Objective To evaluate the role of spinal phosphatidyl-inositol 3-kinase/Akt (PI3k/Akt) signaling pathway in the maintenance of bone cancer pain (BCP) in rats and its relationship with microglial activation.Methods Forty healthy female Sprague-Dawley rats,weighing 180-200 g,were randomly divided into 5 groups (n =8 each):sham operation group (group S) ; PI3K inhibitor LY294002 group (group L) ; group BCP; BCP + dimethyl sulfoxide (DMSO) group (group BCP + D) ; BCP + LY294002 group (group BCP + L).BCP was induced by inoculating Walker 256 mammary gland carcinoma cells into the medullary cavity of the left tibia.At 7-9 days after inoculation,LY294002 2.5 μg/10 μl was injected intrathecally in L and BCP + L groups,normal saline 10 μl was injected intrathecally in S and BCP groups,and 5％ DMSO 10 μl was injected intrathecally in BCP+ D group once a day.Mechanical paw withdrawal threshold (MWT) was measured at 1 day before inoculation and 1,3,5,7,8 and 9 days after inoculation.The rats were sacrificed after MWT was measured on day 9 after inoculation and the L4-6 segments of the spinal cord were removed to determinate the activation of spinal microglia using immunofluorescence.Results Compared with group S,MWT was significantly decreased,and the activation of spinal microglia was increased in BCP,BCP + D and BCP+ L groups.Compared with BCP and BCP + D groups,MWT was significantly increased,and the activation of spinal microglia was decreased in BCP + D group.Conclusion Spinal PI3K/Akt signaling pathway is involved in the maintenance of BCP possibly through activating microglia in spinal dorsal horns of rats.

Objective To evaluate the role of c-Jun N-terminal kinase (JNK)/monocyte chemoattractant protein-1 (MCP-1) signaling pathway in the spinal cord in the maintenance of bone cancer pain (BCP) in rats.Methods Fifty female Sprague-Dawley rats,weighing 150-180 g,were randomly divided into 5 groups (n =10 each) using a random number table:sham operation group (group S),sham operation + JNK inhibitor SP600125 (group SP),BCP group,BCP + dimethyl sulfoxide (DMSO) group,and BCP+ SP600125 group (group BCP+ SP).BCP was induced by injecting Walker 256 mammary gland cancer cells into the bone marrow of the left tibia.On 10-12 days after BCP,SP600125 10 μg(10 μ1) was injected intrathecally once a day in SP and BCP + SP groups,and 5％ DMSO 10 μl was injected intrathecally once a day in BCP + DMSO group.Mechanical paw withdrawal threshold (MWT) was measured at 1 day before BCP and 3,6,9,10,11 and 12 days after BCP.After measurement of MWT at 12 days after BCP,the rats were sacrificed and L4-6 segments of the spinal cord were removed for determination of MCP-1 expression by using immuno-histochemistry and Western blot.Results Compared with S group,MWT was significantly decreased at 6-12 days after BCP,MCP-1 expression was upregulated in BCP,BCP + DMSO and BCP + SP groups (P ＜ 0.01),and no significant change was found in the parameters mentioned above in SP group (P ＞ 0.05).Compared with BCP group,MWT was significantly increased at 10-12 days after BCP,MCP-1 expression was down-regulated in BCP + SP group (P ＜ 0.01),and no significant change was found in the parameters mentioned above in BCP + DMSO group (P ＞ 0.05).Conclusion JNK/MCP-1 signaling pathway in the spinal cord may be involved in the maintenance of BCP in rats.

