异丙酚对内毒素诱导人脐静脉内皮细胞过氧亚硝基阴离子生成的影响

Objective To investigate the effects of propofol on the production of peroxynitrite anion (ONOO-) in lipopelysaccharide (LPS)-induced human umbilical vein endothelial cell (HUVEC) injury. Methods HUVECs were provided by centre for preservation of cell strains of special species, Wnhan University, and were assigned to one of 7 groups (n = 5 each). In group Ⅰ no additive was added to HUVECs. In group Ⅱ , Ⅲ ,HUVECs were incubated with LPS 0.1, 1.0, 10 μg/ml. In group Ⅴ, Ⅵ HUVECs were incubated with propofol 4 or40 μg/ml + LPS 10 μg/ml. In group Ⅶ HUVECs were incubated with intralipid (solvent for propofol) 40 μg/ml + LPS 10 μg/ml. After 6 h incubation nitrotyrosine protein in HUVECs was detected by immunocytochemical staining and immunoblot assay. Lactate dehydrogeuase (LDH) release and cell viability were measured. Results LPS significantly increased the expression of nitrotyrosine, enhanced LDH release and decreased live cell numbers in a concentration-dependent manner. These changes were significantly attenuated by propofoi especially 40 μg/ml. Conclusion Propofol can protect HUVECs from LPS-induced injury through inhibiting the production of ONOO-.     

盐酸戊乙奎醚对内毒素性急性肺损伤大鼠肺组织Toll样受体4mRNA和Toll样受体2mRNA表达的影响

Objective To investigate the effect of penehyclidine (PHCD) on Toll-like receptor 4 (TLR4)mRNA and Toll-like receptor 2 (TLR2) mRNA expression in the lung tissue in rats with acute lung injury induced by lipopolysaccharide (LPS) .Methods Sixty healthy SD rats of both sexes weighing 200-220 g were randomly divided into 5 groups ( n = 12 each) :control group (group C) , LPS group and P1-3 groups. Acute lung injury was induced by intraperitoneal (IP) LPS 8 mg/kg in LPS and P1-3 groups. PHCD 0.3, 1.0 and 3.0 mg/kg were given IP after LPS administration in P1-3 groups. The animals were anesthetized at 6 h after IP LPS. Blood samples were collected for determination of serum TNF-α and IL-6 concentrations ( by ELISA) and then sacrificed, the lungs were immediately removed for determination of TLR4 mRNA and TLR2 mRNA expression (by RT-PCR), and microscopic examination. Results LPS significantly increased TLR4 mRNA and TLR2 mRNA expression in the lung tissue and serum TNF-α and IL-6 concentrations. PHCD 1.0 or 3.0 mg/kg significantly inhibited LPS-induced increase in TLR4 mRNA and TLR2 mRNA expression in the lung tissue and serum TNF-α and ILr6 concentrations.The lung histopathologic damage was significantly ameliorated in P2 and P3 groups as compared with group LPS.Conclusion PHCD can protect the lungs against LPS-induced acute lung injury through inhibiting TLR4 mRNA and TLR2 mRNA expression in the lung tissue and reducing the inflammatory response.     

盐酸戊乙奎醚对内毒素性急性肺损伤大鼠肺组织Toll样受体4mRNA和Toll样受体2mRNA表达的影响

Objective To investigate the effect of penehyclidine (PHCD) on Toll-like receptor 4 (TLR4)mRNA and Toll-like receptor 2 (TLR2) mRNA expression in the lung tissue in rats with acute lung injury induced by lipopolysaccharide (LPS) .Methods Sixty healthy SD rats of both sexes weighing 200-220 g were randomly divided into 5 groups ( n = 12 each) :control group (group C) , LPS group and P1-3 groups. Acute lung injury was induced by intraperitoneal (IP) LPS 8 mg/kg in LPS and P1-3 groups. PHCD 0.3, 1.0 and 3.0 mg/kg were given IP after LPS administration in P1-3 groups. The animals were anesthetized at 6 h after IP LPS. Blood samples were collected for determination of serum TNF-α and IL-6 concentrations ( by ELISA) and then sacrificed, the lungs were immediately removed for determination of TLR4 mRNA and TLR2 mRNA expression (by RT-PCR), and microscopic examination. Results LPS significantly increased TLR4 mRNA and TLR2 mRNA expression in the lung tissue and serum TNF-α and IL-6 concentrations. PHCD 1.0 or 3.0 mg/kg significantly inhibited LPS-induced increase in TLR4 mRNA and TLR2 mRNA expression in the lung tissue and serum TNF-α and ILr6 concentrations.The lung histopathologic damage was significantly ameliorated in P2 and P3 groups as compared with group LPS.Conclusion PHCD can protect the lungs against LPS-induced acute lung injury through inhibiting TLR4 mRNA and TLR2 mRNA expression in the lung tissue and reducing the inflammatory response.     

