mitochondrial membrane potential detection dye; ROS: reactive oxygen species; CON: control group; CAE: caerulein treatment; CAESUC-COA: caerulein + exogenously added succinyl-CoA; CAE SP: caerulein + succinylphosphonate; SIRT5OE CAE: mutant AR42J cells overexpressing SIRT5 after caerulein stimulation; K118R-SUCLA2OE CAE: mutant AR42J cells overexpressing SUCLA2_K118R after caerulein stimulation; K118R-SUCLA2OE_SIRT5OE CAE: mutant AR42J cells overexpressing SIRT5 and SUCLA2_K118R at the same time after caerulein stimulation.). Figure 7 In E, the mitochondrial membrane potential of AR42J cells stimulated by caerulein decreased, and the increase of succinyl-CoA exacerbated this change, while SP treatment alleviated this change. Figure 7 In F, ROS levels increased in AR42J cells stimulated by caerulein, and the increase of succinyl-CoA exacerbated this change, while SP treatment alleviated this change. Figure 7 In G, overexpression of SIRT5 and K118R_SUCLA2 can slow down the changes in protein succinylation level and ROS level induced by caerulein, and slow down the changes in mitochondrial membrane potential decrease induced by caerulein (the magnification of JC1 and protein succinylation staining is 100 times, and the magnification of ROS is 10 times).

[0213] 2.2 Assessment of mtDNA release

[0214] Select cell samples as in step 2.1, use MitoBright LT Red (DOJINDO, MT11) and Hoechst 33342 (Beyotime C1025) to stain mitochondria and nuclei, respectively, and use anti-dsDNA antibody immunofluorescence to observe the distribution of mtDNA.

[0215] Immunofluorescence results of mtDNA distribution in AR42J cells overexpressing SIRT5 and K118R_SUCLA2 after caerulein stimulation Figure 8 As shown in A (mtDNA: mitochondrial DNA; Control: control group; CAE: caerulein treatment; SIRT5OE: mutant AR42J cells overexpressing SIRT5; K118R_SUCLA2OE: mutant AR42J cells overexpressing SUCLA2_K118R; SIRT5OE K118R_SUCLA2OE: mutant AR42J cells overexpressing both SIRT5 and SUCLA2_K118R.). Figure 8In A (green represents dsDNA antibody staining, blue represents DAPI cell nucleus staining, and red represents MitoTracker mitochondrial staining, and the magnification is 100 times), caerulein stimulation can induce a significant increase in the release of mtDNA in the cytoplasm of wild-type AR42J cells, while overexpression of SIRT5 or K118R_SUCLA2 can reduce the release of mtDNA, and simultaneous overexpression of SIRT5 and K118R_SUCLA2 can further reduce the release of mtDNA.

[0216] 3.RT-PCR

[0217] Select cell samples as in step 2.1, use Total RNA Extraction Reagent (Vazyme, R401-01) to isolate total RNA, and use HiScriptⅢ1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, R312-01) to reverse transcribe into cDNA. RT-PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) and gene-specific primers (see Table 3), and ACTIN was selected as the internal reference gene.

[0218] Table 3 Gene-specific primer sequences

[0219]

[0220]

[0221] 3.1 Quantitative analysis of AR42J cytoplasmic mtDNA levels

[0222] Cytoplasmic, nuclear, and mitochondrial fractions were isolated, and qPCR for D-Loop and TERT was performed to quantify cytoplasmic mtDNA levels.

[0223] The quantitative analysis results of AR42J cytoplasmic mtDNA levels are shown in Figure 8 As shown in B (mtDNA relativeexpression: relative expression of mitochondrial DNA; Control: control group; CAE: caerulein treatment; SIRT5 OE CAE: mutant AR42J cells overexpressing SIRT5 after caerulein stimulation; K118R SUCLA2 OE CAE: mutant AR42J cells overexpressing SUCLA2_K118R after caerulein stimulation; SIRT5 OE K118R SUCLA2 OE CAE: mutant AR42J cells overexpressing SIRT5 and SUCLA2_K118R after caerulein stimulation.). Figure 8 In B, caerulein stimulation led to an increase in the level of mtDNA in the cytoplasm of wild-type AR42J cells, while overexpression of SIRT5 or K118R_SUCLA2 reduced the level, but there was no significant difference between the co-overexpression group and the single overexpression group.

