OBJECTIVES: This study aimed to investigate the effect of new biomimetic micro/nano surfaces on the osteoclastic differentiation of RAW264.7 macrophages by simulating natural osteons for the design of concentric circular structures and modifying graphene oxide (GO). METHODS: The groups were divided into smooth titanium surface group (SS), concentric microgrooved titanium surface group (CMS), and microgroove modified with GO group (GO-CMS). The physicochemical properties of the material surfaces were studied using scanning electron microscopy (SEM), contact-angle measurement, atomic force microscopy, X-ray photoelectron spectroscopy analysis, and Raman spectroscopy. The effect of the modified material surface on the cell biological behavior of RAW264.7 was investigated by cell-activity assay, SEM, and laser confocal microscopy. The effect on the osteoclastic differentiation of macrophages was investiga-ted by tartrate-resistant acid phosphatase (TRAP) immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) experiments. RESULTS: Macrophages were arranged in concentric circles along the microgrooves, and after modification with GO, the oxygen-containing groups on the surface of the material increased and hydrophilicity increased. Osteoclasts in the GO-CMS group were small in size and number and had the lowest TRAP expression. Although it promoted the proliferation of macrophages in the GO-CMS group, the expression of osteoclastic differentiation-related genes was lower than that in the SS group, and the difference was statistically significant (P<0.05). CONCLUSIONS Concentric circular microgrooves restricted the fusion of osteoclasts and the formation of sealing zones. Osteomimetic concentric microgrooves modified with GO inhibited the osteoclastic differentiation of RAW 264.7 macrophages.
Graphite/pharmacology* ; Titanium/pharmacology* ; Haversian System ; Macrophages ; Cell Differentiation ; Oxides/pharmacology* ; Surface Properties

OBJECTIVE: To investigate the effect of the chemoprotectant tempol on the anti-tumor activity of cisplatin (DDP). METHODS: The cellular toxicity of tempol in human colon cancer SW480 cells and mouse colon cancer CT26 cells were evaluated using MTT and cell counting kit-8 assays. CalcuSyn software analysis was used to determine the interaction between tempol and DDP in inhibition of the cell viability. A subcutaneous homograft mouse model of colon cancer was established. The mice were randomly divided into control group, tempol group, cisplatin group and tempol + DDP treatment group with intraperitoneal injections of the indicated agents. The tumor size, body weight and lifespan of the mice were measured, and HE staining was used to analyze the cytotoxic effect of the agents on the kidney and liver. Immunohistochemistry and Western blotting were performed to detect the expression of Bax and Bcl2 in the tumor tissue, and TUNEL staining was used to analyze the tumor cell apoptosis. The level of reactive oxygen species (ROS) in the tumor tissue was determined using flow cytometry. RESULTS: Tempol showed inhibitory effects on the viability of SW480 and CT26 cells. CalcuSyn software analysis showed that tempol had a synergistic anti-tumor effect with DDP (CI < 1). In the homograft mouse model, tempol treatment alone did not produce obvious anti-tumor effect. HE staining showed that the combined use of tempol and DDP alleviated DDP-induced fibrogenesis in the kidneys, but tempol also reduced the anti-tumor activity of DDP. Compared with the mice treated with DDP alone, the mice treated with both tempol and DDP had a significantly larger tumor size ( < 0.01) and a shorter lifespan ( < 0.05). Tempol significantly reversed DDP-induced expression of Bax and Bcl2 in the tumor tissue and tumor cell apoptosis ( < 0.001), and obviously reduced the elevation of ROS level in the tumor tissue induced by DDP treatment ( < 0.05). CONCLUSIONS Tempol can attenuate the anti-tumor effect of DDP while reducing the side effects of DDP. Caution must be taken and the risks and benefits should be carefully weighed when considering the use of tempol as an anti-oxidant to reduce the toxicities of DDP.
Animals ; Antineoplastic Agents ; Antioxidants ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Cisplatin ; Cyclic N-Oxides ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Mice ; Spin Labels

