Effect of schisandrin B on lung mRNA expression of transforming growth factor-β1 signal transduction molecule in rat lungs exposed to silica

Objective To investigate the effects of schisandrin B (Sch-B) on expression of transforming growth factor-β1 (TGF-β1) and signal transduction molecule mRNA in rat lungs exposed to SiO2,and explore the intervention mechanism of Sch-B on pulmonary fibrosis induced by SiO2. Methods Ninety six Wistar rats were randomly divided into control (normal saline) group, SiO2 group and SiO2 plus Sch-B group.The rats were exposed to SiO2 by direct tracheal instillation to establish the silicotic animal models. SiO2 group and SiO2 plus Sch-B group were treated with 1ml SiO2 (50 mg/ml) for each rat From the first day after model establishment, SiO2 plus Sch-B group were orally given Sch-B (80 mg/kg) a day, control group and silica group were orally given olive oil. On the 3rd, 7th,14th and 28th days after treatment, 8 rats in each group were sacrificed and samples were collected. The histo-pathological examination of lung was performed by HE staining. The expression levels of TGF-β1 、TGFβR Ⅱ and Smad4 mRNA in the lung tissues were detected by RT-PCR. Results The results of histo-pathological examination showed that in SiO2 group, lung tissues were injured obviously; the alveolar inflammation with alveolus interval edema and inflammation cell infiltration appeared on the 3rd and 7th days; the alveolus interval became thicker, became thicker, fibroblast and collagen matiix increased markedly on 14th day; the alveolar structure was damaged, alveolar wall thickened obviously,collagen aggradation and pulmonary fibrosis displayed on 28th day. The alveolar inflammation and pulmonary fibrosis in SiO2 plus Sch-B group were significantly less than those in SiO2 group. The expressions levels of TGF-β1、TGFβR Ⅱ and Smad4 mRNA (TGF-β1: 1.03±0.31 、1.33±0.39、1.08±0.26、0.82±0.16,TGF-βR Ⅱ:0.65 ±0.11、0.80 ±0.16、0.83 ±0.24、0.62 ±0.15, Smad4: 0.87 ±0. 15、0.68 ±0.11、0.78 ±0.19、0.30 ±0.08) in SiO2group were significantly higher than those in the control group (TGF-β1:0.59±0.22、0.55 ±0.25、0.56±0.20、0.55 ±0.12,TGR-βR Ⅱ:0.28 ±0.13、0.31 ±0. 15、0.34 ±0.15、0.27 ±0.09,Smad4:0.23 ±0.11、0.40 ±0. 12、0.39 ±0.12、0.18±0.06)(P＜0.01 or P＜0.05), but the expression level of TGF-β1 mRNA was the highest on the 7th day. The expression levels of TGF-β1 and Smad4 mRNA (TGF-β1: 0.68 ±0.28、0.88 ±0.25、0.75 ±0.11、0.61 ±0. 14,Smad4:0.25 ±0.12、0.45 ±0.09、0.44 ±0.07、0.21 ±0.04) in SiO2 plus Sch-B group were significantly lower than those in SiO2 group (P＜0.01 or P＜0.05),but there were no significant differences of the TGFβR Ⅱ mRNA expression levels between SiO2 group and SiO2 plus Sch-B group. Conclusion Sch-B can reduce the pulmonary fibrosis induced by SiO2 through inhibition of the mRNA express of TGF-β1 and Smad4 in the lung tissue, modulating the TGF-β1/Smad4 signal transduction pathway and inhibiting the target gene activation.     

细胞外信号调节蛋白激酶1/2信号通路对SiO2活化的肺成纤维细胞转化生长因子-β1表达的影响

目的 探讨细胞外信号调节蛋白激酶(ERK)信号通路对矽尘诱导的人胚肺成纤维细胞(HELF)中转化生长因子(TGF-β1)表达的影响及调控作用.方法 收集矽肺患者支气管肺泡灌洗液中的肺泡巨噬细胞(AM),在体外将AM分别用不含SiO2的DMEM培养液和含SiO2(50 μg/ml)的DMEM 培养液培养作用18 h后,将收集的AM条件培养上清液与HELF共同孵育.通过采用免疫细胞化学法检测应用ERK1/2抑制剂PD98059干预后SiO2活化的矽肺患者AM上清介导的HELF中TGF-β1表达的改变.结果 SiO2处理组HELF中TGF-β1的表达量(0.3227±0.0238)明显高于空白对照组(0.1637±0.0196)及AM对照组(0.2406±0.0225),而PD98059干预组TGF-β1的表达量(0.2711±0.0229)明显低于SiO2处理组,差异均有统计学意义(P＜0.05).结论 PD98059对由SiO2刺激的矽肺患者AM条件培养上清介导的HELF中TGF-β1的表达有一定抑制作用,ERK信号途径在一定程度上可能介导SiO2刺激的HELF细胞因子表达.

