Effects of Chaihu-Shugan San on behavior and hippocampal neurogenesis in depression model mice / 中华行为医学与脑科学杂志

Objective:To explore the effects of Chaihu-Shugan San (CSS) on the behavior and neurogenesis function of depression model mice induced by chronic unpredictable mild stress (CUMS).Methods:Thirty clean grade healthy male C57BL/6 adult mice were randomly divided into control group (Con group), model group (CUMS group) and Chaihu-Shugan San treatment group (CSS group), with 10 mice in each group.The mice in CUMS group and CSS group were given CUMS intervention to establish depression model. At the same time of modeling, the mice in CUMS group and CSS group were given distilled water and CSS(2.7 g/kg) by gavage respectively.While the mice in Con group were only given equal volume distilled water by gavage without CUMS stimulation.After the intervention, the depressive-like behavior of mice was evaluated by increased body weight, sugar water preference test (SPT), forced swimming test (FST) and tail suspension test (TST). The number of newborn neurons was detected by immunofluorescence staining. The mRNA expression levels of brain-derived neurotrophic factor (BDNF), fibroblast growth factor 2 (FGF2) and spindle and kinetochore-associated protein 2(SKA2) in mice hippocampus were detected by qRT-PCR.Statistical analysis was performed by SPSS 22.0 software. One-way ANOVA was used for multi group comparison, and Tukey test was used for pairwise comparison.Results:(1) After modeling, there was significant difference in body weight increment among the three groups ( F=8.859, P <0.05). The body weight increment of CUMS group was lower than those of Con group and CSS group (both P< 0.05). There were significant differences in sugar water preference rate, tail suspension immobility time and swimming immobility time among the three groups ( F=10.544, 12.957, 8.095, all P<0.05). The sugar water preference rate in CUMS group was lower than that in Con group ((87.46±2.78)%, (93.90±3.31)%, P<0.05), and that in CSS group was higher than that in CUMS group ((91.65±2.61)%)( P<0.05). The tail suspension immobility time ((198.00±27.57) s) and swimming immobility time ((322.20±46.98) s) in CUMS group were higher than those in Con group ((138.80±38.50) s, (238.50±50.51) s, both P<0.05). The tail suspension immobility time ((139.00±21.29) s) and swimming immobility time ((265.20±44.90) s) in CSS group were lower than those in CUMS group (both P<0.05). (2) Immunofluorescence showed that there was significant difference in the number of newborn neurons labeled by BrdU and NeuN in the dentate gyrus of hippocampus among the three groups ( F=9.486, P<0.05). The number of double labeled cells (31.66±3.21) in CUMS group was lower than that in Con group(63.66±15.17) and CSS group (58.00±6.00) (both P<0.05). (3) RT-PCR results showed that the mRNA levels of BDNF, FGF2, SKA2 in hippocampal dentate gyrus of the three group were significantly different( F=14.522, 9.337, 8.701, all P<0.05). The levels of BDNF mRNA (0.79±0.06), FGF2 mRNA (0.74±0.18) and SKA2 mRNA (0.52±0.32) in the dentate gyrus of hippocampus in CUMS group were lower than those in Con group (BDNF mRNA (1.03±0.10), FGF2 mRNA (1.04±0.11), SKA2 mRNA (1.05±0.37), all P<0.05). Compared with CUMS group, the mRNA levels of BDNF (1.07±0.80), FGF2 (1.30±0.29) and SKA2 (1.40±0.55) in CSS group were higher (all P<0.05). Conclusion:CSS can alleviate the depressive like behavior of depression model mice, which may be related with increasing the mRNA expression levels of BDNF, FGF2, SKA2 and promoting the proliferation of neural stem cells in hippocampus.

Upregulation of microRNA-520a-3p inhibits the proliferation, migration and invasion via spindle and kinetochore associated 2 in gastric cancer.

