Horticultural experiment and water maze test-related urinary metabolomics study on the intervention of D-galactose-induced aging rats
Urine metabolomics study on the effects of ethanol extract of astragalus on D-galactose-induced aging rats Abstract: To study the effects of astragalus on D-galactose-induced aging model rats, and to explore the anti-aging mechanism of Astragalus membranaceus. SD rats were randomly divided into 5 groups, namely, blank group, model group, low, medium and high doses of jaundice (50, 100 and 200 mg·kg−1, respectively), and subcutaneous injection of D-galactose (100 mg· The kg-1 method was used to establish a subacute aging rat model, and the spatial learning and memory ability (Morris water maze) and open-field test of aging rats were evaluated. The NMR data of the model rat urine was collected by metabolomics technique and combined with multivariate statistical analysis to explore the anti-aging mechanism. The results showed that all three doses of Astragalus membranaceus could improve the behavioral ability of aging rats. Multivariate statistical analysis showed that the low, medium and high doses of Astragalus membranaceus could cause different degrees of callback in the urine of aging rats, such as citric acid, pyruvic acid, lactic acid, pantothenic acid, trimethylamine and β-hydroxybutyric acid. Regulation of metabolic pathways such as energy metabolism, glucose metabolism, and intestinal metabolism indicates that Astragalus plays an anti-aging role associated with these pathways. Key words: Astragalus; anti-aging; behavioral research; metabolomics Astragalus is the dry root of Scutellaria baicalensis Georgi, which has the effects of clearing away heat and dampness, purging fire, detoxification, hemostasis and fetus. Modern scholars' research on its pharmacological activities mainly focuses on antibacterial, antiviral, anti-inflammatory, anti-cancer, anti-oxidation, free radical scavenging and protection against ischemia-reperfusion injury. Recent studies have shown that aqueous extract of Astragalus membranaceus can increase the thymus index of mice with rapid aging and increase the activity of antioxidant enzymes in mice. The extract of Astragalus membranaceus can improve oxidative damage and neuroinflammation in mice with Alzheimer's disease. And memory damage, but the study of Astragalus on D-galactose-induced aging rat model has not been reported. Aging is a degenerative change in the body tissues and organ functions with age, and is the result of a combination of factors such as genes, environment and lifestyle. With age, the risk of some diseases such as Alzheimer's disease, cardiovascular disease, type 2 diabetes, and cancer increases significantly. The use of natural products with anti-aging activities such as traditional Chinese medicines and botanicals is becoming more and more extensive, which is of great significance for the accumulation of anti-aging knowledge. Metabolomics is a new systematic method to study the changes of the body's metabolites. It can fully understand the pathological process and the metabolic pathways of the body by revealing the changes of metabolic products in the metabolic process. At present, nuclear magnetic resonance spectroscopy (NMR)-based metabolomics has become a powerful tool widely used in basic biology, molecular pathology, clinical diagnosis, biomedicine and environmental science. Due to the complexity of the aging process, the current mechanism of aging is not fully understood. In recent years, the application of high-throughput technologies, including metabolomics techniques, has provided new explanations for the pathophysiological mechanisms of aging and identifies specific markers associated with aging in different species. The D-galactose-induced aging model is currently the most widely used. The model has many aging manifestations such as shortened life span, learning and memory impairment, neurological degeneration, immunity and low fertility, and many forms of organ and physiology and biochemistry in various organs. Indicators show similar changes to natural aging, widely used in aging mechanisms research and pharmacodynamic evaluation of antioxidants and anti-aging drugs. Many studies have shown that the self-release of D-galactose-induced aging rats is reduced (the field test) and the spatial memory capacity is also damaged (water maze test). In this paper, D-galactose-induced aging rat model was used to verify the anti-aging effect of Astragalus membranaceus by behavioral investigation. The changes of urine metabolites in rats were determined by 1HNMR-based metabolomics method. The mechanism of aging provides a theoretical basis for the development and utilization of Astragalus. Materials and Methods The medicinal material Astragalus membranaceus was purchased from the Liuquan Chinese Herbal Medicine Cooperative of Lingchuan County, Shanxi Province, and was identified as the dry root of Scutellaria baicalensis Georgi by Professor Qin Xuemei from the Modern Research Center of Traditional Chinese Medicine of Shanxi University. The samples were preserved in the modern research of traditional Chinese medicine in Shanxi University. center. Animal SPF male SD rats, body weight (180±20) g, provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal license number SCXK (Beijing) 2012-0001; animal breeding room maintained temperature (23±1.5) °C, relative humidity (45 ± 15)%. Animals began to experiment after 1 week of adaptation. Instruments and reagents Bruker 600-MHz AVANCE III NMR detector (Brook, Germany); Sartorius BSA124S analytical balance (Sartorius, Germany); Morris water