Effect of acupuncture at Renying(ST 9)on gene expression profile of hypothalamus in spontaneously hypertensive rats

INTRODUCTION

2 Pierdomenico SD,Di Nicola M,Esposito AL,et al.Prognostic value of different indices of blood pressure variability in hypertensive patients.Am J Hypertens 2009;22(8):842-847.

Acupuncture has been accepted as an ancient curative modality in Oriental Medicine and has been shown to exhibit clinical therapeutic effects on hypertension.6Furthermore,whereas the long term use of drug therapy may produce a multiplicity of side effects or even drug resistance,which negatively impact therapeutic efficacy,acupuncture,on the other hand,is associated with a very low(0.13%)incidence of side effects,7high safety,and optimal effects.In our previous research,we utilized the twirling-rotating needling technique of acupuncture,which has been shown to demonstrated a significant anti-hypertensive effect and,we found,was able to reduce the both the systolic and diastolic pressure in spontaneously hypertensive(SH)rats.8Many additional studies have also provided various explanations of the antihypertensive mechanism underlying the Renying(ST 9)acupuncture effect,9,10including the potential decrease in plasma renin,aldosterone,and angiotensinⅡactivity,or potential increase in the excretion of sodium in addition to changes in plasma norepinephrine and endorphin levels.11

Taken together,we employed gene chip technique and observed how acupuncture on Renying(ST 9)influences the expressions of genes,and used the twirling-rotating needling technique of acupuncture which has a significant anti-hypertensive effect.Our results suggest that acupuncture at Renying(ST 9)could significantly lower blood pressure of SH rats,and the data from DNA microarray analyses clearly showed between-group differences in up-and down-regulated genes expression.It is anticipated that understanding the the mechanisms of acupuncture at Renying(ST 9)will provide accurate and efficient strategies for prevention,diagnosis and control.Nevertheless,further studies are needed to confirm our results.

MATERIALS AND METHODS

Ethics statement

All procedures for animal experiments were conducted in accordance with the World Health Organization International Guiding Principles for Biomedical Research Involving Animals,15and were approved by the Animal Care and Use Committee at Beijing University of Chinese Medicine.

Animals preparation

A total of 18 specified pathogen free(SPF)9-week-old male SH rats weighing 180-200 g and 9 body weight-matched specified pathogen free SPF male WKY rats were obtained from Beijing Vital River Laboratory Animal Technology Co.,Ltd.,License number:SCXK(Beijing)200223(Beijing,China).Experimental animals were raised in clean cabinets and were provided free access to water and food.A controlled environment with a temperature of(20±1)℃,humidity of 50%,and 12-h light-dark cycle was maintained throughout the whole study.SH rats were obtained from outbred WKY males with marked elevation of blood pressure(BP)mated to females with slightly elevated BP.

Grouping

The 18 SH rats(BP≧140 mm Hg)were randomly divided into 2 groups by random number table:Renying(ST 9)group(n=9)with acupuncture treatment at Renying(ST 9),and model control group(n=9)without treatment.Body weight-matched WKY rats with normal BP were used as blank control group(n=9).Single blind method was used in the experiment,and the experiment operator knew the grouping and intervention.The experimental protocols are shown in Figure 1.

Intervention

All rats in the three groups were loosely immobilized in a specially made restrainer with all four limbs exposed.Renying(ST 9)is located in the anterior region of the neck,at the same level as the superior border of the thyroid cartilage,anterior to the sternocleidomastoid muscle,and over the common carotid artery.16

Figure 1 Schematic diagram for methodologies

WKY:Wistar-Kyoto;SH:spontaneously hypertensive;KEGG:Kyoto Encyclopedia of Genes and Genomes;qRT-PCR:quantitative reverse transcription-polymerase chain reaction.

For treatment of the Renying(ST 9)group,the needles(0.16 mm×7 mm,purchased from Suzhou Acupuncture Goods Co.,Ltd.,Suzhou,China)were directly inserted into Renying(ST 9)bilaterally to approximately 4-5 mm.After the insertion,the reducing twirling17(rotate the needle counter clockwise for 360°and then clockwise for 360°,with the thumb moves backward heavily and forward gently,60 times per min)was applied for 1 min;the needle was then retained for 19 min after manipulation.The acupuncture intervention was performed once a day for 28 d.All intervention was provided by the same individual.Rats in the blank control and model control groups were immobilized in the same restrainer for 20 min without acupuncture intervention.

Measurement of blood pressure

Following acupuncture,the systolic pressure of the caudal artery was measured in a controlled environment at a temperature of(22±2)℃.Sober rats were preheated at 36℃for 15 min and then the systolic pressure was measured using a noninvasive blood pressure instrument from Chengdu TME Technology Co.,Ltd.,(Chengdu,China)when the rats were quiet and conscious.Each rat was measured 3 times and the average BP was recorded.During the experiments,all animals were handled with extreme caution to minimize stress to the rats.BP was recorded at 1,5,9,13,17,21,25,and 28 d after acupuncture.

Sample collection and RNA extraction

The hippocampi were rapidly dissected 2 h after the measurement of caudal artery BP on the 28th day following treatment,frozen in liquid nitrogen,and stored at－80℃until used.Total RNA was extracted using RNeasy mini kit(Qiagen GmbH,Hilden,Germany)according to the manufacturer's instructions.Total RNA was estimated and quantitated by spectrophotometry using a NanoDropND-1000(Thermo Scientific,Waltham,MA,USA)and the integrity was assessed via formaldehyde-denatured agarose gel electrophoresis using an Agilent 2100 bio-analysis system(Agilent Technologies,Santa Clara,CA,USA).

