| ObjectiveIn order to research the effect of acupuncture at TaiChong point (LR3) and non-acupoint on the blood pressure in Spontaneously Hypertensive Rats (SHR), and investigate, in whole level of protein expression, the central nerve mechanism of acupuncturing TaiChong point (LR3) to treat SHR, which provide the experimental clue for searching the specific indicators of biological effects that connect to the meridian effect, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technologies were used and the differentially expressed proteins in the medulla oblongata of SHR were also separated and identified.Methods309-week-old male SHR were randomly divided into model group, TaiChong group and non-acupoint group, each group involve 10 SHR. And another normal control group involves 10 SD rats of normal blood pressure with the same weight with SHR. In the TaiChong group, acupuncture bilateral Taichong points (located between the 1st and the 2nd metatarsal of dorsal hind); while in non-acupoint group acupuncture bilateral non-acupoint (located at the indentation between the 3rd and 4th metatarsal of dorsal hind). As rats awake, in Taichong group and non-acupoint group, daily acupuncture 5 minutes per times, twisting nedlle 80±5 times/min frequently, Twist nedlle 180±5 degrees in rang. In Taichong group, the needle towards Yongquang but not penetrate or reach the plantar skin, rather than in non-acupoint group, needle perpendicular to the skin and penetrate the skin.5 minutes after acupuncture, using BP-6 non-invasive blood pressure measuring system for tail arterial blood pressure measurement in rats. Acupuncture treatment continnues totally 7 weeks, all the rats brains were removed at the end, then separated in the ice for the medulla oblongata. And the medulla oblongata is cracked, to Sonication, desalted, seperated for proteins with the 2-DE. Geting the gel images with Typhoon9200 scanner, analyzing the differences between the various groups of protein with Imagesaster 6.0. With MALDI-TOF-TOF tandem MS analysis, protein mass image was obtained. And with the GPS software, to search and identify protein in the MASCOT database identification, then used enzyme-linked immunosorbent assay (ELISA) test to further verify the identification of the protein.Results1. The influence of acupuncture at Taichong point (LR3) or non-acupoints on the blood pressure SHR'blood pressure. (1) Comparison results among groups: The original, before acupuncture, systolic blood pressure of Taichong group, and the non-acupoint group and model group were not significantly different (all P> 0.05). The 1st day in acupuncture, in Taichong group, and the non-acupoint group and model group, systolic blood pressure between the rats were not significantly different (all P> 0.05). The next day in acupuncture, compared with the model group and non-acupoints group, systolic blood pressure of Taichong group significantly decreased (P<0.01), prompting that systolic blood pressure of Taichong group started to decrease in the next day in acupuncture. The 3rd day in acupuncture, compared with model group, systolic blood pressure of the non-acupoint group were significantly higher, suggesting that the acupuncture at non-acupoint may be a non-benign stimulus, resulting in a significantly increased of systolic blood pressure in rats. On the 4th in acupuncture, systolic blood pressure of Taichong group and the non-acupoint group and model group were not significantly different (all P> 0.05). On the 5th day of acupuncture, compared with non-acupoint, Taichong group's blood pressure decreased significantly (P<0.01). In acupuncture 6th, 7th day, compared with the model group and non-acupoints group, Taichong group' systolic blood pressure were significantly lower (P<0.01), suggested acupuncture at Taichong points begin to stabilize the blood pressure level in the 5th. In different time periods, in the model group, Taichong group and non-Meridian Group, systolic blood pressure were significantly higher than normal group (P<0.01), prompted acupuncture at Taichong or non-acupoint were not able to restore blood pressure to normal levels in this study.(2) Comparison results in each group: Compared with the 1st day, Taichong group systolic blood pressure decreased significantly in the next day (P<0.01); but on the 3rd and the 4th day, the systolic blood pressure were increased in small rate (P> 0.05), and then began to decrease at the 5th day (P<0.01), until the 6th day,7th day the reducing of systolic blood pressure remained stable (P<0.01), suggested acupuncture at Taichong points begin to stabilize the blood pressure level in the 5th. Non-acupoints group'systolic blood pressure were not significantly different in different time (P> 0.05). Compared with the 3rd day, the model rats'systolic blood pressure significantly increased on the 6th day (P<0.01); compared with the 4th day, model group's systolic blood pressure were significantly increased in the 6th day and the 7th days (P<0.01), prompting that systolic blood pressure of model group was rising. In different time, in nomal group, systolic blood pressure between the rats were not significantly different (all P> 0.05).2. The two-dimensional gel electrophoresis (2-DE) image of rat's medulla in each group, and its analysis. In this study,2-DE image were successfully obtained for normal SD rats, SHR and the SHR after acupuncturing Taichong or non-acupoint; and 621±30 proteins in normal SD rats'medulla,621±20 proteins in SHR's medulla, 621±40 proteins in medulla of SHR after acupuncturing Taichong,621±20 proteins in medulla of SHR after acupuncturing non-acupoint were found also. Compared with SHR in model group,88 proteins were found differentially expressed in medulla from Taichong group (expression ratio of protein was beyond 2 folds); 91 proteins were found differentially expressed in medulla oblongata from non-acupoints group (expression ratio of protein was beyond 2 folds),47 proteins