| Twirling Reinforcing-Reducing Manipulation(TRRM)is regarded as the key factor to achieve the efficacy of acupuncture.As one of the most commonly used TRRM during the clinical treatment,TRRM has long been the controversial focus in the field of acupuncture and moxibustion at home and abroad.Relevant clinical and experimental studies have shown that TRRM has control effects on various diseases,and can achieve the overall regulation of the body's life activities by a variety of pathways including neuro-endocrine-immune system,peripheral and central protein expression,cell activity,cell cytokines,signal transduction and sympathetic nerve discharge and so on.Due to many factors that are unfavorable to TRRM application,such as complex technique,nonstandard operating practices and inconsistent treatment effect,many scholars do not pay attention to TRRM or operate improperly in clinical and experimental studies at home and abroad,which directly affect the exertion of acupuncture in achieving its best curative effect.Essential hypertension(EH)is one of the most predisposing factors of cerebrovascular disease.Its main complications including cerebral vascular sclerosis,cognitive decline,and stroke,which not only has characteristics of high morbidity,high recurrence rate,low reversibility,and cure rate,but also lay a heavy burden on families and society.Studies have shown that the pathogenesis of EH is complex,and it is the result of a combination of genetic,environmental,stress,obesity,and high-salt diets.The central nervous system,especially sympathetic nerve activity and brain renin-angiotensin system,etc.plays a key role in the formation and development of hypertension.In the earlier stage of our research,based on the study of hypertension,we found that TRRM has a better antihypertensive effect and obtain much scientific research results on the peripheral antihypertensive mechanism of blood pressures(BPs)reduction and the protective mechanisms of heart,kidney,aorta and other target organs by TRRM intervention.However,the central mechanism of BPs regulation and the mechanism of protection against hypertensive cerebral injury by TRRM are still insufficient.The positron emission computed tomography,which can dynamically and intuitively reflect the changes in the morphological structure and function of the central brain regions at the molecular level in the non-invasive state,providing technology and platform for the study of the central activation effect of hypertension by TRRM intervention.Objective:To further elucidate the central mechanism of TRRM in BPs regulation,in this study,we selected spontaneously hypertensive rats(SHRs)as an EH animal model and observed the central activation effect of brain regions in SHRs by TRRMs interving "Taichong",based on the 18F-FDG-PET imaging.The key brain regions were screened and its physiological and pathological associations with BPs regulation were analyzed,for the purpose of further revealing the potential central mechanism of TRRM in the regulation of BPs,which also provided new targets and reliable theoretical support for the prevention and treatment of hypertension in clinical application.Methods:"Experiment 1":The effect of TRRM on the regulation of blood pressure in SHRs.Forty male SHRs were randomly and equally divided into 4 groups,a twirling reinforcing(B)group,a twirling reducing(X)group,a single-needle acupuncture(Z)group and a model(M)group(n=10/group).Ten Wistar rats were assigned to the control(K)group.The rats in the X,B and Z groups respectively received needling at bilateral Taichong(LR3)with twirling reducing manipulation,twirling reinforcing manipulation and single-needle acupuncture manipulation,while the rats in the K and M groups received no treatment just the same fixed stimulation as the X,B and Z groups.All interventions were administered in the afternoon between14:00-16:00,once daily for 28 days with 1-day intervals between every two weeks.The BPs of all rats were measured repeatedly on lday before acupuncture and on the 3rd,8th,13th,23rd,27th days at 8:00-12:00 AM,respectively.The purpose of this study is to observe the effect of different TRRMs on BPs changes in SHR."Experiment 2":The effects of PET imaging in SHRs by TRRM intervention for 14 days.The 18F-FDG-PET imaging scans were conducted on the days 1,14 and 28.All rats were sent to the PET Experimental Centre of the Chinese Academy of Sciences before 8:00.Cerebral images were acquired using PET after 24 hours of fasting.The experimental procedures was as follows:(1)Blood glucose was measured in samples from tail vein bleeds.(2)The rats were allowed to rest for 20 min in a dark and quiet room.(3)A tracer(18F-FDG)was injected via the tail vein for imaging,and the rats in the B,X and Z groups were accordingly subjected to RRTM intervention during this process.(4)PET was performed using a primate PET scanner.(5)The preprocessing and data analysis of the PET images.Combining experimental data:the changes of BPs in SHRs by TRRM intervention for 14 days in experiment 1 and the PET imaging data on days 1 and 14,we mainly investigated the central activation effect of TRRM on the brain regions of SHRs for 14 days,and to elucidate the potential central mechanism of short-term TRRM intervention on the regulation of blood pressure."Experiment 3":The effects of PET imaging in SHRs by TRRM intervention for 28 days.The experimental method is the same as experiment 2.Combining experimental data:the changes of BPs in SHRs by TRRM intervention for 28 days in experiment 1 and the PET imaging data on days 1 and 28,we mainly investigated the central activation effect of TRRM on the brain regions of SHRs for 28 days,and to elucidate the potential central mechanism of long-term TRRM intervention on the regulation of blood pressure."Experiment 4":The central mechanism of the effects on the hypothalamic RAS in SHRs by TRRM intervention.Fifty-six male SHRs were randomly and equally divided into 4 groups,a twirling reinforcing(B)group,a twirling reducing(X)group,a single-needle acupuncture(Z)group and a model(M)group(n=14/group).Fourteen Wistar rats were assigned to the control(K)group.The rats in the X,B and Z groups