Microarray image analysis

Microarray analysis was performed and each sample repeated 3 times in each group with the Gene Chip Rat Gene 2.0 ST Array(Affymetrix,Santa Clara,CA,USA),which contains 610 400 distinct probes corresponding to 16 771 well-annotated genes.Total RNA from each sample was used for labeling and array hybridization as follows:(a)preparation of the Poly-A RNA Controls;(b)first-strand cDNA synthesis;(c)sond-strand cDNA synthesis;(d)cRNA synthesis byin vitrotranscription;(e)2nd-cycle cDNA synthesis;(f)RNase H hydrolysis;(g)single-stranded cDNA fragmentation and labeling;(h)array hybridization using the Affymetrix Gene Chip 645 System followed by washing with the Affymetrix Gene Chip 450 System;and(i)array scanning using the Affymetrix Gene Chip 7G microarray scanner.The labeling and hybridization steps were carried out according to manufacturer protocol(Gene Chip WT PLUS Reagent Kit,P/N 902280,Affymetrix,Santa Clara,CA,USA).

Microarry data analysis

Scanned images(CEL format)were then imported into Affymetrix Expression console software(Affymetrix,Santa Clara,CA,USA)for grid alignment and expression data analysis.Expression data were normalized through quantile normalization using the Robust Mul-tichip Average(RMA+SKETCH)algorithm included in the Affymetrix Expression console software.CHP files(Affymetrix,Santa Clara,CA,USA)were generated after normalization.The obtained 30 gene expression level files were imported into the Transcriptome Analysis console for further analysis.Gene expression profiles of the model control group were compared to those from the blank control or Renying(ST 9)group.Genes exhibiting altered expression were selected according to the criteria:P＜0.05and fold changes≥1.5.

Gene functional annotation

The Database for Annotation,Visualization,and Integrated Discovery(DAVID)v6.7 functional clustering tool(http://david.abcc.ncifcrf.gov/)(Leidos Biomedical Research,Inc.,Frederick,MD,USA)was used to identify over-represented ontology groups among the gene expression profiles and to group differentially expressed genes(DEGs)into functional categories.The biological process,cellular component,and molecular functions were analyzed simultaneously.DEGs of the model control group(vsblank control group)or Renying(ST 9)group(vsmodel control group)datasets were selected.Gene Ontology Biological Process was selected as the functional annotation category for this analysis.

Kyoto encyclopedia of genes and genomes(KEGG)pathway analysis

The DEGs were next categorized more specifically according to KEGG biological pathways.We used Fisher's exact test to select the significant pathway,and the threshold of significance was defined by thePvalue and false discovery rate.Biological pathway analysis suggested related pathways that were significantly modulated among Renying(ST 9)treatedvsSH groups.

Validation of selected DEGs using quantitative reverse transcription-polymerase chain reaction(qRT-PCR)analysis

Total RNA was separately extracted from the 30 individual samples using the RNeasy mini kit in accordance with the manufacturer's instructions.The first-strand cDNAs were synthesized from the mRNA of each of the three groups using a reverse transcription kit(transcriptor First Strand cDNA Synthesis Kit,Life Technologies,New York City,NY,USA).The reaction was performed using the program:25℃for 10 min,55℃for 30 min,85℃for 5 min,and a hold at 4℃.The real-time PCR reactions were carried out using a 7900HT Real-Time PCR instrument(Applied Biosystems,Foster city,CA,USA).The amplification reaction(20 μL mixture)contained 2 μL cDNA template,10 μL Power SYBR Green PCR Master Mix(P/N 4367659,Applied Biosystems,Foster city,CA,USA),3 μL primers,and 5 μL water.RT-PCR reactions were carried out as follows:1 cycle of 10 min at 95℃,40 cycles of 15 s at 95℃,and 1 min at 60℃.After cycling,a melting protocol was performed of 15 s at 95℃,15 s at 60℃,and 15 s at 95℃.Every sample was tested three times.After the end of the experiment,RQ Manager1.2.1(Applied Biosystems,Foster city,CA,USA)and Data Assist V3.0 software(Applied Biosystems,Foster city,CA,USA)were used for the calculation of Ct value.Relative expression of the RT-PCR product was calculated using the comparative 2－ ΔΔCtmethod.All data were analyzed using SAS 9.0 statistical software(SAS Institute Inc.,Cary,NC,USA).Table 1 lists the sequences of the primers employed for RT-PCR and their anticipated PCR product size.The endogenous control Gapdh was used for normalization.The differences in the gene expression level between the model control group and the Renying(ST 9)group were compared usinga studentt-test;a probability ofP＜0.05 was considered to be statistically significant.

Table 1 Sequences of primers employed for RT-PCR and their anticipated PCR product size

Notes:RT-PCR:reverse transcription-polymerase chain reaction;abbr.:abbreviation.

