Acupuncture Ameliorates Depression-Like Behaviors Through Modulating the Neuroinflammation Mediated by TLR4 Signaling Pathway in Rats Exposed to Chronic Restraint Stress.

Recently, emerging evidence has identified that stress-induced activation of neuroinflammation is considered to be one of the most prevalently precipitating factors in the pathogenesis of depression. Data from clinical trials and experimental findings has verified the efficacy and safety of acupuncture in the prevention and treatment of depression. However, the mechanism of the preventive effect of acupuncture for depression has not been fully elucidated. The current study aimed to investigate the preventive effect and mechanism of acupuncture through modulating the neuroinflammation mediated by toll-like receptor 4 (TLR4) signaling pathway in rats exposed to chronic restraint stress (CRS). All rats were subjected to CRS for 21 days, with the exception of rats in control group. One hour before CRS, rats in acupuncture group were exposed to acupuncture at Baihui (GV20) and Yintang (GV29). The depression-like behaviors were evaluated by body weight assessment and sucrose preference test at 0, 7, 14, and 21 days. The expression of activated microglia in hippocampus was detected by immunofluorescence. The expression of key proteins on TLR4 signaling pathway of TLR4, MyD88, TRAF6, NF-κB p65, TNF-α, and mRNA of TLR4 in the hippocampus was detected by western blot and real-time quantitative polymerase chain reaction to investigate the effect of acupuncture on stress-induced activation of neuroinflammation. The present study provided evidence that acupuncture exerted potential preventive effect that might be mediated in part by suppressing the neuroinflammation induced by TLR4 signaling pathway, which may be a promising treatment target to improve current treatments for depression.

Effects of acupuncture on regulating the hippocampal inflammatory response in rats exposed to post-traumatic stress disorder.

Data from clinical and experimental studies have verified the efficacy and safety of acupuncture in the treatment of post-traumatic stress disorder (PTSD). However, the concrete mechanism has not been well elucidated. The stress-induced activation of inflammatory response is involved in the development and pathogenesis of PTSD. Here, we aimed to investigate the effects of acupuncture on regulating the hippocampal inflammatory response in rats exposed to PTSD. Forty male rats were randomly divided into control, model, acupuncture and sertraline group. Within 1 day after adaptive feeding, all rats were exposed to single prolonged stress (SPS), except for the rats in the control group. Rats in acupuncture group were exposed to acupuncture intervention at the acupoints of Baihui (GV20) and Yintang (GV29), 20 min once per day for 15 days. Rats in sertraline group were exposed to a suspension of sertraline and distilled water (0.2 mg/ml), once per day for 15 days continuously. Body weight and elevated plus maze experiment were detected at different time-points to evaluate the behavioral changes of rats. HE staining method was used to observe the basic pathological morphological changes in hippocampus. Immunofluorescence staining method was used to observe the activation of hippocampal microglia. The content of IL-6 and IL-1ß in serum were detected by ELISA method. Compared with the control group, the body weight of rats in model group significantly decreased on 8 days, and the percentage of time in open arms and open arm entries decreased significantly on 15 days after SPS procedures, which indicated that SPS induced PTSD-like behavior in rats. Acupuncture exerted therapeutic effect. Simultaneously, the result of HE staining confirmed that SPS induced hippocampal morphological changes in SPS rats. Notably, acupuncture reversed the reduction and pathological injury to some extent. The results have also shown that acupuncture intervention effectively reversed the activated microglia of the hippocampus in rats. Moreover, the expression of IL-1ß in serum was significantly decreased by acupuncture intervention. In summary, the present study demonstrated that the role of acupuncture in eliminating PTSD-like behavior might be connected with reversing the pathological process of the inflammatory response mediated by the activation of microglia induced by SPS.

Enhancement of growth, antioxidative status, nonspecific immunity, and disease resistance in gibel carp (Carassius auratus) in response to dietary Flos populi extract.

