Effect of different therapies of Chinese medicine on the expressions of c-Fos and c-Jun proteins in hippocampus of rats with post-stroke depression

BACKGROUND ： c-fos and c-jun, the important immediate early genes （IEG）, are regarded as the markers for the location and function of neuronal activity, as well as the third signal messengers, they couple the stress stimulation and the gene expression in neuron, and hippocampus is involved in the process of signal transmission after stress stimulation induced depression. OBJECTIVE： To observe the therapeutic effects of Bushen Yiqi （tonifying kidney to benefit qi）, Huoxue Huayu （promoting blood circulation to dissipate blood stasis） and Ditan Kaiqiao （eliminating phlegm for resuscitation） on the expressions of c-Fos and c-Jun proteins in hippocampus and spontaneous behaviors of rats with post-stroke depression （PSD）, and compare the results with those of fluoxetine, which is known to have definite effect on depression. DESIGN： A randomized controlled tna SETTING ： Zhejiang College of Traditional Chinese Medicine MATERIALS ： The trial was completed in Zhejiang College of Traditional Chinese Medicine from January to July in 2003. Fifty-six healthy adult Wistar male rats of clean grade, weighing （250±50） g, were randomly divided into 7 groups with 8 rats in each group： control group, model group, forced swimming group, Bushen Yiqi group; Huoxue Huayu, Ditan Kaiqiao group and fluoxetine group. The Bushen Yiqi Tang contained Renshen, Huangqi, Heshouwu, Gouqi, Shudi, etc., crude drugs 1 800 g/L. The Huoxue Huayu Tang contained Danshen, Chuanxiong, Chishao, Yujin, etc., crude drugs 3 600 g/L. The Ditan Kaiqiao Tang contained Banxia, Danxing, Changpu, Yuanzhi, etc., crude drug 1 000 g/b METHODS： ① Except the control group and forced swimming group, rats in the other groups were made into PSD models by deligating the bilateral common carotid artedes permanently. ② Rats in the control group, model group and forced swimming group were intragastncally perfused by saline （3 mL for each time）; those in the Bushen Yiqi group, Huoxue Huayu, Ditan Kaiqiao group and fluoxetine group were intragastncally perfused with Bushen Yiqi Tang （18 g/kg）, Huoxue Huayu Tang （9 g/kg）, Ditan Kaiqiao Tang （9 g/kg） and fluoxetine （2.5 mg/kg） respectively, once a day. ③ At 55 days after model establishment, rats in the forced swimming group were managed according to the Porsolt＇s method. They were placed in water for 15 minutes, and then taken out and dned, no moving-time within 5 minutes was recorded at drying and 24 hours after drying. ④ Measurement of spontaneous behaviors： Except the forced swimming group, the spontaneous behaviors and activities （including horizontal and vertical movements） of rats were observed with the Open-Field method at 28, 42 and 56 days after administration in the other groups. ⑤ The expressions of c-Fos and coJun proteins in hippocampus were determined with the immunohistochemical method, the relative sectional area ratio and average objective gray value of c-Fos and c-Jun positive cells in hip- pocampus were measured with the computerized image analytical system. MAIN OUTCOME MEASURES： The spontaneous behaviors of rats, the relative sectional area ratio and average objective gray value of c-Fos and c-Jun positive cells in hippocampus were observed. RESULTS： Of the 56 rats, 1 died in the forced swimming group, and finally 55 rats were involved in the analysis of results. ① Results of spontaneous activities： At 28 days, the times of crossing movements were obviously fewer in the model group and fluoxetine group [（69.00±37.01）, （98.11 ±36.68） times/3 minutes] than in the control group [（128.44±16.85） times/3 minutes, P 〈 0.01, 0.05], but those in the Bushen Yiqi group, Huoxue Quyu group and Ditan Kaiqiao group had no obvious differences as compared with those in the control group （P 〉 0.05）. At 42 and 56 days, the times of crossing movements were obviously more in the Bushen Yiqi group, Huoxue Quyu group and Ditan Kaiqiao group [（106.44±31.24）, （117.20±23.95）, （134.80±28.18）, （136.36±40.95） times/3 minutes; （117.33±35.91）, （129.60 ±23.78）, （131.90 ±26.81）, （136.09±28.34） times/3 minutes] than in the model group [（64.00±17.51）, （72.86±20.68） times/3 minutes, P 〈 0.01]. The times of rearing movements had no obvious differences among the groups for the three times （P 〉 0.05）. ② The no moving-time within 5 minutes 24 hours after drying was obviously longer than that at drying in the forced swimming group. ③ The average objective gray values of c-Fos positive cells were not obviously different in the Bushen Yiqi group and Ditan Kaiqiao group from the control group （P 〉 0.05）, but lower in the model group than in the control group （69.84±9.82, 75.78±5.89, P 〈 0.01）, and higher in the forced swimming group than in the control group （85.97±10.99, P 〈 0.01）; all higher in the fluoxetine group, Bushen Yiqi group, Huoxue Quyu group and Ditan Kaiqiao group than in the model group （81.27±10.73, 74.04±8.34, 83.29±9.89, 70.14±4.92, P 〈 0.05-0.01）. The average objective gray values of c-Jun positive cells were obviously lower in the Bushen Yiqi group than in the control group （68.11 ±6.89, 79.58±5.86, P 〈 0.01）, but all higher in the other groups than in the control group （84.68±7.15, 81.34 ±8.36, 97.51±10.55, 85.68±9.25, 86.19±10.98, P 〈 0.05-0.01）; Those were obviously higher in the fluoxetine group, Huoxue Quyu group and Ditan Kaiqiao group than in the model group （P 〈 0.05-0.01 ）, lower in the Bushen Yiqi group than in the model group （P 〈 0.05）, all obviously lower in the Bushen Yiqi group, Huoxue Quyu group and Ditan Kaiqiao group than in the fluoxetine group （P 〈 0.01）. The relative sectional area ratios of c-Fos and c-Jun positive cells had no obvious differences among the groups （P 〉 0.05）. CONCLUSION ： The methods of Bushen Yiqi, Huoxue Quyu and Ditan Kaiqiao can effectively treat PSD in rats, and the results were equivalent with those of fluoxetine, the actions of the above-mentioned drugs may correlated with their regulation to c-Fos and c-Jun expressions in hippocampus. PSD animal models can be successfully established by both permanent deligation of bilateral common carotid arteries and forced swimming, and the models induced by the former has similar basic cerebrovascular lesions as human stroke in clinic.