Polymyxin B antagonizing biological activity of lipopolysaccharide

Objective ： To investigate the mechanism of polymyxin B （ PMB ） antagonizing the biological activity of Hipopolysaccharide （LPS）. Methods： The affinity of PMB for LPS and lipid A was assayed by biosensor, and the neutralization of PMB for LPS （2 ng/ml ） was detected by kinetic turbidimetric limnins test. The releases of TNF-α and IL-6 in murine peritoneal macrophages （PMφ） after exposure to LPS （ 100 ng/ml） were detected, and the expression levels of TLR4, TNF-α and IL-6 mRNA in PMφ induced by LPS （100 ng/ml） were measured by RT-PCR. Results： PMB had high-affinity to LPS and lipid A with dissociation equilibrium constants of 18.9 nmol/L and 11.1 nmol/L, respectively, and neutralized LPS in a dosedependent manner. Furthermore, PMB could markedly inhibit the expressions of TLR4, TNF-α and IL-6 mRNA and the release of cycokines in LPS-stimniated murine PMφ. Conclusions： PMB neutralizes LPS and inhibites the expression and release of cycokines in macrophages, in which the affinity of PMB for lipid A plays an important role.     

盐酸戊乙奎醚对内毒素性急性肺损伤大鼠肺组织Toll样受体4mRNA和Toll样受体2mRNA表达的影响

目的 探讨盐酸戊乙奎醚对内毒索性急性肺损伤大鼠肺组织Toll样受体4(TLR4)mRNA和Toll样受体2(TLR2)mRNA表达的影响.方法 健康SD大鼠60只,雌雄不拘,体重200～220g,采用随机数字表法,将大鼠随机分为5组(n=12),对照组(C组)、LPS组和低、中、高剂量盐酸戊乙奎醚组(P1组～P3组).C组腹腔注射生理盐水2ml;LPS组腹腔注射LPS 8mg/kg;P1组～P3组分别腹腔注射LPS 8 mg/kg和盐酸戊乙奎醚0.3、1.0和3.0 mg/kg.给药结束后6 h时开胸,心室取血,并取肺组织,采用ELISA法测定血清TNF-α和Ib-6的浓度,RT-PCR法测定肺组织TLR4 mRNA和TLR2 mRNA 的表达水平,并观察肺组织病理学结果.结果 与C组比较,LPS组、P1组～P3组血清TNF-α、IL-6浓度和肺组织TLR4 mRNA、TLR2 mRNA表达均升高(P<0.05);与LPS组比较,P2组和P3组血清TNF-α、IL-6浓度和肺组织TLR4 mRNA、TLR2 mRNA表达均降低(P<0.05),P1组上述指标差异无统计学意义(P>0.05);与P1组比较,P2组和P1组血清TNF-α、IL-6浓度和肺组织TLR4 mRNA、TLR2 mRNA表达均降低(P<0.05);P2组和P3组血清TNF-α、IL-6浓度和肺组织TLR4 mRNA、TLR2 mRNA表达比较差异无统计学意义(P>0.05).P2组和P3组肺组织病理学损伤程度明显轻于LPS组.结论 盐酸戊乙奎醚可通过下调肺组织TLR4 mRNA和耵JR2 mRNA的表达,降低炎性反应,从而减轻大鼠内毒素性急性肺损伤.

