Effect of electroacupuncture at pericardium meridian on D-ser and NMDAR in acute phase of MCAO rats

YUAN Liu-mei, XIA Yun, LU Xiao-ye, LIU Lei, ZHOU Ying, LOU Bi-dan

1.Hunan University of Traditional Chinese Medicine, Changsha 410007, China

2.The First Affiliated Hospital of Hunan University of Traditional Chinese Medicine, Changsha 410007, China

3.Guangdong Nanhua Vocational College of Industry and Commerce, Guangzhou 510507, China

4.Changsha Health Vocational College, Changsha 410100, China

Keywords:

ABSTRACT

1.Introduction

Acute ischemic stroke (AIS) is the most common type of stroke in China [1], and its pathogenesis and treatment methods have been the focus of research.Excitatory toxicity mediated by N-methyl-Daspartic acid receptor (NMDAR) is a hot topic in stroke research.D-serine (D-Ser), as the main endogenous ligand of the ' glycine site ' on the NR1 subunit of NMDAR, can regulate the function of NMDAR[2].Based on the theory of heart and brain governing spirit[3], the previous study of our research team[4-7]confirmed that electroacupuncture at pericardium meridian can improve the symptoms of neurological deficits in MCAO rats, the therapeutic mechanism is closely related to the regulation of D-Ser synthesis and release, regulating the expression of NR1, NR2A, NR2B, and affecting intracellular Ca2+content.On the basis of previous studies,this experiment further explored the effect of electroacupuncture on D-ser content and NMDAR expression in brain tissue of MCAO rats in the acute phase by adding exogenous D-Ser reagent.

2.Materials and methods

2.1 Experimental animals and grouping

Thirty-six SPF healthy adult male SD rats, weighing 250-300 g, 3-4 months old, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.They were randomly divided into the following 6 groups : normal group, sham operation group, model group, EA pericardium meridian group, model+D-Ser group, EA pericardium meridian+D-Ser group.Treat animals in strict accordance with animal ethics.

2.2 Main reagents and instruments

Conventional chemical reagents (Shanghai Sinopharm, China),skim milk powder (Beijing Pulilai, China, No.P1622), RIPA lysate (Shanghai Beyotime, China, No.P0013B), NR1 (Abyntek,No.A7677), NR2A (Proteintech, USA, No.19953-1-AP), NR2B(Proteintech, USA, No.21920-1-AP), β-actin (Proteintech, USA,No.66009-1-Ig).HRP goat anti-mouse IgG (Proteintech, USA,Item No.SA00001-1), HRP goat anti-rabbit IgG (Proteintech,USA, Item No.SA00001-2), Standard Serine (Sigma, USA, Item No.S0450000),Exogenous D-Ser reagents (MCE, Batch No.:HY-100808), formic acid (Sigma, Batch No.: F0507), acetonitrile(Sigma, Batch No.: 34851), shaker (Kylin-Bell, Jiangsu, China,Batch No.: TS-1), desktop refrigerated centrifuge (Xiangyi, Hunan,China, Batch No.: H1650R), electrophoresis apparatus (Beijing,China, Liuyi, Batch No.: DYY-6C), electrophoresis tank (Beijing,China, Liuyi, Batch No.: DYCZ-24DN), transfer membrane apparatus (Beijing, China, Liuyi, Batch No.: DYCZ-40D).Vortex mixer (Kylin-Bell, Jiangsu, China, Item No.: GL-88B), magnetic stirrer (Rex, China, Item No.: JB-13), precision PH meter (Rex,China, Item No.: PHS-3C), electronic balance (Shuangjie, USA,Item No.: JJ224BC), biological sample homogenizer (Aosheng,Hangzhou, China, Item No.: BioPrep-24), SDZ-II Huatuo electroacupuncture therapeutic apparatus (Suzhou Medical Appliance Factory Co., Ltd.), liquid quality instrument (Shimadzu,Item No.: LC-MS-MS-8050).

2.3 Model preparation

The rat middle cerebral artery occlusion model was selected.The preparation method was based on the Zea Longa[8]method, and the common carotid artery insertion method was used.The rats were awake after anesthesia and scored.The scoring criteria were based on Zea Longa 's 5-point neurobehavioral scoring criteria: those with a score of 1-3 were included in the experiment, and the rest were excluded.The sham operation group was not inserted after separation of blood vessels.

