Application of combination of middle-jiao-regulating decoction and prostaglandin E2 in treatment of NEC

Abstract

The invention provides an application of combination of a middle-jiao-regulating decoction and prostaglandin E2 in treatment of NEC. Active ingredients of the middle-jiao-regulating decoction are prepared from the following raw materials: codonopsis pilosula, rhizoma zingiberis, honey-fried licorice root and bighead atractylodes rhizome, and the mass ratio of codonopsis pilosula to rhizoma zingiberis to honey-fried licorice root to bighead atractylodes rhizome is (1-2): (1-2): (1-2): (1-2). A large number of researches and experiments find that the Zhongzhong decoction and the prostaglandin E2 have a remarkable synergistic effect when being used for treating neonatal NEC, and the combination of the Zhongzhong decoction and the prostaglandin E2 in low dosage can remarkably reduce the severity and incidence of neonatal mouse NEC diseases, improve the intestinal barrier function and effectively increase the neonatal mouse NEC survival rate, and has a very good treatment effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of Lizhong decoction combined with prostaglandin E2 in treating NEC. Background Art

[0002] Necrotizing enterocolitis (NEC) is an acute intestinal inflammatory necrotizing disease that seriously threatens the life of newborns and often occurs in very low birth weight (VLBW) infants. The current clinical guidelines for the treatment of NEC mainly include routine supportive treatments such as fasting, gastrointestinal decompression, and the use of antibiotics.

[0003] Although TCM does not have a clear name for NEC, it can be classified into the categories of "fullness, distension, and distention" and "intestinal wind and bloody stool" in combination with its main sites of disease occurrence in the terminal ileum and ascending colon, as well as typical gastrointestinal symptoms such as abdominal distension, vomiting, diarrhea, or blood in the stool. The etiology and pathogenesis are indeed "deficiency in the root and excess in the symptoms". Deficiency is the internal cause of disease, which often occurs in premature infants, especially VLBW infants, because of their inadequate congenital endowment and delicate internal organs, which are very easy to cause deficiency and coldness in the middle jiao. Excess is the external cause of disease, such as improper feeding leading to milk and food damaging the spleen and stomach, infection and its mediated inflammatory response, which can easily disturb the qi mechanism, causing NEC in children with ascending and descending disorders and qi stagnation and blood stasis. In terms of TCM etiology and pathogenesis, whether it is aimed at the excess pathogenesis, the application of raw chrysophanol, compound danshen injection, Qinghua decoction, compound dachengqi decoction, etc., and the application of specific electromagnetic spectrum auxiliary treatment for the deficiency pathogenesis, all have achieved good clinical efficacy. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide the use of Lizhong Decoction combined with prostaglandin E2 in the treatment of NEC. The two can synergize in the treatment of NEC, and a low-dose combination can achieve a good therapeutic effect.

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

[0007] The first aspect of the present invention is to provide a Chinese medicine composition combined with prostaglandin E2 for use in the preparation of a drug for preventing and / or treating neonatal necrotizing enterocolitis, wherein the active ingredients of the 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 mass ratio of Codonopsis pilosula, dried ginger, licorice root and Atractylodes macrocephala is (1-1.5):(1-1.5):(1-1.5):(1-1.5).

[0009] In some embodiments, the mass ratio of Codonopsis pilosula, dried ginger, licorice 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] In some embodiments, the step (3) is concentrated to a concentration of 0.5 g to 5 g of crude drug per ml of the drug solution.

[0016] The second aspect of the present invention is to provide a combined drug for preventing and / or treating neonatal necrotizing enterocolitis, comprising a Chinese medicine composition and prostaglandin E2, wherein the Chinese medicine composition and the prostaglandin E2 are independent dosing units, or the Chinese medicine composition and the prostaglandin E2 together form a combined dosing unit;

[0017] 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).

[0018] In some embodiments, the mass ratio of Codonopsis pilosula, dried ginger, licorice root and Atractylodes macrocephala is (1-1.5):(1-1.5):(1-1.5):(1-1.5).

[0019] In some embodiments, the mass ratio of Codonopsis pilosula, dried ginger, licorice and Atractylodes macrocephala is 1:1:1:1.