Abstract：

Objective To investigate the effect of penehyclidine (PHCD) on Toll-like receptor 4 (TLR4)mRNA and Toll-like receptor 2 (TLR2) mRNA expression in the lung tissue in rats with acute lung injury induced by lipopolysaccharide (LPS) .Methods Sixty healthy SD rats of both sexes weighing 200-220 g were randomly divided into 5 groups ( n = 12 each) :control group (group C) , LPS group and P1-3 groups. Acute lung injury was induced by intraperitoneal (IP) LPS 8 mg/kg in LPS and P1-3 groups. PHCD 0.3, 1.0 and 3.0 mg/kg were given IP after LPS administration in P1-3 groups. The animals were anesthetized at 6 h after IP LPS. Blood samples were collected for determination of serum TNF-α and IL-6 concentrations ( by ELISA) and then sacrificed, the lungs were immediately removed for determination of TLR4 mRNA and TLR2 mRNA expression (by RT-PCR), and microscopic examination. Results LPS significantly increased TLR4 mRNA and TLR2 mRNA expression in the lung tissue and serum TNF-α and IL-6 concentrations. PHCD 1.0 or 3.0 mg/kg significantly inhibited LPS-induced increase in TLR4 mRNA and TLR2 mRNA expression in the lung tissue and serum TNF-α and ILr6 concentrations.The lung histopathologic damage was significantly ameliorated in P2 and P3 groups as compared with group LPS.Conclusion PHCD can protect the lungs against LPS-induced acute lung injury through inhibiting TLR4 mRNA and TLR2 mRNA expression in the lung tissue and reducing the inflammatory response.     

谷氨酰胺抑制人外周血单个核细胞细胞因子表达的机制

Objective To investigate the mechanism that glutamine (Gin) downregulates the cytokine expression in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PMBCs). Methods PMBCs were extracted from healthy volunteer by density gradient centrifugation, the cells were divided into two parts. The first part of PMBCs was pretreated with Gin of the concentration of 0, 8,15 mmol/L for 0.5 h and 2.0 h respectively, then stimulated by LPS for 4. 0 h. Cells and supematants were collected. The second part of PBMCs was divided into group A, B and C. Group A and B were preteated with HSP70 blocker (Quereetin) for 1.0 h, then were stimulated by LPS for 4. 0 h. Cells and supematants were also collected. The release of TNF-α and IL-10 was analyzed via enzyme-linked immunosorbent assay (ELISA) and HSP70 via Western Blot. In this experiment, the effect of Quercetin on TNF-α,IL-10 and HSP70 expression in human PBMCs was assessed. Results Gin led to an increase in HSP70 expression, and decreased TNF-α, IL-10 release at 4. 0 h after LPS stimulation when 8 mmol/L glutamine pretreated for 0.5 h and 2. 0 h, 15 mmol/L glutamine pretreated for 0.5 h (P < 0.05). The expression level of HSP70 was significantly decreased, however, the expression of TNF-α and IL-10 was enhanced in Quercetin group (P <0.05). Conclusion The effect of glutamine attenuating cytokine release in PBMCs is related to the enhancement of HSP70 expression.     

右美托咪啶对脂多糖诱导大鼠外周血单核细胞Toll样受体4mRNA表达的影响

目的 探讨右美托咪啶对脂多糖(LPS)诱导大鼠外周血单核细胞Toll样受体4(TLR4)mRNA表达的影响.方法 健康雄性Wistar大鼠40只,取外周血分离培养单核细胞,采用随机数字表法,将其随机分为5组(n=8),A组:阴性对照;B组:单核细胞中加入LPS(终浓度为1μg/ml);C组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为0.5 ng/ml);D组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为5.0 ng/ml);E组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为50.0 ng/ml).孵育24 h后,收集上清液,采用ELISA法测定TNF-α、IL-1β和IL-6的浓度,采用RT-PCR法测定TLR4 mRNA的表达.结果 与A组比较,B组TNF-α、IL-1p、IL-6的浓度升高,TLR4 mRNA表达上调(P＜0.01);与B组比较,C组、D组和E组TNF-α、IL-1β、IL-6的浓度降低,TLR4 mRNA表达下调(P＜0.05或0.01);与C组比较,D组和E组TNF-α、IL-1β、IL-6的浓度降低(P＜0.01),TLR4 mRNA表达差异无统计学意义(P＞0.05);D组和E组各指标比较差异无统计学意义(P＞0.05).结论 右美托咪啶可通过下调TLR4 mRNA表达,抑制TLR4的合成,从而抑制LPS诱导大鼠外周血单核细胞TNF-α、IL-1β和IL-6的生成与释放.