Gene abbr.GADPH Ephx2 Chi3l1 5-HT1A Klk1 Cbs Primer Front Rear Front Rear Front Rear Front Rear Front Rear Front Rear Length(bp)217 124 120 152 159 Oligonucleotide sequences5'-3'5'-CAGGGCTGCCTTCTCTTGTG-3'5'-ACCAGCATCACCCCATTTGA-3'5'-TTCGACCTTGACGGAGTGC-3'5'-GCGCCAAGTAGGAAGTCTCT-3'5'-AGGTCACAGGAAGCATTTGG-3'5'-TGAAGGGTCATAGGGGTGGT-3'5'-TGATCTCGCTCACTTGGCTC-3'5'-CGGGATATAGAAAGCGCCGA-3'5'-GCCAGTCTCGGGTTATTGGA-3'5'-CGCAGAGGTATTTCGTGAAGC-3'5'-AAGCTGAAGGAGAAGTGCCC-3'5'-CGGTGGGGATGAAGTCGTAG-3'136

RESULTS

Blood pressure changes following acupuncture

BP measurements taken on 1,5,9,13,17,21,25,and 28 d in each group are shown in Figure 2.By the 28th day after treatment the systolic BP in the model control group had increased from(168.7±2.9)to(186.6±3.4)mm Hg,showing a steady trend over the 1,5,9,13,17,21,25,and 28 d following experiment initiation to reach a significantly higher level(P＜0.01)than the same-aged WKY rats[(124.9±2.3)mm Hg].In contrast,compared with the modelcontrol group,systolic BP in the Renying(ST 9)group markedly decreased at the 9th d(P＜0.05)and the 13th,17th,21st,25th,and 28th d(P＜0.01)during treatment,indicating that acupuncture at Renying(ST 9)could significantly lower the blood pressure of SH rats(Figure 2).

Microarry data analysis

A total of 139 genes were found to be regulated during the process of EH and identified as DEGs.Of these,91 genes were down-regulated and 48 were up-regulated(P＜0.05;fold change≥1.5)between the model and blank control groups.A total of 159 DEGs were also found to be regulated during the process of Renying(ST 9)intervention,among which 80 were down-regulated and 79 were up-regulated(P＜0.05;fold change≥1.5)between the Renying(ST 9)and model control groups.

To further look into the differences in gene expression in these groups,we analyzed overlaps of the genes with Venn Diagrams(Figure 3).Among the up-regulated genes in the model control group,28 were down-regulated in the Renying(ST 9)group(Figure 3A).The epoxide hydrolase 2,cytoplasmic(Ephx2),chitinase3-like 1(Chi3l1),Acsm3,Tstd1,MGC108823,Naip5,RGD1310641,Wdr46,Pus71,and Znrd1as1 genes exhibited significant up-regulation in the model control group but were down-regulated in the Renying(ST 9)group(Table 2).Among the down-regulated genes in the model control group,42 were up-regulated in the Renying(ST 9)group(Figure 3B).5-hydroxytryptamine 1A receptor(5-HT1A),kallikrein 1(Klk1),cystathionine beta-synthase(Cbs),Ifitl,Cd4,Pnpla1,Zcchc9,Ppp2r4,Tnnt1,and Slc11a1 exhibited significant down-regulation in the model control group but were up-regulated in the Renying(ST 9)group(Table 3).

Gene functional annotation

Figure 2 Effects of Renying(ST 9)on systolic blood pressure of SH rats

Blank control group:WKY rats with normal BP(n=9),Model control group:without treatment(n=9),Renying(ST 9)group:with acupuncture treatment at Renying(ST 9)(n=9).SH:spontaneously hypertensive.aP＜0.01,versus blank control group;bP＜0.05,cP＜0.01,versus model control group.n=9 animals per group.

GO functional annotation was next used to determine whether the gene expression changes associated with acupuncture treatment were related to different biologi cal processes that may contribute to the development of hypertension in the SH rats.We used the DAVID 6.7b module from DAVID Bioinformatics Resources(http://david.abcc.ncifcrf.gov/)to perform GO analysis.The DEGs could be clustered in different biological process,molecular function,and cellular component categories and the frequency(number)of genes involved in response to stimulus,cellular process,binding,organnelle,cell part,receptor activity,metabolic process and catalytic activity in SH was identified(Figure 4).

Figure 3 Venn diagram of differential expressed genes in the model control group(vsthe blank control group)and the Renying(ST 9)group(vsthe model control group)

A:28 overlapping genes up-regulated in the MvsK group and down-regulated in the RvsM group;B:42 overlapping genes down-regulated in the M vs K group and up-regulated in the RvsM group.MvsK:the model control group compares with the blank control group;RvsM:the Renying(ST 9)group compares with the model control group.Purple circles indicate the numbers of genes up-or down-regulated in the MvsK group(left);green and blue circles represent the numbers of down-or up-regulated genes in the RvsM group(right).

KEGG pathway analysis

Further functional pathway analysis showed that compared with the blank control group,DEGs in the model control group were involved in 26 KEGG pathways(P＜0.05)(Table 4).Furthermore,compared with the model control group,DEGs in the Renying(ST 9)group were mainly associated with 19 KEGG pathways(Table 5).

