Study On The Mechanism Of Signal Transduction Pathway In Target Brain Region By Acupuncture To Reduce Blood Pressure Of Spontaneously Hypertensive Rats

BackgroundEssential Hypertension(EH)is one of the most concerned diseases in the world.As we all know,hypertension has a high incidence rate,many complications,and a high mortality rate,which seriously endangers human health.According to statistics,about 40%of people over the age of 25 suffer from hypertension worldwide,including nearly 1 billion patients with essential hypertension,and by 2025 this number is expected to reach 1.56 billion.Hypertension is also a major risk factor for cardiovascular disease,such as stroke,coronary heart disease and cognitive impairment,and hypertension and its complications cause more than 10.4 million deaths each year.At present,oral medicine is the main treatment method in clinical practice,but there are many drawbacks in drug treatment,such as large side effects,poor compliance,and ineffective efficacy.Traditional Chinese medicine has an absolute advantage in the treatment of hypertension.Acupuncture has been used for more than 3,000 years as an effective treatment.Its therapeutic effect is remarkable,its control is considerable,its operation is simple,and it has certain target organs such as heart,brain,kidney and blood vessels with two-way adjustment and protection.In recent years,acupuncture and blood pressure reduction has attracted the attention of many scholars.The United States,Germany and other countries have carried out a number of large-scale acupuncture treatment of primary hypertension clinical trials.A number of clinical randomized controlled trials have shown that acupuncture at Tai Chong can regulate patients with essential hypertension.The basis of this research is based on the results of our previous experiments using brain functional imaging technology(PET,fMRI,etc.)on the specific research of acupoints,and established a theoretical model of the acupoint-brain-related acupoint mechanism.As a living organism,the therapeutic stimulation of acupuncture points passes through the regulation and integration of the brain’ s information processing center,and then acts on the target organs to present a therapeutic effect.According to this theoretical model,we used PET-CT imaging technology combined with proteomics technology in the early experiments to start from the medulla,and explored the central mechanism of acupuncture at Taichong.In the central nervous system of SHR,some target brain regions related to the blood pressure reduction of Taichong were more accurately located.It was found that acupuncture at Taichong can affect the content of 11 proteins in the medulla of SHR and achieve the effect of blood pressure reduction.qPCR and Western Blot experiments showed that 6 of the 11 proteins were consistent with the previous results.Thus,we propose a new mechanism of acupuncture in BP reduction which acupuncture at Taichong activates the brain region of concentrated target effects in the brain and lowers blood pressure by affecting key protein synthesis within it.However,in the previous issue,we only studied the antihypertensive effect of a single acupoint and the synthetic changes of the protein in the effect brain region,and there are more signal pathways to be discovered.The combination of acupuncture points is the basis of acupuncture treatment.The combination of multiple acupoints with the same or similar effects has the enhancing effect of acupuncture.The comparison of the antihypertensive effect of Taichong combined with Taixi and single point of Taichong or Taixi in this study is to clarify the relationship between the compatibility effect of acupuncture and the onset of acupuncture.To explore these changes,it is necessary to directly examine the activation of the cellular pathway in the acupuncture effect and understand its effect on the regulation of gene expression.Therefore,it is necessary to use transcriptome sequencing technology to explore the performance of acupuncture effects at the gene level and cell signal transduction pathway,and to explore the scientific basis for more acupuncture effect mechanisms.Research on this aspect has not been carried out abroad,and there are few reports in this area in China.ObjectiveIn this study,the second-generation central antihypertensive drug moxonidine was used as a control.The combination of Taichong and Taixi