Plasma,metabolomics,study,of,Buyang,Huanwu,Decoction,improving,learning,andmemory,in,D-gal,model,mice,based,on,GC-MS

YU Ming-hui, XUE Ao, ZHAO De-ping, XU Yan-ming,2, XUE Hui, JIANG Jing, SUN Hui-feng✉ , ZHANG Ning,2✉

1. College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, China

2. College of Jiamusi, Heilongjiang University of Chinese Medicine, Jiamusi 154007, China

3. Heilongjiang Provincial Institute of Drug Inspection, the Research Institute for Biosafety Inspection, Harbin 150088, China

Keywords:Buyang Huanwu Decoction Alzheimer"s disease GC-MS Plasma metabolomics

ABSTRACT Objective: To study the protective effect of Buyang Huanwu Decoction on the learning and memory ability of D-gal induced aging mice using GC-MS metabolomics method. Methods:Twenty-four three-month-old kunming animals were selected as experimental samples and randomly divided into control group, model group and Buyang Huanwu Decoction according to their body weight. The memory level of experimental animals was detected by novel body recognition test, and the neuron structure of experimental animals was detected by HE staining. The plasma of each group of experimental animals was quantitatively analyzed by gas chromatography-mass spectrometry, and the important metabolites and main metabolic pathways in the process of pathological changes were traced by using plasma metabolism.Results: In HE staining, compared with blank group, hippocampal neurons in model group were disordered and morphologically abnormal. Compared with the model group, the hippocampal neurons in Buyang Huanwu Decoction group arranged regularly and had normal morphology. Compared with blank group, the index of new object recognition in model group was significantly decreased (P＜0.05); Compared with model group, the new object recognition index of Buyang Huanwu Decoction group was significantly increased (P＜0.05). Five different metabolites of AD were identified by GC-MS, which were L-pyroglutamate, lysine,pyrophosphoric acid, creatinine and α-lactose. Conclusion: Buyang Huanwu Decoction has a significant effect on D-gal model mice, and can effectively improve their learning and memory level. The mechanism may be related to glutathione metabolism and aminophyl biosynthesis.✉Corresponding author: SUN Hui-feng, Doctor, Professer; ZHANG Ning, Doctor,Professer.E-mail: huifengsun@hotmail.com; zhangning0454@163.com.

Alzheimer"s disease[1,2](AD) it is the most common cause of dementia in the elderly and a major health problem for elderly patients worldwide. At present, there are many hypotheses about the pathogenesis of AD, but there is no effective treatment. Studies have shown that the pathogenesis of AD is related to oxidative stress, and oxidative stress-induced apoptosis may be a potential factor leading to AD[3],Improving oxidative stress and apoptosis can alleviate nerve damage caused by AD[4]. Traditional Chinese medicine believes that the disease is located in the brain, closely related to the heart, liver, spleen and kidney. The disease is based on deficiency and standard solid. The basic pathogenesis is the reduction of essence deficit and marrow, deficiency of qi and blood, the heart does not contain god, god loses its position, and the appearance and spirit are separated from the development of dull syndrome[5]. Buyang Huanwu Decoction from the Qing Dynasty Wang Qingren"s "Medical Forest Correcting Mistakes", made of raw astragalus membranaceus, angelica tail, red peony root, chuanxiong,peach kernel, red flower, dragon, combined and used, will greatly replenish the vitality, blood circulation, collaterals, remove blood stasis, all diseases can be cured[6].It has been reported that Buyang Huanwu Decoction can play a neuroprotective role by maintaining the integrity of the blood-brain barrier[7],it can also inhibit brain inflammation and reduce neuronal apoptosis in AD model mice[8].Relevant studies have shown that Buyang Huanwu Decoction has a certain protective effect on brain neurovascular units in AD model mice[9], but the effect of D-gal on plasma metabolites and other aspects has not been reported.

Metabonomics combines advanced analytical technology platform with multivariate statistical analysis to scientifically explain the therapeutic effect and mechanism of TCM on diseases by analyzing the changes of metabolic profile of the body[10,11].GC-MS has the advantages of mature and stable technology, good selectivity,relatively perfect database, comprehensive information provided by one analysis and more accurate qualitative analysis in metabonomics research[12].In this study, D-gal model and GC-MS metabolomics were used to explore the changes and pathways of plasma metabolites after buyanghuwu Decoction treatment, providing a new scientific basis for clinical use.

