Application of antibacterial peptide in treatment of pancreatic diseases and intestinal diseases caused by pancreatic diseases

Abstract

The invention provides application of an antibacterial peptide in preparation of a medicine for treating, preventing or relieving pancreatic diseases or related intestinal diseases caused by the pancreatic diseases. The antibacterial peptide comprises REG3B, LL-37 and RNASE1 (Ribonucleic Acid SE1), has a treatment effect on pancreatitis and pancreatitis-mediated intestinal injury and inflammation, and can regulate pancreas-intestine interaction.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to antimicrobial peptide REG3B and Orai1 inhibitors and their use in preparing drugs for treating pancreatic diseases. Background Art

[0002] Acute pancreatitis (AP) is a painful, potentially life-threatening inflammatory disease of the exocrine pancreas with a high morbidity and health burden. Gallstones and alcohol abuse contribute to 70% of AP cases, with other causes including hypertriglyceridemia, certain medications, and trauma. The severity and prognosis of AP depend on the extent of local pancreatic damage and the development of persistent organ failure. The mortality rate of severe acute pancreatitis (SAP) can be as high as 30%. Studies have shown that inadequate pancreatic juice, electrolyte, and bicarbonate secretion increases patients' risk of pancreatitis. Pancreatitis-associated toxins can contribute to impaired pancreatic juice secretion and the pathogenesis of pancreatitis. Key mediators, including the cystic fibrosis transmembrane conductance regulator (CFTR), which is primarily expressed in pancreatic ductal epithelial cells (PDECs), have been shown to mediate bicarbonate and fluid secretion, thereby influencing the severity of pancreatitis. Multiple studies have demonstrated that impaired pancreatic ductal secretion can lead to pancreatic acinar cell damage, exacerbating AP.

[0003] On the other hand, pancreatitis-associated intestinal inflammation and dysbiosis can also directly affect the severity and prognosis of AP. Pancreatitis-induced intestinal mucosal damage and impaired intestinal barrier function can allow the translocation of facultative pathogenic strains of bacteria into the inflamed pancreas, leading to subsequent pancreatic necrosis, infection, and death.

[0004] Although there have been studies on the pathogenesis of acute pancreatitis and the regulation of PDECs' interactions between the pancreas and the intestine, there is currently no effective early targeted treatment. Summary of the Invention

[0005] Based on the technical problems that need to be solved in the existing technology, the applicant has discovered a new target that can effectively treat or alleviate pancreatitis, especially acute pancreatitis and related diseases caused by it.

[0006] The technical solution of this application specifically includes:

[0007] Use of antimicrobial peptides in preparing drugs for treating, preventing or alleviating pancreatic diseases or related intestinal diseases caused by pancreatic diseases.

[0008] Furthermore, the antimicrobial peptide is selected from C-type lectin antimicrobial peptides, cathelicidins family antimicrobial peptides or RNase A family antimicrobial peptides.

[0009] Further preferably, the antimicrobial peptide is REG3B, LL-37 or RNASE1.

[0010] The technical solution of the present application also includes the use of Orai1 inhibitors in the preparation of drugs for treating, preventing or alleviating related intestinal diseases caused by pancreatic diseases.

[0011] Furthermore, the pancreatic disease includes pancreatitis, pancreatic damage or impaired pancreatic juice secretion.

[0012] Furthermore, the pancreatitis includes acute pancreatitis or chronic pancreatitis.

[0013] Further preferably, the acute pancreatitis is acute biliary pancreatitis.

[0014] Furthermore, the related intestinal diseases caused by pancreatic diseases are intestinal inflammation caused by pancreatitis, intestinal dysbiosis or pancreatico-intestinal dialogue diseases.

[0015] Furthermore, the intestinal diseases caused by pancreatic diseases include duodenitis, colitis, intestinal mucosal damage, intestinal barrier function damage or intestinal flora imbalance caused by pancreatitis

[0016] Furthermore, the Orai1 inhibitor includes a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule hybridizing with an Orai1 nucleic acid molecule, or a mutated Orai1 protein.

[0017] Further preferably, the Orai1 inhibitor is GSK-7975A, CM4620 or CM5480.

[0018] The present application also provides a pharmaceutical composition for treating, preventing or alleviating pancreatic diseases or related intestinal diseases caused by pancreatic diseases, which comprises antimicrobial peptides and / or Orai1 inhibitors.

[0019] Furthermore, the antimicrobial peptide in the pharmaceutical composition is selected from C-type lectin antimicrobial peptides, cathelicidins family antimicrobial peptides or RNase A family antimicrobial peptides.

[0020] Furthermore, the antimicrobial peptide in the pharmaceutical composition is REG3B, LL-37 or RNASE1.

[0021] Furthermore, the pharmaceutical composition also contains an Orai1 inhibitor.

[0022] Furthermore, the Orai1 inhibitor in the pharmaceutical composition is a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule hybridizing with an Orai1 nucleic acid molecule, or a mutated Orai1 protein.

[0023] Furthermore, the pharmaceutical composition further comprises a carrier for delivering the antimicrobial peptide and / or Orai1 inhibitor.

[0024] Furthermore, the vector may be a viral vector or a lipid nanoparticle.

[0025] Effects of the Invention

[0026] This application can significantly alleviate pancreatitis symptoms in a clinically representative acute pancreatitis model by knocking out or inhibiting the function of the Orai1 gene in PDECs, including the treatment and relief of pancreatic inflammation, local damage, and impaired pancreatic secretion. It also has therapeutic effects on intestinal damage related to pancreatic disease, especially damage to the duodenum and colon, inflammation, impaired intestinal barrier function, and intestinal dysbiosis. Furthermore, antimicrobial peptides such as REG3B, RNASE1, and LL-37, secreted by Orai1, also have beneficial effects on pancreatic diseases and pancreatic-related intestinal damage, providing new targets and treatment methods for the treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A is the effect of intracellular Orai1 inhibitor on Ca2+ induced by POA and NaT, respectively. 2+ Signal impact situation.

[0028] Figure 1 B shows the effects of intracellular Orai1 inhibitors on mitochondrial membrane potential induced by POA and NaT, respectively.

[0029] Figure 1 C is a schematic diagram of the experimental procedure for Orai1 deletion in PDECs.

[0030] Figure 1 D shows the expression of Orai1 in primary pancreatic ductal cells.

[0031] Figure 1 E shows the effect of pancreatic ductal cell-specific Orai1 deficiency on mitochondrial membrane potential.

[0032] Figure 2 A and B are schematic diagrams of the experimental procedure for isolating pancreatic ductal cells using magnetic beads.

[0033] Figure 2 C shows the results of quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection.

[0034] Figure 3 A is a schematic diagram of the POA-induced AP model experiment.

[0035] Figure 3 B shows the effect of Orai1 deficiency in POA model PDECs on serum amylase and lipase levels.

[0036] Figure 3 C is a representative image of hematoxylin and eosin-stained pancreatic sections in the POA model.

[0037] Figure 3 D is the pancreatic histological severity score in the POA model.

[0038] Figure 3 E is the expression of proinflammatory cytokines in the POA model.

[0039] Figure 4 A is a schematic diagram of the NaT-induced AP model experiment.

[0040] Figure 4 B shows the effect of Orai1 deficiency in NaT model PDECs on serum amylase and lipase levels.

[0041] Figure 4 C is a representative image of hematoxylin and eosin-stained pancreatic sections in the NaT model.

[0042] Figure 4 D is the pancreatic histological severity score in the NaT model.

[0043] Figure 4 E is the expression of proinflammatory cytokines in the NaT model.

