Exploring the Mechanisms of Electroacupuncture-Induced Analgesia through RNA Sequencing of the Periaqueductal Gray.

Electroacupuncture (EA) can relieve various pains. However, its mechanism in terms of the transcriptome is still not well-known. To explore the full profile of EA-induced molecular modification in the central nerve system, three twins of goats were selected for a match-paired experiment: EA stimulation (60 Hz, 30 min) and none-EA (control). Goats in the EA group showed an increased (p < 0.05) nociceptive threshold compared with the control goats. Experimental goats were sacrificed at 4 h of the experiment, and the periaqueductal grays were harvested for RNA sequencing. As a result, 2651 differentially expressed genes (1803 up-regulated and 848 down-regulated genes) were found and enriched in 30 Kyoto Encyclopedia of Genes and Genomes pathways and 149 gene ontology terms. EA-regulated five neuropeptide genes (proenkephalin, proopiomelanocortin, preprodynorphin, diazepam-binding inhibitor and proprotein convertase 1 inhibitor) were validated with quantitative PCR. Furthermore, up-regulated glutamate receptors, glutamate transporters, γ-aminobutyric acid (GABA) receptors, GABA transporters, synaptotagmins or mitogen-activated protein kinase (MAPK) genes might contribute to EA-induced analgesia through regulating the glutamatergic synapse, GABAergic synapse, MAPKs, ribosome or ubiquitin-proteasome pathways. Our findings reveal a full profile of molecular modification in response to EA and provide a solid experimental framework for exploring the mechanisms underlying EA-induced analgesia.

[Physiological Characteristics and Nitrogen and Phosphorus Uptake of Myriophyllum aquaticum Under High Ammonium Conditions].

Ammonium nitrogen (NH4+-N) at high concentrations is toxic to plants. In order to explore the NH4+-N tolerance of Myriophyllum aquaticum (M. aquaticum) and its ability of nitrogen (N) and phosphorus (P) uptake, this study used a nutrient solution with three NH4+-N levels (70, 210, 420 mg·L-1) to incubate M. aquaticum for 21 d. The characteristics of plant physiology and N and P uptake of M. aquaticum were measured. At NH4+-N of 70 mg·L-1, M. aquaticum grew healthily, and shoot height and biomass linearly increased with the increase incubation time. Relative shoot height and biomass of M. aquaticum were 40.56 cm and 17.82 g·hole-1 on day 21, respectively. Compared to the control with 70 mg·L-1 ammonium, malondialdehyde (MDA) content of M. aquaticum was significantly increased; chlorophyll and soluble sugar contents were also high at NH4+-N of 210 mg·L-1. M. aquaticum suffered from the NH4+-N stress. However, the stress of 210 mg·L-1 NH4+-N did not affect its normal growth and there was no significant difference in the relative growth rate of the shoot height and biomass compared with the control. At NH4+-N of 420 mg·L-1, MDA contents of M. aquaticum doubled and the shoot height and biomass growth rate were only 27.4% and 17.9% of those for 70 mg·L-1 NH4+-N, indicating that M. aquaticum was subjected to serious stress that caused unhealthy growth or even death. At three NH4+-N levels, the ranges of N and P content of M. aquaticum were 30.7-53.4 mg·g-1 and 3.8-7.7 mg·g-1, respectively, which indicated that M. aquaticum had a high uptake capacity of N and P. M. aquaticum is an ideal wetland plant that has a good application prospect for constructed wetlands in biological treatment of high-ammonia wastewater.

Chemical-based species classification of rhubarb using simultaneous determination of five bioactive substances by HPLC and LDA analysis.

