Use of high-density lipoprotein in the preparation of a medicament for treating acute pancreatitis

Abstract

The present invention discloses the use of high-density lipoprotein in the preparation of a medicament for preventing or treating acute pancreatitis. The medicament for treating acute pancreatitis provided by the present invention contains high-density lipoprotein, which can effectively reduce the inflammatory response, inflammatory cell infiltration and necrosis of pancreatic acinar cells in patients with acute pancreatitis. The medicament for treating acute pancreatitis provided by the present invention contains high-density lipoprotein, which can selectively accumulate in the pancreatic tissue of patients with acute pancreatitis. High-density lipoprotein not only has the effect of treating acute pancreatitis itself, but also high-density lipoprotein-related nano-biological preparations can be used to carry other anti-inflammatory drugs for targeted treatment of acute pancreatitis, having good clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of high-density lipoprotein in the preparation of drugs for treating acute pancreatitis. Background Art

[0002] Acute Pancreatitis (AP) is an inflammatory disease caused by the injury and necrosis of pancreatic acinar cells. In severe cases, it can develop into systemic inflammatory response syndrome (SIRS) and be accompanied by organ dysfunction. Severe Acute Pancreatitis (SAP) is a serious condition that can develop SIRS and persistent organ failure (persistent OF), with a mortality rate as high as 36 - 50%. SIRS and infected pancreatic necrosis (IPN) are important factors leading to organ failure (OF) and the main cause of the high mortality rate of AP. However, there is currently a lack of effective drugs for clinically controlling SIRS and IPN.

[0003] HDL (high-density lipoprotein) is a lipoprotein with a small volume and high density in human plasma, with a diameter of 8 - 10 nm and a density of 1.063 - 1.210 g / mL. HDL is mainly composed of proteins and lipids, and the protein and lipid content and types in different subtypes of HDL are different. Under physiological conditions, the proteins in HDL mainly include apolipoproteins, enzymes, lipid transport proteins, etc. The lipids in HDL mainly include phospholipids, sphingolipids, and neutral lipids. Among them, apolipoprotein A-1 (apo A-1) is the main structural and functional protein of HDL, accounting for about 70% of the protein content of HDL. Phospholipids and cholesterol are the main lipids of HDL, accounting for about 68% of the lipid content of HDL. For a long time, the research on HDL by scientific researchers has focused on the field of cardiovascular diseases mainly coronary atherosclerotic heart disease. Research shows that reverse cholesterol transport (RCT) and other lipid transports are important biological functions of HDL. HDL prevents and treats coronary atherosclerotic-related diseases by reverse transporting cholesterol in atherosclerotic plaque macrophages. However, there is currently no report on the research of HDL in treating AP. Summary of the Invention

[0004] To solve the problems existing in the prior art, the present invention provides the use of high-density lipoprotein in the preparation of a drug for preventing or treating acute pancreatitis.

[0005] To achieve the above object, the specific solution of the present invention is as follows:

[0006] The present invention provides the use of high-density lipoprotein in the preparation of a drug for preventing or treating acute pancreatitis.

[0007] Further, in the above technical solution, the high-density lipoprotein is the high-density lipoprotein in the blood of healthy humans.

[0008] Further, in the above technical solution, the high-density lipoprotein contains, but is not limited to, apolipoprotein A-1 (apoA-1), apolipoprotein A-2 (apo A-2), paraoxonase 1 (PON1), phospholipid transfer protein (PLTP), phospholipids, sphingolipids, and cholesterol.

[0009] Further, in the above technical solution, the high-density lipoprotein is prepared into a drug preparation of a single component, or combined with other drugs to prepare a compound drug preparation as a carrier.

[0010] Further, in the above technical solution, the dosage form of the drug preparation includes: injection, tablet, pill, capsule or powder.

[0011] Further, in the above technical solution, the intake method of the drug preparation includes: oral administration, intravenous injection or subcutaneous injection.

[0012] Further, in the above technical solution, the intake dose of the drug preparation is 1 to 100 mg / kg body weight.

[0013] Preferably, the intake dose of the drug preparation is 10 to 50 mg / kg body weight.

[0014] The present invention also provides a drug for acute pancreatitis, comprising high-density lipoprotein.

[0015] Beneficial effects

[0016] (1) The drug for treating acute pancreatitis provided by the present invention contains high-density lipoprotein, which can effectively reduce the inflammatory response, inflammatory cell infiltration and necrosis of pancreatic acinar cells in patients with acute pancreatitis.

[0017] (2) The drug for treating acute pancreatitis provided by the present invention contains high-density lipoprotein, which can selectively accumulate in the pancreatic tissue of patients with acute pancreatitis. High-density lipoprotein not only has the effect of treating acute pancreatitis itself, but also high-density lipoprotein-related nanobiological preparations can be used to transport other anti-inflammatory drugs for targeted treatment of acute pancreatitis, having good clinical application prospects.

[0018] (3) High-density lipoprotein belongs to the endogenous lipoprotein in healthy human bodies, with high safety, no toxic or side effects, reducing the research cost and R & D cycle of drugs for treating acute pancreatitis; meanwhile, high-density lipoprotein also has the activities of anti-atherosclerosis and regulating blood lipids, especially having great potential for treating hyperlipidemic pancreatitis.

[0019] (4) The present invention develops a new medical use for endogenous high-density lipoprotein in healthy human bodies, providing a new solution for the R & D of drugs for clinical treatment of acute pancreatitis. Description of the Drawings

[0020] Figure 1 are the levels of endogenous HDL in the sera of mice in the Con group and the AP group and the levels of endogenous HDL, apo A-1 and 2, and SAA in the pancreatic tissues; among them, Figure 1 A shows the levels of endogenous HDL in the sera of mice in the Con group and the AP group detected by ELISA (n = 6). Figure 1 B shows the levels of endogenous HDL in the pancreatic tissues of mice in the Con group and the AP group detected by ELISA (n = 6). Figure 1 C shows the levels of apo A-1 and 2 in the pancreatic tissues of mice in the Con group and the AP group detected by DIA quantitative proteomics (n = 6). Figure 1 D shows the levels of SAA in the pancreatic tissues of mice in the Con group and the AP group detected by DIA quantitative proteomics (n = 6). The significant differences are respectively indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0021] Figure 2 are the distributions of FITC-labeled HDL in the pancreatic tissues and adjacent organs of mice in the Con group and the AP group; among them, Figure 2 A shows the FITC fluorescence pictures of the abdomens of mice in the Con+FITC-HDL group and the AP+FITC-HDL group (from left to right) and the average FITC fluorescence intensity of the pancreatic tissues of mice collected by a small animal in vivo imaging system (Spectrum in vivo imaging system, IVIS) 6 hours after injection (n = 6). Figure 2 B shows the FITC fluorescence pictures of pancreatic tissue sections collected by a fluorescence microscope of the pancreatic tissues of mice (n = 4). The significant differences are respectively indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0022] Figure 3 are that HDL is a necessary factor for the selective aggregation of FITC-HDL in the pancreatic tissues of AP mice; among them, Figure 3At 6 hours after injection, FITC fluorescence images of the abdominal regions of mice in the Con+FITC group, Con+FITC-HDL group, AP+FITC group, and AP+FITC-HDL group (from left to right) were collected by IVIS. The pancreatic tissues of the four groups of mice were taken out under light-proof conditions, and FITC fluorescence images of the pancreatic tissues of the four groups of mice were collected by IVIS (n = 1).