Abstract：

Objective To investigate the effect and regulation of extracellual signal-regulated kinase (ERK1/2)signaling pathway on the expression of transforming growth factor-β1 (TGF-β1) in human embryonic lung fibroblasts induced by SiO2. Methods Human alveolar macrophages were collected from a silicotic patient by bronchoalveolar lavage and in the presence or absence of SiO2 (50 ug/ml) exposition for 18h, and then the conditioned supernatants were used to incubate HELF. The expressions of TGF-β1 of the HELF acted with the conditioned AM supernatant fluid were detected with the immunocytochemistry method after treatment with PD98059 of inhibitor of ERK. Results The expression of TGF-β1 in HELF of the SiO2 treatment group(OD value is 0.322 7±0.023 8)exceed blank group (OD value is 0.163 7±0.019 6) and AM control group(OD value is 0.240 6±0.022 5) by the immunocytochemistry method. But the expression of TGF-β1 had reduction in some extent in the PD98059 intervention group (OD value is 0.271 l±0.022 9). The values were statistically different (P＜0.05). Conclusion ERK inhibitor PD98059 have inhibition effect on the expression of transforming growth factor-pi and expression of cytokine of human embryonic lung fibroblasts stimulated by SiO2, The study indicate that the proliferation and collagen prodction of HELF activated by SiO2 are mediated by ERK/MAPK signal pathway in some extent. PD98059 may antagonizes silica-induced lung fibrosis by inhibiting the expression of transforming growth factor-β1.     

石英尘和博莱霉素诱导的大鼠肺纤维化模型的比较研究

目的 比较石英尘和博莱霉素诱导的大鼠肺纤维化模型中的肺泡炎和早期纤维化改变,并对其机制进行探讨.方法 将大鼠随机分为SiO2组(14只,气管内注入40mg/ml SiO2混悬液1 ml)、BLM组(14只,气管内注入5 mg/kg BLM A5)和对照组(14只,气管内注入1 ml无菌生理盐水).在造模后7、14 d每组各处死7只动物,取肺组织病理切片行HE染色,对肺泡炎进行计分,行饱和苦味酸天狼猩红染色,采用图像分析系统测得各组胶原面积后,进行定量分析,免疫组织化学技术检测肺组织中CD68和TNF-α蛋白表达情况,采用图像分析计算累积吸光度值,进行半定量分析.结果 (1)HE染色光学显微镜下可见,BLM组7d时肺泡炎(肺泡炎评分2.814±0.832)最明显,偏振光下显示BLM组14d时肺纤维化[胶原面积(1284.57±554.72)μm2)]最严重,均明显高于对照组和同期SiO2组,差异有统计学意义(P＜0,05).(2)免疫组化结果显示,BLM组7 d时TNF-α表达最高(17.100±1.831),明显高于对照组(0.420±0.020)、SiO2组7 d(7.909±1.275)及BLM组14d(13.506±1.454),差异均有统计学意义(P＜0.05);在SiO2组14 d时TNF-α表达为22.778±2.512,明显高于BLM组(14 d)、对照组及SiO2组(7 d),差异有统计学意义(P＜0.05).14 d时,SiO2组CD68表达明显高于对照组、BLM组(14 d)及SiO2组(7d),差异有统计学意义(P＜0.05).结论 BLM诱导的大鼠肺损伤模型的早期肺泡炎重于SiO2诱导的大鼠肺损伤模型,纤维化进程早于SiO2诱导的大鼠肺损伤模型,TNF-α在两种模型的病程中均起着重要的作用,而巨噬细胞更为持续地参与了SiO2诱导的肺纤维化.

Abstract：

Objective To compare the pulmonary alveolitis and the early fibrosis of pulmonary fibrosis induced by quartz dust and bleomycin in rats, and investigate their mechanism. Methods The female rats were divided into three groups: control group exposed to normal saline by the trachea; SiO2 group exposed to SiO2 by the trachea; BLM group exposed to BLM A5 by the trachea. Each half of the animals were sacrificed on the 7th andl4th day after expoasure. The lungs of rats were collected to observe pulmonary alveolitis by HE staining and to observe fibrosis by saturated picric acid sirius red staining. The expression of tumor necrosis factor-α (TNF-α) and CD68 in pulmonary tissues were analyzed quantitatively by immunohistochmistry and image analysis system. Results (1) The alveolitis and pulmonary fibrosis of rats in both SiO2 group and BLM group were became more serious gradually over time, HE staining under light microscope showed that BLM group on the 7th day had the most obvious alveolitis (2.814±0.832), the saturated picric acid sirius red staining under polarized light showed that BLM group on the 14th day had the worst pulmonary fibrosis (1284.57±554.72), which were significantly higher than those (103.69±18.29 and 111.78±37.45) in control group and SiO2 group on the 7th day (P＜0.05). (2) The results of immunohistochmistry examination indicated that the expression (17.100±1.831) of TNF-α in the BLM group on the 7th day was significantly higher than those (0.451 ±0.441, 7.909±1.275 and 13.506±1.454) in control group, SiO2 group on 7th day and BLM group on 14th day (P＜0.05). The expression (22.778 ±2.512) of TNF-a in the SiO2 group on the 14th day was significantly higher than those in control group, SiO2 group on 7th day and BLM group on 14th day (P＜0.05).The expression (134.941 ±35.951) of CD68 in the SiO2 group on the 14th day was significantly higher than those in control group,SiO2 group on 7th day and BLM group on 14th day(P＜0.05).Conclusion The early alveolitis of BLM-induced lung injury model was more serious than that of SiO2-induced lung injury model,and the fibrosis process of BLM-induced lung injury model was earlier than that of SiO2-induced lung injury model.TNF-α plays an important role in the conrse of both models.but macrophages is involved in Si06nduced pulmonary in a more continuous way than in BLM-induced pulmonary fibrosis.     

