Application of combination of middle-jiao-regulating decoction and melatonin in treatment of NEC

Abstract

The invention provides an application of a middle-jiao-regulating decoction and melatonin in treatment of NEC, the active components of the middle-jiao-regulating decoction are prepared from the following raw materials: codonopsis pilosula, dried ginger, honey-fried licorice root and bighead atractylodes rhizome, and the mass ratio of codonopsis pilosula to dried ginger to honey-fried licorice root to bighead atractylodes rhizome is (1-2): (1-2): (1-2): (1-2). In combination with years of clinical experience and a large amount of research, the inventor of the invention finds that the melatonin and the middle warmer regulating decoction have an obvious synergistic effect when being combined for preventing and treating neonatal NEC, the intestinal tissue injury of neonatal mouse NEC can be effectively reduced by combining the melatonin and the middle warmer regulating decoction in low dosage, the intestinal barrier function is improved, and the effect of preventing and treating neonatal NEC is achieved. The occurrence rate of the NEC is reduced; and the survival rate of the NEC is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and relates to the application of Lizhong decoction combined with melatonin in treating NEC. Background Art

[0002] Necrotizing enterocolitis (NEC) is an acute inflammatory necrotizing disease of the intestine that is a serious threat to the life of newborns. In newborns with necrotizing enterocolitis, three factors are usually present in the small intestine: persistent intestinal ischemic damage, bacterial colonization, and intestinal substrate (such as enteral feeding). Infants with NEC may present with intestinal obstruction with abdominal distention, gastric biliary residual (after feeding) with gradual vomiting of bile, or bloody stools under the naked eye or microscope, and sepsis may manifest as lethargy, unstable body temperature, increased episodes of apnea, and metabolic acidosis.

[0003] The etiology and pathogenesis of NEC are not yet fully understood. The mainstream view is that premature birth and a series of complications of premature infants (such as asphyxia, respiratory distress syndrome, respiratory failure, and patent ductus arteriosus, etc.) are one of the main causes of NEC. Superimposed improper feeding, hypoxia-ischemia, infection and mediated inflammatory response affect the blood supply to the intestinal mucosa, leading to local ischemia of the mucosa, weakening intestinal peristalsis, and accumulation of food in the intestinal cavity, affecting intestinal function and accelerating bacterial reproduction, which in turn causes ischemia-hypoxia and inflammatory damage to the intestinal wall, ultimately leading to intestinal mucosal bleeding, erosion and necrosis. For the treatment of NEC, the current clinical guidelines mainly include routine supportive treatments such as fasting, gastrointestinal decompression and the use of antibiotics.

[0004] Data show that the incidence of NEC complications in extremely premature infants aged 22 to 25 weeks in my country has not shown a decreasing trend from 2019 to 2021, suggesting that existing prevention and treatment strategies can no longer further reduce the incidence of NEC. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide an application of a Chinese medicine composition combined with melatonin in the treatment of NEC. The Chinese medicine composition combined with melatonin can synergistically enhance the efficacy, and a good therapeutic effect can be achieved by low-dose combination.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions.

[0007] The first aspect of the present invention provides the use of a traditional Chinese medicine composition combined with melatonin in the preparation of a medicament for preventing and / or treating neonatal necrotizing enterocolitis, wherein the active ingredients of the traditional Chinese medicine composition are prepared from the following raw materials: codonopsis pilosula, dried ginger, roasted licorice and atractylodes macrocephala, and the mass ratio of codonopsis pilosula, dried ginger, roasted licorice and atractylodes macrocephala is (1-2):(1-2):(1-2):(1-2).

[0008] In some embodiments, the active ingredients of the traditional Chinese medicine composition are prepared from the following raw materials: Codonopsis pilosula, dried ginger, licorice and Atractylodes macrocephala, and the mass ratio of Codonopsis pilosula, dried ginger, licorice and Atractylodes macrocephala is (1-1.5):(1-1.5):(1-1.5):(1-1.5).

[0009] In some embodiments, the active ingredients of the traditional Chinese medicine composition are prepared from the following raw materials: Codonopsis pilosula, dried ginger, licorice root and Atractylodes macrocephala, and the mass ratio of Codonopsis pilosula, dried ginger, licorice root and Atractylodes macrocephala is 1:1:1:1.

