Application of Lizhong Decoction in the treatment of neonatal necrotizing enterocolitis

Abstract

The present invention provides the use of Lizhong Decoction in the treatment of neonatal necrotizing enterocolitis, wherein the active ingredients of the Lizhong Decoction 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). The inventors, based on their own many years of clinical treatment experience and a large number of studies, found that Lizhong Decoction 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 increase survival rate. In addition, Lizhong Decoction has no obvious toxic side effects when used to treat neonatal necrotizing enterocolitis and is highly safe.

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 from Zhang Zhongjing's Treatise on Febrile and Miscellaneous Diseases, known for warming the middle-Jiangsu, dispelling cold, replenishing Qi, and strengthening the spleen. It's made with equal parts of ginseng, dried ginger, roasted licorice, and white atractylodes (Atractylodes macrocephala). In clinical practice, ginseng is often replaced with codonopsis.

[0003] Necrotizing enterocolitis (NEC) is a life-threatening acute inflammatory and necrotizing intestinal disease in newborns, often occurring 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 the incidence in VLBW infants is close to 5%, also showing an upward trend. More seriously, surviving infants are prone to digestive and neurological sequelae, placing a heavy burden on their families and society.

[0004] Regarding the treatment of NEC, current clinical guidelines mainly include routine supportive treatments such as fasting, gastrointestinal decompression, and the use of antibiotics. Recent data on the survival of extremely premature infants in the United States show that the mortality rate decreased from 2005 to 2021, but 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 in 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 the inflammatory response mediated by it 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, thereby causing ischemia and hypoxia of the intestinal wall, inflammatory damage, and ultimately leading 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 the patent ductus arteriosus in premature infants with hemodynamic compromise, and breastfeeding and its standardized feeding strategies, are all developed based on the current understanding of the pathogenesis.

[0005] Recent data from the my country Neonatal Collaborative Network indicates that the incidence of NEC complications in extremely premature infants born between 22 and 25 weeks of gestation has not decreased from 2019 to 2021, suggesting that existing prevention and treatment strategies are no longer sufficient to further reduce the incidence of NEC. Therefore, new treatment options for neonatal NEC are urgently needed. 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, roasted liquorice 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, roasted liquorice 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, adding 6 to 10 times the volume of water of the total weight of the raw materials to soak, and then boiling and extracting; (2) repeating step (1) 1 to 2 times; (3) combining the extracts, centrifuging, taking the supernatant, and concentrating to obtain.

[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 milliliter 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 a use of a traditional Chinese medicine composition for the preparation of a medicament for 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 liquorice root, and Atractylodes macrocephala. Based on the inventors' years of clinical experience and extensive research, they have found that the traditional Chinese medicine composition has a significant therapeutic effect on NEC, effectively reducing the degree of NEC tissue damage in newborn mice, improving intestinal barrier function, significantly reducing the incidence of NEC, and increasing survival rates. Furthermore, the traditional Chinese medicine composition has no significant toxic side effects when used to treat neonatal necrotizing enterocolitis, and is highly safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This study is to conduct an acute toxicity test on the effect of oral administration of LZT on the 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 are shown.

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

[0023] Figure 5 is the survival rate of NEC in newborn mice in 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] Figure 9 LZT treatment can significantly reduce the permeability of intestinal tissue in neonatal NEC mice.

[0028] Figure 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 specific conditions are not specified, are generally carried out under conventional conditions or 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. 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 sage Zhang Zhongjing. It is prepared by mixing ginseng, dried ginger, roasted licorice root, 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 pathogenesis of NEC, which is deficiency, we selected codonopsis, dried ginger, roasted licorice root, and white atractylodes in the ratio of (1-2):(1-2):(1-2):(1-2), preferably 1:1:1:1.

[0032] The following describes the method in conjunction with 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, Radix Glycyrrhizae Preparata, and Atractylodes macrocephala are mixed according to the amount used, and then 10 times the volume of distilled water relative to the total amount of the mixed raw materials is added to soak for 45 minutes, 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 and centrifuged at 10,000×g for 15 minutes. Finally, the supernatant after centrifugation is concentrated to 1g of the raw drug per milliliter of the medicinal liquid, 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 the Guangdong Medical Laboratory Animal Center. This study was approved by the Guangdong Medical Laboratory 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.; Dried ginger (B25864), Shanghai Yuanye Biotechnology Co., Ltd.; and Radix Glycyrrhizae uralensis (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 for establishing the neonatal NEC model are as follows: 8-10 day old C57 / BL6 mice were separated from their mothers and housed in a neonatal incubator. Formula milk was administered orally via oral cannula using a clean, sterile silicone tube, with a volume of 200 μl per 5 g body weight, for 2-3 minutes each time, five times daily. Hypoxia (5% O₂, 95% N₂) and hypothermia (refrigerated at 4°C) were administered at 8:00 and 20:00 daily for 10 minutes each. LPS (10 mg per kg body weight) was also added during the second gavage each day. The modeling cycle lasted four 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 saline was given during modeling; LZT treatment group (LZT): LZT was given by gavage during modeling, referring to the NEC model gavage method, with 200 μl per 5 g body weight, and gavage was performed through oral intubation using a clean sterile silicone tube, and the time was controlled within 2 to 3 minutes.

