[Down-regulation of Ech1 decreases the adhesion ability of mouse hepatocarcinoma Hca-F cells].

To examine the differential expression pattern of Ech1 protein in mouse Hca-F and Hca-P hepatocarcinoma cell lines with high and low rates of lymphatic metastasis, respectively, and to investigate the relationships between Ech1 expression and adhesion of Hca-F cells. Fluorescence two-dimensional difference in-gel electrophoresis (2D DIGE) and mass spectrometry were used to detect Ech1 expression. Ech1 gene silencing was achieved by stable transfection of Hca-F cells with a plasmid vector harboring short hairpin RNA (shRNA) targeting Ech1, pGPU6/GFP/Neo-shRNA-Ech1. Ech1 mRNA and protein expressions were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting analysis, respectively. Adhesive properties of cells were assessed by hematoxylin-eosin staining and fluorimetric detection of extracellular matrix (ECM) proteins. Endogenous Ech1 protein level was remarkably higher in the highly metastatic Hca-F cell line than in the Hca-P cell line (2.7-fold by 2D DIGE; 1.5-fold by Western blotting). shRNA-induced silencing of Ech1 significantly reduced the adhesion ability of Hca-F cells, as evidenced by decreased absorbance values of fibronectin and collagen I (Hca-F cells vs. pGPU6/GFP/Neo-shRNA-Ech1 cells: 1.42+/-0.26 vs. 1.01+/-0.27 and 1.14+/-0.07 vs. 0.90+/-0.09, respectively; P less than 0.05). Down-regulation of Ech1 can inhibit the adhesive capacity of metastatic Hca-F cells.

[Expression of enoyl CoA hydratase 1 reduces cell proliferation and migration in mouse hepatocarcinoma cells].

OBJECTIVE: To study the expression of enoyl CoA hydratase 1 (ECH1) and the effect when down-regulation of ECH1 gene expression in mouse hepatocarcinoma cell. METHODS: Immunofluorescence was used for detecting the expression of ECH1, and stably transfected Hca-F cells with pGPU6/GFP/Neo-shRNA-ECH1 expression plasmids. Cell proliferation was assessed by Cell counting kit-8 (CCK8) assay. The Boyden-transwell assay (8 µm pore size) was performed to analyze the inhibitory effect of shRNA on Hca-F cell migration and invasion. RESULTS: ECH1 expression was obtained in the cytoplasm and upregulated expression in Hca-F cells than that in Hca-P cells. The down-regulation of ECH1 could inhibit the cell proliferation of Hca-F cells, decrease the number of cell pass through Transwell (27.07 ± 17.49) compared with scramble-negative (72.38 ± 18.83) and Hca-F controls (59.06 ± 30.33), decrease the migration capacities of Hca-F cells, increase the ratio of Hca-F cells in S phase (86.1%) compared with scramble-negative (75.8%) and Hca-F controls (66.2%) and decrease the ratio of G(1) phase (9.4%) compared with scramble-negative (24.2%) and Hca-F controls (30.3%). CONCLUSION: ECH1 serves as a potential critical factor attributes to tumor lymphatic metastasis.

[Localization and expression of CLIC1 in hepatocarcinoma ascites cell lines with high or low potentials of lymphatic spread].

OBJECTIVE: To study the localization and expression of CLIC1 in mouse hepatocarcinoma ascites cell lines with different metastatic potentials. METHODS: Mouse hepatocarcinoma ascites models (a high potential of lymphatic metastasis cell line-Hca-F, and a low potential of lymphatic metastasis cell line-Hca-P) were investigated using fluorescent two-dimensional difference-gel electrophoresis (2-D DIGE) and mass spectrometry for detecting the localization and expression of CLIC1. Immunofluorescence, immunocytochemistry and Western blot were used to assess CLIC1 protein status in the two cell lines. RESULTS: CLIC1 expression was obtained in the cytoplasm and plasma membrane of cells in both cell lines. 2-D DIGE showed that CLIC1 was overexpressed in Hca-F cells, 1.6 folds higher than that of the Hca-P cells. Hca-F cells also had a higher integral membrane CLIC1 in the Hca-P cells. CONCLUSIONS: Although CLIC1 expression is detected in both Hca-F and Hca-P cell lines, a higher protein expression level is present in Hca-F cells. CLIC1 may play an important role in tumor metastasis.

Effect of 17ß-oestradiol on T-type calcium channels in the lateral habenula.