[0010] In some embodiments, the dosage form of the Chinese medicine composition is decoction, granules, tablets, capsules, or pills.

[0011] In some embodiments, the dosage form of the Chinese medicine composition is a decoction, and the preparation method of the Chinese medicine composition comprises the following steps: (1) taking Codonopsis pilosula, dried ginger, roasted licorice and Atractylodes macrocephala in proportion, soaking them in water 6 to 10 times the total weight of the raw materials, then boiling and extracting, and collecting the extract; (2) repeating the boiling and extraction of the Chinese medicine residue in step (1) 1 to 2 times, and collecting the extract; (3) combining the extracts, centrifuging, taking the supernatant, and concentrating to obtain the decoction.

[0012] In some embodiments, the soaking time in step (1) is 0.5 h to 2 h.

[0013] In some embodiments, the extraction time in step (1) is 30 min to 45 min.

[0014] In some embodiments, the centrifugation condition in step (3) is 8000 g to 12000 g for 10 min to 30 min.

[0015] The second aspect of the present invention provides a combined drug for preventing and / or treating neonatal necrotizing enterocolitis, wherein the active ingredients of the drug include melatonin and the traditional Chinese medicine composition as described above.

[0016] In some embodiments, the Chinese medicine composition and the melatonin are each an independent administration unit, or the Chinese medicine composition and the melatonin together form a combined administration unit.

[0017] The inventors of the present invention have combined their own many years of clinical experience and a large number of studies to find that melatonin and the Chinese medicine composition have obvious synergistic effects when used in combination to prevent and treat neonatal NEC. The low-dose combination of the two can effectively reduce the intestinal tissue damage of neonatal NEC, improve the intestinal barrier function, reduce the incidence of NEC and increase the survival rate of NEC. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1These are the survival rate test results of NEC in each group of newborn mice.

[0019] Figure 2 Detection results of NEC tissue damage degree in each group of newborn mice.

[0020] Figure 3 These are the results of FITC-dextran concentration detection in the plasma of neonatal mice with NEC in each group.

[0021] Figure 4 These are the results of the expression level detection of intestinal barrier protein ZO-1 in the intestinal tissue of newborn mice with NEC in each group. DETAILED DESCRIPTION

[0022] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0023] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0024] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0025] The following is a description with reference to specific implementation methods.

[0026] The Chinese medicine composition described in the following examples is named Lizhong Decoction, which is a classic prescription for warming the middle and dispelling cold, replenishing qi and strengthening the spleen in "Treatise on Febrile and Miscellaneous Diseases" written by the medical saint Zhang Zhongjing. It is composed of ginseng, dried ginger, roasted licorice, and white atractylodes in equal amounts of 1:1:1:1. Considering that ginseng is often replaced by codonopsis in clinical practice, combined with the function of Lizhong Decoction and the basic pathological characteristics of NEC, we selected codonopsis, dried ginger, roasted licorice, and white atractylodes in the amount of (1-2):(1-2):(1-2):(1-2) to prepare Lizhong Decoction, preferably 1:1:1:1.

[0027] In the following examples, the Lizhong Decoction is prepared from the following raw materials: 9g of Codonopsis pilosula, 9g of dried ginger, 9g of Atractylodes macrocephala, and 9g of Radix Glycyrrhizae Preparata. The preparation method is as follows: (1) Codonopsis pilosula, dried ginger, Rhizoma Glycyrrhizae Preparata and Atractylodes macrocephala are mixed according to the amount used, and distilled water with a volume of 10 times the total amount of the mixed raw materials is added to soak for 45 minutes, and then heated and boiled for 45 minutes, and the decoction is filtered out; (2) 8 times the volume of distilled water is added to the remaining residue again, heated and boiled for 45 minutes, and the decoction is filtered out; (3) The two decoctions are combined, centrifuged at 10,000×g for 15 minutes, and finally the supernatant after centrifugation is concentrated to contain 1g of crude drug per milliliter of the drug solution, filtered through a 0.22μm sterile filter membrane, and stored at 4°C for later use.

[0028] Example 1

[0029] This example studies the effect of the combined use of Lizhong Decoction (LZT) and melatonin (MEL) in the treatment of NEC.