[0047] Specimen collection and processing

[0048] Twelve 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 Scientific, Cat#18912014) by gavage (750 mg / kg) and then killed by cervical dislocation 4 hours later. After killing mice, under sterile conditions: (1) blood was collected by decapitation into sodium heparin anticoagulant tubes and centrifuged at 3000×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 with ophthalmic scissors. The intestinal tube from the lower end of the duodenum to the ileocecal part was removed, and the 0.5 cm of intestinal tube near the ileocecal part was fixed in 4% formaldehyde solution.

[0049] Observation indicators and methods

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

[0051] General condition and 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 are 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 mouse intestinal tissue

[0059] 1) Sampling: Fix fresh intestinal tissue samples with 4% paraformaldehyde for at least 24 hours. Remove the fixed tissue and trim the target area with a scalpel in a fume hood. Place the trimmed tissue and corresponding label in a dehydration box.

[0060] 2) Dehydration: Place the dehydration box into the hanging 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: Embed the wax-soaked samples with an embedding machine; place the melted wax into the embedding frame, and before the wax solidifies, take the sample out of the dehydration box and place it into the embedding frame and label it; 。 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, and slice to a thickness of 4 μm. Then, flatten the slices on a 40°C warm water plate in a microtome, transfer them to a glass slide, and bake them in a 60°C oven for about 6 hours. Store them at room temperature for later use.

[0063] HE staining

[0064] 1) Dewaxing and hydrating paraffin sections: First, place the sections in xylene for 20 minutes, repeat once, then place them in anhydrous ethanol for 10 minutes, repeat once, followed by 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes. Finally, rinse with distilled water.

[0065] 2) Hematoxylin staining of cell nuclei: Place the sections in Harris hematoxylin solution for approximately 5 minutes, then rinse with tap water. Then, differentiate with 1% hydrochloric acid alcohol solution for a few seconds, immediately rinse with tap water, and finally, with 0.6% ammonia solution to reverse the blueing, and rinse with tap water.

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

[0067] 4) Dehydration and mounting: First, place the sections in 95% alcohol for 5 minutes, repeat once, then place in absolute ethanol for 5 minutes, repeat once, and then place in xylene for 5 minutes, repeat once, to dehydrate and make them transparent. At this time, remove the sections from the xylene and let them dry slightly. Then, mount the sections with neutral gum, taking care to avoid the formation of bubbles.

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

[0069] Western blot assay

[0070] Total protein extraction

[0071] Place an appropriate amount of tissue in a grinder and add the corresponding amount of lysis buffer in proportion, then grind it with a grinder to fully lyse it; 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) Prepare SDS-PAGE gel: Clamp the aligned glass plates and secure them vertically on a rack. Prepare the separating gel solution. After adding TEMED, immediately shake well, fill the gel, and seal with water. Wait until a refraction line appears between the water and the gel, then wait approximately 5 minutes for the gel to fully solidify and discard the upper water layer. Prepare the stacking gel using the same process as for the separating gel. Fill the remaining space with stacking gel and insert a comb into the stacking gel. Once the stacking gel solidifies, gently remove the comb. Rinse with deionized water, place the gel in an electrophoresis tank, add sufficient electrophoresis buffer, and begin electrophoresis.

[0074] 2) Protein loading: Use a microinjector to draw up 30 μg of sample and slowly add it to the sample well. Reserve one well for adding 5 μl of protein marker.

[0075] 3) Separation Electrophoresis: Turn on the power supply and adjust the voltage to 80V. Run the electrophoresis for about 30 minutes. After the stacking gel has run, adjust the voltage to 120V and run the electrophoresis for about 60 minutes (stop the electrophoresis when the bromofen blue reaches about 1 cm from the bottom of the separation gel).

