Application of middle-jiao-regulating decoction in treatment of neonatal necrotizing enterocolitis

Abstract

The invention provides an application of a middle-jiao-regulating decoction in treating neonatal necrotizing enterocolitis, 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 treatment experience and a large amount of research, the inventor finds that the Zhongzhong decoction has an obvious treatment effect on NEC, the NEC tissue damage degree of newborn rats can be effectively reduced, the intestinal barrier function is improved, the NEC occurrence rate is remarkably reduced, and the survival rate is increased. In addition, the middle warmer regulating decoction has no obvious toxic and side effects when being used for treating neonatal necrotizing enterocolitis, and is high in safety.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of Lizhong decoction in treating neonatal necrotizing enterocolitis. Background Art

[0002] Lizhong Decoction is a classic prescription for warming the middle and dispelling cold, replenishing qi and strengthening the spleen in the Treatise on Febrile and Miscellaneous Diseases written by the medical saint Zhang Zhongjing. It is made of ginseng, dried ginger, roasted licorice, and white atractylodes in equal proportions of 1:1:1:1. In clinical practice, ginseng is often replaced by codonopsis.

[0003] Necrotizing enterocolitis (NEC) is an acute intestinal inflammatory necrotizing disease that seriously threatens the life of newborns. It often occurs in very low birth weight (VLBW) infants. The global incidence of NEC in VLBW infants is 7% and is on the rise. Data from my country show that the incidence of NEC in VLBW infants is close to 5% and is also on the rise. What is more serious is that surviving infants are very likely to have digestive and nervous system sequelae, which imposes a heavy burden on families and society.

[0004] For the treatment of NEC, the current clinical guidelines mainly include routine supportive treatments such as fasting, gastrointestinal decompression and antibiotic use. The recent survival status of extremely premature infants in the United States shows that the mortality rate has decreased from 2005 to 2021, but it has not decreased since 2015. The cause 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 its mediated inflammatory response, by affecting the blood supply to the intestinal mucosa, lead to local ischemia of the mucosa, weaken intestinal peristalsis, and accumulate food in the intestinal cavity, affecting intestinal function and accelerating bacterial reproduction, which in turn causes ischemia and hypoxia of the intestinal wall, inflammatory damage, and ultimately leads to intestinal mucosal bleeding, erosion and necrosis. Existing preventive measures, such as the use of glucocorticoids in mothers of premature infants, the use of ibuprofen to close patent ductus arteriosus in premature infants with hemodynamic changes, and breastfeeding and its standardized feeding strategies, are all developed based on the current understanding of the pathogenesis.

[0005] Combined with the recent data from the my country Neonatal Collaboration Network, the incidence of NEC complications in extremely premature infants aged 22 to 25 weeks has not decreased from 2019 to 2021, suggesting that existing prevention and treatment strategies can no longer further reduce the incidence of NEC. Therefore, this field needs to develop new treatment options for neonatal NEC. Summary of the invention

[0006] Based on this, the object of the present invention is to provide the use of Lizhong Decoction in treating neonatal necrotizing enterocolitis.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions.

[0008] The present invention provides an application of a traditional Chinese medicine composition in preparing a medicine for preventing and / or treating neonatal necrotizing enterocolitis. 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 (1-2):(1-2):(1-2):(1-2).

[0009] 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).

[0010] In some embodiments, the mass ratio of Codonopsis pilosula, dried ginger, licorice and Atractylodes macrocephala is 1:1:1:1.

[0011] In some embodiments, the dosage form of the Chinese medicine composition is decoction, granules, tablets, capsules, or pills.

[0012] 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, and then boiling them for extraction; (2) repeating step (1) 1 to 2 times; (3) combining the extracts, centrifuging, taking the supernatant, and concentrating to obtain the decoction.

[0013] In some embodiments, the soaking time in step (1) is 0.5 h to 2 h.

[0014] In some embodiments, the extraction time in step (1) is 30 min to 45 min.

[0015] In some embodiments, the centrifugation condition in step (3) is 8000 g to 12000 g for 10 min to 30 min.

[0016] 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.

[0017] In some embodiments, the Chinese medicine composition has at least one of the following effects: reducing intestinal tissue damage and improving intestinal barrier function.