[0023] Figure 4 To reveal the biological functions closely related to endogenous HDL in the pancreatic tissues of AP mice by GO enrichment analysis (GOEA); Figure 4 A shows the proteins with significant changes in the pancreatic tissues of AP group mice screened by DIA quantitative proteomics (compared with Con group mice), with the criterion of p < 0.05 (n = 6); Figure 4 B shows that 158 proteins (R 2 > 0.9) related to the level of endogenous HDL in the pancreatic tissues of AP mice were classified according to Log2(AP / Con)Ratio > 0 or < 0 (n = 6); Figure 4 C is a GO enrichment analysis diagram, with the criterion of FDR < 0.05 (the red circles represent the GO analysis results of 52 up-regulated proteins, and the blue circles represent the GO analysis results of 106 down-regulated proteins; the size of the circles represents the intensity of the GO analysis results).

[0024] Figure 5 For the serum enzyme indexes and pathological damage conditions of pancreatic tissues in each group of mice; among them, Figure 5 A shows the effect of 20 mg / mL HDL on the serum amylase level of AP mice detected by ELISA (n = 6). Figure 5 B shows the effect of 20 mg / mL HDL on the serum lipase level of AP mice detected by ELISA (n = 6). Figure 5 C shows the HE staining images (100X) of pancreatic tissue sections of mice in the Con group, AP group, and HDL group. The significant differences are represented as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0025] Figure 6 For the mRNA expression of pro-inflammatory cytokines in pancreatic acinar 266-6 cells of each group of mice; among them, Figure 6 A shows the effect of gradient concentrations of STC on the viability of mouse pancreatic acinar 266-6 cells measured by the CCK-8 method; Figure 6Group B: Detection of TNF-α mRNA expression in pancreatic acinar 266-6 cells of mice in the Normal group, STC group, and HDL group by Real-time PCR (n = 4). Panel 6C: Detection of IL-6 mRNA expression in pancreatic acinar 266-6 cells of mice in the Normal group, STC group, and HDL group by Real-time PCR (n = 4). Statistically significant differences are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0026] Figure 7 Are the levels of pro-inflammatory cytokines in the sera and pancreatic tissues of mice in each group; Figure 7 Panel A: Detection of the effect of 20 mg / mL HDL on the mRNA level of TNF-α in the sera of AP mice by ELISA (n = 6). Figure 7 Panel B: Detection of the effect of 20 mg / mL HDL on the mRNA level of IL-6 in the sera of AP mice by ELISA (n = 6). Figure 7 Panel C: Detection of the effect of 20 mg / mL HDL on the mRNA level of IL-8 in the sera of AP mice by ELISA (n = 6). Figure 7 Panel D: Detection of the effect of 20 mg / mL HDL on the TNF-α mRNA expression in the pancreatic tissues of AP mice by Real-time PCR technique (n = 6). Figure 7 Panel E: Detection of the effect of 20 mg / mL HDL on the IL-6 mRNA expression in the pancreatic tissues of AP mice by Real-time PCR technique (n = 6). Figure 7 Panel F: Detection of the effect of 20 mg / mL HDL on the IL-8 mRNA expression in the pancreatic tissues of AP mice by Real-time PCR technique (n = 6). Statistically significant differences are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0027] Figure 8 Are the infiltrations of macrophages and neutrophils in the pancreatic tissues of mice in each group; Statistically significant differences are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0028] Figure 9 Are the effects of HDL on the levels of TNF-α and IL-6 in the supernatants of LPS-induced mouse Raw264.7 cells; Among them, Figure 9 Panel A: Determination of the effect of gradient concentrations of LPS (O55:B5) on the viability of mouse Raw264.7 cells by CCK-8 assay. Figure 9 Panel B: Determination of the effect of gradient concentrations of LPS (O111:B4) on the viability of mouse Raw264.7 cells by CCK-8 assay. Figure 9C shows the effect of 2 mg / mL HDL on the TNF-α level in the supernatant of mouse Raw264.7 cells induced by 1 μg / mL LPS (O55:B5) and LPS (O111:B4) detected by ELISA. Figure 9 D shows the effect of 2 mg / mL HDL on the IL-6 level in the supernatant of mouse Raw264.7 cells induced by 1 μg / mL LPS (O55:B5) and LPS (O111:B4) detected by ELISA. Statistically significant differences are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0029] Figure 10 GO enrichment analysis reveals the functions of multiple organelles regulated by HDL in the pancreatic tissue of AP mice; among them, Figure 10 A is to screen the significantly changed proteins in the pancreatic tissue of mice in the HDL group (compared with mice in the AP group), with p < 0.05 as the standard (n = 6). Figure 10 B is a GO enrichment analysis diagram, with FDR < 0.05 as the standard (the red circle represents the GO analysis results of 228 up-regulated proteins, and the blue circle represents the GO analysis results of 486 down-regulated proteins; the size of the circle represents the intensity of the GO analysis results).

[0030] Figure 11 Protein expression and distribution of key HDL receptors SR-B1 and ABCG1 in the pancreatic tissue of AP mice; among them, Figure 11 A is to detect the protein expression of SR-B1 in the pancreatic tissue of mice in the Con group, AP group and HDL group by Western Blotting (n = 3). Figure 11 B is to detect the protein expression of ABCG1 in the pancreatic tissue of mice in the Con group, AP group and HDL group by Western Blotting (n = 3). Figure 11 C shows the protein expression and expression location of SR-B1 and ABCG1 in the pancreatic tissue of mice in the Con group, AP group and HDL group by immunofluorescence of pancreatic tissue (n = 3), and the IOD values of SR-B1 and ABCG1 in pancreatic tissue samples are calculated using Image-Pro Plus 6.0 software. Statistically significant differences are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0031] Figure 12 STC induces an increase in the protein expression of SR-B1 in mouse pancreatic acinar 266-6 cells.