[0044] Figure 5 A is a schematic diagram of the experimental procedure for RNA sequencing analysis of FACS-sorted pancreatic ductal cells.

[0045] Figure 5 B shows the gating strategy used to sort DBA+ pancreatic duct cells and the purity of the sorted cells.

[0046] Figure 5 C and D are volcano plots of differentially expressed genes (DEGs).

[0047] Figure 5 E, F, and G are the results of DEGs identified by Venn diagram.

[0048] Figure 6 A is a representative image of duodenal filling in the ethanol + POA induced model.

[0049] Figure 6 B is the quantitative statistical results of duodenal filling in the ethanol + POA induced model.

[0050] Figure 6 C is a representative image of duodenal filling in the NaT-induced model.

[0051] Figure 6D is the quantitative statistical result of duodenal filling in the NaT-induced model.

[0052] Figure 6 E is a schematic diagram of the experimental protocol for evaluating intestinal permeability.

[0053] Figure 6 F and G are the quantitative statistical results of blood fluorescence intensity in ethanol+POA and NaT-induced models, respectively.

[0054] Figure 7 A shows representative images and histological scores of hematoxylin and eosin-stained duodenal and colon sections in the ethanol + POA model.

[0055] Figure 7 B shows representative images and quantitative statistical results of CD45 immunohistochemical staining of the duodenum and colon in the ethanol + POA model.

[0056] Figure 7 C is the expression levels of pro-inflammatory cytokines in the duodenum and colon in the ethanol + POA model.

[0057] Figure 7 D is the expression level of tight junction protein genes in the duodenum and colon in the ethanol + POA model.

[0058] Figure 8 A shows representative images and histological scores of hematoxylin and eosin-stained duodenal and colon sections in the NaT-induced model.

[0059] Figure 8 B shows representative images and quantitative statistical results of CD45 immunohistochemical staining of the duodenum and colon in the NaT-induced model.

[0060] Figure 8 C is the expression levels of pro-inflammatory cytokines in the duodenum and colon in the NaT-induced model.

[0061] Figure 8 D is the expression level of tight junction protein genes in the duodenum and colon in the NaT-induced model.

[0062] Figure 9 A and B are the statistical results of Simpson diversity index of duodenum and cecum in the ethanol + POA model, respectively.

[0063] Figure 9 C and D are the principal component analysis results of the duodenal and cecal microbiota, respectively.

[0064] Figure 9 E and F are bacterial abundance maps of the duodenal microbiome at the family and genus levels.

[0065] Figure 9G and H are bacterial abundance diagrams of the cecal microbiome at the family and genus levels.

[0066] Figure 10 A and B show the relative abundance of Shigella and Lactobacillus in the duodenum.

[0067] Figure 10 C, D, and E show the relative abundance of Lactobacillus, Desulfovibrio, and Allobacterium in the cecum.

[0068] Figure 11 A is a schematic diagram of the liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis experimental strategy.

[0069] Figure 11 B is a heat map of representative proteins identified in mouse pancreatic juice.

[0070] Figure 11 C is the Venn diagram identification result.

[0071] Figure 11 D is the relative abundance of proteins belonging to antimicrobial peptides and digestive enzymes.

[0072] Figure 12 A is a schematic diagram of the experimental strategy for exogenous supplementation of REG3B in AP mice.

[0073] Figure 12 B shows the changes in serum amylase and lipase.

[0074] Figure 12 C shows representative images and histological scores of hematoxylin and eosin-stained pancreatic sections.

[0075] Figure 12 D is the expression levels of proinflammatory cytokines Tnf and Il1b in the pancreas detected by qPCR.

[0076] Figure 12 E shows representative images and histological scores of hematoxylin and eosin-stained duodenal sections.

[0077] Figure 12 F shows the CD45 immunohistochemical staining and quantitative statistical results of duodenal sections.

[0078] Figure 12 G is the expression level of proinflammatory cytokines in the duodenum.

[0079] Figure 12 H is the expression level of tight junction protein genes in the duodenum.

[0080] Figure 13 A shows representative images and histological scores of hematoxylin and eosin-stained colon sections.

[0081] Figure 13 B is the CD45 immunohistochemical staining and quantitative statistical results of colon sections.

[0082] Figure 13 C is the expression level of proinflammatory cytokines in the colon.

[0083] Figure 13 D is the expression level of tight junction protein genes in the colon. DETAILED DESCRIPTION

[0084] The present application is described in detail below. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0085] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of this application shall be as defined by the attached claims.

[0086] The inventors of the present application have discovered that by removing Orai1 from pancreatic ductal epithelial cells (PDECs) or inhibiting its gene function, the severity of pancreatitis and related intestinal diseases caused by it can be effectively alleviated. Based on this, the present application provides the use of Orai1 inhibitors in the preparation of drugs for treating, alleviating or preventing pancreatic diseases and related intestinal diseases caused by them.

[0087] In the present application, Orai1 is a calcium release-activated calcium channel protein and a channel-forming subunit of SOCE (store-operated calcium entry). Its main function is to allow calcium ions to enter the cell by binding to the endoplasmic reticulum calcium sensor STIM1 after intracellular calcium ions are released.

[0088] Pancreatitis-associated toxins, such as bile acids, alcohol, and their non-oxidative metabolites, can induce Ca2+ in PDECs. 2+Long-term elevation of calcium leads to mitochondrial calcium overload and cell damage. Orai1, a key calcium regulator expressed in pancreatic ductal cells, protects against calcium-dependent pancreatic acinar cell death.

[0089] In the present application, the Orai1 inhibitor can inhibit the expression, content or activity of the Orai1 gene or RNA or the protein encoded by it.

[0090] In a specific embodiment, the Orai1 inhibitor comprises a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule that hybridizes with an Orai1 nucleic acid molecule, or a mutated Orai1 protein.

[0091] In the present application, examples of the nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNA (sirna), and short hairpin RNA (shRNA). Such inhibitory nucleic acid molecules can be designed to target any region of the Orai1 nucleic acid molecule. In some embodiments, the antisense RNA, sirna, or shRNA hybridizes with a sequence within the nucleic acid molecule or mrna molecule of Orai1 and reduces the expression of the Orai1 protein in the subject's cells. In some embodiments, the Orai1 inhibitor comprises an antisense molecule that hybridizes with the Orai1 nucleic acid molecule or mrna molecule and reduces the expression of the Orai1 protein in the subject's cells. In some embodiments, the Orai1 inhibitor comprises a sirna that hybridizes with the Orai1 nucleic acid molecule or mrna molecule and reduces the expression of the Orai1 protein in the subject's cells. In some embodiments, the Orai1 inhibitor comprises a shRNA that hybridizes with the Orai1 nucleic acid molecule or mrna molecule and reduces the expression of the Orai1 protein in the subject's cells.

[0092] In the present application, the inhibitory nucleic acid molecule may comprise RNA or DNA, or both RNA and DNA. The inhibitory nucleic acid molecule may also be linked or fused to a heterologous nucleic acid sequence (e.g., a heterologous nucleic acid sequence in a vector) or a heterologous marker. For example, the inhibitory nucleic acid molecule may be within a vector comprising the inhibitory nucleic acid molecule and the heterologous nucleic acid sequence or as an exogenous donor sequence comprising the inhibitory nucleic acid molecule and the heterologous nucleic acid sequence. The inhibitory nucleic acid molecule may also be linked or fused to a heterologous marker. The marker may be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, enzyme, or fluorophore quencher). Such markers may be detected by spectroscopy, photochemistry, biochemistry, immunochemistry, or chemical means. Such markers include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The marker may also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme. The term "marker" may also refer to a "tag" or a hapten that selectively binds to a conjugated molecule so that the conjugated molecule is used to generate a detectable signal when subsequently added with a substrate. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, Ha, Flag or 3xflag, 6xhis or polyhistidine, glutathione-S-transferase (Gst), maltose binding protein, epitope tags, or the fc portion of an immunoglobulin. Many labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent, and chemiluminescent substrates, and other labels.