MicroRNAs (miR) serve important roles in the development and progression of tumors by targeting different genes. miR-520a-3p reported in lung and breast cancers as a tumor suppressor gene. However, the expression and functional significance of miR-520a-3p is not completely understood in gastric cancer (GC). In the present study, it was demonstrated that the expression levels of miR-520a-3p were significantly downregulated in GC tissues and cells using RT-qPCR. In addition, downregulated expression of miR-520a-3p was associated with the clinical stage of the tumor and invasion in patients with GC. Furthermore, overexpression of miR-520a-3p significantly inhibited cell proliferation, invasion and migration in SGC-7901 and MGC-803 GC cell lines using proliferation, wound healing and cell invasion assays. Spindle and kinetochore associated 2 (SKA2) was upregulated in GC cells using western blot analysis and a target gene of miR-520a-3p; miR-520a-3p mimics significantly reduced SKA2 expression. In addition, upregulation of SKA2 protein expression SKA2 reversed the miR-520a-3p-mediated inhibition of SGC-7901 cell proliferation, migration and invasion. In conclusion, miR-520a-3p functioned as a tumor suppressor gene by targeting SKA2 in GC cell lines, and may serve as a novel prognostic and potential therapeutic marker.

SKA2 mediates invasion and metastasis in human breast cancer via EMT.

Spindle and kinetochore­associated protein 2 (SKA2) is essential for regulating the progression of mitosis. In recent years, SKA2 upregulation has been detected in various human malignancies and the role of SKA2 in tumorigenesis has received increasing attention. However, the expression and functional significance of SKA2 in breast cancer are not completely understood. To study the effects of SKA2 on breast cancer, the expression levels of SKA2 in breast cancer tissues and cell lines were evaluated by western blotting, reverse transcription­quantitative polymerase chain reaction and immunohistochemical staining. The results demonstrated that SKA2 expression was increased in breast cancer tissues and cells, and SKA2 overexpression was associated with clinical stage and lymph node metastasis. Functional investigations revealed that SKA2 knockdown in breast cancer cells significantly reduced migration and invasion, and resulted in the decreased expression levels of matrix metalloproteinase (MMP)2 and MMP9. Furthermore, the typical microtubule arrangement was altered in SKA2 small interfering RNA (siSKA2)­transfected cells. Reduced levels of SKA2 also downregulated the expression of epithelial­mesenchymal transition proteins, including fibronectin, N­cadherin and vimentin, whereas there were no alterations in the protein expression levels of E­cadherin. Conversely, upregulation of SKA2 decreased the expression levels of E­cadherin, and increased N­cadherin, fibronectin and vimentin levels. Notably, it was demonstrated that E­cadherin was translocated from the cytoplasm to the nucleus in siSKA2­transfected cells. These results demonstrated that SKA2 may be associated with breast cancer metastasis, and siSKA2 inhibited the invasion and metastasis of breast cancer via translocation of E­cadherin from the cytoplasm to the nucleus.

Peroxisome proliferator-activated receptor-α-mediated transcription of miR-301a and miR-454 and their host gene SKA2 regulates endothelin-1 and PAI-1 expression in sickle cell disease.

Endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1) play important roles in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show higher levels of placenta growth factor (PlGF) in SCD correlate with increased plasma levels of ET-1, PAI-1, and other physiological markers of PH. PlGF-mediated ET-1 and PAI-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively little is understood regarding post-transcriptional regulation of PlGF-mediated expression of ET-1 and PAI-1. Herein, we show PlGF treatment of endothelial cells reduced levels of miR-301a and miR-454 from basal levels. In addition, both miRNAs targeted the 3'-UTRs of ET-1 and PAI-1 mRNAs. These results were corroborated in the mouse model of SCD [Berkeley sickle mice (BK-SS)] and in SCD subjects. Plasma levels of miR-454 in SCD subjects were significantly lower compared with unaffected controls, which correlated with higher plasma levels of both ET-1 and PAI-1. Moreover, lung tissues from BK-SS mice showed significantly reduced levels of pre-miR-301a and concomitantly higher levels of ET-1 and PAI-1. Furthermore, we show that miR-301a/miR-454 located in the spindle and kinetochore-associated protein-2 (SKA2) transcription unit was co-transcriptionally regulated by both HIF-1α and peroxisome proliferator-activated receptor-α (PPAR-α) as demonstrated by SKA2 promoter mutational analysis and ChIP. Finally we show that fenofibrate, a PPAR-α agonist, increased the expression of miR-301a/miR-454 and SKA2 in human microvascular endothelial cell line (HMEC) cells; the former were responsible for reduced expression of ET-1 and PAI-1. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-301a/miR-454 expression can ameliorate PH and lung fibrosis by reduction in ET-1 and PAI-1 levels in SCD.