2.4 Intraperitoneal injection

EA pericardium meridian+D-Ser group and model+D-Ser group:The exogenous D-Ser reagent was injected intraperitoneally once at 160 mg/kg 30 min after the success of inserting the suture.Then according to the method of applying subject factors to be treated accordingly.

2.5 Electroacupuncture treatment

The rats were fixed by binding method.According to the animal acupoint map of‘experimental acupuncture and moxibustion’,the EA pericardium meridian group and the EA pericardium meridian+D-Ser group were treated with electroacupuncture once 24 h after successful modeling.Tianquan and Quze acupoints on the paralyzed side of the pericardium meridian were selected as one group, and Neiguan and Daling acupoints were selected as one group for electroacupuncture treatment.Select Huatuo brand electroacupuncture therapeutic apparatus, dilatational wave, output current of 0.5-1 mA, frequency of 15 Hz, intensity of local tissue tremor for the degree of treatment for 30 min.The remaining groups were only tied and fixed for 30 min without intervention.

2.6 Behavioral score of rats

The behavioral score of rats was evaluated according to the standard of Sun Jingfang 's 'animal experiment methodology'.The highest score was 11 points, which was evaluated twice at 6 h after successful modeling and before sampling.The higher the score,the more serious the motor dysfunction of rats, which was used to evaluate the damage and recovery of motor function after cerebral ischemia in rats.

2.7 Detection methods and indicators

2.7.1 Detection of D-Ser content in ischemic brain tissue by liquid chromatography tandem mass spectrometry

The sample was extracted and the standard substance solution was prepared.The standard substance mother solution was taken and water was added to prepare the standard substance solution with a concentration of 1, 2, 5, 10, 20, 50 and 100 ng/mL, respectively.The standard substance solution was filtered through a 0.22 μm filter and analyzed by HPLC-MS/MS; UPLC conditions : liquid chromatography model: LC30; chromatographic column: Waters ACQUITY UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm); mobile phase: A: 0.1% formic acid aqueous solution, column temperature: 35 ℃; B: acetonitrile, flow rate : 0.3 mL/min, injection volume:1 μL; mass spectrometry conditions: mode: ESI, positive ion mode analysis, scanning mode: multiple reaction monitoring (MRM), dry gas: nitrogen 10.0 L/min, heating gas: air 10.0 L/min, collision gas:argon 270 kPa, atomizing gas: 3.0 L/min, interface temperature:200 ℃, DL tube temperature: 280 ℃, dwell time: 150 ms, heating module temperature: 300 ℃, delay time: 3 ms, interface voltage:1.0 KV; the actual sample detection was carried out, and different treatment tissues were used to detect the content of serine in the tissues, and different contents were detected in the tissues.

2.7.2 Detection of NR1, NR2A and NR2B protein expression levels by Western Blot

Firstly, protein was extracted and about 0.025 g tissue samples were cut.After ice cracking, centrifugation and other treatments, the supernatant was denatured with loading buffer.SDS-PAGE gel was prepared.After the gel was solidified, 2 μL of marker was placed in the first hole, and 20 μL of denatured protein was placed in the remaining hole.Start electrophoresis, voltage constant 75 V, time 130 min ; according to the molecular weight were cut, prepared filter paper, NC membrane into the transfer buffer, according to the 'filter paper-NC membrane-glue-filter paper' order at 300 mA constant current transfer membrane; wash the membrane with 1×PBST once; 5% skim milk powder prepared with 1×PBST, immersed in the film, sealed at room temperature for 1.5 h ; the first antibody was diluted with 1×PBST according to a certain proportion, and the membrane was incubated with the first antibody overnight.The next day, it was placed at room temperature for 30 min, washed with 1×PBST for 10 min, and repeated 3 times; the HRP-labeled secondary antibody was diluted with 1×PBST, and the membrane was incubated with the secondary antibody for 1.5 h.The membrane was washed with 1 PBST for 15 min and repeated three times.Finally, the ECL color exposure, imaging in gel imaging system.

2.8 Statistical processing

All data were input into the computer and processed by SPSS 26.0 statistical software.The measurement data were expressed as mean ±standard deviation.When the data met the normal distribution and homogeneity of variance, the analysis of variance LSD or SNK method was used for comparison between multiple groups.When the normal distribution is satisfied and the homogeneity of variance is not satisfied, the Games-Howell method is adopted.When the data do not conform to normal distribution, non-parametric test is used;when the difference normal test was consistent, paired sample t test was used for intra-group comparison; otherwise, paired sample rank sum test is used.P＜0.05 was considered statistically significant.