[0032] Figure 13 The SR-B1 antagonist ITX5061 inhibits the down-regulating effect of HDL on the levels of pro-inflammatory factors in the serum and pancreatic tissue of AP mice; among them, Figure 13A was for detecting the levels of TNF-α in the sera of mice in the Con group, AP group, HDL group, and ITX5061 group by ELISA (n = 6); Figure 13 B was for detecting the levels of IL-6 in the sera of mice in the Con group, AP group, HDL group, and ITX5061 group by ELISA (n = 6); Figure 13 C was for detecting the mRNA expression of TNF-α in the pancreatic tissues of mice in the Con group, AP group, HDL group, and ITX5061 group by Real-time PCR (n = 4). Figure 13 D was for detecting the mRNA expression of IL-6 in the pancreatic tissues of mice in the Con group, AP group, HDL group, and ITX5061 group by Real-time PCR (n = 4). The statistically significant differences were respectively expressed as: *p < 0.05, **p < 0.01, ***p < 0.001.

[0033] Figure 14 To knockdown SR-B1 and inhibit the down-regulation effect of HDL on the mRNA expressions of TNF-α and IL-6 in mouse pancreatic acinar 266-6 cells induced by STC; among them, Figure 14 A was for screening the knockdown efficiency of 4 kinds of SR-B1 plasmid DNAs on SR-B1 in mouse pancreatic acinar 266-6 cells by Real-time PCR technology; Figure 14 B was for verifying the knockdown effect of SR-B1-901 plasmid DNA on SR-B1 in mouse pancreatic acinar 266-6 cells by Western Blotting technology; Figure 14 C was for detecting the mRNA expression of TNF-α in mouse pancreatic acinar 266-6 cells in the Normal group, STC group, HDL group, and shRNA / SR-B1 group by Real-time PCR technology. Figure 14 D was for detecting the mRNA expression of IL-6 in mouse pancreatic acinar 266-6 cells in the Normal group, STC group, HDL group, and shRNA / SR-B1 group by Real-time PCR technology. The statistically significant differences were respectively expressed as: *p < 0.05, **p < 0.01, ***p < 0.001. Specific implementation manners

[0034] The following non-limiting examples can enable those of ordinary skill in the art to more comprehensively understand the present invention, but do not limit the present invention in any way.

[0035] Example 1

[0036] The mouse model of AP was induced by the combined injection of caerulein (Cae) and lipopolysaccharide (LPS). C57BL / 6 mice were fasted and water-deprived for 12 hours before modeling. Cae at a concentration of 20 μg / mL was intraperitoneally injected 7 times (50 μg / kg body weight) at 1-hour intervals. At the same time as the 7th injection of Cae, LPS O111:B4 at a concentration of 1 mg / mL (10 mg / kg body weight) was intraperitoneally injected. C57BL / 6 mice were divided into 2 groups according to the random table method. ① Control group (Con): Healthy mice were intraperitoneally injected with normal saline; ② Model group (AP): Healthy mice were intraperitoneally injected with Cae and LPS.

[0037] At 24 hours after the first injection of Cae, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg / kg), and blood samples and pancreatic samples were collected. The inferior vena cava was bluntly dissected, and a 1 mL syringe was inserted into the inferior vena cava to slowly draw blood. The blood samples were left standing at room temperature for 1 hour, and the serum was taken after centrifugation at 5000 rpm for 10 minutes at 4°C. The pancreas was located according to the anatomical position of the mouse and the pancreatic tissue samples were collected. The pancreatic tissue was homogenized with PBS, and then the homogenate was transferred to a centrifuge and centrifuged at low speed for 5 minutes to obtain the pancreatic tissue homogenate.

[0038] ELISA was used to detect the HDL levels in the serum and pancreatic tissue homogenate of mice. Using the standard products of 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0 mmol / L as the abscissa and the OD value as the ordinate, a graph was plotted on graph paper to establish a standard curve. The content of HDL in each test sample was calculated according to the curve equation. The HDL levels in the serum and pancreas of the Con group and AP group mice are shown in Figure 1 Figures A and 1B.

[0039] The collected pancreatic samples were subjected to DIA quantitative proteomics detection. Next, proteomics data processing was carried out. The Spectronaut X software was used to process the results of DIA quantitative proteomics. The target proteins were extracted using the proteomics database established in the laboratory. Other parameters were default set at the peptide and protein levels with FDR < 1%. Figure 1 Figures C and 1D show the levels of apoA-1 and 2 and SAA in the pancreatic tissue of mice determined by DIA quantitative proteomics.

[0040] From Figure 1It can be seen that compared with the Con group mice, the serum endogenous HDL level of the AP group mice was significantly decreased, while the endogenous HDL, apoA-1 and 2, and SAA levels in the pancreatic tissue were significantly increased. Apo A-1 and apo A-2 are the main structural and functional proteins of HDL. The content of apo A-1 accounts for about 70% of the HDL protein, while the content of apo A-2 accounts for about 15 - 20% of the HDL protein. Under the acute inflammatory state, apoA-1 in HDL will be replaced by SAA. Therefore, the changes in the levels of apo A-1 and 2 and SAA indirectly reflect the changes in the HDL level. The above results suggest that the endogenous HDL in the blood circulation of AP mice aggregates in the pancreatic tissue.

[0041] Example 2

[0042] FITC-labeled HDL (FITC-HDL) was injected intraperitoneally into the Con group mice and the AP group mice, which were respectively denoted as the Con+FITC-HDL group and the AP+FITC-HDL group. The mice were anesthetized with isoflurane, and the abdominal FITC fluorescence pictures of the Con group mice and the AP group mice were collected using a small animal in vivo imaging system (Spectrum in vivo imaging system, IVIS) 6 hours after injecting FITC-HDL. Then, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg / kg), and the pancreatic tissue and its adjacent organs were taken out under light-proof conditions. The FITC fluorescence pictures of the pancreatic tissue and its adjacent organs were collected again using a small animal in vivo imager. The collected pictures were imported into the Living Image software and the average FITC fluorescence intensity values in the abdomen and pancreatic tissue of the Con group mice and the AP group mice were calculated. The average FITC fluorescence intensity value reflects the content of FITC-HDL in the pancreatic tissue of the mice. The content of FITC-HDL in the pancreas and its adjacent organs of the Con group and the AP group mice is as Figure 2 shown in A.

[0043] The pancreatic tissue of the mice was embedded with OCT embedding medium and frozen in a -80 °C refrigerator for 10 - 20 minutes. Then, the pancreatic tissue of the mice embedded with OCT embedding medium was taken out from the -80 °C refrigerator, and frozen sections were made using a cryostat under light-proof conditions. Next, the cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and sealed with a coverslip. Finally, the FITC fluorescence intensity in the pancreatic tissue of the mice was observed under a fluorescence microscope, and the FITC fluorescence intensity value of the pancreatic tissue was calculated using the Image-ProPlus 6.0 software to reflect the content of FITC-HDL in the pancreatic tissue. The content of FITC-HDL in the pancreatic tissue of the Con group and the AP group mice is asFigure 2 as shown in B.