[0093] In the present application, the gene editor comprises a DNA gene editor and a RAN gene editor. The gene editor comprises a gene editing protein and an optional gRNA. Preferably, the gene editing protein is a Cas protein. In the present application, suitable Cas proteins include, for example, wild-type Cas9 protein and wild-type CPF1 protein (such as fnCPF1). The Cas protein can have complete cutting activity to produce double-strand breaks in the Orai1 genomic nucleic acid molecule, or it can be a nicking enzyme that produces single-strand breaks in the Orai1 genomic nucleic acid molecule. Additional examples of Cas proteins include, but are not limited to, Cas1, Cas1b, Cas2, Cas3, Cas4, Cas5, Cas5e (Casd), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, Casf, Casg, Cash, Csy1, Csy2, Csy3, Cse1 (Casa), Cse2 (Casb), C se3 (Case), Cse4 (Casc), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, Csax, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4 and cu1966, and homologs or modified versions thereof.

[0094] In a specific embodiment of the present application, the Orai1 inhibitor is GSK7975A, CM4620 or CM5480.

[0095] The present application provides the use of Orai1 inhibitors in treating, preventing or alleviating related intestinal diseases caused by pancreatic diseases.

[0096] The present application provides the use of an Orai1 inhibitor in the preparation of a drug for treating, preventing or alleviating related intestinal diseases caused by pancreatic diseases.

[0097] The present application further provides the use of antimicrobial peptides in treating, preventing or alleviating pancreatic diseases or related intestinal diseases caused by pancreatic diseases.

[0098] Pancreatic juice contains digestive enzymes and antimicrobial peptides, which are secreted from the pancreas into the intestinal lumen. Proteomic analysis of mouse pancreatic juice revealed that Orai1 in PDECs potentially regulates these components, which are digestive enzymes and antimicrobial peptides. These enzymes are secreted from the pancreas into the intestinal lumen.

[0099] The antimicrobial peptides described herein, also known as host defense peptides, are a class of small peptides with antimicrobial activity that are induced in insects. They have a molecular weight of approximately 2,000 to 7,000 and are composed of 20 to 60 amino acid residues. Most of these active peptides possess strong alkalinity, thermal stability, and broad-spectrum antimicrobial properties. Currently, over 2,700 antimicrobial peptides have been discovered, originating from bacteria, archaea, protozoa, fungi, plants, and animals.

[0100] The antimicrobial peptides described in the present application can be selected from C-type lectin antimicrobial peptides, cathelicidins family antimicrobial peptides or RNase A family antimicrobial peptides.

[0101] C-type lectins (CTLs) are a large superfamily of proteins containing one or more C-type lectin-like domains. They can bind to a variety of endogenous and exogenous ligands, primarily glycoconjugates. CTLs interact with various endogenous and exogenous ligands and participate in important physiological processes such as immune defense, immune homeostasis, and immune surveillance to maintain homeostasis. As important pattern recognition receptors (PRRs), CTLs play a crucial role in innate and adaptive antimicrobial immune responses and in protecting against microbial infections.

[0102] Cathelicidins are a family of antimicrobial peptides found in mammals that contain a conserved cathelin domain. They are primarily derived from macrophages, neutrophils, natural killer cells, and epithelial cells of the skin, intestines, respiratory tract, and ocular surface of primates, ungulates, and rodents. Cathelicidins contain a C-terminal cationic antimicrobial domain that is only active after the N-terminal "cathelin" portion of the peptide is released. Cathelicidins not only possess strong resistance against common Gram-positive and Gram-negative bacteria, fungi, and viruses, but also exhibit activity against many clinically isolated drug-resistant strains. LL-37 is the only antimicrobial peptide belonging to the cathelicidins family currently found in humans.

[0103] Ribonucleases (RNases) are a class of nucleic acid hydrolases that regulate nucleic acid metabolism and protein synthesis by degrading their substrate RNA. RNase A-type RNases, also known as pancreatic ribonucleases, are a class of small, alkaline, secreted proteins, exemplified by bovine pancreatic RNase A, that specifically degrade pyrimidine bases. They comprise a group of homologous proteins isolated from various vertebrates. Some proteins in the RNase A family possess specialized biological functions beyond RNA hydrolysis, such as antibacterial, antifungal, and antiparasitic activities, and the reduction of RNA viral infections. Examples include RNase 1, EDN / RNase 2, ANG / RNase 5, RNase 7, and RNase 8, which have been identified from the human genome as RNases with enzymatic activity.

[0104] In some specific embodiments, the antimicrobial peptide is REG3B, LL-37 or RNASE1. REG3B is a key component of pancreatic juice, mediated by Orai1, and expressed by pancreatic acinar and islet cells in the pancreas and neuroendocrine and Paneth cells in the small intestine.

[0105] The pancreatic diseases described in this application include pancreatitis, pancreatic injury, impaired pancreatic juice secretion and other related diseases.

[0106] Pancreatitis in this application includes acute pancreatitis and chronic pancreatitis, preferably acute pancreatitis. In some specific embodiments, the acute pancreatitis is acute biliary pancreatitis.

[0107] The related intestinal diseases caused by pancreatic diseases described in this application include intestinal inflammatory diseases caused by pancreatitis, intestinal dysbiosis diseases or pancreatico-intestinal dialogue diseases.

[0108] In some specific embodiments, intestinal inflammation caused by intestinal ischemia-reperfusion during acute pancreatitis (AP) often leads to multiple organ dysfunction and aggravates the severity of AP.

[0109] In some embodiments, the exocrine pancreas plays a key role in mediating the severity of pancreatitis as well as the associated intestinal damage and inflammation.

[0110] In a specific embodiment, the intestinal disease includes an intestinal disease of the duodenum or colon. The pancreas and duodenum are anatomically close, and pancreatitis usually affects the duodenum, resulting in varying degrees of secondary inflammation.

[0111] In a specific embodiment, the related intestinal diseases caused by pancreatic diseases include duodenitis, colitis, intestinal mucosal damage, intestinal barrier function damage or intestinal flora imbalance caused by pancreatitis.

[0112] In this application, the pancreas-intestinal dialogue refers to the complex biological communication between the pancreas and intestinal organs based on a bidirectional pathway mediated by cellular, soluble, and neurohormonal effects. For example, pancreatic inflammation can induce intestinal inflammation, intestinal mucosal damage, and impaired intestinal barrier function, leading to the translocation of facultative pathogenic strains of bacteria into the inflamed pancreas, further triggering subsequent pancreatic necrosis, infection, and death. Intestinal microbial imbalance can promote the development of AP by destroying the intestinal barrier, triggering local or systemic inflammation, promoting bacterial translocation, and changing microbial metabolites.

[0113] The present application provides a pharmaceutical composition for treating, preventing or alleviating pancreatic diseases or related intestinal diseases caused by pancreatic diseases, which contains antimicrobial peptides.

[0114] In a specific embodiment, the antimicrobial peptide is REG3B, LL-37 or RNASE1.

[0115] In some embodiments, the pharmaceutical composition further comprises an Orai1 inhibitor.

[0116] In a specific embodiment, the Orai1 inhibitor is a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule that hybridizes with an Orai1 nucleic acid molecule, or a mutated Orai1 protein.

