Use of butyric acid and its derivatives or pharmaceutically acceptable salts thereof in preparing products for treating hypoxic-ischemic encephalopathy

Abstract

The present invention relates to the field of medical technology. The present invention provides use of butyric acid and its derivatives or pharmaceutically acceptable salts thereof in preparing products for treating hypoxic-ischemic encephalopathy. For the first time, butyric acid-producing bacteria and abnormal butyric acid metabolic pathways were found in feces of HIBD model rats. Further studies have found that the use of butyric acid and its derivatives or pharmaceutically acceptable salts thereof to intervene in newborn HIBD model rats can restore pathological damage to the cerebral cortex and hippocampus of the HIBD rats and behavioral studies related to learning and memory, and promote the recovery of intestinal contents and metabolic pathway-related flora, indicating that butyric acid and its derivatives or pharmaceutically acceptable salts thereof have the use of preparing products for alleviating, assisting in the treatment or treating hypoxic-ischemic encephalopathy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to use of butyric acid and its derivatives or pharmaceutically acceptable salts thereof in preparing products for treating neonatal hypoxic-ischemic encephalopathy. Background Art

[0002] Neonatal hypoxic-ischemic encephalopathy (HIE) refers to hypoxic-ischemic damage to the brain caused by perinatal asphyxia. Hypoxic-ischemic brain damage (HIBD) is a serious complication of asphyxia, with a high mortality and disability rate. Neonatal HIBD is brain damage caused by perinatal asphyxia, which can cause permanent neurological damage in severe cases and is one of the common causes of nervous system damage in children. Clinically, it is mainly manifested as impaired consciousness, changes in muscle tone and primitive reflexes, and severe symptoms such as convulsions, cerebral edema, and intracranial hypertension may occur.

[0003] Previous clinical and animal model studies on neonatal hypoxic-ischemic encephalopathy (HIE) have not yet found any research or reports on the use of butyrate or its pharmaceutically acceptable derivatives to alleviate or treat neonatal hypoxic-ischemic brain damage. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a use of butyric acid and its derivatives or pharmaceutically acceptable salts thereof in the preparation of products for treating hypoxic-ischemic encephalopathy.

[0005] To this end, the present invention provides the following technical solutions:

[0006] Use of butyric acid and its derivatives or pharmaceutically acceptable salts thereof in preparing products for alleviating, assisting in the treatment or treating hypoxic-ischemic encephalopathy.

[0007] Optionally, the hypoxic-ischemic encephalopathy includes hypoxic-ischemic brain injury.

[0008] Optionally, the hypoxic-ischemic encephalopathy includes immature cerebral hypoxic-ischemic encephalopathy;

[0009] Optional, including neonatal hypoxic-ischemic encephalopathy.

[0010] Optionally, the hypoxic-ischemic encephalopathy includes hypoxic-ischemic brain damage in the immature brain;

[0011] Optional, including neonatal hypoxic-ischemic brain damage.

[0012] Optionally, the use includes use in preparing a product for alleviating, assisting in the treatment or treating pathological damage to the cerebral cortex and / or hippocampus caused by hypoxic-ischemic encephalopathy.

[0013] Optionally, the use includes use in preparing products for alleviating, assisting in the treatment or treating behavioral disorders related to learning and memory caused by hypoxic-ischemic encephalopathy.

[0014] Optionally, the use includes use in preparing products for alleviating, assisting in the treatment or treating the intestinal contents and / or metabolic pathway-related flora disorders caused by hypoxic-ischemic encephalopathy.

[0015] Optionally, use in the preparation of a product for increasing low butyrate concentration levels in the cerebral cortex caused by hypoxic-ischemic encephalopathy.

[0016] Optionally, the butyric acid and its derivatives or pharmaceutically acceptable salts thereof include butyric acid, sodium butyrate, potassium butyrate or calcium butyrate;

[0017] Optionally, the product includes medicine or food;

[0018] Optionally, the food includes functional food or health care product.

[0019] A pharmaceutical preparation for relieving, assisting in the treatment or treating hypoxic-ischemic encephalopathy, comprising butyric acid and its derivatives or pharmaceutically acceptable salts thereof as active ingredients;

[0020] Optionally, it also includes pharmaceutical excipients or carriers permitted by the preparation;

[0021] Optionally, the pharmaceutical preparation may be in the form of a liquid preparation or a solid preparation;

[0022] Optionally, the pharmaceutical preparation includes injection, tablet, capsule or granule.