[0044] IVIS results showed that compared with the mice in the Con+FITC-HDL group, the level of FITC-HDL in the pancreatic tissues of the mice in the AP+FITC-HDL group was significantly increased, while there was no significant difference in the level of FITC-HDL in the spleen, liver and small intestine. The FITC immunofluorescence results of pancreatic tissues showed that, consistent with the IVIS results, compared with the mice in the Con+FITC-HDL group, the level of FITC-HDL in the pancreatic tissues of the mice in the AP+FITC-HDL group was significantly increased. The above results suggest that FITC-HDL selectively accumulates in the pancreatic tissues of AP mice.

[0045] To prove that HDL is a necessary factor for the selective accumulation of FITC-HDL in the pancreatic tissues of AP mice, we injected FITC and FITC-HDL into the mice in the Con group and the AP group for 6 hours respectively, and divided them into 4 groups: Con+FITC group, Con+FITC-HDL group, AP+FITC group and AP+FITC-HDL group. The mice were anesthetized with isoflurane, and the FITC fluorescence images of the abdomen of the 4 groups of mice were collected using IVIS. Then, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg / kg), and the pancreatic tissues were taken out under light-proof conditions, and the FITC fluorescence images of the pancreatic tissues were collected again using a small animal in vivo imager. The FITC-HDL content in the pancreatic tissues of the 4 groups of mice was as Figure 3 shown.

[0046] IVIS results showed that the FITC fluorescence intensity in the pancreatic tissues of the mice in the Con+FITC group and the AP+FITC group was significantly lower than that of the mice in the Con+FITC-HDL group and the AP+FITC-HDL group. The above results suggest that HDL is a necessary factor for the selective accumulation of FITC-HDL in the pancreatic tissues of AP mice, and FITC itself does not accumulate in the pancreatic tissues of AP mice like FITC-HDL.

[0047] Example 3

[0048] This example explored what biological functions the endogenous HDL that accumulates in the pancreatic tissues of AP mice plays.

[0049] The proteins in the pancreatic tissues of mice in the Con group and the AP group were detected by DIA quantitative proteomics technology, and bioinformatics analysis was performed on the proteomics results. Protein numbers were used from the UniProt knowledgebase database. The Gene Ontology (GO) Database (www.webgestalt.org) was used to analyze the cellular components (CC), biological processes (BP), and molecular functions (MF) regulated by differentially expressed proteins. The Cytoscape and GMT Database were used for GO enrichment analysis of the GO analysis results.

[0050] Figure 4 A. The results of DIA quantitative proteomics showed that compared with the mice in the Con group, 925 proteins were significantly increased and 2749 proteins were significantly decreased in the pancreatic tissues of mice in the AP group (P<0.05); Figure 4 B. Through Pearson correlation test, 158 proteins were screened out from the above 3674 proteins and were related to the endogenous HDL level in the pancreatic tissue (R 2 >0.9). Among them, 52 proteins were increased in the pancreatic tissues of mice in the AP group and were positively correlated with the endogenous HDL level in the pancreatic tissue; 106 proteins were decreased in the pancreatic tissues of mice in the AP group and were negatively correlated with the endogenous HDL level in the pancreatic tissue. Figure 4 C. We performed GO enrichment analysis (GOEA) on the GO analysis results of 52 increased proteins and 106 decreased proteins through the Cytoscape software to obtain the GO enrichment analysis map. The GOEA results showed that the endogenous HDL in the pancreatic tissues of AP mice was positively correlated with immune response and wound regulation, while negatively correlated with ribonucleotide metabolic process, mitochondrial membrane function, and tricarboxylic acid cycle. The above results suggest that the endogenous HDL in the pancreatic tissues of AP mice is closely related to the inflammatory response and mitochondrial function of the pancreatic tissues.

[0051] Example 4

[0052] The Cae and LPS combined injection was used to induce the AP mouse model. Mice were fasted and water-deprived for 12 hours before modeling, and were intraperitoneally injected with 20 μg / ml Cae 7 times (50 μg / kg body weight) at 1-hour intervals. At the same time as the 7th injection of Cae, 1 mg / mL LPS O111:B4 (10 mg / kg body weight) was intraperitoneally injected. 20 mg / mL HDL (100 mg / kg body weight) was intraperitoneally injected 18 hours after the 1st injection of Cae. C57BL / 6 mice were divided into 3 groups according to the random table method. ① Control group (Con): healthy mice were intraperitoneally injected with normal saline; ② Model group (AP): healthy mice were intraperitoneally injected with Cae and LPS; ③ Treatment group (HDL): mice in the model group were intraperitoneally injected with HDL.

[0053] The levels of serum amylase and lipase in mice of each group were detected by ELISA. First, prepare the standard solution. Take 8 1.5 mL centrifuge tubes. Add 900 μL of specimen diluent to the first tube, and add 500 μL of specimen diluent to the second to eighth tubes. Add 100 μL of the standard solution at 2000 U / L to the first tube, mix well on a vortex mixer, and then aspirate 500 μL with a pipette and transfer it to the second tube. Repeat the two-fold dilution in this way, and aspirate 500 μL from the seventh tube and discard it. The eighth tube is the blank control. Then, add 100 μL of the standard solution and 100 μL of the serum or cell supernatant sample to be tested to the pre-prepared 96-well plate, mix well on an oscillator for 3 minutes, and incubate at 37 °C for 60 minutes. Wash the plate 6 times, and finally blot dry on a paper towel. Add 50 μL of ddH2O and 50 μL of the working solution of the first antibody to each well, except for the blank well. Mix well on an oscillator for 3 minutes and incubate at 37 °C for 20 minutes. Wash the plate 6 times, and finally blot dry on a paper towel. Add 100 μL of the working solution of the enzyme-labeled antibody to each well and incubate at 37 °C for 10 min. Wash the plate 6 times, and finally blot dry on a paper towel. Add 100 μL of the substrate working solution to each well (operate in the dark), and incubate at 37 °C in the dark for 2 - 15 minutes. Add 100 μL of the termination solution to each well and gently mix manually. Detect the OD value with an enzyme-labeled instrument at a wavelength of 450 nm within 30 minutes. Establish a standard curve and calculate the content of each sample to be tested according to the curve equation. The levels of serum Amylase and Lipase in mice of the Con group, AP group, and HDL group are as Figure 5 shown in Figures 5A and 5B.

[0054] The pancreatic tissues were paraffin-embedded, fixed in 4% paraformaldehyde, and the pancreatic tissues were stained with HE. The HE staining results of the pancreatic tissues of mice in the Con group, AP group, and HDL group are as Figure 5 shown in Figure 5C.