Abstract：

Objective To investigate the effects of different concentrations of dexmedetomidine on the expression of Toll-like receptor 4 (TLR4) mRNA in rat peripheral blood monocytes exposed to lipopolysaccharide ( LPS ). Methods Peripheral blood monocytes isolated from male Wistar rats were seeded in 24-well plate in RPMI 1640 liquid culture medium in CO2 incubator at 37 ℃ and 5% CO2 for 2 h, and were randomly divided into 5 groups ( n = 8 each): group A negative control; group B was exposed to LPS 1 μg/ml and C, D and E groups were exposed to LPS 1 μg/ml + dexmetomidine 0.5, 5.0 and 50.0 ng/ml respectively. The monocytes were then incubated for 24 h. The concentrations of TNF-α, IL-1β and IL-6 in the supernatant of the cultured monocytes were detected by ELISA. The expression of TLR4 mRNA in the monocytes was detected by RT-PCR.Results Exposure to LPS significantly increased the expression of TLR4 mRNA and the concentrations of TNF-α, IL-1β and IL -6 in group B as compared with group A ( P ＜ 0.01 ). Dexmedetomidine attenuated the LPS-induced increase in the expression of TLR 4 mRNA and the concentrations of TNF-α, IL-1β and IL-6 in a dose-dependent manner ( P ＜0.05or 0.01 ). Conclusion Dexmedetomidine can inhibit the synthesis of TLR4 and inhibit the secretion and dilivery of TNF-α, IL-1β and IL-6 by down-regulating the gene expression of TLR4 in rat peripheral blood monocytes exposed to LPS.     

脂多糖对小鼠肺成纤维细胞活化的影响

目的 评价脂多糖(LPS)对小鼠肺成纤维细胞活化的影响.方法 原代培养的小鼠肺成纤维细胞,接种于96孔培养板,采用随机数字表法,将其随机分为2组,正常对照组(C组,外=6)不作任何处理,LPS组(n=24)加入LPS 1μg/ml,分别于LPS孵育3、6、24、72 h时取6孔(C组于培养72 h时),收集细胞,采用实时PCR法测定Ⅰ型前胶原mRNA、α-平滑肌肌动蛋白(α-SMA)mRNA、Toll样受体4(TLR4)mRNA和整合素β1 mRNA的表达水平.结果 与C组比较,LPS组LPS孵育3、6和24 h时Ⅰ型前胶原mRNA、α-SMA mRNA、TLR4 mRNA和整合素β1 mRNA的表达水平差异无统计学意义(P＞0.05),LPS孵育72 h时上述指标表达水平上调(P＜0.05).结论 在急性肺损伤的早期LPS一方面可直接活化小鼠肺成纤维细胞,导致肺纤维化;另一方面可上调TLR4和整合素β1的表达,增加细胞对LPS的反应性,从而加速小鼠肺成纤维细胞的活化,促进肺纤维化.

Abstract：

Objective To investigate the effect of lipopolysaccharide (LPS) on the activation of mouse lung fibroblasts. Methods Primary cultured mouse lung fibroblasts were incubated in 96 well plates and randomly divided into 2 groups: control group ( group C, n = 6) and LPS group ( n = 24). The fibroblasts were cultured for 72 h in group C. LPS 1 μg/ml was added and then the fibroblasts were incubated for 72 h in group LPS. The expression of type Ⅰ procollagen mRNA, α-smooth muscle actin (α-SMA) mRNA, Toll-like receptor 4 (TLR4)mRNA and integrin β1 mRNA was determined using real-time PCR at 3, 6, 24 and 72 h of incubation (6 wells at each time point). Results Compared with group C, there was no significant change in the expression of type Ⅰ procollagen mRNA, α-SMA mRNA, TLR4 mRNA and integrin β1 mRNA at 3, 6 and 24 h of incubation ( P ＞0.05), but the parameters mentioned above were significantly up-regulated at 72 h of incubation in group LPS ( P ＜ 0.05). Conclusion In the early acute lung injury, LPS leads to pulmonary fibrosis through activating lung fibroblasts directly, and also accelerates the activation of lung fibroblasts and promotes the process of pulmonary fibrosis through up-regulating the expression of TLR4 and integrin β1 in mice.     

曲尼司特对环孢素A诱导的人肾小管上皮细胞向间充质转变的抑制作用

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups: (1)In the control group, cells were treated without any medicine; (2) The cell were treated with CsA (4. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/L) and tranilast (100μmol/L); (4) The cells were treated with tranilast (100 μmol/L) alone for 72 h.Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The irmmunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA + Tranilast group was higher than the CsA group, while the expression of α-SMA and OPN in the CsA + Tranilast group was lower than the CsA group. Western blotting showed that protein expression level of E-cadherin in CsA group was dramatically lower than that in the control group (P＜0. 05), while that of α-SMA and OPN in CsA group was significantly higher than in the control group (P＜0.05). The protein expression level of E-cadherin in HK-2 cells in the CsA + Tranilast group was markedly higher than in the CsA group (P＜0.05), and that of α-SMA and OPN in CsA + Tranilast group was significantly lower than in the CsA group (P＜0. 05). Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.     