OBJECTIVES: To investigate the role of tetramethylpyrazine(TMP) nitrone in proliferation and differentiation of neural stem cells (NSCs). METHODS: We separated and cultivated the original generation of NSCs from cerebral cortex of 14 days rat embryo, and the phenotype characteristics of the third-generation NSCs was tested by immunofluorescence. The experiment was divided into control group, β-mercaptoethanol positive control group, tetramethylpyrazine nitrone group and tetramethylpyrazine nitrone + ethylene glycol tetraacetic acid(EGTA) group (=4). The third-generation cultivation of NSCs was used in the experiment. The effect of tetramethylpyrazine nitrone on the number of NSCs proliferation was determined by BrdU and MTT, and the differentiation of NSCs was determined by Western blot. RESULTS: The primary NSCs was isolated successfully, neurospheres with typical NSCs morphology and expressing nestin was formed at 3-5 days. As BrdU and MTT assay results shown, compared with the control group andβ-mercaptoethanol positive control group, the NSCs proliferation numbers of tetramethylpyrazine nitrone group increased significantly(<0.05). The results of Western blot showed that the neuronal differentiation rate of NSCs was increased significantly in both the tetramethylpyrazine nitrone group and tetramethylpyrazine nitrone + EGTA group, and the differentiation rate of NSCs in tetramethylpyrazine nitrone + EGTA group increased more significantly(<0.05). CONCLUSIONS Tetramethylpyrazine nitrone can significantly enhance the proliferation and neuronal differentiation rate of NSCs. Decrease in extracellular Ca can promote the differentiation of NSCs into neurons induced by tetramethylpyrazine nitrone. Ca signaling plays an important role in the differentiation of NSCs into neurons.
Animals ; Calcium Signaling ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Neural Stem Cells ; cytology ; drug effects ; Nitrogen Oxides ; pharmacology ; Pyrazines ; pharmacology ; Rats

OBJECTIVETo explore the autophagy of RPMI8226 cells induced by As(2)O(3) and its possible mechanisms.

METHODSRPMI8226 was incubated with different concentration of As(2)O(3) for different time, and the inhibiting rate was calculated by MTT method. The autophagic rate of RPMI8226 cells incubated with different concentration of As(2)O(3) was determined by FACS. The change of cells ultrastructure was observed by transmission electron microsopy (TEM). After incubation with different concentration of As(2)O(3), the expression of Beclin-1 on RPMI8226 was detected by RT-PCR and Western blot.

RESULTSDifferent concentration of As(2)O(3) could significantly inhibit the proliferation of RPMI8226 cells (P < 0.05), and the inhibitory effect was in dose- and time-dependent way in a certain range. the autophagic rate increased with the increasing of drug concentration and prolonging of action time (P < 0.05). TEM results revealed a typical autophagosome in RPMI-8226 cell treated by As(2)O(3) for 48 hours. Beclin-1 was up-regulated in RPMI 8226 cells when treated with different concentration of As(2)O(3) for 48h (P < 0.05).

CONCLUSIONAs(2)O(3) can induce autophagy of RPMI8226 cells, and the mechanism may be associated with the upregulation of Beclin-1.

Apoptosis Regulatory Proteins ; metabolism ; Arsenicals ; pharmacology ; Autophagy ; drug effects ; Beclin-1 ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Membrane Proteins ; metabolism ; Oxides ; pharmacology ; Up-Regulation

OBJECTIVETo explore the effect of magnetic iron nanoparticles ( Fe₃O₄- MNP) in combination with arsenic trioxide and adriamycin on apoptosis and autophagy of Raji cells, a non-Hodgkin's lymphoma (NHL) cell line.

METHODSThe growth inhibition rate of Raji cells was analyzed by MTT assay, the cells apoptosis and intracellular concentration of ADM were determined by flow cytometry (FCM), the expression levels of apoptosis-related proteins such as BCL-2, NFκB, Survivin, BAX, P53, and Caspase-3, and related to autophagy-proteins, such as LC3, Beclin-1, and P62/SQSTM1 were detected by Western blot.

RESULTSThe growth inhibition of Raji cells in the group of ADM + As₂O₃were higher than that in the group of ADM or As₂O₃alone, however, lower than that in the group of Fe₃O₄- MNP combined with ADM and As₂O₃(ADM+As₂O₃+ MNP) (P < 0.05). The apoptotic rate and accumulation of intracellular ADM in the group of Fe₃O₄- MNP combined with ADM and As₂O₃were significantly higher than those in control, ADM, As₂O₃, and ADM plus As₂O₃groups (P < 0.05). The upregulation of BAX, P53 and Caspase-3 expression and the down regulation of BCL-2, NFκB, and Survivin expression at protein level were more remarkable in the group of ADM+As₂O₃ + MNP, compared with the other groups (P < 0.05). Moreover, the expressions of LC3 and Beclin-1 proteins in the group of ADM+As₂O₃+ MNP were higher, while the expression of P62/SQSTM1 was lower than that in other groups (P < 0.05).

CONCLUSIONThe Fe3O4 - MNP combined with ADM and As₂O₃can increase the antitumor efficacy on Raji cells by promoting apoptosis and inducing autophagy. It would be a promising strategy for malignant lymphoma therapy.