This study investigated the effects of dietary Flos populi extract (FPE) on the growth, antioxidation capability, innate immune response, and disease resistance in gibel carp. A total of 480 fish were fed with five different diets containing 0, 0.5, 1.0, 1.5, or 2.0 g kg-1 FPE (designated as control, D0.5, D1.0, D1.5, or D2.0 groups) for 45 days. The fish were challenged with A. hydrophila after the feeding trial. Compared with the control, the feed efficiency (FE), weight gain (WG), final body weight (FBW), and specific growth rate (SGR) were significantly improved in groups D1.0 and D1.5. Dietary FPE significantly increased serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities, as well as glutathione (GSH) content. The contents of protein carbonyl (PCC) and malondialdehyde (MDA) in serum decreased significantly. Additionally, FPE supplementation in diets resulted in significant improvement in serum lysozyme (LZM) and myeloperoxidase (MPO) activities, as well as immunoglobulin M (IgM) and complement 3 (C3) concentrations. The hepatic antioxidant enzymes (CAT and SOD) activities increased, whereas content of MDA decreased in fish treated with dietary FPE than those of control both pre- and post-challenged. After 12 h-challenge, an obvious downregulation of hepatic Kelch-like-ECH-associated protein 1 (Keap1), splenic tumor necrosis factor-α (TNF-α), interleukin (IL)-8, IL-1ß, and toll-like receptor 2 (TLR2) mRNA levels was observed in fish treated with dietary FPE, whereas hepatic Nrf2 transcription level was upregulated compared to the control. Furthermore, compared to group D0.5, higher relative percent survival (RPS) was observed in gibel carp fed dietary 1.0-2.0 g/kg FPE. Our results reveal that FPE supplemented diet has a stimulatory effect on antioxidant capacity and nonspecific immune response, along with improved growth performance and enhanced resistance against A. hydrophila infection in juvenile gibel carp.

Non-invasive Urine Test for Molecular Classification of Clinical Significance in Newly Diagnosed Prostate Cancer Patients.

Objective: To avoid over-treatment of low-risk prostate cancer patients, it is important to identify clinically significant and insignificant cancer for treatment decision-making. However, no accurate test is currently available. Methods: To address this unmet medical need, we developed a novel gene classifier to distinguish clinically significant and insignificant cancer, which were classified based on the National Comprehensive Cancer Network risk stratification guidelines. A non-invasive urine test was developed using quantitative mRNA expression data of 24 genes in the classifier with an algorithm to stratify the clinical significance of the cancer. Two independent, multicenter, retrospective and prospective studies were conducted to assess the diagnostic performance of the 24-Gene Classifier and the current clinicopathological measures by univariate and multivariate logistic regression and discriminant analysis. In addition, assessments were performed in various Gleason grades/ISUP Grade Groups. Results: The results showed high diagnostic accuracy of the 24-Gene Classifier with an AUC of 0.917 (95% CI 0.892-0.942) in the retrospective cohort (n = 520), AUC of 0.959 (95% CI 0.935-0.983) in the prospective cohort (n = 207), and AUC of 0.930 (95% 0.912-CI 0.947) in the combination cohort (n = 727). Univariate and multivariate analysis showed that the 24-Gene Classifier was more accurate than cancer stage, Gleason score, and PSA, especially in the low/intermediate-grade/ISUP Grade Group 1-3 cancer subgroups. Conclusions: The 24-Gene Classifier urine test is an accurate and non-invasive liquid biopsy method for identifying clinically significant prostate cancer in newly diagnosed cancer patients. It has the potential to improve prostate cancer treatment decisions and active surveillance.

Genetically predicted selenium is negatively associated with serum TC, LDL-C and positively associated with HbA1C levels.

BACKGROUND & AIMS: Pervious epidemiological evidence on the associations of selenium, zinc with lipid profile and glycemic indices was contradictory. The aim of this study was to investigate whether selenium and zinc were casually associated with lipid profile and glycemic indices using mendelian randomization (MR) analysis. METHOD: A two-sample MR was used to evaluate the causal-effect estimations. Summary statistics for selenium, zinc, lipids and glycemic indices were retrieved from previous large-scale genome-wide association study (GWAS). Single nucleotide polymorphisms (SNPs) that independently and strongly associated with the selenium and zinc were selected as the instrumental variables. The casual estimates were calculated using inverse variance weighted method (IVW), with weighted median, MR-Egger, and MR-PRESSO test as sensitivity analysis, respectively. RESULTS: In the standard IVW analysis, per SD increment in selenium was associated with an 0.077 mmol/L decrease of TC (95 %CI: -0.102,-0.052) and 0.074 mmol/L of LDL-C (95 %CI: -0.1,-0.048). Suggestive casual associations were found between selenium and insulin or HbA1c. With IVW method, per SD increase in selenium was associated with an 0.023 mmol/L increase of insulin (95 %CI: 0.001,0.045), and an 0.013 mmol/L increase of HbA1c (95 %CI: 0.003,0.023). The results were robust in the sensitivity analysis. Zinc was not casually associated with any of lipid and glycemic markers. CONCLUSION: Our MR analysis provides evidence of the potential causal effect of Se on beneficial lipid profile, including decreased TC and LDL-C. Furthermore, suggestive casual evidence was suggested between Se and increased serum HbA1c levels. Careful consideration is required for the protective effects of Se supplementation. No casual-effect association was found between Zn and any indices of the lipid and glucose parameters.