3.Results

3.1 Behavioral scores of rats in each group

As shown in Table 1, (1) Before treatment : Compared with the normal group and the sham operation group, the behavioral scores of each group increased after modeling (P＜0.05), indicating that the modeling was successful ; compared with the model group, the behavioral scores of the model+D-Ser group and the EA pericardium meridian+D-Ser group were increased (P＜0.05), indicating that exogenous injection of D-Ser aggravated the behavioral disorders of rats.(2) After treatment: Compared with the model group, there was no significant difference in the EA pericardium meridian group (P＞0.05),the behavioral scores of the model+D-Ser group and the EA pericardium meridian+D-Ser group were still increased(P＜0.05);Compared with the model+D-Ser group, the behavioral score of the EA pericardium meridian+D-Ser group was not statistically significant (P＞0.05 ).(3) Intra-group comparison before and after treatment: Except for the normal group and the sham operation group, the behavioral scores of each group were slightly increased, and the difference was not statistically significant(P＞0.05).

Tab1 Comparison of behavioral scores of rats in each group (scores, n=6, )

Tab1 Comparison of behavioral scores of rats in each group (scores, n=6, )

Note: Compared with the normal group and the sham operation group,*P＜0.05; compared with the model group, #P＜0.05.

groups Before treatment After treatment normal group 0.000±0.000 0.000±0.000 sham operation group 0.000±0.000 0.000±0.000 model group 6.667±0.817*7.000±0.632*EA pericardium meridian group 6.500±1.049*7.500±1.225*model+D-Ser group 8.000±1.265*#8.333±0.817*#EA pericardium meridian+D-Ser group 8.500±0.837*#8.667±1.033*#

3.2 Comparison of D-Ser content in brain tissue of rats in each group

As shown in Table 2 and Figure 1, there was no significant difference between the normal group and the sham operation group(P＞0.05);compared with the normal group, the D-Ser content of each group increased to varying degrees after modeling (P＜0.05), and the model+D-Ser group increased most significantly; compared with the model group, the content of D-Ser in the model+D-Ser group was further increased (P＜0.05), while there was no statistical significance in the EA pericardium meridian group and the EA pericardium meridian+D-Ser group (P＞0.05);compared with the model+D-Ser group, the content of D-Ser in the EA pericardium meridian+D-Ser group decreased (P＜0.05).

Tab2 Comparison of D-Ser content in brain tissue of rats in each group ()

Tab2 Comparison of D-Ser content in brain tissue of rats in each group ()

Note: Compared with normal group, *P＜0.05; compared with the model group, #P＜0.05; compared with model+D-ser group, ^P＜0.05.

groups n D-Ser normal group 6 246.365±72.304 sham operation group 6 252.030±22.940 model group 6 496.540±11.553*EA pericardium meridian group 6 428.733±55.838*model+D-Ser group 6 645.430±61.863*#EA pericardium meridian+D-Ser group 6 539.454±30.896*^

Fig1 D-Ser content of each group

3.3 Comparison of NR1 protein expression in each group of rats

As shown in Table 3 and Figure 2, there was no significant difference in NR1 protein expression between the normal group and the sham operation group (P＞0.05); compared with the normal group, the protein expression of NR1 in each group was increased after modeling (P＜0.05), and the model+D-Ser group was the most significant; compared with the model group, the protein expression of NR1 in the EA pericardium meridian group was significantly decreased (P＜0.05), the protein expression of NR1 in the model+DSer group was relatively increased (P＜0.05), and the EA pericardium meridian+D-Ser group was not statistically significant (P＞0.05).Compared with the model+D-Ser group, the expression of NR1 protein in the EA pericardium meridian+D-Ser group decreased(P＜0.05).

Tab3 Comparison of NR1 protein expression of rats in each group ()

Tab3 Comparison of NR1 protein expression of rats in each group ()

Note: Compared with normal group, *P＜0.05; compared with the model group, #P＜0.05; compared with model+D-ser group, ^P＜0.05.

groups n NR1 normal group 6 0.173±0.008 sham operation group 6 0.185±0.030 model group 6 0.520±0.017*EA pericardium meridian group 6 0.272±0.012*#model+D-Ser group 6 0.612±0.018*#EA pericardium meridian+D-Ser group 6 0.502±0.035*^