Validation of selected DEGs using qRT-PCR analysis

To validate the microarray results,representative genes were selected and their expression levels in the hypothalamus were evaluated by real-time qPCR(Figure 5).These genes were selected based on fold change differences,previous association with BP regulation,and/or involvement in processes or pathways that may influence BP.The expression levels of Chi3l1(P＜0.01)and Ephx2(P＜0.01),were significantly higher in the model control group compared to the blank control group,whereas they were significantly lower in the Renying(ST 9)group when compared to the model control group(P＜0.01 for each).The expression levels of Klk1(P＜0.01),Cbs(P＜0.01),and 5-HT1A(P＜0.01)were significantly lower in the model control group compared to the blank control group,whereas they were also significantly increased in the Renying(ST 9)group when compared to the model control group(P＜0.01,P＜0.01,andP＜0.05,respectively).These findings demonstrated that the regulation patterns determined by RT-PCR correlated with those determined using array analysis.

Table 2 Genes up-regulated in model control group(compared with blank control group)but down-regulated in Renying(ST 9)group(compared with model control group)

Notes:FC1:gene expression level in model control group/gene expression level in blank control group;FC2:gene expression level in Renying(ST 9)group/gene expression level in model control group.Positive values indicate higher expression in model samples,while negative values indicate lower expression in Renying(ST 9)samples.

Pvalue 0.001 560 0.000 052 0.000 051 0.000 469 0.059 352 0.003 731 0.003 452 0.001 369 0.005 589 0.008 218 0.003 259 0.004 353 0.012 170 0.000 638 Gene abbreviation Ephx2 Chi3l1 Acsm3 Tstd1 MGC108823 Naip5 Agmo RGD1310641 Wdr46 Kcnip3 Pus71 Znrd1as1 RT1-CE5 Retsat FC1 1.83 3.07 4.25 2.68 2.26 2.53 1.81 1.67 2.07 1.75 2.04 2.21 1.55 1.65Pvalue 0.002 049 0.000 578 0.000 789 0.000 785 0.039 058 0.035 820 0.000 978 0.000 149 0.003 555 0.004 926 0.000 127 0.005 369 0.047 569 0.004 488 FC2－1.81－2.91－4.3－2.73－2.64－2.38－1.79－2.18－2.13－1.65－2.04－1.97－1.95－1.93

Table 3 Genes down-regulated in model control group(compared with blank control group)but up-regulated in Renying(ST 9)group(compared with model control group)

Notes:FC1:gene expression level in model control group/gene expression level in blank control group;FC2:gene expression level in Renying(ST 9)group/gene expression level in model control group.Positive values indicate higher expression in model samples,while negative values indicate lower expression in Renying(ST 9)samples.

Gene abbreviation 5-HT1A Klk1 Cbs Ifitl Cd4 Pnpla1 Zcchc9 Ppp2r4 Tnnt1 Slc11a1 Apold1 Setd4 Creg1 Cbr1 Vom2r57 Ooep FC1－1.55－1.66－1.53－2.98－2.78－2.32－2.56－2.4－2.28－2.47－1.83－1.81－2.06－14.74－1.69－1.74Pvalue 0.010 598 0.004 164 0.001 249 0.025 860 0.010 701 0.003 224 0.002 598 0.000 073 0.000 427 0.000 423 0.001 519 0.004 867 0.000 757 0.000 002 0.007 212 0.002 004 FC2 1.54 1.9 1.61 2.82 2.77 2.72 2.55 2.49 2.46 2.17 2.08 2.00 1.93 14.53 1.92 1.88Pvalue 0.011 944 0.001 888 0.000 733 0.002 877 0.009 877 0.003 110 0.001 036 0.000 094 0.000 063 0.001 104 0.009 646 0.003 162 0.000 840 0.000 004 0.000 929 0.000 489

DISCUSSION

Hypertension represents an important public health issue worldwide because of its high prevalence and concomitant effect on increased disease risk.18A number of animal and clinical studies have reported the efficacy of acupuncture in reducing hypertension6,19-21and studies have indicated that the involvement of nitric oxide(NO),22neurotransmitters,23aldosterone,endothelin,and angiotensinⅡ24in the brain all appear to contribute to the antihypertensive effects of acupuncture.25,26However,each of these studies evaluated only a single factor.In comparison,microarray analysis enables the parallel analysis of the expression of a very large number of genes encompassing a substantial fraction of the entire genome.Therefore,we conducted a microarray experiment to assay the effects of acupuncture on SH rats.Notably,in our study we found specific and significant alterations in the expression profile of acupuncture in SH rats.These findings support the premise that the regulation of transcription by acupuncture on Renying(ST 9)represents a key for understanding the efficacy of this treatment.

萍萍一直站在门口，那门也一直没有关上，抓住门框的手现在还抓着，她这样的姿态像是在等着我立刻离开似的，我就说：“你是不是要我马上就走？”

In the present study,SH rats have been used as a genetic model for essential hypertension because it exhibits specific and uniform genetic predisposition,allowing the study of causal factors,mechanisms,and pathologies of hypertension as well as its behavioral consequences.This model has also been used for comparing the efficacy of proposed therapeutic interventions for the development of clinical treatments.27Our results showed that,compared with the blank control group,rats in the model control group developed significantly increased BP.Furthermore,compared with the model control group,we identified a total of 139 genes demonstrating significantly altered expression in the Renying(ST 9)group,among which 91 showed decreased expression whereas 48 exhibited increased expression.