[0117] In some specific embodiments, the Orai1 inhibitor is GSK7975A, CM4620 or CM5480.

[0118] In some embodiments, the pharmaceutical composition further comprises a carrier for delivering the Orai1 inhibitor and / or antimicrobial peptide.

[0119] In some embodiments, the vector is a viral vector or a lipid nanoparticle.

[0120] In the present application, the viral vector may be, for example, an adeno-associated virus (Aav) vector, a lentiviral vector, an adenoviral vector, a retroviral vector, a herpes virus, an Sv40 vector, a poxvirus vector, or the like.

[0121] Example

[0122] This application provides general and / or specific descriptions of the materials and experimental methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight. All reagents or instruments used without manufacturer indication are commercially available conventional reagents.

[0123] The animals and main experimental methods used in the examples of this application are as follows.

[0124] animal . Orai1 f / f Mice were donated by Professor Xiao Bo from West China Hospital of Sichuan University. Sox9-CreERT2 mice were purchased from Jackson Laboratory. Orai1 mouse line Duct-specific deletion of Orai1 f / f The mice were crossed with Sox9-CreERT2 mice. Mouse littermates (Orai1f / f ) served as a control. All mice were maintained in a controlled environment at 22°C with a 12-hour light-dark cycle and provided with standard laboratory chow with free access to food and water. All mice used in this study were 8 to 12 weeks of age, and both male and female mice were used in all experiments.

[0125] PDEC isolation After incubation with FITC-conjugated bifluorolectin antibody (FL-1031, Vector Laboratories) and anti-FITC microbeads (130-048-701, Miltenyi Biotech), the antibody-positive cell fraction was separated using an MS chromatography column (130-042-201, Miltenyi Biotech). The unlabeled cells that passed through, representing the enriched PDECs, were collected and their purity was tested by qPCR and immunofluorescence.

[0126] Fluorescence microscopy to detect intracellular Ca 2+ and mitochondrial potential Cytosolic Ca 2+ The measurement of mitochondrial membrane potential can be carried out as follows (Wen et al. Gastroetnerology 2018): pancreatic duct epithelial cells or H6C7 cells are incubated in standard HEPES solution and loaded with 4 μM high affinity Ca 2+ The dye Fluo-4-AM (#F14201, Invitrogen) was incubated at room temperature for 0.5 hours. After transferring the cells to the perfusion chamber, the cells were imaged using a Leica SP8 confocal microscope (Leica). Ca was recorded at baseline. 2+The signal was stimulated for 60 seconds, followed by 1mM NaT, 200μM POA stimulation, and then 10μM Orail inhibitor (such as GSK-7975A) was applied at 100 seconds. To measure mitochondrial membrane potential, pancreatic duct epithelial cells or H6C7 cells were incubated in standard HEPES solution and loaded with 100nmol / L TMRM (#T668; Invitrogen) and cultured at room temperature for 0.5 hours. The cells were then transferred to a perfusion chamber and imaged using a Leica SP8 confocal microscope. The mitochondrial membrane potential was recorded for 60s at baseline and then stimulated with 1mM NaT, 200μM POA, and GSK-7975A (5 and 10μM). Fluorescence intensity (F) was analyzed using ImageJ software (National Institute of Health, Bethesda, MD) and normalized to baseline levels (F0).

[0127] Induction and treatment of experimental acute pancreatitis .right Mice were intraperitoneally injected with 100 mg / kg tamoxifen (T5648, Sigma) daily for 5 days to generate PDECs-specific Orai1-deficient mice. Experimental AP was induced after a 7-day interval. Caerulein hyperirritant pancreatitis was induced 10 hours later by intraperitoneal injection of 100 μg / kg caerulein (HY-A0190, MedChemExpress). Alcohol-induced AP was originally proposed by Huang et al. Mice received two intraperitoneal injections of 150 mg / kg palmitoleic acid (P9417, Sigma) and 1.35 g / kg ethanol, one hour apart. To mitigate ethanol-induced peritoneal irritation, 200 μL of saline was pretreated before the ethanol / POA treatment. In some experiments, 100 μg / kg REG3B (HY-P76003, MedChemExpress) was intraperitoneally injected immediately after the palmitoleic acid and ethanol injections. Biliary AP was induced by retrograde biliary ductal infusion of 2% sodium taurocholate (86339, Sigma), as previously described by Perides et al. The procedure was as follows: Mice were anesthetized with an intraperitoneal injection of 50 mg / kg pentobarbital. Subsequently, a small midline laparotomy was performed using aseptic technique, and the bile and pancreatic duct was exposed and cannulated through the duodenum using a 0.4 mm diameter needle connected to an infusion catheter. The proximal common bile duct was then clamped with a microvascular clamp. Subsequently, the mice were infused with 2% NaT at a rate of 7.5 μL / min (Harvard Apparatus) with an injection dose of 2 μL / g body weight. After infusion, the abdominal wall and skin were sutured separately. To ensure postoperative comfort, the mice were placed on a heating pad until they regained consciousness, and 0.05 mg / kg buprenorphine was given immediately after surgery to relieve pain. The researchers carefully observed the mice for physical activity, grooming behavior, alertness level, responsiveness to stimuli, and any potential indicators of pain. All mice were humanely killed at the designated time points.

[0128] Methods for determination of serum amylase and lipase Serum was obtained by centrifugation at 3000 g for 15 min at 4°C. Serum amylase and lipase were measured using amylase and lipase detection kits (C016-1-1 / A054-1-1, Nanjing Jiancheng Bioengineering Institute).

[0129] Histology, immunohistochemistry. Mouse pancreatic and intestinal tissues were fixed with 4% paraformaldehyde and then embedded in paraffin. 4 μm paraffin sections were taken for hematoxylin-eosin (H&E) staining and immunohistochemistry. Pancreatic injury was evaluated according to the scoring criteria previously reported in the literature. Duodenal and colonic injuries were evaluated according to the scoring criteria previously described by Chiu et al. and Williams et al., respectively. For immunohistochemistry, paraffin sections were dewaxed, rehydrated, and then boiled in citric acid antigen extraction solution (E673001-0250, Sangon Biotech) to extract antigens. The sections were then incubated with CD45 antibody (70257S, Signalling) overnight at 4°C and observed using an immunohistochemistry kit (SK-4105, Vector Laboratories).

[0130] Quantitative reverse transcription polymerase chain reaction Total RNA from pancreatic and intestinal tissues was extracted using Trizol reagent (15596018CN, Invitrogen) and then converted into cDNA using PrimeScript RT Master Mix (RR036B, TaKaRa). RT-qPCR was performed using TB Green Premix Ex Taq (RR420A, TaKaRa) in a QuantStudio 6Flex Real-Time PCR System (Applied Biosystems). 2-ΔΔ All primers were purchased from Sangon Biotech (Shanghai, China), and the primer sequences are shown in Table 1.

[0131] Table 1

[0132]

[0133]

[0134] Flow cytometry and fluorescence-activated cell sorting PDECs isolated from mice were stained with the following surface marker antibodies: Violet 510-conjugated Ghost Dye (13-0870, TONBO biosciences) and FITC-conjugated Dolichos biflorus agglutinin (FL-1031, Vector Laboratories). Cells were acquired on a BD LSRFortessa (BD Biosciences) and analyzed using FlowJo software, version 10.8.1 (Tree Star). To obtain PDECs, live DBA+ cells were sorted using a BD FACSAria III (BD Biosciences).