Apoptosis ; Arsenicals ; pharmacology ; Autophagy ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Doxorubicin ; pharmacology ; Ferric Compounds ; pharmacology ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Nanoparticles ; Oncogene Proteins, Fusion ; metabolism ; Oxides ; pharmacology

Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Cell Line, Tumor ; drug effects ; Humans ; Leukemia ; pathology ; Oxides ; pharmacology

OBJECTIVETo investigate the anticancer efficacy and the hepatic and renal toxicity of As2O3-lipiodol emulsion via transarterial embolization in a rabbit VX2 liver tumor model.

METHODSVX2 tumors were implanted in rabbit livers successfully, followed by transarterial embolization with high-dose As2O3(5 mg/kg with 0.2 mL lipiodol, n=10), low-dose As2O3(1 mg/kg with 0.2 mL lipiodol, n=10), and control(0.2 mL lipiodol, n=10). The growth ratios and microvessel densities(MVDs) of the tumors were estimated by multi-row spiral CT and CD34 immunohistochemical staining, respectively. Hepatic and renal function was also evaluated by means of blood biochemical analysis.

RESULTSThe growth ratios of the tumors differed significantly among three groups(P<0.01). The high-dose and low dose group showed significantly lower tumor growth ratios[44.05%(-36.40%~64.60%), 95.20%(-11.60%~159.40%)] than control group[145.55%(98.90%~250.30%), all P<0.05]. The MVDs of the tumors were significantly lower in the high-dose(21.4±10.6) and low-dose group(34.1±12.0) than those in control group(57.9±16.1,all P<0.05). The levels of blood ALT and AST obtained 28 days after transarterial embolization were significantly lower in the high-dose[(25.50±12.37)U/L,(24.25±10.89)U/L] and low-dose group[(45.00±14.04)U/L,(35.22±11.86)U/L] than in control group[(79.12±30.52)U/L,(75.25±25.89)U/L, all P<0.05].

CONCLUSIONAs2O3-lipiodol emulsion via transarterial embolization has anticancer effect without significant hepatic and renal functional damage in rabbit VX2 liver tumors.

Animals ; Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Embolization, Therapeutic ; Emulsions ; pharmacology ; Ethiodized Oil ; pharmacology ; Liver Neoplasms, Experimental ; drug therapy ; Oxides ; pharmacology ; Rabbits ; Tomography, Spiral Computed

The present study was aimed to investigate the roles of renal sympathetic nerve and oxidative stress in the development of foot shock-induced hypertension. Ninety rats were divided into 6 groups (the number of each group was 15): control group, foot shock group, denervation of renal sympathetic nerve group, denervation of renal sympathetic nerve + foot shock group, Tempol treatment + foot shock group, denervation of renal sympathetic nerve + Tempol treatment + foot shock group. Rats were received electrical foot shock for 14 days (2-4 mA, 75 V, shocks of 50-100 ms every 30 s, for 4 h each session through an electrified grid floor every day). Renal sympathetic ablation was used to remove bilateral renal sympathetic nerve in rats (rats were allowed to recover for one week before the beginning of the foot shock procedure). The antioxidant Tempol was injected intraperitoneally at 1 h before foot shock. Systolic blood pressure was measured at 1 h after foot shock on day 0, 3, 7, 10 and 14. Contents of thiobarbituric acid reactive substance (TBARS), renin, angiotensin II (AngII) and glutathione peroxidase (GSH-Px) in plasma were measured by ELISA after 14-day foot shock. The results showed that systolic blood pressure of foot shock group was significantly increased (P < 0.05) compared with that of control group from day 7 to day 14 of foot shock. Denervation of renal sympathetic nerve and/or Tempol treatment significantly reduced the increase of systolic blood pressure induced by foot shock. Levels of TBARS, renin and AngII in plasma were increased significantly in foot shock group compared with that of control group (P < 0.05). Plasma GSH-Px concentration was decreased in foot shock group rats compared with that of control group (P < 0.05). Denervation of renal sympathetic nerve and/or tempol treatment significantly reduced the increase in TBARS, renin, AngII levels induced by foot shock in comparison with that of foot shock group (P < 0.05), but had no effects on the reduction of GSH-Px concentration. The results suggest that renal sympathetic nerve may play an important role in the development of foot shock-induced hypertension, and renal sympathetic nerve may influence oxidative stress and directly or indirectly activate renin-angiotensin-aldosterone system, so the foot shock-induced high blood pressure may be maintained and hypertension may therefore be produced.
Animals ; Antioxidants ; pharmacology ; Blood Pressure ; Cyclic N-Oxides ; pharmacology ; Denervation ; Electric Stimulation ; Hypertension ; physiopathology ; Kidney ; innervation ; Oxidative Stress ; Rats ; Renin-Angiotensin System ; Spin Labels ; Sympathetic Nervous System ; physiology

OBJECTIVETo investigate whether brain reactive oxygen species mediate sympathoexcitation and arterial pressure elevation in DOCA-salt hypertensive rats.