The pathogenesis of EH is believed to result from imbalances and disturbances in the flow ofQiand blood,YinandYangof the viscera.The imbalance leads to complex pathological changes of wind,fire,phlegm,stasis,and deficiency.For Renying(ST 9),a major acupoint,regulatingQiand blood,eliminating wind through activating blood circulation,and regulating the function of liver and spleen are considered to represent the treatment principles.Renying(ST 9)is located near the internal carotid sinus,under the outer membrane of the carotid sinus blood vessels,a tissue that contains the pressure sensors.Notably,the arterial baroreceptor reflex acts as one of the main mechanism of the central nervous system for regulating peripheral cardiovascular function and plays an important role in maintaining the stability of the body's arterial pressure.Our study showed that,after acupuncture treatment,the BP of the Renying(ST 9)group decreased significantly from the ninth day compared with that of the model control group,suggesting that acupuncture could reduce the BP of SH.Next,to identify genes re-sponsive to this treatment that may indicate the mechanism of action of acupuncture treatment,a comparison of gene expression in the hypothalami of the model group with the Renying group was performed.Our results showed that approximately 159 genes were differentially expressed following acupuncture treatment,among which we identified that 79 were upregulated and 80 were downregulated.This indicated that acupuncture stimulation at Renying(ST 9)could alter the transcriptional activity of the hypothalamus in SH rats.We further identified 19 pathways as being potentially regulated by acupuncture treatment using KEGG pathway analysis.KEGG pathway selection provides a platform for integrating and elucidating useful data.For example,such pathway matching indicated that the mitogen activated protein kinase(MAPK)signaling pathway was possibly involved in the antihypertensive activity of acupuncture in rats.MAPK signal transduction pathways are involved in the control of diverse cellular events including cell proliferation,differentiation,and apoptosis.28MAPKs have been implicated in cardiomyocyte and vascular smooth muscle cell(VSMC)proliferation,hypertrophy,and differentiation,which represent key responses in the pathology of cardiovascular diseases such as hypertension,congestive heart failure,left ventricular hypertrophy,and atherosclerosis.29Thus,our data suggested that acupuncture might regulate BP through the MAPK signaling pathway,which is consistent with the results of previous studies by ourselves and others,showing that the MAPK signaling pathway was activated by acupuncture in the brain.30Further functional studies are therefore required to dissect the role of the MAPK signaling pathway in the therapeutic effects of acupuncture.

Figure 4 Gene ontology(GO)terms according to biological processes,molecular functions and cellular components

A-C:model control group versus blank control group.A:cellular components;B:biological processes;C:molecular functions.D-F:Renying(ST 9)group versus model control group.D:cellular components;E:biological processes;F:molecular functions.Blank control group:WKY rats with normal BP(n=9),model control group:without treatment(n=9),Renying(ST 9)group:with acupuncture treatment at Renying(ST 9)(n=9).GO terms are ordered by the number of transcripts calculatedP-values and fold-change are shown.Differentially expressed transcripts involved in the term(count)withP＜0.05 and fold-change＞1.5 were included.

Table 4 KEGG pathways of altered genes in model control group compared with blank control group

Note:KEGG:Kyoto Encyclopedia of Genes and Genomes.

KEGG pathway rno00590:arachidonic acid metabolism rno04612:antigen processing and presentation rno04514:cell adhesion molecules Rno00260:glycine,serine and threonine metabolism rno05332:graft-versus-host disease rno05330:allograft rejection rno00830:retinol metabolism rno04940:type I diabetes mellitus rno05320:autoimmune thyroid disease rno05416:viral myocarditis rno00650:butanoate metabolism rno00564:glycerophospholipid metabolism rno04144:endocytosis rno04620:Toll-like receptor signaling pathway rno04670:leukocyte transendothelial migration rno04740:olfactory transduction rno04080:neuroactive ligand-receptor interaction Gene CBR1,CYP4F4,PLA2G2A,Ephx2,ALOX5 RT1-CE1,RT1-CE4,RT1 CE5,CD4,WDR46 RT1-CE1,RT1-CE4,RT1-CE5,CD274,CD4,CLDN10 PSAT1,GLDC,Cbs RT1-CE1,RT1-CE4,RT1-CE5 RT1-CE1,RT1-CE4,RT1-CE5 ALDH1A1,RETSAT,UGT2B RT1-CE1,RT1-CE4,RT1-CE5 RT1-CE1,RT1-CE4,RT1-CE5 RT1-CE1,RT1-CE4,RT1-CE5 ACSM3,HMGCS2 GPD1,PLA2G2A RT1-CE1,RT1-CE4,RT1-CE5 TLR7,TLR8 CYBB,CLDN10 OLR513,OLR1673,OLR1467,OLR1686,OLR1378,OLR1733 HTR1A,PRSS1

Table 5 KEGG pathways of altered genes in Renying(ST 9)group compared with model control group

Note:KEGG:Kyoto Encyclopedia of Genes and Genomes.