姜黄素对大鼠肺间质纤维化影响的机制研究

Objective To observe the possible mechanism and inhibitory effects of curcumin on pulmonary fibrosis induced bleomycin in rats at the fibrosing stage. Methods 80 male Sprague-Dawley rats were random divided into 4 groups (20 rats in each group). Rats in the fibrosis model group, the prednisone group and the curcumin group were induced by instilled bleomycin through tracheal, rats in the control group with same volume normal saline. Since the 15th day after bleomycin administration, the curcumin group and prednisone group were given curcumin (300 mg/kg) or prednisone (5mg/kg) per day by intragastric administration, respectively. The normal control group and the model group were given 1% sodium carboxymethyl cellulose ( 10ml/kg). Six rats of each group were random sacrificed on the 21st, 28th, 42nd and 56th days after bleomycin administration. The histological changes of the pulmonary were evaluated by H. E and Masson dyeing. The expressions of transforming growth factor-β1 (TGF-β1), platelet-derived growth factor (PDGF) and hydroxyproline in the tissue of pulmonary were assessed by immunohistochemistry and digestion method. Results Pulmonary fibrosis and hydroxyproline level in the curcumin group were obviously reduced as compared with the model group on the 42nd and 56th day[42 d:1. 28 ±0. 61 vs 2. 28 ±0. 39,P <0. 01 ;(1.73 ±0. 22)mg/g vs (2.50 ±0. 37) mg/g, P <0.01;56 d:1.00 ±0.59 vs 1.73 ±0.36, P< 0. 05; ( 1.57 ± 0. 36) mg/g vs (2. 20 ± 0. 42) mg/g, P < 0. 01 ], and it was also lower than that in prednisone group on the 42nd day( P < 0. 05 ). The expression of TGF-β1 and PDGF in the curcumin group were obviously lower than that in the model group on the 28th, 42nd and 56th day[28 d:TGF-β1 :3642. 05 ±839. 31 vs 5067. 35 ±738. 39, P <0. 05 ;PDGF:2957. 55 ±739. 16 vs 4457. 75 ±568. 39, P <0. 05;42 d: TGF-β1: 2689. 73 ± 529.22 vs 4089. 50 ± 619. 37, P < 0. 01; PDGF: 2834. 46 ± 567. 16 vs 3239. 52 ±628. 26, P <0. 01 ;56 d:TGF-β1: 1968.57 ±408. 36 vs 2968.20 ±498.42, P <0. 01 ;PDGF: 1083.36 ±381.35 vs 2019. 40 ±412. 36, P <0. 01 ], which was lower than that in prednisone group on the 42nd and 56th day (42 d,TGF-β1 :3529. 07 ±981.35,PDGF:2618. 34 ±813. 34;56 d,TGF-β1 :2530. 83 ±439. 37,PDGF: 1738. 35 ±536. 62, Pall <0. 05 ) , and it had no obvious difference compared with control group on the 56th day ( P > 0. 05 ). Conclusion Curcumin could alleviate bleomycin-induced pulmonary fibrosis in rats at the fibrosing stage by inhibiting the expressions of TGF-β1 and PDGF.     