was compared with single point of Taichong or Taixi.On the one hand,it was to explore the central effect difference between central antihypertensive drugs and acupuncture to find the difference between the key brain regions activated by blood pressure and the central neurobiological effects,and explore the difference between the two blood pressure pathways and the antihypertensive target at the level of the cell signal transduction pathway,which is beneficial to acupuncture and blood pressure reduction.On the other hand,this study explores the time-effect characteristics of acupuncture to lower blood pressure,in order to guide clinical practice.MethodsForty-eight male spontaneously hypertensive rats were randomly divided into model group,Taichong group,Taixi group,Taichong+Taixi group,non-acupoint group and moxonidine group.Another 8 male WKY rats served as normal controls.Taichong group,Taixi group,Taichong+Taixi group and non-acupoint group were continuously acupuncture for 7 days.The moxonidine group was intragastrically administered at a dose of 0.04 mg/kg,continuous gavage for 7 days,but model group and normal group only performs the grasping operation and does not perform acupuncture treatment.The blood pressure of each group was compared at 30,60,and 90 min after the first day of treatment,and the effect brain area of acupuncture immediately decompressed was explored by PET-CT scan;the blood pressure of each group was compared at 24,48,and 72 h after 7 days of continuous treatment.PET-CT scan explores the effector brain area of hypotension after acupuncture treatment.In addition,24 hours after treatment,the rats were removed from the medulla,hypothalamus,thalamus,cerebellum,hippocampus and sensory cortex;the mRNA in the brain region was extracted,and the key brain regions were detected by transcriptome sequencing to find the signal transduction in key brain regions.The pathway was further verified by qPCR using the results of transcriptome sequencing.ResultsThe results of immediate acupuncture treatment showed:1)Compared with the SHR group,the Taichong,Taixi,Taichong+Taixi groups had the effects of reducing systolic and diastolic blood pressure at 30 and 60 min,while Taichong+Taixi group can still reduce the systolic and diastolic blood pressure of SHR at 90 min;2)Compared with the SHR group,the moxonidine group can reduce the systolic and diastolic blood pressure of SHR 30-90 min after gavage;3)Compared with the SHR group,the sham acupuncture group had no significant antihypertensive effect at 30-90 min.4)Compared with the sham acupuncture group,the Taichong group and the Taixi group had significant effects on reducing systolic and diastolic blood pressure at 30 and 60 min.The Tai Chong+Taixi group had a significant effect on reducing systolic and diastolic blood pressure at 30-90 min.The results of 7-day acupuncture treatment showed:1)Compared with the SHR group,the Taichong group,Taixi group,Taichong+Taixi group had the effects of reducing systolic and diastolic blood pressure at 24 and 48 hours after treatment,and Taichong+Taixi group can reduce the systolic and diastolic blood pressure of SHR at 72h after treatment;2)Compared with SHR group,moxonidine group can reduce SHR systolic blood pressure and diastolic blood pressure after 24-72h;3)Compared with the SHR group,the sham acupuncture group had no significant antihypertensive effect after 24-72h;4)Compared with the sham acupuncture group,the Taichong and Taixi groups had significant effects in reducing systolic and diastolic blood pressure at 24 and 48 h after treatment,while the Taichong+Taixi group reduced systolic and diastolic blood pressure 24-72 h after the course of treatment.The results of PET-CT scanning on the changes of glucose metabolism in the brain area immediately after acupuncture showed that compared with the WKY group,the glucose metabolism in the brain area of the SHR group was significantly reduced in the motor cortex,sensory cortex and visual cortex,but the glucose metabolism in the rat brain region was obvious increased in the thalamus,ventral tibial cortex,dorsal thalamus_lateral nucleus group,etc.;Compared with the SHR group,the glucose metabolism in the brain region of the Taichong group was found in the granular island cortex and anterior olfactory nucleus,but the elevated glucose metabolism was found in the anterior cerebellum and the posterior cerebellum;compared