INTRODUCTION: Rhubarb is a traditional Chinese medicine derived from the rhizome of three species: Rheum tanguticum, Rheum palmatum and Rheum officinale. There are several species that are often misidentified as rhubarb. Taxonomical identification of these various species can be challenging. We have developed an HPLC-based species classification to identify rhubarb. OBJECTIVE: The objective of this study was to develop a simple HPLC method for the simultaneous determination of bioactive compounds and identification of medicinal rhubarb rhizome and non-medicinal species. METHODOLOGY: Quantitative analysis was performed on a C18-column using 0.05 M aqueous phosphoric acid and acetonitrile as the mobile phase under gradient conditions with ultraviolet detection at 280 nm. The method was validated with respect to linearity, accuracy, precision, and recovery. Statistical analysis was used to classify different groups of species. RESULTS: All calibration curves showed good linearity (r ≥ 0.9995). The method showed good repeatability with intra- and inter-day standard deviations of less than 1.13% and 1.32%, respectively. The accuracy and recovery of all marker compounds were in the ranges of 98.0 to 102.6% and 99.21 to 102.04%, respectively. Seventeen peaks were selected, and 39 known and 57 unknown samples were classified into five species based on linear discriminant analysis with an accuracy of 100%. CONCLUSION: A chemical-based species classification method of rhubarb using simultaneous determination of bioactive compounds by HPLC was developed with 39 known samples of five different species and successfully applied to identify 57 unknown samples collected from Korea and China.

[Effect of Shenmai injection on expression of hypoxia-inducible factor-1alpha in hypoxic-ischemic brain damage: experiment with rats].

OBJECTIVE: To investigate the effects of Shenmai injection containing active principles of Ginseng and ophiopogon root on the expression of hypoxia-inducible factor 1-alpha (HIF-1alpha) in brain after hypoxic-ischemic brain damage (HIBD). METHODS: 108 neonatal SD rats were randomly divided into 2 equal groups: (1) Shenmai group (Group SM), undergoing ligation of the right common carotid artery to establish HIBD models, breathing immediately a mixed gas with 8% oxygen and 92% nitrogen for 2 hours to cause HI insult, and then injected intraperitoneally with Shenmai injection 10 mg/kg once a day for 7 days, and (2) normal saline (NS) group (Group NS) undergoing ligation of the right common carotid artery to establish HIBD models, breathing immediately a mixed gas with 8% oxygen and 92% nitrogen for 2 h, and then injected intraperitoneally with NS 10 mg/kg once a day for 7 days. Another 54 neonatal rats underwent sham operation but did not undergo hypoxia as control group (Group C), 2, 12, and 24 hours, and 3, 7, and 14 days after HI insult 9 rats from each group were killed with their right hippocampal tissues taken out. Flow cytometry was used to examine the apoptotic rate of the hippocampal neurons. RT-PCR was used to detect the mRNA expression of HIF-1alpha. RESULTS: (1) The apoptosis rate of the right hippocampal tissues began increase 2 h after Hi insult, peaked 24 h after HI, then gradually decreased, and almost returned to the original levels 14 d after HI. There was no significant differences in apoptosis rates 14d after HI among the 3 groups (all P > 0.05). The neuron apoptosis rates 12 h, 24 h, 3 d, and 7 d after HI of Group SM were all significantly lower than those of Group NS (e.g 24 h: (11.95 +/- 1.13)% vs (16.80 +/- 1.44)%, all P < 0.05). (2) The HIF-1alpha mRNA expression level in right brain began to increase 2 h after HI, peaked 24 h after HI, then gradually decreases, and returned to the original level 14 d after Hi in both Group SM and Group NS; The HIF-1alpha mRNA expression in right brain 12 h, 24 h, 3 d, and 7 d after HI of Group SM were all significantly higher than those of Group NS (e.g 24 h: (44.32 +/- 4.03)% vs (35.63 +/- 3.73)%, all P < 0.05). CONCLUSION: The HIF-1alpha mRNA expression in brain tissue is up-regulated after HI insult. Shenmai injection helps increase the mRNA expression of HIF-1alpha in brain and reduces the apoptosis of hippocampus neurons after HI insult.

[Effects of electroacupuncture on behavior, plasma COR and expressions of PKA and PKC in hippocampus of the depression model rat].