[0224] 3.2 Alterations of cGAS-STING signaling pathway in AR42J cells

[0225] AR42J cells were selected as in step 2.1 and co-cultured with rat macrophage NR8383 cells. The mRNA levels of cGAS, STING, IRF3 and IFN-β were determined by RT-PCR for the AR42J cell samples obtained after co-culture in the same manner as in “Example 11.1.1 Caerulein Treatment”.

[0226] The RT-PCR results of the mRNA levels of cGAS, STING, IRF3, and IFN-β after AR42J cells were co-cultured with NR8383 cells are shown in Figure 8 C. Figure 8 D. Figure 8 E and Figure 8 F (relative expression: relative expression; NR8383: rat macrophage NR8383 cells; NR8383-AR42J: rat macrophage NR8383 cells and AR42J cells co-culture group; NR8383-AR42J-CAE: rat macrophage NR8383 cells and AR42J cells co-culture group stimulated by caerulein; NR8383-SIRT5_OE AR42J-CAE: rat macrophage NR8383 cells stimulated by caerulein were co-cultured with AR42J cells overexpressing SIRT5; NR8383-K118MUT_SUCLA2_OE_AR42J+CAE: rat macrophage NR8383 cells stimulated by caerulein were co-cultured with mutant AR42J cells overexpressing SUCLA2_K118R; NR8383-SIRT5_OE+K118MUT_SUCLA2_OE_AR42J+CAE: rat macrophage NR8383 cells stimulated by caerulein were co-cultured with mutant AR42J cells overexpressing both SIRT5 and SUCLA2_K118R. ). Figure 8 C. Figure 8 D. Figure 8 E and Figure 8In F, caerulein stimulation induced a significant increase in the mRNA levels of key inflammatory factors such as cGAS, STING, IRF3, and IFN-β in the co-culture system, while SIRT5 or K118R_SUCLA2 overexpression inhibited the expression of the above inflammatory factors to varying degrees, among which the co-overexpression group had the most significant downregulation of STING. These results suggest that the SIRT5-SUCLA2 axis can inhibit the activation of pancreatic acinar cells to macrophages by inhibiting the release of mtDNA.

[0227] 4. IFN-β biochemical detection

[0228] The AR42J cell model obtained in "Example 11.1.1 Caerulein Treatment" was selected, and 10 μM succinyl-CoA or 5 mM SP was used for exogenous intervention. The plasma IFN-β level of mice with acute pancreatitis was detected using a mouse interferon β enzyme-linked immunosorbent assay kit (Beyotime, PI568).

[0229] Figure 8 G: ELISA results of the effect of exogenous intervention on IFN-β levels in AR42J cells after caerulein stimulation Figure 8 G (IFN-β: interferon β; CON: control group; CAE: caerulein treatment group; CAE SUC-COA: caerulein treatment group with exogenous addition of succinyl-CoA; CAE SP: caerulein treatment group with exogenous addition of succinyl-phosphonate.). Figure 8 In G, the IFN-β level of AR42J cells was significantly increased after caerulein stimulation, succinyl-CoA could further promote this change, while SP could effectively reduce the IFN-β level.

[0230] 5. Immunohistochemistry

[0231] Select the mice of "Example 42.1 Construction of succinylation-related exogenous intervention acute pancreatitis mouse model" and perform the same operation as "2.3 Immunohistochemistry in Example 4". Select cGAS antibody, STING antibody and IRF3 antibody for staining, and the magnification is 40 times.