[0055] As Figure 5As shown in Figures A and 5B, the ELISA test results showed that compared with the mice in the Con group, the levels of serum Amylase and Lipase in the mice in the AP group were significantly increased, while HDL significantly decreased the levels of serum Amylase and Lipase in the mice in the AP group. As Figure 5 shown in Figure C, the HE staining results of the pancreatic tissue indicated that HDL significantly reduced the infiltration of inflammatory cells, edema, bleeding, and necrosis of acinar cells in the pancreatic tissue of the mice in the AP group. The above results suggest that HDL can improve the damage of pancreatic acinar cells in AP mice and reduce the infiltration of inflammatory cells in the pancreatic tissue.

[0056] Example 5

[0057] 780 μM of STC was used to act on mouse pancreatic acinar 266-6 cells for 6 hours to induce a damage model of mouse pancreatic acinar 266-6 cells. 2 mg / mL of HDL was added to the culture medium containing mouse pancreatic acinar 266-6 cells 6 hours before the action of STC. Mouse pancreatic acinar 266-6 cells were divided into 3 groups. ① Normal group (Normal): Mouse pancreatic acinar 266-6 cells were added to DEME medium containing 10% FBS; ② Model group (STC): Mouse pancreatic acinar 266-6 cells were added with STC; ③ Treatment group (HDL): Mouse pancreatic acinar 266-6 cells in the model group were added with HDL.

[0058] The CCK-8 method was used to determine the effect of different gradient concentrations (0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 μM) of sodium taurocholate (STC) on the viability of mouse pancreatic acinar 266-6 cells. STC was allowed to act for 6 hours (6 replicates for each concentration). The IC50 value of STC on mouse pancreatic acinar 266-6 cells was as Figure 6 shown in Figure A.

[0059] The pro-inflammatory levels of mouse pancreatic acinar 266-6 cells in each group were analyzed by Real-time PCR. First, the total RNA in the cells was extracted. The purity of the total RNA of each sample was analyzed. 2 μL of the total RNA solution of each sample was taken to measure the OD260 / OD280 of each sample. The total RNA samples with OD260 / OD280 values between 1.8 and 2.2 were used for subsequent cDNA synthesis. Then, the total RNA amount of each sample was unified to 1 μg, and reverse transcription reaction was carried out according to the reaction system of the EVO M-MLV reverse transcription kit, and the cDNA was stored at -80 °C. Finally, Real-time PCR reaction was carried out on the cDNA of each sample. The levels of pro-inflammatory cytokines in mouse pancreatic acinar 266-6 cells in each group were as shown in Figures 6B and 6C.

[0060] Figure 6The CCK-8 results showed that the IC50 value of STC on mouse pancreatic acinar 266-6 cells was 780 μM. Figure 6 B and 6C showed that HDL reduced the expression of pro-inflammatory cytokine mRNA in STC-induced mouse pancreatic acinar 266-6 cells.

[0061] Example 6

[0062] The levels of pro-inflammatory cytokines in the sera of mice in the Con group, AP group, and HDL group were detected by ELISA. The specific method was referred to Example 4.

[0063] The levels of pro-inflammatory cytokines in the pancreas of mice in the Con group, AP group, and HDL group were detected by Real-time PCR. The specific method was referred to Example 5.

[0064] Figure 7 A-7C showed that the ELISA results showed that compared with the mice in the Con group, the levels of TNF-α, IL-6, and IL-8 in the sera of mice in the AP group were significantly increased, while HDL significantly reduced the levels of TNF-α, IL-6, and IL-8 in the sera of mice in the AP group. Figure 7 D-7F showed that the Real-time PCR analysis results showed that compared with the mice in the Con group, the mRNA expressions of TNF-α, IL-6, and IL-8 in the pancreatic tissues of mice in the AP group were significantly increased, while HDL significantly inhibited the mRNA expressions of TNF-α, IL-6, and IL-8 in the pancreatic tissues of mice in the AP group.

[0065] Example 7

[0066] Immunohistochemical staining of pancreatic tissues of each group of mice. The paraffin sections of pancreatic tissues were immersed in xylene and gradient ethanol, and then washed with distilled water for 1 minute. The paraffin sections were placed in 0.01M sodium citrate antigen retrieval solution and retrieved under high pressure for 4 minutes. After retrieval, take out the antigen retrieval box, open the lid, and let it stand at room temperature for 60 minutes. After antigen retrieval, dry the slides and mark the tissue area with an immunohistochemistry pen. In the slide washing box, rinse the pancreatic tissue sections with PBS twice, 3 minutes each time. Add an appropriate amount of endogenous peroxidase blocker to the slide samples and incubate at room temperature for 15 minutes, then wash with PBS 3 times, 5 minutes each time. Dropwise add the working solution of goat serum for blocking and incubate at room temperature for 30 minutes, then pour it out without washing. Dropwise add the primary antibody and incubate at 4°C overnight (16 - 24 hours), then wash with PBS 3 times, 5 minutes each time. Dropwise add the biotin-labeled secondary antibody IgG and incubate at room temperature for 20 minutes; wash with PBS 3 times, 5 minutes each time. Dropwise add the working solution of streptavidin labeled with horseradish peroxidase and incubate at room temperature for 15 minutes; wash with PBS 3 times, 5 minutes each time. Dropwise add an appropriate amount of freshly prepared DAB chromogenic working solution and incubate at room temperature for 5 minutes; rinse with tap water 2 times and place in PBS for use. Drop the hematoxylin staining solution on the slides for about 40 - 50 seconds, then rinse with tap water for 2 minutes. Immerse in acid ethanol for 5 seconds and rinse with tap water for 2 minutes. Blue with ammonia water for 7 seconds, then immerse in gradient ethanol and xylene for mounting. Finally, collect pictures of pancreatic tissues through an ordinary optical microscope and calculate the IOD value of pancreatic tissue samples using Image-Pro Plus 6.0 software. The expression of MPO and F4 / 80 in pancreatic tissues of mice in the Con group, AP group, and HDL group (100X, n = 4) is as Figure 8 shown.

