Research method of influence mechanism of succinic acid on necrotizing enterocolitis

Abstract

The invention discloses a method for researching an influence mechanism of succinic acid on necrotizing enterocolitis, and relates to the technical field of biomedical research. Comprising the following steps: S1, animal model establishment: selecting newborn mice, and randomly dividing the newborn mice into four groups; s2, intervention period: continuously processing for 3-5 days, and recording the body weight, the survival rate and the survival state every day; s3, sample collection: killing the mouse, taking intestinal tissues, and sub-packaging for different detections; s4, multi-dimensional detection is carried out; and S5, data analysis: integrating phenotype, pathology and molecular data, and verifying the function of the succinic acid-SUCNR1 axis. According to the invention, a complete technical system for researching the action mechanism of the succinic acid-SUCNR1 axis in necrotizing enterocolitis is constructed, and a complete evidence chain from phenomenon observation to mechanism clarification is formed through organic combination of animal model construction, molecular mechanism verification and pathological evaluation, so that the reliability of a research conclusion is ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical research, and in particular to a research method for the influence mechanism of succinic acid on necrotizing enterocolitis. BACKGROUND

[0002] Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in preterm infants, characterized by acute onset of intestinal ischemia and necrosis. The global incidence of NEC is 7%, and the mortality rate of most cases is about 25%, but in the most severe NEC cases, the mortality rate within 48 hours after diagnosis can be as high as 80%.

[0003] It is increasingly recognized that the complications of NEC can extend to other organs including the lungs and brain in addition to the harmful effects on the intestine. The pathogenesis of NEC is not clear, NEC is a complex disease caused by multiple factors, such as the immaturity of neonatal intestinal motility and intestinal mucosal barrier, abnormal immune response, intestinal flora imbalance, etc.; for a long time, the induction of excessive inflammation has been considered to play a role in NEC, and it has been found in both human and animal studies of NEC.

[0004] There is currently a lack of targeted treatment and prevention measures, especially a lack of treatment strategies targeting the cause itself; in recent years, metabolites such as succinic acid have been found to be abnormally elevated in NEC, but the specific mechanism of action of succinic acid in NEC is not clear. SUCNR1, as the natural receptor of succinic acid, may mediate the inflammatory signaling pathway. The prior art lacks a systematic research method to integrate animal models, molecular detection and pathological evaluation to reveal the function of the succinic acid-SUCNR1 axis. SUMMARY

[0005] The purpose of the present application is to solve the shortcomings in the prior art, and a research method for the influence mechanism of succinic acid on necrotizing enterocolitis is proposed.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: The research method for the influence mechanism of succinic acid on necrotizing enterocolitis comprises the following steps: S1: animal model establishment: select newborn mice and randomly divide them into 4 groups, as follows: ① Control group: normally raised; ② NEC model group: induced by artificial feeding, hypoxic cold stimulation, etc. ③ NEC+succinic acid intervention group: on the basis of the NEC model, intraperitoneally injected or orally administered exogenous succinic acid; ④ NEC+succinic acid+SUCNR1 antagonist group: succinic acid and SUCNR1 antagonist 7a are administered at the same time; S2: Intervention period: continuous treatment for 4 days, record body weight, survival rate and survival status every day; S3: Sample collection: sacrifice mice, take intestinal tissue, and sub-pack for different detection, as follows: Part of the tissue is frozen at -80℃ for molecular biology detection; Part of the tissue is fixed with paraformaldehyde for HE staining; Part of the tissue is used for succinic acid content determination; S4: Multi-dimensional detection: Histopathology: HE staining to evaluate intestinal injury score; Metabolite level: succinic acid determination kit to quantify intestinal tissue succinic acid content; Protein expression: WB to detect SUCNR1 receptor protein, ELASA to detect inflammatory factors; Gene expression: RT-qPCR to analyze NLRP3, TNF-α, IL-1β, IL-6 mRNA; S5: Data analysis: integrate phenotype, pathology, and molecular data to verify the function of succinic acid-SUCNR1 axis.