Fig2 NR1 protein expression of rats in each group

3.4 Comparison of NR2A and NR2B protein expression in each group of rats

As shown in Table 4 and Figure 3, there was no significant difference in NR2A and NR2B protein expression between the normal group and the sham operation group (P＞0.05); compared with the normal group, the expression of NR2A and NR2B protein in each group increased after modeling (P＜0.05);compared with the model group, the expression of NR2A and NR2B protein in the EA pericardium meridian group and the EA pericardium meridian+D-Ser group decreased (P＜0.05), and the decrease in the EA pericardium meridian group was greater than that in the EA pericardium meridian+D-Ser group; the expression of NR2A and NR2B protein in the model+D-Ser group was further enhanced than that in the model group (P＜0.05).The protein expression of NR2A and NR2B in the EA pericardium meridian+D-Ser group was lower than that in the model+D-Ser group (P＜0.05).groups NR2A NR2B

Tab4 Comparison of NR2A and NR2B protein expression of rats in each group()

Tab4 Comparison of NR2A and NR2B protein expression of rats in each group()

Note: Compared with normal group, *P＜0.05; compared with the model group, #P＜0.05; compared with model+D-ser group, ^P＜0.05.

normal group 0.165±0.048 0.157±0.039 sham operation group 0.212±0.087 0.178±0.031 model group 0.447±0.031*0.437±0.038*EA pericardium meridian group 0.272±0.048*#0.232±0.038*#model+D-Ser group 0.577±0.008*#0.592±0.026*#EA pericardium meridian+D-Ser group 0.370±0.025*#^0.365±0.026*#^

Fig3 NR2A and NR2B protein expression of rats in each group

4.Discussion

The early onset of ischemic stroke is mainly due to wind, fire,phlegm, blood stasis on the brain collaterals, blocking the orifices[9].Chinese medicine believes that the heart governs the blood and vessels, and stores the spirit.Heart in the position of the viscera is very important, is regarded as 'official monarch '.The brain is formed by the convergence of the brain marrow, so it is called the'sea of the marrow', and it is also the place of the gods, so it is called the ' house of the gods '.The heart and brain jointly dominate and command people 's life activities and spiritual activities.Both are closely related to each other in physiology and pathology.When the physiological function of heart is normal, the heart-qi is abundant to promote the blood to the brain, so that the brain can be nourished.It can be seen that ' gods are hidden in the brain and occur in the heart', and the heart and brain are connected to each other.Some modern diseases can also reflect the pathological relationship between them,such as 'brain heart syndrome'.The pericardium meridian can assist in completing the work of the heart governing blood vessels and gods, and at the same time can ' replace the heart to receive evil '.Pericardial diseases are mainly caused by blood vessels.Vessel is connected with blood-qi, although it is not directly related to the brain, but acupuncture pericardium meridian could regulate the heart-qi, promote the running of blood, so that blood could supply to the brain, moisten brain orifices, so as to treat stroke and other brain-related diseases.Therefore, the pericardium meridian, heart and brain are closely related.Clinical studies[10] have confirmed that electroacupuncture at pericardium meridian has a definite effect on patients with cerebral infarction.

NMDAR is an important excitatory neurotransmitter in the central nervous system.Its excitation and activation are important targets in the mechanism of cerebral ischemia injury.When NMDAR is overactivated, the intracellular Ca2+level increases, resulting in excitatory toxicity and nerve damage, therefore, the activation of NMDAR has an important effect on the process of neuronal damage after cerebral ischemia[11].NMDAR is an important ionotropic glutamate receptor,but the activation of some ionotropic glutamate receptors (iGluRs) is not entirely dependent on glutamate, on the GluN1/GluN2 NMDAR,glycine or D-Ser acts synergistically with glutamate as a mandatory co-agonist to regulate the level of NMDAR activity[12].As an important neurotransmitter of glial cells, D-Ser regulates synaptic transmission and neuronal excitability by exciting NMDA receptors.Foreign studies[13] found that after 3 h of D-serine administration,the intracellular and extracellular concentration of D-Ser increased significantly, suggesting that exogenous D-Ser can affect D-Ser in brain tissue, thereby affecting the activity of NMDAR, but there are few related studies in China.