2.1 Animal

Twenty-four 3-month-old Kunming mice, 12 male and 12 female,weighing (25±5) g, were selected and purchased from Liaoning Changsheng Biological Co., LTD., certificate Number: SCXK (Liao)2015-0001. The whole experiment process followed the experimental animal management and environmental protection related norms.

2.2 Source and dose of buyang huanwu decoction

Astragalus membranaceus, Angelica tail, red peony root,earthworm, Ligusticum chuanxiong, Safflower, and peach seed were mixed in the prescribed proportion (6:2:2:2:2:2:1), provided by jiamusi College of Heilongjiang University of Chinese Medicine.The dose was 18.2 g·kg-1·d-1, which was converted by raw dosage.

2.3 The main instrument

New object recognition (RD-1121-NR-M, Shanghai Shift Information Technology Co., LTD.); Paraffin embedding machine(SYD-B, Shenyang Yude); Rotary slicer (RM2016, LEICA,Germany); Electric thermostatic water bath (DK-2000-IIIL, Tianjin Tester Instrument Company); Gas chromatograph Agilent 6890N,mass spectrometer Agilent 5975B, elastic quartz capillary column(DB-5MS, Agilent).

2.4 Grouping and Administration

The experimental mice were randomly divided into 3 groups according to body weight. Control group and Model group were given double steam water by intragastric administration. Buyang Huanwu Decoction (BYHWT) group was given buyang Huanwu decoction liquid (18.2 g·kg-1·d-1) by gavage. Control group was injected subcutaneously with normal saline, and the other groups were injected subcutaneously with D-gal (125 mg·kg-1·d-1), which were modeled and given at the same time for consecutive 8 weeks.At week 8, animal behavior experiment was performed, and plasma samples were collected after the experiment.

2.5 New Object Recognition

Follow the literature[13],The mice were first put into an empty box to familiarize themselves with the environment, and then two identical small cubes were placed on both sides of the box for training. After 5 min of training, one of the small cubes was replaced by a small ball, and the contact time of the experimental mice to the small ball was recorded as Tn and Tf within 5 min. The recognition index = (Tn-Tf)/(Tn+Tf) was used to represent the object recognition ability of mice (reference). The recognition index indicated the learning and memory ability of the mice.

2.6 HE Dyeing

The sampling was fixed with 4% paraformaldehyde perfusion for 24 h, washed with slow water for one night, dehydrated with gradient ethanol and xylene and other reagents, and then soaked in melted wax. Then it was embedded, frozen, sliced 5-8 mm, dewaxed,dehydrated, and finally dyed. Dyeing solution I 5-7 min; Washed;Dyeing solution II, washing; Color enhancer Ⅲ. After dehydration and drying, the slices were sealed and the pathological changes of hippocampus were observed under microscope.

2.7 GC-MS Metabonomics

2.7.1 Sample processing

After the behavioral experiment, blood was collected from the eyeballs of each group and put into the heparinized EP tube for centrifugation and plasma separation. Add 300 μL methanol: acetone(1:1) to 100 μL plasma sample, swirl for 60 s, and incubate for 10 min. Centrifugal. Take 200 μL supernatant into 1.5 mL centrifuge tube, dry and derivate. 60 μL methoxamine hydrochloride was added,kept at 70 ℃ for 1h, then 60 μLMSTFA(containing 1%TMCS) was added, and incubated at 70 ℃ for 1 h. Finally, 60 μL 0.1 mg/mL n-heptane (containing 22ane) was added as internal standard, and the sample was injected by GC-MS.

2.7.2 Chromatographic conditions and mass spectrometry conditions

Plasma metabolomics analysis was performed on a 6890N-5975BGC-MS system using a DB-5MS column (30 m×0.25 mm,0.25 μm, agilent, MA, USA). The temperature program was initially set to 70 ℃ and held for 4 min, rising to 310 ℃ at 15 ℃/min and then held for 4 min. The auxiliary temperature and injector temperature are 280 ℃ and the injection volume is 1 μL (4:1 splitter ratio). The flow rate of helium carrier gas was set at 20 mL/min. MS scan range is between M/Z45-800. EI source temperature maintained at 230 ℃.