[0023] The technical solution of the present invention has the following advantages:

[0024] 1. The use of butyric acid and its derivatives or pharmaceutically acceptable salts thereof provided by the present invention in the preparation of products for alleviating, assisting in the treatment or treating hypoxic-ischemic encephalopathy. For the first time, butyric acid-producing bacteria and abnormal butyric acid metabolic pathways were found in the feces of HIBD model rats. Further studies found that the use of butyric acid and its derivatives or pharmaceutically acceptable salts thereof to intervene in newborn HIBD model rats can restore pathological damage to the cerebral cortex and hippocampus of HIBD rats and behavioral changes related to learning and memory, and promote the recovery of intestinal contents and metabolic pathway-related flora, indicating that butyric acid and its derivatives or pharmaceutically acceptable salts thereof have the use of preparing products for alleviating, assisting in the treatment or treating hypoxic-ischemic encephalopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 It is H&E staining of the cerebral cortex and hippocampus tissues of rats in each group in the experimental examples of the present invention;

[0027] Figure 2 It is the result of the representative passage on the 5th day of the water maze test of rats in each group in the experimental example of the present invention and the result of the representative lane on the 6th day of the test;

[0028] Figure 3 It is the escape latency test result of each group of rats in the experimental example of the present invention; compared with the sham operation group, *<0.05; compared with the HIBD model group, #<0.05, ##<0.01;

[0029] Figure 4 It is the test result of the number of times rats in each group crossed the platform in the experimental example of the present invention; compared with the sham operation group, * indicates p<0.05; compared with the HIBD model group, # indicates p<0.05, and ## indicates p<0.01;

[0030] Figure 5 is the detection result of butyrate concentration in the cerebral cortex of rats in each group of rats (except HIBD+normal saline group) in the experimental example of the present invention; the error bar represents SD, and compared with the sham operation group, * represents p<0.05; compared with the HIBD model group, # represents p<0.05, and ## represents p<0.01;

[0031] Figure 6 This is a heat map of significantly different metabolites in feces of rats in the sham operation group and the HIBD group in the experimental example of the present invention;

[0032] Figure 7 It is a bar graph of the abundance of fecal flora in rats of each group of rats (except the HIBD+normal saline group) in the experimental examples of the present invention. DETAILED DESCRIPTION

[0033] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0034] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0035] Experimental example

[0036] 1. Animals and groups

[0037] Newborn rats (Wistar rats, 7-day-old, either sex, SPF grade, purchased from Vital River) were randomly divided into sham operation group, HIBD model group, HIBD+normal saline group, and HIBD+sodium butyrate group.

[0038] Sham operation group: except for unilateral common carotid artery ligation and hypoxia, other conditions were the same as those of the HIBD model group.

[0039] HIBD model group: The ischemic hypoxia model refers to the classic Rice-Vannucci method (Rice JE 3rd, VR, Brierley JB. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol. 9, 131-141 (1981).): After the rats were anesthetized with chloral hydrate, they were fixed on a sterile animal operating table, and a midline incision (5-10 mm) was made in the neck. After the skin was cut, the left anterior cervical muscle group was finely separated with ophthalmic forceps to expose the carotid sheath. The common carotid artery was gently separated from the vagus nerve with a glass needle, and the common carotid artery was ligated with a single line of 5-0 suture and the wound was sutured after ensuring that there was no blood flow. The entire operation process was controlled within 5 minutes. After the operation, the rats were placed in a cage next to the mother rat to rest for 1 hour, and the young rats were placed in a closed hypoxia box containing 8% O2 and 92% N2 for 2 hours.

[0040] HIBD+normal saline group: From the day when HIBD model was successfully established, rats were gavaged with normal saline at a dose of 100 μL, once a day for 2 consecutive weeks.

[0041] HIBD+sodium butyrate group: Since the day of successful HIBD model establishment, sodium butyrate (100 mg / kg) was intragastrically administered at a dose of 100 μL, once a day for 2 consecutive weeks.

[0042] 2. Detection methods and results

[0043] (1) Sequencing and mass spectrometry

[0044] The intestinal contents (feces) of rats in the HIBD model group (6 rats) and the sham operation group (6 rats) were detected by 16s high-throughput sequencing and GC-MS mass spectrometry. Figure 6As shown, compared with the sham operation group, the butyrate metabolic pathway (KEGG) and related metabolites in the feces of rats in the HIBD group were downregulated. It was found that the number of butyrate-producing bacteria in the feces of rats in the HIBD model group was significantly reduced and the butyrate metabolic pathway was abnormally enriched.

[0045] (2) H&E staining of rat cerebral cortex and hippocampus

[0046] After 2 weeks of sodium butyrate treatment, the rats in each group (3 in each group) were killed, and the brain tissues were fixed with formalin and sliced ​​for H&E staining. Figure 1 As shown in the data, compared with the HIBD model group, the pathological damage of the cerebral cortex and hippocampus tissues of the rats in the HIBD+normal saline group did not show any improvement, while the pathological damage of the cerebral cortex and hippocampus tissues of the rats in the HIBD+sodium butyrate group was improved, indicating that sodium butyrate intervention can alleviate or treat the pathological damage of the cerebral cortex and hippocampus tissues of newborn HIBD rats.

