Objective To analyze the expression of ROCK2 in osteosarcoma tissues and explore its effect on the invasion and migration of osteosarcoma cells and related mechanism. Methods Real-time fluorescent quantitative PCR, Western blot and immunohistochemical staining were used to detect the expression of ROCK2 in osteosarcoma tissues. The migration and invasion of osteosarcoma cells were analyzed by wound-healing and Transwell assays after the knockdown of ROCK2 expression. The effect of reducing ROCK2 expression on metastasis in vivo was tested by tail vein metastasis experiment. We detected the expression of EMT-related proteins in osteosarcoma cells, and added the EMT inducer TGF-β to osteosarcoma cells with down-regulated ROCK2 expression to detect the changes of cell invasion and migration. Results The expression of ROCK2 in osteosarcoma tissues was significantly higher than that in corresponding adjacent tissues (P < 0.05). After down-regulating the expression of ROCK2 in osteosarcoma cells, the invasion and migration abilities in vivo and in vitro were significantly inhibited (P < 0.05). The expression of EMT-related protein E-cadherin decreased, while N-cadherin and Vimentin proteins increased significantly. Inducing EMT could attenuate the inhibitory effect of down-regulating ROCK2 on the invasion and migration of osteosarcoma cells. Conclusion The expression of ROCK2 is increased in osteosarcoma tissues, which promotes the invasion and migration of osteosarcoma cells by inducing EMT. ROCK2 may be a potential biomarker for targeted therapy of osteosarcoma.

Objective To explore the protective effect and mechanism of vitamin D combined with puerarin on liver fibrosis.Methods The rats were divided into normal control group (C),tetrachloromethone group (CCl4),vitamin D group (V),puerarin group(P) and vitamin D combined with puerarin group(V+P).After 8 weeks,the rats were sacrificed and blood and liver samples were collected.The level of blood hyaluronic acid(HA) was tested.The hydroxyproline(Hyp) level in the liver was measured.The liver paraffin sections were made and examined by the sirius red staining.The mRNA levels of collagen Ⅰ and collagen Ⅲ in the liver tissue were detected by RT-PCR,and the levels of NF-κB and TNF-α in the liver were detected by Western blot.Results The CCl4 group appeared obvious liver fibrosis.The liver fibrosis degree was significantly improved in the group V,P and V+P,the blood HA level and liver Hyp level were reduced.The mRNA levels of collagen Ⅰ and collagen Ⅲ as well as the protein levels of NF-κB and TNF-α in the liver were significantly decreased.Among them.The liver fibrosis improvement degree in the V+P group was most significant.Conclusion Vitamin D combined with puerarin can protect rat liver fibrosis induced by CCl4 and its mechanism may be related with reducing the activation of hepatic stellate cells(HSC) and decreasing the collagenous fibers secretion.

Rosmarinic acid (RA) is a natural polyphenolic compound that exists in many medicinal species of Boraginaceae and Lamiaceae. The previous studies have revealed that RA had therapeutic effects on hepatocellular carcinoma (HCC) in the H22-xenograft models by inhibiting the inflammatory cytokines and NF-κB p65 pathway in the tumor microenvironment. However, its molecular mechanisms of immunoregulation and pro-apoptotic effect in HCC have not been fully explored. In the present study, RA at 75, 150, and 300 mg/kg was given to H22 tumor-bearing mice via gavage once a day for 10 days. The results showed that RA can effectively inhibit the tumor growth through regulating the ratio of CD4⁺/CD8⁺ and the secretion of interleukin (IL)-2 and interferon-γ, inhibiting the expressions of IL-6, IL-10 and signal transducer and activator of transcription 3, thereby up-regulating Bax and Caspase-3 and down-regulating Bcl-2. The underlying mechanisms involved regulation of immune response and induction of HCC cell apoptosis. These results may provide a more comprehensive perspective to clarify the anti-tumor mechanism of RA in HCC.
Animals ; Apoptosis ; Boraginaceae ; Carcinoma, Hepatocellular ; Caspase 3 ; Cytokines ; Interleukin-10 ; Interleukin-6 ; Interleukins ; Lamiaceae ; Mice ; STAT3 Transcription Factor ; Therapeutic Uses ; Tumor Microenvironment

To investigate the effects of gossypol acetic acid (GA) on proliferation and apoptosis of the macrophage cell line RAW264.7 and further understand the possible underlying mechanism responsible for GA-induced cell apoptosis, RAW264.7 cells were treated with GA (25~35 micromol/L) for 24 h and the cytotoxicity was determined by MTT assay, while apoptotic cells were identified by TUNEL assay, acridine orange/ethidium bromide staining and flow cytometry. Moreover, mitochondrial membrane potential (DeltaPsi(m)) with Rhodamine 123 and reactive oxygen species (ROS) with DCFH-DA were analyzed by fluorescence spectrofluorometry. In addition, the expression of caspase-3 and caspase-9 was assessed by Western Blot assay. Finally, the GA-induced cell apoptosis was evaluated by flow cytometry in the present of caspase inhibitors Z-VAD-FMK and Ac-LEHD-FMK, respectively. GA significantly inhibited the proliferation of RAW264.7 cells in a dose-dependent manner, and caused obvious cell apoptosis and a loss of DeltaPsi(m) in RAW264.7 cells. Moreover, the ROS production in cells was elevated, and the levels of activated caspase-3 and caspase-9 were up-regulated in a dose-dependent manner. Notably, GA-induced cell apoptosis was markedly inhibited by caspase inhibitors. These results suggest that GA-induced RAW264.7 cell apoptosis may be mediated via a caspase-dependent mitochondrial signaling pathway.
Animals ; Antineoplastic Agents, Phytogenic/*pharmacology ; Apoptosis/*drug effects ; Cell Line ; Cell Proliferation/*drug effects ; Dose-Response Relationship, Drug ; Gossypol/*analogs & derivatives/pharmacology ; Membrane Potential, Mitochondrial/*drug effects ; Mice ; Mice, Inbred BALB C ; Reactive Oxygen Species/*metabolism ; Signal Transduction/*drug effects

OBJECTIVETo explore the effect and molecular mechanism of the unconventional prefoldin RPB5 interactor (URI) in hepatocellular carcinoma HepG2 cells.

