[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.

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.

[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.

Electroacupuncture pretreatment exhibits anti-depressive effects by regulating hippocampal proteomics in rats with chronic restraint stress.

The clinical effect of electroacupuncture on depression is widely recognized. However, the signal transduction pathways and target proteins involved remain unclear. In the present study, rat models of chronic restraint stress were used to explore the mechanism by which electroacupuncture alleviates depression. Rats were randomly divided into control, model, and electroacupuncture groups. Chronic restraint stress was induced in the model and electroacupuncture groups by restraining rats for 28 days. In the electroacupuncture group, electroacupuncture pretreatment at Baihui (GV20) and Yintang (GV29) acupoints was performed daily (1 mA, 2 Hz, discontinuous wave, 20 minutes) prior to restraint for 28 days. Open field tests and body weight measurements were carried out to evaluate the depressive symptoms at specific time points. On day 28, the crossing number, rearing number, and body weights of the model group were significantly lower than those in the control group. Behavior test results indicated that rat models of depressive-like symptoms were successfully established by chronic restraint stress combined with solitary raising. On day 28, an isobaric tag for a relative and absolute quantitation-based quantitative proteomic approach was performed to identify differentially expressed proteins in hippocampal samples obtained from the model and electroacupuncture groups. The potential function of these differential proteins was predicted through the use of the Cluster of Orthologous Groups of proteins (COG) database. Twenty-seven differential proteins (uncharacteristic proteins expected) were selected from the model and electroacupuncture groups. In addition to unknown protein functions, COG are mainly concentrated in general prediction function, mechanism of signal transduction, amino acid transport and metabolism groups. This suggests that electroacupuncture improved depressive-like symptoms by regulating differential proteins, and most of these related proteins exist in nerve cells.

Sophoridine exerts an anti-colorectal carcinoma effect through apoptosis induction in vitro and in vivo.

AIMS: To further investigate the anti-colorectal carcinoma (CRC) effect of Sophoridine (SRI) which is a quinolizidine alkaloid extracted from a traditional Chinese medicine (TCM) Sophora alopecuroides L. and detect the mechanism involved, provide some basis for the development of S. alopecuroides L. MAIN METHODS: The anti-proliferation of SRI in human colorectal cells SW480 were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. The potential mechanism of anti-proliferation was also investigated using apoptosis assays. The rate of apoptosis cells was detected also. The apoptosis-related proteins cysteinyl aspartate specific protease (caspase), caspase-3, caspase-7, caspase-9, and poly-ADP-ribose-poly-merase (PARP) were determined by western blotting analysis. In animal studies, nude mice were subcutaneously injected with SW480 cells in the armpit to establish the xenograft tumors and administrated with different drugs (control, 5-Fu, SRI H, and SRI L). The general state of health of the mice and the growth of tumors were observed and the inhibitory rate was calculated. The pathology and ultrastructure of xenograft tumors treated with SRI were observed also. KEY FINDINGS: SRI significantly inhibited the growth of SW480 cells, and the administration of SRI significantly inhibited the growth of xenograft tumors without apparent toxicity. SRI's mechanism of action involved the induction of apoptosis. SIGNIFICANCE: These results suggest that SRI produces obvious anti-tumor effects in vitro and in vivo. It supports the viability of developing SRI as a novel therapeutic prodrug for CRC treatment, as well as providing a method for identifying new anti-tumor drugs in TCM.

Apigenin inhibits proliferation and induces apoptosis in human multiple myeloma cells through targeting the trinity of CK2, Cdc37 and Hsp90.

BACKGROUND: Multiple myeloma (MM) is a B-cell malignancy that is largely incurable and is characterized by the accumulation of malignant plasma cells in the bone marrow. Apigenin, a common flavonoid, has been reported to suppress proliferation in a wide variety of solid tumors and hematological cancers; however its mechanism is not well understood and its effect on MM cells has not been determined. RESULTS: In this study, we investigated the effects of apigenin on MM cell lines and on primary MM cells. Cell viability assays demonstrated that apigenin exhibited cytotoxicity against both MM cell lines and primary MM cells but not against normal peripheral blood mononuclear cells. Together, kinase assays, immunoprecipitation and western blot analysis showed that apigenin inhibited CK2 kinase activity, decreased phosphorylation of Cdc37, disassociated the Hsp90/Cdc37/client complex and induced the degradation of multiple kinase clients, including RIP1, Src, Raf-1, Cdk4 and AKT. By depleting these kinases, apigenin suppressed both constitutive and inducible activation of STAT3, ERK, AKT and NF-κB. The treatment also downregulated the expression of the antiapoptotic proteins Mcl-1, Bcl-2, Bcl-xL, XIAP and Survivin, which ultimately induced apoptosis in MM cells. In addition, apigenin had a greater effects in depleting Hsp90 clients when used in combination with the Hsp90 inhibitor geldanamycin and the histone deacetylase inhibitor vorinostat. CONCLUSIONS: Our results suggest that the primary mechanisms by which apigenin kill MM cells is by targeting the trinity of CK2-Cdc37-Hsp90, and this observation reveals the therapeutic potential of apigenin in treating multiple myeloma.