曲尼司特对环孢素A诱导的人肾小管上皮细胞向间充质转变的抑制作用

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups: (1)In the control group, cells were treated without any medicine; (2) The cell were treated with CsA (4. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/L) and tranilast (100μmol/L); (4) The cells were treated with tranilast (100 μmol/L) alone for 72 h.Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The irmmunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA + Tranilast group was higher than the CsA group, while the expression of α-SMA and OPN in the CsA + Tranilast group was lower than the CsA group. Western blotting showed that protein expression level of E-cadherin in CsA group was dramatically lower than that in the control group (P＜0. 05), while that of α-SMA and OPN in CsA group was significantly higher than in the control group (P＜0.05). The protein expression level of E-cadherin in HK-2 cells in the CsA + Tranilast group was markedly higher than in the CsA group (P＜0.05), and that of α-SMA and OPN in CsA + Tranilast group was significantly lower than in the CsA group (P＜0. 05). Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.     

曲尼司特对环孢素A诱导的人肾小管上皮细胞向间充质转变的抑制作用

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups: (1)In the control group, cells were treated without any medicine; (2) The cell were treated with CsA (4. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/L) and tranilast (100μmol/L); (4) The cells were treated with tranilast (100 μmol/L) alone for 72 h.Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The irmmunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA + Tranilast group was higher than the CsA group, while the expression of α-SMA and OPN in the CsA + Tranilast group was lower than the CsA group. Western blotting showed that protein expression level of E-cadherin in CsA group was dramatically lower than that in the control group (P＜0. 05), while that of α-SMA and OPN in CsA group was significantly higher than in the control group (P＜0.05). The protein expression level of E-cadherin in HK-2 cells in the CsA + Tranilast group was markedly higher than in the CsA group (P＜0.05), and that of α-SMA and OPN in CsA + Tranilast group was significantly lower than in the CsA group (P＜0. 05). Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.     

肝脏缺血/再灌注后Toll样受体4表达情况及丙泊酚对其表达的影响

Objective To observe the changes in TLR4 expression during hepatic ischemia/reperfusion (I/R) and the effects of propofol on the expression of TLR4 induced by I/R injury in rats.Methods Fifty six male SD rats were randomly divided into min before isehemia in P groups,and the same volume of sodium lactate Ringer's solution was infused in sham group and I/R groups.Plasma ALT,AST,TNF-α levels were measured,and the expression of Tlr4 was detected 2,6,24 h after reperfusion.Results The levels of plasma ALT,AST and TNF-α were lower,and Tlr4 expression was weaker in P groups than those in I/R groups (P＜0.05).Conclusion Propofol decreases hepatic ischemia/reperfusion (I/R)-induced Tlr4 expression and exerts property of liver protection.     

Effect of isoflurane on expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA In the hippocamapus of immature rats

Obiective To investigate the effect ofisoflurane on expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA in the hippocampus of immature rats.Methods sixty-four 7-clay-old SD rats were randomly assigned into 2 groups(n=32 each):control group(group C)and isoflurane group(group S).group S was exposed to 1.5% isoflurane for 6 h while group C to air.Fore animals were killed before anesthesia(T0,baseline),at 2,4,6 h(T1-3)of isoflurane anesthesia and 4,6,12 and 24 h after anesthesia(T4-7).The hippocampi were immediately removed for determimation of the expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA by RT-PCR.Results Compared with group C,the expression of IL-1β mRNA at T1-5,IL-6 mRNA at T2.3 and TNF-α mRNA at T1-6 in the hippocampus was upregulated in group S.Conclusion The expression of IL-1β mRNA,IL-6 mRNA and TNF-β mRNA was elevated in the hippocampus of immature rats after being exposed to isoflurane.     