[0020] In some embodiments, the dosage form of the Chinese medicine composition is decoction, granules, tablets, capsules, or pills.

[0021] 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.

[0022] In some embodiments, the soaking time in step (1) is 0.5 h to 2 h.

[0023] In some embodiments, the extraction time in step (1) is 30 min to 45 min.

[0024] In some embodiments, the centrifugation condition in step (3) is 8000 g to 12000 g for 10 min to 30 min.

[0025] In some embodiments, the step (3) is concentrated to a concentration of 0.5 g to 5 g of crude drug per ml of the drug solution.

[0026] After extensive research and experiments, the present invention found that the traditional Chinese medicine composition combined with prostaglandin E2 has a significant synergistic effect in the treatment of neonatal NEC. The low-dose combination of the two can significantly reduce the severity and incidence of NEC in newborn mice, improve intestinal barrier function, and effectively increase the survival rate of NEC in newborn mice, with a good therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The survival rates of NEC in newborn mice in the LZT-5.0, LZT-20, PGE2-25, PGE2-100, and LZT5-PGE2-25 groups.

[0028] Figure 2 This is the survival rate of newborn NEC in LZT5-PGE2-25, LZT5-PGE2-100 and LZT20-PGE2-25 groups.

[0029] Figure 3 These are the results of detecting the degree of NEC tissue damage in each group of newborn mice.

[0030] Figure 4 The results of FITC fluorescence detection in the plasma of NEC of newborn mice in each group.

[0031] Figure 5 These are the results of the detection of ZO-1 expression levels in the ileum tissues of neonatal mice with NEC in each group. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] 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.

[0035] The following is a description with reference to specific embodiments.

[0036] 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.

[0037] 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.

[0038] Example 1

[0039] This example studies the effect of the combined use of Lizhong Decoction (LZT) and prostaglandin E2 (PGE2) in the treatment of NEC.

[0040] 1. Materials and Methods

[0041] 1. Research subjects

[0042] 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.

[0043] 2. Main Reagents

[0044] 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; prostaglandin E2 (D2250015), Sigma-Aldrich, USA; 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.

[0045] 3. Experimental Methods

[0046] Animal experiments

[0047] Establishment of a necrotizing enterocolitis model in neonatal mice

[0048] The specific steps of establishing the neonatal 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 via 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.

[0049] Animal grouping and treatment

[0050] Normal control group (BF): mother mice were fed in the same cage; treatment control group (SOL): phosphate buffer saline was given during the modeling process; LZT treatment group (LZT): LZT was given by gavage during the modeling process, referring to the gavage method of NEC model, 200 μl was given per 5 g body weight, and gavage was performed through the mouth 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; PGE2 treatment group (PGE2): PGE2 was given 30 minutes before the first addition of LPS to induce NEC during the modeling process, and mice were injected with PGE2 intraperitoneally (ip). Only one drug was given during the entire modeling process; LZT combined with PGE2 treatment group: LZT gavage + PGE2 ip injection treatment were performed during the modeling process.

[0051] Specimen collection and processing

[0052] 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, 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.

[0053] Observation indicators and methods

[0054] 1) General condition and body weight of newborn mice

[0055] General condition and body weight changes of mice: General condition includes spirit, reaction, activity status, food intake, abdominal distension, vomiting, gastric retention and changes in stool characteristics; weigh the mice before the first feeding every day and record the death.

[0056] 2) Changes in the gross morphology of intestinal tissue

[0057] 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.

[0058] 3) Histopathological scoring

[0059] 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.

[0060] Table 1 Scoring criteria for intestinal tissue pathology

[0061]

[0062] Preparation of paraffin sections of neonatal rat intestinal tissue

[0063] 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;

[0064] 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;

[0065] 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;

[0066] 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.

[0067] HE staining

[0068] 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;

[0069] 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;

[0070] 3) Eosin staining of cytoplasm: Place the slices in eosin staining solution and stain for about 2 minutes;

[0071] 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;

[0072] 5) Use a microscope to collect and analyze images.

[0073] Western blot detection

[0074] Total protein extraction

[0075] 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.