[Protective effect of Oviductus Ranae capsules on the reproductive organs of aged mice].

OBJECTIVE: To observe the protective effect of Oviductus Ranae (OR) capsules on the reproductive organs in an aged mouse model established by D-galactose injection. METHODS: Forty-eight female Kunming mice were randomly divided into 4 equal groups, namely the high- and low-dose OR groups, diethylstilbestrol (DT) group, and model group. The mice received subcutaneous injection of D-galactose for 6 weeks to establish aging models. Another 12 mice were injected daily with normal saline (NS) to serve as the normal control group. From the third week of the experiment, the mice were given oral OR at low or high doses (in the OR groups) or vegetable oil (in the model or control groups) till the sixth week. In the last two weeks, the vaginal smears were obtained from the mice for evaluating the changes of the vaginal keratinocytes and counting the days of estrus. After completion of drug administration, all the mice were sacrificed and the serum content of estradiol (E(2)) was detected by radioimmunoassay, with the ovarian and uterine indices determined. The ovarian and uterine pathologies were observed using HE staining, and SOD and MDA activities in the ovary and uterus were also assessed. RESULTS: OR obviously increased E(2) level and the ovarian and uterine indices in the aged mice, also alleviating the pathological change of the ovary and uterus. OR substantially depressed MDA content and enhanced SOD activity in the ovary and uterus. CONCLUSION: OR has definite antioxidative effects and ameliorates the degenerative changes of the reproductive organs in mouse models of aging.

[Effects of Yifuning soft gelatin capsules on pathology and biomechanical indexes of ovariectomied rats].

OBJECTIVE: To study the effects of Yifuning soft gelatin capsules (YFN) on pathology and biomechanical indexes of ovariectomied female rats. METHOD: Sixty female Sprague-Dawley rats without pregnancy and delivery were used,50 of them were ovariectomized and randomly divided into 5 groups: ovariectomy(OVX), OVX with diethylstilbestrol tables (DT), OVX with YFN (high dose,middle dose and low dose), the others were sham-operated group. The rats began to take the rats drugs in the fifth week after the operation. After 12 weeks of treat ment the rats were killed, and the bones were collected to inspect. The pathology of bone was observed with the HE stain and the area of trabecular was detected. The electronic testing device was used to detect the biomechanical properties. RESULT: The results indicated that the rat model of postmenopausal osteoporosis (PMO) was a success. Compared with the model group, after application of the drugs, the observation of HE showed different repairing around trabecular. The biomechanical parameters also increased significantly. CONCLUSION: YFN can effectively prevent the loss of bone in OVX rats and strengthen bone quality, and has a good effect on PMO oostmenopausal osteoporosis.

[Inhibitory effect of Biejiajianwan pills on tumor growth and proliferating cell nuclear antigen expression in mice bearing H22 cell-induced tumor].

OBJECTIVE: To study the anti-tumor effect of Biejiajianwan pills and explore its mechanism. METHODS: Mice bearing neoplasm induced by H22 cell inoculation were randomized into 4 groups, namely the negative control group, positive control group (with cyclophosphamide treatment), and high- and low-dose Biejiajianwan pill group. The tumor-bearing mice received corresponding treatments for 15 consecutive days, after which their thymuses and spleens were isolated and weighed to calculate the thymus and spleen indices. The tumor mass was also weighed and the tumor inhibition rate calculated. Immunohistochemistry was performed to evaluate the expression intensity of proliferating cell nuclear antigen (PCNA) in the tumor tissue. RESULTS: The pills significantly inhibited the growth of the implanted tumor and reduced the expression of PCNA in the tumor. CONCLUSION: One of the possible mechanisms of the anti-tumor effects of Biejiajian pills might be associated with the inhibition of PCNA expression in the tumor.