二氧化硅刺激矽肺肺泡巨噬细胞上清介导的人胚肺成纤维细胞Ⅲ型胶原和Ⅲ型前胶原的表达

目的 探讨经SiO2刺激的肺泡巨噬细胞(AM)通过人胚肺成纤维细胞(HELF)对Ⅲ型胶原(CⅢ)和Ⅲ型前胶原(pCⅢ)表达的影响及抗TGF-β1抗体的干预作用.方法 收集矽肺患者AM,将其分为加入SiO2粉尘悬液的处理组和仅加入无血清培养液的对照组.培养18 h后获取培养上清.将原代培养的HELF分为:(1)处理组:加入经SiO2刺激的AM上清;(2)对照组:加入未经SiO2刺激的AM上清;(3)空白对照组:仅加入含体积分数为3%胎牛血清的DMEM;(4)处理组+抗转化生长因子β1(TGF-β1)抗体干预组:在处理组的基础上加上抗TGF-β1抗体(10 μg/ml);(5)对照组+抗TGF-β1抗体干预组:在对照组的基础上加上抗TGF-β1抗体(10 μg/ml).前3组分别培养6、12、18、24、36、48 h,后2组分别培养18、24、36 h,用免疫细胞化学法检测HELF中pCⅢ的表达,用免疫印迹(Western blot)法检测HELF条件培养上清中CⅢ的表达.结果 与对照组(0.1212±0.0079、0.1414±0.0058、0.1620±0.0081、0.1965±0.0103、0.1715±0.0116)相比,处理组12、18、24、36和48 h pCⅢ表达(0.1423±0.0107,0.1624±0.0011,0.1925±0.0056,0.2421±0.0097,0.2102±0.0103)明显升高,差异有统计学意义(P＜0.05或P＜0.01).与对照组(0.2296±0.0121、0.2778±0.0116、0.3367±0.0269、0.3722±0.0214)相比,处理组12、18、24、36、48 hCⅢ含量(0.2559±0.0061、0.3249±0.0110、0.4171±0.0193、0.5441±0.0452、0.4751±0.0252)明显增高,差异均有统计学意义(P＜0.05或P＜0.01).与同期相对应的非干预组相比,18、24、36 h抗TGF-β1抗体干预组CⅢ和pCⅢ均明显降低,差异均有统计学意义(P＜0.01或P＜0.05).结论 SiO2经AM的介导,诱导HELF高表达CⅢ和pCⅢ,部分效应是通过TGF-β1起作用.

Abstract：

Objective To study the effects of supernatant of alveolar macrophages (AM) exposed to SiO2 on the expression of type Ⅲ collagen and type Ⅲ procollagen in human lung fibroblasts (HELF) and the intervention effects of anti-TGF-β1 antibody Methods AMs collected from a silicotic by bronchoalveolar lavage were divided into 2 parts, one part was exposed to SiO2 and other part served as control. The supernatant was obtained from AMs cultured for 18 h. HELF were divided into (1) exposure group, which was added with supernatant from AMs exposed to SiO2; (2) control group, which was added with the supernatant from AMs not exposed to SiO2; (3) blank control group, which was added with DMEM; (4) exposure group plus anti-TGF-β1antibody (10μg/ml); (5) control group plus anti-TGF-β1 antibody (10 μg/ml). (1)-(3) groups were cultured for 6, 12, 18, 24, 36, 48h, respectively. (4)-(5) groups were cultured for 18, 24, 36, respectively.Immunocytochemical test and Western blot assay were used to detect pC Ⅲ expression levels in HELF and C Ⅲ expression levels in the supernatant of HELF culture, respectively. Results The pC Ⅲ expression levels of exposure group were 0.1423 ±0.0107,0.1624±0.0011,0.1925 ±0.0050,0.2421 ±0.0097 and 0.2103 ±0.0103,respectively, which were significantly higher than those (0.1212±0.0079、0.1414±0.0058、0.1620±0.0081、0.1965±0.0103 、0.1715±0.0116) of control group (P＜0.05 or P＜0.01). The C Ⅲ levels of exposure group were (0.2559±0.0061、0.3249±0.0110、0.4171±0.0193、0.5441 ±0.0452、0.4751±0.0252), respectively, which were significantly higher than control group (0.2296±0.0121、0.2778±0.0116、0.3367±0.0269、0.3722±0.0214). The pC Ⅲ and C Ⅲ expression levels of exposure plus anti-TGF-β1 antibody group were significantly lower than those of control plus anti-TGF-β1 antibody group (P＜0.05 or P＜0.01).Conclusion AMs exposed to SiO2 can induce the elevated pC Ⅲ and C Ⅲ expression levels in HELF by TGF-β 1 to some extent.     

