Electroacupuncture of the Baihui and Shenting acupoints for vascular dementia in rats through the miR-81/IL-16/PSD-95 pathway.

Background: There is currently no effective treatment for vascular dementia (VaD). Scalp electroacupuncture (EA) has served clinically as an alternative treatment for VaD, but its mechanism is still unclear. In this study, we investigated the effect of EA at the Baihui (GV 20) and Shenting (GV 24) acupoints on spatial learning and memory ability, and the expression level of microRNA-81 (miR-81), interleukin-16 (IL-16), and postsynaptic density protein-95 (PSD-95) in the frontal cortex of VaD rats. Methods: Male Sprague-Dawley rats were randomly divided into four groups, sham, VaD, non-acupuncture (non-AP) and EA group. The VaD model was established by permanent bilateral occlusion of the common carotid arteries. Morris Water Maze was used to assess the rats' spatial learning and memory. Immunochemistry (IHC), quantitative reverse transcription polymerase chain reaction (qRT-PCR), and western blot analysis were performed to detect the expression level of miR-81, IL-16, and PSD-95. Finally, luciferase assay was used to determine the effect of miR-81 on IL-16 expression in PC12 cells. Results: The space exploration experiment of MWM showed the time and distance of the rat's activities around the platform were decreased in the EA group. Compared to the VaD and non-AP group, the number of terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL)-positive frontal cortical neurons was significantly decreased in EA group. The number of the PSD-95-positive cells and the miR-81 expression level in the frontal cortical in the EA group was dramatically increased in comparison with the other groups. In the PC12 cell validation experiment, IL-16 expression level was reduced under the condition of the miR-81 mimic treatment, while increased in the miR-81 inhibitor group. The PSD-95 protein level was up-regulated in the small interfering (si)RNA-IL16 group compared to the NC-IL16 groups with or without oxygen/glucose deprivation/reperfusion (OGD/R) conditions (P<0.05). However, this was abolished by miR-81 mimic. Conclusions: In VaD rats, EA may improve spatial learning and memory through miR-81/IL-16/PSD-95 pathway.

Study on the mechanism of Yupingfeng powder in the treatment of immunosuppression based on UPLC⁃QTOF⁃MS, network pharmacology and molecular biology verification.

AIMS: The current study aims to investigate the effect of Yupingfeng (YPF) powder on immunosuppression, and explore the possible mechanisms. MAIN METHODS: Firstly, the monomer components of YPF powder were analyzed by UPLC-QTOF-MS combined with UNIFI automatic analysis platform, then the mechanism of YPF on immunosuppressive treatment was investigated using network pharmacological method, and finally the prediction was verified in a Candida albicans (Can)-induced immunosuppressive BALB/c mouse model. KEY FINDINGS: 98 monomer compounds in YPF were obtained. Through virtual analysis and screening on the oral utilization and drug likeness properties of the components, 47 effective components were got. 9 core targets obtained were enriched in IL-17 signaling pathway. In the mouse model, YPF could reduce the number of Can and alleviate Can-induced inflammation in the kidney effectively, upregulate Can-induced low proportion of CD4+/CD8+ of splenic lymphocytes, and increase Can-induced low activity of IL-17 pathway. SIGNIFICANCE: These results demonstrate that YPF could improve the immunity of Can-induced immunosuppression in BALB/c mice through upregulating the activity of IL-17 pathway.

Historical anthropogenic contributions to mercury accumulation recorded by a peat core from Dajiuhu montane mire, central China.