Effect of isoflurane on expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA In the hippocamapus of immature rats

Obiective To investigate the effect ofisoflurane on expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA in the hippocampus of immature rats.Methods sixty-four 7-clay-old SD rats were randomly assigned into 2 groups(n=32 each):control group(group C)and isoflurane group(group S).group S was exposed to 1.5% isoflurane for 6 h while group C to air.Fore animals were killed before anesthesia(T0,baseline),at 2,4,6 h(T1-3)of isoflurane anesthesia and 4,6,12 and 24 h after anesthesia(T4-7).The hippocampi were immediately removed for determimation of the expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA by RT-PCR.Results Compared with group C,the expression of IL-1β mRNA at T1-5,IL-6 mRNA at T2.3 and TNF-α mRNA at T1-6 in the hippocampus was upregulated in group S.Conclusion The expression of IL-1β mRNA,IL-6 mRNA and TNF-β mRNA was elevated in the hippocampus of immature rats after being exposed to isoflurane.     

异丙酚对脂多糖诱导的小鼠巨噬细胞表达释放高迁移率族蛋白1的抑制作用

Objective To explore the inhibition effect of propofol on HMGB1 expression and releasing from mouse macrophage cell RAW264.7 induced by LPS. Methods Detect the time course of HMGB1 expression of RAW264.7 cells using RT-PCR and western blot. Then cells were divided into three groups: control, 250μg/L LPS stimulated, 250μg/L LPS stimulated simultaneously with 50μmol/L propofol. HMGB1 expression and releasing were detected using RT-PCR and western blot when cell were stimulated until the time the HMGB1 expression got to the highest level. Results 16 h after stimulated by LPS, the HMGB1 expression got to the highest level. When LPS stimulated with 50μmol/L propofol, the expression and releasing of HMGB1 by RAW264.7 were inhibited (P<0.05);16 h after stimulated by LPS, the HMGB1 mRNA expression got to the highest level,When LPS stimulated with 50μmol/L propofol, the expression and releasing of HMGB1 mRNA by RAW264.7 were inhibited Significantly,in each group ,the changes of HMGB1 Protein are the same to that of HMGB1 mRNA. Conclusion Propofol can inhibit the expression and releasing of HMGB1 by RAW264.7, which may be play a role in protect of sepsis by LPS.     

异丙酚预先给药对内毒素诱导大鼠肾小球血管内皮细胞通透性升高的影响

目的 探讨异丙酚预先给药对脂多糖(LPS)诱导大鼠肾小球血管内皮细胞通透性升高的影响.方法 分离、培养SD大鼠肾小球血管内皮细胞,以1×106/ml的密度接种于24孔培养板(200 μl/孔)和transwell小室(100 μl/室),采用随机数字表法,将其随机分为6组(n=10),正常对照组(C组)不作任何处理;脂肪乳对照组(I 组)加入10%脂肪乳4 μg/ml;异丙酚组(P组)加入异丙酚4μg/ml;LPS组(L组):加入LPS 10μg/ml;LPS+脂肪乳组(L+I组)加入10%脂肪乳4 μg/ml及LPS 10μg/ml;LPS+异丙酚组(L+P组)加入异丙酚4μg/ml 及LPS 10 μg/ml.于加入LPS前30 min加入脂肪乳或异丙酚,药物的浓度均为终浓度.加入LPS后6 h,收集细胞,采用逆转录-聚合酶链反应测定血管内皮细胞生长因子(VEGF)mRNA表达水平;收集上清液,采用酶联免疫吸附法测定VEGF浓度;测定血管内皮细胞通透性.结果 与C组比较,L组、L+I组和L+P组VEGF mRNA表达上调,上清液中VEGF浓度和血管内皮细胞通透性增加(P＜0.05),而I组和P组上述指标差异无统计学意义(P＞O.05).与L组比较,L+P组VEGF mRNA表达下凋,上清液中VEGF浓度和血管内皮细胞通透性降低(P＜0.05),L+I组上述指标差异无统计学意义(P＞0.05).上清液中VEGF浓度与血管内皮细胞通透性呈正相关(r=0.833,P＜0.05).结论 异丙酚预先给药可抑制LPS诱导大鼠肾小球血管内皮细胞通透性升高,其机制与下调VEGF表达有关.

