Animal model study on quercetin alleviating lipopolysaccharide acute lung injury through scap / srebp2 / nlrp3 pathway

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a research method for reducing an animal model of lipopolysaccharide acute lung injury through a SCAP / SREBP2 / NLRP3 pathway by quercetin, which comprises the following steps: S1, grouping and processing of experimental animals; S2, cell culture and grouping thereof; S3, lung wet / dry ratio (W / D) of the experimental animals is evaluated to assess lung edema and bronchoalveolar lavage fluid (BALF) is collected; S4, lung tissue pathology of the experimental animals is observed; S5, ELISA is used to detect levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1beta (IL-1beta); S6, Western blot is used to detect pathway proteins; S7, qPCR is used to detect expression of related nucleic acids; S8, flow cytometry is used to detect cell mortality, and statistical processing is performed using GraphPad Prism 9.0 software for statistical analysis. The application expresses the role of quercetin in mouse acute lung injury and the regulation and influence of the SCAP / SREBP2 / NLRP3 pathway-mediated inflammatory response through an experimental method, thereby providing a theoretical basis for treatment of ARDS.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically an animal model research method for quercetin to alleviate lipopolysaccharide-induced acute lung injury via the SCAP / SREBP2 / NLRP3 pathway. Background Technology

[0002] Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinical syndromes characterized by refractory hypoxemia caused by a variety of pathogenic factors. As an acute inflammatory disease, it mainly manifests as inflammatory damage to lung tissue, accompanied by non-cardiac dyspnea, severe hypoxemia, and pulmonary edema. It has a high morbidity and mortality rate. Long-term research has not yet yielded a definitive conclusion on its pathophysiological mechanism. Early identification of ARDS is the key to diagnosis and treatment. After long-term research, the academic community has developed the widely accepted "Berlin definition," which effectively guides how to manage this clinical critical illness.

[0003] Pneumonia and sepsis are important factors inducing acute lung injury. During inflammation, neutrophils release a large number of inflammatory factors, which amplify to form an inflammatory storm response, leading to damage to the alveolar epithelium and pulmonary microvascular endothelium, causing diffuse lung injury. Edema fluid containing inflammatory cells forms in the alveolar spaces, which in turn leads to pulmonary edema and hyaline membrane formation, resulting in impaired alveolar gas exchange.

[0004] Studies have shown that NLRP3 inflammasome activation initiates the secretion of mature IL-1β by cells, promoting inflammatory processes and oxidative stress, and exacerbating endothelial damage. Research has confirmed that SREBP2 plays a key role in the blood flow-mediated activation of NLRP3 inflammasomes that are prone to atherosclerosis, leading to the release of inflammatory factors and exacerbating vascular endothelial cell inflammation. Inhibition of SREBP2 can effectively reduce NLRP3 expression.

[0005] Quercetin, a natural bioflavonoid, possesses anti-fibrotic, antiviral, anticancer, anti-inflammatory, and antioxidant properties. Studies have shown that quercetin can effectively alleviate inflammatory damage by reducing SCAP and SREBP2 levels. Furthermore, research has confirmed that quercetin can coordinate NLRP3 inflammasome signaling, inhibiting lipopolysaccharide-induced NLRP3 inflammation-related protein expression and subsequent inflammatory responses. However, the role and mechanism of quercetin in ARDS remain unclear. Therefore, this study investigates the role of quercetin in acute lung injury in mice and its regulatory effect on the SCAP / SREBP2 / NLRP3 pathway-mediated inflammatory response, providing a theoretical basis for ARDS treatment. To this end, this invention provides an animal model research method for quercetin to alleviate lipopolysaccharide-induced acute lung injury via the SCAP / SREBP2 / NLRP3 pathway. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies and solve the problems mentioned in the background, this invention proposes an animal model research method for quercetin to alleviate lipopolysaccharide-induced acute lung injury via the SCAP / SREBP2 / NLRP3 pathway.