were found differentially expressed in medulla oblongata from normal group (expression ratio of protein was beyond 2 folds).3. Differences in protein expression in the medulla of rats that dentified. (1) In medulla of Taichong group, compared with model group,5 proteins were down-regulated (expression ratio of protein was beyond 2 folds):Synapsin-1, NAD dependent deacetylase, electron transfer flavin protein and myelin basic protein; 6 proteins were up-regulated (expression ratio of protein was beyond 2 folds): glutamate dehydrogenase, glutathione S transferase, aldehyde dehydrogenase 2, Rho GDP dissociation inhibitor, DJ-1 protein and superoxide dismutase. (2) In medulla of non-acupoint group, compared with model group,1 proteins were up-regulated (expression ratio of protein was beyond 2 folds):electron transfer flavoprotein; 3 proteins were down-regulated (expression ratio of protein was beyond 2 folds): Heat shock protein 90, the Ubiquitin hydrolase isozyme and DJ-1 protein. (3)In medulla of Taichong group, compared with non-acupoints group,7 proteins were down-regulated (expression ratio of protein was beyond 2 folds):Heat shock protein 90, Synapsin-1, pyruvate kinase, NAD dependent deacetylases enzyme, protein kinase C inhibitor protein 1, Ubiquitin hydrolase isozymeand myelin basic protein; 6 proteins were up-regulated (expression ratio of protein was beyond 2 folds):glutamate dehydrogenase, aldehyde dehydrogenase 2, glutathione S transferase, Rho GDP dissociation inhibitor, DJ-1 protein and superoxide dismutase. (4) In medulla of model group, compared with normal group,6 proteins were down-regulated (expression ratio of protein was beyond 2 folds):pyruvate kinase, glutamate dehydrogenase, glutathione S transferase, aldehyde dehydrogenase 2, Ubiquitin hydrolase isozyme and superoxide dismutase; 7 proteins were up-regulated (expression ratio of protein was beyond 2 folds):Heat shock protein 90, Synapsin-11, NAD dependent deacetylase, electron transfer flavin protein, protein kinase C inhibitor protein 1, and myelin basic protein. (5) In medulla of Taichong group, compared with normal group,3 proteins were down-regulated (expression ratio of protein was beyond 2 folds):superoxide dismutase and myelin basic protein;4 proteins were up-regulated (expression ratio of protein was beyond 2 folds): Heat shock protein 90, Rho GDP dissociation inhibitor, DJ-1 protein and superoxide dismutase. (6) In medulla of non-acupoint group, compared with the normal group, 4 proteins were down-regulated (expression ratio of protein was beyond 2 folds): glutamate dehydrogenase, aldehyde dehydrogenase 2, glutathione S transferase and over superoxide dismutase; 7 proteins were up-regulated (expression ratio of protein was beyond 2 folds):Heat shock protein 90, synapsin 1, NAD dependent deacetylase, protein kinase C inhibitor protein 1, Ubiquitin hydrolase isozyme, DJ-1 protein and myelin basic protein.5. results of ELISA(1) The expression changes of glutathione S transferase in medullary among each group. In the medulla model group, compared with normal group, the expression of glutathione S transferase was significantly down-regulated (P<0.01). After acupuncture treatment, the expression of glutathione S transferase in the medulla of Taichong group were significantly up-regulated, compared with the model group, and the non-acupoint group, (P<0.01); but the expression of glutathione S transferase in the medulla of non-acupoint group has no significant difference form the model group (P> 0.01), and lower than the normal group (P<0.01). (2) The expression changes of aldehyde dehydrogenase in the medulla among each group. In the medulla model group, compared with normal group, the expression of aldehyde dehydrogenase was significantly down-regulated (P<0.01). After acupuncture treatment, the expression of aldehyde dehydrogenase in the medulla of Taichong group were significantly up-regulated, compared with the model group, and the non-acupoint group, (P<0.01); but the expression of aldehyde dehydrogenase in the medulla of non-acupoint group has no significant difference form the model group (P> 0.01), and lower than the normal group (P<0.01) (3) The expression changes of protein kinase C in medullary among each group. The expression of protein kinase C in the medulla of model group has no significant difference form the nomal group (P> 0.01); After acupuncture treatment, expression of protein kinase C in medullary of Taichong group has no significant changes from the model group, normal group, and the non-acupoint group (all P> 0.01). ELISA results of Glutathione S transferase, aldehyde dehydrogenase are consistent with the two-dimensional gel electrophoresis; meanwhile the ELISA results of protein kinase C are inconsistent with the results of two-dimensional gel electrophoresis of protein kinase C inhibitor protein 1.Conclusion1. Acupuncture Taichong can reduce the hypertension of SHR, but were not able to restore blood pressure to normal levels in this study.2. acupuncture at non-acupoints didn't have an obvious effect on decreasing the blood pressure in SHR, even cause increase in SHR hypertension.3. Acupuncture Taichong Point can down-regulated expressions of the proteins that promote excitatory neurotransmitter, induced inflammatory response, oxidative phosphorylation and induce oxidative stress protein, and up-regulate amino acid oxidation chaperones and energy metabolism and oxidative stress in SHR' medulla. Its mechanism is a multi-channel, multi-link, the combined effect of multiple targets.4. Acupuncture non-acupoint can down-regulated expressions of the proteins that promote energy metabolism, and up-regulate amino acid oxidation, protein catabolism and expressions of protein that induced inflammatory response in medullary of SHR. The mechanism is less means, fewer links, fewer target Point of simple effect.5. Acupoints effect and its mechanism has some specificity.
|
PDF Full Text Request