[0030] 1. Materials and Methods

[0031] 1. Research subjects

[0032] Experimental animals: SPF grade C57 / BL6 pregnant mice were purchased from Guangdong Medical Experimental Animal Center. This study has been approved by the Guangdong Medical Experimental Animal Ethics Committee.

[0033] 2. Main Reagents

[0034] Phosphate buffered saline (PBS), Invitrogen, USA; Anti-ZO-1 (ab216880, 195 kDa), abcam, UK; Anti-β-Actin (ab179467, 42 kDa), abcam, UK; Goat anti-rabbit IgG-HRP (ab6721), abcam, UK; PageRuler Prestained Protein Ladder (26616) (10-170 KDa), Thermo scientific, USA; Reduced SDS-PAGE 5× Loading Buffer, Kangwei Century Biotechnology Co., Ltd.; Total protein extraction kit (P1250), Applygen, Beijing, China; Melatonin (S1204), Selleck Chemicals, China; Atractylodes macrocephala (B25582), Shanghai Yuanye Biotechnology Co., Ltd.; Codonopsis pilosula (B26097), Shanghai Yuanye Biotechnology Co., Ltd.; Dried ginger (B25864), Shanghai Yuanye Biotechnology Co., Ltd.; Roasted liquorice (B27088), Shanghai Yuanye Biotechnology Co., Ltd.

[0035] 3. Experimental Methods

[0036] Animal experiments

[0037] Establishment of a necrotizing enterocolitis model in neonatal mice

[0038] The specific steps of the neonatal mouse NEC model are as follows: C57 / BL6 mice aged 8 to 10 days are separated from their mothers and raised in a neonatal mouse incubator. Formula milk is administered orally with a clean sterile silicone tube through an oral cannula in an amount of 200 μl per 5 g body weight, and the time is controlled at 2 to 3 minutes per time, 5 times a day. Hypoxia (5% O2, 95% N2) and low temperature (placed in a 4°C refrigerator) treatment are given at 8:00 and 20:00 every day, each for 10 minutes. At the same time, LPS is added during the second oral administration of the day (the amount of LPS is calculated as 10 mg per 1 kg body weight). The modeling cycle is 4 days.

[0039] Animal grouping and treatment

[0040] Normal control group (BF): mother mice were fed in the same cage; treatment control group (SOL): phosphate buffer saline was given for treatment; LZT treatment group (LZT): LZT was given by gavage during the modeling process, referring to the gavage method of NEC model, 200 μl per 5 g body weight, and gavage was performed through oral intubation with a clean sterile silicone tube. The time was controlled at 2 to 3 minutes. Each group received a single gavage once a day at 8 am; MEL treatment group (MEL): melatonin was injected intraperitoneally (ip) into mice, once a day during the entire modeling process, and the injection was given 1 hour before the first gavage of the day; combined treatment group (LZT-MEL): LZT gavage + ip injection of MEL were performed during the modeling process.

[0041] Specimen collection and processing

[0042] 12 hours after the last hypoxia combined with hypothermia treatment, mice with poor mental state were directly killed by cervical dislocation; mice with no obvious mental abnormalities were injected with FITC-dextran (Thermo Fisher, Cat#18912014) by gavage (750 mg / kg) and killed by cervical dislocation 4 hours later. After killing the mice, under sterile conditions: (1) blood was collected by decapitation into a sodium heparin anticoagulant tube and centrifuged at 3,000×g for 10 minutes at 4°C to separate plasma; (2) the abdominal skin was cleaned and disinfected under sterile conditions, and the abdominal cavity was opened along the midline of the abdomen with ophthalmic scissors, and the intestinal tube from the lower end of the duodenum to the ileocecal part was removed, and 0.5 cm of the intestinal tube near the ileocecal part was retained and fixed in 4% formaldehyde solution.

[0043] Observation indicators and methods

[0044] 1) General condition and body weight of newborn mice

[0045] General condition and body weight changes of mice: General condition includes spirit, reaction, activity status, food intake, abdominal distension, vomiting, gastric retention, changes in stool characteristics, etc.; weigh the mice before the first feeding every day and record the death.

[0046] 2) Changes in the gross morphology of intestinal tissue

[0047] Gross morphological changes of intestinal tissue: After opening the abdominal cavity, observe the color changes of the intestinal tissue, intestinal gas accumulation, necrosis and bleeding with the naked eye and record them.