[0007] Preferably, in S1, the establishment of the NEC animal model includes artificial feeding, hypoxia treatment, and cold stimulation; the hypoxia treatment is 95% N2 environment exposure for 90 seconds, and the cold stimulation is 4℃ environment exposure for 10 minutes, repeated twice a day for 4 days.

[0008] Preferably, in S2, the intervention method includes applying succinic acid intervention by gavage with formula milk containing 100mmol / L exogenous succinic acid, with a dose of 30ul / g.

[0009] Preferably, in S4, the detection of inflammatory indicators includes TNF-α, IL-1β, IL-6, and NLRP3 inflammasome at mRNA and protein levels.

[0010] Preferably, in S4, the mRNA level is detected by RT-qPCR, and the primers are directed against TNF-α, IL-1β, IL-6, and NLRP3 genes.

[0011] Preferably, in S4, the protein level is detected by ELASA, including standard gradient dilution and OD value determination; intestinal pathological changes are detected by HE staining and double-blind pathological scoring.

[0012] Preferably, in S4, the succinic acid level is quantified by intestinal tissue succinic acid determination, as follows: Tissue lysis: add 100μL BeyoLysis Buffer A to 10mg tissue TM Buffer A, grind on ice; Centrifugation: 4℃, 12000xg centrifugation for 5 minutes, take supernatant; Working solution preparation: Amplex Red reaction working solution is specifically 70 muL of detection buffer, 2 muL of Amplex Red, 2 muL of enzyme solution A / B, 2 muL of auxiliary factor and 2 muL of substrate; Detection: 96-well plate sample addition, standard curve concentration 0-500 muM, determination of A570 absorbance.

[0013] Preferably, in the S4, HE staining and pathological score are specifically as follows: Fixation: paraformaldehyde fixation for 24-48 hours; Dehydration and embedding: gradient ethanol dehydration and paraffin embedding; Section staining: section thickness 4 muM, hematoxylin staining for 5 minutes after deparaffinization, hydrochloric acid alcohol differentiation and eosin re-staining; Covering observation: neutral gum covering, and photographing under a microscope at 40x and 100x magnifications; Double-blind scoring: 0-4 points, and NEC is diagnosed when the score is greater than or equal to 2.

[0014] Preferably, in the S4, RNA extraction and RT-qPCR are specifically as follows: Grinding extraction: tissue is added with QLS lysis solution and magnetic beads for grinding extraction; RNA purification: after centrifugation, the same volume of ethanol is added, centrifuged, and eluted from a column, and dissolved in enzyme-free water; cDNA synthesis: after removing gDNA, M-MLV reverse transcription is performed; qPCR system: SYBR Green method, and primers include internal references; Data calculation: 2^(-Delta Delta Ct) method is used to analyze expression differences.

[0015] Preferably, in the S4, Western Blot is specifically as follows: Lysis solution: RIPA plus protease inhibitor and phosphatase inhibitor; Electrophoresis and membrane transfer: 80V electrophoresis for 120 minutes by SDS-PAGE gel, and membrane transfer is performed at 400mA for 30 minutes; Antibody incubation: primary antibody incubation at 4℃ overnight, and secondary antibody incubation at room temperature for 1 hour; Development: chemiluminescence method detection, and ImageJ analysis of bands.

[0016] The present application has the following beneficial effects: 1. The present application constructs a complete technical system for researching the mechanism of the succinic acid-SUCNR1 axis in necrotizing enterocolitis, and forms a complete evidence chain from phenomenon observation to mechanism elucidation through the organic combination of animal model construction, molecular mechanism verification and pathological evaluation, thereby ensuring the reliability of the research conclusion.

[0017] 2. The present application not only provides a specific research method, but also establishes a standard paradigm for studying the interaction between metabolites and receptors. By correlating changes in succinic acid content, receptor expression regulation, inflammatory response activation, and pathological damage, a technical route is provided for similar research.