吡非尼酮抗百草枯中毒小鼠肺纤维化的研究

目的 探讨吡非尼酮(PF)对百草枯(PQ)中毒小鼠肺纤维化的治疗作用,为临床治疗提供理论依据.方法 雄性ICR小鼠90只,随机分为正常对照组、PQ组、地塞米松组、25、50和100 mg/kgPF组,每组15只.正常对照组小鼠一次性空腹灌胃给予生理盐水,2 h后给予质量分数为1%羧甲基纤维素(CMC)灌胃,再每天定时空腹灌胃同等量CMC;PQ组、地塞米松组及各PF剂量组小鼠给予PQ100mg/kg一次性灌胃染毒,灌胃后2 h,再每天定时PQ组给予0.02ml/10 gCMC灌肠,PF组给予PF(25、50、100 mg/kg)和地塞米松(0.02 ml/10 g)灌胃,每天1次,共49 d.计算肺系数,HE染色,光学显微镜下观察肺组织病理改变;测定肺组织羟脯氨酸(HYP)含量,转化生长因子(TGF-β1)的mRNA表达水平、蛋白表达水平,支气管肺泡灌洗液(BALF)中的TGF-β1蛋白含量.结果 PQ组3 d生存率为53.33%,25、50、100 mg/kg PF组3 d生存率分别为46.67%、73.33%、86.67%,地塞米松组3 d生存率为80%,地塞米松组、50、100 mg/kg PF组3 d生存率明显高于PQ组和25 mg/kg PF组,差异有统计学意义(P＜0.05).25、50及100mg/kg PF组小鼠肺系数均明显低于PQ组,差异有统计学意义(P＜0.05).地塞米松组肺组织中HYP含量为(50.95±11.65)mg/g,25、50、100mg/kg PF组HYP含量分别为(44.52±9.48)、(43.27±6.01)、(40.82±5.90)mg/g,较PQ组[(74.27±3.68)mg/g]明显下降,差异均有统计学意义(P＜0.01).地塞米松组BALF中TGF-β1蛋白含量为(22.03±7.27)mg/ml,25、50、100 mg/kg PF组TGF-β1蛋白含量分别为(55.33±17.50)、(27.75±5.84)、(21.31±6.82)mg/ml,与PQ组[(52.52±15.51)mg/ml]相比,明显降低,差异有统计学意义(P＜0.01);100 mg/kg PF组肺组织TGF-β1mRNA表达水平与PQ组相比,明显下降,差异有统计学意义(P＜0.01)与PQ组比较,地塞米松组,50、100mg/kgPF组肺组织中TGF-β1蛋白表达下降,差异有统计学意义(P＜0.01).结论 PF可以减少百草枯中毒小鼠肺组织胶原沉积,减轻肺部纤维化程度.

Abstract：

Objective To study the curative effects of pirfenidone (PF)on pulmonary fibrosis induced by paraquat (PQ) in mice and to provide the theoretical basis for clinical treatment. Methods Ninety adult healthy male ICR mice were randomly divided into six groups: control group, PQ group , 2 mg/kg Dexamethasone group, 25 mg/kg PF group, 50 mg/kg PF group and 100 mg/kg PF group, there were 15 mice in each group. The corresponding volume of normal saline was given to the each mouse in control group according to the weight, after 2 h 0.1% CMC was given to the each mouse of control group one time by intragastric administration, then the CMC was administrated at regular time until sacrifice. All mice for other 5 groups were exposed to 100 mg/kg PQ by intragastric administration. At 2 h after exposure to PQ, 0.02 ml/10 g dexamethasone and 25、50、100 mg/kg PF were given to mice for dexamethasone group and for 3 PF groups by intragastric administration each day for 49 days, respectively. The lung coefficient was calculated and pathological changes of lung tissue were observed by HE staining for each mouse. The hydroxyproline (HYP)level in lung tissue was measured for each mouse. The mRNA level of and the protein level of TGF-β1 in lungtissue for each mouse were determined, and the protein level of TGF-β1 in the bronchus-alveolus lavage fluid (BALF) of each mouse was detected. Results The survival rates on the 3rd day in PQ group, 3 PF groups and dexamethasone group were 53.33%, 46.67%, 73.33%, 86.67% and 80%, respectively. The survival rates on the 3rd day in dexamethasone group, 50 mg/kg and 100 mg/kg PF groups were significantly higher than those of PQ group and 25 mg/kg PF group (P＜0.05). The lung coefficients of 3 PF groups were significantly lower than that of the PQ group (P＜0.05). The lung tissue HYP levels of dexamethasone group and 3 PF groups were 50.95±11.65, 44.52±9.48, 43.27±6.01 and 40.82±5.90 mg/g respectively, which were significantly lower than that (74.27±3.68) of PQ group(P＜0.01 ). The TGF-β1 protein levels of BALF in dexamethasone group, 50 and 100 mg/kg PF groups were 22.03±7.27, 27.75±5.84 and 21.31 ±6.82 ng/ml respectively, which were significantly lower than that(52.52±15.51 ) ng/mlof PQ group(P＜0.01 ). The expression level of TGF-β1 mRNA in 100 mg/kg PF group decreased significantly, as compared with PQ group (P＜0.01). Conclusion PF could reduce the collagen deposition and pulmonary fibrosis induced by PQ in mice lungs.     