[0135] RNA sequencing According to the above method, Orai1f / f and PDECs were isolated from mouse pancreas, and total RNA was extracted. Because RNA concentrations in the processed PDECs were low, library construction and RNA sequencing (RNA-seq) were performed using the Switching Mechanism for 5' Ends of RNA Transcripts (SMART) technology, performed by OE Biotechnology. Gene expression was normalized to exon per million fragments mapped per kilobase. Differentially expressed genes were identified using a combined log2 (fold change) > 0.5 and P < 0.05 criteria.

[0136] Magnetic resonance imaging (MRI) . Twenty-four hours after the mice were intraperitoneally injected with a mixture of 1.35 g / kg ethanol and 150 mg / kg POA, the exocrine and exocrine pancreatic secretions in vivo were observed. 18 hours before the MRI examination, the mice were allowed to drink water freely. Exocrine pancreatic secretion was measured at 3 Tesla using an 8-channel phased array radio frequency coil of PhilipsMRI. All mice were subcutaneously injected with 1% pentobarbital (50 mg / kg, ip). The mice were first scanned with coronary artery T2WI. The sequence was acquired using the following image parameters: TR / TE 1000 / 60ms; matrix 324x 339; field of view 130x 104mm; slice thickness 2mm; 12 slices. The mice were then scanned with a coronary T2 map. The sequence was acquired using the following image parameters: TR / TE1000 / 12ms; matrix 288x 232; field of view 120x 120mm; slice thickness 2mm; 6 slices. Mice were injected with 200 μg / kg of secretin (HY-P1244A, MedChemExpress) via the tail vein. T2WI and T2 mapping coronary artery scans were repeated as described above. The time from injection to MRI was within 6 minutes. After scanning, images were analyzed using pseudocolor enhancement. An ROI of the same size and cross-section of the duodenum was selected for each mouse, and T2WI signal intensity and relaxation time were measured. T2WI signal intensity and relaxation time were evaluated before and after secretin stimulation. At least five ROIs were selected and the average was calculated.

[0137] FITC-dextran permeability assay . Intestinal permeability was assessed by intraluminal administration of FITC-dextran 4000 (46944, Sigma). Specifically, mice were orally administered FITC-dextran (250 mg / kg body weight) by gavage 4 hours before blood collection. The concentration of FITC-dextran in plasma was repeatedly measured using a fluorimeter (ARVO, Perkin-Elmer). A standard curve was established using FITC-dextran solutions of different dilutions. The absorption of 25 μL plasma samples or standard solutions was then evaluated in a fluorimeter at 488 nm.

[0138] 16S rRNA gene amplicon sequencingTotal genomic DNA samples were extracted using the OMEGA Soil DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer's instructions and stored at −20°C for further analysis. The quantity and quality of the extracted DNA were determined using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. The V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR using the forward primer (5'-ACTCCTACGGGAGGCAGCA-3') and the reverse primer (5'-GGACTACHVGGGTWTCTAAT-3'). 16S rRNA gene amplicon sequencing was performed using the NovaSeq platform (Shanghai, China) using the NovaSeq 6000SP reagent kit (500 cycles).

[0139] Bioinformatics analysis of the microbiome . Microbiome bioinformatics was performed using QIIME2 (version 2022.11) with slight modifications according to the official tutorial (https: / / docs.qiime2.org / 2022.11 / tutorials / ). Briefly, the raw sequence data were demultiplexed using the demux plugin and the primers were cut using the cutadapt plugin. The sequences were then quality filtered, denoised, merged, and chimeras removed using the DADA2 plugin. Non-singleton amplicon sequence variants (ASVs) were aligned with mafft and used to construct phylogeny with fasttree2. The classification sklearn naive Bayes classifier in the feature classifier plugin was used to assign classifications to ASVs for the SILVA version 138.1 database. Sequence data analysis was mainly performed using QIIME2 and R packages (v3.2.0). The gut microbiome was analyzed using the Personalbio Cloud platform ( https: / / www.genescloud.cn ). α-diversity indices at the ASV level, such as the Simpson index, were calculated using the ASV table in QIIME2 and displayed as box plots. ASV-level ordination abundance curves were generated to compare the richness and evenness of ASVs between samples. β-diversity analysis was performed using the Bray-Curtis metric to investigate structural variations in microbial communities between samples and visualized using principal coordinate analysis (PCoA). Taxonomic composition and abundance were analyzed using QIIME2 and displayed as box plots.

[0140] Pancreatic juice collectionMice were given 200 μg / kg of secretin (HY-P1244A, MedChemExpress) via retroorbital injection. The common bile and pancreatic duct was exposed, and a 1.5 mL EP tube connected to a 0.4 mm diameter needle was inserted through the duodenum to collect pancreatic juice for 1 hour.

[0141] LCMS / MS analysis . Protein extraction and digestion were performed using a proteomics sample preparation kit (PF-226089, BioMS), and peptide desalting was performed using a peptide purification kit (PD-23150, BioMS). The peptide components were redissolved in 10 μL of 0.1% FA solution. The samples were separated using a U3000 liquid chromatography system and a nanoViper C18 column (75 μm × 250 mm, 2 μm). The mobile phase consisted of water and 0.1% FA as solvent A and 80% acetonitrile and 0.1% FA as solvent B. A gradient elution from 1% to 35% was used, with a total run time of 90 minutes and a flow rate of 1.2 μL / min. The peptide mixture was analyzed and identified using an Orbitrap Exploris 480 mass spectrometer in high sensitivity mode. The instrument parameters were as follows: each full scan was set to high-speed data-dependent scanning, and the scan time was 60 minutes. The primary scan had a resolution of 120,000, a mass range of 350–1200 m / z, an AGC target of 3e6, and a maximum injection time of 50 ms. The secondary scan had a resolution of 30,000, collision energies set to 25%, 30%, and 35%, an AGC target of 2e5, a maximum injection time of 50 ms, and an 80 DIA scan window. Direct DIA database searching was performed (Version 17.2.230208., Quasar). The database used was uniprot-human-81803-20230327.fasta. The search parameters were set as follows: protein databases from the respective species, trypsin digestion with a maximum of two cleavage sites missing, and precursor and fragment ion mass tolerances of 10 ppm and 0.02 Da, respectively. Carbamidomethyl (C) was set as a fixed modification, and oxidation (M) and acetyl (n-terminus) were set as variable modifications. The false discovery rate (FDR) of peptides and proteins was controlled to be below 1.0%, and at least one unique peptide was identified for each protein. The q-value threshold was <1.0% when importing DIA data.

[0142] Statistical analysis. Data are expressed as mean ± SEM. All statistical analyses were performed using Prism 8.0 (GraphPad software). Comparisons between two groups were performed using an unpaired two-tailed Student's T test. To compare data from multiple groups, one-way analysis of variance followed by a Tukey post-test was used. In experiments with more than one independent variable, comparisons were assessed by two-way analysis of variance with a post hoc Tukey's multiple comparison test. P < 0.05 was considered statistically significant.

[0143] Study Approval All animal studies have been approved by the Animal Ethics Committees of Shanghai General Hospital (2019-A019-01) and Peking Union Medical College Hospital (XHDW-2023-154).