[0018] 3. The present application clearly demonstrates the core role of the succinic acid-SUCNR1 axis in NEC; reveals a new mechanism of NEC pathogenesis; provides a new biomarker for NEC and discovers potential therapeutic targets. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Technical roadmap of the research method of the effect of succinic acid on necrotizing enterocolitis proposed by the present application; Figure 2 Comparison and analysis chart of intestinal succinic acid content of NEC patients and model mice proposed by the present application; Figure 3 Expression upregulation of SUCNR1 receptor in NEC model and succinic acid intervention effect chart proposed by the present application; Figure 4 Multi-index evaluation chart of succinic acid aggravating NEC pathological phenotype through SUCNR1 proposed by the present application; Figure 5 Molecular evidence chart of succinic acid-SUCNR1 axis activating NLRP3 inflammatory pathway proposed by the present application. DETAILED DESCRIPTION

[0020] The technical solutions of the present application will be further described in detail below in combination with specific embodiments.

[0021] Example 1: The research method of the effect of succinic acid on necrotizing enterocolitis, comprising the following steps: S1: Animal model establishment: select newborn mice (such as C57BL / 6 strain), and randomly divide them into 4 groups, as follows: ① Control group (Ctrl): normally raised; ② NEC model group (NEC): induced NEC by artificial feeding, hypoxia cold stimulation, etc. ③ NEC+succinic acid intervention group (NEC+Succ): exogenous succinic acid is administered by gavage on the basis of the NEC model; ④ NEC+succinic acid+succinic acid receptor 1 antagonist group (NEC+Succ+7a): succinic acid and SUCNR1 antagonist 7a are administered simultaneously; S2: Intervention period: continuous treatment for 4 days, daily record of body weight, survival rate and survival status; S3: Sample collection: sacrifice mice, take intestinal tissue (such as ileocecal), and sub-pack for different detection as follows: Part of the tissue is frozen at -80℃ for molecular biology detection (WB, RT-qPCR, ELASA); Part of the tissue is fixed with paraformaldehyde for HE staining; Part of the tissue is used for succinic acid content determination; S4: Multi-dimensional detection: Histopathology: HE staining to evaluate intestinal injury score; Metabolite level: succinic acid determination kit to quantify intestinal tissue succinic acid content; Protein expression: WB detection of SUCNR1 receptor protein, ELASA detection of inflammatory factors (TNF-α, IL-1β, IL-6); Gene expression: RT-qPCR analysis of NLRP3, TNF-α, IL-1β, IL-6 mRNA; S5: Data analysis: integrate phenotype, pathology, and molecular data to verify the function of succinic acid-SUCNR1 axis.

[0022] In S5, the succinic acid level is quantified by intestinal tissue succinic acid determination as follows: Dissolve Amplex Red and detection buffer, mix well after equilibration to room temperature, and store other reagents in ice bath; Amplex Red reaction working solution preparation: (96-well plate: 80ul / well 100ul per well more than 20ul 100ulx96 wells=9.6ml); Intestinal tissue sample, 100ul BeyoLysisTM Buffer A for Metabolic Assay per 10mg tissue, grind thoroughly; 4℃ 12000xg centrifuge for 3-5min, take supernatant for subsequent experiment (all operations are carried out on ice), and the prepared tissue sample can be stored at -20℃ or -80℃ if it cannot be detected immediately; Standard preparation: take 10ul succinic acid standard solution (10nM) and add 190ul BeyoLysisTM Buffer A for Metabolic Assay to prepare a succinic acid standard solution with a concentration of 500uM. Take 500uM succinic acid standard solution (0, 0.8, 2, 4, 8, 12, 16, 20ul) and add to the 96-well plate, and correspondingly add BeyoLysisTM Buffer A for Metabolic Assay to 20ul. At this time, the standard curve concentrations are 0, 20, 50, 100, 200, 300, 400, and 500uM, respectively.

[0023] Sample loading: take 1-20ul sample or diluted sample into 96-well plate, and add the detection lysate accordingly, make up to 20ul, and set up blank control wells containing only detection lysate; Sample total dilution factor n: if the sample is diluted by 10 times in this step, add 5ul of diluted sample, n=10x20 / 5=40.

[0024] Pre-experimental sample concentration gradient setting: stock solution, 10 times, 20 times, 50 times, 100 times, 200 times, 400 times, 500 times.