METHODSThe cDNA sequence and shRNA of URI were obtained and sub-cloned into eukaryotic expression vectors. Then those vectors were transfected into HepG2 cells to obtain stable transfection cell line. The cell proliferation and anchor-independent growth in URI-overexpressing and knockdown HepG2 cells were determined by CCK-8 and soft agar colony assay. Flow cytometry was applied to detect the cell cycle and apoptosis of γ-ray irradiated cells. Apoptosis related genes were detected by Western blot.

RESULTSThe pCDNA3.1-URI and pGPU6-URIi eukaryotic expression vectors were constructed successfully and corresponding stable transfection cell lines were obtained. Cell proliferation rates of the HepG2, pCDNA3.1-URI-HepG2 and pGPU6-URIi-HepG2 cells were (588.78 ± 32.12)%, (959.33 ± 58.8)% and (393.93 ± 39.7)%, respectively (P < 0.05). The number of cell clones of HepG2, pCDNA3.1-URI-HepG2 and pGPU6-URIi-HepG2 cells were 43 ± 7, 85 ± 5 and 20 ± 4 (P < 0.05), respectively. After γ-ray irradiation, the URI-overexpressing cell line showed a significantly lower apoptosis rate and G(2)/M phase arrest than those in the URI-depleted cell line (P < 0.05). In the HepG2 cells, the relative protein expression levels of URI, Bax and Bcl-2 were 0.92 ± 0.03, 1.11 ± 0.13 and 0.82 ± 0.01 (P < 0.05). In the pCDNA3.1-URI-HepG2 cells, the relative protein expression levels of URI, Bax and Bcl-2 were 1.79 ± 0.12, 0.48 ± 0.01 and 2.20 ± 0.30 (P < 0.05), respectively. In the pGPU6-URIi-HepG2 cells, the relative protein expression levels of URI, Bax and Bcl-2 were 0.50 ± 0.04, 1.52 ± 0.20 and 0.38 ± 0.01 (P < 0.05), respectively. The expression of Bax was down-regulated and Bcl-2 was up-regulated in the URI-overexpressing cell line. However, on the contrary, expression of Bax was up-regulated and Bcl-2 was down-regulated in the URI-depleted cell line.

CONCLUSIONSURI may promote the growth of hepatocellular carcinoma cells via inhibition of cell proliferation and reducing the apoptosis in hepatocellular carcinoma cells in vitro. After the impairment of URI expression, the proliferation ability of hepatocellular carcinoma cells is suppressed and the ability to resist γ-ray irradiation is reduced. URI may become a potential new target for cancer therapy of hepatocellular carcinoma.

Apoptosis ; Carcinoma, Hepatocellular ; metabolism ; Cell Cycle ; Cell Proliferation ; Down-Regulation ; Genetic Vectors ; Hep G2 Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Liver Neoplasms ; RNA, Small Interfering ; Transfection

Humans ; Bacteremia ; Cross Infection ; Methicillin-Resistant Staphylococcus aureus ; Retrospective Studies ; Sepsis ; Staphylococcal Infections/epidemiology* ; Staphylococcus aureus ; Tertiary Care Centers ; China

Objective The resting-state functional magnetic resonance imaging (rs-fMRI) method was used to observe brain activity and its functional connection upon electroacupuncture stimulation at bilateral uterine acupoints (EX-CA1), as well as to investigate the mechanism of acupuncture in the treatment of gynecological diseases. Methods Twenty-two healthy female subjects were stimulated by electroacupuncture at bilateral uterine acupoints; rs-fMRI data of the brain were acquired and standardized. Degree centrality (DC), amplitude of low-frequency fluctuation (ALFF), and regional homogeneity (ReHo) were used to analyze local spontaneous brain activity via acupuncture. An independent component analysis was used to evaluate the functional connectivity of the resting brain networks after acupuncture. Results Analytical results showed that the neural activity intensity of the precuneus lobe, orbitofrontal cortex, lingual gyrus, amygdala, and posterior central gyrus decreased after acupuncture (voxel P < 0.001, cluster P < 0.05). Functional connectivity analysis revealed weakened auditory and right frontal-parietal networks (voxel P < 0.001, cluster P < 0.05), enhanced visual network (voxel P < 0.001, cluster P < 0.05), and synergistic auditory network and hypothalamic-pituitary system. Conclusion Significant differences in neural activity and functional connectivity in specific brain regions were observed after acupuncture intervention at uterine acupoints; the hypothalamic-pituitary system also showed various active states in different brain regions. It is speculated that the effective mechanism of acupuncture at uterine acupoints is related to the regulation of reproductive hormones, emotional changes, and somatic sensations. Therefore, the methods used in this study could clarify the neural mechanism of uterine-point acupuncture in the treatment of gynecological diseases and may serve as a reference for other studies pertaining to acupuncture.