KEGG pathway rno04612:antigen processing and presentation rno05332:graft-versus-host disease rno05330:allograft rejection rno04940:type I diabetes mellitus rno05320:autoimmune thyroid disease rno04514:cell adhesion molecules(CAMs)rno05416:viral myocarditis rno04144:endocytosis rno00260:glycine,serine and threonine metabolism rno00830:retinol metabolism rno04920:adipocytokine signaling pathway rno00590:arachidonic acid metabolism rno04640:hematopoietic cell lineage rno04660:T cell receptor signaling pathway rno04080:neuroactive ligand-receptor interaction rno00230:purine metabolism rno04810:regulation of actin cytoskeleton rno04740:olfactory transduction rno04010:MAPK signaling pathway Gene RT1-CE16,RT1-CE4,RT1-CE5,CD4,WDR46,RT1-N2 RT1-CE16,RT1-CE4,RT1-CE5,RT1-N2 RT1-CE16,RT1-CE4,RT1-CE5,RT1-N2 RT1-CE16,RT1-CE4,RT1-CE5,RT1-N2 RT1-CE16,RT1-CE4,RT1-CE5,RT1-N2 RT1-CE16,RT1-CE4,RT1-CE5,CD4,RT1-N2 RT1-CE16,RT1-CE4,RT1-CE5,RT1-N2 RT1-CE16,LOC684122,RT1-CE4,RT1-CE5,RT1-N2 PSAT1,Cbs ALDH1A1,RETSAT CD36,POMC CBR1,Ephx2 CD36,CD4 RHOC,CD4 HTR1A,PTGDRL,NMUR2 POLR3K,ATIC RHOC,FGF1 OLR329,OLR1704,OLR470,OLR4,OLR1611,OLR951 PRKCA,FGF7,FGF16,MKNK2,FGF12,MAX,BDNF,MAP3K5,ATF4,PLA2G12A,RASGRP2,PLA2G2A,GADD45B

The identification of another pathway in our research,"Cell adhesion molecules(CAMs)",suggests that the process of acupuncture activates intercellular signal transduction.Many cell adhesion molecules are localized at synaptic sites in neuronal axons and dendrites.These molecules,which bridge pre-and postsynaptic specializations,provide a mechanical link between cells.31Studies indicate that interactions between specific CAMs are able to control synapse formation,regulate dendritic spine morphology,modify synaptic receptor function,and modulate synaptic plasticity.32Based on our data,we suggest that acupuncture may activate the CAM pathway to mediate the transmission of information between neurons.Our results show that the CAM pathway-related genes RT1-CE16,RT1-CE4,RT1-CE5,Cd4,and RT1-N2 were differently expressed after acupuncture.This suggests that acupuncture may regulate intercellular signal transduction in SH hypothalamus tissue through specific genes such as those listed above.

In the DAVID gene functional classifications,the categories entitled"cellular process","biological adhesion","metabolic process","membrane","binding","receptor activity",and"transporter activity"were found to change in response to acupuncture in the model and Renying(ST 9)groups.Our research suggests that the process of acupuncture activates both intercellular and intracellular signal transduction.In particular,the local electrical activity of the cell membrane,interactions with the cytoskeleton,or the activation of certain receptors may play a part in activating these process.33Based on our data,we speculate that interactions between cells and re-arrangement of the cytoskeleton occur subsequent to acupuncture and that signals are transferred from one cell to another;the message is then relayed by the cells internally to lead to the transcription of new mRNA transcription and the synthesis of macromolecular proteins.34

In addition,five candidate genes related to inflammation and neurotransmitter release(Chi3l1,Ephx2,Klk1,and 5-HT1A,Cbs,respectively),identified as DEGs based on the results of our microarray analysis,were further validated using real-time PCR.The results obtained were consistent with the gene-chip findings,indicating that the data determined by gene-chip technology were reliable.

Inflammation has been recognized to play a crucial pathophysiological role in hypertension and in cardiovascular disease and participates in many processes that contribute to the development of elevated BP.35In the vasculature,inflammation has been shown to enhance the proliferation of smooth muscle cells and plays a role in vascular remodeling.35-37Accordingly,we expected that inflammatory-related genes would likely be identified in our experiment.Consistent with these expectations,we found that the expression of several primary genes related to inflammatory response was changed in the hypothalamus consequent to hypertension or acupuncture treatment.

Chi3l1 has been implicated as an EH candidate gene.The encoded protein,also termed cartilage glycopro-tein-39,represents an inflammatory glycoprotein involved in endothelial dysfunction,cell attachment and migration,reorganization,and tissue remodeling as a response to endothelial damage.38Chi3l1 has been shown to act as an adhesion and migration factor for vascular cells and is segregated by differentiated VSMCs.39-41The participation of Chi3l1 in inflammatory states and vascular processes implies that Chi3l1 may have the ability to regulate BP.Notably,the current study showed a significant increase of Chi3l1 in the model control group compared to the blank control group,along with a significant decrease following treatment.It has also been reported that EA can reduce systolic and diastolic BP and attenuate the cardiac damage associated with hypertension,as represented by cardiac apoptosis,fibrosis,and hypertrophy.42Therefore,we speculate that Renying(ST 9)had positive effects on cardiovascular remodeling and BP regulation in SH rats though influencing Chi3l1 expression in the hypothalamus.

Ephx2 encodes a soluble epoxide hydrolase that metabolizes epoxyeicosatrienoic acids(EETs).Therefore,the activity level of Ephx2 determines the concentration of EETsin vivo.Physiological concentrations of EETs decrease cytokine-induced endothelial cell adhesion molecule expression;in addition,EETs prevent leukocyte adhesion to the vascular wall by a mechanism involving inhibition of the transcription factors NF-κB and IκB kinase.43EETs are considered endogenous antihypertensive substances that modulate the Ca2+and K+ion channels as well as gene expression and produce vasorelaxation in addition to their anti-inflammatory effects.44The gene chip results obtained here were parallel to those of most other studies.45,46The enhanced expression of the Ephx2 gene in the hypertensive hypothalamus might therefore lead to a reduced amount of EETs and be conducive to the development of hypertension in SH rats.47In turn,the Ephx2 gene in the hypothalamus was significantly decreased in the Renying(ST 9)group compared with the model group.Clinical studies have shown that acupuncture can exert a protective effect against the production and expression of inflammatory cytokines and adhesion molecules,effectively blocking the inflammatory reaction and reducing BP.48Therefore,we speculate that the reduction in Ephx2 expressionviaRenying(ST 9)may have led to an attenuation of vascular remodeling and induction of blood vessel vasodilation by potentiating the anti-inflammatory and anti-proliferative effects of EETs.