[0144] Example 1 Orai1 in PDECs for Ca 2+ and the effect on mitochondrial membrane potential

[0145] First, the intracellular Ca 2+ Whether the signal is mediated by Orai1. Human normal pancreatic ductal epithelial cell line H6C7 cells were used and treated with a potent Orai1 inhibitor GSK-7975A and the cells were detected using the above method. Internal Ca 2+ and mitochondrial potential The above experiments found that inhibition of Orai1 in pancreatic ductal cells can prevent intracellular Ca 2+ values ​​and preserves mitochondrial membrane potential in response to pancreatitis-associated toxins. Figure 1 In A, human pancreatic ductal cells (HPDE6c7) were stimulated with 200 μM palmitoleic acid (POA) or 1 mM sodium taurocholate (NaT). 2+ Tracing results showed that inhibition of Orai1 by 10 μM GSK-7975A significantly reduced the intracellular Ca2+ expression induced by POA or NaT. 2+ Signal (n = 10-22 per group). Mitochondrial dysfunction may be related to intracellular Ca 2+ We next evaluated the effect of Orai1 inhibition on mitochondrial membrane potential. Figure 1 TMRM traces in B show that 200 μM POA or 1 mM NaT induced a significant decrease in mitochondrial membrane potential ΔΨM, with CCCP (which inhibits mitochondrial ATP production) serving as a control. GSK-7975A demonstrated a concentration-dependent inhibitory effect on POA- or NaT-induced decreases in ΔΨm, indicating that mitochondrial membrane potential gradually increased with increasing GSK-79750A (n = 12-17 per group). This suggests that both NaT and POA significantly reduce mitochondrial membrane potential, potentially leading to a loss of ATP production, and that Orai1 inhibition maintains this decrease in mitochondrial membrane potential in a dose-dependent manner.

[0146] Furthermore, this result was verified in primary isolated PDECs. f / f The mouse line was fused with a tamoxifen-inducible Cre gene carrying the Sox9 promoter (Sox9-Cre ERT2 ) hybridization to selectively delete Orai1 in PDECs (like Figure 1 C). Seven days after tamoxifen injection, the magnetic beads-based biflorus lectin (DBA + )-positive cell sorting to isolate and enrich primary PDECs. ( Figure 2 A and B are schematic diagrams of the experimental procedure for isolating pancreatic ductal cells using magnetic beads. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect acinar, ductal, and endocrine markers to determine the purity of PDECs (n = 3 per group). Data are presented as mean ± SEM (e.g. Figure 2 C). The expression of Orai1 in purified primary pancreatic ductal cells was detected by qPCR (n=3 per group), and it was found that DBA + The expression of Orai1 in cells was reduced by 70% (e.g. Figure 1 (D). Isolated mouse pancreatic ductal cells were challenged with 200 μM POA or 1 mM NaT in HEPES buffer. Tracking changes in ΔΨm showed that pancreatic ductal cell-specific Orai1 deletion improved ΔΨm impaired by POA or NaT (n = 17-34 per group). Data are presented as mean ± SEM. *P < 0.05 compared with the control group (e.g., Figure 1 This result shows that the loss of Orai1 in primary PDECs preserved the mitochondrial membrane potential compared with the control group.

[0147] Collectively, these data suggest that inhibition or deletion of Orai1 in PDECs can prevent the intracellular Ca 2+ The sustained increase in mitochondrial membrane potential was maintained by pancreatitis-related toxins.

[0148] Example 2 Effect of Orai1 deficiency in PDECs on acute pancreatitis

[0149] The effect of Orai1 in PDECs on the severity of pancreatitis was studied using the AP experimental model.

[0150] Pancreatitis was induced by intraperitoneal injection of ethanol and POA to simulate alcohol-induced AP. f / f and Mice (n = 5-11 mice per group) were intraperitoneally injected with 150 mg / kg palmitoleic acid plus 1.35 g / kg ethanol every hour to simulate acute pancreatitis induced by ethanol and palmitoleic acid (POA). The experimental procedure was as follows: Figure 3 As shown in A. The experimental results showed that the loss of Orai1 in PDECs led to a significant decrease in serum amylase and lipase levels (as shown in Figure 3 Pancreatic histological scores and subscores of edema, inflammatory infiltration, and necrosis were evaluated, and the results showed that local pancreatic damage was partially alleviated ( Figure 3 C is a representative image of hematoxylin and eosin-stained pancreatic sections. Figure 3 D is the histological severity score including subscores of edema, inflammation, and necrosis). In addition, the expression of proinflammatory cytokines in the pancreas, including tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β), was assessed by qPCR and the results showed that these cytokines were also significantly downregulated (e.g. Figure 3 E).

[0151] Furthermore, NaT was retrogradely infused into the bile-pancreatic duct to simulate biliary AP, which is also one of the most common causes of AP. f / f and In mice (n = 5-7 mice per group), biliary AP was simulated by retrograde infusion of 2% sodium taurocholate (NaT) into the bile and pancreatic ducts to induce biliary acute pancreatitis. The experimental procedure was as follows: Figure 4 A. The experiment also observed a significant decrease in serum amylase and lipase levels (such as Figure 4 Local pancreatic damage was also alleviated, and the pancreatic histological score was improved, with a decrease in the subscores of edema, inflammatory infiltration, and necrosis (as shown in Figure 2). Figure 4 C and 4D). The expression of proinflammatory cytokines such as TNF-α and IL-1β in the pancreas was downregulated (as shown in Figure 4 E).

[0152] Taken together, these data demonstrate that Orai1 deletion in PDECs prevents local pancreatic injury and inflammation, i.e., attenuates pancreatitis, in two clinically representative AP models.

[0153] Example 3 Effect of Orai1 deficiency in PDECs on restoring impaired pancreatic juice secretion and intestinal epithelial permeability

[0154] DBA of pancreas by flow cytometry + Transcriptome analysis was performed to examine whether the loss of Orai1 would lead to changes in the intrinsic gene characteristics of PDECs. f / f and FACS sorted pancreatic ductal cells from mice (n=3 per group) were subjected to RNA sequencing (RNA-seq) analysis. The experimental procedure is shown in the following figure. Figure 5 As shown in A. DBA was confirmed by flow cytometry analysis. +Successful enrichment and purity of cells. 83% of live cells after sorting were DBA, compared to 1.84% of live cells in unsorted pancreatic single cell suspension. + Ductal cells (eg Figure 5 The volcano plot showed that compared with the control group (no pancreatitis), pancreatitis induced a total of 1078 differentially expressed genes (DEGs), of which 588 were upregulated and 490 were downregulated (as shown in Figure 2B). Figure 5 C). Orai1 with AP f / f Compared with mice with AP There were 300 DEGs in mice, of which 129 were upregulated and 171 were downregulated (e.g. Figure 5 D). The Venn diagram further showed that only 75 DEGs were identified when the DEGs induced by pancreatitis alone were compared with those induced by Orai1 deficiency in PDECs (Fig. Figure 5 E). Further analysis showed that the down-regulated genes in pancreatitis alone and those in AP were Compared with the upregulated genes in mice, only 32 DEGs were identified (e.g. Figure 5 F). In contrast, the expression of β-catenin in pancreatitis alone was compared with that in patients with AP. Among the genes downregulated in mice, 28 DEGs were found (e.g. Figure 5 Among the identified DEGs, only a limited number of genes were functionally explained in association with the protective effects observed in AP, suggesting that the protective effects observed upon Orai1 depletion in PDECs may not be mediated by changes in PDEC-intrinsic signaling.