Effects of silicotic alveolar macrophages exposed to SiO2 on the expression of type Ⅲ collagen and type Ⅲ procollagen in human lung fibroblasts

Objective To study the effects of supernatant of alveolar macrophages (AM) exposed to SiO2 on the expression of type Ⅲ collagen and type Ⅲ procollagen in human lung fibroblasts (HELF) and the intervention effects of anti-TGF-β1 antibody Methods AMs collected from a silicotic by bronchoalveolar lavage were divided into 2 parts, one part was exposed to SiO2 and other part served as control. The supernatant was obtained from AMs cultured for 18 h. HELF were divided into (1) exposure group, which was added with supernatant from AMs exposed to SiO2; (2) control group, which was added with the supernatant from AMs not exposed to SiO2; (3) blank control group, which was added with DMEM; (4) exposure group plus anti-TGF-β1antibody (10μg/ml); (5) control group plus anti-TGF-β1 antibody (10 μg/ml). (1)-(3) groups were cultured for 6, 12, 18, 24, 36, 48h, respectively. (4)-(5) groups were cultured for 18, 24, 36, respectively.Immunocytochemical test and Western blot assay were used to detect pC Ⅲ expression levels in HELF and C Ⅲ expression levels in the supernatant of HELF culture, respectively. Results The pC Ⅲ expression levels of exposure group were 0.1423 ±0.0107,0.1624±0.0011,0.1925 ±0.0050,0.2421 ±0.0097 and 0.2103 ±0.0103,respectively, which were significantly higher than those (0.1212±0.0079、0.1414±0.0058、0.1620±0.0081、0.1965±0.0103 、0.1715±0.0116) of control group (P＜0.05 or P＜0.01). The C Ⅲ levels of exposure group were (0.2559±0.0061、0.3249±0.0110、0.4171±0.0193、0.5441 ±0.0452、0.4751±0.0252), respectively, which were significantly higher than control group (0.2296±0.0121、0.2778±0.0116、0.3367±0.0269、0.3722±0.0214). The pC Ⅲ and C Ⅲ expression levels of exposure plus anti-TGF-β1 antibody group were significantly lower than those of control plus anti-TGF-β1 antibody group (P＜0.05 or P＜0.01).Conclusion AMs exposed to SiO2 can induce the elevated pC Ⅲ and C Ⅲ expression levels in HELF by TGF-β 1 to some extent.     

EGFL7基因沉默对裸鼠胃癌血管生成的影响

Objective To investigate the effect of short hairpin RNA (shRNA) targeting at epidermal growth factor-1 ike domain 7 ( EGFL7 ) gene on angiogenesis of stomach carcinoma and its mechanism in nude mice.Methods shRNA targeting at EGFL7 gene was constructed and transfected into SGC-7901 cells (pshEGFL7 group), meanwhile, the cells transfected with vector plasmids were used as control group.Positive clones were selected and the transplanted tumor animal models were constructed in nude mice, and the growth and volume of tumors were observed.After 8 weeks, EGFL7 mRNA and protein in transplanted tumor tissues were detected.Moreover, Anti-CD34, VEGF and TSP1 were stained by the immuno- chemistry method, and MMP-2 and TIMP2 mRNA were detected by RT-PCR.Results EGFL7 mRNA was significantly down regulated in pshEGFL7 group.In psh EGFL7 group, the tumor volume was 1.86 ± 0.65 cm3,MVD was 20.84 ± 6.38, while in control group, the tumor volume was 4.86 ± 1.15 cm3, MVD was 39.48±9.01.In EGFL7 group, TSP1 protein presented positive, and VEGF protein presented weakly positive or negative.The expression of MMP-2 mRNA decreased, TIMP2 mRNA increased in pshEGFL7 group, and it was significantly different compared with control group( P ＜0.01 ).Conclusion RNA interference targeting at EGFL7 gene can balance TSP1/VEGF, through regulating the expression of MMP-2/TIMP2 to impair angiogenesis of stomach cancer in nude mice.     

EGFL7基因沉默对裸鼠胃癌血管生成的影响

Objective To investigate the effect of short hairpin RNA (shRNA) targeting at epidermal growth factor-1 ike domain 7 ( EGFL7 ) gene on angiogenesis of stomach carcinoma and its mechanism in nude mice.Methods shRNA targeting at EGFL7 gene was constructed and transfected into SGC-7901 cells (pshEGFL7 group), meanwhile, the cells transfected with vector plasmids were used as control group.Positive clones were selected and the transplanted tumor animal models were constructed in nude mice, and the growth and volume of tumors were observed.After 8 weeks, EGFL7 mRNA and protein in transplanted tumor tissues were detected.Moreover, Anti-CD34, VEGF and TSP1 were stained by the immuno- chemistry method, and MMP-2 and TIMP2 mRNA were detected by RT-PCR.Results EGFL7 mRNA was significantly down regulated in pshEGFL7 group.In psh EGFL7 group, the tumor volume was 1.86 ± 0.65 cm3,MVD was 20.84 ± 6.38, while in control group, the tumor volume was 4.86 ± 1.15 cm3, MVD was 39.48±9.01.In EGFL7 group, TSP1 protein presented positive, and VEGF protein presented weakly positive or negative.The expression of MMP-2 mRNA decreased, TIMP2 mRNA increased in pshEGFL7 group, and it was significantly different compared with control group( P ＜0.01 ).Conclusion RNA interference targeting at EGFL7 gene can balance TSP1/VEGF, through regulating the expression of MMP-2/TIMP2 to impair angiogenesis of stomach cancer in nude mice.     