[0007] The technical solution adopted by this invention to solve its technical problem is: the animal model research method for quercetin to alleviate lipopolysaccharide-induced acute lung injury via the SCAP / SREBP2 / NLRP3 pathway, as described in this invention, and the research method is as follows:

[0008] S1: Grouping and treatment of laboratory animals;

[0009] S2: Cell culture and its grouping;

[0010] S3: Assessment of pulmonary edema and collection of bronchoalveolar lavage fluid (BALF) by measuring the wet-to-dry ratio (W / D) of lungs in experimental animals;

[0011] S4: Pathological observation of lung tissue in experimental animals;

[0012] S5: ELISA method was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β);

[0013] S6: Western blot detection of pathway proteins;

[0014] S7: qPCR detection of relevant nucleic acid expression;

[0015] S8: Flow cytometry was used to detect cell death rate and statistical analysis was performed using GraphPadPrism 9.0 software.

[0016] Preferably, the method for grouping and treating experimental animals in S1 is as follows:

[0017] A1: Forty male C57BL / 6 mice were randomly divided into a control group, an LPS group, an LPS+QUE group, and a QUE group, with 10 mice in each group. LPS was lipopolysaccharide and QUE was quercetin.

[0018] A2: Mice in the LPS+QUE group and QUE group were injected intraperitoneally with 100 mg / kg of quercetin solution, while the control group was injected with an equal volume of PBS buffer.

[0019] A3: One hour later, mice in the four groups were anesthetized with 50 mg / kg pentobarbital. The mice were then intubated. Mice in the LPS group and LPS+QUE group were instilled with 5 mg / kg LPS in 100 μl via endotracheal intubation. Meanwhile, the control group and QUE group were instilled with the same volume of sterile PBS buffer according to the above procedure.

[0020] A4: After infusion, hold the mouse upright for 2-3 minutes to allow the solution to fully settle in the lungs. Sacrifice the mouse 24 hours after modeling and collect the specimen for testing.

[0021] Preferably, the cell culture and grouping in S2 are as follows:

[0022] B1: HUVECs cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in a 37°C, 5% CO2 cell culture incubator. HUVECs cells refer to human umbilical vein endothelial cells.

[0023] B2: The cells were divided into four groups: control group, LPS group, LPS+QUE group and QUE group. The LPS+QUE group and QUE group were first treated with quercetin (30 mmol / L) for 1 h, and then the LPS group and LPS+QUE group were treated with LPS (1 μg / ml) for 24 h. Finally, the cells from the above four groups were collected for further experiments.

[0024] Preferably, in S3, the wet-to-dry weight ratio (W / D) of the experimental animal lungs is used to assess pulmonary edema: the left lung tissue of the four groups of mice in S1 is taken after sacrifice, placed on pre-weighed tin foil and weighed for wet weight (W), and then the sample is placed in an 80℃ oven for 48 hours until constant weight is achieved, and its dry weight (D) is weighed. The wet-to-dry weight ratio of the lung tissue is calculated by W / D; in S3, the bronchoalveolar lavage fluid (BALF) is collected: the four groups of mice in S1 are anesthetized and intubated, and the lungs are flushed with 1mL PBS buffer 3-5 times to collect BALF. The obtained BALF is centrifuged at 1500rpm at 4℃ for 15min and the supernatant is collected. The collected supernatant is stored in a -80℃ refrigerator for later use.

[0025] Preferably, the pathological observation of the lung tissue of the experimental animals in S4 is as follows:

[0026] C1: Take the right lung of a mouse and immerse it in 4% paraformaldehyde for 24 hours;

[0027] C2: The specimens were then dehydrated with ethanol solutions of different concentrations, embedded in paraffin, cut into 5-7 μm thick sections, dewaxed with xylene, and stained with hematoxylin and eosin (HE).

[0028] C3: After staining, dehydrate and mount the slides, then observe the morphology of mouse lung tissue under a microscope.

[0029] Preferably, in S5, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) are detected by ELISA: following the instructions of the ELISA kit, the levels of TNF-α, IL-6, and IL-1β in the BALF (bronchoalveolar lavage fluid) and cell culture medium (supernatant) of each group of mice in S3 are measured.

[0030] Preferably, the Western blot detection pathway proteins in S6 are as follows:

[0031] D1: Mouse lung tissue and HUVECs cells were collected, and proteins were extracted from the lung tissue and HUVECs cells using RIPA buffer containing PMSF. The protein concentration was determined using a BCA kit.