[0018] The present invention provides an application of a Chinese medicine composition in the preparation of a medicine for 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 liquorice and Atractylodes macrocephala. The inventors have found, based on their own years of clinical treatment experience and a large number of studies, that the Chinese medicine composition has a significant therapeutic effect on NEC, can effectively reduce the degree of NEC tissue damage in newborn mice, improve intestinal barrier function, significantly reduce the incidence of NEC, and improve the survival rate. In addition, the Chinese medicine composition has no obvious toxic side effects when used to treat neonatal necrotizing enterocolitis, and has high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is an acute toxicity test of LZT administered orally to mice, and its effect on body weight and weight gain of newborn mice.

[0020] Figure 2 The survival rates of newborn mice with NEC in the BF, SOL, LZT-1.25, LZT-2.5, and LZT-5 groups are shown.

[0021] Figure 3 The survival rates of newborn mice with NEC in the BF, SOL, LZT-5, LZT-10, and LZT-20 groups.

[0022] Figure 4 The survival rates of newborn mice with NEC in the BF, SOL, LZT-20, LZT-40, and LZT-80 groups are shown.

[0023] Figure 5 The survival rates of newborn mice with NEC in the BF, SOL, LZT-80, and LZT-160 groups.

[0024] Figure 6 LZT treatment can significantly improve NEC tissue damage in newborn mice.

[0025] Figure 7 LZT treatment can significantly reduce the severity of NEC tissue damage in newborn mice.

[0026] Figure 8 LZT treatment can significantly reduce the incidence of NEC.

[0027] Fig. 9 LZT treatment can significantly reduce the permeability of intestinal tissue in neonatal NEC mice.

[0028] Fig.10 LZT treatment can significantly increase the expression level of intestinal barrier proteins in the intestinal tissue of neonatal NEC mice. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] The Chinese medicine composition of the present invention 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 prepared by 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 select 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.

[0032] The following is a description with reference to specific embodiments.

[0033] In the following examples, the Lizhong Decoction (LZT) 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 dosage, 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.

[0034] Example 1

[0035] This example studies the therapeutic effect of Lizhong Decoction on NEC.

[0036] 1. Materials and Methods

[0037] 1. Research subjects

[0038] 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.

[0039] 2. Main Reagents

[0040] 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. Atractylodes macrocephala (B25582), Shanghai Yuanye Biotechnology Co., Ltd.; Codonopsis pilosula (B26097), Shanghai Yuanye Biotechnology Co., Ltd.; Dry ginger (B25864), Shanghai Yuanye Biotechnology Co., Ltd.; Roasted licorice root (B27088), Shanghai Yuanye Biotechnology Co., Ltd.

[0041] 3. Experimental Methods

[0042] Animal experiments

[0043] Establishment of a necrotizing enterocolitis model in neonatal mice

[0044] 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 gavage every day (the amount of LPS is calculated as 10 mg per 1 kg body weight). The modeling cycle is 4 days.

[0045] Animal grouping and treatment

[0046] Normal control group (BF): mother mice were fed in the same cage; treatment control group (SOL): phosphate buffer was given during modeling; LZT treatment group (LZT): LZT was given by gavage during modeling, referring to the gavage method of NEC model, with 200 μl per 5 g body weight, and gavage was performed through oral intubation with a clean sterile silicone tube, and the time was controlled at 2 to 3 minutes.

[0047] Specimen collection and processing

[0048] 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.

[0049] Observation indicators and methods

[0050] 1) General condition and body weight of newborn mice

[0051] 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.

[0052] 2) Changes in the gross morphology of intestinal tissue

[0053] 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.

[0054] 3) Histopathological scoring

[0055] 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.

[0056] Table 1 Scoring criteria for intestinal tissue pathology

[0057]

[0058] Preparation of paraffin sections of neonatal rat intestinal tissue

[0059] 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;

[0060] 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;

[0061] 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;

[0062] 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.

[0063] HE staining

[0064] 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;

[0065] 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;

[0066] 3) Eosin staining of cytoplasm: Place the slices in eosin staining solution and stain for about 2 minutes;

[0067] 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;

[0068] 5) Use a microscope to collect and analyze images.

[0069] Western blot detection

[0070] Total protein extraction

[0071] 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.