Mercury (Hg) accumulation records spanning the last 16,000 years before present (yr BP, relative to AD 1950) were derived from a peat core collected from Dajiuhu mire, central China. The natural Hg concentration and accumulation rate (free from anthropogenic influence) were 135.5 ± 53.9 ng g(-1) and 6.5 ± 4.5 µg m(-2) yr(-1), respectively. The increase in Hg flux that started from a core depth of 96.5 cm (3358 cal yr BP) is independent of soil erosion and organic matter content. We attribute this to an increase in atmospheric Hg deposition derived from regional anthropogenic activities. Anthropogenic Hg accumulation rates (Hg-ARA) in the pre-industrial period peaked during the Ming and the early Qing dynasties (582-100 cal yr BP), with Hg-ARA of 9.9-24.6 and 10.7-24.4 µg m(-2) yr(-1), respectively. In the industrial interval (post∼1850 AD), Hg-ARA increased progressively and reached 32.7 µg m(-2) yr(-1) at the top of the core. Our results indicate the existence of regional atmospheric Hg pollution spanning the past ∼3400 years, and place recent Hg enrichment in central China in a broader historical context.

MicroRNA Profiling Response to Acupuncture Therapy in Spontaneously Hypertensive Rats.

MicroRNAs (miRNAs) are a group of endogenous noncoding RNAs that play important roles in many biological processes. This study aimed to check if miRNAs were involved in the response to acupuncture in rats. Microarray analysis was performed to compare the miRNA expression profiles of medulla in spontaneously hypertensive rats (SHRs) treated with or without acupuncture. Our microarray analysis identified 222 differentially expressed miRNAs in the medulla of SHRs treated with acupuncture at taichong acupoint. Among these miRNAs, 23 miRNAs with a significant difference were found in acupuncture-treated SHRs compared to untreated rats. These 23 miRNAs could regulate 2963 target genes which were enriched in at least 14 pathways based on our bioinformatic analysis. miRNA-339, miR-223, and miR-145 were downregulated in the medulla of SHRs compared to normotensive rats. Notably, these miRNAs were upregulated to basal levels in the medulla of SHRs treated with acupuncture at taichong in comparison with SHRs receiving acupuncture at nonacupoint group or SHRs without any treatment. Our findings have revealed significant changes of a panel of selective miRNAs in hypertensive rats treated at taichong acupoint. These data provide insights into how acupuncture elicits beneficial effects on hypertension.

Acupuncture may exert its therapeutic effect through microRNA-339/Sirt2/NFκB/FOXO1 axis.

Recently, we have found that a number of microRNAs (miRNAs) and proteins are involved in the response to acupuncture therapy in hypertensive rats. Our bioinformatics study suggests an association between these miRNAs and proteins, which include miR-339 and sirtuin 2 (Sirt2). In this paper, we aimed to investigate whether Sirt2 was a direct target of miR-339 in neurons. In human SH-SY5Y cells, the luciferase assay implied that Sirt2 was likely a target of miRNA-339. Overexpression of miR-339 downregulated Sirt2 expression, while knockdown of miR-339 upregulated Sirt2 expression in human SH-SY5Y cells and rat PC12 cells. In addition, overexpression of miR-399 increased the acetylation of nuclear factor-κB (NF-κB) and forkhead box protein O1 (FOXO1) in SH-SY5Y cells, which are known targets of Sirt2. Our findings demonstrate that miR-339 regulates Sirt2 in human and rat neurons. Since Sirt2 plays a critical role in multiple important cellular functions, our data imply that acupuncture may act through epigenetic changes and subsequent action on their targets, such as miRNA-339/Sirt2/NF-κB/FOXO1 axis. Some physiological level changes of neurons after altering the miR-339 levels are needed to validate the suggested therapeutic role of miR-339/Sirt2/NF-κB/FOXO1 axis in response to acupuncture therapy in the future work.

Adenosine as a probing tool for the mechanistic study of acupuncture treatment.

To date, acupuncture has been widely used despite a lack of solid clinical evidence in the East and West. However, there are few validated in vitro models for the mechanistic studies of acupuncture. We hypothesized that adenosine could be used as a probing tool in the mechanistic studies of acupuncture because of its critical role in the action of acupuncture. Subsequently, we tested this hypothesis using both in vitro and in vivo experiments. First, we found that adenosine stimulation mimicked the effect of acupuncture on microRNA profiling (including miR-339, miR-145 and miR-451) and protein level (including Sirt2) in nerve growth factor-induced differentiated PC12 cells. These miRNA and proteins have been found to be regulated by acupuncture treatment in the brain of spontaneously hypertensive rats. Next, we found that adenosine stimulation downregulated miR-339 expression through adenosine A1 receptor-mediated pathway. Finally, we showed that the concentration of adenosine was actually decreased in the brain of spontaneously hypertensive rats after acupuncture treatment at Taichong acupoint. Taken together, these findings suggest that adenosine could be used as a useful probing tool for acupuncture mechanistic studies, while more validation studies are certainly warranted.

