Although there has been some discordance as to the efficacy of acupuncture in hypertension 
, our results indicate that acupuncture at the Taichong (LR3) acupoint does alleviate high blood pressure due to essential hypertension, though this treatment alone is not able to bring blood pressure down to normal levels. When discussing the efficacy of acupuncture as a treatment for hypertension, it is important to distinguish between the short-term and long-term effect of acupuncture on hypertension. Our study, like most others, reports the short-term effect of acupuncture (directly following treatment) on blood pressure. Due to the proteomic nature of our study and the necessary euthanasia of the rats, the long-term effect of acupuncture on blood pressure regrettably could not be studied.
There are remarkable gender differences in many physiological parameters in health and disease. To avoid the interfering effect of female sex hormones on blood pressure, we have chosen male animals in this study. Studies have revealed sex hormones play a role in the regulation of blood pressure and development of hypertension 
. The prevalence of hypertension is predicted to increase more among women than men. When rats were fed with an 8% NaCl diet, female rats became less hypertensive than male rats 
, which suggests that female sex hormones protect against the development of hypertension.
As stabilization of blood pressure in the Taichong group takes 5 days, the lag time between acupuncture treatment and stabilization of blood pressure suggests activation of specific metabolic pathways, causing changes at the protein expression level that contribute to the alteration at the cellular level. Changes at the cellular level eventually induce reduction in blood pressure at the systemic level. Analysis of the effects of acupuncture at the Taichong point in SHRs reveals a complex scenario of dramatic changes in abundance of various cellular proteins. Analysis of the fourteen statistically significant differentially proteins between the groups provided insights into the potential mechanisms through which acupuncture may reduce hypertension. As acupuncture is a general treatment with “pleiotropic” responses, the simultaneous activation of multiple therapeutic mechanisms is expected. The results are in accordance with our belief that multiple mechanisms play a role in alleviation of hypertension. Due to the complexity and underlying discord relating to the pathogenic factors associated with essential hypertension, this discussion will address the major changes in the proteomic/peptidomic analysis and how they may tie in with the incomplete reversal of hypertension pathogenesis in SHRs. The potential mechanisms involved include: reduction of oxidative stress, sympathetic modulation via synapsin-1, as well as NO level modulation.
As shown in & , protein expression in the model SHR rats is significantly different from those of normal (non-SHR) rats. The proteomic response that contributes to the SHR phenotype and reduction of hypertension in Taichong-needled rats includes the modulation of seven proteins related to oxidative stress, including SOD, ALDH2, GSTM5, GLUD1, protein DJ-1, HSP90α, and α-ETF. Many studies have reported the involvement of oxidative stress in hypertension, but questions have been raised as to whether oxidative stress causes hypertension, or if hypertension causes oxidative stress: a “chicken or egg” scenario. Oxidative stress results in the formation of reactive oxygen species (ROS), which are present in low levels in normal cells and functions and play an important role in vascular biology in regards to cell signaling and vascular contraction-relaxation. Delano et al. and other authors 
reported that oxidative stress might be a more global condition not only confined to vascular tissues, and more recently a number of reports have been published relating to the modulatory effect of ROS on hypertension in the brain.
Of these proteins, SOD, ALDH2, GSTM5, DJ-1 and GLUD1 were up-regulated, while electron α-ETF and HSP90α are down-regulated with Taichong treatment when compared to the model SHR rats. SOD, a protein involved in the dismutation of the superoxide anion into oxygen and hydrogen peroxide, is an important antioxidant defense in most cells. GSTM5 is involved in the conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles, resulting in the detoxification of many oxidative stress proteins. GLUD1, primarily a mitochondrial matric enzyme, is involved in oxidative deamination and ammonia detoxification. ALDH2, another mitochondrial protein, functions to catalyze the oxidation of aldehydes and is also necessary for the bioactivation of organic nitrates, which are molecules with high vasodilator potency. The DJ-1 protein protects against oxidative stress and hydrogen peroxide based cell death, especially in neurons. HSP90α is a heat shock protein that promotes structural maintenance cell cycle control, and signal transduction, especially during stressful conditions; while α-ETF is a mitochondrial matrix protein that functions as a primary electron acceptor for primary dehydrogenase.