[0032] D2: After quantification, add loading buffer, denature in boiling water for 10 min, and store at -20℃ for later use;

[0033] D3: Prepare a 10% SDS-PAGE gel, add 50 μg of protein sample to each well, transfer to a PVDF membrane after electrophoresis, block with TBST containing 5% skim milk powder for 1 h, add the corresponding primary antibody and incubate overnight at 4℃; wash the membrane 3 times with TBST buffer, add secondary antibody and incubate at 37℃ for 1 h, wash the membrane 3 times with TBST buffer, develop and expose with ultrasensitive luminescent solution, and analyze the gray values ​​of protein bands using ImageJ software system.

[0034] Preferably, the qPCR detection involves related nucleic acid expression:

[0035] E1: Mouse lung tissue and HUVECs were collected, and RNAiso was added and mixed thoroughly. After precipitation with isopropanol and washing with 75% ethanol, the mixture was dissolved in DEPC water. The concentration was detected using a nucleic acid analyzer, and the RNA was reverse transcribed into cDNA.

[0036] E2: Primer sequence: SCAP upstream 5'-GCATCACAGCCCTTGTCTTC-3'

[0037] Downstream 5'-CACTGTGTTGCTGCTGCTGTA-3'; SREBP2 upstream

[0038] 5'-GTCGATCAAGTCAGCAGCCAAG-3' downstream

[0039] 5'-TTGGCCTGAGGTTTCACCAAG-3'; NLRP3 upstream

[0040] 5'-TACGGCCGTCTACGTCTTCT-3' downstream

[0041] 5'-CGCAGATCACACTCCTCAAA-3'; GAPDH upstream

[0042] 5'-TGTGTCCGTCGTGGATCTGA-3' downstream

[0043] 5'-TTGCTGTTGAAGTCGCAGGAG-3'.

[0044] Preferably, in step S8, the cell death rate was detected by flow cytometry using the Annexin V-FITC / PE apoptosis detection kit. HUVECs cells were resuspended in 500 µl of binding buffer and then incubated with FITC-labeled Annexin V (5 µl) and PE (5 µl) at room temperature in the dark for 15-20 min. The cell death rate was then determined by flow cytometry. Statistical analysis in step S8 was performed using GraphPadPrism 9.0 software. Quantitative data were expressed as mean ± standard deviation (x±s). One-way ANOVA was used for comparisons between groups, and LSD-t test was used for pairwise comparisons within groups. P < 0.05 was considered statistically significant.

[0045] The beneficial effects of this invention are as follows:

[0046] 1. The present invention describes an animal model research method for quercetin to alleviate lipopolysaccharide-induced acute lung injury via the SCAP / SREBP2 / NLRP3 pathway. This method experimentally expresses the role of quercetin in acute lung injury in mice and its regulatory effect on the inflammatory response mediated by the SCAP / SREBP2 / NLRP3 pathway, providing a theoretical basis for the treatment of ARDS. Attached Figure Description

[0047] The invention will now be further described with reference to the accompanying drawings.

[0048] Figure 1 HE staining is used to observe pathological changes in lung tissue;

[0049] Figure 2 The effect of quercetin on lung edema and inflammatory response in LPS-induced model mice;

[0050] Figure 3 It is used to detect the nucleic acid and protein levels of SCAP / SREBP2 / NLRP3 in mouse lung tissue;

[0051] Figure 4 The effects of quercetin on HUVECs cell activity and inflammatory factor levels;

[0052] Figure 5 It involves detecting the levels of SCAP / SREBP2 / NLRP3 nucleic acids and proteins in HUVECs; Detailed Implementation

[0053] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0054] The research method is as follows:

[0055] S1: Grouping and treatment of laboratory animals;

[0056] S2: Cell culture and its grouping;

[0057] S3: Assessment of pulmonary edema and collection of bronchoalveolar lavage fluid (BALF) by measuring the wet-to-dry ratio (W / D) of lungs in experimental animals;

[0058] S4: Pathological observation of lung tissue in experimental animals;

[0059] S5: ELISA method was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β);

[0060] S6: Western blot detection of pathway proteins;

[0061] S7: qPCR detection of relevant nucleic acid expression;

[0062] S8: Flow cytometry was used to detect cell death rate and statistical analysis was performed using GraphPadPrism 9.0 software.

[0063] The method for grouping and treating experimental animals in S1 is as follows:

[0064] A1: Forty male C57BL / 6 mice were randomly divided into a control group, an LPS group, an LPS+QUE group, and a QUE group, with 10 mice in each group. LPS was lipopolysaccharide and QUE was quercetin.