一个新型的研究领域从兴起到成熟的过程伴随着参与该领域的研究人员的增多。利用分析软件对所检索的文献作者数量进行统计，如表2所示,看出1990-2018年，情报信息机构中共有393名作者参与发表了知识服务研究的相关文献，其中2009年是发文作者数量最多的年份，2007-2016年每年的作者数量都稳定在20人以上，表明这一时间段该领域的研究进入了成熟期。另外每年的论文作者数量基本都是新增作者，一方面表明该领域研究比较活跃，总有新增的人员参与；另一方面表明该领域的作者大多没有对知识服务工作进行持续性的研究。从这个意义上来说，这种人员参与的研究模式不利于这项研究工作的深入开展，以及事业的可持续发展。

Tissue Klk1 represents an intricate endogenous pathway involved in hypertension,inflammation,cardiovascular homeostasis,cytokine release,prostacyclin and nitric oxide production,and cell proliferation in the brain.49Klk1 is produced by mast cells and platelets during the initial inflammation process.50Klk1 is able to stimulate the production and release of inflammatory mediators such as eicosanoids,adhesion molecules,cytokines,prostaglandins,NO,and free radicals.51Numerous studies have reported that Klk1 exhibits a wide spectrum of beneficial effects by decreasing blood pressure.52Our results demonstrate a significant decrease of Klk1 in the model control group compared to the blank control group,which is consistent with the clinical finding that hypertensive patients also exhibit lower levels of Klk1.53Furthermore,needling Renying(ST 9)increased the expression of Klk1 and clinical studies have shown that acupuncture significantly decreased the levels of proinflammatory cytokines in the hypothalamus.54Together,these results suggest that acupuncture exhibits anti-hypertension-like effects and that its mechanism of action appears to involve the inhibition of proinflammatory cytokines.55Therefore,we speculate that Renying(ST 9)might improve the state of SH by decreasing the expression of Klk1.

Central and peripheral blood flow is modulated in part by the action of neurotransmitters within the complex neuronal networks of the hypothalamus.In particular,previous electrophysiologic studies have shown that the hypothalamus comprises an important component of the cardiovascular center,which adjusts the sympathovagal balancevianeurotransmitters to regulate the cardiovascular system.56

5-hydroxytryptamine(5-HT;serotonin)is a biogenic monoamine with a molecular weight of 176 Da that can modulate cardiovascular responses.In the central nervous system(CNS),5-HT1A receptors play an important role in mediating the signals from this neurotransmitter.57Excitation of the brain stem and spinal cord by 5-HT1A stimulation from descending pathways can suppress sympathetic activity,reduce BP,and slow heartrate.58Furthermore,theactivation of 5-HT1A receptors in the brain stem causes a potent and selective suppression of the hypertensive and sympathoexcitatory response evoked by stimulation of the dorsomedial hypothalamic nucleus.59

In turn,H2S functions as a neuromodulator that enhances N-methyl-D-aspartate receptor-mediated currents and modulates the sympathetic nervous system.60Furthermore,through its role as a kind of endothelium-derived relaxing factor,H2S has been shown to improve the diastolic function of the peripheral vasculature.H2S levels are regulated to a large part by the CNS,within which Cbs serves as the principal enzyme involved in H2S production.

[5]Aaron Smith, Monica Anderson, Automation in Everyday Life, Oct. 4, 2017, http://www.pewinternet.org/2017/10/04/automation-in-everyday-life/.

Our data showed that the expression of Cbs and 5-HT1A both decreased in the model control group when compared with the blank control group whereas after needling Renying(ST 9),both genes were significantly elevated.It has been reported that the serotonin produced in the EA-activated nucleus raphe pallidus neurons that project to the rostral ventrolateral medulla(rVLM)contributes,through a 5-HT1A mechanism,to EA-mediated inhibition of rVLM sympathetic premotor neurons and acupuncture modulation of the excitatory reflexes.61During acupuncture,somatic sensory nerve-evoked input,acting through a number of neurotransmitter systems in several cardiovascular regions of the brainstem,is thought to essentially restore altered neuronal activity back toward a stable baseline.62Consistent with this model,the current study showed that acupuncture at Renying(ST 9)influences the expression of factors(5-HT1A and Cbs)with stimulatory and inhibitory effects on neurotransmitter signaling in the CNS to alter the processing of sensory information.This,ultimately,impacts autonomic outflow and thus cardiovascular function,eventually normalizing BP by lowering elevated BP.

张连长:“还有意见以后再提,给你的半分钟过了!第一排听我口令,向前一步——走!向右——转!你们都跟着他,把麻袋收集到仓库去!”