[0076] 4) Transfer: Cut the target protein strip based on the protein marker. Next, prepare the PVDF membrane by soaking it in methanol for 1-5 minutes, then equilibrate it with transfer buffer for 10 minutes. Simultaneously, place the filter paper and sponge in the transfer buffer. After soaking, start with the positive electrode, then add the sponge, filter paper, PVDF membrane, gel, filter paper, and sponge. Close the negative electrode, place the membrane in the electroporation tank, and turn on the power supply for transfer. The transfer current and time are 300 mA 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 and wash the membrane with TBST for 5 minutes, repeat six times. Incubate with primary antibody at the desired antibody concentration, diluting the ZO-1 protein concentration to 1:1000 in TBST. Incubate overnight at 4°C.

[0079] 7) Secondary antibody incubation: Wash the membrane with TBST for 5 minutes, repeat 6 times, and incubate with secondary antibody, diluted in TBST according to the desired antibody concentration, and incubate at room temperature for 2 hours (the concentration of the secondary antibody used was 1:4000);

[0080] 8) Color Development: Wash the membrane in TBST for 5 minutes, repeat 6 times, and develop using an ECL color development kit. Depending on the size of the membrane, take equal amounts of Solution A and Solution B, mix thoroughly, and use filter paper to absorb some of the wash solution from one corner of the membrane. Place the membrane on clean plastic wrap and cover with the mixture. Develop for 1-5 minutes, then absorb the color solution. Fold the plastic wrap over the membrane, sandwiching it between the plastic wrap, and place in a dark box. Observe the brightness of the fluorescence in a darkroom to determine the exposure time. Develop the membrane and fix it.

[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. The plasma FITC-dextran concentration was calculated based on the standard curve.

[0084] Acute toxicity test

[0085] Acute toxicity test grouping: Forty SPF-grade newborn C57 / BL6 mice aged 7 to 10 days, of either sex, were purchased from the Guangdong Provincial Laboratory 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 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 and administration route: Based on the improvement in survival of neonatal NEC rats with LZT in the NEC model, the optimal dose of 20 g / kg was selected as the medium-dose group, with 2.5 g / kg and 160 g / kg as the low-dose and high-dose groups, respectively. Using the same gavage protocol as the NEC model, 200 μl per 5 g body weight was administered via oral cannula using a clean, sterile silicone tube for 2-3 minutes. Each group received a single gavage at 8:00 AM daily for observation of toxic reactions. To achieve a single dose of 160 g / kg in the high-dose group, the 1 g / ml LZD stock solution was further concentrated to 4 g / ml using a 200 μl per 5 g body weight dose. The CON group received the same volume of phosphate buffered saline by gavage. Following administration, the newborn rats were housed with their mothers and fed normally.

[0087] Acute toxicity test observation indicators:

[0088] (1) Observation of toxicity symptoms and general vital signs: Observe and record the animal's general condition, coat color, activity, gait, demeanor, stool, urine, etc. Observe the occurrence of poisoning symptoms, the time of occurrence and duration, 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 daily during the observation period. 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 to 16h), the animals were transferred to the dissection room and killed by cervical dislocation. 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, sectioned, stained with HE (Haematoxylin & Eosin), and subjected to microscopic histopathological examination. If no obvious abnormalities were observed by naked eye, 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 examination contents shall be the same as those in item (3).

[0092] Data statistics and analysis

[0093] Unless otherwise noted, all data are presented as mean ± standard deviation. Student's t-test was used for comparisons between two groups. For three or more groups, Kruskal-Wallis paired comparison test or one-way analysis of variance coupled with Bonferroni multiple comparison test was used for statistical analysis. Survival curves were analyzed using Kaplan-Meier estimates and the log-rank test. P values ​​≤ 0.05 were considered statistically significant.

[0094] 2. Experimental Results

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

[0096] Although LZT has been used clinically for over 2,000 years, there are no records of its use in treating neonatal NEC. Therefore, referring to the requirements of the Technical Guidelines for Single-Dose Toxicity Studies of Drugs issued by the State Food and Drug Administration, and building on the aforementioned research, we conducted an acute toxicity study of LZT administered orally to neonatal mice. Three daily dose concentration gradients of 2.5 g / kg (LZT-L), 20 g / kg (LZT-M), and 160 g / kg (LZT-H) were selected, representing 4.3, 33.3, and 266.6 times the recommended human dose, respectively. A phosphate buffered saline solution was administered orally as a control group (CON).

