CN107648248A - Lamp-dish flower acetic is preparing the application in treating NLRP3 relevant disease medicines - Google Patents

Abstract

The invention discloses the application of scutellarin in the preparation of medicines for treating NLRP3-related diseases. In the present invention, it is found that scutellarin, as an inhibitor of NLRP3 inflammasome, can inhibit the activation of NLRP3 inflammasome and cell pyroptosis. Scutellarin can significantly inhibit the release of pro-inflammatory factor IL-1β and danger signal molecule HMGB1 induced by LPS+ATP, inhibit cell pyroptosis, and effectively reduce the death caused by bacterial sepsis. Drugs for NLRP3 inflammasome activation and pyroptosis-related diseases caused by endogenous DAMPs. The present invention discovers the new activity of scutellarin, which is developed for the prevention and treatment of acquired inflammatory diseases and autoimmune diseases caused by hereditary NLRP3 mutations, hereditary cryopyrin-associated periodic fever syndrome, infectious inflammatory diseases and neurological diseases. Drugs for degenerative diseases provide the rationale.

Description

Application of scutellarin in the preparation of medicines for treating NLRP3-related diseases

technical field

The invention belongs to the fields of biomedicine and medicine, and particularly relates to the application of scutellarin in the preparation of medicines for treating NLRP3-related diseases.

Background technique

Scutellarin (also known as scutellarin) is a flavonoid isolated from scutellarin and other Chinese herbal medicines ("Pharmacopoeia of the People's Republic of China" 2010 edition), with a molecular formula of C ₂₁ H ₁₈ O ₁₂ , and its chemical structure is as follows shown. Scutellarin is yellow needle-like crystal, soluble in dimethyl sulfoxide and ethanol, insoluble in water. Clinically, it is mainly used for the treatment of ischemic cardiovascular and cerebrovascular diseases, such as angina pectoris, myocardial infarction, stroke, cerebral thrombosis and paralysis after cerebrovascular disease, etc., and has obvious curative effect. In addition, studies have shown that scutellarin has obvious antioxidant effects, can effectively eliminate the generation of oxygen free radicals, and thus exert a protective effect on cardiovascular and cerebrovascular diseases.

NLRP3 is one of the danger signal receptors in innate immune cells (including macrophages), which can be activated by various pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to form multi-subunit inflammasomes. At the same time, it induces inflammatory death of cells - pyroptosis, which triggers a strong inflammatory response, which is an important part of the body's innate immune defense system.

Although the NLRP3 inflammasome is an important part of innate immunity, its abnormal activation and dysfunction are closely related to the pathological process of a variety of acquired inflammatory diseases and autoimmune diseases caused by inherited NLRP3 mutations. A variety of metabolic diseases and immune disorders, such as obesity, type 2 diabetes (T2D), atherosclerosis, gout, etc., are all related to abnormal activation of NLRP3 induced by endogenous DAMPs . In obese mice and human adipose and liver tissues, the expression of NLRP3 inflammasome components and elevated caspase-1 activity are directly related to the symptoms of T2D in obese individuals; islet amyloid polypeptide (IAPP) may be associated with NLRP3 in T2D Associated with abnormal activation. Cholesterol crystals can cause the destruction of macrophage lysosomes and activate NLRP3 inflammasome and the secretion of interleukin-1 (IL-1) family cytokines, which is an important cause of atherosclerosis. Endogenous sodium urate crystals (MSU) can also cause NLRP3 activation in macrophages, leading to the release of inflammatory cytokines such as interleukin-1β (IL-1β) and inflammatory responses, which are closely related to the occurrence of gout (gout). strong association. Hereditary cryopyrin-associated periodic fever syndrome (CAPS) is associated with gain-of-function mutations in NLRP3 that constitutively activate NLRP3, resulting in persistent activation of caspase-1 and excessive secretion of IL-1β and IL-18. In addition, the occurrence and progression of neurodegenerative diseases and brain damage are also related to NLRP3: such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, etc., may It is related to the activation of NLRP3 induced by endogenous DAMPs (such as misfolded proteins) and the production of inflammatory cytokines such as IL-1β; recent studies have shown that ischemic brain injury is also associated with the activation of NLRP3. It is worth noting that the occurrence and development of sepsis is also associated with abnormal activation of inflammasomes (including NLRP3) and excessive pyroptosis. Bacterial infection not only produces a large amount of PAMP in the body, but also the bacteria themselves can release ATP, and can also induce monocytes/macrophages to release ATP, thereby activating the activation of inflammasomes such as NLRP3, causing a strong inflammatory response in the acute phase, leading to innate immune cells (including Excessive pyroptosis of macrophages) and a significant increase in suppressive immune cells eventually suppress the immune function of the body and make patients enter an immunosuppressive state in the late stage of sepsis; at the same time, pyroptosis of parenchymal cells in organs may also be the cause of organ dysfunction. important cause of obstacles. Therefore, inhibiting the activation of NLRP3 inflammasome and the occurrence of pyroptosis will help alleviate the above-mentioned inflammatory diseases and provide new therapeutic approaches.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide the application of scutellarin in the preparation of drugs for the treatment of NLRP3-related diseases. As an inhibitor of NLRP3 inflammasome, the scutellarin can effectively inhibit The activation and pyroptosis of cells can be used to prepare medicines for treating NLRP3-related diseases.

