| ObjectiveThis study is based on the effect of renin angiotensin aldosterone system(RAAS)on the regulation of hypertension,which is a well-known pathogenesis of essential hypertension,to further study the effect of gene methylation modification on RAAS.It has been reported that acupuncture and moxibustion at Taichong can also regulate blood pressure,which is related to the expression of RAAS related proteins.However,there is no report that acupuncture and moxibustion at Taichong can regulate the methylation of RAAS gene.Therefore,on the basis of a large number of studies,we speculated that acupuncture at Taichong could regulate the methylation of RAAS gene in hypothalamic paraventricular nucleus,and regulate the blood pressure of essential hypertensive rats by affecting the excitability of sympathetic nervous system,Try to find a series of changes related to essential hypertension from the gene level.This study explored the molecular mechanism of the occurrence and development of essential hypertension,and further explored the therapeutic mechanism of acupuncture effect,so as to provide experimental basis for modern acupuncture theory.MethodsTwenty four hypertensive rats(systolic blood pressure≥160mmhg)were randomly divided into three groups(8 rats in each group):(1)model group(2)Acupoint group(3)Non acupoint group.Eight normal male Wistar rats served as blank control group.Essential hypertensive rats were purchased from Beijing Weitong Lihua Experimental Animal Technology Co.,Ltd.,which has similar pathogenesis with human essential hypertension patients.Referring to experimental acupuncture and moxibustion,acupoint Atlas of experimental animals and acupoint positioning method of simulated human body,the acupoint group received acupuncture intervention at Taichong Point on both sides of rats,while the non acupoint group received acupuncture intervention at the depression between the third and fourth metatarsal bones on the dorsal foot of rats.Acupuncture methods:in the acupoint group and non acupoint group,the rats were fixed with cloth cover/fixator,and the acupoint area was routinely disinfected.The 0.25 mm x 13 mm stainless steel filiform needle was directly punctured into the bilateral acupoints,and the pingbu pingxie technique was used.The needle was retained for 30 minutes,once a day(acupuncture intervention was given at 9:00-12:00 a day),for 14 consecutive days.The rats in the model group and the blank control group were fixed for 30 minutes at the same time without acupuncture.Electronic balance was used to record the body weight of rats in each group after acupuncture intervention for 14 consecutive days;Bp-6 animal noninvasive blood pressure test system was used to record the systolic blood pressure,diastolic blood pressure and mean arterial pressure of rats in each group after acupuncture intervention for 14 consecutive days;The plasma concentrations of AGT,AngⅡ and ACE were detected by ELISA after 14 days of acupuncture intervention;Genomic bisulfite sequencing(wgbs)was used to analyze the methylation of RAAS related essential hypertension gene in hypothalamic paraventricular nucleus(PVN)of rats after 14 days of acupuncture intervention;RT qPCR was used to detect the expression of Agt and Ace mRNA in hypothalamus of rats in each group after 14 days of acupuncture intervention;ELISA was used to detect the protein concentration of AGT,AngⅡ and ACE in hypothalamus of rats in each group after 14 days of acupuncture intervention.Results1.The results of body weight measurement showed that there was no difference in body weight between the normal group and the model group during the experiment;compared with the model group,the body weight of the acupoint group and the non-acupoint group decreased(P<0.001),and the decrease degree of the acupoint group was more obvious,and there was a marked difference versus the non-acupoint group(P<0.05).2.The results of blood pressure measurement showed that acupuncture at Taichong could decrease the blood pressure of rats.Compared with the blank control group,the blood pressure of the model group was significantly increased,and there was no effect on the blood pressure of the model group before and after restraint(P<0.001);On the first day before the intervention,the blood pressure of each group was similar.On the seventh day after the intervention,the systolic blood pressure of the acupoint group decreased to(120.918 ±9.853)mmHg,and the blood pressure of non acupoint group also decreased to(138.837 ± 17.756)mmHg,which was significantly different from that of the model group(P<0.001).After 14 days of intervention,the systolic blood pressure of the acupoint group decreased to(114.503±9.514)mmHg,and the blood pressure of non acupoint group also decreased(152.280 9±18.022)mmHg,which was significantly different from that of the model group(P<0.001);Compared with the acupoint group,the decrease of systolic blood pressure in the non acupoint group was smaller(P<0.001).The blood pressure in the acupoint group and non acupoint group decreased most significantly after 7 days of acupuncture intervention(P<0.001);Compared with the acupoint group,the blood pressure in the non acupoint group decreased slightly,and after 7 days of acupuncture intervention,the blood pressure could not be maintained at a certain level,showing an upward trend.3.The results of ELISA demonstratedthat:ACE protein content of the acupoint group and non acupoint group were(0.316±0.452)ng/ml and(1.508 ± 0.850)ng/ml respectively,which were significantly different from those of the hypertension model group(6.797 ± 1.000)ng/ml(P<0.001),while the ANGII protein content of the acupoint group and non acupoint group were(14.386 ±8.961)ng/ml and(25.376±8.865)ng/ml respectively,which were significantly different from those of the hypertension model group(101.828±22.160)ng/ml(P<0.001).There was no significant difference in the concentration of AGT protein between the two groups.4.The results of hypothalamic gene methylation showed that the degree of promoter methylation in Agt gene model group was(0.10240±0.0942)compared with the blank control group,the methylation level was lower(P<0.001);The methylation level of promoter in Ace gene model group was 0.1797(0.0049,0.6919),which was