[0067] Immunofluorescence staining of pancreatic tissues of each group of mice. Fresh tissues were taken from anesthetized animals and fixed in 4% paraformaldehyde for 24 hours, and dehydrated with 20% and 30% sucrose solutions respectively. The tissues were taken out of the 30% sucrose solution, and the surface was blotted dry with filter paper. The tissues were placed in an OCT embedding cassette for embedding and stored at -80 °C. Sections were made using a cryostat. The sections were air-dried at room temperature for 24 hours and then stored at -20 °C for subsequent experiments. After the frozen sections were taken out of -20 °C, they were rewarmed at room temperature for 30 minutes. The tissue area was circled with an immunohistochemistry pen. In a slide washing box, the sections were rinsed 3 times with PBS for 3 minutes each time. The washing solution was discarded, 0.01M sodium citrate buffer was poured in, and the slide was placed in a microwave oven and microwaved twice for 6 minutes each time, at medium-low heat, 92 - 98 °C, avoiding boiling. After microwave treatment, it was cooled at room temperature for 30 minutes, the antigen retrieval solution was discarded, and then washed 2 times with PBS for 3 minutes each time, gently shaken on a shaker. The washing solution was discarded, and the slides were air-dried at room temperature. 1 drop of 10% goat serum blocking solution was added to each sample and blocked at 37 °C for 60 minutes under constant temperature conditions. The blocking solution was blotted dry without washing, the primary antibody was added, and the samples were placed in a wet box and incubated overnight at 4 °C. After the sections were taken out of the refrigerator, they were rewarmed at 37 °C for 30 minutes, and the slides were washed 4 times with PBS for 5 minutes each time. The PBS was aspirated, 1 drop of the diluted secondary antibody was added to each sample, and incubated at 37 °C in the dark for 1 hour. The slides were washed 4 times with 1×PBS for 5 minutes each time. The remaining PBS was poured out, DAPI was added to stain the cell nuclei for 3 minutes, the remaining DAPI was aspirated, and the slides were sealed. Finally, fluorescence images of pancreatic tissues (100X, n = 4) were collected by a fluorescence microscope, and the IOD values of pancreatic tissue samples were calculated using Image-Pro Plus 6.0 software. The expression of Ly6G in the pancreatic tissues of mice in the Con group, AP group, and HDL group was as Figure 8 shown.

[0068] Figure 8 The immunohistochemical results showed that compared with the Con group of mice, the protein expressions of F4 / 80 and MPO in the pancreatic tissues of the AP group of mice were significantly increased, while HDL significantly inhibited the protein expressions of F4 / 80 and MPO in the pancreatic tissues of the AP group of mice. The immunofluorescence results showed that compared with the Con group of mice, the protein expression of Ly6G in the pancreatic tissues of the AP group of mice was significantly increased, while HDL significantly inhibited the protein expression of Ly6G in the pancreatic tissues of the AP group of mice. The above results suggest that HDL can reduce the inflammatory response in the pancreatic tissues of AP mice.

[0069] Example 8

[0070] A mouse Raw264.7 cell injury model was induced by treating the cells with 1 μg / mL LPS for 24 hours. 2 mg / mL HDL was added to the culture medium containing mouse Raw264.7 cells 6 hours before the LPS treatment. Mouse Raw264.7 cells were divided into 3 groups. ① Normal group: Mouse Raw264.7 cells were added to DMEM medium containing 10% FBS; ② Model group (LPS): Mouse Raw264.7 cells were added to LPS; ③ Treatment group (HDL): Mouse Raw264.7 cells in the model group were added to HDL.

[0071] The CCK-8 method was used to determine the effects of different gradient concentrations (0, 0.125, 0.25, 0.5, 1, 2, 4 μg / mL) of LPS (O55:B5) and LPS (O111:B4) on the viability of Raw264.7 cells. Raw264.7 cells were plated in 96-well plates for 12 hours, approximately 20,000 cells / well. Then the cells were taken out of the incubator, and the prepared CCK-8 solution was added, 110 μL / well, and incubated in the incubator for 1 - 4 hours. The absorbance values of each well were measured at 450 nm using a microplate reader. The effects of different concentrations of LPS (O55:B5) and LPS (O111:B4) on the viability of Raw264.7 cells are shown as Figure 9 shown in Figures A and 9B.

[0072] ELISA was used to detect the levels of pro-inflammatory cytokines TNF-α and IL-6 in the supernatants of Raw264.7 cells in each group. The specific method was referred to Example 4. The levels of TNF-α and IL-6 in the supernatants of Raw264.7 cells in the Normal group, LPS group, and HDL group are shown as Figure 9 shown in Figures C and 9D.

[0073] Figure 9 As shown in Figures A and 9B by the CCK-8 results, LPS (O55:B5) and LPS (O111:B4) were not toxic to Raw264.7 cells, and LPS (O55:B5) and LPS (O111:B4) at each concentration promoted the proliferation of Raw264.7 cells. Figure 9 As shown in Figures C and 9D by the ELISA detection results, HDL significantly reduced the levels of TNF-α and IL-6 in the supernatants of Raw264.7 cells induced by LPS (O55:B5) and LPS (O111:B4). The above results suggest that HDL inhibits the inflammatory response induced by LPS-activated macrophages.

[0074] Example 9

[0075] DIA quantitative proteomics was used to detect the protein levels in the pancreatic tissues of mice in each group, and the proteins with significant changes in the pancreatic tissues of mice in the HDL group were screened out. Then, GO analysis was performed on the above-mentioned proteins with significant changes, and GOEA was performed on the GO analysis results using Cytoscape software to obtain a GO enrichment analysis map. The proteins with significant changes in the pancreatic tissues of mice in the HDL group (compared with the mice in the AP group, P<0.05) are as Figure 10 shown in A; then, GO analysis was performed on the above-mentioned proteins with significant changes, and GOEA was performed on the GO analysis results using Cytoscape software to obtain a GO enrichment analysis map as Figure 10 shown in B.

[0076] Figure 10 A shows that the results of DIA quantitative proteomics indicate that compared with the mice in the AP group, 218 proteins in the pancreatic tissues of mice in the HDL group were significantly increased, and 486 proteins were significantly decreased (P<0.05); Figure 10 B shows that the results of GOEA show that HDL promotes the aggregation of plasma lipoprotein particles in the pancreatic tissues of AP mice (Plasma Lipoprotein Particle), while inhibiting purine ribonucleoside metabolism (Purine Ribonucleoside Metabolism), ribosome subunit function (Ribosome SubunitFunction), endoplasmic reticulum function (Integral Endoplasmic Reticulum), cytoplasmic vesicle membrane (CytoplasmicVesicle Membrane), endosome transport (Endosome Transport Localization), cellular complex function (Cellular Complex Function), and organic catabolism (Organic Catabolic Process). The above results show that HDL regulates the functions of multiple organelles in the pancreatic acinar cells of AP mice, including mitochondria, ribosomes, endosomes, endoplasmic reticulum, Golgi apparatus, and cytoplasmic vesicles.

[0077] Example 10

[0078] SR-B1 and ABCG1 are the main receptors for HDL to transport cytoplasmic membrane and intracellular lipids, and are also the main receptors for HDL to regulate various cellular inflammatory responses.

