| ObjectiveTo study the mechanism of acupuncture at Taichong and Taixi lowering the blood pressure and screen the key proteins though observing general situation,pathomorphological,molecular biology and proteomics four levels,through observing acupuncture Taichong,Taixi spontaneous hypertensive rats weight,blood pressure,brain HE staining,brain Nissl staining,TMT-based proteomics and its validation.Finally,trying to find the mechanism of antihypertensive effect of acupuncture on spontaneously hypertensive rats and screening the key proteins,in order to elucidate the mechanism and experimental basis of antihypertension by acupuncture.MethodsSpontaneous hypertensive rats(SHRs)were randomly divided into 3 groups according to randomized block design with the original blood pressure as stratification by STATA.The model group,Chongxi group and non-acupoint group,12 in each group.Wistar-Kyoto rats(WKY)were not grouped.Acupuncture points: Taichong(bilateral),too Creek(bilateral),non-point(bilateral).Operation method: Rat was acupuncture awake,twirling and "Pingbupingxie",acupuncture treatment start at 8:30 am daily.Acupuncture Left and right at the same time(Perpendicular insertion).Acupuncture points on the left firstly(2.5min),then acupuncture right acupuncture points(2.5min),5 minutes in total.Acupuncture 1 times a day,every Sunday suspension,acupuncture for 4 weeks,a total of 24 times.The twisting frequency is 80 ± 5 times / min and the twisting amplitude is 180 ± 5 degrees.When acupuncture the loose black socks to cover the upper head and neck of rats,exposing lower limb.In a dark environment,rats can be quiet and stable,making acupuncture easy and protecting the operator from being scratched or bitten by the rats.Acupuncture is operated by the same person.The control and model groups were not acupunctured,however,covered with black socks and grasped for 5 min.In the experiment,the body weight of the rats was recorded before and after the experiment.The blood pressure of each group was recorded before the experiment,the end of the first week,the end of the second week,the end of the third week and the end of the experiment.Tissues were taken immediately after the experiment.HE staining and Nissl staining were used to observe the general pathological structures of the hypothalamus in rats.TMT quantitative proteomics was used to analyze the protein expression of hypothalamus in each group to find the differential proteins.Western blot was used to verify the expression of key differentially expressed proteins.Results 1.Change in body weight of ratsDuring the experiment,the weight of rats in each group increased.The body weight gain before and after the experiment: the difference between the model group and the blank group was not statistically significant(P> 0.05),The body weight gain of the model group before and after the experiment was higher than that of the red river group and the non-hole group(P <0.05).There was no significant difference between Chongxi group and non-acupoint group(P> 0.05).2.Blood pressure measurement results of rats in each groupBefore the experiment,the blood pressure of the normal group was less than that of the other three groups(P <0.05),and there was no significant difference between the other groups.When blood pressure was measured in the first weekend,the blood pressure in the normal group was less than that in the other three groups(P <0.05).There was no significant difference in the other groups between the two groups.And there was no significant difference between the other groups.When blood pressure was measured in the second weekend,the blood pressure in the normal group was less than that in the other three groups(P <0.05).The blood pressure of Chongxi group was less than that of non-acupoint group(P < 0.05),and there was no significant difference between the other groups.When blood pressure was measured in the third weekend,the blood pressure in the normal group was less than that in the other three groups(P <0.05).The blood pressure of Chongxi group was less than that of non-acupoint group(P <0.05),the blood pressure of Chongxi group was less than that of model group(P <0.05).There was no significant difference between model group and non-acupoint group.When blood pressure was measured in the Fourth weekend,the blood pressure in the normal group was less than that in the other three groups(P <0.05).The blood pressure of Chongxi group was less than that of non-acupoint group(P <0.05),the blood pressure of Chongxi group was less than that of model group(P <0.05).There was no significant difference between model group and non-acupoint group.There was no correlation between