Inhibition of growth and metastasis of triple-negative breast cancer targeted by Traditional Chinese Medicine Tubeimu in orthotopic mice models

Objective： Triple-negative breast cancer（TNBC） is highly invasive and metastatic, which is in urgent need of transformative therapeutics. Tubeimu（TBM）, the rhizome of Bolbostemma paniculatum（Maxim.） Franquet, is one of the Chinese medicinal herbs used for breast diseases since the ancient times. The present study evaluated the efficacy, especially the anti-metastatic effects of the dichloromethane extract of Tubeimu（ETBM） on TNBC orthotopic mouse models and cell lines.Methods： We applied real-time imaging on florescent orthotopic TNBC mice model and tested cell migration and invasion abilities with MDA-MB-231 cell line. Digital gene expression sequencing was performed and Kyoto Encyclopedia of Genes and Genomes（KEGG） analysis applied to explore the pathways influenced by ETBM.Moreover, quantitative real-time polymerase chain reactions（q RT-PCR） and Western blot were delivered to confirm the gene expression changes.Results： ETBM exhibited noticeable control on tumor metastasis and growth of TNBC tumors with no obvious toxicity. In compliance with this, it also showed inhibition of cell migration and invasion in vitro. Its impact on the changed biological behavior in TNBC may be a result of decreased expression of integrin β1（ITGβ1）, integrin β8（ITGβ8） and Rho GTPase activating protein 5（ARHGAP5）, which disabled the focal adhesion pathway and caused change in cell morphology.Conclusions： This study reveals that ETBM has anti-metastatic effects on MDA-MB-231-GFP tumor and may lead to a new therapeutic agent for the integrative treatment of highly invasive TNBC.