[0079] Western Blotting was used to detect the protein expressions of SR-B1 and ABCG1 in the pancreatic tissues of mice in each group. The pancreatic tissues were ground in liquid nitrogen, and then the ground pancreatic tissue powder was transferred to a homogenization tube containing RIPA lysis buffer. It was mixed 5 times with a 1 mL pipette and homogenized for 15 seconds. After homogenization, the homogenate and all precipitates were aspirated with a pipette into a new 1.5 mL EP tube and centrifuged at 14000 g at 4 °C for 10 minutes. The supernatant was collected as the total protein extract of the pancreatic tissue, mixed well and stored in aliquots. The BCA method was used to determine the protein concentration of each group of samples. The protein concentrations of each group of samples were diluted to the same concentration to unify the protein loading amount. A certain volume of protein loading buffer was added to each group of protein samples, vortexed and mixed well, and then boiled in boiling water for 10 minutes to denature the protein, and stored in aliquots. According to the SDS-PAGE rapid gel preparation instructions, gels with corresponding concentrations were prepared based on the molecular weight of the target protein. The separating gel added with TEMED was mixed well, added to the gel plate with a 1 mL pipette, flattened with ddH2O, and left standing at room temperature for 25 minutes. The ddH2O was poured off and blotted dry with filter paper. TEMED was added to the stacking gel, manually mixed, added to the gel plate with a 1 mL pipette, a comb was inserted, and left standing at room temperature for 25 minutes. It was wrapped with plastic wrap and stored at 4 °C for later use. The gel plate slot was assembled, leak-tested, then filled with electrophoresis buffer, loaded with samples, and the sample information was recorded. The gel plate was placed in the electrophoresis tank, and the corresponding volume of electrophoresis buffer was added according to the number of gel plates. According to the molecular weight of the target protein, the electrophoresis voltage was selected until the bromophenol blue reached the bottom of the gel, and electrophoresis was stopped. The membrane was cut, and ice chips, ice packs, and transfer buffer were prepared. The PVDF membrane was activated with methanol for 2 min, and the activated PVDF membrane was placed in the transfer buffer for soaking. The gel strip was transferred to filter paper, covered with the PVDF membrane to prepare a sandwich structure. Transfer buffer was added, and the appropriate transfer current and time were selected according to the molecular weight of the target protein. After the transfer was completed, the PVDF membrane was taken out and washed with 1xTBST for 5 minutes. 5 mL of blocking solution was added to each band, and blocked at room temperature with gentle shaking for 2 hours, then washed with 1xTBST for 8 minutes, twice. According to the antibody instructions ratio SR-B1 (1:5000) and ABCG1 (1:5000), the primary antibody was prepared with 5% BSA and incubated at 4 °C for 12 - 24 hours. Washed with 1xTBST (containing Tween) for 8 minutes, three times. According to the antibody ratio in the instructions (1:5000), the secondary antibody was prepared with 5% BSA and incubated at room temperature with gentle shaking for 1 hour. Washed with 1xTBST (containing Tween) for 8 minutes, three times. The ECL luminescent solution was prepared according to the ratio of solution A to solution B = 1:1. The band was taken out from 1xTBST, placed in the Tanon luminescence instrument, then 200 μL of luminescent solution was dropped on it and gently shaken evenly, and the instrument was operated to obtain a photo. The Image J software was used to analyze the gray value of the protein band, with β-actin as the reference value. The protein expressions of SR-B1 and ABCG1 in the pancreatic tissues of the Con group, AP group, and HDL group were as Figure 11 shown in Figures A and 11B.

[0080] The distribution of SR-B1 and ABCG1 in pancreatic tissues of mice in each group was detected by immunofluorescence. The distribution of SR-B1 and ABCG1 in pancreatic tissues of the Con group, AP group, and HDL group was as Figure 11 shown in C.

[0081] Figure 11 As shown in A and 11B by Western Blotting results, compared with the mice in the Con group, the protein expression of SR-B1 in the pancreatic tissues of the AP group mice was significantly increased, while the protein expression of ABCG1 was significantly decreased. Figure 11 As shown in C by immunofluorescence results, both SR-B1 and ABCG1 were distributed in the cell membranes and cytoplasm of pancreatic acinar cells. At the same time, compared with the mice in the Con group, the fluorescence intensity of SR-B1 in the pancreatic tissues of the AP group mice was significantly increased, while the fluorescence intensity of ABCG1 was significantly decreased. The high expression of SR-B1 protein in the pancreatic tissues of AP mice indicates its more binding to HDL and plays a more important role in the regulation of inflammatory responses in the pancreatic tissues of AP mice by HDL.

[0082] To further prove the high expression of SR-B1 in pancreatic acinar cells of AP mice, we explored the effect of STC on the protein expression of SR-B1 in mouse pancreatic acinar 266-6 cells at the cellular level.

[0083] Mouse pancreatic acinar 266-6 cells were divided into 3 groups. ① Normal group: Mouse pancreatic acinar 266-6 cells were added to DEME medium containing 10% FBS; ② Model group (STC): Mouse pancreatic acinar 266-6 cells were added with STC; ③ Treatment group (HDL): Mouse pancreatic acinar 266-6 cells in the model group were added with HDL.

[0084] 780 μM of STC was selected to act on mouse pancreatic acinar 266-6 cells for 6 hours to induce a cell injury model of mouse pancreatic acinar 266-6 cells. 2 mg / mL of HDL was added to the medium containing mouse pancreatic acinar 266-6 cells 6 hours before the action of STC.

[0085] The protein expression of SR-B1 in mouse pancreatic acinar 266-6 cells in each group was detected by Western Blotting. The protein expression of SR-B1 in mouse pancreatic acinar 266-6 cells in the Normal group, STC group, and HDL group (n = 1) was as Figure 12 shown. Compared with the Normal group, the protein expression of SR-B1 in mouse pancreatic acinar 266-6 cells in the STC group was increased.

[0086] We demonstrated at the cellular level that stimulation with STC increased the protein expression of SR-B1 in mouse pancreatic acinar 266-6 cells, which was consistent with the increased protein expression of SR-B1 in the pancreatic tissue of AP mice.

[0087] ITX5061 is an SR-B1 antagonist that can block the binding of HDL to SR-B1. To demonstrate that SR-B1 is the key receptor for HDL to regulate the inflammatory response in the pancreatic tissue of AP mice, we explored the effect of the SR-B1 antagonist ITX5061 on the regulation of the inflammatory response in the pancreatic tissue of AP mice by HDL.

[0088] An AP mouse model was induced by combined injection of Cae and LPS. Mice were fasted and water-deprived for 12 hours before modeling, and were intraperitoneally injected with 20 μg / ml Cae 7 times (50 μg / kg body weight) at 1-hour intervals. At the same time as the 7th injection of Cae, 1 mg / mL LPS O111:B4 (10 mg / kg body weight) was intraperitoneally injected. 20 mg / mL HDL (100 mg / kg body weight) was intraperitoneally injected 18 hours after the 1st injection of Cae. 5 mg / mL ITX5061 (30 mg / kg body weight) was intraperitoneally injected 18 hours and 1 hour before the 1st injection of Cae, respectively. C57BL / 6 mice were divided into 4 groups according to the random table method. ① Control group (Con): healthy mice were intraperitoneally injected with normal saline; ② Model group (AP): healthy mice were intraperitoneally injected with Cae and LPS; ③ Treatment group (HDL): mice in the model group were intraperitoneally injected with HDL; ④ SR-B1 inhibitor group (ITX5061): mice in the treatment group were intraperitoneally injected with ITX5061.