[0065] A2: Mice in the LPS+QUE group and QUE group were injected intraperitoneally with 100 mg / kg of quercetin solution, while the control group was injected with an equal volume of PBS buffer.

[0066] A3: One hour later, mice in the four groups were anesthetized with 50 mg / kg pentobarbital. The mice were then intubated. Mice in the LPS group and LPS+QUE group were instilled with 5 mg / kg LPS in 100 μl via endotracheal intubation. Meanwhile, the control group and QUE group were instilled with the same volume of sterile PBS buffer according to the above procedure.

[0067] A4: After infusion, hold the mouse upright for 2-3 minutes to allow the solution to fully settle in the lungs. Sacrifice the mouse 24 hours after modeling and collect the specimen for testing.

[0068] The cell culture and grouping in S2 are as follows:

[0069] B1: HUVECs cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in a 37°C, 5% CO2 cell culture incubator. HUVECs cells refer to human umbilical vein endothelial cells.

[0070] B2: The cells were divided into four groups: control group, LPS group, LPS+QUE group and QUE group. The LPS+QUE group and QUE group were first treated with quercetin (30 mmol / L) for 1 h, and then the LPS group and LPS+QUE group were treated with LPS (1 μg / ml) for 24 h. Finally, the cells from the above four groups were collected for further experiments.

[0071] In S3, the wet-to-dry weight ratio (W / D) of the lungs in experimental animals was used to assess pulmonary edema: the left lung tissue of the four groups of mice in S1 was taken after sacrifice, placed on pre-weighed tin foil, and the wet weight (W) of the lungs was measured. Then, the sample was placed in an 80℃ oven for 48 hours until constant weight was achieved, and its dry weight (D) was measured. The wet-to-dry weight ratio of the lung tissue was calculated by W / D. In S3, the bronchoalveolar lavage fluid (BALF) was collected: the four groups of mice in S1 were anesthetized and intubated. The lungs were flushed with 1mL PBS buffer 3-5 times to collect BALF. The obtained BALF was centrifuged at 1500rpm at 4℃ for 15min, and the supernatant was collected. The collected supernatant was stored in a -80℃ refrigerator for later use.

[0072] The pathological observation of lung tissue from the experimental animals in S4 is shown below.

[0073] C1: Take the right lung of a mouse and immerse it in 4% paraformaldehyde for 24 hours;

[0074] C2: The specimens were then dehydrated with ethanol solutions of different concentrations, embedded in paraffin, cut into 5-7 μm thick sections, dewaxed with xylene, and stained with hematoxylin and eosin (HE).

[0075] C3: After staining, dehydrate and mount the slides, then observe the morphology of mouse lung tissue under a microscope.

[0076] In S5, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were detected by ELISA: following the instructions of the ELISA kit, the levels of TNF-α, IL-6, and IL-1β in the BALF (bronchoalveolar lavage fluid) and cell culture medium (supernatant) of each group of mice in S3 were measured.

[0077] The Western blot detection pathway proteins in S6 are shown below:

[0078] D1: Mouse lung tissue and HUVECs cells were collected, and proteins were extracted from the lung tissue and HUVECs cells using RIPA buffer containing PMSF. The protein concentration was determined using a BCA kit.

[0079] D2: After quantification, add loading buffer, denature in boiling water for 10 min, and store at -20℃ for later use;

[0080] D3: Prepare a 10% SDS-PAGE gel, add 50 μg of protein sample to each well, transfer to a PVDF membrane after electrophoresis, block with TBST containing 5% skim milk powder for 1 h, add the corresponding primary antibody and incubate overnight at 4℃; wash the membrane 3 times with TBST buffer, add secondary antibody and incubate at 37℃ for 1 h, wash the membrane 3 times with TBST buffer, develop and expose with ultrasensitive luminescent solution, and analyze the gray values ​​of protein bands using ImageJ software system.

[0081] The qPCR detection of related nucleic acid expression:

[0082] E1: Mouse lung tissue and HUVECs were collected, and RNAiso was added and mixed thoroughly. After precipitation with isopropanol and washing with 75% ethanol, the mixture was dissolved in DEPC water. The concentration was detected using a nucleic acid analyzer, and the RNA was reverse transcribed into cDNA.

