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Objective To investigate the effect of intrathecal CLP257 on the bone cancer pain in rats.Methods Forty adult female Sprague-Dawley rats,weighing 180-200 g,were divided into 4 groups (n=10 each) using a random number table:sham operation group (group S),bone cancer pain group (group BCP),dimethyl sulfoxide (DMSO) group,and CLP257 group.Bone cancer pain was induced by inoculating Walker 256 mammary gland carcinoma cell suspension (about 1× 105cells) 10 μl into the medullary cavity of the left tibia.On 7th-9th days after establishment of the model,5％ DMSO 10 μl was injected intrathecally once a day in group DMSO,and 10 μg/μl CLP257 10 μl was injected intrathecally once a day in group CLP257.The mechanical paw withdrawal threshold (MWT) was measured on 1 day before establishment of the model (T0),on 1st-6th days after establishment of the model (T1-6),and at 4 h after intrathecal administration on 7th-9th days after establishment of the model (T7-9).After the last intrathecal administration,the L4-6 segments of the spinal cord were removed for determination of the expression of potassium chloride cotransporter 2 (KCC2) protein and mRNA by Western blot and fluorescent quantitative real-time polymerase chain reaction,respectively.Results Compared with group S,the MWT was significantly decreased,and the expression of KCC2 protein and mRNA was down-regulated in BCP and DMSO groups,and the MWT was significantly decreased (P＜0.05),and no significant change was found in the expression of KCC2 protein and mRNA in group CLP257 (P＞0.05).Compared with group BCP,the MWT was significantly increased,and the expression of KCC2 protein and mRNA was up-regulated in group CLP257 (P＜0.05),and no significant change was found in the parameters mentioned above in group DMSO (P＞0.05).Conclusion Intrathecal CLP257 can attenuate the bone cancer pain in rats.
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Aim To investigate the changes in the ex-pression of WNK1 in spinal cord of a rat model with bone cancer pain. Methods Female SD rats, weig-hing 170 ~200 g, were randomly divided into three groups:normal control group (group C, n=3), sham operation group ( group S, n =3 ) and bone cancer pain group ( group BCP, n =24 ) . Group C was not given any treatment, and group S was injected into the bone marrow of left tibia with 5 μl PBS solution while group BCP with 5 μl WALKER 256 mammary gland cancer cell suspension (approximately 1 × 105 cells). Mechanical paw withdrawal threshold ( MWT ) was measured at d1 before inoculation ( baseline) and d3, 6,9,10,11,12 after inoculation. Group S and C were sacrificed at d 12 while group BCP at d 3 ,6 ,9 ,12 after inoculation and spinal cord ( L4~6 ) were removed at different time points for detection of WNK1 mRNA ex-pression by qRT-PCR and WNK1 protein expression by Western blot. Results Compared with group C and S,group BCP’ s MWT started to decrease since d 3 ( P0. 05 ) while the protein expression upregulated since d6 and also showed an in-creasing trend to d 12 ( P<0. 01 ) . Conclusion The expression of WNK1 in spinal cord of a rat model with bone cancer pain increased abnormally, which may be involved in the occurrence and maintenance of a rat model with bone cancer pain.