The purpose of the present invention is achieved through the following technical scheme: the application of scutellarin in the preparation of medicines for treating NLRP3-related diseases.

The NLRP3-related diseases include acquired inflammatory diseases and autoimmune diseases caused by inherited NLRP3 mutations, hereditary cryopyrin-associated periodic fever syndromes (cryopyrin-associated periodic syndromes), infectious inflammatory diseases and neurodegenerative diseases.

The acquired inflammatory diseases and autoimmune diseases caused by genetic NLRP3 mutations are metabolic diseases and immune disorders, including obesity, type 2 diabetes (T2D), atherosclerosis and gout (gout) .

The infectious inflammatory diseases include sepsis, endotoxemia, and cholestasis.

The neurodegenerative diseases include multiple sclerosis, Alzheimer's disease and Parkinson's disease.

Scutellarin inhibits NLRP3 inflammasome activation, pyroptosis, caspase-1 activation, IL-1β (pro-inflammatory factor IL-1β) secretion, HMGB1 (high mobility group box B1) secretion, Inhibiting the formation of ASC plaque (ASC speck), inhibiting the formation of ASC oligomers, anti-systemic bacterial infection, and/or anti-bacterial sepsis drugs.

The systemic bacterial infection is preferably intraperitoneal bacterial infection, and scutellarin inhibits the activation and maturation of caspase-1 by inhibiting LPS sensitization and ATP-induced activation of NLRP3 inflammasome in macrophages and pyroptosis The release of inflammatory factors such as IL-1β and HMGB1, thereby reducing the death caused by abdominal bacterial infection.

The bacteria are Gram-negative pathogenic bacteria; preferably Escherichia coli; more preferably Escherichia coli DH5α.

The drugs for treating NLRP3-related diseases or inhibiting the activation of NLRP3 inflammasomes include pharmaceutically acceptable excipients and other active ingredients that play a compatible and synergistic role, and can be in various dosage forms, such as tablets, granules, capsules, Dropping pills, sustained-release preparations, oral liquids, injections, etc.

In the present invention, bone marrow-derived macrophages (BMDM) are sensitized (priming) by LPS, and then stimulated by a second signal such as ATP to activate NLRP3 inflammasome, leading to the activation of caspase-1, and promoting the formation of mature IL-1β and HMGB1 Secretion, formation of ASC speck (ASC speck) and ASC oligomers; scutellarin pretreatment of LPS-activated BMDM cells can significantly inhibit the activation of caspase-1, inhibit the secretion of mature IL-1β, HMGB1 and the secretion of ASC speck and Formation of ASC oligomers. After intragastric administration of scutellarin in mice, the death caused by intraperitoneal injection of live bacteria can be significantly reduced. Therefore, scutellarin can inhibit systemic inflammation and sepsis and other diseases by inhibiting the activation of inflammasome and the secretion of IL-1β.