[0083] E2: Primer sequences: SCAP upstream 5'-GCATCACAGCCCTTGTCTTC-3' downstream 5'-CACTGTGTTGCTGCTGCTGTA-3'; SREBP2 upstream 5'-GTCGATCAAGTCAGCAGCCAAG-3' downstream 5'-TTGGCCTGAGGTTTCACCAAG-3'; NLRP3 upstream 5'-TACGGCCGTCTACGTCTTCT-3' downstream 5'-CGCAGATCACACTCCTCAAA-3'; GAPDH upstream 5'-TGTGTCCGTCGTGGATCTGA-3' downstream 5'-TTGCTGTTGAAGTCGCAGGAG-3'.

[0084] In S8, cell death rate was detected by flow cytometry using the Annexin V-FITC / PE apoptosis detection kit. HUVECs cells were resuspended in 500 µl of binding buffer and then incubated with FITC-labeled Annexin V (5 µl) and PE (5 µl) at room temperature in the dark for 15-20 min. Cell death rate was then measured by flow cytometry. Statistical analysis in S8 was performed using GraphPad Prism 9.0 software. Quantitative data are expressed as mean ± standard deviation (x±s). One-way ANOVA was used for comparisons between groups, and LSD-t test was used for pairwise comparisons within groups. P < 0.05 was considered statistically significant.

[0085] Pathological damage to mouse lung tissue:

[0086] 24 hours after modeling, the HE staining results are as follows: Figure 1 show, Figure 1 A control group and Figure 1 The alveolar structure in the DQUE group was basically intact, the septa were not thickened, and there was no obvious alveolar interstitium; compared with the control group. Figure 1 In the BLPS group mice, the normal alveolar structure was significantly disrupted, with obvious hemorrhage and inflammatory cell infiltration, and thickened septa; Figure 1 Comparison of BLPS group mice Figure 1 In the CLPS+QUE group, lung tissue structure damage was reduced, alveolar structure was visible, and hemorrhage and inflammatory cell infiltration were reduced.

[0087] Mouse lung tissue wet / dry weight ratio:

[0088] like Figure 2 A shows that the W / D ratio of lung tissue can effectively reflect the degree of pulmonary edema. Compared with the control group, the W / D ratio of the LPS group was significantly increased (P<0.05). Compared with the LPS group, the W / D ratio of the LPS+QUE group was decreased (P<0.05). This indicates that using QUE can alleviate the degree of pulmonary edema in LPS-induced model mice. Figure 2 A: Wet / dry weight ratio of lung tissue.

[0089] Total protein and total cell count in bronchoalveolar lavage fluid:

[0090] As shown in 2B-C, BALF was collected from anesthetized mice 24 hours after modeling. Compared with the control group, the protein content and total cell number of BALF in the LPS group were significantly increased (P<0.05); compared with the LPS group, the protein content and total cell number of BALF in the LPS+QUE group were significantly decreased (P<0.05). This indicates that supplementing with QUE can reduce pulmonary vascular permeability and alleviate pulmonary inflammatory response in LPS-modeled mice. Figure 2 B: Total number of cells in BALF; Figure 2 C: Total protein concentration in BALF.

[0091] Levels of inflammatory factors in bronchoalveolar lavage fluid:

[0092] like Figure 2 As shown in the DF, a cascade of amplified inflammatory responses can occur during ALI; changes in pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in lung tissue reflect the body's inflammation level; ELISA results showed that in a mouse model of acute lung injury, IL-1β, IL-6, and TNF-α were significantly elevated in BALF, while injection of QUE significantly reduced the expression levels of these inflammatory factors (P < 0.05). Figure 2 D: Level of TNF-α in BALF; Figure 2 E: Levels of IL-1β in BALF; Figure 2 F: IL-6 level in BALF.

[0093] like Figure 3 As shown in AC, qPCR results indicated that, compared with the control group, the expression of SCAP, SREBP2, and NLRP3 was increased in the LPS group (P<0.05), while the expression of SCAP, SREBP2, and NLRP3 was decreased in the LPS+QUE group compared with the LPS group (P<0.05); similarly, as Figure 3 As shown in the DE, Western blot results indicated that compared to the control group, the expression of SCAP / SREBP2 / NLRP3 signaling pathway-related proteins (SCAP, SREBP2, NLRP3) was increased in the LPS group (P<0.05); compared to the LPS group, the expression levels of these proteins were decreased in the LPS+QUE group (P<0.05). Figure 3 A: SCAP nucleic acid expression level; Figure 3 B: SREBP2 nucleic acid expression level; Figure 3 C: NLRP3 nucleic acid expression level; Figure 3 D: Results of Western blot analysis of mouse lung tissue; Figure 3 E: Relative expression level of SCAP protein; Figure 3 F: Relative expression level of SREBP2 protein; Figure 3 G: Relative expression level of NLRP3 protein.