Auricular acupressure reduces anxiety levels and improves outcomes of in vitro fertilization: a prospective, randomized and controlled study.

The study was to explore whether auricular acupressure (AA) can relieve anxiety during the period from trans-vaginal oocyte retrieval to the embryo transfer in IVF treatment and whether AA can improve the outcomes of IVF. 305 infertile patients with tubal blockage who were referred for IVF were included. The women were randomized into a control group with 102 cases, a Sham-AA group with 102 cases and an AA group with 101 cases. The anxiety levels were rated with Spielberger's State Trait Anxiety Inventory and the Amsterdam Preoperative Anxiety and Information Scale. Data of clinical pregnancy rate (CPR), implantation rate (IR) and live birth rate (LBR) were obtained. The levels of neuropeptide Y (NPY) and transforming growth factor alpha (TGF-alpha) in the follicular fluids were detected with ELISA. After treatment, in AA group, the levels of state anxiety, preoperative anxiety and need-for-information were significantly lower, whereas CPR, IR, LBR and NPY levels in the follicular fluids were markedly higher than Sham-AA group and control group. We concluded that AA could help to reduce anxiety levels associated with IVF and improves the outcomes of IVF partly through increasing the levels of NPY in the follicular fluids.

The effect of PN-1, a Traditional Chinese Prescription, on the Learning and Memory in a Transgenic Mouse Model of Alzheimer's Disease.

Traditional Chinese Medicine (TCM) is a complete medical system that has been practiced for more than 3000 years. Prescription number 1 (PN-1) consists of several Chinese medicines and is designed according to TCM theories to treat patients with neuropsychiatric disorders. The evidence of clinical practice suggests the benefit effects of PN-1 on cognitive deficits of dementia patients. We try to prove and explain this by using contemporary methodology and transgenic animal models of Alzheimer's disease (AD). The behavioral studies were developed to evaluate the memory of transgenic animals after intragastric administration of PN-1 for 3 months. Amyloid beta-protein (A ß ) neuropathology was quantified using immunohistochemistry and ELISA. The western blotting was used to detect the levels of plasticity associated proteins. The safety of PN-1 on mice was also assessed through multiple parameters. Results showed that PN-1 could effectively relieve learning and memory impairment of transgenic animals. Possible mechanisms showed that PN-1 could significantly reduce plaque burden and A ß levels and boost synaptic plasticity. Our observations showed that PN-1 could improve learning and memory ability through multiple mechanisms without detectable side effects on mice. We propose that PN-1 is a promising alternative treatment for AD in the future.

Proteomic response to acupuncture treatment in spontaneously hypertensive rats.

Previous animal and clinical studies have shown that acupuncture is an effective alternative treatment in the management of hypertension, but the mechanism is unclear. This study investigated the proteomic response in the nervous system to treatment at the Taichong (LR3) acupoint in spontaneously hypertensive rats (SHRs). Unanesthetized rats were subject to 5-min daily acupuncture treatment for 7 days. Blood pressure was monitored over 7 days. After euthanasia on the 7(th) day, rat medullas were dissected, homogenized, and subject to 2D gel electrophoresis and MALDI-TOF analysis. The results indicate that blood pressure stabilized after the 5th day of acupuncture, and compared with non-acupoint treatment, Taichong-acupunctured rat's systolic pressure was reduced significantly (P<0.01), though not enough to bring blood pressure down to normal levels. The different treatment groups also showed differential protein expression: the 2D images revealed 571 ± 15 proteins in normal SD rats' medulla, 576 ± 31 proteins in SHR's medulla, 597 ± 44 proteins in medulla of SHR after acupuncturing Taichong, and 616 ± 18 proteins in medulla of SHR after acupuncturing non-acupoint. In the medulla of Taichong group, compared with non-acupoint group, seven proteins were down-regulated: heat shock protein-90, synapsin-1, pyruvate kinase isozyme, NAD-dependent deacetylase sirtuin-2, protein kinase C inhibitor protein 1, ubiquitin hydrolase isozyme L1, and myelin basic protein. Six proteins were up-regulated: glutamate dehydrogenase 1, aldehyde dehydrogenase 2, glutathione S-transferase M5, Rho GDP dissociation inhibitor 1, DJ-1 protein and superoxide dismutase. The altered expression of several proteins by acupuncture has been confirmed by ELISA, Western blot and qRT-PCR assays. The results indicate an increase in antioxidant enzymes in the medulla of the SHRs subject to acupuncture, which may provide partial explanation for the antihypertensive effect of acupuncture. Further studies are warranted to investigate the role of oxidative stress modulation by acupuncture in the treatment of hypertension.