body weight and blood pressure(P> 0.05).3.The brain morphological results of rats in each group.Hypothalamus: The histopathology results of hypothalamus HE staining and Nissl staining of rats in each group showed no obvious pathological changes compared with those in control group,such as neuron nuclear karyopyknosis,deeply stained(anachromasis),nucleus disappear and so on.However,compared with the control group,the total number of nerve cells and the number of Nissl body in the three spontaneously hypertensive rats(SHRs)reduce to varying degrees.The total number of hypothalamic neuronal cells and the number of Nissl bodies from more to less are: control group,Chongxi group,nonacupoint group,model group.4.Results of proteomics experimentsProteomics results showed that a total of 6548 proteins were identified,of which 124 were statistically significant in the spontaneous hypertensive rats(SHRs)model group and the control WKY rats.In the model group,spontaneous hypertensive rats 73 protein expression increased,(Nme1、 Blvra、 Crabp1、 Cbs、 O08559、 Cbr1、 Ces1d、Mettl7a、 Tppp3、 Flot1、 Sult1d1、 Ube3c、 Pank2、 Flot2、 F1M086、 Afmid、Lrrc8d、 Lgals3 、 Krt8 、 Acp1 、 Fgfr1op2 、 Ecel1、 Kcnq2 、 Dguok 、 Ephx2、Wdr24、 Map3k5、 Ormdl3、 Cartpt、 Sept11、 Rab11fip4、 Prph、 Lrfn2、 Alb、Rab3b、 Fabp7、 Fam127b、 Spta1、 Tinf2、 Mecr、 Cpped1、 Cd200、 Clic6、 Vwa1、 Dda1、 Pik3c2a、 Hyi、 Dock10、 Gldc、 Srp72、 Gnas、 Metap2、 Ndufv3、Il1rapl1、 Iqgap1、 Nop58、 P01836、 Hnrnpab、 Smarca5、 Gstt1、 Stk26、 Sun1、 Synm、 Sh3bp5、 Pdlim3、 P20762、 Dynlt1、 Ssr1、 Golga5、 Rbfa、 Krt5、 Ada、 Lpcat1),51 proteins expression decreased(Retsat 、 Alb 、 Kyat1 、Eps15l1、 Vezf1、 Rpe、 Mvd、 Rdh13、 Abhd10、 Pym1、 Hapln2、 RGD1559441、Shoc2、 Enpp6、 Cox6a1、 Glul、 Tspan3、 Hist2h2ab、 Scn4b、 Qdpr、 Tmem170a、 Naxd、 Actr6、 Robo1、 Glra1、 Mug1、 Cd200、 Ca2、 RGD1561636、 Ppox、Trim36、 Igkc 、 Cbr4 、 Reep1 、 Cox7 c 、 Agpat5 、 Tmem38 a 、 Gpaa1 、 Spock1、Psd3、 Bad、 Lin7b、 Pcdh19、 Igg-2a、 Fbxl15、 Grin2b、 Cldn11、 Snca、 Fau、Stmn1、 Phospho1).Compared with the model group,Chongxi group had 69 proteins statistically significant differences,and the expression of 42 proteins in Chongxi group was increased(Fam171a2、 Dmtn、 Nrgn、 Cox6a1、 Gng8、 Cldn10、 Slc12a5、 Tfip11、 Scn4b、 Dlgap3、 Pikfyve、 Plxna2、Map4k2、 Th、 Btbd17、 Hist3h2bb、 Wdr81、 LOC100174910、 Cnep1r1、 Slc17a7、 Fam210b、 Slc4a4、 Tubb6、 Krt222、 Necab1、 Prl8a7、 Q8R462、 Btbd8、Lrrfip1 、 Psd3 、 Lrrc7 、 Srrm2 、 Tmem38 a 、 Prkcb 、 Sv2 b 、 Mesdc1 、 Cyc1 、LOC679794 、 Iglon5 、 Cplx1 、 Hapln1 、 Dynlt3),27 proteins expression decreased(Ece1、 P02262、 Lgals3、 Gfap、 Flnc、 Slc7a2、 Apoc1、 Cp、 Rit1、Fabp7、 Krt5、 Btf3l4、 Prph、 Krt10、 Mgst1、 Krt2、 Ces1d、 Synm、 Pdlim3、Slc7a11、 Cartpt、 Glipr2、 Sh3bp5、 Fcgbp、 Sult1a1、 Anxa1、 Gstt1).Compared with the model group,the non-acupoint group had 30 proteins with statistically significant difference,and 26 proteins were increased in non-acupoint group(LOC361985、 Fam171a2、 Map4k2、 Dpf2、 Tada1、 Lmbrd1、 Lrrfip1、 Hist3h2bb、 Palmd、 Pcp4l1、 Ctnnal1、 Mto1、Ubr2、 Akt1s1、 RGD1311703、 Pde10a、 Ankrd17、 Coa4、 Cdc26、 Hmgn2、 Fmo1、LOC100174910 、 Gcc1 、 Rbfa 、 Timmdc1 、 Dynlt3),4 proteins expression decreased(Mobp、 Fth1、 Plekha1、 Tinf2).There were 14 differentially expressed proteins in the model group,control group and Chongxi group(Cox6a1、 Scn4b、 Psd3、 Tmem38a、 Lgals3、 Fabp7、 Krt5、 Prph、 Ces1d、 Synm、 Pdlim3、 Cartpt、 Sh3bp5、 Gstt1).5.Western blot analysis of differential proteins and Repeatability.By computer-based random number method,five differential proteins(Lgals3 、 EPHX2 、 Bad 、 Blvra 、 Vezf1)were selected from the control group and the model group and used to verify the repeatability of the TMT experiment.From the above-mentioned 14 common differential proteins,Lgals3,Fabp7 and Prph were selected to be verified by Western blot in accordance with the literature and the results of TMT.Repeatability verification results: The results of TMT experiments and Western blot experiments showed that the above five proteins showed the same trend through two verifications.Therefore,the repeatability of the TMT experiment can be regarded as good and can be the next step to verify key proteins.The key proteins: The three key proteins in TMT experiments and Western blot experiments showed the same trend.Conclusion:1.Acupuncture can control the weight gain of rats,but no matter the body weight of Chongxi group and non-acupoint group were decreased,and the blood pressure of non-acupoint group were not reduced.It cannot be considered that the control of body weight is one of the mechanisms of antihypertensive.2.Acupuncture Taichong and Taixi can Lower the blood pressure of spontaneously hypertensive rats(SHRs),but it cannot be returned to normal levels.3.Acupuncture Taichong