Compared with the prior art, the present invention has the following advantages and effects:

1. In the present invention, scutellarin is an inhibitor of NLRP3 inflammasome, which can significantly inhibit the activation and pyroptosis of NLRP3 inflammasome in LPS-sensitized and ATP-induced macrophages, and finally inhibit the activation of caspase-1 and The release of inflammatory factors such as mature IL-1β and HMGB1 can effectively reduce the death caused by bacterial sepsis such as abdominal bacterial infection. The above-mentioned novel activities and uses of scutellarin have not been reported in open literature.

2. In the present invention, scutellarin inhibits the activation of NLRP3 inflammasome and pyroptosis, thereby inhibiting the release of pro-inflammatory factor IL-1β and danger signal molecule HMGB1. It has a potential therapeutic effect on the clinical treatment of diseases related to abnormal activation of NLRP3, and can be used in the development of related drugs.

3. In the present invention, it is first found that scutellarin can inhibit the activation of NLRP3 inflammasome and pyroptosis: scutellarin can significantly inhibit the pro-inflammatory factor IL-1β and the danger signal molecule HMGB1 induced by LPS+ATP. Released, inhibits pyroptosis and can effectively reduce the death of mice caused by bacterial infection. Currently, there is no specific drug that can effectively prevent bacterial infection or endogenous DAMP-induced NLRP3 inflammasome activation and pyroptosis. As an inhibitor of NLRP3 inflammasome, scutellarin is used for the preparation of drugs for preventing and treating NLRP3 inflammasome activation and pyroptosis caused by infection or endogenous DAMP, and is useful for the clinical treatment of NLRP3-related diseases (such as gout; Hereditary Cryopyrin-related periodic fever syndrome; neurological diseases and brain injuries: such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, etc.; infectious inflammatory diseases, such as sepsis.) etc. have important Significance, can be used for related drug development.

Description of drawings

Figure 1 is the western blot analysis and bead-based immunoassay (CBA) of the inhibitory effect of scutellarin on LPS+ATP-induced mouse bone marrow-derived macrophage (BMDM) inflammasome activation Analysis of the level of IL-1β in the culture supernatant; wherein, Figure A is the western blot analysis figure; Figure B is the level of IL-1β in the culture supernatant.

Figure 2 shows the distribution of ASC and NLRP3 and the formation and immunoblotting of ASC plaque (ASCspeck) by immunofluorescence microscopy analysis of scutellarin on LPS+ATP-induced mouse bone marrow-derived macrophages (BMDM) Analysis of the formation of ASC oligomers; among them, panel A is the immunofluorescence microscopic analysis of ASC and NLRP3, panel B is the histogram of the percentage of cells containing ASC plaques, and panel C is the western blot of ASC oligomer formation diagram.

Fig. 3 is the propidium iodide (PI) staining fluorescence figure and western blot analysis figure of the inhibitory effect of scutellarin (scutellarin) on LPS+ATP-induced mouse bone marrow-derived macrophage (BMDM) cell pyroptosis; Among them, panel A is a fluorescence micrograph stained with propidium iodide (PI), panel B is an analysis chart of the percentage of pyroptotic cells in total cells, and panel C is an analysis chart of Western blot.

Fig. 4 is an analysis graph showing that scutellarin reduces the death rate of mice with abdominal bacterial infection.

Detailed ways

The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

Materials used in the examples:

Scutellarin was purchased from Guangdong Institute of Drug Control. Lipopolysaccharide (LPS), Adenosine Triphosphate (ATP), Propidium Iodide (PI), Hoechst 33342, Dimethyl Sulfoxide (DMSO), Sodium Deoxycholate, Bis(N-Hydroxysuccinimidyl Suberate) (disuccinimidyl suberate) was purchased from Sigma-Aldrich Company. Cell complete medium DMEM, penicillin/streptomycin, fetal bovine serum (FBS) were purchased from ThermoFisher Company. Anti-caspase-1 antibody was purchased from Santa Cruz Company. Anti-NLRP3 antibody was purchased from Adipogen AG. Anti-IL-1β, HMGB1, ASC, β-tubulin (β-tubulin antibodies are all products of Cell Signaling Technology Company. Cytometric Bead Array (CBA) Mouse IL-1βFlex Set Kit was purchased from BD Company. Trichloroacetic acid (TCA ) was purchased from Guangzhou Chemical Reagent Factory. In vitro cell experiments, scutellarin was dissolved in DMSO, and in vivo experiments, scutellarin was dissolved in PBS solution containing 2% (v/v) Tween-80.