[Effect of moderate acupuncture-stimulation of "Taichong" (LR 3) on blood pressure and plasma endothelin-1 levels in spontaneous hypertension rats].

OBJECTIVE: To observe the effect of different strength of acupuncture stimulation on blood pressure and plasma endothelin (E)-1 in spontaneous hypertension rats (SHR) ,so as to seek a better acupuncture parameter for clinical treatment of hypertension. METHODS: Twenty-eight 9-week-old SHRs were randomized into mild-stimulation group, moderate-stimulation group, strong-stimulation group and model group (n = 7 in each group). Seven normotensive SD rats served as a normal control group. Acupuncture stimulation with mild, moderate and strong stimulation was applied to bilateral "Taichong" (LR 3) for 5 min, once daily for 7 days. Blood pressure (BP) was determined by using a non-invasive BP-6 detection system. Plasma ET-1 was assayed by radioimmunoassay. RESULTS: Compared with the model group, the systolic pressure of the moderate-stimulation group on the 6th and 7th day was decreased significantly after acupuncture of "Taichong" (LR 3) (P<0. 01), being significantly lower than that of the mild- and strong-stimulation groups (P<0. 01). In comparison with the normal control group, plasma ET-1 level in the model group was increased significantly (P<0.01), while compared with the model group, only that in the moderate-stimulation group was down-regulated considerably (P<0. 01). No significant differences were found between the mild-stimulation and model groups, between the strong-stimulation and model groups, and between the mild-stimulation and strong-stimulation groups in plasma ET-1 level (P>0. 05). CONCLUSION: Moderate-stimulation of "Taichong" (LR 3) can lower blood pressure and plasma EA-1 level in spontaneous hypertension rats. The reduced level of plasma ET-1 may be one of its mechanisms underlying improving hypertension.

Protective action of tetramethylpyrazine phosphate against dilated cardiomyopathy in cTnT(R141W) transgenic mice.

AIM: Dilated cardiomyopathy (DCM) is the most common cause of heart failure, and pharmacological intervention is not currently available. Here we investigate the effect of tetramethylpyrazine phosphate (TMPP) on the progression of DCM in the cTnT(R141W) transgenic mouse model. METHODS: The cTnT(R141W) transgenic mice aged 2 months were divided into model group and TMPP group, whereas age-matched nontransgenic mice were used as wild-type control. TMPP 45 mg.kg(-1).d(-1) was administered for 7 months. Following assessment of cardiac function by echocardiography, cardiac tissues were prepared for histology and electron microscopy. Levels of molecular markers for cardiomyocyte hypertrophy and fibrosis were detected by RT-PCR. Expression of structural proteins of the sarcomere and intercalated disc was determined by Western blot. RESULTS: TMPP significantly prevented cardiac dilatation and dysfunction with the development of DCM, and decreased mortality by 54%. TMPP decreased HW/BW ratios and expression of hypertrophic markers BNP and ACTA1, as well as reduced interstitial collagen deposition and expression of profibrotic markers Col1a1 and Col3a1. TMPP attenuated ultrastructural disruption caused by cTnT(R141W) expression and decreased expression of structural proteins myotilin and E-cadherin which were up-regulated in the cTnT(R141W) heart. Moreover, TMPP reduced the mRNA expression of Calm1 and Camk2b in the cTnT(R141W) heart. CONCLUSION: Our results suggest that TMPP could be a promising drug for prevention and treatment of DCM.