However,previous studies have been limited in their focus on targeting specific cytokines and hormones that were expected to be involved in the process of acupuncture enhancement.In contrast,gene chip technology,characterized by high sensitivity and throughput,represents a method capable of detecting the expression of numerous genes simultaneously,thus providing a platform for the concurrent analysis of multiple genes influencing hypertension.12This method thus also serves as a powerful means of gaining insight into the acupuncture's antihypertensive mechanism and has been used to detect genes that are differentially expressed between SH and Wistar-Kyoto(WKY)rats.13,14Accordingly,in the current study we employed a gene chip technique that enabled us to observe the expression of several thousand genes simultaneously in order to observe how acupuncture on Renying(ST 9)broadly influences gene expression.In particular,we examined the effect of acupuncture at Renying(ST 9)on essential hypertension,investigated the changes of gene expression in the hypothalamus of rats after acupuncture treatment using microarray analysis,and predicted the associated biological mechanisms by identifying candidate genes and other specific genes exhibiting differential expression between control,hypertensive,and treated animals.

In conclusion,our data suggest that acupuncture at Renying(ST 9)could significantly lower the BP of hypertensive rats and affect their gene expression profile in the hypothalamus,in particular that of 5 hypertension-related genes.Acupuncture may counter hypertension in various ways.We speculate that the identified target genes and their associated signal transduction pathways,which are associated with inflammation and neurotransmitter release,may be involved in the anti-hypertensive mechanism of acupuncture at Renying(ST 9).

25 Tjen-A-Looi SC,Guo ZL,Li M,et al.Medullary GAB Aergic mechanisms contribute to electroacupuncture modulation of cardiovascular depressor responses during gastric distention in rats.Am J Physiol Regul Integr Comp Physiol 2013;304(5):R321-R332.

REFERENCE

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（1）根据动态效应分析得到的结论：水资源消耗对自身的动态冲击作用最大，城镇化程度越高的省份，水资源消耗对自身的冲击越小；人口城镇化程度较高的省份，对水资源消耗影响的动态效应表现为负向冲击作用，反之则表现为正向冲击作用，且人口城镇化程度越高，其作用就越小；经济城镇化较低的省份会对水资源消耗产生正向冲击作用，反之会产生负向冲击作用，且经济城镇化程度越高的省份，负向冲击作用就越大；全国大部省份的产业城镇化对水资源消耗都有正向冲击作用，只有少部分省份的产业城镇化会对水资源消耗起到负向冲击作用。

With the dramatic improvement of average life expectancy in recent years and trend toward increasing age of the general population,hypertension has become a major public health problem.Notably,essential hypertension (EH), which comprises approximately 90%-95%of hypertension cases and affects over 1 billion adults worldwide,1represents a predisposing risk factor for stroke,myocardial infarction,congestive heart failure,and arterial aneurysm,as well as the leading cause of chronic renal failure in humans.2Furthermore,recent studies have shown that most forms of hypertension are also associated with a wide variety of functional changes in the hypothalamus,3with a complicated pathogenesis that mainly involves the hypothalamus-pituitary-adrenal cortex axis4and sympathetic nerve-adrenal medulla system.5Accordingly,current research efforts worldwide have focused on the search for a novel and effective decompression method to combat the pervasive effects of hypertension.

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在Wnt细胞极性信号通路中，Wnt蛋白与Fzd蛋白结合后激活下游小GTP酶，其在细胞极化和细胞骨架重排等方面发挥作用。此外，也有研究[9]证实，Wnt细胞极性信号通路与内皮细胞迁移、增殖密切相关。

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研究表明，CLA可以通过激活PPARγ途径调节炎症反应。Yu等[18]使用CLA处理巨噬细胞，发现CLA异构体混合物能降低细胞因子如IL-1和IL-6的生成，认为CLA具有抗炎症反应至少是部分依赖于过氧化物酶体增殖物激活受体(PPAR)的调节，Yang等[19]也发现cis-9,trans-11 CLA具有类似的作用。Bassaganya-Riera等[20]的研究表明，CLA可以通过激活猪结肠PPARγ降低炎性因子TNF-α的产生，从而调控结肠炎的发作时间及损伤程度。

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同样，风险分析、评估也存在难点。沈崇德举例称，如风险指数到底是多少？因认识的差异，仁者见仁，智者见智。

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2）泥浆宜选用化学泥浆。化学泥浆是一种水溶性，易混合的粉末颗粒聚合物，在水中充分溶解后成半透明糊状，黏度大，在孔内沉淀杂质速度非常快。钻具在孔内钻进时，泥浆总是保持清澈透明，钻具钻杆表面干净，在孔内由于钻具连续运动，化学泥浆和水的混合越均匀黏度就越强，凝聚也就越快，可快速在孔壁周围形成一层薄透明糊状保护层且无毒无污染。

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文艺复兴运动送葬了“黑暗的中世纪”，为欧洲社会乃至整个人类社会开启了一个崭新的时代。随着欧洲工业革命的到来，近代西方的社会结构发生了巨大变化，近代西方社会管理实践和思想也发生了根本性的变化。一般认为，近代以来的西方社会管理主要经历了三个阶段：一是以行政权与王权的分化为特点的社会管理的独立与起始阶段；二是以改善社会管理方式、提高管理效率、增长社会财富为特点的社会管理的规范与发展阶段；三是以调整社会结构、实现社会公正、推进全面福利为特点的社会管理的转型与完善阶段。

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