[0048] 3) Histopathological scoring

[0049] The fixed ileum specimens were stained with HE and scored by two pathologists in a double-blind manner. The scoring criteria were shown in Table 1. A histological score of ≥2 was defined as NEC.

[0050] Table 1 Scoring criteria for intestinal tissue pathology

[0051]

[0052] Preparation of paraffin sections of neonatal rat intestinal tissue

[0053] 1) Sampling: Fix the fresh intestinal tissue samples with 4% paraformaldehyde for no less than 24 hours; take the fixed tissue, trim the target area with a scalpel in a fume hood, and then place the trimmed tissue and the corresponding label in a dehydration box;

[0054] 2) Dehydration: Place the dehydration box into the basket of the dehydrator and dehydrate in the following order: 75% alcohol dehydration for 4 hours, 85% alcohol dehydration for 2 hours, 90% alcohol dehydration for 2 hours, 95% alcohol dehydration for 1 hour, anhydrous ethanol dehydration for 30 minutes, a second anhydrous ethanol dehydration for 30 minutes, alcohol benzene dehydration for 5-10 minutes, xylene dehydration for 5-10 minutes, a second xylene dehydration for 5-10 minutes, soft wax dipping for 1 hour, hard wax dipping for 1 hour;

[0055] 3) Embedding: Use an embedding machine to embed the wax-soaked samples; put the melted wax into the embedding frame, and before the wax solidifies, take the samples out of the dehydration box and put them into the embedding frame and label them; 。 Freeze on a freezing table, take out and trim the wax block after solidification;

[0056] 4) Sectioning: Place the trimmed wax block on a microtome with a slice thickness of 4 μm; then flatten the slices on a 40°C warm water surface in a slide spreader, transfer them to a glass slide, and bake them in a 60°C oven for about 6 hours, then store them at room temperature for later use.

[0057] HE staining

[0058] 1) Dewaxing and hydration of paraffin sections: first, place the sections in xylene for 20 minutes, repeat once, then place them in anhydrous ethanol for 10 minutes, repeat once, then place them in 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and finally wash with distilled water;

[0059] 2) Hematoxylin staining of cell nuclei: Place the sections in Harris hematoxylin staining solution for about 5 minutes, then rinse with tap water, then differentiate with 1% hydrochloric acid alcohol differentiation solution for a few seconds, immediately rinse with tap water, and finally blue with 0.6% ammonia solution and rinse with tap water;

[0060] 3) Eosin staining of cytoplasm: Place the slices in eosin staining solution and stain for about 2 minutes;

[0061] 4) Dehydration and sealing: First, place the slices in 95% alcohol for 5 minutes, repeat once, then place them in anhydrous ethanol for 5 minutes, repeat once, and then place them in xylene for 5 minutes, repeat once, to make them dehydrated and transparent. At this time, take the slices out of xylene and dry them slightly, and seal them with neutral gum, taking care to avoid the generation of bubbles;

[0062] 5) Use a microscope to collect and analyze images.

[0063] Western blot detection

[0064] Total protein extraction

[0065] Take an appropriate amount of tissue into a grinder, add a corresponding amount of lysis buffer according to the proportion, and then grind it with a grinder to make it fully lysed; after lysis, centrifuge the lysate at 12000×g for 5 minutes, aspirate the supernatant as the total protein extract and quantify it using a BCA protein quantification kit; after quantification, add 4 times the volume of 5×SDS protein loading buffer and boil at 100℃ for 10 minutes; then, store at -20℃ for later use.

[0066] Western blotting to detect protein expression

[0067] 1) Make SDS-PAGE gel: Clamp the aligned glass plates with a clamp and fix them vertically on the rack; prepare the separation gel solution, and after adding TEMED at the end, immediately shake it well, fill the gel and seal it with water; when a refraction line appears between the water and the gel, wait for about 5 minutes for the gel to fully solidify, and discard the upper water; prepare the concentrated gel, the process is the same as the preparation of the separation gel, fill the remaining space with concentrated gel, and insert the comb into the concentrated gel; after the concentrated gel solidifies, gently pull out the comb; rinse it with deionized water, then put it into the electrophoresis tank, add enough electrophoresis buffer, and start sample electrophoresis;

[0068] 2) Protein loading: Use a microinjector to draw 30ug of sample and slowly add it to the sample well, leaving one well for adding 5μl of protein marker;