[0025] One sample uses 2 EP tubes, one for stock solution and one for 50 times diluted sample (1ul stock solution sample + 49ul lysate) Stock solution Lysis solution Stock solution 20ul 10 fold 2ul 18ul 20 fold 1ul 19ul 50 fold 50 fold stock solution Lysis solution 100 fold 10ul 10ul 200 fold 5ul 15ul 400 fold 2.5ul 17.5ul 500 fold 2ul 18ul If absorbance detection is used, measure A570.

[0026] Among them, in S5, intestinal tissue damage is evaluated by HE staining and pathological scoring, specifically as follows: Put 2ml of paraformaldehyde into a 2ml EP tube, take a small paper strip for later use, and take the intestinal tissue; Fixation: paraformaldehyde fixation for 24-48 hours or more; After dehydration, embed the tissue, place the intestinal tissue flat for subsequent sectioning; Sectioning, prepare blades, tweezers, pencils, brushes, slides, and paper; before sectioning, first cut the wax block cold, place the wax block on the operating table, then align the blades; sweep the brush from bottom to top; Place the slide in a 60°C oven for 2-3 hours, then place it in a 37°C incubator overnight; HE staining, deparaffinization, processing time is xylene I 10min; xylene II 5min; Hydration: 100% ethanol 5min; 95% ethanol 5min; 80% ethanol 5min; 75% ethanol 5min Rinse with running water for 1-2min Staining: 5min; rinse with running water for 1-2min; observe the staining under a microscope; 1% hydrochloric acid alcohol 3-5 seconds; rinse with running water for 1-2min; observe the staining under a microscope; Saturated lithium carbonate 5-10s; rinse with running water for 1-2min; 95% ethanol 1min; eosin staining 1min; 75% ethanol for a few seconds; 95% ethanol I 1 min; 95% ethanol II 1 min; 100% ethanol I 2 min; 100% ethanol II 2 min; Xylene I 5 min; Xylene II 5 min; Mounting: Neutral gum drops were taken on the glass slide, and the cover glass was gradually covered from one end to avoid bubbles, and the plate was dried.

[0027] Observation and pathological score: HE sections were observed under a light microscope for morphological manifestations, and 40x and 100x magnifications were used for observation and photography for image preservation. A double-blind method was used for intestinal injury severity scoring, 0 points: no damage to the intestine; 1 point: distal epithelial shedding; 2 points: intermediate layer villus shedding; 3 points: villus complete necrosis, crypts remaining; 4 points: intestinal transmural necrosis. The score was based on the highest score observed in three to five fields of the sample, and a diagnosis of NEC was confirmed at 2 points and above.

[0028] Among them, in S5, mRNA analysis of inflammatory factors was performed by RNA extraction and RT-qPCR, as follows: Turn on the grinder (-10°C); Homogenate lysis: 10 mg (20 mg) + 300 ul (600 ul) Buffer QLS lysis solution into 2 ml centrifuge tube (deenzyme) + magnetic beads (one big and one small, blue for WB, green for PCR) Grinding (grinder parameters: running speed: 65HZ, time 45s, pause time: 15 seconds, times: 4 times, temperature: -10°C); Centrifuge: (4°C, 12000 rpm, 15 min), prepare items: A, 1.5 ml centrifuge tube (such as C1, C2..) in the centrifuge; B, centrifuge column (pink) in the reagent kit (such as C1, C2...); C, 1.5 ml centrifuge tube (double the amount of the sample), one set of labels (such as C1, C2..), one set of labels detailed final placement -80°C; Prepare deionized water and place on ice; Supernatant + equal volume of anhydrous ethanol, mix well with a shaker, absorb the supernatant into a 1.5 ml EP tube; Transfer 1 ml of the mixed liquid into the centrifuge column with a pipette; Room temperature centrifuge: 12000, 2 min, discard the filtrate, use the original tube to discard the filtrate and then use it again; add 700ul (20mg) Buffer QWB to the centrifugal column, 12000 rpm, 1 min, discard the filtrate; load with Collection Tubes, 12000 rpm, 2 min; load the final EP tube (centrifugal column) 1.5ml; add 50ul (600QLS lysis solution) to the centrifugal column, 40ul (300QLS lysis solution) deenzyme water, room temperature for 3 min; Measurement: adjust 1ul, select RNA, first perform blank detection, concentration less than 200 is discarded; more than 1000 concentration of specimen is added with water and shaken evenly; CDNA Remove gDNA, M-MLV reverse transcription (42℃, 15 min): 10ul system (in the -20℃ refrigerator reverse transcription box), prepare 200ul EP tube; 5x gDNAclear Reaction MIX 2ul Total RNA 1000 / concentration = V volume De-enzyme water 10 ul - 2 ul - V (V - 8 negative) Add volume from large volume to small volume, finger pop and centrifuge.