[0232] Immunohistochemical results of the expression of cGAS, IRF3, and STING in the pancreas of mice with acute pancreatitis Figure 8 H (cGAS: cGAS protein; STING: STING protein; IRF3: interferon regulatory factor 3; SHAM: sham operation group; CAE: caerulein treatment group; CAE+SUC-COA: caerulein treatment group with exogenous addition of succinyl-CoA; CAE+SP: caerulein treatment group with exogenous addition of succinyl-phosphonate.). Figure 8In H, the expression levels of inflammatory markers such as cGAS, STING, and IRF3 in the pancreatic tissue of mice with acute pancreatitis were significantly increased, and exogenous succinyl-CoA treatment further promoted this change, while SP treatment could slow down this change.

[0233] 6. Co-immunoprecipitation

[0234] Select the AR42J cell sample overexpressing SIRT5 and K118R_SUCLA2 obtained in "Example 33.3 Construction of an AR42J cell acute pancreatitis model overexpressing K118R_SUCLA2 and simultaneously overexpressing SIRT5 and K118R_SUCLA2", and perform the same operation as "3.2 Immunoprecipitation in Example 3".

[0235] The immunoprecipitation results of CYC1 protein succinylation level are shown in Fig. 9 E (MOCK: wild-type overexpression of SUCLA2; K118R-SUCLA2-SIRT5_OE: simultaneous overexpression of SIRT5 and K118R SUCLA2 mutant; CAE: caerulein; INPUT: input; IP: immunoprecipitation; PAN-KSUC: protein succinylation; CYC1: cytochrome c1; ACTIN: actin; KSUC: succinylation.). Fig. 9 In E, simultaneous overexpression of SIRT5 and K118R_SUCLA2 can effectively reduce the succinylation modification level of CYC1 protein.

[0236] 7. Mitochondrial Complex III Activity Assay

[0237] The mitochondrial respiratory chain complex III / CoQ-cytochrome C reductase activity detection kit (Solarbio, BC3240) was used to evaluate the activity of mitochondrial complex III. The AR42J cell sample overexpressing SIRT5 and K118R_SUCLA2 obtained in "Example 33.3 Construction of AR42J cell acute pancreatitis model overexpressing K118R_SUCLA2 and overexpressing SIRT5 and K118R_SUCLA2" was selected, 1.0 mL of extract was added to the cell sample, and after homogenization, centrifuged at 4°C and 600g for 10 minutes, the supernatant was transferred to another centrifuge tube, and centrifuged at 4°C and 11000g for 15 minutes. 200 μL of extract was added to the precipitate, and the supernatant was collected after ultrasonic disruption. The absorbance at 550 nm was measured to calculate the activity of mitochondrial complex III.

[0238] The results of the mitochondrial complex III activity assay in AR42J cells overexpressing SIRT5 and K118R_SUCLA2 after caerulein stimulation are shown in Fig. 9 As shown in F. Fig. 9In F, overexpression of SIRT5 and K118R_SUCLA2 alleviated the caerulein-induced decrease in ETC III activity in AR42J cells.

[0239] In summary, the SIRT5-SUCLA2 pathway can regulate the succinylation level of Cyc1, affect the function of the mitochondrial electron transport chain and the release of mtDNA, and play a key role in the occurrence and development of acute pancreatitis inflammatory response and multiple organ failure. Exogenous intervention in succinyl-CoA metabolism may provide a new therapeutic approach for this.

[0240] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. Use of a succinylated protein in the preparation of a product for the diagnosis or treatment of acute pancreatitis, characterized in that: The succinylated proteins include SUCLA2 with succinylated lysine at position 118 and succinylated Cyc1.

2. Use of a succinylated protein as a target for treating acute pancreatitis, characterized in that: The succinylated proteins include SUCLA2 with succinylated lysine at position 118 and succinylated Cyc1.

3. The use according to claim 2, characterized in that: The target is a drug target.

4. A product, characterized in that The active ingredients of the product include SUCLA2 with succinylated lysine at position 118 and succinylated Cyc1; the product has at least one of the following functions: (1) predicting the risk of occurrence and progression of pancreatitis; and (2) serving as a drug target for the treatment of pancreatitis.

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