Could biochar amendment be a tool to improve soil availability and plant uptake of phosphorus? A meta-analysis of published experiments.

As one of the most important nutrients for plant growth, phosphorus was often poorly available in soil. While biochar addition induced improvement of soil structure, nutrient and water retention as well as microbial activity had been well known, and the effect of biochar soil amendment (BSA) on soil phosphorus availability and plant P uptake had been not yet quantitatively assessed. In a review study, data were retrieved from 354 peer-reviewed research articles on soil available P content and P uptake under BSA published by February 2019. Then a database was established of 516 data pairs from 86 studies with and without BSA in agricultural soils. Subsequently, the effect size of biochar application was quantified relative to no application and assessed in terms of biochar conditions, soil conditions, as well as experiment conditions. In grand mean, there was a significant and great effect of BSA on soil available P and plant P uptake by 65% and 55%, respectively. The effects were generally significant under manure biochar, biochar pyrolyzed under 300 °C, soil pH <5 and fine-textured soil, and soils that are very low in available P. Being significantly correlated to soil P availability (R2=0.29), plant P uptake was mostly enhanced with vegetable crops of high biomass yield. Overall, biochar amendment at a dosage up to 10 t ha-1 could be a tool to enhance soil availability and plant uptake of phosphorus, particularly in acid, heavy textured P-poor soils.

Predicting the invasive trend of exotic plants in China based on the ensemble model under climate change: A case for three invasive plants of Asteraceae.

Ageratina adenophora, Eupatorium odoratum, and Mikania micrantha are three highly destructive invasive plants of Compositae in China. Through the screening of SDMs, random forest (RF), gradient boosting model (GBM), artificial neural network (ANN), and flexible discriminant analysis (FDA) with TSS greater than 0.8 are selected to construct a high-precision ensemble model (EM) as the prediction model. We use specimen sites and environmental variables containing climate, soil, terrain, and human activities to simulate and predict the invasion trend of three invasive weeds in China in current, the 2050s, and the 2070s. Results indicate that the highly invasive risk area of three exotic plants is mostly distributed along the river in the provinces south of 30° N. In the future scenario, the three exotic plants obviously invade northwards Yunnan, Sichuan, Guizhou, Jiangxi and Fujian. Climate is the most important variable that affects the spread of three kinds of alien plant invasions. Temperature and precipitation variables have a similar effect on A. adenophora and E. odoratum, while M. micrantha is more sensitive to temperature. It has been reported that Ipomoea batatas and Vitex negundo can prevent the invasion of three invasive plants. Hence, we also simulate the suitable planting areas for I. batatas and V. negundo. The results show that I. batatas and V. negundo are suitable to be planted in the areas where the three weeds show invasion tendency. In the paper, predicting invasion trends of exotic plants and simulating the planting suitability of crops that can block invasion, to provide a practical significance reference and suggestion for the management, prevention, and control of the invasion of exotic plants in China.

Effects of dietary fish meal replacement by fermented moringa (Moringa oleifera Lam.) leaves on growth performance, nonspecific immunity and disease resistance against Aeromonas hydrophila in juvenile gibel carp (Carassius auratus gibelio var. CAS III).

This study was aimed to evaluate the effects of partial replacement of fish meal by fermented moringa leaves (FMLs) on growth performance, serum biochemistry, antioxidant status, nonspecific immunity, and resistance against Aeromonas hydrophila in juvenile gibel carp (Carassius auratus gibelio var. CAS III). Four isonitrogenous and isoenergetic balanced diets, including three FML diets (substituting 20%, 40%, 60% of the fish meal in basal diet, F20, F40 and F60, respectively) and a basal diet (a diet containing 10% fish meal) were used. Each diet was randomly allocated to four fish groups (F20, F40, F60 and control) reared in a recirculating system. After 50 days of the feeding trial, fish were challenged by A. hydrophila. The result revealed that final mean body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), feed efficiency (FE) and survival rate (SR) were significantly increased (P < 0.05) in F20 and F40 groups compared with the control group. Decreased hepatosomatic index (HSI), body crude lipid, serum aspartate transaminase (AST) and serum alanine aminotransferase (ALT) activities, and increased serum alkaline phosphatase (AKP) and serum glutathione peroxidase (GPx) activities were observed in F40 and F60 groups compared with the control and F20 groups. All FMLs-supplemented groups increased (P < 0.05) serum superoxide dismutase (SOD), catalase (CAT) and lysozyme activities, complement component 3 (C3) and serum immunoglobulin M (IgM) concentration, or decreased serum malondialdehyde (MDA) and protein carbonyl (PCC) contents (P < 0.05). After the challenge test, the significant downregulation of toll-like receptors2 (TLR2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-8 mRNA transcription levels was observed in spleens of FMLs supplemented groups. Dietary F40 and F60 showed higher (P < 0.05) relative percent survival (RPS) (48.72% and 43.59%, respectively) against A. hydrophila infection than control. These results indicate that, as a dietary fish meal substitute, FMLs enhance the growth, and antioxidant and immune response, and regulate the expression of immune-related genes and increase disease resistance against A. hydrophila via TLR2 pathway in gibel carp, with greatest effects of 40% fish meal substitution.