[0029] qPCR system: based on SYBR Green method, primers include internal reference (such as GAPDH) and target gene (NLRP3, TNF-α, etc.); Data calculation: use 2^(-ΔΔCt) method to analyze expression difference.

[0030] Among them, in the S5, the SUCNR1 protein is semi-quantified by Western Blot, specifically as follows: Preparation: Prepare lysis solution: RIPA: 500ul (50mg), generally add 500ul; Protease inhibitor: 1:100, (100 is RIPA); Phosphatase inhibitor (phosphorylated protein needs to add phosphatase inhibitor): 1:50, 50 is RIPA; For example, 6 groups, each group has four specimens, 24 samples, plus 2 losses, a total of RIPA: 500x26=13000ul=13ml; protease inhibitor: 13000÷100=130ul; phosphatase inhibitor: 13000÷50=260ul. Add the three liquids to a 15ml EP tube.

[0031] Lysis, add 500ul lysis solution to each tube, after lysis, at least stand for 30min, lysis parameters same as PCR; centrifuge the liquid after lysis in 1.5ml EP tube, after centrifugation, absorb the supernatant into 1.5ml EP tube (clearly marked); dilute loading buffer 5 times, such as 40ul loading buffer + 160ul supernatant; 100℃ water bath, 10min (need to count time); 30 times dilution, prepare 24 200ul EP tubes, mark group number and order. 29ul PBS + 1ul protein supernatant (not cooked), shake and shake evenly; Vibrate, shake and centrifuge the reagents in the BCA extraction box; 96 well plate: 1-8 columns, from 0-2000, add 2 columns, and then 24 samples add 3 columns, add 20ul to each well; Mix BCAA liquid and B liquid in the smart buffer box (room temperature) at 50:1, and add 200ul to each well, such as 200x

(2+3)x8+2 (more than two holes loss)

[0032] In S5, the inflammatory factor protein is detected by ELASA, and the specific steps are as follows: Take out the kit and place it at room temperature for 30 min, and pre-cool PBS; Grind the tissue 50mg+450ul (pre-cooled PBS+protease inhibitor)+magnetic beads with a grinder (65Hz, run 45s, interval 15s, 4 cycles). Stand for 20-30 minutes (as much as possible to crack the liquid); Take the liquid and put it into a centrifuge, 5000xg, centrifuge for 5-10min; Take the supernatant (can be stored in-80C); Standard gradient working solution preparation: add 1ml universal diluent to the freeze-dried standard, stand for 15 minutes, and mix gently after complete dissolution (the concentration is different according to the kit) (the concentration is 1000pg / mL), then according to 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.25pg / mL, 15.62pg / mL, 0pg / mL.

[0033] Dilution method: take 7 EP tubes, add 500ul universal diluent to each tube, 500ul 1000pg / mL standard working solution to the first EP tube, mix evenly to 500pg / ml standard working solution, and follow the same steps. The last tube is directly blank, and no liquid needs to be taken from the second to last tube.

[0034] Add sample; Biotinization detection working solution preparation: 15min before use, centrifuge 100x concentrated biotinization antibody at 1000xg for 1min, dilute 100x concentrated biotinization antibody to 1x working concentration with universal diluent (for example: 10ul concentrated solution+990ul universal diluent), and use it immediately.

[0035] Enzyme binding working solution preparation: 15min before use, centrifuge 100x concentrated enzyme binding at 1000xg for 1min, dilute 100x concentrated HRP enzyme binding with universal diluent to 1x working concentration (for example: 10ul concentrated solution+990ul universal diluent), and use it immediately.