Electroacupuncture pretreatment exhibits antidepressive 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 m A, 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.

Effect on Life Span and Membrane Protein in Red Blood Cells by Integrated Medicine Therapy on Chronic Aplastic Anemia

Objective: To explore the mechanism ofintegrated traditional Chinese and Westernmedicine (TCM--WM ) therapy on chronicaplastic anemia (CAA). Methods: The RBClife span of 30 normal human subjects and 30patients with CAA were measured by sir labelled technique before and after TCM--WMtherapy. The morphology and distribution ofRBC membrane protein granules were observed by freeze fracture etching and transmission electron microscope. Results: The halflife of erythrocytes (RBC TI/2)was shortenedin CAA cases and there was a significant difference compared to healthy control (P <0. 01). After therapy, the RBC life span prolonged and approached the normal level. Before treatment, there existed abnormal in morphology, decrease in amount and uneven indistribution of protein granules in protoplasmicface (PF) and extracellular face (EF) of RBCmembrane. After treatment, the protein granules of RBC membrane was improved and approached to control. Conclusions: The morphology, amount, quality and distribution ofRBC membrane protein granule were closelyrelated to its life span. The therapeutic effectof TCM--WM was better than that of WMalone and it had a function both in stabilizingmembrane protein and extending the RBC lifespan.

Effects of Modified Qing'e Pill(加味青娥丸) on Expression of Adiponectin,Bone Morphogenetic Protein 2 and Coagulation-Related Factors in Patients with Nontraumatic Osteonecrosis of Femoral Head

Objective： To observe the regulation of Chinese herbal medicine, Modified Qing＇e Pill（加味青娥丸, MQEP）, on the expression of adiponectin, bone morphogenetic protein 2（BMP2）, osteoprotegerin（OPG） and other potentially relevant risk factors in patients with nontraumatic osteonecrosis of the femoral head（ONFH）. Methods： A total of 96 patients with nontraumatic ONFH were unequal randomly divided into treatment group（60 cases） and control group（36 cases）. The treatment group were treated with MQEP while the control group were treated with simulated pills. Both groups were given caltrate D. Six months were taken as a treatment course. Patients were followed up every 2 months. The levels of plasma adiponectin, BMP2, OPG, von Willebrand factor（vWF）, von Willebrand factor cleaving protease（vWF-cp）, plasminogen activator inhibitor 1（PAI-1）, tissue plasminogen activator（tPA）, C-reactive protein（CRP）, blood rheology, bone mineral density（BMD） of the femoral head and Harris Hip Score were measured before and after treatment. Results： After 6 months of treatment, compared with the control group, patients in the treatment group had significantly higher adiponectin and BMP2 levels（P〈0.01 and P=0.013, respectively）, lower vWF, PAI-1 and CRP levels（P=0.019, P〈0.01 and P〈0.01, respectively）, and lower blood rheology parameters. BMD of the femoral neck, triangle area and Harris Hip Score in the treatment group were significantly higher than those in the control group. Moreover, plasma adiponectin showed a positive association with BMP2（r=0.231, P=0.003） and a negative association with PAI-1（r=–0.159, P〈0.05）. Conclusions： MQEP may play a protective role against nontraumatic ONFH by increasing the expression of adiponectin, regulating bone metabolism and improving the hypercoagulation state, which may provide an experimental base for its clinical effects.