Example 1

(1) Cell culture and treatment:

L929 cells were purchased from the Cell Bank of Kunming Institute of Zoology, Chinese Academy of Sciences. The cell culture method refers to the operation procedure of Monack laboratory (Stanford University). ¹ × 108 cells are cultured in a Erlenmeyer flask with a bottom area of 500 ^cm2 , and the culture medium is DMEM complete medium (200 mL). Keep frozen for later use.

The culture method of mouse bone marrow-derived macrophages (BMDM): C57BL/6 mice, 6-8 weeks old, female, were purchased from the Experimental Animal Center of Southern Medical University. After sacrificed by cervical dislocation, the femur was removed, and the bone marrow cells were washed out with cold PBS using a syringe. The obtained cells were cultured in a culture dish (the density is 5×10 ⁶ /dish, Medium volume is 10 mL). After culturing for 3 days, add 5 mL of fresh BM-Mac medium; on the 5th day of culturing, replace with 10 mL of fresh BM-Mac medium. On day 7 of culture, cells were collected and cultured in a 6-well plate containing 10% (v/v) FBS and DMEM complete medium containing 100 U/mL penicillin and 100 μg/mL streptomycin at a cell density of 1.2×10 ⁶ /well (2mL culture medium), cultivated for 24 hours. Then change the medium, add DMEM complete medium (2mL) containing 500ng/mL LPS to pretreat for 4 hours, activate BMDM cells, and then culture with different concentrations of scutellarin (0.1, 0.2, 0.4mmol/L) Base (1.2mL) was treated for 1 hour, and finally 3mmol/L ATP was added for 30 minutes.

(2) Detection method of caspase-1 and IL-1β in the supernatant:

After the cells were treated as above, absorb 1.2 mL of the culture supernatant, centrifuge briefly at 300×g for 5 minutes, transfer the supernatant to another centrifuge tube, and add 21 μL of 10% (w/v) sodium deoxycholate solution (final The concentration is 0.15% (w/v)), shake well, then add 94 μL of 100% trichloroacetic acid (final concentration is 7.2% (w/v)) and mix well, then precipitate the protein overnight at 4°C. Then centrifuge at 14000×g for 30 minutes, discard the supernatant. Add 700 μL of cold acetone to the precipitate to wash away the trichloroacetic acid in the precipitate; centrifuge at 14000×g for 5 minutes. Discard the supernatant and repeat the wash twice with the above cold acetone. After the last wash and centrifugation, discard the supernatant; let the residual acetone in the precipitate evaporate at room temperature, and finally dissolve it with protein electrophoresis sample solution. After boiling for 5 minutes, the protein after the above treatment was detected by immunoblotting.

(3) CBA kit detects the content of IL-1β in the supernatant:

BMDM cells were seeded in 24-well plates (density 1.5×10 ⁵ /well), and after overnight, 500ng/mL LPS was induced for 4 hours, and scutellarin (0.1, 0.2, 0.4mmol/L) was added for 1 hour. Then 3mmol/L ATP was added to stimulate for 1 hour, and finally the cell culture supernatant was collected, and the IL-1β content was determined using the CBA Mouse IL-1βFlex Set kit, and the operation steps were carried out according to the kit instructions. Finally, the flow cytometer was used for detection on the machine, and the data was acquired and analyzed through the CELLQuest software.

(4) Results: The degree of inflammasome activation was reflected by the activation level of caspase-1 (p10 fragment) and the release of IL-1β. The expression levels of activated caspase-1(p10) and IL-1β in cell culture supernatant decreased with the dose of scutellarin increasing. It shows that scutellarin dose-dependently inhibits the activation of inflammasome in LPS+ATP-induced BMDM cells (Figure 1). In Figure 1, β-tubulin is the internal reference protein for electrophoresis loading, indicating that the total amount of protein loaded in each lane is equal.