Changes in plant C, N and P ratios under elevated [CO2] and canopy warming in a rice-winter wheat rotation system.

Elevated atmospheric CO2 concentration ([CO2]) can stimulate plant growth through enhanced photosynthetic rate. However, plant C, N and P ratios in response to elevated [CO2] combined with canopy warming in rice-winter wheat rotation system remain largely unknown. Here we investigated the impacts of elevated [CO2] and warming on plant nutrient ratios under open-air conditions. Four treatments including the ambient condition (CK), elevated [CO2] (500 ppm, CE), canopy warming (+2 °C, WA), and the combination of elevated [CO2] and warming (CW) were used to investigate the responses of plant C, N and P ratios in a rice-winter wheat rotation system in southeast China. Results showed that elevated [CO2] increased C:N ratio in whole plant by 8.4-14.3% for both crops, and increased C:P ratio by 11.3% for rice. The changes in ratio were due to an increase in C concentration by 0.8-1.2% and a reduction in N concentration by 7.4-10.7% for both crops, and a reduction in P concentration by 10.0% for rice. Warming increased N allocation in rice leaf and N concentration by 12.4% for rice, resulting in increases in the ratios of N to C and P by 11.9% and 9.7% in rice, but not in wheat. However, CW had no effect on plant C:N ratio in rice, indicating the positive effect of elevated [CO2] could offset the negative impact of warming on C:N ratio. By contrast, CW significantly decreased plant C:P and N:P ratios by 16% due to the increase in P allocation in stem for wheat. These results suggest that impacts of climate change on plant nutrient balance occur through interactions between the effects of climate change on nutrient uptake and allocation, which is important for food quality and productivity under global climate change.

Network Pharmacology Integrated Molecular Docking Reveals the Antiosteosarcoma Mechanism of Biochanin A.

BACKGROUND: As the malignant tumor with the highest incidence in teenagers, osteosarcoma has become a major problem in oncology research. In addition to surgical management, the pharmacotherapeutic strategy for osteosarcoma treatment is an attractive way to explore. It has been demonstrated that biochanin A has an antitumor capacity on multiple kinds of solid tumor, including osteosarcoma. But the precise mechanism of biochanin A against osteosarcoma is still needed to be discovered. OBJECTIVE: To identify the potential therapeutic targets of biochanin A in treating osteosarcoma. METHODS: In present study, an integrated approach including network pharmacology and molecular docking technique was conducted, which mainly comprises target prediction, network construction, gene ontology, and pathway enrichment. CCK8 test was employed to evaluate the cell viability of MG63 cells. Western-blot was used to verify the target proteins of biochanin A. RESULTS: Ninety-six and 114 proteins were obtained as the targets of biochanin A and osteosarcoma, respectively. TP53, IGF1, JUN, BGLAP, ATM, MAPK1, ATF3, H2AFX, BAX, CDKN2A, and EGF were identified as the potential targets of biochanin A against osteosarcoma. Based on the western-blot detection, the expression of BGLAP, BAX, and ATF3 in MG63 cell line changed under the treatment of biochanin A. CONCLUSION: Biochanin A can effectively suppress the proliferation of osteosarcoma and regulate the expression of BGLAP, BAX, and ATF3, which may act as the potential therapeutic targets of osteosarcoma.

[Effect of Acupuncture Intervention on Depression Behavior and Expression of ERK 1/2 and BDNF in Prefrontal Cortex in Chronic Unpredictable Mild Stress Induced Depression Rats].