T-type calcium channels (T-channels) are critical for regulating neuronal excitability. Oestrogen alters neuronal excitability by modulating the expression of T-channels. The lateral habenula (LHb), as a link between the limbic system and midbrain structures, expresses T-channels and ERs. However, little is known about the role of oestrogen with respect to modulating T-channels in the LHb. In the present study, we investigated the distribution of T-channels in 3 LHb subregions (rostral, middle and caudal) in normal female rats. Next, we analysed the influence of 17ß-oestradiol (E2 ) on T-channels in the LHb in ovariectomised (OVX) rats (oil and E2 groups) using whole-cell patch clamp recording and a real-time polymerase chain reaction (PCR). In normal rats, the results obtained showed that the peak of T-type calcium current (IT ) was -474.61 ± 48.33 pA and IT density was -29.11 ± 1.93 pA/pF. The IT peak and IT density on LHb neurones gradually decreased across the rostrocaudal axis. The neuronal firing pattern varied depending on the location: burst firing was dominant (53.85%) in the rostral LHb, whereas tonic firing was dominant (79.31%) in the caudal LHb. In OVX rats, real-time PCR analysis revealed that E2 treatment decreased Cav3.3 mRNA expression in the caudal LHb. Patch clamp recording showed that E2 treatment decreased the peak IT and also reduced the low-threshold spikes (LTS) number, amplitude and width of LTS in the caudal LHb. Taken together, the results obtained in the present study suggest that E2 may inhibit T-channel activity by selectively down-regulating Cav3.3 calcium channel in the caudal LHb, leading to reduced the possibility of burst firing.

[Real-time measurement of noradrenaline release in central nervous system].

In order to investigate the central nervous mechanism and the diseases involved in catecholamine transmitter secretion, the dynamics of catecholamine release is studied in single cell, brain slice or in vivo. Noradrenaline is an important neurotransmitter and modulator in the central nervous system (CNS) and the peripheral nervous system (PNS). In the present paper, we first compared three real-time methods used to measure noradrenaline secretion in single cells (membrane capacitance, amperometry and confocal fluorescence microscopy imaging). Compared to the electrophysiological method and fluorescence microscopy, the basic usage of the carbon fiber electrode (CFE) in neuroscience research was presented as an example. Then, we presented a primary description of ion channels, including voltage-gated Na(+), K(+) and Ca(2+) channels in locus coeruleus (LC) neurons in rat brain slices. Finally, we presented example recordings of combined patch-clamp and amperometry measurements in LC neurons, indicating Ca(2+)-dependent quantal noradrenaline release following Ca(2+) influx through Ca(2+) channels.

Estradiol suppresses neuronal firing activity and c-Fos expression in the lateral habenula.

Estradiol is an ovarian steroid hormone that regulates physiological functions in the central nervous system, including mood, cognition, sleep and mental state. Emerging evidence has revealed that there is an enrichment of cells that express the estrogen receptor in the lateral habenula (LHb) region, however, the precise biological functions of estradiol on neurons in the LHb remain to be elucidated. The present study aimed to determine the effects of estradiol on LHb neurons, by observing neuronal firing activity, and c­Fos mRNA and protein expression levels in the LHb using whole cell recording, reverse transcription­quantitative polymerase chain reaction and western blotting. Ovariectomized female Wistar rats were supplemented with a range of estradiol doses across five groups: Ovariectomized (no treatment); Oil (sesame oil); low estradiol; medial estradiol (ME) and high estradiol. Circulating serum estradiol levels were assessed over a 33 day period following surgery. Estradiol suppressed spontaneous firing activity in LHb neurons (P<0.05, compared with firing rates prior to estradiol treatment), which suggested a role for this hormone in regulating neuronal activity. Estradiol replacement therapy resulted in sustained serum estradiol levels for ~3 weeks after surgery. The mRNA and protein levels of c­Fos were significantly downregulated in LHb tissues from ME rats as compared with Oil animals (P<0.05). In conclusion, the present study demonstrated that estradiol suppresses neuronal activities in the LHb region, suggesting that the LHb may be a potential target for estradiol action.

A potential target for the treatment of Parkinson's disease: effect of lateral habenula lesions.

OBJECTIVE: Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that is caused predominantly by the degeneration of the nigrostriatal dopaminergic pathway. Lateral habenula (LHb) has efferent projections that terminate in the substantia nigra pars compacta (SNpc) and electrical stimulation of the LHb effectively suppresses the activity of dopamine-containing neurons in the SNpc. This study was aimed to investigate whether LHb lesions can ameliorate the syndromes of PD via affecting the activities of SNpc neurons in 6-hydroxydopamine (6-OHDA)-induced PD model rats. METHODS: Concentrations of dopamine (DA) and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum, which is the area projected by the SNpc dopaminergic neurons were assayed by high-performance liquid chromatography (HPLC) coupled with fluorescence detection. The immunohistochemical method was applied to detect the numbers of tyrosine hydroxylase (TH)-positive cells in the substantia nigra. RESULTS: The results showed that LHb lesions induced a significant reduction in apomorphine-induced rotational behavior. The DA, DOPAC and HVA levels in the striatum of PD model rats were increased by the LHb lesions. CONCLUSION: Therefore, we speculate that the LHb lesions induced a significant amelioration in motor disorders via increasing the DA levels in the striatum, which may lead to a potential therapeutic strategy for the treatment of PD.