[0076] Western blotting to detect protein expression

[0077] 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;

[0078] 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;

[0079] 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);

[0080] 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;

[0081] 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;

[0082] 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;

[0083] 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);

[0084] 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;

[0085] 9) Take photos (or scan) and record the analysis results.

[0086] Detection of FITC-dextran concentration in plasma

[0087] 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.

[0088] Data statistics and analysis

[0089] 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.

[0090] 2. Experimental Results

[0091] 1. Low-dose LZT combined with low-dose PGE2 has a synergistic effect on improving the survival rate of neonatal NEC mice

[0092] The inventors' previous studies found that the daily dose of LZT at 20g / kg and the daily dose of PGE2 at 100mg / Kg are the optimal doses for treating NEC, which can optimally improve the survival rate of neonatal NEC mice. According to the optimal doses of the two drugs, in the combined drug treatment group of this embodiment, LZT is set at two gradient daily doses of 5.0g / kg and 20g / kg, and PGE2 is set at two gradient daily doses of 25mg / Kg and 100mg / Kg, and a combined treatment group is set at the same time.

[0093] Kaplan-Meier estimates and log-rank tests were used to analyze the survival rates of breast-fed (BF) and NEC newborn mice after LZT, PGE2, combined treatment (LZT-PGE2), or phosphate buffer control treatment (SOL) (calculation formula: number of surviving mice after modeling / number of mice before modeling*100%). LZT-5.0, LZT-20, PGE2-25, PGE2-100, LZT5-PGE2-25, LZT5-PGE2-100 and LZT20-PGE2-25 represent LZT 5.0g / kg, LZT 20g / kg, PGE2 25mg / Kg, PGE2 100mg / Kg, LZT 5.0g / kg+PGE2 25mg / Kg, LZT 5.0g / kg+PGE2 100mg / Kg and LZT 20g / kg+PGE2 25mg / Kg treatment groups, respectively.

[0094] The results are as follows Figure 1As shown, consistent with the results of previous studies, the survival rate of neonatal NEC in the LZT 20g / kg group was higher than that in the LZT 5.0g / kg group, and the survival rate of neonatal NEC in the PGE2 100mg / Kg group was higher than that in the PGE2 25mg / kg group. The daily dose of LZT at 20g / kg and the daily dose of PGE2 at 100mg / Kg are the optimal doses for the treatment of NEC alone. The survival rate of neonatal NEC in the LZT 5.0g / kg+PGE225mg / kg group was equivalent to that in the LZT 20g / kg group and the PGE2 100mg / Kg group, indicating that LZT and PGE2 can synergize with each other, and the combination of low doses of the two can improve the therapeutic effect and achieve the therapeutic effect of the optimal doses of LZT and PGE2. Compared with the LZT5.0g / kg+PGE225mg / kg group, the low-dose LZT combined with the optimal dose of PGE2 treatment group (LZT 5.0g / kg+PGE2100mg / kg) and the low-dose PGE2 combined with the optimal dose of LZT (LZT 20g / kg+PGE225mg / kg) treatment group showed a trend of further improvement in the survival rate of neonatal NEC, although the difference was not statistically significant ( Figure 2 ).

[0095] The combination index (CI) of LZD and PGE2 was further calculated by the Chou-Talalay method. The results showed that the CI values ​​of LZT 5.0g / kg+PGE2 25mg / kg, LZT 5.0g / kg+PGE2 100mg / kg, and LZT 20g / kg+PGE2 25mg / kg groups were 0.74, 0.58, and 0.58, respectively, all less than 1, indicating that the combined use of low-dose LZD and PGE2 has a synergistic effect.

[0096] Existing studies have found that PGE2 shows a protective effect on NEC, but high doses of PGE2 can cause tissue inflammation and damage. Therefore, low doses of PGE2 and Lizhong Decoction can be used together to treat NEC, while ensuring efficacy and reducing drug side effects.