Abstract：

Objective To investigate the influence of propofol pretreatment on the increased glomerular endothelial cell permeability induced by lipopolysaccharide (LPS) in rats.Methods Glomerular endothelial cells isolated from SD rats were cultured in 24-well plates(200 μl/well) and transwell filters (100 μl/filter) at 1×106/ml and assigned into 6 groups (n=10 each):control group (group C) , introlipid group (group I), propofol group (group P) , LPS group (group L), LPS+introlipid group (group L+I) and LPS+propofol group (group L +P). In group I, 10% introlipid 4 μg/ml was added. In group P, 4 μg/ml propofol was added. In group L, 10 μg/ml LPS was added. In group L+I, 10% introlipid 4 fig/ml combined with 10 μg/ml LPS was added. In group L+ P, 4 μg/ml propofol combined with LPS 10 μg/ml was added. Introlipid or propofol was added 30 min before the administration of LPS and the corresponding concentrations mentioned above were all final concentrations.After 6 h incubation with LPS, the cells were collected for measurement of vascular endothelial growth factor (VEGF) mRNA expression using RT-PCR. The supernatant was collected for determination of the VEGF concentration by ELJSA. The endothelial cell permeability was determined. Results Compared with group C, the expression of VEGF mRNA was up-regulated and the VEGF concentration and endothelial cell permeability were significantly increased in L, L+I and L + P groups (P＜0.05 ) ,but no significant change was found in the parameters mentioned above in I and P groups (P＞0.05). Compared with group L, the expression of VEGF mRNA was downregulated and the VEGF concentration and endothelial cell permeability were significantly decreased in L+P group (P＜0.05), but no significant change was found in the parameters mentioned above in group L+I(P＞0.05). A positive correlation existed between the concentration of VEGF and the permeability of endothelial cells(r= 0.833,P＜0.05).Conclusion Propofol pretreatment can decrease the increased glomerular endothelial cell permeability induced by LPS probably through down-regulation of VEGF expression.     

Isoflurane induces expression of vascular endothelial growth factor through activating protein kinase C in myocardial cells

Objective： Vascular endothelial growth factor （VEGF） plays important roles in establishing collateral circulation of ischemic myocardium. This study aimed to investigate the effect of isoflurane on VEGF expression and the potential intracellular signal transduction pathway in cultured rat myocardial cells in order to further reveal the molecular mechanism of myocardial preservation of isoflurane. Methods： Primary myocardial cells of Sprague-Dawley rats were isolated and cultured. They were divided randomly into control group, isoflurane group, protein kinase C （PKC） inhibitor group and PKC inhibitor＋isoflurane group where cells were respectively incubated without any treatment, treated by 0.5, 1.0 and 1.5 minimum alveolar concentration （MAC） of isoflurane for 6 hours, by PKC inhibitor calphostin C at a final concentration of 50 nmol/L and by 50 nmol/L calphosfin C＋ 1.0 MAC isoflurane for 6 hours. VEGF expression was detected by enzyme-linked immunosorbent assay （ELISA） and the expression levels of PKC isoforms were determined by Western immunoblotting method. Results： Isoflurane increased the VEGF expression in myocardial cells in a dose-dependent way. VEGF levels were significantly higher in 1.0 and 1.5 MAC isoflurane groups than in the control group （both P〈0.01）. The effect of isoflurane on upregulating VEGF expression was blocked by PKC inhibitor calphostin C （P〈0.01）, but calphostin C did not alter VEGF expression （P〉0.05）. Isoflurane induced the activation and translocation of PKC Immunoblotting analysis revealed that the immunoreactivity of PKC ε increased significantly in the membrane fractions and deceased significantly in the kytoplasm fractions for cells treated with 1.0 MAC isoflurane as compared with the untreated cells, but not of PKC a, PKCα and PKCζ （P〈0.01）. Conclusion： Isoflurane induces myocardial cells to release VEGF through activating PKCε from the endochylema to the cytomembrane, suggesting a possible novel mechanism of isoflurane protecting myocardial cells.     

骨髓间充质干细胞对急性出血坏死性胰腺炎大鼠肺组织Toll样受体2/4mRNA表达的影响及意义

目的 研究骨髓间充质干细胞(BMSCs)对急性出血坏死性胰腺炎(AHNP)大鼠肺组织T0ll样受体(TLR)2/4表达的影响并初步探讨其机制.方法 采用逆行胰胆管牛磺胆酸钠注射制造AHNP大鼠模型,动物分为假手术组、胰腺炎组和BMSCs治疗组;流式细胞仪检测BMSCs表面标记阳性细胞率;RT-PCR方法检测肺组织TLR2/4mRNA表达变化;同时观察肺组织形态学改变,进行肺湿/干重比(W/D)测定.结果 与假手术组比较,胰腺炎组大鼠从3h时肺组织TLR2/4mRNA表达开始增高,在12 h时肺组织TLR2/4mRNA表达达到峰值;同时肺损伤加重,肺组织TNF-α浓度升高(P＜0.05).给予BMSCs治疗后,TLR2/4mRNA表达降低,肺损伤程度减轻,肺组织TNF-α浓度降低(P＜0.05).结论 急性出血坏死性胰腺炎时,组织内TLR2和TLR4mRNA表达上调,肺组织损伤加重.BMSCs可以明显抑制AHNP肺组织TLR2/4mRNA的表达,降低肺组织TNF-α浓度,从而减轻肺损伤.