While the proteomic response of oxidative stress proteins demonstrates a complex, interweaved modulation, the predominant up-regulation of enzymes involved in ROS removal suggest an overall decrease in oxidative stress levels intracellularly due to acupuncture. The two oxidative stress related proteins down-regulated in response to acupuncture, HSP90α and α-ETF, play a major role when stressful conditions present, so reduction of oxidative stress should result in the down-regulation of these proteins. Furthermore, many other studies have linked acupuncture with a reduction in oxidative stress 
. ROS have many effects intracellularly as they function in signal transduction. This is the first report to implicate oxidative stress reduction as a possible mechanism of the therapeutic effect of acupuncture in hypertension. Acupuncture at the Taichong point changes the protein expression such that it reverses the changes seen in the proteomics analysis of SHR model compared to the normal rat, moving SHR rats back towards a normal protein expression as it related to hypertension. Additionally, it is interesting to note that multiple mitochondrial proteins have modulated protein levels after acupuncture treatment; additional research must be done to determine if mitochondrial function is in any way linked to the alleviation of hypertension due to acupuncture.
An up-regulation of oxidative stress enzymes and a decrease of oxidative stress may have multiple effects through which blood pressure is modulated. An increase in ROS in the rostral ventrolateral medulla (RVLM) contributes to the neural pathogenesis of hypertension 
. In the RVLM, Nox-induced ROS initiate a forward loop in cross-activation of different receptors and between Nox and mitochondrial ROS 
. Thus, a decrease in ROS due to acupuncture may oppose this pathogenic mechanism of hypertension. Additionally, the caudal ventrolateral medulla (CVLM) provides inhibitory input to the RVLM of the brain. A lack of these inhibitory inputs is believed to in part play a role in the neuropathogenesis of hypertension 
. The increase in oxidative stress seen in hypertension could affect the frequency of these inhibitory inputs, thus the reduction of oxidative stress seen in acupuncture may provide a mechanism through with CVLM inhibitory input to the RVLM is augmented. In addition, paraventricular nucleus (PVN) of the hypothalamus plays a major role in autonomic and neuroendocrine regulation of blood pressure and body fluid homeostasis 
. Functional studies have demonstrated the involvement of PVN in the control of fluid electrolyte homeostasis, feeding behavior, cardiovascular regulation, and stress adaptation 
. It is unclear whether acupuncture affects the regulatory function of these regions through modulation of ROS.
It is technically difficult to oblate specific nuclei from the rat medulla and to collect enough samples for our proteomic and biochemical analysis. Since multiple regions/nuclei of the medulla are involved in blood pressure regulation, we collected the whole medulla for our study. To examine whether the proteins located in specific nuclei of the medulla are related to blood pressure modification by acupuncture, we have conducted a preliminary immunohistochemical study and shown altered MBP and synapsin I expression in some specific regions (e.g. NTS and RVLM) of SHR medulla (data not shown). Further studies are ongoing to investigate which nuclei and what types of neurons and glial cells are involved in the proteomic responses to acupuncture in SHRs.
We would like to state that this study does not provide direct evidence for the involvement of oxidative stress reduction in hypertension. Our results show a strong correlation between acupuncture treatments, upregulation of antioxidant enzymes, and reduction of hypertension. In the future, our laboratory will conduct long term studies to generate direct evidence of this involvement.
ALDH2, an enzyme involved in the activation of NO compounds, plays a role in the bioactivation of multiple drugs used for heart failure, such as glyceryl trinitrate and pentaerythritol tetranitrate. Although acupuncture does not directly lower blood pressure to normal level in SHRs, the resulting up-regulation of ALDH2 suggests that it could be used as a complementary treatment to modern medicine to increase the efficacy of certain nitric oxide based drugs. Additionally, hypertension has been shown to result in decreases of endothelial and neuronal nitric oxide synthase (eNOS & nNOS), while acupuncture has been shown to stop the reduction of nNOS and eNOS in SHRs 
. The increased levels of nNOS as a result of hypertension directly affect NO levels, which is a potent vasodilator, further supporting our hypothesis that acupuncture modulates hypertension through multiple, simultaneously acting pathways.