[0097] 2. The therapeutic effect of low-dose LZT combined with low-dose PGE2 on NEC in neonatal mice

[0098] Further, the daily dose of LZT 5.0g / kg combined with PGE2 25mg / Kg was studied in terms of the degree of tissue damage in neonatal NEC. After the last hypoxia combined with low temperature stimulation treatment for 12h, 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 4h later, they were killed by cervical dislocation. In this embodiment, the number of survival cases of the breast-feeding group (BF), the phosphate buffer treatment control group (SOL) and the low-dose LZT combined with low-dose PGE2 treatment group (LZT5-PGE2-25) after the last hypoxia combined with low temperature stimulation treatment for 12h were 12, 11 and 18 respectively. Since one mouse in each of the surviving mice in the SOL and LZT5-PGE2-25 groups was in poor mental state, FITC-dextran perfusion was not performed and the mice were directly killed. Therefore, the number of mice perfused with FITC-dextran in the BF, SOL, and LZT5-PGE2-25 groups was 12, 10, and 17, respectively. The mice were killed 4 hours 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, Western Blot test; (2) The plasma was obtained for FITC-dextran concentration test.

[0099] (1) Low-dose LZT combined with low-dose PGE2 can significantly reduce the degree of NEC tissue damage in newborn mice

[0100] H&E staining was performed on the pathological sections of the ileum of newborn mice. The severity of NEC disease 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%.

[0101] The results are as follows Figure 3 As shown, a is a representative staining image (H&E staining) of the ileum pathological sections of newborn mice in the breast-fed group, phosphate buffer treatment control group, and low-dose LZT combined with low-dose PGE2 treatment group; scale bar, 50μm; b is the severity of tissue damage; c is the incidence of NEC in newborn mice (damage score ≥ 2). The results showed that a daily dose of LZT 5.0g / kg combined with PGE2 25mg / Kg can significantly reduce the severity and incidence of NEC in newborn mice.

[0102] (2) The protective effect of low-dose LZT combined with low-dose PGE2 on NEC in neonatal mice is related to the improvement of intestinal barrier function

[0103] In order to explore whether LZT 5.0g / kg combined with PGE2 25mg / Kg can alleviate NEC tissue damage in newborn mice and improve intestinal barrier function, FITC-dextran was injected into all surviving newborn 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.

[0104] The results are as follows Figure 4 As shown, compared with the phosphate buffer treated control group, LZT 5.0g / kg combined with PGE2 25mg / Kg can significantly reduce the plasma FITC-dextran concentration, indicating that LZT 5.0g / kg combined with PGE2 25mg / Kg can improve the intestinal barrier function of mice.

[0105] Western Blot technology was further used to analyze the expression level of intestinal tissue tight junction-related biomarker zonula occludens-1 (ZO-1) in the ileum tissue of newborn mice in the breast-fed group, phosphate buffer treatment control group and low-dose LZT combined with low-dose PGE2 treatment group, with 6 mice tested in each group.

[0106] The results show that Figure 5 ), compared with the breast-fed group, the expression of ZO-1 in the ileum tissue of newborn mice in the phosphate buffer treatment control group was significantly decreased, while LZT 5.0g / kg combined with PGE2 25mg / Kg treatment could significantly improve the reduction of ZO-1, indicating that the protective effect of LZT 5.0g / kg combined with PGE2 25mg / Kg treatment on NEC in newborn mice is related to the improvement of intestinal barrier function. Figure 5 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).

[0107] 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.

[0108] 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 prostaglandin E2 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 mass ratio of Codonopsis pilosula, dried ginger, roasted liquorice 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 mass ratio of the codonopsis pilosula, dried ginger, roasted liquorice 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. A combined drug for preventing and / or treating neonatal necrotizing enterocolitis, characterized in that: It comprises a Chinese medicine composition and prostaglandin E2, wherein the Chinese medicine composition and the prostaglandin E2 are independent administration units, or the Chinese medicine composition and the prostaglandin E2 together form a combined administration unit; 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).

7. The combined drug according to claim 6, characterized in that: The mass ratio of Codonopsis pilosula, dried ginger, roasted liquorice and Atractylodes macrocephala is (1-1.5):(1-1.5):(1-1.5):(1-1.5).

8. The combined drug according to claim 7, characterized in that: The mass ratio of the codonopsis pilosula, dried ginger, roasted liquorice and atractylodes macrocephala is 1:1:1:

1.

9. The combined drug according to any one of claims 6 to 8, characterized in that: The dosage form of the Chinese medicine composition is decoction, granules, tablets, capsules and pills.

10. The combined drug according to claim 9, 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.

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