[0036] 1x Washing Solution Preparation: Add 10ml of 20x Washing Solution to 190ml of distilled water (Concentrated washing solution taken from the refrigerator may have crystals, which is normal. It can be placed at room temperature until the crystals are completely dissolved before preparation).

[0037] Remove the required strips from the aluminum foil bag after equilibration to room temperature for 10 minutes, and seal the remaining strips in a resealable bag and return them to 4°C.

[0038] Add samples: Add 100 μL of sample or standard at different concentrations to each well, and add 100 μL of universal diluent to the blank well. Cover with sealing film and incubate at 37°C for 1 hour. (It is recommended to dilute the sample at least 1-fold with universal diluent before adding it to the ELISA plate for testing. For all test samples and standards, it is recommended to set up duplicate wells.)

[0039] Add biotinylated antibody: Remove the ELISA plate, discard the liquid, and do not wash. Add 100 μL of biotinylated antibody working solution directly to each well, cover with sealing film, and incubate at 37°C for 60 minutes.

[0040] Wash the plate: Discard the liquid, add 300 μL of 1x washing solution to each well, pat dry on absorbent paper, and repeat the washing process 3 times. Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film, and incubate at 37°C for 30 minutes.

[0041] Wash the plate: Discard the liquid and wash the plate 5 times using the washing method described above. Add substrate: Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 min.

[0042] Add stop solution: Take out the microplate, add 50 μL of stop solution directly to each well, and immediately measure the OD value of each well at a wavelength of 450 nm.

[0043] Experimental results: like Figure 2 As shown: A. Children with NEC had higher levels of succinic acid in their stools compared to children without NEC. B.NEC model mice had higher levels of succinic acid in their intestinal tissue compared to normal mice; like Figure 3 As shown: A. Immunohistochemical analysis showed that the level of succinate receptor (SUCNR1) in the intestinal tissue of NEC model mice was significantly higher than that in the intestinal tissue of normal mice. B, C, and WB experiments showed that the protein expression of SUCNR1 in the intestinal tissue of NEC model mice was higher than that in the intestinal tissue of normal mice. In the NEC model mice treated with exogenous succinic acid (NEC+Succ), the protein expression of SUCNR1 in the intestinal tissue was significantly higher than that in the intestinal tissue of NEC model mice. As shown in Figure 4 A, B. The expression of succinate receptor (SUCNR1) in the intestinal tissue of mice in the NEC + Succ + 7a group was significantly lower than that in the NEC + Succ group.

[0044] C. Changes in body weight of mice in each group were monitored during modeling: the body weight of mice in the normal group (Ctrl) showed a good growth trend, while the body weight of mice in the NEC group showed stagnation or decrease compared with the normal group, the body weight of mice in the NEC + Succ group was significantly lower than that in the NEC group, and the body weight of mice in the NEC + Succ + 7a group showed no significant change compared with the NEC + Succ group. It is suggested that succinic acid can aggravate the weight loss of mice in the NEC model, but the weight loss aggravated by succinic acid is not improved after treatment with succinate receptor antagonist (7a).

[0045] D. The mortality of mice in each group was monitored during modeling: no mice died in the normal group (Ctrl), the mortality of mice in the NEC group was significantly increased compared with the normal group, the mortality of mice in the NEC + Succ group was increased compared with the NEC group, and the mortality of mice in the NEC + Succ + 7a group was decreased compared with the NEC + Succ group.

[0046] E. The appearance of mice in each group after modeling: the mice in the normal group had normal body size and shiny hair; the mice in the NEC group were smaller than the normal mice, and their hair was darker; the mice in the NEC + Succ group were significantly smaller than the NEC mice, and their hair was not shiny, and obvious skin wrinkles could be seen; the mice in the NEC + Succ + 7a group were slightly larger than the NEC + Succ mice, and their hair was shiny.

[0047] F. Intestinal tissue of mice in each group was dissected after modeling: the intestinal tissue of mice in the normal group showed no hyperemia and swelling; the intestinal tissue of mice in the NEC group showed obvious inflation, and beaded changes could be seen; the intestinal tissue of mice in the NEC + Succ group showed obvious bleeding; the intestinal tissue of mice in the NEC + Succ + 7a group showed less bleeding than that in the NEC + Succ group.