[0069] 3) Separation electrophoresis: Turn on the power supply, adjust the voltage to 80V first, and run the electrophoresis for about 30 minutes. After the concentrated gel is run, adjust the voltage to 120V and run the electrophoresis for about 60 minutes (stop the electrophoresis when the bromofen blue runs to about 1 cm from the bottom of the separation gel);

[0070] 4) Transfer: Cut the target protein strip according to the position of the protein marker; Next, prepare the PVDF membrane: soak it in methanol for 1-5 minutes, then balance it with transfer solution for 10 minutes, and soak the filter paper and sponge in the transfer solution; After soaking, start from the positive electrode, put the sponge, filter paper, PVDF membrane, gel, filter paper, sponge in turn, then close the negative electrode, put it in the electrotransfer tank, and turn on the power to transfer the membrane; the transfer current and time are 300mA and 90 minutes;

[0071] 5) Blocking: After transfer, remove the membrane, rinse gently once in TBST, and block all proteins with 5% skim milk powder at 4°C overnight;

[0072] 6) Primary antibody incubation: discard the blocking solution, wash the membrane with TBST for 5 minutes, repeat 6 times; incubate with primary antibody, dilute the concentration of ZO-1 protein with TBST to 1:1000 according to the required antibody concentration; incubate at 4°C overnight;

[0073] 7) Secondary antibody incubation: Wash the membrane with TBST for 5 minutes, repeat 6 times, incubate with secondary antibody, dilute with TBST according to the required antibody concentration, and incubate at room temperature for 2 hours (the concentration of secondary antibody used is 1:4000);

[0074] 8) Color development: Wash the membrane with TBST for 5 minutes, repeat 6 times, and develop with ECL color development kit. According to the size of the membrane, take appropriate equal amounts of A and B solutions, mix them evenly, use filter paper to absorb some of the washing solution from one corner of the membrane, place it on a clean plastic wrap, cover it with the mixed solution, and develop for 1-5 minutes. Absorb the color development solution, fold the plastic wrap so that the membrane is sandwiched between the plastic wrap, and place it in a dark box; observe the brightness of the fluorescence in a dark room to determine the exposure time, and wash the film for development and fixing;

[0075] 9) Take photos (or scan) and record the analysis results.

[0076] Detection of FITC-dextran concentration in plasma

[0077] The absorbance intensity of FD-70 was measured using a multifunctional microplate reader (BioTek, USA) with an excitation wavelength of 493 nm and an emission wavelength of 518 nm. Finally, the plasma FITC-dextran concentration was calculated based on the standard curve.

[0078] Data statistics and analysis

[0079] Unless otherwise stated, all data are presented as mean ± SD, and t-test was used for statistical analysis between two groups. Kruskal-Wallis coupled paired comparison test or one-way analysis of variance coupled with Bonferroni multiple comparison test was used for statistical analysis of three or more groups. Survival curve analysis was performed using Kaplan-Meier estimation and log-rank test. P values ​​≤ 0.05 were considered statistically significant.

[0080] 2. Experimental Results

[0081] 1. Low-dose LZT combined with low-dose MEL can synergistically improve the survival rate of neonatal NEC mice

[0082] The inventors have found through research that LZT at a daily dose of 20 g / kg can optimally improve the survival rate of neonatal mice with NEC; MEL at a daily dose of 10 mg / kg can optimally improve the survival rate of neonatal mice with NEC. Therefore, in this embodiment, two gradients of 5.0 g / kg and 20 g / kg are set for LZT, and two gradients of 2.5 mg / kg and 10 mg / kg are set for MEL. At the same time, the following combined treatment groups are set: LZT 5.0 g / kg-MEL 2.5 mg / Kg group, LZT 5.0 g / kg-MEL 10 mg / Kg group, and LZT 20 g / kg-MEL 2.5 mg / Kg group.

[0083] Kaplan-Meier estimates and log-rank tests were used to analyze the survival rates of breast-fed groups, NEC neonatal mice treated with different doses of LZT, MEL, combined treatment groups, and phosphate buffer-treated control groups.