Cyclovirobuxinum D Alleviates Cardiac Hypertrophy in Hyperthyroid Rats by Preventing Apoptosis of Cardiac Cells and Inhibiting the p38 Mitogen-Activated Protein Kinase Signaling Pathway

Objective： To investigate the underlying mechanisms of cyclovirobuxinum D（Cvb-D） on alleviating cardiac hypertrophy in rats. Methods： Sprague-Dawley rats were randomly divided into 5 groups： control group; levothyroxine-induced cardiac hypertrophy group（model）; levothyroxine-induced cardiac hypertrophy ＋ Cvb-D group（Cvb-D）; levothyroxine-induced cardiac hypertrophy ＋ captopril group（captopril）; levothyroxine-induced cardiac hypertrophy ＋ SB203580 group（SB203580）, n=10 for each group. Rats were daily administered the respective drugs continuously for14 days by gastric gavage. A rat model of cardiac hypertrophy was established by intraperitoneal injection of levothyroxine to investigate whether Cvb-D protects against cardiac hypertrophy by inhibiting the p38 mitogen-activated protein kinase（MAPK） signaling pathway and preventing apoptosis of cardiac cells. Results： Treatment with Cvb-D significantly deceased left ventricle hypertrophy, improved the histopathology, hemodynamic conditions, and cardiac function in rats with cardiac hypertrophy. Compared with the normal control group, in rats with cardiac hypertrophy, expression of bax in the heart and phospho-p38 MAPK protein levels were significantly up-regulated（P〈0.01 or 0.05）, whereas the bcl-2 protein level was downregulated（P〈0.01）. In contrast, Cvb-D treatment reversed the changes in bax and phospho-p38 MAPK protein levels but increased the bcl-2 protein level（P〈0.01 or 0.05）, and these effects were similar to those of captopril and SB203580（a specific p38 MAPK inhibitor） treatment. Furthermore, both Cvb-D, captopril and SB203580 reduced m RNA expression of p38α, p38β, c-fos, and c-jun m RNA, and Cvb-D had a stronger effect（P〈0.01）. Conclusion： These results demonstrate that Cvb-D protects against cardiac hypertrophy, which is possibly mediated by prevention of cardiac cell apoptosis and inhibition of the p38 MAPK signaling pathway.

Inhibition Mechanism of Qingluo Tongbi Granule(清络通痹颗粒)on Osteoclast Differentiation Induced by Synovial Fibroblast and Monocytes Co-Culture in Adjuvant-Induced Arthritic Rats

Objective： To study the mechanism underlying the inhibitory effect of Qingluo Tongbi Granule （清络通痹颗粒, QTG） on osteoclast differentiation in rheumatoid arthritis in rats. Methods： Fibroblast and monocyte co-culture were used to induce osteoclast differentiation in adjuvant-induced arthritic （AIA） rats. Serum containing QTG was prepared and added to the osteoclasts, and activation of the tumor necrosis factor receptorassociated factor 6/mitogen-activated protein kinase/nuclear factor of activated T cells, cytoplasmic1 （TRAF6/ MAPK/NFATcl） pathways was examined. Results： The induced osteoclasts were multinucleated and stained positive for tartrate-resistant acid phosphatase （TRAP） staining. Serum containing QTG at 14.4, 7.2 or 3.6 g/kg inhibited the activation of TRAF6, extracellular regulated protein kinase （ERK）1/2, c-Jun N-terminal kinase （JNK） and p38 and decreased the percentage of cells with nuclear NFATcl in a dose-dependent manner, the high and middle doses exhibited clear inhibitory activity （P〈0.01 and P〈0.05, respectively）. After the addition of MAPK inhibitors, the NFATcl expression showed no significant difference compared with the control group （P〉0.05）. Conclusions： Serum containing QTG could generally inhibit the TRAF6/MAPK pathways and possibly inhibit the NFATcl pathway. In addition, QTG may regulate other signaling pathways that are related to osteoclast differentiation and maturation.