| Objective:In order to further clarify the central effect mechanism of Twirling Reinforcing Reducing Manipulation(TRRM)on blood pressure regulation,we performed a proteomics analysis of spontaneously hypertensive rats(SHRs)treated with acupuncture twirling reinforcing manipulation(TRFM),twirling reducing manipulation(TRDM)and electroacupuncture(EA)on "Taichong"(LR3).Moreover,we used parallel reaction monitoring msvalidation(PRM)to validate the reliability of the differentially expressed proteins(DEPs).This study is to reveal the central antihypertensive mechanism of TRFM and TRDM from the protein molecular level,and to find the potential protein target of acupuncture,to provide experimental basis for the clinical application of TRFM and TRDM in the treatment of hypertension.Methods:56 male SHRs were randomly divided into 4 groups:the model group(M),the twirling reinforcing manipulation group(B),the twirling reducing manipulation group(X)and the electroacupuncture group(E),14 SHRs in each group,and 14 Wistar-Kyoto(WKY)rats were used as blank control group(WKY).The bilateral "Taichong"(LR3)of SHR rats in group B,X and E were used as acupuncture points,and TRFM,TRDM and EA were applied respectively,while WKY and M groups were not treated with acupuncture,but were given the same stimulation of capture and fixation.Acupuncture manipulation was performed once a day for 14 days.The blood pressure was measured on the first day and the 2nd,4th,6th,8th,10th,12th and 14th day.On the 15th day,we collected hypothalamus samples.The morphology of hypothalamic neurons was observed by HE staining,the expression of hypothalamic DEPs was analyzed by Labelfree proteomics,the key DEPs of SHR hypothalamus were verified and analyzed by PRM technique,the contents of AngⅡand Ang(1-7)in hypothalamus were detected by ELISA,the activity of SOD was detected by xanthine oxidase method,and the content of MDA was detected by TBA method.Results:“Experiment 1”:(1)One day before acupuncture(the 0th day),there were no significant differences in systolic BPs between B、X、E and M groups(all P>0.05),indicating that the baseline of BPs in each group was homogeneous and comparable.①Compared with the WKY group,the systolic BPs of M group was significantly increased on the 0th 2nd,4th,6th,8th,10th,12th and 14th days(all P<0.05).②Compared with the M group,the systolic BPs in B,X and E groups were decreased from the 6th day,and decreased significantly on the 6th,8th 10th,12th and 14th days(all P<0.05).③Compared with the B group,the systolic BPs of the X group was decreased significantly on the 10th and 14th days(P<0.05),the E group was decreased significantly on the 14th day."Experiment 2":(1)Compared with the M group,the number of DEPs of the M vs WKY group identified was 417(146 up-regulated,271 down-regulated),the number of DEPs of the B vs M group identified was 130(93 up-regulated,37 down-regulated),the number of DEPs of the X vs M group was 174(107 up-regulated,67 down-regulated),the number of DEPs of the E vs M group was 207(134 up-regulated,73 down-regulated).(2)Gene Ontology(the top ten):in the M vs WKY group,extracellular exosome,regulation of cellular component organization,Golgi apparatus,hydrolase activity,acting on acid anhydrides,in phosphorus-containing anhydrides,cellular response to DNA damage stimulus,protease binding,Lys48-specific deubiquitinase activity,retinoid binding.In the B vs M group,extracellular exosome,mitochondrion,regulation of cellular component organization,purine-containing compound metabolic process,regulation of transport,ribose phosphate metabolic process,transmembrane transport,cytosolic part,cholesterol binding,protein farnesyltransferase complex.The X vs M group includes extracellular exosome,transmembrane receptor protein kinase activity,nucleolus,hydrolase activity,acting on acid anhydrides,in phosphorus-containing anhydrides,RNA binding,cellular amide metabolic process,wound healing,cytosol,cellular nitrogen compound biosynthetic process,cytosolic part.The E vs M group includes extracellular exosome,protein tyrosine phosphatase activity,cytosolic part,mitochondrial inner membrane,nucleoside phosphate metabolic process,regulation of cellular component organization,actin binding,Lys48-specific deubiquitinase activity,endoplasmic reticulum to cytosol transport,mitochondrion.(3)KEGG pathway:in the M vs WKY group,there were Hippo signal pathway,oxidative phosphorylation pathway,serotonergic synapse,cAMP signal pathway,chemokine signal pathway and glucagon signal pathway.In the B vs M group,there was Apelin signal pathway.In the X vs M group,there were Hippo signal pathway and PI3K-Akt signal pathway and Hippo signal pathway and oxidative phosphorylation pathway were related to the E vs M group.(4)IP A analysis:the regulation of acupuncture Taichong was related to cytoplasmic tissue,cytoskeleton tissue,microtubule dynamics,neuronal development and neurite formation.The key genes involved were A2M,RAB33A,DGKG,SCL13A5,CCDC85A and CRLF1 up-regulated,while RANBP10,SARAE,MYL9,M1c and BEX1 genes down-regulated"Experiment 3":The results of PRM verification of differential proteins were consistent with the results of proteomics.There are differences in the expression of different proteins regulated by different acupuncture interventions.TRFM can significantly up-regulate the expression of Hebp2 and PSPH and down-regulate the expression of Dyrk1A;TRDM can significantly up-regulate the expression of Rab10 and PSPH;EA can significantly up-regulate the expression of Hebp2,CHL1,NENF,POMGnT2,POMGnT2 and down-regulate the expression of Dyrk1A."Experiment 4":Compared with the WKY group,the content of AngⅡ in the M group was significantly higher than that in the group WKY(P<0.05),and the content of AngⅡ in group B,X and E was significantly lower than that in the group M(P<0.05).Compared with the WKY group,the content of Ang(1-7)in the M group decreased significantly(P<0.05),and compared with the M group,Ang(1-7)in B,X and E groups increased after intervention and increased more significantly in X and E groups(P<0.05).Compared with the WKY group,the expression of SOD activity in the M group decreased significantly(P<0.01),and the expression of SOD activity in B,X and E groups increased significantly compared with the M group(P<0.01).Compared with the B group,SOD activity in X and E groups increased more(P<0.01).Compared with the WKY group,the content of MDA in the M group increased significantly(P<0.01).Compared with group M,and the content of MDA in B,X and E groups decreased significantly after intervention,and that in groups X and E significantly decreased(P<0.01).Compared with group B,the content of MDA in groups X and E increased more(P<0.01).Conclusion:1.TRFM,TRDM and EA can significantly lower the SBP of SHR,and.TRDM and EA have the better antihypertensive effects in terms of treatment,and the systolic blood pressure decreased significantly from the 6th day.2.TRFM,TRDM and EA can reduce blood pressure by affecting the expression of DEPs in hypothalamus of SHR.There are differences in DEPs and pathways affected by different acupuncture techniques.3.The expressions of Hebp2,CHL1,Rab10,NENF,POMGnT2,Dyrkla,PSPH and NDUFAF4 were regulated in different degrees by TRFM,TRDM and EA intervention.It is speculated that the central effect of acupuncture Taichong(LR3)regulating blood pressure is related to oxidative stress;neuronal proliferation differentiation and regeneration;insulin resistance;neuronal excitability and vascular endothelial function.4.TRFM,TRDM and EA can regulate the contents of AngⅡ and Ang(1-7)in hypothalamus of SHR,increase SOD activity and reduce the content of lipid oxidation product MDA.TRDM and EA have better effects than TRFM.The effect of acupuncture on regulating blood pressure may be related to regulating the contents of AngⅡ and Ang(1-7)and reducing oxidative stress injury in the hypothalamus. |
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