[0084] The results are as follows Figure 1 As shown in the figure, LZT-5.0, LZT-20, MEL-2.5, MEL-10, LZT5-MEL2.5, LZT5-MEL10, and LZT20-MEL2.5 respectively represent LZT 5.0g / kg treatment group, LZT 20g / kg treatment group, MEL 2.5mg / Kg treatment group, MEL 10mg / Kg treatment group, LZT 5.0g / kg + MEL 2.5mg / Kg combined treatment group, LZT 5.0g / kg + MEL 10mg / Kg combined treatment group, and LZT 20g / kg + MEL 2.5mg / Kg combined treatment group.

[0085] The results showed that the effect of the LZT5-MEL2.5 combined treatment group in improving the survival rate of neonatal NEC was comparable to that of the LZT-20 treatment group and the MEL-10 treatment group, indicating that low-dose LZT combined with low-dose MEL (LZT5-MEL2.5) had an equivalent effect on improving the survival rate of neonatal NEC as the optimal dose of LZT alone and MEL alone. Furthermore, compared with LZT5-MEL2.5, the low-dose LZT combined with the optimal dose of MEL treatment group (LZT5-MEL10) and the low-dose MEL combined with the optimal dose of LZT treatment group (LZT20-MEL2.5) had a trend of further improving the survival rate of neonatal NEC, but the difference was not statistically significant ( Figure 1 ).

[0086] The combination index (CI) of LZD and PGE2 was further calculated by the Chou-Talalay method. The results showed that the CI values ​​of the LZT 5.0g / kg+MEL 2.5mg / Kg combined treatment group, the LZT 5.0g / kg+MEL 10mg / Kg combined treatment group, and the LZT 20g / kg+MEL 2.5mg / Kg combined treatment group were 0.63, 0.61, and 0.49, respectively, all less than 1, indicating that low-dose LZD and low-dose MEL combined treatment of NEC can synergistically enhance efficacy.

[0087] 2. The therapeutic effect of low-dose LZT combined with low-dose MEL on NEC in neonatal mice

[0088] The daily dose of LZT 5.0g / kg+MEL 2.5mg / Kg was further studied in the treatment of NEC in neonatal mice. 12 hours after the last hypoxia combined with hypothermia stimulation, mice with poor mental state were killed by cervical dislocation; mice with no obvious abnormal mental state were injected with FITC-dextran by gavage, and 4 hours later, they were killed by cervical dislocation. In this example, the number of surviving cases in the breast-feeding group (BF), the phosphate buffer treatment control group (SOL) and the low-dose LZT combined with low-dose MEL treatment group (LZT5-MEL 2.5) after the last hypoxia combined with hypothermia stimulation treatment 12 hours were 12, 11 and 18 respectively. There was no obvious mental abnormality in each group of mice, and FITC-dextran was perfused. Therefore, the number of mice perfused with FITC-dextran in the BF, SOL, and LZT5-PGE2-25 groups was 12, 11, and 18, respectively. The mice were killed 4 h after FITC-dextran perfusion. The killed mice: (1) The corresponding intestinal tissues were obtained for the following tests: H&E staining, injury score, NEC incidence, intestinal barrier permeability test, and Western Blot test; (2) The plasma was obtained for FITC-dextran concentration test.

[0089] (1) Low-dose LZT combined with low-dose MEL can significantly reduce the degree of NEC tissue damage in newborn mice

[0090] H&E staining was performed on the pathological sections of the ileum of newborn mice. The severity of NEC tissue damage in newborn mice was evaluated according to the standards described in Table 1, and the NEC incidence was calculated, NEC incidence = number of mice with NEC after modeling / number of mice before modeling*100%.

[0091] like Figure 2 As shown, a is a representative staining (H&E staining) of the ileum pathological sections of newborn mice in the breast-fed group, phosphate buffer-treated control group, and low-dose LZT combined with low-dose MEL treatment group; scale bar, 50 μm. b is an evaluation of the severity of tissue damage. c is the incidence of NEC in newborn mice (injury score ≥ 2). The results showed that compared with the phosphate buffer-treated control group, the daily dose of LZT 5.0 g / kg combined with MEL 2.5 g / kg could significantly reduce the severity and incidence of NEC tissue damage in newborn mice.