[0072] Western blotting to detect protein expression

[0073] 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;

[0074] 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;

[0075] 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);

[0076] 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;

[0077] 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;

[0078] 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;

[0079] 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);

[0080] 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;

[0081] 9) Take photos (or scan) and record the analysis results.

[0082] Detection of FITC-dextran concentration in plasma

[0083] 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.

[0084] Acute toxicity test

[0085] Acute toxicity test grouping: 40 SPF-grade newborn 7-10-day-old C57 / BL6 mice of either sex were purchased from Guangdong Experimental Animal Center and randomly divided into a control group (CON), a low-dose LZT group (LZT-L), a medium-dose LZT group (LZT-M), and a high-dose LZT group (LZT-H). Referring to the pre-experimental dose, the daily doses of the low-dose, medium-dose, and high-dose LZT were 2.5 g / kg, 20 g / kg, and 160 g / kg, respectively, administered once. The CON group was given the same volume of phosphate buffer.

[0086] Acute toxicity test dose setting basis and administration route: According to the improvement of LZT on NEC survival rate in the neonatal rat NEC model, the optimal dose of 20g / kg was selected as the medium dose group, 2.5g / kg and 160g / kg were low dose group and high dose group respectively. Referring to the NEC model gavage method, according to the amount of 200μl per 5g body weight, gavage was performed through oral intubation with a clean sterile silicone tube, and the time was controlled at 2-3 minutes. Each group was gavaged once a day at 8 am to observe toxic reactions. According to the amount of 200μl per 5g body weight, in order to make the high-dose group single medication reach 160g / kg, the 1g / ml LZD storage solution was further concentrated to 4g / ml for gavage. The CON group was gavaged with the same volume of phosphate buffer. After administration, the newborn mice were fed normally in the same cage with the mother mice.

[0087] Acute toxicity test observation indicators:

[0088] (1) Observation of toxic symptoms and general vital signs: Observe and record the general condition, coat color, activity, gait, demeanor, stool, urine, etc. of the animals. Observe the occurrence of poisoning symptoms, the time and duration of occurrence, and recovery status, and record dead or dying animals.

[0089] (2) Body weight observation: The animals were closely observed for 6 hours after administration on the day of administration (D0), and observed once a day during the observation period after administration. The animals were weighed using an electronic balance on the day before administration (-D1), the first day (D1), the third day (D3), the fifth day (D5), the tenth day (D10), and the fourteenth day (D14).

[0090] (3) Treatment at the end of the observation period: After weighing on D14, the animals were separated from their mothers and fasted but not watered. On the second day of fasting (fasting time 12h-16h), the animals were transferred to the dissection room and killed by dislocating the cervical vertebra. Gross pathological observations were performed to observe changes in various organs (including but not limited to the heart, liver, spleen, lungs, kidneys, adrenal glands, brain, stomach, intestines, testicles, prostate, ovaries, and uterus). If changes in the volume, color, texture, etc. of an organ were found, the changed organ was fixed with 10% neutral formalin, embedded in paraffin, sliced, stained with HE (Haematoxylin & Eosin), and subjected to histopathological examination under a microscope. If no obvious abnormality was observed by naked eye observation, tissue fixation and histopathological examination were omitted.

[0091] (4) Examination of dead and dying animals during the experiment: If an animal is found to be dying during the experiment, it shall be actively killed and autopsied; if an animal is found dead, an autopsy shall be performed promptly. The autopsy shall be conducted for gross pathological observation, and the contents of the examination shall be the same as those in item (3).

[0092] Data statistics and analysis

[0093] 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.

[0094] 2. Experimental Results

[0095] 1. Oral administration of LZT is non-toxic to newborn mice

[0096] Although LZT has been used clinically for more than 2,000 years, there is no record of LZT treating neonatal NEC. Therefore, the present invention refers to the relevant requirements of the technical guidelines for drug single-dose toxicity research issued by the State Food and Drug Administration, and on the basis of the above research, further conducts an acute toxicity test of LZT orally administered to neonatal mice, selecting three daily dose concentration gradients of 2.5g / kg (LZT-L), 20g / kg (LZT-M) and 160g / kg (LZT-H), which are equivalent to 4.3, 33.3 and 266.6 times the recommended human dose, respectively; phosphate buffer was taken orally as the treatment control group (CON).