Objective To evaluate the role of spinal microglial C-C chemokine receptor type 2 (CCR2) in the maintenance of bone cancer pain (BCP) in rats.Methods Fifty unmated female Sprague-Dawley rats,aged 2 months,weighing 160-180 g,were randomly divided into 5 groups (n =10 each):sham operation group (group Ⅰ),sham operation + RS102895 (CCR2 antagonist) group (group Ⅱ),BCP group (group Ⅲ),BCP + dimethylsulfoxide (DMSO) group (group Ⅳ),and BCP + RS102895 group (group Ⅴ).The rats were anesthetized with intraperitoneal chloral hydrate.BCP was induced by intra-tibial inoculation of 1 × 105 Walker 256 mammary gland carcinoma cells into the medullary cavity of the left tibial metaphysis.On 10-12 days after operation,3 μg/μl RS102895 10 μd was injected intrathecally once a day in Ⅱ and Ⅴ groups,10％ DMSO 10 μl was injected intrathecally once a day in Ⅳ group,and normal saline 10 μl was injected intrathecally once a day in Ⅰ and Ⅲ groups.Mechanical paw withdrawal threshold was measured at 1 day before operation and 3,6,9,10,11 and 12 days after operation.After measurement of pain threshold at day 12 after operation,the lumbar segments (L4-6) of the spinal cord were removed for determination of the level of ox-42 (spinal microglial activation marker) (by immuno-histochemistry) and contents of IL1-β,IL-6 and TNF-α (by ELISA).Results Compared with group Ⅰ,mechanical paw withdrawal threshold was significantly decreased at 6-12 days after operation,the number of ox-42 positive cells and contents of IL1-β,IL-6 and TNF-α were increased in Ⅲ,Ⅳ and Ⅴ groups,and no significant change was found in the parameters mentioned above in group Ⅱ.Compared with group Ⅲ,mechanical paw withdrawal threshold was significantly increased at 10-12 days after operation,the number of ox-42 positive cells and contents of IL1-β,IL-6 and TNF-α were decreased in group Ⅴ,and no significant change was found in the parameters mentioned above in group Ⅳ.Conclusion Spinal microglial CCR2 is involved in the maintenance of BCP via activating microglia and promoting the release of inflammatory cytokines of rats.

OBJECTIVETo research the regulation of transmembrane transport activity of paclitaxel influenced by fangchinoline in MDR1-MDCK II cells.

METHODPaclitaxel, one of the substrate of P-gp, was selected as the model drug. Verapamil hydrochloride was adopted as the active control to investigate the bilateral transport activity of paclitaxel regulated by fangchinoline in MDR1-MDCK II cells. RP-HPLC was applied to determine the concentration of paclitaxel in the transporting medium, which was used to calculate apparent permeability coefficient of paclitaxel across MDR1-MDCK I1 monolayer cells.

RESULTThe efflux rate of paclitaxel was faster than the absorption rates across the MDR1-MDCK II monolayer cells with highly expressed P-gp. The absorption rates of paclitaxel combinated with fangchinoline and verapamil hydrochloride respectively were remarkably increased and the efflux rate was decreased. The reversal effect of the fangchinoline was stronger than the verapamil hydrochloride with the same molar concention.

CONCLUSIONFangchinoline can apparently decrease the efflux of paclitaxel and inhibit the multidrug resistance of antitumor drug mediated by P-gp.

ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Animals ; Benzylisoquinolines ; pharmacology ; Biological Transport ; drug effects ; Cell Line ; Cell Membrane ; drug effects ; metabolism ; Cell Membrane Permeability ; drug effects ; Dogs ; Humans ; Models, Biological ; Paclitaxel ; pharmacokinetics ; Plant Extracts ; pharmacokinetics

As a micro-wound and target-aimed technology without special limitation, Electric Pulses have been widely researched in tumor treatment and the effects have been demonstrated by a series of experiments, yet the mechanism has not been explained clearly. In this experiment, energy controllable steep pulse (ECSP) was used to treat nude mice bearing human ovarian tumor, and the result was compared with that of the control group. The expression of an important coagulant factor-tissue factor (TF) was analyzed, as TF was also a tumor indicator of invasion and metastasis, the result may indicate the relationship among ECSP, thrombosis and tumor invasion. In this study, to shed light on the mechanism of tumor treatment in electrical fields, nude mice bearing ovarian tumors were randomly divided into the treated group and the untreated group. We treated the former group and took out the tumor instantly. The thrombosis and necrosis of ovarian tumor were observed under microscope. The expression of TF was analyzed by SP immunohistochemistry and RT-PCR. Lower level of TF expression was noticed in the tumor tissue treated by ECSP, and more apparent thrombosis was also seen in this group. The results make it clear that ECSP can accelerate thrombosis and consume coagulant factors such as TF, and that low expression of TF in tumor tissue can cut out the signal paths of tumor invasion. So it is suggested that ECSP may restrain tumor invasion and metastasis by modulating thrombosis.
Animals ; Electric Stimulation Therapy ; methods ; Electromagnetic Fields ; Electroporation ; methods ; Female ; Humans ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Ovarian Neoplasms ; metabolism ; therapy ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Thromboplastin ; biosynthesis ; genetics