METHODSDOCA-salt hypertensive model was established in male SD rats by subcutaneous injection of DOCA after uninephrectomy and drinking 1% NaCl solution for 4 weeks. The baseline mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) were recorded in the rats under mild anesthesia, and MAP changes following intravenous hexamethonium injection were observed. The responses of MAP, HR and RSNA to intracerebroventricular administration of tempol (20 µmol/L in 10 µl) were evaluated; plasma NE level was measured with ELISA, and ROS level and NAD(P)H oxidase activity in the hypothalamus were detected using chemiluminescence assay.

RESULTSMAP and plasma NE levels were significantly increased in DOCA-salt rats as compared with those in the control group (P<0.01). In DOCA-salt hypertensive rats, intravenous hexamethonium injection induced a blood pressure reduction 240% of that in control rats, and significantly increased the levels of superoxide anion and NAD(P)H oxidase activity in the hypothalamus. Intracerebroventricular microinjection of tempol also resulted in more significant changes of MAP, HR and RSNA in DOCA-salt rats than in the control group (P<0.01).

CONCLUSIONSympathoexcitation due to increased NAD(P)H oxidase-derived ROS levels in the hypothalamus may mediate arterial pressure elevation in DOCA-salt hypertensive rats.

Animals ; Antioxidants ; Arterial Pressure ; Blood Pressure ; Brain ; metabolism ; Cyclic N-Oxides ; pharmacology ; Desoxycorticosterone ; Desoxycorticosterone Acetate ; Disease Models, Animal ; Heart Rate ; Hypertension ; Kidney ; innervation ; Male ; NADPH Oxidases ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Sodium Chloride ; Spin Labels ; Superoxides ; metabolism ; Sympathetic Nervous System

This study was aimed to explore the regulation of arsenic trioxide (As₂O₃) on imbalance between adipogenic and osteogenic differentiation of BM-MSC from patients with aplastic anemia(AA). The BM-MSC from AA patients were separated and purified, placed into the adipogenic and osteogenic differentiation culture system, then added the As₂O₃, CsA, As₂O₃combined with CsA were added to corresponding differentiation culture system, the concentration of As₂O₃and CsA were set at 0.001 µmol/ml and 2.5 mmol/ml respectively, the cells were divided into As₂O₃group, the CsA group, combined group and control group (no drug). The cell morphological observation, oil red 'O' staining, Von-Kossa staining, and RT-PCR were used to detect corresponding differentiation marks. The results indicated that in respect to adipogenic differentiation, cellular morphology observing and oil red 'O' staining showed that the rate of adipocyte differentiation in As₂O₃group was (18.3 ± 1.9)%, which was lower than the (91.8 ± 2.7)% in the CsA group and (92.1 ± 1.2)% in control group (P < 0.05), there was no significant difference in comparison with (8.3 ± 1.9)% in the combined group (P > 0.05), but the rate of differentiation in CsA group was higher than that in combined group (P < 0.05), and there was no significant difference in comparison wtih control group. RT-PCR showed that the LPL-mRNA expression level in As₂O₃group were significantly lower than that in the CsA group and the control group (P < 0.05), no difference was observed while compared with the combined group (P > 0.05), but the LPL-mRNA expression level in CsA group was significantly higher than that in the combined group (P < 0.05). In terms of osteogenetic differentiation, the calcium deposition in As₂O₃group and combined group was obviously observed while rarely in the CsA group and the control group when detected by the Von-Kossa staining. OST-mRNA expression level in As₂O₃group were higher than that in CsA group and the control group (P < 0.05), while compared with the combined group, there was no significant difference (P > 0.05), but the OST-mRNA expression level in the CsA group was lower than that in the combined group (P < 0.05). It is concluded that As₂O₃can significantly enhance the ability of BM-MSC from AA patients to differentiate into osteoblast, also can inhibit the adipogenic differentiation, in contrast, the CsA can not promote the osteoblast differentiation of BM-MSC from AA patients.
Adipocytes ; cytology ; drug effects ; Anemia, Aplastic ; pathology ; Arsenicals ; pharmacology ; Bone Marrow ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Osteoblasts ; Osteogenesis ; drug effects ; Oxides ; pharmacology