[0048] G. HE staining results of intestinal tissue of mice in each group: the basal layer of intestinal tissue of normal mice was complete, and the intestinal villi were normal; the basal layer of intestinal tissue of NEC mice was thin, and the intestinal villi were obviously inflated; the basal layer of intestinal tissue of mice in the NEC + Succ group was significantly thinned, and the intestinal villi were shed; the basal layer of intestinal tissue of mice in the NEC + Succ + 7a group was thicker than that in the NEC + Succ group, and the pathological changes of intestinal villi were reduced.

[0049] ​H. The results of HE score of each group of mice: the HE score of intestinal tissue of normal group mice was 0-1, the HE score of intestinal tissue of NEC group mice was 2-3, the HE score of intestinal tissue of NEC+Succ group mice was 3-4, and the HE score of intestinal tissue of NEC+Succ+7a group mice was 2-3.

[0050] As shown in Figure 5 A. RT-qPCR was used to analyze the changes of NLRP3 inflammasome mRNA. The expression of NLRP3 inflammasome mRNA in the normal group was low, the expression in the NEC group increased, the expression of NLRP3 inflammasome mRNA in the NEC+Succ group was significantly increased compared with the NEC group, and the expression of NLRP3 inflammasome mRNA in the NEC+Succ+7a group was reduced compared with the NEC+Succ group.

[0051] B, C, D. RT-qPCR was used to analyze the changes of pro-inflammatory factors TNF-α, IL-1β, IL-6 mRNA. The expression of TNF-α, IL-1β, IL-6 mRNA in the normal group was low, the expression in the NEC group increased, the expression of TNF-α, IL-1β, IL-6 mRNA in the NEC+Succ group was significantly increased compared with the NEC group, and the expression of TNF-α, IL-1β, IL-6 mRNA in the NEC+Succ+7a group was reduced compared with the NEC+Succ group.

[0052] F, G. ELASA was used to determine the changes of TNF-α, IL-1β, IL-6 protein levels. The expression of TNF-α, IL-1β, IL-6 mRNA in the normal group was low, the expression in the NEC group increased, the expression of TNF-α, IL-1β, IL-6 mRNA in the NEC+Succ group was significantly increased compared with the NEC group, and the expression of TNF-α, IL-1β, IL-6 mRNA in the NEC+Succ+7a group was reduced compared with the NEC+Succ group.

[0053] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can make equivalent replacement or change according to the technical solution and the inventive concept of the present application within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.​

Claims

1. A research method for studying the mechanism of succinic acid's effect on necrotizing enterocolitis, characterized by, Includes the following steps: S1: Animal Model Establishment: Newborn mice were selected and randomly divided into 4 groups, as follows: ① Control group: Normal feeding; ②NEC model group: NEC was induced through artificial feeding, hypoxia and cold stimulation, etc. ③ NEC + succinic acid intervention group: exogenous succinic acid was injected intraperitoneally or administered orally on the basis of the NEC model; ④ NEC + succinic acid + succinic acid receptor 1 antagonist group: Simultaneous administration of succinic acid and SUCNR1 antagonist 7a; S2: Intervention period: Continuous treatment for 4 days, with daily recording of weight, survival rate and survival status; S3: Sample Collection: Mice were euthanized, intestinal tissue was collected, and aliquoted for different tests, as detailed below: Some tissues were cryopreserved at -80°C for molecular biological assays. Some tissues were fixed with paraformaldehyde for HE staining; Some tissues were used for succinic acid content determination; S4: Multi-dimensional detection: Histopathology: HE staining was used to assess intestinal injury scores; Metabolite levels: Succinate assay kit quantifies succinate content in intestinal tissue; Protein expression: Western blot was used to detect SUCNR1 receptor protein, and ELISA was used to detect inflammatory factors; Gene expression: RT-qPCR analysis of NLRP3, TNF-α, IL-1β, and IL-6 mRNA; S5: Data Analysis: Integrate phenotypic, pathological, and molecular data to validate the function of the succinic acid-SUCNR1 axis.