This experimental study was designed to investigate the effects and the expressions of microvessel density (MVD), vascular endothelial growth factor (VEGF) on the transplanted tumor in the rat model with Walker-256 after energy controllable steep pulse(ECSP). The experiment revealed that the steep pulse electrical field has better effect on tumor, compared with the control. The positive cell staining intensity of VEGF in the control group was significantly higher than that in ECSP group (P < 0.05). The number of MVD in the tumor tissues of ECSP group was significantly lower than that of tumor control group (P < 0.05). These results showed that ECSP could inhibit the growth and angiogenesis of tumor and its pathway is to down-regulate the expression of VEGF possibly.
Animals ; Carcinoma 256, Walker ; blood supply ; therapy ; Electric Conductivity ; Electric Stimulation Therapy ; methods ; Electromagnetic Fields ; Electroporation ; methods ; Female ; Male ; Neovascularization, Pathologic ; Rats ; Rats, Wistar ; Vascular Endothelial Growth Factor A ; metabolism

Pulsed electric fields (PEFs) with fixed frequency, width and gradually increased peak value of voltage was applied to 30 healthy rabbit liver tissues. The specific aims were to explore the feasibility of establishing a model of in vivo PEFs distribution in healthy rabbit liver tissues and to provide important references for clinical electrochemotherapy and for electrotransfer. Repeated experiment and self-comparison statistics design were implemented. The rabbit underwent the experiment under intravenous anesthesia and their liver tissues, after exposure to PEFs, were sent for HE staining. Necrotic borderline was visible 3 days after PEFs application, the necrotic shape of concentric circle was evident around the electrodes under optical microscope at lower voltage, as voltage increasing, two necroses in the shape of concentric circle gradually enlarged; nuclei with chromatin condensation, fragmentation and lysis alterations were seen in the middle region between the needles; concentric circles changed into ellipse fusiform and finally overlaped each other forming irregular necrosis contours. Cell cavitation and tissues ischemia were also observed within electric field. The shape of tissue necrosis from the experiment was noted to correlate with theoretic simulation of electric field distribution. Therefore, rabbit liver tissues can be a good carrier for in vivo modeling of electric field distribution when the lethal effects of PEFs in tissues are investigated. PEFs also show safety for the surrounding normal tissue while causing damage or injury to the target area therapeutically.
Animals ; Electricity ; adverse effects ; Electrochemotherapy ; Liver ; pathology ; Male ; Models, Theoretical ; Necrosis ; Rabbits

Purpose To observe the thrombolytic effect of different ultrasonic frequencies combined with urokinase-containing targeted microbubble contrast agent on rabbit femoral artery,to explore the main influencing factors,and to determine the potential indicators related to recurrent embolism in the microcirculation.Materials and Methods Unilateral femoral arterial thrombosis models were established in the selected 72 New Zealand white rabbits,which were randomly divided into 12 groups,6 rabbits in each group.This study was performed on an experimental combination of three factors and different levels,including different ultrasonic frequencies (1.6,2.2,2.8 MHz),different ultrasonic irradiation time (30 and 60 min),and different urokinase dose (3 and 6 mg).Thrombolysis with urokinase-containing targeted microbubble was performed under low frequency ultrasonic assisted irradiation,the recanalization status of blood vessels was observed,and recurrent embolism in the microcirculation was confirmed by HE staining.Furthermore,rabbit blood samples were collected,with indicators,including 6-keto-prostaglandin F1a (6-keto-PGF1a),Thromboxane B2 (TXB2),P/T ratio (6-keto-PGF1a/TXB2) and P-selectin (SP) being detected.Results All the vessels were recanalized.There was no occurrence of recurrent embolism in the group with ultrasonic frequency of 2.2 MHz,radiation time of 30 min,and urokinase dose of 3 mg.Rabbits' blood vessels were observed to be not completely recanalized in other groups,accompanied with different degree of recurrent embolism in the microcirculation.6-keto-PGF 1 a content of the rabbits in the group without recurrent embolism obviously increased after thrombolysis,and the difference was statistically significant (P＜0.05).While,there was no statistical difference in other indicators (P＞0.05).Conclsion The thrombolysis with ultrasound frequency of 2.2 MHz,irradiation time of 30 min,and urokinase dose of 3 mg could achieve complete recanalization of blood vessels.Under the certain conditions of ultrasonic frequency,irradiation time and urokinase dosage,thrombus can be effectively dissolved.However,there may be risk of recurrent embolism in the microcirculation during thrombolysis process.The increase of 6-keto-PGF 1a content has a certain effect on reducing the formation of recurrent embolism in the microcirculation.