2.8 Statistical processing

The CDF format file was exported on the mass spectrometer, and converted to Abf format by ABF. Converter4.0.0. The comparison analysis, noise reduction, peak alignment, peak merging and normalization were performed on MSDIAL4.70 software, and the data were exported to Area. PCA analysis, OPLS-DA analysis,VIP Predictive and replacement tests were performed using online software metabolic analyzer (www.metaboanalyst.ca) and SIMCA14.1 software. Behavioral data were processed by GraphPad Prism5 statistical software. The data were expressed as mean ±standard deviation (±s), and P＜0.05 was statistically significant.

3.1 New object recognition experiment

The experimental results are shown in Table 1 and Figure 1.Compared with the blank group, the new object recognition index of the model group decreased (P＜0.05). The new object recognition index of Buyang Huanwu Decoction group was higher than that of model group (P＜0.05).

Tab 1 Identification index of new objects in each group(n=8,±s)

Tab 1 Identification index of new objects in each group(n=8,±s)

Note: Compared with Control group, *represented P＜0.05; Compared with the Model group, # represents P＜0.05.

Group Identify the index Control 0.333±0.129 Model 0.129±0.077*BYHWT 0.265±0.107#

Fig 1 Identification index of new objects in each group(n=8,±s )

3.2 Pathophysiological change

Results As shown in Figure 2, neurons in the hippocampus of mice in the blank group were neat, with a large number of neurons, clear nucleoli and no nuclear pyknosis. In the model group, the neurons in the hippocampus were disordered, some neurons were lost, and the nerve cells were shrunked, nucleoli were not obvious, and solid shrinkage was obvious. Compared with the model group, neurons in the hippocampus of Buyang Huanwu Decoction group were arranged more orderly, with more neurons, more normal cell shapes,clearer nucleoli, and no obvious nuclear pyknosis.

3.3 Superposition diagram of total ion flow in mouse plasma quality control samples

As shown in Figure 3 below, by analyzing the overlap phenomenonof the ion flow diagram of QC sample, it can be seen that all peaks are well distinguished and retain the same degree of time. On this basis,different metabolites in Control group, Model group and BYHWT group were selected by unidimensional statistical analysis and multivariate statistical analysis, and the next research was carried out.

Fig 2 Pathological morphology of mouse hippocampal neurons(HE stain,×40)

Fig 3 Superposition diagram of total ion flow in plasma tissue quality control samples from model mice

3.4 PCA score graph and system stability analysis

The PCA score chart (Figure 4) is shown below. PCA analysis of the Control group, Model group and BYHWT group showed that the separation phenomenon of loci difference between the Model group and Control group was particularly prominent, indicating that the plasma metabolites of D-Gal Model mice were significantly different. The difference between the BYHWT group and the Model group was significant, indicating that the endogenous metabolite levels changed significantly after the intervention of The TCM prescription Buyang Huanwu Decoction.

Fig 4 PCA score of Control group, Model group and BYHWT group

3.5 Discriminant Analysis of Orthogonal Partial Least Squares Method (OPLS-DA)

In order to further find the differential metabolites between each group, plasma samples of Control group, Model group, Model group and BYHWT group were analyzed by OPLS-DA analysis. The results were shown in Figure 5 and 6 below. The OPLS-DA score showed that the separation between Model group and Control group,and between BYHWT group and Model group was obvious and the degree of aggregation was good. The surface metabolites were significantly different between Model group and Control group,and between BYHWT group and Model group. In the OPLS-DA displacement diagram, R2X=0.602 and R2Y =0.868 were compared between the Model group and the Control group, and R2X=0.564 and R2Y=0.96 were compared between the BYHWT group and the Model group, indicating that the Model has a good fitting effect.The intercept of Q2on the Y-axis between the Model group and the Control group was -0.24, and the intercept of Q2on the Y-axis between the BYHWT group and the Model group was -0.539,indicating that the analysis Model was reliable and stable.

Fig 5 OPLS-DA of Model group and Control group and OPLS-DA permutation test chart

Fig 6 OPLS-DA of BYHWT group and Model group and OPLS-DA permutation test chart

3.6 Results of differential metabolite analysis

In this study, OPLS-DA was used to systematically analyze the plasma data of model group and blank group, Buyang Huanwu Decoction group and model group. VIP Predictive and t test were used to find the variables between each group and directly screen the metabolites between each group (VIP＞1.0, P＜0.05). As shown in Table 2, five differential metabolites were found under the above treatment methods, which were L-pyroglutamate, Lysine,Pyrophosphoric Acid, Creatinine and α-Lactose. Compared with blank group, creatinine, α -lactose and lysine contents in model group were significantly increased, while L-pyroglutamate and pyrophosphoric acid contents were significantly decreased; Compared with the model group, the contents of creatinine, α-lactose and lysine in Buyang Huanwu Decoction group were significantly reduced to normal level,while the contents of L-pyroglutamate and pyrophosphoric acid were increased, which were adjusted back to normal level.