Example 2

(1) Detection of ASC plaque formation:

BMDM cells were seeded in glass-bottom culture dishes (density 5×10 ⁵ /well). After overnight, 500ng/mL LPS was induced for 4 hours, scutellarin 0.4mmol/L was added for 1 hour, and then 3mmol/L ATP was stimulated for 1 hour, and the culture medium was discarded at last, 1 mL of 4% (w/v) paraformaldehyde was added to each well, fixed at room temperature for 15 minutes, and then 2 mL of cold methanol was added to each well to permeabilize at -20°C for 10 minutes. Block with blocking solution for 1 hour at room temperature, add primary antibody (100 μL/well), incubate overnight at 4°C, add CF568-goat rabbit anti-IgG and CF488A-goat anti-mouse IgG without cross-reaction after washing (both purchased from Biotium USA) Company), incubated at room temperature for 1 hour, stained with Hoechst 33342 for 10 minutes and protected from light, observed and photographed with a Zeiss inverted fluorescence microscope.

(2) ASC oligomer detection:

Seed BMDM cells in a 6-well plate (density 1×10 ⁶ /well). After overnight, induce with 500ng/mL LPS for 4 hours, add scutellarin 0.4mmol/L for 1 hour, and finally discard the supernatant , Add 500 μL pre-cooled PBS containing 0.5% (v/v) TritonX-100 to each well. Centrifuge the cell lysate at 4°C, 6000×g for 5 minutes, discard the supernatant, add pre-cooled PBS to wash twice, then resuspend in 200 μL of PBS, directly add suberic acid bis(N-hydroxysuccinimide) ester) to a final concentration of 2 mmol/L, and reacted at room temperature for 30 minutes. Centrifuge at 6000×g for 15 minutes at 4°C to absorb the supernatant, add 25 μL of 2× electrophoresis loading buffer, boil for 5 minutes, and detect the protein after the above treatment by immunoblotting.

(3) Results: Figure 2 shows the distribution of ASC and NLRP3 and the distribution of ASC plaques (ASC speck ) formation and immunoblot analysis of ASC oligomer formation. It can be seen from this figure that scutellarin can significantly inhibit the formation of ASC plaques and ASC oligomers in BMDM cells induced by LPS+ATP, further indicating that scutellarin can inhibit the activation of NLRP3 inflammasome.

Example 3

(1) Cell culture and treatment:

BMDM cells were seeded in 24-well plates (density 1.5×10 ⁵ /well), after overnight, induced with 500ng/mL LPS for 4 hours, and added scutellarin (0.1, 0.2, 0.4mmol/L) for 1 hour , then add 3mmol/L ATP to stimulate for 1 hour, and finally add PI (2μg/mL) and Hoechst 33342 (5μg/mL) for staining at room temperature for 10 minutes, place it under a Zeiss inverted fluorescence microscope to observe and take pictures.

(2) Detection method of HMGB1 in the supernatant:

After the cells were treated as above, absorb 1.2 mL of the culture supernatant, centrifuge briefly at 300×g for 5 minutes, transfer the supernatant to another centrifuge tube, and first add 21 μL of 10% (w/v) sodium deoxycholate solution (final The concentration is 0.15% (w/v)), shake well, then add 94 μL of 100% trichloroacetic acid (final concentration is 7.2% (w/v)) and mix well, then precipitate the protein overnight at 4°C. Then centrifuge at 14000×g for 30 minutes and discard the supernatant. Add 700 μL of cold acetone to the precipitate to wash away the trichloroacetic acid in the precipitate; centrifuge at 14000×g for 5 minutes. Discard the supernatant and repeat the wash twice with the above cold acetone. After the last wash and centrifugation, discard the supernatant; let the residual acetone in the precipitate evaporate at room temperature, and finally dissolve it with protein electrophoresis sample solution. After boiling for 5 minutes, the protein after the above treatment was detected by immunoblotting.