lower than that in blank control group(P=0.044);The results showed that the methylation level of Ace gene promoter log2 ratio(normal control group/model group)was 4.744,and the methylation level of Ace2 gene promoter log2 ratio(normal control group/model group)was 4.411;The methylation level of AGT gene promoter log2 ratio(normal control group/model group)was 2.792,that of agtr1a gene promoter log2 ratio(normal control group/model group)was 6.964,and that of Scn 1b gene promoter log2 ratio(normal control group/model group)was-2.715.The promoter methylation degree of Agt gene acupoint group was(0.4272±0.2120)compared with the model group,the level of methylation increased(P=0.004).The methylation level of Ace gene promoter in acupoint group was 0.7606(0.4484,0.9163),which was higher than that in model group(P=0.002).Compared with the model group,the methylation degree of Ace gene promoter log2 ratio(model group/acupoint group)and Agt gene promoter log2 ratio(model group/acupoint group)in the acupoint group were-3.722 and-4.513,respectively;The methylation degree of Ace gene promoter log2 ratio(model group/non acupoint group)was-3.744,and that of AGT gene promoter log2 ratio(model group/non acupoint group)was-3.192.5.RT qPCR was used to detect the expression of Agt and Ace mRNA in hypothalamus.The results showed that the mRNA cycle threshold of AGT gene model group was(7.4470±0.2015)compared with the blank control group,the mRNA expression level was significantly higher(P<0.001);In the acupoint group,the circulating threshold of promoter mRNA was(5.39933 ± 0.1697)compared with the model group,the mRNA expression level was significantly lower(P<0.001);In the non acupoint group,the threshold of promoter mRNA cycle was(5.35433±0.2299)compared with the model group,the mRNA expression level was significantly lower(P<0.001).In ACE gene model group,the threshold of promoter mRNA cycle was(12.02300±0.4158)compared with the blank control group,the mRNA expression level was significantly higher(P=0.001);In the acupoint group,the circulating threshold of promoter mRNA was(7.5360 ± 0.7226)compared with the model group,the mRNA expression level was significantly lower(P<0.001);In the non acupoint group,the threshold of promoter mRNA cycle was(11.3400 ±0.3236)compared with the model group,the mRNA expression level was significantly lower(P<0.001).6.The results of ELISA showed that the concentration of AGT protein was(299.0639±0.259)ng/ml,which was higher than that of the blank control group(P=0.049);The histone concentration was(201.2279 ± 25.6820)ng/ml compared with the model group,the protein concentration decreased(P=0.017);The concentration of non acupoint histone was(230.1576 ± 11.8072)ng/ml compared with the model group,the protein concentration decreased(P=0.103).The histone concentration of ACE protein model was(158.6562 ± 1.1636)ng/ml compared with the blank control group,the protein concentration increased significantly(P<0.001);The histone concentration of acupoints was(118.5098 ±6.8672)ng/ml compared with the model group,the protein concentration decreased significantly(P<0.001).The histone concentration of ANGII protein model was(443.8186±13.0676)ng/ml,which was significantly higher than that of the blank control group(P<0.001);The histone concentration was(179.3201±7.1996)ng/ml compared with the model group,the protein concentration decreased significantly(P<0.001);The non acupoint histone concentration was(192.3393±14.5024)ng/ml compared with the model group,the protein concentration decreased significantly(P<0.001).Conclusion1.In this study,essential hypertension rats were used to simulate human essential hypertension.In 1963,Okamoto mated male Wistar Kyoto rats with significant hypertension symptoms with female Wistar Kyoto rats with mild hypertension symptoms.Since then,siblings mated and spontaneously hypertensive rats were continuously selected for culture.It has no obvious renal injury with stable hypertension,and can be used in experimental research.2.Acupuncture at Taichong Point can reduce the weight of primary hypertension rats.The experimental results show that acupuncture at Taichong Point can regulate the blood pressure of rats,which may be related to the control of the weight of rats.After 14 days of intervention,the weight of rats in the acupoint group decreased more than that in the non acupoint group,indicating that acupuncture at Taichong Point can indeed regulate the weight growth of rats,It is speculated that acupuncture at Taichong can regulate blood volume and blood pressure.3.After 7 days of acupuncture at Taichong Point,the blood pressure of primary hypertension rats was decreased,and the amplitude and stability of blood pressure in the acupoint group were stronger than those in the non acupoint group.Taichong Point is the place where Qi,blood and meridians gather.The results confirmed that acupuncture at Taichong could indeed reduce the blood pressure of primary hypertension rats,and the effect of the acupoint group was better than that of the non acupoint group,which was consistent with the results of similar literature.4.This study also confirmed the effect of Taichong Point Acupuncture on peripheral RAAS protein index of primary hypertensive rats,which is consistent with the results of similar literature,and speculates that it may be related to the local RAAS of central hypothalamus.5.Acupuncture at Taichong can increase the methylation level of ACE and AGT gene promoter in hypothalamic paraventricular nucleus.The experimental results show that acupuncture at Taichong can regulate the methylation level of RAAS related genes,change the methylation level of promoter sequence,and affect the local tissue of hypothalamus.6.Acupuncture at Taichong Point can regulate the expression of RAAS in hypothalamus.The results suggest that acupuncture intervention can increase the methylation level of ACE and AGT gene promoter in hypothalamus,and affect the peripheral RAAS and blood volume by affecting the RAAS in central area,so as to achieve the purpose of regulating blood pressure.This may be one of the deeper mechanisms of acupuncture at Taichong Point down regulating the peripheral circulation of ACE,AngⅡand other hypertension factors. |
PDF Full Text Request