OBJECTIVE: To investigate the effect of acupuncture intervention on the depression behavior and expression of extracellular signal-regulated protein kinases (ERK 1/2), p-ERK 1/2 and brain-derived neurotrophic factor (BDNF) in the prefrontal cortex of chronic unpredictable mild stress (CUMS) induced depression rats, so as to explore its antidepressant mechanism. METHODS: Sixty-four male SD rats were randomly divided into control, model, acupuncture, Fluoxetine, model ＋ Dimethyl sulfoxide (DMSO), model ＋ PD 98059(an ERK pathway inhibitor), acupuncture ＋ PD 98059 and Fluoxetine ＋ PD 98059 groups (n＝8 rats in each). The CUMS depression model was established by using chronic mild and unpredictable stress methods for 21 days. Manual acupuncture stimulation was applied to "Baihui" (GV 20) and "Yintang" (GV 29) for 10 min before modeling, once daily for 21 days. Fluoxetine hydrochloride suspension (1.8 mg•kg－1•d－1) was given to rats of the Fluoxetine group and Fluo-xetine ＋ PD 98059 group by gavage 30 min before CUMS. PD 98059 (dissolved in DMSO, 10 µL) was administered to rats of model ＋ PD 98059 group, acupuncture ＋ PD 98059 and Fluoxetine ＋ PD 98059 group, and DMSO (10 µL) to rats of model ＋ DMSO group by intracerebroventricular injection 1 h before CUMS. Sucrose consumption test was carried out to evaluate the depressive behavior. Western blot was performed to detect the expression of ERK 1/2, p-ERK 1/2 and BDNF of prefrontal cortex. RESULTS: Compared with the control group, the sucrose consumption and the expression levels of p-ERK 1/2 and BDNF protein in the prefrontal cortex were significantly reduced in the model and model＋DMSO group (P<0. 01). After the intervention, modeling induced decrease of the sucrose consumption, and p-ERK 1/2 and BDNF expression was significantly up-regulated in both acupuncture and Fluoxetine groups (P<0.01, P<0.05), but not in the model＋PD 98059, Fluoxetine ＋PD 98059 and acupuncture＋PD 98059 groups (P>0.05). No significant differences were found among the model＋PD 98059, Fluoxetine ＋PD 98059 and acupuncture＋PD 98059 groups in the sucrose consumption, and ERK 1/2, p-ERK 1/2 and BDNF expression levels (P>0.05), and in the expression levels of ERK 1/2 protein among the 8 groups (P>0.05). CONCLUSION: Acupuncture intervention has an anti-depressive role in CUMS induced depression rats, which may be related to its effects in up-regulating the expression of p-ERK 1/2 and BDNF in the prefrontal cortex tissue.

Antidepressant-Like Effects of Acupuncture-Insights From DNA Methylation and Histone Modifications of Brain-Derived Neurotrophic Factor.

Sensitive and stable biomarkers that facilitate depression detection and monitor the antidepressant efficiency are currently unavailable. Thus, the objective is to investigate the potential of DNA methylation and histone modifications of brain-derived neurotrophic factor (BDNF) in monitoring severity and antidepressive effects of acupuncture. The depression rat model was imitated by social isolation and chronic unpredicted mild stress (CUMS). The expression of serum BDNF was detected by enzyme-linked immunosorbent assay (ELISA), the hippocampal BDNF, acetylation levels in histone H3 lysine 9 (acH3K9), and HDAC2 by Western blot, the hippocampal mRNA of BDNF by RT-polymerase chain reaction (PCR). The DNA methylation patterns of the promoter I of BDNF was detected by MS-PCR. We investigated that the expression of BDNF in serum and hippocampus were significantly downregulated compared with controls. The same trend was found in mRNA of BDNF. Notably, acupuncture reversed the downregulation of BDNF in serum and hippocampus and mRNA of BDNF compared with model group. Acupuncture reversed the CUMS-induced downregulation of hippocampal acH3K9. On the contrary, the CUMS-induced upregulation of hippocampal HDAC2 in model group was significantly reversed by acupuncture. Collectively, the antidepressant effect of acupuncture might be mediated by regulating the DNA methylation and histone modifications of BDNF, which may represent novel biomaker for detection of depression and monitoring severity and antidepressive effects.

Genome-wide transcriptome analysis of hippocampus in rats indicated that TLR/NLR signaling pathway was involved in the pathogenisis of depressive disorder induced by chronic restraint stress.

Data from clinical investigations and laboratory fundings have provided preliminary evidence for the effectiveness and safety of acupuncture therapy in depression. However, the mechanisms underlying the antidepressant response of acupuncture are not fully elucidated. To elucidate the potential effects of acupuncture for depression on the hippocampal genome-wide transcriptome at the molecular level, we evaluated the transcriptomic profile of depression rats under treatment of acupuncture, and fluoxetine. We identified a very significant effect of acupucture intervention, with 107 genes differentially expressed in acupuncture vs. model group; while 41 genes between fluoxetine vs. model group. Notably, the 54 differentially expressed genes between acupuncture and fluoxetine showed the significantly different effect between acupuncture and fluoxetine. Through GO (gene ontology) functional term and KEGG (kyoto encyclopedia of genes and genomes) pathway analysis, we identified that the upregulation of gene sets were related to inflammatory response, innate immunity and immune response. We found that toll-like receptor signalling pathway and NOD like receptor signalling pathway were associated with the function of inflammatory response, innate immunity and immune response. Importantly, acupuncture reversed the upregulation of gene sets that were related to inflammatory response, innate immunity and immune response (including toll-like receptor signalling pathway and NOD like receptor signalling pathway), which might be critical for the pathogenesis of depression and provide evidence for the antidepressive effects of acupuncture by regulating inflammatory response, innate immunity and immune response via toll-like receptor signalling pathway and NOD like receptor signalling pathway.