The expression and meaning of TGF-β and TGIF in endometrosis

Objective To explore the role of TGF-βand TGIF in the pathogenesis of endometriosis. Methods The expression of TGF-β and TGIF was detected by immunohistochemistry method in the ectopic and eutopic endometrium of 30 cases with endometriosis (ec-topic endometrium group and eutopic endometrium group) and in the normal endometrium of 40 cases without endometriosis (control group). Result The expression of TGF -β in ectopic endometrium group was significantly higher than that in eutopic endometrium group and control group (P < 0.05). There was no significant difference in the expression of TGF- βbetween eutopic endometrium group and control group(P > 0.05). The expression of TGIF in ectopic endometrium group was significantly lower than that in eutopic endometrium group and control group( P <0.05). There was no significant difference in the expression of TGIF between eutopic endometrium group and control group(P > 0.05). There were negative correlation between the expressions of TGF - β and TGIF in ectopic endometrium group and eutopic endome-trium group(rs= - 0.769, - 0.549, P < 0.05). Conclusion The abnormal expression of TGF-β and TGIF in ectopic endometrium of pa-tients with endometriosis may be associated with the genesis and progression of endometriosis.     

矽肺患者氧化应激指标及外周血单核细胞NF-κB水平的变化

目的 观察矽肺患者氧化应激指标及外周血单核细胞NF-κB水平的变化,探讨矽肺发生发展的机制.方法 选择某铸造厂接触矽尘作业工龄在1年以上的工人200例为接尘组,该厂2008年住院及门诊随访的矽肺患者130例为矽肺组,32例0+病例为观察对象组,同时选择某酒店服务人员100例为对照组.分别测定超氧化物歧化酶(SOD)、血清中谷胱甘肽过氧化物酶(GSH-Px)、一氧化氮合酶(NOS)活力,一氧化氮(NO)、丙二醛(MDA)含量及总抗氧化能力(T-AOC),外周血单核细胞核蛋白中NF-κB水平.结果 与对照组比较,接尘组和矽肺组NO含量明显升高,SOD活力明显降低,差异均有统计学意义(P＜0.01).与对照组及接尘组比较,矽肺组T-AOC水平、NOS活力、MDA含量均明显升高,差异均有统计学意义(P＜0.01).与对照组[(223.360±46.838)U/ml]比较,接尘组及矽肺组GSH-Px活力[(231.164±36.484)、(270.469±39.228)U/ml]明显升高,且矽肺组GSH-Px活力明显高于接尘组,差异均有统计学意义(P＜0.05,P＜0.01).与观察对象组[(256.906±21.418)U/ml]和Ⅰ期矽肺组[(259.594±34.790)U/ml]比较,Ⅲ期矽肺组GSH-Px活力[(290.750±39.129)U/ml]明显升高,差异均有统计学意义(P＜0.05).与对照组[(59.71±9.27)ng/L]比较,接尘组及矽肺组NF-κB水平[(72.06±9.12)、(85.25±11.64)ng/L]明显升高,且矽肺组NF-κB水平明显高于接尘组,差异均有统计学意义(P＜0.01).血清中GSH-Px活力与矽肺分期呈正相关(r=0.507,P＜0.01).外周血单核细胞核蛋白NF-κB水平与矽肺分期、年龄、GSH-Px活力、NO含量呈正相关,差异均有统计学意义(r值分别为0.376、0.243、0.233、0.221,P＜0.01).结论 机体氧化和抗氧化系统的失衡与矽肺的发生发展有关,并与NF-κB的活化一致.