respectively received needling at bilateral Taichong(LR3)with twirling reducing manipulation,twirling reinforcing manipulation and single-needle acupuncture manipulation,while the rats in the K and M groups received no treatment just the same fixed stimulation as the X,B and Z groups.All interventions were administered in the afternoon between14:00-16:00,once daily for 14 days with 1-day intervals between two weeks.The BPs of all rats were measured repeatedly on 1day before acupuncture and on the 3rd,8th,13th days at 8:00-12:00 AM,respectively.On the 14th day,rats were anesthetized and then sacrificed to be taken the hypothalamus tissue,the pathological changes of hypothalamus were observed by HE staining,and the expression of hypothalamic RAS axis components were detected by ELISA and RT-qPCR.The purpose of this study is to further investigate the central mechanism of the antihypertensive effects of TRRM intervention for 14 days.Results:"Experiment 1":One day before acupuncture,there were no significant differences in systolic BPs between B?X?Z and M groups(all P>0.05),indicating that the baseline of BPs in each group was homogeneous and comparable.Compared with the K group,the systolic BPs in M?Z?B and X groups were significantly increased on the lday before and 3rd,8th,13th,18th,23rd,27th days(all P<0.01).Compared with M group,systolic BPs in X group had significantly decreased on the 3rd day(P<0.05),and on the 8th,13th,18th,23rd and 27th days(all P<0.01).Compared with the Z group,systolic BPs in B group was significantly decreased on the 8th day(P<0.05),and on the 13th,18th,23rd,and 27th days(all P<0.01);and X group decreased significantly on the 8th,13th,18th,23rd,and 27th days(all P<0.01).Compared with the B group,the systolic pressure in the X group was significantly decreased on the 13th,18th,23rd,and 27th days(all P<0.01).The results showed that the antihypertensive effects of TRRM intervention for 14 days and 28 days were both X group>B group>Z group."Experiment 2":The PET results showed that on the 1st day,compared with the M group,the K group showed significantly increased glucose metabolism mainly in the brain regions of striatum,cingulate cortex,visual cortex,corpus callosum,cerebellum,and sensory cortex.Higher rates of glucose metabolism in the striatum,thalamus,sensory cortex and motor cortex were found in the Z versus the M groups.Compared with the Z group,the B group exhibited increased glucose metabolism in the cerebellum,thalamus,parietal cortex and visual cortex,while the X group exhibited higher levels of glucose metabolism mainly in the hippocampus,cerebellum,and visual cortex.On the 14th day,compared with the M group,the K group showed significantly increased glucose metabolism mainly in the brain regions of thalamus,cerebellum,striatum and visual cortex.Higher rates of glucose metabolism in the cerebellum,midbrain,and hippocampus were found in the Z versus the M groups.Compared with the Z group,the B group exhibited increased glucose metabolism in the cerebellum,hippocampus,midbrain,thalamus,visual cortex,sensory cortex and motor cortex,while the X group exhibited higher levels of glucose metabolism mainly in the hypothalamus,medulla oblongata,cerebellum,hippocampus and insular cortex of the brain."Experiment 3":The PET results showed that on the 28th day,compared with the M group,the K group showed significantly increased glucose metabolism mainly in the brain regions of striatum,cingulate cortex,visual cortex,prelimbic cortex,olfactory cortex and motor cortex.Higher rates of glucose metabolism in the striatum,dorsal thalamus,sensory cortex,septal nucleus,internal capsule and corpus callosum were found in the Z versus the M groups.Compared with the Z group,the B group exhibited increased glucose metabolism in the cerebellum,dorsal thalamus,parietal cortex,visual cortex,hippocampus and midbrain,while the X group exhibited higher levels of glucose metabolism mainly in the medulla oblongata,hippocampus,olfactory bulb and septal nucleus of the brain."Experiment 4":Compared with the M group,the BP showed a significant decrease in the Z,B and X groups(all P<0.01),and the expression of the elevating-BP axis components in RAS were significantly decreased(all P<0.01),while the expression of the lowering-BP axis components in RAS were significantly increased(all P<0.01).Furthermore,the X group showed the better effects of lowering the BP and beneficially regulating the RAS than that of other groups(all P<0.01).Conclusion:1.TRRM can significantly lower the BP of SHR,and the antihypertensive effects of TRRM intervention for 14 days and 28 days were both X group>B group>Z group,moreover,the twirling reducing manipulation has the best antihypertensive effects in terms of treatment cycle and clinic effect,followed by the twirling reinforcing manipulation and the single-needle manipulation.2.TRRM intervention for 14 days can exert its central regulation of BP in SHR by increasing the level of glucose metabolism in multiple target brain regions such as hypothalamus,medulla oblongata,hippocampus,cerebellum,insular cortex,midbrain,thalamus,and visual cortex,and its potential central mechanism may be related to the neurotransmitters that exist in each of the target regions,especially the hypothalamic RAS has a more regulatory core role.3.TRRM intervention for 28 days can achieve the central regulation of BP in SHR by increasing the level of glucose metabolism in multiple target brain regions such as medulla oblongata,hippocampus,cerebellum,parietal cortex,olfactory bulb,septal nucleus,midbrain,thalamus and visual cortex and other brain regions,and its potential central mechanism may be mostly related to the neurotransmitters that exist in the brain regions of medulla oblongata,hippocampus,cerebellum and parietal cortex.4.TRRM intervention can exert its antihypertensive effect in SHR and achieve its protective effect on the hypothalamus neurons through beneficially regulating the expression of hypothalamic RAS axis components of SHR,which may be one of the underlying mechanisms that could explain the antihypertensive effect by TRRM intervention for 14 days. |
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