Differential intensity-dependent effects of pulsed electromagnetic fields on RANKL-induced osteoclast formation, apoptosis, and bone resorbing ability in RAW264.7 cells.

Pulsed electromagnetic fields (PEMF) have been proven to be effective for promoting bone mass and regulating bone turnover both experimentally and clinically. However, the exact mechanisms for the regulation of PEMF on osteoclastogenesis as well as optical exposure parameters of PEMF on inhibiting osteoclastic activities and functions remain unclear, representing significant limitations for extensive scientific application of PEMF in clinics. In this study, RAW264.7 cells incubated with RANKL were exposed to 15 Hz PEMF (2 h/day) at various intensities (0.5, 1, 2, and 3 mT) for 7 days. We demonstrate that bone resorbing capacity was significantly decreased by 0.5 mT PEMF mainly by inhibiting osteoclast formation and maturation, but enhanced at 3 mT by promoting osteoclast apoptosis. Moreover, gene expression of RANK, NFATc1, TRAP, CTSK, BAX, and BAX/BCL-2 was significantly decreased by 0.5 mT PEMF, but increased by 3 mT. Our findings reveal a significant intensity window for low-intensity PEMF in regulating bone resorption with diverse nature for modulating osteoclastogenesis and apoptosis. This study not only enriches our basic knowledge for the regulation of PEMF in osteoclastogenesis, but also may lead to more efficient and scientific clinical application of PEMF in regulating bone turnover and inhibiting osteopenia/osteoporosis. Bioelectromagnetics. 38:602-612, 2017. © 2017 Wiley Periodicals, Inc.

Protective effects of Oviductus Ranae-containing serum on oxidative stress-induced apoptosis in rat ovarian granulosa cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Oviductus Ranae (OR) is a traditional Chinese medicine derived from Rana temporaria chensinensis David, and is known to have a wide variety of pharmacological effects. AIM OF THE STUDY: To investigate the function and mechanism of OR-containing serum in protecting rat ovarian granulosa cells from hydrogen peroxide (H2O2)-induced oxidative damage. MATERIALS AND METHODS: H2O2-treated granulosa cells were pretreated with OR-containing serum, and viability and proliferation assays were carried out using Cell Counting Kit-8 (CCK-8). Apoptotic granulosa cells were observed microscopically using 4',6-diamidino-2-phenylindole (DAPI), and the apoptotic ratio was quantified via Annexin V/ propidium iodide (PI) staining combined with flow cytometry. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) in the cells were measured using 2,7-dichlorofluorescein diacetate (DCFH-DA) and rhodamine 123, respectively, and analyzed by flow cytometry. Mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, and p38, and other apoptosis-related proteins (p53, Bcl-2, Bax, caspase-9, caspase-3), were detected by western blot analysis, and the related mRNA levels were detected using reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The results revealed that treatment with OR-containing serum reduced apoptosis and mitochondrial membrane damage in H2O2-treated granulosa cells. The OR-containing serum interfered with H2O2-induced intracellular generation of ROS and loss of ΔΨm, which typically lead to apoptosis. Furthermore, the OR-containing serum down-regulated pro-apoptotic proteins such as p53, Bax, caspase-9, and caspase-3, while up-regulating the anti-apoptotic protein Bcl-2. Finally, the OR-containing serum increased phosphorylation of ERK1/2, and reduced JNK and p38 phosphorylation. CONCLUSIONS: OR-containing serum protected rat ovarian granulosa cells against H2O2-induced apoptosis, by reducing ROS production and improving mitochondrial membrane potential, through down-regulation of negative regulators of proliferation, activation of ERK1/2, and inhibition of the activity of JNK and p38.

Mechanisms Underlying the Antidepressant Response of Acupuncture via PKA/CREB Signaling Pathway.