[0089] The levels of pro-inflammatory cytokines in the sera of mice in each group were detected by ELISA, and the specific method was referred to Example 4. The levels of TNF-α and IL-6 in the sera of mice in the Con group, AP group, HDL group, and ITX5061 group were as Figure 13 shown in Figures A and 13B.

[0090] The levels of pro-inflammatory cytokines in the pancreatic tissues of mice in each group were detected by Real-time PCR, and the specific method was referred to Example 5. The levels of TNF-α and IL-6 mRNA in the pancreatic tissues of mice in the Con group, AP group, HDL group, and ITX5061 group were as Figure 13 shown in Figures C and 13D.

[0091] ELISA results showed that HDL significantly downregulated the levels of TNF-α and IL-6 in the serum of AP mice, while ITX5061 inhibited the downregulatory effect of HDL on the levels of TNF-α and IL-6 in the serum of AP mice. Real-time PCR results showed that HDL significantly downregulated the mRNA expressions of TNF-α and IL-6 in the pancreatic tissues of AP mice, while ITX5061 inhibited the downregulatory effect of HDL on the mRNA expressions of TNF-α and IL-6 in the pancreatic tissues of AP mice. The above results suggested that weakening the binding of SR-B1 to HDL would inhibit the alleviating effect of HDL on the inflammatory response in the pancreatic tissues of AP mice, which also indicated that SR-B1 was the key receptor for HDL to alleviate the inflammatory response in the pancreatic tissues of AP mice.

[0092] We explored the effect of knocking down SR-B1 on the downregulation of TNF-α and IL-6 mRNA expressions in mouse pancreatic acinar 266-6 cells induced by STC at the cellular level.

[0093] Mouse pancreatic acinar 266-6 cells were divided into 4 groups. ① Normal group (Normal): Mouse pancreatic acinar 266-6 cells were added to DEME medium containing 10% FBS; ② Model group (STC): Mouse pancreatic acinar 266-6 cells were added to STC; ③ Treatment group (HDL): Mouse pancreatic acinar 266-6 cells in the model group were added to HDL. ④ SR-B1 knockdown group (shRNA / SR-B1): STC-induced mouse pancreatic acinar 266-6 cells with SR-B1 knockdown were modeled and added to HDL.

[0094] shRNA / SR-B1 transfected cells. The shRNA / SR-B1 was introduced into mouse pancreatic acinar 266-6 cells by cell transfection technology. The steps of shRNA / SR-B1 transfection: The cells were seeded in 6-well plates for culture, and the transfection experiment was carried out when the cells grew to 60-80% confluence. Respectively, 3 μg of NC plasmid DNA, 3 μg of SR-B1-332 plasmid DNA, 3 μg of SR-B1-806 plasmid DNA, 3 μg of SR-B1-901 plasmid DNA, 3 μg of SR-B1-1096 plasmid DNA and 3 μg of GAPDH plasmid DNA were added to 100 μL of serum-free DMEM medium. 36 μL of Lipofectamine 2000 was added to 600 μL of serum-free DMEM medium; mixed well and incubated at room temperature for 5 minutes. Respectively, 100 μL of serum-free DMEM medium containing Lipofectamine 2000 was added to the serum-free DMEM medium containing NC, SR-B1-332, SR-B1-806, SR-B1-901, SR-B1-1096 and GAPDH plasmid DNA (ratio: 3 μg plasmid DNA: 6 μL Lipofectamine 2000). Incubated at room temperature for 30 minutes to form a plasmid-Lipofectamine 2000 complex. The cells were taken out of the cell culture incubator, the culture medium in the 6-well plate was aspirated, washed once with PBS, 1.8 mL of DMEM medium containing 10% FBS was added to each well first, and then 200 μL of DNA-Lipofectamine 2000 complex was added to each well. The 6-well plate was gently shaken back and forth to evenly cover the cells with the DNA-Lipofectamine 2000 complex. After transfection for 12 hours, it was replaced with DMEM medium containing 10% FBS and continued to culture for 36 hours. The knockdown efficiency of SR-B1-332, SR-B1-806, SR-B1-901 and SR-B1-1096 plasmid DNA was detected by Real-time PCR as Figure 14 shown in A.

[0095] The expression of SR-B1 protein in mouse pancreatic acinar 266-6 cells in the Normal group and shRNA / SR-B1 group was detected by Western Blotting, as Figure 14 shown in B.

[0096] The levels of pro-inflammatory cytokines in mouse pancreatic acinar cells of each group were detected by Real-time PCR. The levels of TNF-α and IL-6 mRNA in mouse pancreatic acinar cells of the Normal group, STC group, HDL group and shRNA / SR-B1 group were as Figure 14 shown in C and 14D.

[0097] Figure 14A shows that the knockdown efficiency of SR-B1 in mouse pancreatic acinar 266-6 cells by SR-B1-901 plasmid DNA screened by Real-time PCR technology is the highest. Figure 14 B verifies the knockdown effect of SR-B1-901 plasmid DNA on SR-B1 in mouse pancreatic acinar 266-6 cells by Western Blotting technology. Figure 14 C and 14D show that Real-time PCR results show that HDL significantly downregulates the mRNA expression of TNF-α and IL-6 in STC-induced mouse pancreatic acinar 266-6 cells, while knockdown of SR-B1 inhibits the downregulation of TNF-α and IL-6 mRNA expression in STC-induced mouse pancreatic acinar 266-6 cells by HDL. The above results suggest that SR-B1 is a key receptor for HDL to downregulate the mRNA expression of TNF-α and IL-6 in STC-induced mouse pancreatic acinar 266-6 cells.

Claims

1. Use of high-density lipoprotein in the preparation of a medicament for treating acute pancreatitis.

2. The application according to claim 1, wherein The high-density lipoprotein is the high-density lipoprotein in the blood of healthy individuals.

3. The application according to claim 2, characterized in that The high-density lipoprotein contains apolipoprotein A-1, apolipoprotein A-2, paraoxonase 1, phospholipid transfer protein, phospholipids, sphingolipids and cholesterol.

4. The application according to claim 1, characterized in that The high-density lipoprotein is prepared into a pharmaceutical preparation of a single component.

5. The application according to claim 4, characterized in that, The dosage form of the pharmaceutical preparation is an injection.

6. The application according to claim 4, characterized in that The administration method of the pharmaceutical preparation is intraperitoneal injection.

7. The application according to claim 4, characterized in that, The administration dosage of the pharmaceutical preparation is 100 mg / kg body weight.

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