[0094] HUVECs cell viability and inflammatory factor levels:

[0095] like Figure 4 As shown in AF, CCK-8 and flow cytometry results indicated that compared with the control group, the LPS group showed significantly decreased cell viability and significantly increased cell mortality (P<0.05); compared with the LPS group, the LPS+QUE group showed significantly increased cell viability and decreased cell mortality (P<0.05). Figure 4 G-IELISA results showed that, compared with the control group, the levels of IL-1β, IL-6 and TNF-α in the cell culture medium of the LPS group were significantly increased (P<0.05); compared with the LPS group, the levels of inflammatory factors in the LPS+QUE group were decreased (P<0.05). Figure 4 A: Cell death rate; Figure 4 B: Control group; Figure 4 C: LPS group; Figure 4 D: LPS+QUE group; Figure 4 E: QUE group; Figure 4 F: Cell activity; Figure 4 G: Level of TNF-α in HUVECs supernatant; Figure 4H: Level of IL-1β in HUVECs supernatant; Figure 4 I: Level of IL-6 in HUVECs supernatant.

[0096] HUVECs cell SCAP / SREBP2 / NLRP3 levels:

[0097] like Figure 5 As shown in AC, qPCR results indicated that compared to the control group, the expression of SCAP, SREBP2, and NLRP3 was increased in the LPS group (P<0.05), while the expression of SCAP, SREBP2, and NLRP3 was significantly decreased in the LPS+QUE group compared to the LPS group (P<0.05). Figure 5 D-GWestern blot results showed that, compared with the control group, the expression of SCAP, SREBP2 and NLRP3 in the cell culture medium of the LPS group was increased (P<0.05); compared with the LPS group, the expression of related proteins in the LPS+QUE group was decreased (P<0.05). Figure 5 A: SCAP nucleic acid expression level; Figure 5 B: SREBP2 nucleic acid expression level; Figure 5 C: NLRP3 nucleic acid expression level; Figure 5 D: Results of Western blot analysis of mouse lung tissue; Figure 5 E: Relative expression level of SCAP protein; Figure 5 F: Relative expression level of SREBP2 protein; Figure 5 G: Relative expression level of NLRP3 protein.

[0098] The terms "front," "back," "left," "right," "top," and "bottom" all refer to the figures in the accompanying drawings. Figure 1 Based on the perspective of the observer, the side of the device facing the observer is defined as the front, the left side of the observer is defined as the left, and so on.

[0099] In the description of this invention, it should be understood that the terms "center", "longitudinal", "lateral", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting the scope of protection of this invention.