[0097] (1) Grading of acute toxicity of oral LZT

[0098] As shown in Table 2, in terms of the occurrence of death events, no deaths occurred in the newborn mice in the CON and LZT-L, LZT-M, and LZT-H groups during the entire experimental period, indicating that the acute oral maximum tolerated dose (MTD) of LZT for newborn mice is >320g / Kg, and it is non-toxic according to the acute toxicity classification.

[0099] Table 2

[0100]

[0101] (2) Effects of oral LZT on the general condition of newborn mice

[0102] In terms of general condition, there was no significant difference between the LZT-L group and the CON group within 6 hours and 14 days after administration. Furthermore, compared with the CON group, although the LZT-M and LZT-H groups were quieter and less active than the CON group within 4 hours after administration, the animals had normal reflexes and normal reactions, and no abnormal manifestations of other neuropsychiatric systems were observed. 4 hours after administration and 14 days after administration, the general conditions of the LZT-M and LZT-H groups were consistent with those of the CON group.

[0103] (3) Effects of oral LZT on body weight and organs of newborn mice

[0104] The effects of LZT-L, LZT-M, LZT-H and CON on body weight were observed 1 day before gavage (-D1), and 1 day (D1), 3 days (D3), 5 days (D5), 10 days (D10) and 14 days (D14) after gavage. ns indicates P>0.05, and CV indicates weight gain, i.e., D14 minus -D1 body weight.

[0105] Compared with the CON group, there was no significant difference in body weight change within 14 days after treatment in the LZT-L, LZT-M, and ZLT-H groups, and there was no significant difference in weight gain between the groups on the 14th day after treatment and on the day before treatment (Table 3 and Figure 1 ).

[0106] Table 3

[0107]

[0108] On the 14th day after LZT treatment, all surviving animals were separated from their mothers and fasted overnight (12h-16h). The mice were dissected the next day, and the heart, liver, spleen, lung, kidney, adrenal gland, brain, stomach, intestine, testicle, prostate, ovary, uterus and other major organ tissues of the mice were observed with the naked eye, and no abnormalities were found.

[0109] The above results show that oral administration of LZT has no toxic side effects on newborn mice and has good safety.

[0110] 2. Oral administration of LZT can significantly improve the survival rate of neonatal NEC mice

[0111] According to the conversion formula of human and mouse body surface area, the dosage of mice in the current prescription is 1g / kg, and the following gavage gradient (daily dose) is set: 1.25g / kg, 2.5g / kg, 5.0g / kg, 10g / kg, 20g / kg, 40g / kg, 80g / kg, 160g / kg. According to the amount of 200μl per 5g body weight, in order to make the single dose of high-dose group reach 80g / kg and 160g / kg, the 1g / ml LZD storage solution is further concentrated to 2g / ml and 4g / ml for gavage.

[0112] Kaplan-Meier estimation and log-rank test were used to analyze the survival rate of breast-fed group (BF) and NEC newborn mice treated with different doses of LZT or phosphate buffered saline treatment control group (SOL). The survival rate was calculated as: number of mice surviving after modeling / number of mice before modeling * 100%. LZT-1.25, LZT-2.5, LZT-5, LZT-10, LZT-20, LZT-40, LZT-80, LZT-160 represent daily doses of 1.25 g / kg, 2.5 g / kg, 5 g / kg, 10 g / kg, 20 g / kg, 40 g / kg, 80 g / kg, 160 g / kg, respectively. *P<0.05; **P<0.01.

[0113] The experimental results showed that compared with the control group, the daily dose of 20g / kg and 40g / kg could significantly improve the survival rate of neonatal NEC mice ( Figure 2 to Figure 5 ). This indicates that LZT has a significant therapeutic effect on NEC in neonatal mice, and 20g / kg and 40g / kg are the optimal doses.

