| Objective:Based on the previous research,this study explored further in the effect of the twisting for reinforcing and reducing methods in acupuncture on the mechanismin the central effect of blood pressure reducing and regulating on spontaneously hypertensive rats(SHR).By applying the proteomics technique in the screening of differential proteins in the cerebellum of rats,the key proteins and pathways involved in the development of hypertension were selected in this study.The PRM technique was used in verifying and analyzing the proteins,in order to explain the mechanism of antihypertensive effects of the twisting for reinforcing and reducing methods in acupuncture towards SHR.At the same time,by detecting the expressions of neurotransmitters which areclosely related to the incidence of hypertensionin the cerebellar tissues of rats in each group,this study aimed to explore the new mechanism of the effect of the twisting for reinforcing and reducing methodsin reducing blood pressure,find out the better methods of acupuncture intervention,and to enrich the existing research.In addition,this study aimed to provide a possible theoretical and experimental basis for the central mechanism of action of the twisting for reinforcing and reducing methods in acupuncture with multiple targets,levels and approaches,and to provide a possible idea for the treatment of hypertension with new clinical techniques.Methods:Fifty-six male SHR were randomly divided into four groups:the twisting-for-reinforcing method group(B),twisting-for-reducing method group(X),electroacupuncture group(E),and the model group(M),with 14 rats in each group,and 14 Wistar-Kyoto(WKY)rats were used as a blank group(WKY).The rats in groups B,X and E were treated withthe twisting for reinforcing and reducing methods and electroacupuncture on bilateral Taichong point(LV 3),and the groups W and M were not treated with any acupuncture methods but were given the grasping and fixation stimulation.Methods:acupuncture on bilateral Taichong point(LV 3)at 14:00-16:00 everyday.Twisting on the left point for 3 minutes and then on the right point for 3 minutes,once a day for 14 days.The rate for twisting was 60/min,with the range of 360°.The blood pressure of rats was checked during 8:00-12:00 on the previous day,and 4th,8th,12th and 14th day of acupuncture.On the 15th day of the experiment after the end of all treatments,the cerebella of the rats were selected and HE staining was used to observe the shapes of the cerebella in each group.Lable free proteomics technology was used to screen the differential proteins and pathways of the cerebella.The PRM technology was used to verify the key proteins,and ELISA technology was used to detect the content of GABA and Glu in the cerebella.Results:"Experiment 1":(1)there was little change of systolic blood pressure values in group WKY at the 0,4th,8th,12th and 14th days,with no statistical difference when comparing with other groups(P<0.05).There was no significant difference of the systolic blood pressure values among groups B,X,E and M on the 0 day(the previous day of acupuncture)(all P>0.05),which indicate that the baselines of the four groups were equal and comparable.①Compared with group M,the systolic blood pressure values of groups B,X,and E decreased significantly from the 8th,12th,and 14th days(all P<0.05).②Compared with group B,the systolic blood pressure values of group X decreased on the 8th day of acupuncture(P<0.05).Groups X and E decreased on the 12th and 14th days of acupuncture(P<0.05).(2)The molecular layer cells in the WKY group are small in shape and loosely arranged.Purkinje cells are densely arranged and uniform in size,with regular boundaries,and the nuclei are oval or circular.The cells in the granular layer are arranged closely and the overall layer is arranged.The cerebellar Purkinje cells in group M were loosely arranged,the number decreased and the gap widened,and the granular cell layer was loose.Compared with the group M,the Purkinje cell layers in the three treatment groups B,X,and E were arranged more neatly,and the granular cell layers were arranged orderly and tightly."Experiment 2":(1)Compared with the group M,the number of differential proteins identified by M vs WKY was 397(203 up-regulated and 194 down-regulated).The number of differential proteins identified by B vs M was 96(42 up-regulated and 54 down-regulated).The number of differential proteins identified by X vs M was 216(88 up-regulated,128 down-regulated).The number of differential proteins identified by E vs M was 133(72 up-regulated and 61 down-regulated).(2)GO function results show that the pathogenesis of hypertension and mechanism of acupuncture in reducing blood pressure mainly involves the following aspects:The biological process mainly involves metabolism,immune response,multi-cellular component tissues or biological generation,etc.The cellular component mainly involves organelles,extracellular regions,cell membranes and synapses.The molecular function mainly on molecular sensor activity,catalytic activity,binding and transport activity,etc.(3)The KEGG enrichment pathway showed that compared with group WKY,there are 41 pathways involved by differential proteins which are statistically significant(P<0.05)in group M:Ras signaling pathway,mTOR signaling pathway,cAMP signaling pathway,insulin resistance,oxidative phosphorylation are closely related to the pathogenic mechanism of hypertension.Compared with group M,there were 12 pathways with significant protein enrichment(P<0.05)in group B,which are mainly related to vascular smooth muscle contraction,oxidative phosphorylation and VEGF signaling pathway.Compared with group M,there were 11 pathways with significant protein enrichment(P<0.05)in group X,which are mainly related to gap junctions.Compared with group M,there were 15 pathways with significant protein enrichment(P<0.05)in group E,which are mainly related to oxidative phosphorylation.(4)The results of PRM verification show that:Among all the differential proteins,13 proteins were selected for PRM verification,and it was found that the overall trend of the PRM verification results and the Lable free quantitative results were consistent.The KEGG annotations of differential proteins are in the regulation of lipolysis in adipocytes,autophagy,gap junctions,phosphoinositide metabolism,spliceosomes,lysosomes,thermogenesis,PPAR signaling pathways,and RNA transport,etc."Experiment 3":Compared with the groupM,the group WKY had significant differences in the content of GABA and Glu in the cerebella(P<0.01).Compared with the model group,the GABA content in the cerebellar tissue of the twisting-for-reinforcing method group,twisting-for-reducing method group and electroacupuncture group increased significantly(P<0.01),and the Glu content was significantly reduced(P<0.01).Compared with the twisting-for-reinforcing method group and electroacupuncture group,the twisting-for-reducing method group had the best effect.There was no statistical difference(P>0.05)between the twisting-for-reinforcing method group and electroacupuncture group.Conclusion:1.Both the twisting for reinforcing and reducing methods in acupuncture and electroacupuncture can significantly reduce SHR blood pressure and reduce brain damage.The effect of pressure reduction by twisting for reducing method is the most obvious,and the range of pressure reduction by it is the most significant.2.Different acupuncture methods have different screening and activated pathways for cerebellar differential proteins.The pathogenesis of SHR may be closely related to Ras signaling pathway,oxidative phosphorylation pathway,mTOR signaling pathway,cAMP signaling pathway and insulin resistance pathway.The antihypertensive mechanism of acupuncture may be closely related to vascular smooth muscle contraction,VEGF signaling pathway,oxidative phosphorylation and gap junction pathway.3.The decrease of GABA content in the SHR cerebella and the increase of Glu content may be one of the central pathogenesis of SHR.Acupuncture reduces blood pressure by adjusting the content of GABA and Glu in the cerebella.The effect of the twisting-for-reducing method group is the most significant. |
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