[0100] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for studying an animal model of quercetin alleviating lipopolysaccharide-induced acute lung injury via the SCAP / SREBP2 / NLRP3 pathway, characterized in that... The research method is as follows: S1: Grouping and treatment of laboratory animals: A1: Forty male C57BL / 6 mice were randomly divided into a control group, an LPS group, an LPS+QUE group, and a QUE group, with 10 mice in each group. LPS was lipopolysaccharide and QUE was quercetin. A2: Mice in the LPS+QUE group and QUE group, which had been intraperitoneally injected with 100 mg / kg of quercetin solution, and mice in the control group, which had been injected with an equal amount of PBS buffer, were obtained. A3: 1 hour later, four groups of mice that had been anesthetized with 50 mg / kg pentobarbital were obtained. Then, mice in the LPS group and LPS+QUE group that had been intubated and infused with 5 mg / kg LPS in a total volume of 100 μl were obtained. Mice in the control group and QUE group that had been infused with the same volume of sterile PBS buffer were obtained according to the above procedure. A4: After infusion, hold the mouse upright for 2-3 minutes to allow the solution to fully settle in the lungs. 24 hours after modeling, collect the euthanized mice and collect the specimens for testing. S2: Cell culture and grouping: B1: HUVECs cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in a 37°C, 5% CO2 cell culture incubator. HUVECs cells refer to human umbilical vein endothelial cells. B2: The cells were divided into 4 groups: control group, LPS group, LPS+QUE group and QUE group. The LPS+QUE group and QUE group were first treated with quercetin for 1 h at a concentration of 30 mmol / L. Then the LPS group and LPS+QUE group were treated with LPS for 24 h at a concentration of 1 μg / ml. Finally, the cells from the above four groups were collected for further experiments. S3: Assessment of pulmonary edema and bronchoalveolar lavage fluid (BALF) collection in experimental animals using the wet-to-dry ratio (W / D): Left lung tissue was collected from the four groups of mice in S1 after sacrifice. The wet weight of the lung tissue was measured by placing it on pre-weighed aluminum foil. The sample was then placed in an 80℃ oven for 48 hours until constant weight was achieved, and its dry weight was measured. The wet-dry ratio of the lung tissue was calculated by W / D. Bronchoalveolar lavage fluid (BALF) collection in S3: Four groups of mice in S1 that had been anesthetized and intubated were collected. The lungs were rinsed with 1 mL of PBS buffer 3-5 times to collect BALF. The collected BALF was centrifuged at 1500 rpm at 4℃ for 15 min, and the supernatant was collected. The collected supernatant was stored in a -80℃ freezer for later use. S4: Pathological observation of lung tissue in experimental animals: C1: Take the right lung of a mouse and immerse it in 4% paraformaldehyde for 24 hours; C2: The specimens were then dehydrated with ethanol solutions of different concentrations, embedded in paraffin, cut into 5-7 μm thick sections, dewaxed with xylene, and stained with hematoxylin and eosin. C3: After staining, dehydrate and mount the slides, then observe the morphology of mouse lung tissue under a microscope; S5: ELISA method for detecting tumor necrosis factor-α, interleukin-6, and interleukin-1β levels: Following the instructions of the ELISA kit, the levels of TNF-α, IL-6, and IL-1β in the BALF and cell culture medium of each group of mice in S3 were measured. S6: Western blot detection of pathway proteins: D1: Mouse lung tissue and HUVECs cells were collected, and proteins were extracted from the lung tissue and HUVECs cells using RIPA buffer containing PMSF. The protein concentration was determined using a BCA kit. D2: After quantification, add loading buffer, denature in boiling water for 10 min, and store at -20℃ for later use; D3: Prepare a 10% SDS-PAGE gel, add 50 μg of protein sample to each well, transfer to a PVDF membrane after electrophoresis, block with TBST containing 5% skim milk powder for 1 h, add the corresponding primary antibody and incubate overnight at 4℃; wash the membrane 3 times with TBST buffer, add secondary antibody and incubate at 37℃ for 1 h, wash the membrane 3 times with TBST buffer, develop and expose with ultrasensitive luminescent solution, and analyze the gray values ​​of protein bands using ImageJ software system. S7: qPCR detection of related nucleic acid expression: E1: Take mouse lung tissue and HUVECs, add RNAiso and mix thoroughly. After precipitation with isopropanol and washing with 75% ethanol, dissolve in DEPC water and detect its concentration with a nucleic acid analyzer. Reverse transcribe the RNA into cDNA. E2: Primer sequence: SCAP upstream 5'-GCATCACAGCCCTTGTCTTC-3' Downstream 5'-CACTGTGTTGCTGCTGCTGTA-3'; SREBP2 upstream 5'-GTCGATCAAGTCAGCAGCCAAG-3' downstream; 5'-TTGGCCTGAGGTTTCACCAAG-3'; NLRP3 upstream 5'-TACGGCCGTCTACGTCTTCT-3' downstream 5'-CGCAGATCACACTCCTCAAA-3'; GAPDH upstream 5'-TGTGTCCGTCGTGGATCTGA-3' downstream 5'-TTGCTGTTGAAGTCGCAGGAG-3'; S8: Flow cytometry detection of cell death rate and statistical analysis using GraphPad Prism 9.0 software: Cell death rate was determined by flow cytometry using the Annexin V-FITC / PE apoptosis detection kit. HUVECs were resuspended in 500 µl of binding buffer and then incubated with FITC-labeled Annexin V and PE at room temperature in the dark for 15–20 min. Cell death rate was then measured by flow cytometry. Statistical analysis was performed using GraphPad Prism 9.0 software. Quantitative data are expressed as mean ± standard deviation (x±s). One-way ANOVA was used for comparisons between groups, and LSD-t test was used for pairwise comparisons within groups. P < 0.05 was considered statistically significant.

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