[0114] 3. Therapeutic effect of oral LZT on NEC in neonatal mice

[0115] Since 20g / kg and 40g / kg daily doses are the optimal doses for reducing the mortality of neonatal NEC, a lower daily dose of 20g / kg was further used, and gavage was performed once a day at 8 am as the LZT treatment group (LZT). At the same time, a breast-fed group (BF) and a phosphate buffer treatment control group (SOL) were set up to observe the alleviation of the degree of tissue damage in neonatal NEC. 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 mice in the breast-fed group, phosphate buffer treatment control group and LZT treatment group 12 hours after the last hypoxia combined with hypothermia stimulation treatment was 12, 10 and 17, respectively. There was no obvious abnormality in the mental state of the mice in each group. All mice were killed by cervical dislocation 4 hours after intragastric injection of FITC-dextran, and the corresponding intestinal tissues were taken for the following tests: H&E staining, injury score, NEC incidence, intestinal barrier permeability test, Western Blot test; plasma was taken for FITC-dextran concentration test.

[0116] (1) Oral administration of LZT can reduce the degree of NEC tissue damage in newborn mice

[0117] Figure 6 Representative staining images (H&E staining) of ileum pathological sections of newborn mice in the breast-fed group, phosphate buffered saline treatment control group, and LZT treatment group; scale bar, 50 μm. The results showed that LZT treatment could significantly improve the tissue damage of NEC in newborn mice.

[0118] The severity of intestinal tissue damage in each group of mice was evaluated according to the criteria described in Table 1. Figure 7 The results showed that compared with the breast-fed group, the severity of tissue damage in the phosphate buffer-treated control group was significantly increased; while compared with the phosphate buffer-treated control group, LZT treatment could significantly reduce the severity of NEC tissue damage in newborn mice.

[0119] The incidence of NEC in the phosphate buffer treated control group and the LZT treated group was further calculated. The incidence of NEC = the number of mice with NEC after modeling / the number of mice before modeling * 100%. The incidence of NEC in newborn mice in the phosphate buffer treated control group and the LZT treated group (injury score ≥ 2) is shown in the following table. Figure 8 As shown, LZT treatment can significantly reduce the incidence of NEC.

[0120] In conclusion, LZT treatment can significantly reduce the severity and incidence of NEC in newborn mice.

[0121] (2) The protective effect of oral LZT on NEC in neonatal mice is related to the improvement of intestinal barrier function

[0122] To investigate whether LZT alleviates NEC tissue damage in neonatal mice and improves intestinal barrier function, FITC-dextran was injected into all surviving neonatal mice by oral gavage 4 hours before sacrifice. The fluorescence readings in plasma were detected 4 hours after oral gavage, and the intestinal barrier permeability was evaluated by measuring the FITC-dextran concentration in plasma.

[0123] The results are as follows Fig. 9 As shown in the figure, compared with the breast-fed group, the FITC-dextran concentration in the plasma of mice in the phosphate buffer-treated control group was significantly increased. However, compared with the phosphate buffer-treated control group, the FITC-dextran concentration in the plasma of mice in the LZT-treated group was significantly decreased. This indicates that LZT treatment can significantly reduce the plasma FITC-dextran concentration and improve the intestinal barrier function.

[0124] Western Blot was used to detect the expression of zonula occludens-1 (ZO-1) in the ileum tissue of newborn mice in the breast-fed group, phosphate buffered saline treatment control group and LZT treatment group, with 6 mice in each group for detection. ZO-1 is a biomarker related to tight junctions in intestinal tissue.

[0125] like Fig.10 (The left side is the WB representative graph, and the right side bar scatter plot is the quantification result of the expression level of ZO-1 detected by WB, n=6) as shown, LZT treatment can significantly increase the expression level of intestinal barrier proteins in the intestinal tissue of neonatal NEC mice and significantly improve the reduction of ZO-1, indicating that the protective effect of LZT on neonatal NEC is related to the improvement of intestinal barrier function.

[0126] 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.

[0127] 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 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 claim 1, 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, soaking them in water 6 to 10 times the total weight of the raw materials, and then boiling them for extraction; (2) repeating step (1) 1 to 2 times; (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. The use according to claim 5, characterized in that In step (3), the solution is concentrated to contain 0.5 g to 5 g of crude drug per ml of the solution.

10. The use according to any one of claims 1 to 9, characterized in that: The traditional Chinese medicine composition has at least one of the following effects: reducing intestinal tissue damage and improving intestinal barrier function.

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