Effects of phytoestrogen on mitochondrial structure and function of hippocampal CA1 region of ovariectomized rats.

The present study was undertaken to evaluate whether estrogen deprivation might lead to mitochondrial alteration of hippocampal neurons of ovariectomized (OVX) rats, and to evaluate the protective effect of estrogen and phytoestrogen on the mitochondrial alteration. First, OVX rats were used to mimic the pathologic changes of neurodegeneration of postmenopausal female, and we looked into the alteration of the mitochondrial ultrastructure and ATP content of hippocampal CA1 region after ovariectomy on different phase by transmission electron microscope (TEM) and reversed-phase high-performance liquid chromatography (HPLC), and found the best phase points of the alteration of the mitochondrial ultrastructure and ATP content. Next, estrogen and phytoestrogen were administered to the OVX rats for the protective effects on the mitochondrial ultrastructure and ATP content. Meanwhile, the density, size, shape, and distribution parameters of mitochondrial ultrastructure were analyzed according to the morphometry principle. The experimental results presented that (1) The alteration of mitochondrial ultrastructure elicited by ovariectomy worsened with the days going on, and the changes were the most noteworthy in volume density (Vv), average surface area (S), specific surface area (delta), and particle dispersity (Clambdaz) on 12th day (P < 0.05 or P < 0.01). Moreover, there was no statistical significance of the numerical density (Nv) among the five groups in the first step experiment. (2) The treatment with estrogen, genistein (Gs), and ipriflavone (Ip) significantly reversed the effect elicited by ovariectomy on Vv, S, delta, Clambdaz, Nv, and particle average diameter (D) of mitochondria of hippocampal CA1 region (P < 0.05). (3) Furthermore, ATP content of hippocampal CA1 region after ovariectomy declined significantly on 7th day (P < 0.05), and estrogen and phytoestrogen could reverse the alteration (P < 0.05). Taken together, these results revealed that phytoestrogen may have a protective role against the neurodegeneration after menopause via protecting mitochondrial structure and functions. Phytoestrogen may be a good alternative as a novel therapeutic strategy for menopausal syndrome.

Differential effects of melatonin on hippocampal neurodegeneration in different aged accelerated senescence prone mouse-8.

OBJECTIVES: A purpose of this study is to compare the differential effects of melatonin on hippocampal neurodegeneration in accelerated senescence prone mouse-8 (SAMP8) which is initiated treatment at different age. METHODS: The 4-months old SAMP8 mice were injected subcutaneously with melatonin (1 mg/kg/day) for 4 months. Similar treatments were performed in the 7-months old mice. When the animals were complete 11-months old, a series of tests were performed. Y maze test and Eight-arm radial maze task were used to assess cognitive performance. Hippocampal pyramidal cells were estimated by Nissl's staining. By using Gomori's methenamine silver methods, the methenamine silver staining granules (MSSG) were observed in area CA1 of hippocampus. A computer-assisted morphometric study was carried out on the ultrastructure of perikaryal CA1 pyramidal cell mitochondria. The volume density (Vv), surface density (Sv), numerical density (Nv) and mean volume (V) of the mitochondria were calculated. RESULTS: Melatonin treatment obviously reduced the deposition of MSSG and elevated hippocampal pyramidal cell number while improving the learning and memory deficits of SAMP8. The mice initiated treatment from 4-months old exhibited a greater response to melatonin supplementation than 7-months old mice. It also decreased mean volume (V) and significantly elevated the Sv and Nv of the mitochondria in hippocampal CA1 region. However, 7-months old mice showed little effects on it. CONCLUSIONS: Our results indicate that the protective effects of melatonin on hippocampal neurodegeneration of SAMP8 are age dependent.