Abstract：

Objective To investigate the effect of bone mesenchymal stem cells on Toll-like receptors (TLR) 2/4 expression in the lungs of rats with acute hemorrhagic necrotizing pancreatitis (AHNP). Methods Seventy SD male rats were randomly divided into sham-operation group (n=10), AHNP group(n=30) and MSCs-treated group(n=30). Masc rate of BMSCs with surface mark were measured by flow cytometer. TLR2/4mRNA expression in the the lung were measured by RT-PCR, and The ratio of Wet/dry and lung histological changs were observed. Results TLR2/4 mRNA could be detected in the lungs with low values in sham-operation group, markedly increased in 3 h, and peaked in 12 h in AHNP group (P＜0.05). Lung injuries were aggravated and the levels of TNF-α in the lung were increased (P＜0. 05) . Treatment with MSCs could effectively inhibit TLR2/4 mRNA expression and relieve lung injuries. The levels of TNF-α in the lung were decreased (P＜0.05). Conclusions The expression of TLR2/4 mRNA is increased in the lungs in AHNP and the lung injuries are aggravated. MSCs could markedly inhibit TLR2/4 mRNA expression in the lungs in AHNP, which would lead to relief of lung injury.     

异丙酚对脂多糖诱导的小鼠巨噬细胞表达释放高迁移率族蛋白1的抑制作用

Objective To explore the inhibition effect of propofol on HMGB1 expression and releasing from mouse macrophage cell RAW264.7 induced by LPS. Methods Detect the time course of HMGB1 expression of RAW264.7 cells using RT-PCR and western blot. Then cells were divided into three groups: control, 250μg/L LPS stimulated, 250μg/L LPS stimulated simultaneously with 50μmol/L propofol. HMGB1 expression and releasing were detected using RT-PCR and western blot when cell were stimulated until the time the HMGB1 expression got to the highest level. Results 16 h after stimulated by LPS, the HMGB1 expression got to the highest level. When LPS stimulated with 50μmol/L propofol, the expression and releasing of HMGB1 by RAW264.7 were inhibited (P<0.05);16 h after stimulated by LPS, the HMGB1 mRNA expression got to the highest level,When LPS stimulated with 50μmol/L propofol, the expression and releasing of HMGB1 mRNA by RAW264.7 were inhibited Significantly,in each group ,the changes of HMGB1 Protein are the same to that of HMGB1 mRNA. Conclusion Propofol can inhibit the expression and releasing of HMGB1 by RAW264.7, which may be play a role in protect of sepsis by LPS.     

甲泼尼龙预干预脊髓缺血再灌注损伤对热休克蛋白27和肿瘤坏死因子α表达的影响

Objective To observe the effect of methylprednisolone (MP) pre-intervention on ex-pressions of heat shock protein 27 (HSP27) and tumor necrosis factor alpha (TNF-α) in cells in rat spinal cord following ischemia-reperfusion injury. Methods One hundred and fifty male Sprague-Dawley (SD) rats were randomly divided into 3 equal groups: group A (control) in which the abdominal aorta was exposed without any treatment, group B in which the abdominal aorta was clipped for 30 minutes before reperfusion for 3 bours to establish a model of ischemia- reperfusion injury, and group C in which intravenous MP injection was conducted 30 minutes before the establishment of the ischemia-reperfusion injury model. Three hours later the spinal cords were harvested. Pathological changes of spinal cord cells were observed with HE staining and expressions of HSP27 and TNF-α in spinal cord cells were observed with immunohistochemical staining. The motor function of hind-limbs before was evaluated before sample harvest. The data were analyzed with SPSS software. Results There were significant differences between groups A and B in the expressions of TNF-α and HSP27. Compared with group B, the expression of TNF-α decreased and HSP27 increased in group C, with statistically significant differences between the 2 groups. The motor function score of hind-limbs decreased in group B but improved in group C. Conclusions Since MP can decrease the expression of TNF-α and up-regulate the expression of HSP27, it has a potency of neuro-protection. Spinal cord ischemia-reperfusion injury can be avoided or decreased after MP pre-intervention.