2. The method for studying the mechanism of succinic acid's effect on necrotizing enterocolitis according to claim 1, characterized in that, In S1, the establishment of the NEC animal model includes artificial feeding, hypoxia treatment, and cold stimulation; hypoxia treatment is exposure to 95% N2 environment for 90 seconds, and cold stimulation is exposure to 4℃ environment for 10 minutes, repeated twice a day for 4 days.

3. The method for studying the mechanism of succinic acid's effect on necrotizing enterocolitis according to claim 1, characterized in that, In S2, the intervention method includes the application of succinic acid, which is performed by gavage with formula milk containing 100 mmol / L of exogenous succinic acid at a dose of 30 μL / g.

4. The method for studying the mechanism of succinic acid's effect on necrotizing enterocolitis according to claim 1, characterized in that, In S4, the inflammatory markers detected include TNF-α, IL-1β, IL-6, and NLRP3 inflammasome at both mRNA and protein levels.

5. The method for studying the mechanism of succinic acid's effect on necrotizing enterocolitis according to claim 1, characterized in that, In S4, mRNA levels were detected using RT-qPCR with primers targeting the TNF-α, IL-1β, IL-6, and NLRP3 genes.

6. The method for studying the mechanism of succinic acid's effect on necrotizing enterocolitis according to claim 1, characterized in that, In S4, protein levels were detected using ELISA, including serial dilution of standards and determination of OD values; intestinal pathological changes were detected using HE staining and double-blind pathological scoring.

7. The method for studying the mechanism of succinic acid's effect on necrotizing enterocolitis according to claim 1, characterized in that, In step S4, the succinic acid level is quantified by measuring succinic acid in intestinal tissue, as detailed below: Tissue lysis: Add 100 μL BeyoLysis per 10 mg of tissue. TM Buffer A, ground on ice; Centrifugation: Centrifuge at 4℃, 12000×g for 5 minutes, and collect the supernatant; Preparation of working solution: The specific AmplexRed reaction working solution consists of 70 μL of detection buffer, 2 μL of AmplexRed, 2 μL each of enzyme solutions A and B, 2 μL of cofactor, and 2 μL of substrate; Detection: Add sample to 96-well plate, standard curve concentration 0-500μM, and measure A570 absorbance.

8. The method for studying the mechanism of succinic acid's effect on necrotizing enterocolitis according to claim 1, characterized in that, In S4, the HE staining and pathological scoring are as follows: Fixation: Fix with paraformaldehyde for 24-48 hours; Dehydration and embedding: Gradient ethanol dehydration followed by paraffin embedding; Section staining: Sections were 4 μm thick, dewaxed, stained with hematoxylin for 5 minutes, differentiated with hydrochloric acid alcohol, and counterstained with eosin; Mounting and observation: Mount with neutral resin and take pictures under a microscope at 40x and 100x magnification; Double-blind scoring: 0 to 4 points, ≥2 points confirms NEC.

9. The method for studying the mechanism of succinic acid's effect on necrotizing enterocolitis according to claim 1, characterized in that, In step S4, RNA extraction and RT-qPCR are performed as follows: Grinding and extraction: The tissue was ground and extracted by adding QLS lysis buffer and magnetic beads; RNA purification: After centrifugation, add an equal volume of ethanol, elute the centrifuge column, and dissolve in enzyme-free water; cDNA synthesis: After gDNA removal, M-MLV reverse transcription occurs; qPCR system: SYBR Green method, primers include internal control; Data calculation: The 2^(-ΔΔCt) method was used to analyze the expression differences.

10. The method for studying the mechanism of succinic acid's effect on necrotizing enterocolitis according to claim 1, characterized in that, In S4, the Western Blot is as follows: Lysis buffer: RIPA plus protease inhibitor and phosphatase inhibitor; Electrophoretic transfer: SDS-PAGE gel electrophoresis at 80V for 120 minutes, transfer at 400mA for 30 minutes; Antibody incubation: Primary antibody overnight at 4°C, secondary antibody 1 hour at room temperature; Development: Chemiluminescence detection, ImageJ analysis of bands.

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