with the SHR group,the Taixi group Elevated glucose metabolism in the brain area ofrats is found in the posterior cerebellum and visual cortex,but the decrease in glucose metabolism is seen in the anterior olfactory nucleus and nucleus accumbens.Compared with the SHR group,the glucose metabolism in the brain regions of the Taichong+Taixi group is elevated.Found in the medulla oblongata,anterior cerebellum,etc.,but decreased glucose metabolism in the thalamus,dorsal thalamus,etc.;compared with the moxonidine group,the rat brain region of the Taichong+Taixi group increased in glucose metabolism in the auditory cortex,anterior cortex,etc.However,the decrease in glucose metabolism is found in the cingulate cortex,granular island cortex.After the 7-day acupuncture treatment,the results showed that compared with the WKY group,the increased glucose metabolism in the brain region of the SHR group was found in the thalamus,dorsal thalamus,etc.,but the decrease in glucose metabolism was found in the cingulate cortex,the sensory cortex,etc.;compared with the SHR group,too The increased glucose metabolism in the brain area of the rats in the Taichong group was found in the medulla,but the decrease in glucose metabolism was found in the dorsal thalamus and striatum.Compared with the SHR group,the increase in glucose metabolism in the brain region of the Taixi group was seen in the lower extremities,the olfactory cortex.However,the decrease in glucose metabolism is seen in:thalamus,striatum,etc.Compared with the SHR group,the increase in glucose metabolism in the brain region of the Taichong+Taixi group is seen in the granular island cortex,striatum,etc.,but the glucose metabolism is reduced in dorsal thalamus,motor cortex,etc.;compared with the moxonidine group,the increase in glucose metabolism in the brain region of the Taichong+Taixi group is seen in the thalamus,hypothalamus,etc.,but the decrease in glucose metabolism is seen in the basal ganglia,caudate Nuclear,etc.The results of Pathway functional analysis of differentially expressed genes showed that:1)Compared with the WKY group,the differentially expressed genes were mainly enriched in the medulla oblongata:calcium signaling pathway,cGMP-PKG signaling pathway,myocardial contraction,serotoninergic synapses,Γ-aminobutyric acid(GABA)can synapse,oxytocin signaling pathway,aldosterone synthesis and secretion,arrhythmogenic right ventricular cardiomyopathy(ARVC),MAPK signaling pathway,Rapl signaling pathway,tyrosine metabolism,PI3K-Akt Signal pathways,ECM-receptor interactions,etc.Among them,differentially expressed genes related to cardiovascular diseases are enriched:Cacnalf,Tnf,Ttr,Th,Itgal,Cyp17a1,Arrb1,Gabrb1,Slc18a1,Stard6,Cd36,Mylk3,Sv2c,Gadd45b,Adcyl,Mrvil,Itga4,Cypllb3 and the like.In the hypothalamus,differentially expressed genes are mainly enriched in:MAPK signaling pathway,Rapl signaling pathway,calcium signaling pathway,cGMP-PKG signaling pathway,fluid shear stress and atherosclerosis,prolactin signaling pathway,serotoninergic Touch,gamma-aminobutyric acid(GABA)can synapse,aldosterone synthesis and secretion,tyrosine metabolism and the like.Among them,differentially expressed genes related to cardiovascular diseases are enriched:Efna5,Angpt12,Eddra,Erbb2,Nexn,Tacr3,Tnf,Esr1,Grpr,Gabrbl,Hspalb,Atp2b4,Gabrd,Moapl,Pard6g,Isoc2b,Isocl,Map3k5.In the thalamus,differentially expressed genes are mainly enriched in:MAPK signaling pathway,fluid shear stress and atherosclerosis,gamma-aminobutyric acid(GABA)synapses,aldosterone synthesis and secretion,Rapl signaling pathway,serotonin Can be synaptic and so on.Among them,differentially expressed genes related to cardiovascular diseases are enriched:Gstm2,Tnf,Hspalb,Gabra6,Creb314,Isocl,Map3k5,Gstm5,Wfdc6a,Cdh1 and the like.2)Compared with the model group,the Taichong group is mainly enriched in the medulla oblongata:Rapl signaling pathway,arrhythmogenic right ventricular cardiomyopathy(ARVC),fluid shear stress and atherosclerosis,tyrosine Acid metabolism,prolactin signaling pathway,myocardial contraction,calcium signaling pathway,cGMP-PKG signaling pathway,gamma-aminobutyric acid(GABA)synaptic,Hippo signaling pathway,etc.Among them,differentially expressed genes related to cardiovascular diseases are enriched:Armc3,Nppc,Th,Dbh,Ccdc40,Adrald,Gabra6,Tp73,Nr4al,Htr5b and the like.In the hypothalamus,differentially expressed genes are mainly enriched in:PI3K-Akt signaling