[0155] Given the important role of PDECs in regulating pancreatic fluid secretion, it is speculated that the loss of Orai1 may directly affect pancreatic secretion. To test this, secretin-enhanced magnetic resonance imaging (sMRI) was used to quantify the T2-weighted imaging (T2WI) signal near the duodenum of mice. It was found that both alcohol + POA- and nat-induced pancreatitis led to a significant decrease in T2WI signal, indicating that pancreatic secretion was significantly impaired during pancreatitis. f / f Compared with mice, the loss of Orai1 in PDECs did not affect pancreatic secretion at baseline. However, during AP, the loss of Orai1 in pancreatic ductal cells effectively restored impaired pancreatic secretion. 24 hours after pancreatitis induction, secretin (200 μg / kg) was injected through the tail vein, and MRI imaging was recorded within 6 minutes. ROIs of the duodenum of each mouse of the same size and cross-section were selected to measure T2WI signal intensity and relaxation time. T2WI signal intensity and relaxation time were evaluated before and after secretin stimulation. The results are shown in Figure 3. Figure 6These data support the view that Orai1 in PDECs may play a protective role against pancreatitis by directly mediating pancreatic secretion.

[0156] Since some studies have shown that pancreatic exocrine insufficiency significantly increases intestinal permeability in both patients and animal models, we investigated whether the loss of Orai1 in PDECs affects intestinal epithelial permeability. We evaluated the flux of fluorescein isothiocyanate (FITC)-conjugated dextran from the intestinal lumen into the circulation by intragastric administration and showed that pancreatitis led to a significant increase in intestinal epithelial cell permeability (e.g., Figure 6 E). with Orai1 f / f Compared with mice, Orai1 deficiency in PDECs did not affect intestinal epithelial permeability at baseline. However, during AP, this deficiency helped maintain intestinal epithelial integrity and was associated with reduced pancreatitis severity (e.g., Figure 6 F and 6G). Collectively, these data suggest that loss of Orai1 in PDECs does not directly induce a pancreatic ductal cell-intrinsic gene signature but rather protects against pancreatitis by restoring impaired pancreatic secretion and maintaining intestinal epithelial barrier function.

[0157] Example 4 Effects of Orai1 deficiency in PDECs on duodenum and colon injury and inflammation

[0158] Because intestinal epithelial leakage is one of the indicators of intestinal damage and is associated with disease prognosis, pancreatitis-related intestinal damage is a major determinant of AP severity and prognosis. Therefore, we further studied the effect of Orai1 deficiency in PDECs on intestinal damage and inflammation in experimental AP patients. f / f and Mice (n = 4-11 mice per group) were intraperitoneally injected with 150 mg / kg POA plus 1.35 g / kg ethanol every hour to induce ethanol-plus-palmitolenic acid (POA)-induced acute pancreatitis. Pancreatitis induced by the combination of ethanol and POA caused mild but significant tissue damage in the duodenum and colon, as assessed by H&E scores. Orai1 deficiency in PDECs reduced duodenal epithelial damage, colonic crypt damage, and histological severity scores of inflammation in the duodenum and colon (e.g., Figure 7 As shown in A). CD45 immunostaining to assess inflammatory infiltration showed that pancreatitis caused significant inflammation in the duodenum and colon. In PDECs, loss of Orai1 resulted in a decrease in the area of ​​CD45 positive staining ( Figure 7 B). Similarly, RNA from duodenum and colon tissues was analyzed using RT-qPCR, showing that AP In the duodenum and colon of mice, the expression of pro-inflammatory genes Il1b, Il6 and Tnf was downregulated (e.g. Figure 7 C).

[0159] We further investigated the effects of Orai1 deficiency on the intestinal epithelial barrier. An important determinant of epithelial barrier function and paracellular permeability is the maintenance of tight junction expression between cells. Tight junctions are composed of a protein complex that includes occludin, cadherin, and zona occlusae (ZO). We found that Orai1 deficiency in PDECs upregulated the expression of tight junction genes, including cadherin-1 (Cdh1), Zo1, Zo2, and Zo3 in the duodenum and colon, with more profound effects in the duodenum (e.g., Figure 7 D), indicating that Orai1 deficiency is involved in maintaining tight junction integrity, particularly in the duodenum.

[0160] The above results were further verified by different AP experimental models. f / f and In mice (4-10 mice per group), 2% NaT was retrogradely infused into the bile-pancreatic duct to simulate bile AP, induced by biliary acute pancreatitis. The experimental results showed that bile-induced pancreatitis induced by retrograde NaT infusion into the bile-pancreatic duct significantly caused damage and inflammation in the duodenum and colon, while the loss of Orai1 in PDECs alleviated this situation ( Figure 8 CD45 immunostaining showed that Orai1 deficiency in PDECs reduced the significant inflammation in the duodenum and colon induced by pancreatitis (Figure 4A). Figure 8 Similarly, pro-inflammatory genes Il1b, Il6, and Tnf were expressed in Expression was downregulated in the mouse duodenum but not in the colon (e.g. Figure 8 C). Loss of Orai1 in PDECs upregulated the expression of tight junction genes Cdh1, Zo1, Zo2, and Zo3 in the duodenum and colon (as shown in Figure 8 D). Taken together, these results indicate that Orai1 deletion in PDECs can primarily alleviate duodenal epithelial injury and inflammatory responses in two representative AP models.

[0161] Example 5 Effect of Orai1 deficiency in PDECs on the microbiota in the duodenum

[0162] Because the gut microbiota plays a crucial role in multiple physiological processes, dysbiosis is associated with intestinal infections and chronic inflammatory diseases. Furthermore, alterations in the gut microbiome, including intestinal barrier impairment, immune dysfunction, and microbiome dysbiosis, are associated with the development and severity of acute pancreatitis (AP). Therefore, we investigated the impact of Orai1 on intestinal composition during AP by analyzing the microbiome composition of the duodenum and cecal contents of Orai1-deficient mice during ethanol-plus-palmitoleic acid-induced acute pancreatitis. Acute pancreatitis induced by ethanol-plus-palmitoleic acid (POA) was induced by a two-hour intraperitoneal injection of 150 mg / kg palmitoleic acid (POA) and 1.35 g / kg ethanol, and microbiome analysis was performed using collected duodenal and cecal samples (n = 5-8 mice per group). At baseline (without pancreatitis), duodenum Orai1 deficiency did not affect duodenal and colonic α-diversity assessed by Simpson Rarefraction Plot, whereas pancreatitis reduced duodenal microbiome α-diversity. However, pancreatitis had no effect on cecal α-diversity. Orai1 deficiency restored the pathological reduction in alpha-diversity of the duodenal microbiota (e.g. Figure 9 A and 9B). Principal component analysis (PCA) further demonstrated that Orai1 f / f There is a clear separation between the AP and baseline duodenal and cecal microbiota in mice. f / f and The PCA plot of the mouse duodenal microbiota also showed clear separation, but not in the colon (e.g. Figure 9 C and 9D), indicating that Orai1 in PDECs does have regional differences in its impact on the gut microbiota, with more profound effects in the duodenum.

[0163] To further investigate the effect of Orai1 on microbial composition in PDECs, the relative abundance of each taxonomic group was evaluated at different taxonomic levels. It was also found that during alcohol + POA-induced pancreatitis, the loss of Orai1 in PDECs significantly altered the composition of the duodenal microbiota at the family and genus levels (e.g., Figure 9 E and 9F). Specifically, at the family level in the duodenum, pancreatitis induced a significant increase in Enterobacteriaceae and a significant decrease in Lactobacillaceae. Orai1 deficiency in PDECs significantly reversed these changes in Enterobacteriaceae and Lactobacillaceae. At the genus level in the duodenum, pancreatitis induced a significant increase in Shigella and a significant decrease in Lactobacillus and Halomonas. Orai1 deficiency in PDECs similarly reversed these observed changes.