NMDAR is mainly composed of three major subunits : NR1, NR2(NR2A-2D ), NR3 (NR3A、NR3B).NR1 subunit is the basic functional subunit,and the affinity of NR2A and NR2B to glutamate is generally stronger than that of NR2C and NR2D, which is an important regulatory subunit[14].Previous studies[11,14]have shown that NR1 expression increases NMDA-induced excitatory toxicity; NR2B distributed outside the synapse, could cause sustained slow Ca2+influx, resulting in Ca2+overload, play a role in nerve damage ; in contrast, NR2A, which is distributed in the synapse,plays a neuroprotective role.The expression of different subunits of NMDAR is closely related to the period of cerebral ischemia.In this study, the expression of NR2A and NR2B at 1 d was consistent with the previous study[15]that the protein and gene expression of NR2A and NR2B were significantly enhanced during acute ischemia in MCAO rats, resulting in intracellular Ca2+overload and neuronal cell death.

The results of this experiment showed that the symptoms of neurological deficits occurred in rats after modeling.The content of D-Ser and the protein expression of NR1, NR2A and NR2B in ischemic brain tissue increased, and the two were positively correlated.After 1 day of electroacupuncture treatment, the behavioral scores of rats in each group were not improved.On the one hand, acute ischemic stroke is a dynamic pathological process,and the acute brain tissue is in the stage of ischemic brain edema,so the behavioral disorders of rats are still aggravated.On the other hand, due to the short time of electroacupuncture, the amount of stimulation is not enough, and the score is slightly higher, which may be related to the weakness of rats in the acute phase.Stimulating factors such as binding and grasping can also aggravate the overall behavioral disorder of rats.In the previous study of multiple time points, the team found that[6] the behavioral score of the electroacupuncture pericardium meridian group began to decrease gradually after the third day, indicating that electroacupuncture had a cumulative effect.At the same time, this experiment found that after adding exogenous D-Ser in the acute phase, the level of D-Ser in the brain tissue of rats could be increased by the supplement of exogenous D-Ser, which further enhanced the activity of NMDAR and aggravated nerve damage.However, the D-Ser content of the EA pericardium meridian group and the EA pericardium meridian+DSer group showed a downward trend, and the protein expression of NR1, NR2A, and NR2B was significantly reduced.It was confirmed once again that electroacupuncture pericardium can reduce the D-Ser content and reduce the protein expression levels of NR1, NR2A, and NR2B.In addition, compared with the expression levels of D-Ser and NMDAR at the same time, it was found that compared with the model group, the expression of NR1, NR2A and NR2B protein in the electroacupuncture pericardium group was significantly decreased at 1d (P＜0.05), and its down-regulation prior to D-Ser (P＞0.05).Considering that the synthesis and release of D-Ser involves multiple links and is closely related to SR, D-Ser is derived from L-Ser in vivo by SR racemization.Foreign studies have found that[16] in the cerebral cortex culture of SR-targeted missing mice, the level of D-Ser was reduced by about 85%, and the neurotoxicity was significantly reduced.At the same time, the team 's previous studies have shown that[5] electroacupuncture at pericardium meridian could down-regulate the expression of SR protein and inhibit the synthesis and release of D-Ser.Li-zhen WANG et al.[17] found that there was no significant change in the expression of SR in the first 4 days after focal cerebral ischemia, and the gene and protein levels of SR in the ischemic cortex showed a delayed decrease from 6 to 10 days after ischemia.At the same time, the concentration of D-Ser in the ipsilateral cortex also decreased, indicating that the expression of SR and the level of D-Ser in the temporal parietal cortex showed a delayed decrease in the late stage of focal cerebral ischemia.Combined with previous studies[7], it was shown that the content of D-Ser in the pericardium meridian group began to show a stable downward trend after 3 days, while the expression of NR1, NR2A and NR2B protein decreased significantly on 1-3 days, indicating that the effect of electroacupuncture on the pericardium meridian may have a certain delay in the down-regulation of D-Ser.It is speculated that NR1, NR2A and NR2B may be more sensitive to the regulation of electroacupuncture.

In summary, in vitro injection of exogenous D-Ser can aggravate excitatory toxicity in the acute phase.Electroacupuncture at pericardium meridian points can reduce the content of D-Ser in the brain tissue of MCAO rats in the acute phase, reduce the protein expression levels of NR1, NR2A and NR2B, inhibit the activity of NMDAR, and play a role in promoting nerve repair in MCAO rats, and the expression of NR1, NR2A and NR2B decreased more significantly than D-Ser.

Author’s Contribution:

Yuan Liu-mei, Lou Bi-dan: full participation in guiding the experimental operation and writing papers; Xia Yun, Lu Xiao-ye:feeding animals, materials, participate in paper writing; Liu Lei,Zhou Ying: involved in animal modeling and experimental operation.All authors declare no conflict of interest.