pathway,ECM-receptor interaction,platelet activation,hypertrophic cardiomyopathy(HCM).Among them,differentially expressed genes related to cardiovascular diseases are enriched:LOC310926,Fndc3c1 and the like.In the thalamus,differentially expressed genes are mainly enriched in oxytocin signaling pathway,calcium signaling pathway,PI3K-Akt signaling pathway,and ECM-receptor interaction.Among them,differentially expressed genes related to cardiovascular diseases are enriched:Kcnj4,Drdl,Nexn,Adrald,L0C310926 and the like.3)Compared with the model group,the Taixi group is mainly enriched in vascular smooth muscle contraction,cytokine,cytokine receptor interaction,AMPK signaling pathway,PPAR signaling pathway,calcium signaling pathway,splicing,longevity in the medulla oblongata.Regulates the pathway,serotonin synapses,and regulates the actin cytoskeleton.Among them,differentially expressed genes related to cardiovascular diseases are enriched:Ccr5,Rps15a,Shox2,C1ql2,Epgn,Adrald,Hspalb,Wbp11,Vkorcll,Rfxap,Sec1413,Htr5b.In the hypothalamus,differentially expressed genes are mainly enriched in:calcium signaling pathway,VEGF signaling pathway,cGMP-PKG signaling pathway,arachidonic acid metabolism,fluid shear stress and atherosclerosis,proteoglycans in cancer,vascular smooth muscle Contraction,tryptophan metabolism,complement and coagulation cascades,platelet activation,Wnt signaling pathway,myocardial contraction,renin-angiotensin system.Among them,differentially expressed genes related to cardiovascular diseases are enriched:Sphk1,Pde5a,Akr1c14,Akrlc14,Pde5a,Gstm2,Igf2,Actg2,Cyp1b1,Prg4,Collal,Arhgap29,Wnt6,Tpm2,Enpep,Acta2,Col3a1.In the thalamus,differentially expressed genes are mainly enriched in:arrhythmogenic right ventricular cardiomyopathy,fluid shear stress and atherosclerosis,myocardial contraction,platelet activation,cGMP-PKG signaling pathway,adrenergic energy in cardiomyocytes Signaling,oocyte meiosis,tight junctions,complement and coagulation cascades,ether lipid metabolism.Among them,the differentially expressed genes involved in cardiovascular diseases are:Armc3,Ass1,Ccdc40,Cartpt,Adrald,Cfap45,Tmem255a,Tjp3,Scn4b,Lca51,Gstm6,Lmntdl,Cd55,Sec1413,Calml4,Colla2.4)Compared with the model group,Taichong group in the medulla oblongata differentially expressed genes are mainly enriched in cytokine-cytokine receptor interaction,tyrosine metabolism,Huntington’ s disease,transcriptional disorders in cancer,glycan biosynthesis and metabolism,retrograde Endogenous cannabinoid signaling,gamma-aminobutyric acid can synapse.Among them,differentially expressed genes related to cardiovascular diseases are enriched:Chrnb3,Th,Dbh,Wdr63,Gabrd,Gabra6,Nr4a3,LOC689064,Zfp36,Chrna6.In the hypothalamus,differentially expressed genes are mainly enriched in:PI3K-Akt signaling pathway,platelet activation,RAS signaling pathway,Rapl signaling pathway,calcium signaling pathway,cGMP-PKG signaling pathway,cAMP signaling pathway,and adrenergic energy in cardiomyocytes Signaling,vascular smooth muscle contraction,fluid shear stress,and atherosclerosis.Among them are differentially expressed genes associated with cardiovascular disease:Rpl37a,Rps14,LOC310926,LOC681766.In the thalamus,differentially expressed genes are mainly enriched in:cAMP signaling pathway,renin-angiotensin system,cGMP-PKG signaling pathway,MAPK signaling pathway,myocardial contraction,adrenergic signaling in cardiomyocytes,γ-amino Butyric acid(GABA)synapses,neurotrophin signaling pathways,aldosterone synthesis and secretion.Among them,differentially expressed genes related to cardiovascular diseases are enriched:Drdl,Npy,Massl,Bmp3,Adrald,Rasgrpl,Myh7,Gng7,Ntrk1,Nr4a1.Further qPCR experiments showed that the related pathway genes of acupuncture and hypotension were concentrated in the Adcyl gene,Glral gene,Htr5b gene,Th gene and Tubal α gene in the medulla oblongata;Enol gene,Gngl3 gene,Myorg gene,RT1-A3 gene in the hypothalamus,Ccr5 gene and β-globin gene;RT1-A3 gene of the thalamus.Conclusion1)Acupuncture at Tai Chong,Taixi and Taichong+Taixi can reduce the blood pressure of SHR,and the compatibility of acupoints is better than single-point.2)The thalamus is the key brain area of acupuncture and blood pressure newly discovered in this experiment.3)Acupuncture can regulate a number of signal pathways related to blood pressure regulation in the central nervous system.The in-depth mechanism needs to be further studied.