[0164] Comparison of representative bacterial taxa at the genus level in the duodenum and cecum of pancreatitis induced by ethanol plus POA revealed significant changes in relative abundance of taxa in Orai1. f / fand In mice, acute pancreatitis induced by ethanol plus palmitoleic acid (POA) was induced by intraperitoneal injection of 150 mg / kg POA plus 1.35 g / kg ethanol over two hours. The experimental results further found that pancreatitis led to an increase in the abundance of Shigella spp. in the duodenum, while the abundance was significantly reduced when Orai1 was deleted in PDECs (e.g. Figure 10 The abundance of Lactobacillus decreased in ethanol + POA-induced pancreatitis, while the abundance of Lactobacillus increased after Orai1 deletion (as shown in Figure 2A). Figure 10 In the cecum, pancreatitis induced significant changes in microbiota composition at the family and genus levels compared to the duodenal microbiota. Loss of Orai1 induced smaller changes (e.g. Figure 9 G and 9H). Comparative analysis showed that although pancreatitis itself led to a decrease in the abundance of Lactobacillus spp., in Orai1 f / f and In mice, its abundance remains unchanged (e.g. Figure 10 C). In addition, in Orai1 with or without pancreatitis f / f and In mice, the abundance of other genera such as Desulfovibrio and Allobacterium also did not change (e.g. Figure 10 These results indicate that Orai1 deficiency in PDECs primarily protects against duodenal dysbiosis in acute pancreatitis.

[0165] Example 6 Orai1-mediated secretion of antimicrobial peptides

[0166] Given that Orai1 deficiency in pancreatic ductal cells primarily reduces intestinal tissue damage, inflammation, and the composition of the duodenal gut microbiota, we next performed proteomic analysis of pancreatic juice from Orai1-deficient mice in ethanol-plus-POA-induced acute pancreatitis to identify potential components in PDECs mediated by Orai1 that may play an important role in regulating pancreatitis-associated intestinal changes. f / f and Mice (n = 4-8 mice per group) were intraperitoneally injected with 150 mg / kg POA plus 1.35 g / kg ethanol every hour to induce ethanol plus palmitoleic acid (POA)-induced acute pancreatitis. Pancreatic juice was collected 4 hours after pancreatitis induction and analyzed by tandem mass spectrometry (LC-MS / MS) (experimental strategy as described in Figure 11A total of 170 proteins were detected in mouse pancreatic juice. Pancreatitis itself specifically induced 35 downregulated proteins compared with the control group (no pancreatitis), mainly digestive enzymes and antimicrobial peptides. At baseline, loss of Orai1 resulted in limited differential protein changes; however, during pancreatitis, loss of Orai1 selectively upregulated 17 proteins (e.g., Figure 11 B). Further analysis revealed that down-regulated proteins and In the comparison of upregulated proteins in mice, only 11 differentially altered proteins were found (e.g. Figure 11 C). These proteins are mainly divided into two categories: one group belongs to antimicrobial peptides, including regenerating islet-derived 3β (REG3B) and ribonuclease A family member 1 (RNASE1); the other group is digestive enzymes, including amylase α1A (AMY1), carboxypeptidase A1 (CPA1) and carboxypeptidase A2 (CPA2) (as shown in Figure 2). Figure 11 Therefore, it is speculated that the secretion of antimicrobial peptides, especially REG3B, RNASE1 and cathelicidins (LL-37), may be potential regulatory factors in pancreatic juice, and Orai1-mediated secretion of antimicrobial peptides can affect the severity of pancreatitis and its related intestinal damage and inflammation.

[0167] Example 7 Role of Antimicrobial Peptides in Pancreatitis and Related Intestinal Damage and Inflammation

[0168] Further research was conducted to investigate the effects of exogenous supplementation of antimicrobial peptides such as REG3B on experimental AP. f / f and In mice, acute pancreatitis induced by ethanol plus POA was induced by intraperitoneal injection of 150 mg / kg POA and 1.35 g / kg ethanol over a two-hour period. REG3B (100 μg / kg, intraperitoneal injection) was administered immediately after the last ethanol and POA injection (n = 4-10 mice per group), and a control group was set up. Samples were analyzed 24 hours after the first injection (experimental strategy as shown in Figure 12 The results showed that exogenous supplementation of REG3B could reduce the expression of Orai1 f / f Serum amylase and lipase in mice. No additional reduction was observed in mice (e.g. Figure 12 As assessed by histopathological severity scores and the expression of proinflammatory cytokines, pancreatic local lesions showed similar changes after exogenous REG3B supplementation (as shown in Figure 2B). Figure 12 C and 12D).

[0169] Next, we evaluated the effect of REG3B on pancreatitis-associated intestinal damage and inflammation in the duodenum and colon. Similarly, we observed that REG3B supplementation reduced intestinal damage in these two regions, as assessed by H&E scores. In mice, no further reduction was observed (e.g. Figure 12 E and Figure 13 Intestinal inflammation, as measured by CD45 immunostaining, was also reduced (Figure 4A). Figure 12 F and 13B). In response to REG3B supplementation, the expression of proinflammatory cytokines such as Il1b, Il6, and Tnf, as well as the expression of genes related to duodenum and colonic tight junctions, also showed similar changes (as shown in Figure 12 G, 12H and Figure 13 C and 13D). Collectively, these data suggest that supplementation of REG3B provides substantial protection against AP, ameliorating the severity of pancreatitis and associated intestinal damage and inflammation.

[0170] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application in any other manner. Any person skilled in the art may utilize the above disclosed technical content to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the present application.

Claims

1. Use of an antimicrobial peptide in the preparation of a medicament for treating, preventing, or alleviating pancreatic diseases or related intestinal diseases caused by pancreatic diseases, wherein the antimicrobial peptide is preferably a C-type lectin antimicrobial peptide, a cathelicidins family antimicrobial peptide, or an RNase A family antimicrobial peptide, and further preferably, the antimicrobial peptide is REG3B, LL-37, or RNASE1.

2. Use of Orai1 inhibitors in the preparation of drugs for treating, preventing or alleviating related intestinal diseases caused by pancreatic diseases.

3. The use according to claim 1 or 2, wherein The pancreatic diseases include pancreatitis, pancreatic damage, and impaired pancreatic juice secretion.

4. The use according to claim 3, wherein The pancreatitis includes acute pancreatitis or chronic pancreatitis, and preferably the acute pancreatitis is acute biliary pancreatitis.

5. The use according to claim 1 or 2, wherein The related intestinal diseases caused by pancreatic diseases are intestinal inflammation caused by pancreatitis, intestinal dysbiosis or pancreatico-intestinal dialogue diseases.

6. The use according to claim 1 or 2, wherein The related intestinal diseases caused by pancreatic diseases include duodenitis, colitis, intestinal mucosal damage, intestinal barrier function damage or intestinal flora imbalance caused by pancreatitis.

7. The use according to claim 1, wherein The Orai1 inhibitor includes a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule hybridizing with an Orai1 nucleic acid molecule, or a mutated Orai1 protein. Preferably, the Orai1 inhibitor is GSK-7975A, CM4620, or CM5480.

8. A pharmaceutical composition for treating, preventing or alleviating pancreatic diseases or related intestinal diseases caused by pancreatic diseases, comprising an antimicrobial peptide, preferably a C-type lectin antimicrobial peptide, a cathelicidins family antimicrobial peptide or an RNase A family antimicrobial peptide, and more preferably, the antimicrobial peptide is REG3B, LL-37 or RNASE1.

9. The pharmaceutical composition according to claim 8, further comprising an Orai1 inhibitor, preferably the Orai1 inhibitor is a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule hybridizing with an Orai1 nucleic acid molecule, or a mutated Orai1 protein.

10. The pharmaceutical composition according to claim 8 or 9, further comprising a carrier for delivering the antimicrobial peptide and / or Orai1 inhibitor.

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