OBJECTIVE: To probe into the mechanism of acupuncture for treatment of depression. METHODS: Thirty-two healthy SD male rats were randomly divided into a normal group, a model group, an electroacupuncture (EA) group and a Maprotiline group. The depression rat model was made in the latter three groups, and from the second day of the experiment EA was given at Baihui (GV 20), "Yintang" (EX-HN 1), "Zusanli" (ST 36) and "Fenglong" (ST 40) in the EA group, once every other day; the rats in the Maprotiline group were treated with oral administration of Maprotiline hydrochroride, once each day. After treatment of 3 weeks, changes of behaviors, plasma cortisol (COR) level and expressions of protein kinase A (PKA) and protein kinase C (PKC) in hippocampus were observed in the rats. RESULTS: In the depression model rats, the body weight increased slowly, and horizontal and vertical activities and consumption of sugar liquid significantly decreased; plasma cortisol content significantly increased; expressions of PKA and PKC in the hippocampus significantly reduced. In the rats of EA group, the score of behaviors, the consumption of sugar liquid and the increase of body weight were not significantly different to those in the model group, but the plasma cortisol level significantly decreased and closed to the normal level, and positive expressions of PKA and PKC in the hippocampus could be effectively reversed. In the Maprotiline group, the consumption of sugar liquid significantly increased and plasma cortisol level significantly decreased, and expressions of PKA and PKC in the hippocampus increased as compared with those in the model group. CONCLUSION: The depression model rat has dysfunction of the hypothalamus-pituitary-adrenal axis (HPAA) and EA can regulate functions of HPAA. The mechanism is possibly carried out by regulating functions of relative enzymes in the signal transduction pathway in hippocampal cells.

[Effects of Shenfu injection on hypoxic-ischemic brain damage: experiment with neonatal rats].

OBJECTIVE: To investigate the effect of Shenfu (ginseng and aconite root) injection on hypoxic-ischemic brain damage (HIBD). METHODS: Sixty 7-day-old Sprague-Dawley rats undergoing ligation of left common carotid artery and then put into a container with 8% O2 and 92% N(2) for 2 h so as to establish HIBD models, were randomly divided into 3 groups: Shenfu injection pretreatment group (since 4 days before the experiment Shenfu injection 10 ml/kg was injected intraperitoneally once a day for 4 days), Shenfu injection treatment group [Shenfu injection 10 ml/kg was injected intraperitoneally immediately after hypoxic-ischemia (HI) insult once a day for 7 days], and control group (normal saline 10 ml/mg was injected intraperitoneally immediately after HI insult once a day for 7 days). Twenty neonatal rats underwent sham operation as control group. The 4 groups were further divided into subgroups of 6 rats according to the time points: 2 hours before and 2 hours, 12 hours, 24 hours, 3 days, 7 days, 14 days, and 28 days after HI insult. 3, 7, 14, and 28 days after the HI insult the body weight was observed and the survival rate was observed 28 d after the HI insult. At different time points the rats of different subgroups were killed and their brains were taken out. Flow cytometry was used to calculate the neuron apoptosis rate in the hippocampal CA1 region. RESULTS: The body weight increase levels 3, 7, 14 and 28 days after HI insult of the control group were all significantly less than those of the sham operation group (e.g 7 days: 8.8 g +/- 2.1 g vs 14.0 g +/- 2.9 g, all P < 0.01) and the body weight increase levels 3, 7, 14, and 28 days after HI insult of the control group were all significantly less than those of the Shenfu injection pretreatment group (e.g 7 d: 11.7 g +/- 3.3 g) and Shenfu injection treatment group (e.g 7 d: 10.9 g +/- 2.7 g, P < 0.01 or P < 0.05). The survival rate 28 d after HI insult of the control group was 60%, significantly less than those of other groups (all P < 0.05), and there was no significant difference in the survival rate among the other groups (all P > 0.05). Compared with the sham operation group the neuron apoptosis rates of the hippocampal CA1 region of the Shenfu injection treatment group and Shenfu injection pretreatment group began to increase 2 hours after HI insult, peaked 24 hours after, then gradually decreased, and recovered to normal 14 days after. The neuron apoptosis rates 2, 12, 24, 72 hours, and 7 days after HI insult of the Shenfu injection pretreatment group were all significantly lower than those of the control group (e.g 24 hours: 16.0% +/- 4.2% vs 11.9% +/- 2.3% vs 18.1%, P < 0.01 or P < 0.05), and the neuron apoptosis rates 72 hours and 7 days after HI insult of the Shenfu injection treatment group were significantly lower than those of the control group. CONCLUSION: Shenfu injection can enhance the physical development and elevate the survival rate of neonatal rats with HI insult, and significantly prevents apoptosis of the hippocampus neurons from HI insult.