Protein kinase A (PKA)/cAMP response element-binding (CREB) protein signaling pathway, contributing to impaired neurogenesis parallel to depressive-like behaviors, has been identified as the crucial factor involved in the antidepressant response of acupuncture. However, the molecular mechanisms associated with antidepressant response of acupuncture, neurogenesis, and depressive-like behaviors ameliorating remain unexplored. The objective was to identify the mechanisms underlying the antidepressant response of acupuncture through PKA signaling pathway in depression rats by employing the PKA signaling pathway inhibitor H89 in in vivo experiments. Our results indicated that the expression of hippocampal PKA-α and p-CREB was significantly downregulated by chronic unpredicted mild stress (CUMS) procedures. Importantly, acupuncture reversed the downregulation of PKA-α and p-CREB. The expression of PKA-α was upregulated by fluoxetine, but not p-CREB. No significant difference was found between Acu and FLX groups on the expression of PKA-α and p-CREB. Interestingly, H89 inhibited the effects of acupuncture or fluoxetine on upregulating the expression of p-CREB, but not PKA-α. There was no significant difference in expression of CREB among the groups. Conclusively, our findings further support the hypothesis that acupuncture could ameliorate depressive-like behaviors by regulating PKA/CREB signaling pathway, which might be mainly mediated by regulating the phosphorylation level of CREB.

Antidepressant-like effects of acupuncture involved the ERK signaling pathway in rats.

BACKGROUND: The extracellular signal-regulated kinase (ERK) signaling pathway is considered to be associated with the pathogenesis and treatment of depression. Acupuncture has been demonstrated to ameliorate depression-related behavior and promote neurogenesis. In this study, we explored the role of the ERK signaling pathway in the antidepressant-like effects of acupuncture in rats exposed to chronic unpredictable mild stress (CUMS). METHODS: Eighty male Sprague-Dawley rats were randomly divided into eight groups: control group, model group, model + Acupuncture group (Acu group), model + fluoxetine group (FLX group), model + DMSO group (DMSO group), model + PD98059 group (PD group), model + Acupuncture + PD98059 group (Acu + PD group) and model + fluoxetine + PD98059 group (FLX + PD group). Except for the control group, all rats were subjected to 3 weeks of CUMS protocols to induce depression. Acupuncture was carried out for 10 min at acupoints of Baihui (GV-20) and Yintang (GV-29) each day during the experimental procedure. The ERK signaling pathway was inhibited using PD98059 through intracerebroventricular injection. The depression-like behaviors were evaluated using the sucrose intake and open-field tests. The protein levels of ERK1/2, phosphor (p)-ERK1/2, cAMP response element-binding protein (CREB), p-CREB and brain-derived neurotrophic factor (BDNF) in the hippocampus were examined using western blot. RESULTS: Acupuncture ameliorated the depression-like behaviors and dysfunction of the ERK signaling pathway in the hippocampus of CUMS rats. PD98059 pretreatment inhibited the improvements brought about by acupuncture on the ERK signaling pathway. CONCLUSIONS: Taken together, our results indicated that acupuncture had a significant antidepressant-like effect on CUMS-induced depression model rats, and the ERK signaling pathway was implicated in this effect.

[Effect of Electroacupuncture Intervention on Behavior Changes and Levels of Hippocampal Transforming Growth Factor beta 3 and Basic Fibroblast Growth Factor Proteins in Depression Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on behavior changes and the adundance levels of transforming growth factor beta 3 (TGF-beta 3) and basic fibroblast growth factor (bFGF) proteins in the hippocampus of rats with chronic unpredictable mild stress (CUMS)-induced depression, so as to explore its mechanisms underlying improvement of depression. METHODS: Forty Sprague-Dawley rats were randomly divided into the following groups: control, model, EA, and medication (Fluoxetine), n = 10 in each group. The depression model was established by CUMS combined with solitary raising for 28 days. EA (2 Hz, 0.6 mA) was applied to "Baihui" (GV 20) and "Yintang" (GV 29) for 20 mm, once daily before CUMS every day. The rats of the medication group were given with Fluoxetine (10 mg/kg, 5 mL/kg) before CUMS every day. The behavioral changes (crossing and rearing locomotion) were detected by using open field tests. The expression levels of TGF-beta 3 and bFGF proteins of the bilateral hippocampus tissues were detected using biotin label-based antibody protein chips. Results Compared to the control group, the crossed grid-square numbers and rearing times were significantly decreased in the model group (P<0.01). Following EA and medication interventions, the CUMS induced decreases of the crossed grid-square number and rearing times were notably reversed in both EA and medication groups (P<0.01), suggesting an amelioration of depression after the intervention. The relative expression level of hippocampal TGF-beta 3 was down-regulated (fold change = 0.48, vs. the control group) and that of bFGF up-regulated (fold change= 1.36, vs the control group) in the model group. In both the EA and medication groups, the down-regulated TGF-beta 3 expression and the up-regulated bFGF protein expression were suppressed (TGF-beta 3: fold change = 1.61, 1.6 and bFGF: fold change = 0.61, 0.45, vs. the model group respectively). CONCLUSION: EA can improve the depression-like state in depression rats which may be associated with its effect in up-regulating hippocampal TGF-beta 3 protein level and down-regulating bFGF protein expression via promoting neurogenesis.