[0097] (1) Acute toxicity classification of oral LZT

[0098] As shown in Table 2, no deaths occurred in the newborn mice in the CON, 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 > 320 g / Kg, and it is classified as 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 statistically significant difference in body weight changes in the LZT-L, LZT-M, and ZLT-H groups within 14 days after treatment, and there was no significant difference in weight gain between the groups on the 14th day after treatment compared with 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, lungs, kidneys, adrenal glands, brain, stomach, intestines, testicles, prostate, ovaries, uterus and other major organs and tissues of the mice were observed with the naked eye, and no abnormalities were found.

[0109] The above results indicate that oral administration of LZT has no toxic side effects on newborn mice and is safe.

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

[0111] According to the body surface area conversion formula for humans and mice, the dosage for mice in this prescription was 1g / kg, and the following gavage gradient (daily dose) was set: 1.25g / kg, 2.5g / kg, 5.0g / kg, 10g / kg, 20g / kg, 40g / kg, 80g / kg, and 160g / kg. Based on a volume of 200μl per 5g body weight, in order to achieve a single dose of 80g / kg and 160g / kg in the high-dose group, the 1g / ml LZD stock solution was further concentrated to 2g / ml and 4g / ml for gavage, respectively.

[0112] Kaplan-Meier estimates and log-rank tests were used to analyze the survival rates of breast-fed (BF) and neonatal NEC mice treated with different doses of LZT or the phosphate-buffered saline (PBS)-treated control group (SOL). Survival rate was calculated as: number of surviving mice at the end of modeling / number of mice before modeling × 100%. LZT-1.25, LZT-2.5, LZT-5, LZT-10, LZT-20, LZT-40, LZT-80, and 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, and 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 ( Figures 2 to 5 ). This shows 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] Because daily doses of 20g / kg and 40g / kg were the optimal doses for reducing NEC mortality in neonatal mice, a lower daily dose of 20g / kg was administered orally once daily at 8:00 AM as the LZT-treated group (LZT). A breastfed group (BF) and a phosphate buffer saline-treated control group (SOL) were also established to observe their ability to alleviate NEC tissue damage in neonatal mice. Twelve hours after the last hypoxia-hypothermia treatment, mice with poor mental state were killed by cervical dislocation. Mice with no significant mental abnormalities were given FITC-dextran by oral gavage and then killed by cervical dislocation 4 hours later. In this example, the number of surviving mice in the breast-fed group, phosphate buffer-treated control group, and LZT-treated group 12 hours after the last hypoxia combined with hypothermia stimulation was 12, 10, and 17, respectively. The mice in each group showed no obvious abnormalities in mental state. All mice were intragastricly injected with FITC-dextran and then sacrificed by cervical dislocation 4 hours later. The corresponding intestinal tissues were collected for the following tests: H&E staining, injury score, NEC incidence, intestinal barrier permeability test, and Western Blot analysis; plasma was collected for FITC-dextran concentration detection.

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

[0117] Figure 6 Representative images (H&E staining) of ileum pathological sections from neonatal mice in the breast-fed group, phosphate-buffered saline control group, and LZT-treated group; scale bar, 50 μm. The results showed that LZT treatment significantly improved NEC tissue damage in neonatal 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; whereas compared with the phosphate buffer-treated control group, LZT treatment could significantly reduce the severity of NEC tissue damage in neonatal mice.

[0119] The incidence of NEC in the phosphate buffer treated control group and the LZT treated group was further calculated as follows: NEC incidence = number of mice with NEC after modeling / number of mice before modeling * 100%. The incidence of NEC in newborn mice (lesion score ≥ 2) in the phosphate buffer treated control group and the LZT treated group 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 neonatal 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, all surviving neonatal mice were given FITC-dextran by gavage 4 hours before sacrifice. The fluorescence readings in the plasma were detected 4 hours after gavage, and the intestinal barrier permeability was evaluated by measuring the FITC-dextran concentration in the plasma.

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

[0124] Western blotting was then used to examine the expression of zonula occludens-1 (ZO-1) in the ileum of newborn mice in the breast-fed, phosphate-buffered saline-treated, and LZT-treated groups, with six mice in each group. ZO-1 is a biomarker associated with tight junctions in intestinal tissue.

[0125] like Figure 10 (The left side is the WB representative image, 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 mice is related to the improvement of intestinal barrier function.

[0126] The technical features of the above-described embodiments can be combined arbitrarily. 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-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended 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, wherein 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, and the preparation method of the Chinese medicine composition comprises the following steps: (1) taking codonopsis pilosula, dried ginger, roasted liquorice root and atractylodes macrocephala in proportion, adding water with a volume of 6 to 10 times the total weight of the raw materials to soak, and then boiling and extracting; (2) repeating the 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 centrifugation 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 milliliter 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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