[0047] (3) Experiments on learning and memory-related behaviors

[0048] After 2 weeks of sodium butyrate treatment, rats from each group (3 in each group) were subjected to a water maze test (the experimental rats were placed in a circular pool with opaque, non-toxic white pigment added to the water, which contained a hidden escape platform. The experiment included positioning navigation and spatial exploration. Positioning navigation was to randomly place animals from the four quadrants of the water maze, allowing them to swim in the water and find the escape platform, and use video tracking software to record the time (escape latency) and path for the animals to find the hidden platform. Spatial exploration was to remove the escape platform after positioning navigation, observe the number of times and time the animals crossed the original platform position, and examine the animals' memory of the original platform.) The results of the representative pathways on the 5th day of the experiment and the results of the representative lanes on the 6th day of the experiment are shown in Figure 2. Figure 2 The escape latency test results of rats in each group are shown in Figure 3 As shown in Figure 2, the number of times the rats in each group crossed the previous platform position is as follows: Figure 4 Data processing: Data were analyzed using GraphPad Prism (Version 9.2.0) and SPSS 20.0 (IBM, USA). Student's t-test or analysis of variance was used for comparison between groups.

[0049] By comparison, it can be concluded that compared with the HIBD model group, there was no difference in the learning and memory-related behavioral experimental results of rats in the HIBD+normal saline group, while there was a significant difference in the learning and memory-related behavioral experimental results of rats in the HIBD+sodium butyrate group, indicating that sodium butyrate intervention can restore the learning and memory-related behaviors of newborn HIBD rats.

[0050] (4) Butyrate content in cerebral cortex

[0051] After 2 weeks of sodium butyrate treatment, rats from each group (except the HIBD+saline group) (6 rats in each group) were taken for quantitative detection of butyrate content in the cerebral cortex by liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis (UPLC-ESI-MS / MS analysis was used in this experiment to qualitatively and quantitatively detect the target metabolites. The specific analysis conditions and analysis methods are as follows. Chromatographic conditions: injection volume: 5 μL; flow rate: 0.35 mL / min; mobile phase: A (0.1% formic acid-water solution), B (acetonitrile); gradient elution method: 0 min A / B (90:10, V / V), 1 min A / B (90:10, V / V), 2 min A / B (75:25, V / V), 6 min A / B (65:35, V / V), 6.5 min A / B (5:95, V / V), 7.8 min A / B (5:95, V / V), 7.81 min A / B (90:10, V / V), 8.5min A / B (90:10, V / V). Mass spectrometry conditions: curtain gas: 35 (psi); collision-activated dissociation (CAD) parameters: medium; positive ion spray voltage: 5500V; negative ion spray voltage: -4500V; ion source temperature: 450℃; column temperature: 40℃; ion source: Gas1:50 (psi); Gas2:60 (psi). ), Data processing: According to parameters such as sampling and (or) dilution multiples, the metabolite concentration information is further calculated to finally obtain the absolute content information of each metabolite in the actual sample. The calculation formula is as follows: Sample metabolite content (ng / g) = N1*N2*(C*V) / M); Note: C: Concentration value calculated by substituting the peak area of ​​metabolites in the sample into the standard curve (ng / mL); V: fixed volume: 0.3mL; M: weighed sample mass (g); N1: dilution factor (1); N2: conversion factor (1: ratio of the derivatization amount of sample to that of standard).

[0052] The results are as follows Figure 5 As shown, it is shown that sodium butyrate can pass through the blood-brain barrier into the brain and increase the butyrate concentration in the target organ brain of newborn HIBD rats.

[0053] (5) Intestinal contents and bacterial flora related to metabolic pathways

[0054] After 2 weeks of sodium butyrate treatment, rats from each group (except the HIBD+normal saline group) (5 rats in each group) were taken, and the intestinal contents (feces) of the rats in the HIBD model group (5 rats) and the sham operation group (9 rats) were detected by 16s high-throughput sequencing and GC-MS mass spectrometry. Figure 7As shown in the figure, compared with the sham operation group, the butyrate-producing bacteria in the feces of rats in the HIBD group were downregulated and upregulated after butyrate intervention, indicating that sodium butyrate intervention can promote the recovery of intestinal contents and metabolic pathway-related flora in HIBD.

[0055] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of butyric acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for alleviating, assisting in the treatment or treating the disturbance of intestinal contents and / or metabolic pathways-related flora caused by perinatal cerebral hypoxia-ischemia.

2. The use according to claim 1, characterized in that The butyric acid or pharmaceutically acceptable salts thereof include butyric acid, sodium butyrate, potassium butyrate and calcium butyrate.

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