[Effect of Acupuncture Intervention on c-jun N-terminal Kinase Signaling in the Hippocampus in Rats with Forced Swimming Stress].

OBJECTIVE: To observe the effect of acupuncture on c-jun N-terminal Kinase (JNK) signaling in the hippocampus in rats with forced-swimming stress, so as to reveal its underlying mechanism in relieving depression-like motor response. METHODS: Forty-eight Sprague-Dawley rats were randomly divided into 8 groups as control, control + JNK inhibitor (SP 600125) , model, model + SP 600125, acupuncture, acupuncture + SP 600125, Fluoxetine (an anti-depressant) , and Fluoxetine + SP 600125 (n = 6 in each group). The depression-like behavior (immobility) model was established by forcing the rat to swim in a glass-cylinder and solitary raise. Acupuncture stimulation was applied to "Baihui" (GV-20) and "Yintang" (GV 29) for 20 min before forced swimming and once again 24 h later.. The rats of the Fluoxetine and Fluoxetine+ SP 600125 groups were treated by intragastric administration of fluoxetine 10 mL (1.8 mg)/kg before forced swimming and once again 24 h thereafter. The rats of the model + SP 600125 and acupuncture + SP 600125 groups were treated by intraperitoneal injection of SP 600125 (10 mg/kg) 90 min before forced swimming and 30 min before acupuncture intervention, respectively. The immobility duration of rats in the water glass-cylinder was used to assess their depression-like behavior response. The expression levels of protein kinase kinase 4 (MKK 4), MKK 7, JNK, and phosphorylated JNK (p-JNK) in the hippocampus were detected by Western blot. RESULTS: Compared to the control group, the duration of immobility, and the expression levels of hippocampal MKK 4, MKK 7, and p-JNK proteins were significantly increased in the model group (P < 0.01). While in comparison with the model group, the duration of immobility in the model + SP 600125, acupuncture, acupuncture + SP 600125, Fluoxetine and Fluoxetine + SP 600125 groups, the expression levels of hippocampal MKK 4 and MKK 7 proteins in the Fluoxetine + SP 600125 group, and those of p-JNK protein in the acupuncture, acupuncture + SP 600125, model + SP 600125, Fluoxetine and Fluoxetine + SP 600125 groups were considerably decreased (P < 0.05, P < 0.01). No significant differences were found between the control and control + SP 600125 groups and among the model + SP 600125, acupuncture, acupuncture + SP 600125, Fluoxetine and Fluoxetine + SP 600125 groups in the duration of immobility (P > 0.05), and in the expression level of p-JNK protein (P > 0.05). No significant changes were found in the expression levels of JNK among the 8 groups (P > 0.05). CONCLUSION: Acupuncture stimulation of GV 20 and GV 29 is effective in relieving depression-like motor response in forced-swimming stress rats, which may be closely associated with its effects in down-regulating the expression of hippocampal p-JNK protein.

Yifuning postpones ovarian aging through antioxidant mechanisms and suppression of the Rb/p53 signal transduction pathway.

Yifuning is a traditional Chinese medicine recipe that has been used for many years in China for its effects on treating climacteric syndrome in women. The present study aimed to demonstrate the effects and underlying molecular mechanism of Yifuning on the ovaries of aging rats. Selected aging rats were administered different doses of Yifuning (1.0 or 2.0 g/kg by lavage), and after 6 weeks the rats were sacrificed. The activit of indicators of oxidative stress in the serum were measured. The expression levels of 8-oxo-2'-deoxyguanosine (8-OHDG) and p53 in the ovaries were examined using immunohistochemistry. The expression levels of the corresponding genes and proteins were detected by reverse transcription­quantitative polymerase chain reaction and western blotting analyses, respectively. The results indicated that Yifuning significantly prevented ovarian failure, as indicated by improvements in estrous cycling, reproductive organ weights and sex hormone serum levels. Yifuning significantly increased the levels of superoxide dismutase, glutathione peroxidase, catalase and reduced malondialdehyde and hydrogen peroxide levels. Yifuning reduced DNA damage in the ovaries by reducing the expression of 8­OHDG and p53. Treatment with Yifuning significantly reduced the age­induced p19, p53, p21 and Rb activity in the ovaries. The present study demonstrates that Yifuning prevents ovarian failure and the mechanism involved is partly associated with antioxidants and suppression of the Rb/p53 signal transduction pathway.