and Taixi can inhibit the reduction of the total number of cells and Nissl bodies in the hypothalamus.Physiological mechanisms include the protection the hypothalamus and regulation of the N-methyl-D-aspartate(NMDA)receptor,the level of medullalion and the content of brain-derived neurotrophic factor(BDNF)in paraventricular nucleus of hypothalamus and inhibit the transcription of Toll-like receptor 4(TLR4)signaling pathway Proinflammatory cytokines.Finally,via the various mechanisms mentionedabove,the blood pressure is reduced.4.The 124 differentially expressed proteins in the model group were mainly related to the activities of anionic transmembrane transporters,structural molecules,metal ion transmembrane transporters,inorganic cation transmembrane transporters,and secondary activities of transmembrane transporters,Involved in the establishment of vesicle localization,vesicle localization,regulation of neurotransmitter levels,transport of neurotransmitters,establishment of organelle localization,organelle localization,learning or memory,cognition,monovalent inorganic cation transport,synaptic transmission,The response to the metal ions is centered on the intermediate filaments,the middle silk cytoskeleton,the clathrincoated vesicles,the vesicle membrane,the cytoplasmic vesicle membrane,the postsynaptic density,the coated vesicles,the cell projection membrane,the axis Protrusion,protein-DNA complexes,secretory granules,extracellular matrix proteins,cell bodies and neuronal cell bodies.These proteins and their biological processes,molecular functions,cellular components and pathways involved are most likely the pathogenesis of hypertension in spontaneously hypertensive rats(SHRs).5.The result of comparison between Chongxi group and model group: The difference proteins were involved in the glutathione metabolic pathway,amyotrophic lateral sclerosis(ALS)pathway,cytochrome P450 metabolic pathway of exogenous chemicals,phosphatidylinositol signaling pathway,Parkinson’s disease pathway,myocardial contractile pathway,drug Metabolic pathways,gap junctional pathways,pancreatic secretory pathways,leukocyte trans-endothelial migration pathways,tight junctional pathways,systemic lupus erythematosus pathways,Alzheimer’s disease pathways,and Huntington’s disease pathways.A total of 14 signal transduction pathways.These pathways may be the mechanism by which acupuncture at Taichong and Taixi can reduce the blood pressure in the Hypothalamus of spontaneously hypertensive rats,including the effects of anti-oxidation and detoxification through the regulation of the glutathione metabolic pathway,the exogenous Chemical cytochrome P450 metabolic pathway may produce relaxation of vascular smooth muscle,angiogenesis and inhibition of inflammation,antiapoptosis has a regulatory role,probably through the phosphatidylinositol signaling pathway on cell division,apoptosis and other regulation and inhibition,may Under the action of gap junctional pathway,vascular smooth muscle cell proliferation has a regulatory effect and regulates tight junctions between cells.However,the results also showed that these proteins were associated with a series of neurodegenerative diseases,suggesting that acupuncture at Taichong and Taixi might improve this series of diseases.6.The main mechanisms of lower blood pressure of acupuncture at Taichong and Taixi involved the possible 14 proteins(Cox6a1、 Scn4b、Psd3、 Tmem38a、 Lgals3、 Fabp7、 Krt5、 Prph、 Ces1d、 Synm、 Pdlim3、 Cartpt、 Sh3bp5、 Gstt1).Lgals3,Fabp7 and Prph were selected to be verified by Western blot in accordance with the literature and the results of TMT.These three proteins are considered as potential key or targeting proteins in the hypothalamus of mechanisms of acupuncture antihypertensive.And verify in the next part.7.The repeatability of TMT experiment was established.The three key proteins mentioned above showed similar trends in TMT and Western blot experiments.8.The mechanism of antihypertensive of acupuncture at Taichong and Taixi in spontaneously hypertensive rats(SHRs)can be considered as mainly involving hypothalamic axons,growth regulation of dendrites,inhibition of inflammation and regulation of synapse formation;regulation of lipid metabolism,further reduction of inflammatory pathway activation,reduction of vascular endothelial damage and adjustment of sleep arousal cycles and reduction of anxiety;Inhibits the apoptosis of nerve cells,reduction of neurotoxicity of peripheral proteins,regulation of axonal growth extension,maintenance of synaptic stability,and diseases related to the regulation of nervous system autoimmunity.. |
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