maze (Shanghai Xinsoft); 实验field experimental box (Shanghai Xinsoft). HPLC analysis by methanol chromatography (Fisher); D-galactose (Amresco), sodium trimethylsilylpropionate (TSP, Cambridge Isotope Laboratories Inc., MA), NMR reagent heavy water (Norell, Landisville, USA) , Wahaha pure water. Preparation method of Astragalus membranaceus extract 20 g of Astragalus membranaceus powder was taken and extracted twice with 10 times of 60% ethanol and distilled water as a solvent for 2 hours, and the extracts were combined twice. The solvent was recovered under reduced pressure and lyophilized to obtain a powder. Take the appropriate amount of Astragalus membranaceus aqueous extract and alcohol extract, and determine the content of baicalin according to the pharmacopoeia method, and use the content as the basis for solvent selection. Animal experiments 50 male Sprague-Dawley rats were randomly divided into a blank group, a model group, and a low, medium and high dose group of jaundice, with 10 rats in each group. The model group and the low, medium and high doses of Astragalus were injected subcutaneously with D-galactose 100 mg·kg −1 for 10 weeks, and the blank group was injected with the same volume of normal saline. The low, medium and high doses of Astragalus were administered orally at doses of 50, 100 and 200 mg·kg −1 per day. The blank group and the model group were given an equal volume of distilled water per day for 10 weeks. Behavioral investigation Open field experiment The open field experiment was carried out in the 10th week of modeling. The rats were placed in the center of the test field of the open field behavior. After 2 minutes of adaptation, the number of uprights, resting time and crossing number of the rats were observed within 4 minutes. After the Morris water maze experiment was completed, the Morris water maze experiment was started. The XR-XM101 Morris water maze video analysis system was used for testing. The experimental equipment consisted of a circular pool and an automatic video analysis system. The circular pool is made of ABS with a diameter of 160 cm and a height of 50 cm. The pool is divided into 4 quadrants with a water depth of 30 cm. The platform is located in the middle of any quadrant and the surface is l 2 cm below the water surface. The water temperature is maintained at around 25 °C. The camera above the pool simultaneously recorded the trajectory of the rat. The experimental data was obtained by analysis and processing by Supermaze software. The water labyrinth experiment environment requires quietness, avoids all external disturbances, keeps the room quiet, the temperature is suitable, the light is soft, and the reference objects outside the labyrinth remain unchanged for the rats during the experiment. The experimental procedure was carried out according to the method of Morris et al. The tests included: 1 Positioning navigation test, which was used to measure the ability of rats to acquire water and maze learning and memory, that is, to record their latency. After 5 days of training, a quadrant was randomly selected during training to place the rat in the pool wall, forcing the rats to learn to find a platform below the water surface. The time required to find the platform within 60 s is recorded in the computer. If the platform cannot be found within 60 s, it can be directed to the platform and stay on the platform for 20 s, with an incubation period of 60 s. Train 4 times a day, each training interval 10 minutes. The system automatically records multiple parameters; 2 Space exploration experiment, which is used to measure the ability of the rats to maintain the platform space position after learning to find the platform. After the end of the navigation test, the platform is removed, and the rat is placed in the water at the entrance of the pool. The number of times of crossing the original platform within 60 s and the activity distance and activity time in the target quadrant can be recorded and compared. Data collection and processing was performed by the Morris Water Maze System software. Sample collection and preparation and determination conditions Urine sample collection and storage: All rats were placed in metabolic cages from 18:00 on the 10th weekend to 8:00 am on the following day, and urine was collected on ice, 12 000 r·min−1 Centrifuge and take the supernatant and store at −80 °C. Sample preparation: Thaw the sample, shake it, centrifuge at 12000 r·min−1 for 10 min at 4 °C, take 0.4 m L of urine, place it in a nuclear magnetic tube, add PBS (p H 7.0) 0.2 m L, then add D2O. Nuclear magnetic measurements were performed at 0.1 m L for 10 min. Samples were measured on a 600 MHz NMR (25 °C) instrument using a NOE-2D Spectroscopy (NOSEY) pulse sequence. The measurement frequency is 600.13 MHz, the number of scans is 64, the spectral width is 12345.7 Hz, the pulse time is 14 s, the sampling time is 2.654 s, the delay time is 1.0 s, the sampling data point is 65536, the FID resolution is 0.188 Hz, the sampling interval is 40.5 s, and the internal standard is TSP. 1H NMR Spectrogram Data Processing and Analysis Nuclear magnetic spectra were processed using Mest Re Nova (version 8.0.1, Mestrelab Research, Santiago de Compostella, Spain). Manual phase and baseline adjustment for all spectra. In the map, the chemical shift δ 0.00 of TSP is used as the standard for calibration, and the chemical shift interval δ0.00～9.40 is segment-integrated with δ 0.04, where δ 4.60～5.00 (residual water peak, urea peak) is not integrated. The integral data is normalized and imported into Excel software for the next step of multivariate