[0092] (2) The protective effect of low-dose LZT combined with low-dose MEL on NEC in neonatal mice is related to the improvement of intestinal barrier function

[0093] In order to explore whether the effect of LZT 5.0g / kg combined with MEL 2.5mg / Kg in alleviating NEC tissue damage in neonatal mice is related to the improvement of intestinal barrier function, FITC-dextran was injected into all surviving neonatal mice with no obvious abnormalities in mental state by gavage 4 hours before sacrifice. The mice were killed 4 hours later and plasma was collected for FITC-dextran concentration detection. The FITC-dextran concentration was calculated by measuring the fluorescence readings in the plasma, thereby evaluating the intestinal barrier permeability.

[0094] The results are as follows Figure 3 As shown, compared with the phosphate buffer treated control group, the daily dose of LZT 5.0g / kg combined with MEL 2.5g / kg could significantly reduce the plasma FITC-dextran concentration of mice, indicating that LZT 5.0g / kg combined with MEL 2.5g / kg could improve the intestinal barrier function of mice.

[0095] The expression level of zonula occludens-1 (ZO-1), a biomarker related to intestinal tissue tight junctions, in the ileum tissue of each group of newborn mice was further analyzed by Western Blot technology, with 6 mice in each group.

[0096] like Figure 4As shown, compared with the breast-fed group, the expression of intestinal barrier protein ZO-1 in the ileum tissue of newborn mice in the phosphate buffer treatment control group was significantly reduced, while LZT 5.0g / kg combined with MEL 2.5mg / Kg could significantly improve the reduction of ZO-1, indicating that the protective effect of LZT 5.0g / kg combined with MEL 2.5mg / Kg on NEC in newborn mice is related to the improvement of intestinal barrier function. Figure 4 In the figure, a is a representative figure of Western Blot detection, and b is a result figure of quantification of the expression level of ZO-1 detected by Western Blot (n=6).

[0097] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0098] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a Chinese medicine composition combined with melatonin in the preparation of a medicament for preventing and / or treating neonatal necrotizing enterocolitis, characterized in that: The active ingredients of the traditional Chinese medicine composition are prepared from the following raw materials: codonopsis pilosula, dried ginger, roasted licorice and atractylodes macrocephala, and the mass ratio of codonopsis pilosula, dried ginger, roasted licorice and atractylodes macrocephala is (1-2):(1-2):(1-2):(1-2).

2. The use according to claim 1, characterized in that The active ingredients of the traditional Chinese medicine composition are prepared from the following raw materials: codonopsis pilosula, dried ginger, roasted licorice and atractylodes macrocephala, and the mass ratio of codonopsis pilosula, dried ginger, roasted licorice and atractylodes macrocephala is (1-1.5):(1-1.5):(1-1.5):(1-1.5).

3. The use according to claim 2, characterized in that The active ingredients of the traditional Chinese medicine composition are prepared from the following raw materials: codonopsis pilosula, dried ginger, roasted licorice root and atractylodes macrocephala, and the mass ratio of codonopsis pilosula, dried ginger, roasted licorice root and atractylodes macrocephala is 1:1:1:

1.

4. The use according to any one of claims 1 to 3, characterized in that: The dosage form of the Chinese medicine composition is decoction, granules, tablets, capsules and pills.

5. The use according to claim 4, characterized in that The dosage form of the Chinese medicine composition is a decoction. The preparation method of the Chinese medicine composition comprises the following steps: (1) taking codonopsis pilosula, dried ginger, roasted liquorice and atractylodes macrocephala in proportion, adding 6 to 10 times the volume of water of the total weight of the raw materials to soak, then boiling and extracting, and collecting the extract; (2) repeating the boiling and extracting of the Chinese medicine residue in step (1) for 1 to 2 times, and collecting the extract; (3) combining the extracts, centrifuging, taking the supernatant, and concentrating to obtain the decoction.

6. The use according to claim 5, characterized in that The soaking time in step (1) is 0.5h to 2h.

7. The use according to claim 5, characterized in that The extraction time in step (1) is 30 min to 45 min.

8. The use according to claim 5, characterized in that The centrifugation condition in step (3) is 8000g-12000g for 10min-30min.

9. A combined drug for preventing and / or treating neonatal necrotizing enterocolitis, characterized in that: The active ingredients of the medicine include melatonin and the traditional Chinese medicine composition according to any one of claims 1 to 8.

10. The drug according to claim 9, characterized in that The Chinese medicine composition and the melatonin are respectively independent administration units, or the Chinese medicine composition and the melatonin together form a combined administration unit.

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