The synapsins are a family of phosphoproteins present in neurotransmitter vesicles that are essential to normal neurotransmitter release at the synaptic cleft. Studies have shown that double knockout (synapsin-1 and synapsin-2) mice have a lower basal
blood pressure than normal mice, and additional studies from the same group showed that the same double knockout mice did not respond with progressive hypertension to treatment with cyclosporine A as is normally seen in mice, indicating that the synapsins play an integral role in control of blood pressure 
. As shown from our proteomic panel, synapsin-1 is significantly down-regulated in the Taichong group when compared to the model group. Down-regulation of synapsin could be another parallel avenue through which acupuncture helps control hypertension in SHRs.
Currently, there are no globally accepted biomarkers that readily differentiate between acupuncture at acupoints and acupuncture at a sham location. One of the biggest issues facing acupuncture research is the lack of a reliable method to determine if acupuncture was properly administered. Identification of reliable biomarker(s) for acupuncture would be a powerful tool for researchers to validate the efficacy of acupuncture treatments and optimize acupuncture regimen. Although the modulation of the medullar levels of oxidative stress enzymes cannot be used as a clinical biomarker for acupuncture, it may be useful for validation of acupuncture in animal studies. Adaptation of these changes in specific protein levels in the plasma, urine, or other easily accessible samples, is the next step in development and validation of these alterations as potential biomarkers for acupuncture. Other reports 
have corroborated our results on a much smaller scale, showing acupuncture does in fact up-regulate SOD and glutamate dehydrogenase, however, our complete proteomic expression panel is a more useful and accurate measure of successful acupuncture as the proteomic response is available for more proteins.
In this study, the Western blot analysis revealed that the expression of synapsin I remained unchanged after acupuncture treatment at non-acupoint but was down-regulated after acupuncture treatment at Taichong point (see ). In contrast, MBP remained unchanged after acupuncture treatment at non-acupoint but was up-regulated after acupuncture treatment at Taichong point. Western blot analysis can provide useful semi-quantitative data on protein expression. However, this commonly used assay can easily generate inconsistence of protein expression data given that most proteins are subject to degradation and modification by a number of factors.
In this study, we have observed some inconsistent expression data of individual proteins/genes when distinct techniques/assays are used. For example, although the results of MBP by Western blot assay were consistent with those by qRT-PCR, but were opposite to those from the 2-D gel analysis. Unlike Western blot and 2D gel assays, RT-PCR assays can generate quantitative data of gene expression at mRNA levels. Theoretically, the expression data of a specific gene at both protein and mRNA levels should be consistent. However, these data may be inconsistent due to the following reasons: a) mRNA is unstable and fragile; b) mRNA can be modified and thus its translation into the protein can be altered; and c) transcription and translation are regulated by a number of epigenetic and genetic factors and these two processes can be uncoupled under some conditions.
The clinical implication of our current study is unclear but may implicate the potential use of antioxidants in hypertension management. Potential sources of excessive ROS in hypertension include nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, mitochondria, cyclooxygenase 1 and 2, cytochrome P450 epoxygenase, xanthine oxidase, endothelium-derived NO synthase, and transition metals 
. A number of epidemiological and clinical data suggests that antioxidant-rich diets or natural antioxidants reduce blood pressure and cardiovascular risk 
, and animal studies have also shown the antihypertensive activity of antioxidants 
. However, the majority of randomized clinical studies using natural antioxidants including include vitamins A, C and E, L-arginine, flavanoids, and mitochondria-targeted agents (coenzyme Q10, acetyl-L-carnitine, and α-lipoic acid have given disappointing or conflicting results 
. Further mechanistic studies are needed to elucidate the role of ROS in the pathogenesis of hypertension and if proper use of antioxidants in combination of conventional antihypertensive agents benefits hypertensive patients.
Our current study focused on the “acute” effect over 1 week and identification of proteomic responses for the short-term effect of acupuncture on hypertension. From that point of view, we thought it to be less important to measure SBP before acupuncture each day, and focus more on the proteomic responses through which changes occur. We knew that to study the proteomic response to acupuncture, the rats would be sacrificed shortly after the experiment. Thus, it would be difficult to completely study the long term (e.g. >1 month) effect and sustainability of acupuncture on hypertension. In the future, we would be interested in performing studies to unfold the long-term effect of acupuncture on hypertension.
In conclusion, this study is the first to report the complete protein expression profile of acupuncture on SHRs, and has shown the potential involvement of oxidative stress as a partial mechanism in reduction of blood pressure by acupuncture therapy. Further studies are warranted to investigate the role of oxidative stress modulation by acupuncture in the treatment of hypertension.