(3) Results: After inducing pyroptosis, the cells can be stained positively by PI, and can promote the release of the danger signal molecule HMGB1. Therefore, pyroptosis can be analyzed by PI staining and detecting the release level of HMGB1 in the supernatant. Fig. 3 is propidium iodide (PI) staining fluorescence image and western blot analysis image of the inhibitory effect of scutellarin on LPS+ATP-induced mouse bone marrow-derived macrophage (BMDM) cell pyroptosis. Red fluorescence (PI staining) indicated that the cells were pyroptotic, and scutellarin dose-dependently inhibited the pyroptosis of BMDM cells induced by LPS+ATP and the release of the death marker HMGB1 in the supernatant. It can be seen from this figure that pretreatment with scutellarin can dose-dependently inhibit ATP-induced pyroptosis, and the release level of HMGB1 in the supernatant decreases with the dose of scutellarin ( FIG. 3C ). In Figure 3C, β-tubulin is the internal reference protein loaded by electrophoresis, indicating that the total amount of protein loaded in each lane is equal.

Example 4

(1) Bacterial culture:

Escherichia coli DH5α was shaken in Luria Broth (LB) medium overnight (37°C), and the next day, 10% (v/v) bacterial solution was used to continue to shake and expand in fresh medium for 3 hours (37°C). The amplified bacterial solution was centrifuged at 1824×g for 10 minutes, washed with PBS, and resuspended in PBS solution for later use. Bacterial density was measured with a micro-volume ultraviolet spectrophotometer (Nanodrop 2000; Thermo Scientific); the corresponding colony-forming units (CFU) were determined by spreading culture on LB agarose plates.

(2) Animal experiments:

C57BL/6 mice, 6-8 weeks old, female, were purchased from the Experimental Animal Center of Southern Medical University. After one week of adaptive breeding in this laboratory, scutellarin-containing The PBS solution was pre-gavaged, and 3 hours later, Escherichia coli (2.5×10 ⁹ CFU/mouse) was intraperitoneally inoculated, and 1 hour later, the above-mentioned PBS solution containing scutellarin was administered to the mice again. In the next 120 hours, the survival of the mice was observed and recorded every 6 hours, and the Kaplan-Meier survival curve of the mice was drawn to analyze the survival rate. The difference analysis of the survival rate of mice was analyzed by Long-rank test, and P<0.05 was considered statistically significant.

(3) Results:

Escherichia coli infection can lead to the death of mice due to infection; while scutellarin can effectively reduce the death rate of mice with abdominal bacterial infection (Figure 4). *P<0.05; **P<0.01.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (9)

1. lamp-dish flower acetic is preparing the application in treating NLRP3 relevant disease medicines.

2. lamp-dish flower acetic according to claim 1 is preparing the application in treating NLRP3 relevant disease medicines, its feature It is：Described NLRP3 relevant diseases are autoimmunity disease caused by acquired diseases associated with inflammation and heredity NLRP3 mutation, Heredity Cryopyrin associated period heat pyrexia syndromes, infective inflammation disease, and/or nerve degenerative diseases.

3. lamp-dish flower acetic according to claim 2 is preparing the application in treating NLRP3 relevant disease medicines, its feature It is：

Described acquired diseases associated with inflammation and heredity NLRP3 mutation caused by autoimmunity disease be obesity, diabetes B, Atherosclerosis and/or gout；

Described infective inflammation disease is pyemia, endotoxemia and/or cholestasis；

Described nerve degenerative diseases are multiple sclerosis, Alzheimer disease and/or Parkinson's disease.

4. lamp-dish flower acetic is preparing the corpusculum activation of suppression NLRP3 inflammation, suppress cell Jiao and die, suppress caspase-1 activation, press down IL-1 β secretions processed, suppress HMGB1 secretions, suppress ASC patches and are formed, and suppress ASC oligomer and are formed, anti-systemic bacterial infection, And/or the application in bacteria resistance medication for treating pyemia.

5. application according to claim 4, it is characterised in that：Described systemic bacterial infection is abdominal cavity bacterial infection.

6. the application according to claim 4 or 5, it is characterised in that：Described bacterium is gram negative pathogenic bacteria.

7. the application according to claim 4 or 5, it is characterised in that：Described bacterium is bacillus coli DH 5 alpha.

8. according to the application described in any one of claim 1~7, it is characterised in that：Described medicine includes pharmaceutically acceptable Auxiliary material.

9. according to the application described in any one of claim 1~7, it is characterised in that：The formulation of described medicine is tablet, particle Agent, capsule, dripping pill, sustained release agent, oral liquid or injection.