statistical analysis. The integral data of the above processing was imported into SIMCA-P13.0 (Umetrics, Sweden) software for principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA) was used. And orthogonal partial least squares discriminant analysis (Orthogonal PLS-DA, OPLS-DA) was used to find differential metabolites between samples. Statistical data were processed by SPSS 17.0 software, expressed as x ± s. Statistical analysis and comparison of behavioral indicators and differential metabolites were performed by one-way analysis of variance and t test. P < 0.05, P < 0.01 was used as the difference. Significant. discuss This study replicated the aging model and caused cognitive dysfunction in rats with a significant decline in learning and memory. Urine is one of the main ways of exporting the end products of the whole body. The changes of metabolites in the urine can not only reflect the characteristics of the body's overall metabolism, but also the external manifestations of local tissue or organ dysfunction. In this study, 1H NMR analysis was used to detect metabolites in more than 20 kinds of urine, including metabolites such as fatty acids, amino acids and organic acids. This study conducted a pairwise comparison between the two groups. It was found that citric acid, lactic acid, pantothenic acid, trimethylamine, α-ketoglutaric acid, pyruvic acid, β-hydroxyisovalerate and β-hydroxybutyric acid were greatly affected by D-galactose. May be a potential biomarker for the D-galactose-induced aging model. After the intervention of Astragalus membranaceus, citric acid, lactic acid, pantothenic acid, pyruvic acid, trimethylamine and β-hydroxybutyric acid have different degrees of callback, and the highest dose effect of jaundice is most significant. Through analysis of potential biomarkers, it was found that jaundice delays aging mainly involving two metabolic pathways: tricarboxylic acid (TCA) cycle and pyruvate metabolism. The ATP produced by the TCA cycle is the main source of energy for tissue cells. The ATP produced in this process is produced via the respiratory chain. The intermediates of the TCA cycle in the model group are reduced in citric acid and α-ketoglutaric acid. , indicating that the body's aging also reduces the level of TCA energy metabolism, and the level of metabolites in it is also reduced accordingly. Studies have shown that if down-regulation of genes involved in energy metabolism leads to reduced energy production, damage to ATP-dependent downstream processes, such as synaptic structure and functional integrity, ubiquitin-dependent protein degradation pathways, both of which are in Alzheim Abused in AD (AD). Pyruvic acid is a tri-keto acid produced in the body and plays an important pivotal role in the metabolic linkage of the three major nutrients. Changes in pyruvate have an important role in the evaluation of inflammation in vivo. In the case of inflammation, even if the body preferentially converts pyruvate to lactic acid under aerobic conditions, this phenomenon is called aerobic glycolysis or Warburg effect. The change in pyruvate is closely related to glycolysis and TCA cycle. When D-galactose is stimulated, pyruvic acid is significantly increased due to disorders in glycolysis and TCA cycle. Astragalus can reduce the amount of pyruvic acid in the urine and restore glycolysis and TCA cycle metabolism. Both pyruvate metabolism and TCA cycle occur in the mitochondria, suggesting that jaundice may promote mitochondrial respiration through regulation of the metabolic pathway, protecting mitochondrial function, and ultimately associated with delaying aging. Mitochondria are widely distributed in various eukaryotic cells, and are sites for oxidative metabolism, and are places where carbohydrates, fats, and amino acids are metabolized to release energy. In addition to providing energy for cellular activities, mitochondria also participate in some important metabolic pathways, involved in biochemical functions such as apoptosis and signal transduction regulation. A large number of experimental studies have shown that mitochondria play a vital role in the aging process. The lactic acid content in the model group was increased compared to the blank group. Similar results were seen in dogs with severely cognitive impairment and early-onset AD, with lactic acid levels four times higher than in the normal group. Elevated levels of lactate not only indicate changes in gluconeogenesis, carbohydrates, and energy metabolism, but also indicate glucose metabolism damage in the brain, which is part of the cause of cognitive impairment. This result is consistent with the behavioral results. Trimethylamine is a degradation product of choline and carnitine under the action of intestinal bacterial enzymes. The metabolism of intestinal flora is related to aging-related diseases. The trimethylamine content in the model group increased, indicating that the metabolism of the intestinal flora may be disordered with age. The high dose of Astragalus membranaceus can reduce the content of trimethylamine, indicating that the anti-aging effect of Astragalus membranaceus is related to the regulation of the normal metabolism of intestinal flora. The results of this study showed that Astragalus membranaceus improved the memory impairment and self-release of aging rats. The mechanism of jaundice delaying aging may be related to the regulation of energy metabolism, glucose metabolism and intestinal metabolism. Beverage Sugar,Baking Sugar,High Fructose Corn Syrup,Fructose Sweetener Shandong Bailong Chuangyuan Bio-tech Co.,Ltd. Qingdao Branch , https://www.sdblcycn.com