Tab 2 Differential metabolite results (n=6)

3.7 Enrichment analysis of metabolic pathways

Metabolic pathway enrichment analysis of five different metabolites was performed using KEGG"s metabolite database to identify abnormal metabolic pathways affecting the metabolic network of Alzheimer"s disease mice. The results mainly include the metabolic pathways of glutathione, lysine degradation and aminoyl biosynthesis.

Due to the complexity and heterogeneity of Alzheimer"s disease,there are few biomarkers and therapeutic targets for Alzheimer"s disease currently in clinical application. In this study, we found that the quantity of several metabolites in plasma was affected by D-Gal induction, and these abnormalities were significantly improved by Buyang Huwu Decoction treatment.

L-pyroglutamate is commonly found in the brain and cerebrospinal fluid and helps improve memory[14], L-coke glutamate can inhibit the activities of ACHE, BCHE and Bacel enzymes, thus reducing the cholinergic nerve transmission, which plays an important role in the cognitive impairment of AD and has the effect of improving AD[15]. In this experiment, L-pyroglutamate content was increased to the normal level, indicating that Buyang Huanwu Decoction may improve learning and memory by inhibiting cholinergic neuron transmission. In addition, L-pyroglutamate is an intermediate of glutathione metabolism, and in some critically ill patients,blocking glutathione regeneration causes pyroglutamate acidosis[16].Glutathione[17](GSH) it is the main antioxidant defense molecule in the brain. It is a tripeptide of glutamic acid, cysteine, and glycine, and acts as an antioxidant by providing a reduction of reactive oxygen species[18]. This reaction can occur either through non-enzymatic[19]action or through the catalysis of glutathione peroxidase. In vitro and animal studies have shown that glutathione loss plays an important role in neuron death and involvement in neuron loss in some neurodegenerative diseases such as AD[20],making it a potential therapeutic target to prevent or reduce neurodegeneration.Lysine acetylation is widely present in all organisms and plays an important role in regulating protein synthesis. It is involved in multiple metabolic pathways such as glycolysis and citric acid cycle, and regulates cytoplasmic and mitochondrial functions[21].Studies have shown that mitochondrial dynamics defects can cause AD, Parkinson"s disease (PD), amyotrophic lateral sclerosis (ALS),huntington"s disease (HD) and other pathologies, including inhibiting metabolic state, reducing ATP production, and enhancing oxidative stress[22].Aminoacyl-t RNA synthetase (aaRS) exists widely in cells and catalyzes the esterification reaction between amino acids and corresponding tRNA to generate aminoacyl-tRNA, which provides raw materials for protein synthesis and ensures the accurate transmission of genetic information[23]. Aminoacyl-tRNA synthase mutations cause a variety of neurodegenerative diseases[24], in this experiment, lysine was down-regulated to normal level and KEGG obtained metabolic pathway, suggesting that Buyang Huanwu Decoction may improve AD by promoting lysine acetylation and regulating mitochondrial function and energy metabolism.

In conclusion, Buyang Huanwu Decoction has a good therapeutic effect on D-gal model mice, and its mechanism may be to realize the protective effect on neuron cells by improving oxidative stress,mitochondrial function and energy metabolism. Therefore, we will continue to explore the neurobiological mechanism of Buyang Huanwu Decoction in improving AD, providing new ideas for the development of safe and effective new drugs for the treatment of AD.

Author’s contribution:

Experimental design for the corresponding author Zhang ning,Angus, animal Yu Ming-hui building experimental implementation as the first author, behavioral science experiment teaching for the fourth author Xu Yan-ming, Yu Ming-hui implementation as the first author, pathological dyeing experiment teaching for the third author de-ping zhao, Yu Ming-hui implementation as the first author, GC -MS experiment teaching for the fifth author Xue Hui Jiang Jing and sixth author, The first author is Yu Ming-hui, the second author is Xue Ao and the corresponding author is Zhang Ning. The article was written by Yu Ming-hui.

All author’s declare no conflict of interest.