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Objective To evaluate the effect of lipopolysaccharide (LPS) on the expression of ATP-binding cassette transporter A1 (ABCA1) in alveolar macrophage cells of rats.Methods The alveolar macrophage cells of rats NR8383 were seeded in 6-well plates at a density of 1 × 106 cells/ml (2 ml/well) and randomly divided into 6 groups:control group (group C,n =24),0.2 μg/L LPS group (group L0.2,n =12),2.0 μg/L LPS group (group L2.0,n =12),20.0 μg/L LPS group (group L20.0,n =60),200.0 pg/L LPS group (group L200.0,n =12),and 20.0 μg/L LPS + ABCA1 siRNA group (group L20.0 + siRNA,n =12).The cells were routinely cultured in group C.LPS with the final concentrations of 0.2,2.0,20.0 and 200.0μg/L was added to the culture medium in L0.2,L2.0,L20.0 and I200.0 groups,respectively.In group L20.0 + siRNA,siRNA 50 nmol/L was added to the culture medium and 12 h later LPS 20.0 μg/L was added.In group C,6 wells were chosen for determination of ABCA1 mRNA and protein expression.At 24 h of incubation with LPS 0.2,2.0 and 200.0 μg/L,or at 2,6,12 and 24 h of incubation with LPS 20.0 μg/L,6 wells were chosen and the cell suspension was obtained for measurement of ABCA1 mRNA expression (by real-time fluorescent quantitative PCR),and ABCA1 expression (by flow cytometry).At 12 h of incubation with 20.0 μg/L LPS or with 20.0 μg/L LPS + 50 nmol/L siRNA,6 wells were chosen and the cell suspension was obtained for measurement of TLR4 mRNA expression (by real-time fluorescent quantitative PCR) and TLR4 expression (by flow cytometry).Results Compared with group C,the expression of ABCA1 mRNA and protein was significantly down-regulated in L0.2,L2.0,L20.0 and L200.0 groups,and the expression of ABCA1 mRNA and protein was up-regulated in L20.0 and L20.0 + siRNA groups.In L20.0 group,the expression of ABCA1 mRNA and protein was gradually down-regulated with the prolonging time of incubation with LPS.Compared with group L20.0,the expression of TLR4 mRNA and protein was significantly up-regulated in group L20.0 + siRNA.Conclusion LPS can down-regulate the expression of ABCA1 in alveolar macrophage cells of rats,however,ABCA1 can inhibit the synthesis of TLR4.
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Objective To investigate the effects of triptolide on lipopolysaccharide (LPS)-induced acute lung injury in rats.Methods Sixty-five male SD rats weighing 200-250 g were randomly divided into 5 groups: control group (group C,n =5),LPS group (group L,n =15),different doses of triptolide groups (groups TP1-3,n =15).In group C normal saline was injected iv and 1％ DMSO injected intraperitoneally(ip).In group L LPS 5 mg/kg was injected iv and 1％ DMSO injected ip.In groups TP1-3 LPS 5 mg/kg was injected iv and triptolide 25,50 and 100 μg/kg was injected ip respectively.Blood samples were collected at 1 h before administration and 1,3,6 and 12 h after administration for blood gas analysis.The animals were sacrificed at 12 h after administration.TNF-α concentrations in serum and bronchoalveolar lavage fluid (BALF) were determined by ELISA.The lungs were removed for microscopic examination,evaluation of diffuse alveolar damage (DAD) score and determination of W/D lung weight ratio and the expression of Toll-like receptor4 (TLR4) protein and mRNA.Results Compared with group C,PaO2 was significantly decreased at 3,6 and 12 h after administration,DAD score and W/D lung weight ratio were increased in groups L and TP1-3,TNF-α concentrations in serum and BLAF were increased,expression of TLR4 mRNA and protein was up-regulated in groups L,TP1 and TP2,whlie TNF-α concentrations in serum and BLAF were significantly decreased,expression of TLR4 mRNA and protein was down-regulated in group TP3 ( P ＜ 0.05).Compared with groups L and TP1,PaO2 was significantly increased at 6 and 12 h after administration,DAD score,W/D lung weight ratio and TNF-α concentrations in serum and BLAF were decreased,expression of TLR4 mRNA and protein was down-regulated in groups TP2 and TP3 ( P ＜ 0.05).There was no siginificant difference in blood gas parameters,DAD score and W/D lung weight ratio between group L and group TP1 and between group TP2 and TP3 ( P ＞ 0.05).Compared with group TP2,TNF-α concentrations in serum and BLAF were significantly decreased,expression of TLR4 mRNA and protein was down-regulated in group TP3 (P ＜ 0.05).The lung pathologic injury was reduced in groups TP1-3 as compared with group L.Conclusion Triptolide can attenuate acute lung injury induced by LPS in a dose-dependent manner in rats,and the inhibition of up-regulation of TLR4 expression and release of TNF-α may be involved in the mechanism.