Abstract：

Objective To investigate the change of indicators of oxidative stress in serum and NF-κB in peripheral blood mononuclear cells of patients with silicosis, and explore the mechanism of the development of silicosis. Methods The subjects were divided into (1) 200 workers exposed to SiO2 for at least 1 years in a foundry served as the dust-exposure group; (2) 130 cases with silicosis (Ⅰ phase silicosis 64 cases, Ⅱ phase 46 cases Ⅲ phase 20 cases) served as the silicosis goup; (3) 32 cases with 0+ phase silicosis in the foundry served as the observed group,(4)100 subjects from a hotel served as the control group. The serum including superoxide dismutase (SOD), nitric oxide (NO), serum glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), nitric oxide synthase (NOS), lipid malondialdehyde (MDA) and NF-κB protein levels in peripheral blood mononuclear cells were determined, respectively. Results Compared with the control group,NO levels in dust-exposed group and silicosis group significantly increased, and SOD decreased significantly (P＜0.05 or P＜0.01). Compared with the control group and dust-exposed group, T-AOC, NOS, MDA levels in silicosis group significantly increased (P＜0.05 or P＜0.01). GSH-Px in dust-exposed group and silicosis group were (231.164±36.484) and (270.469±39.228)U/md, respectively which were significantly than that [(223.360±46.838) U/ml] in control group (P＜0.05 or P＜0.01), and there was significant difference of GSHPx between the silicosis group and the dust-exposed group significantly (P＜0.01). GSH-Px level [(290.750±39.129) U/ml] in Ⅲ phase silicosis group were significantly higher than those [(256.906±21.41) and (259.594±34.79) U/ml] in observation group and Ⅰ phase silicosis group (P＜0.05). NF-κB levels [(72.06±9.12) and (85.25±11.64) ng/L] in dust-exposed group and silicosis group were significantly higher than that [(59.71±9.27) ng/L] in control group (P＜0.01), and there was significant difference of between the silicosis group and the dust-exposed group (P＜0.01). There was a positive correlation between serum GSH-Px level and the silicosis stages (r=0.507,P＜0.0l). Also there was a positive correlation between NF-κB level and silicosis stages, age, GSH-Px or NO levels (r=0.376, 0.243, 0.233, 0.221, P＜0.01). Conclusion The imbalance of oxidative and anti-oxidation system and the activation of NF-κB are related with the occurrence and development of silicosis. The monitoring of oxidative stress indicators and NF-κB is beneficial to the prediction and prognosis assessment of silicosis.     

博莱霉素致大鼠肺纤维化原癌基因c-erbB2和肺表皮生长因子受体的表达

目的 探讨肺纤维化模型大鼠肺组织中原癌基因c-erbB2激活和肺表皮生长因子受体(EGFR)的表达.方法 将54只Wistar大鼠随机分成博莱霉素(BLM)组、EGFR拮抗剂Iressa组和对照组,每组18只.用BLM(5 mg/kg)气管注射制造大鼠肺纤维化模型,对照组气管注入生理盐水O.2～0.3ml;Iressa组造模前1 h灌胃给予Iressa(200 mg/kg),BLM组和对照组用生理盐水10 ml/kg灌胃,每组均灌胃5次/周,分别于第1、14、28天处死大鼠.观察大鼠肺组织病理改变,用免疫组化法检测肺组织原癌基因c-erbB2和EGFR的表达.结果 Iressa组大鼠肺组织可见纤维化并炎性细胞浸润,与BLM 组变化规律相同,肺纤维化程度低于BLM组.第28天Iressa组肺纤维化评分(2.17±0.41)明显低于BLM组(3.50±0.84),差异有统计学意义(P＜0.01).各时间点BLM组和Iressa组c-erbB2和EGFR表达均明显高于对照组,差异有统计学意义(P＜0.01).Iressa组的c-erbB2表达与BLM组变化规律一致,均随时间的延长,c-erbB2表达逐渐下降,不同时间的c-erbB2表达比较,差异有统计学意义(P＜0.01).Iressa组的c-erbB2表达与BLM组比较,差异无统计学意义(P＞0.05).Iressa组于第14、28天时肺组织中EGFR表达水平为0.17±0.02和0.28+0.04,明显低于BLM组(0.27+0.04、0.34±0.02),差异有统计学意义(P＜0.01).Iressa组第28天EGFR表达明显高于第14天,差异有统计学意义(P＜0.01).结论 c-erbB2和EGFR在大鼠肺泡炎及肺纤维化不同阶段表达增强,c-erbB2和EGFR可能参与了肺纤维化的发生.

Abstract：

Objective To study the expression of the epidermal growth factor receptor( EGFR) and the oncogene c-erbB2 on pulmonary fibrosis induced by bleomycin( BLM) in rats. Methods Fifty-four Wistar rats were randomly divided into three groups, the pulmonary fibrosis group (BLM), Iressa group and the control group. There were 18 rats in each group. Control group were injected with saline 0.2~0.3 ml in trachea.Iressa group and BLM group were injected with BLM intratracheal. After the fibrosis models were build, Iressa group were given orally Iressa(200 mg/kg)l h before modeling in Iressa group, saline were fed 10 ml/kg in BLM group and control group. The three groups were fed 5 times per week; and were sacrificed after treatment on days 1, 14and 28 respectively. The lungs were harvested for histological studies. Results The lung tissue in Iressa group showed fibrosis and inflammatory cell infiltration, the same as shown in the BLM group. The pulmonary fibrosis score was significantly lower than the BLM group on the 28 th day (2.17±0.41 vs 3.50±0.84, P＜0.01). Compared with the control group, c-erbB2 and EGFR were hyperexpressed significantly both in BLM group and Iressa group at all time points (P＜0.01); c-erbB2 expression had no changes between the Iressa group and the BLM (P＞0.05), that were gradually decreased, and was significantly different at each time point (P＜0.01). EGFR expression was increased gradually on the 14th and 28th day (0.17±0.02 and 0.28±0.04)in Iressa group ,that was significantly lower than the BLM group (0.27±0.04 and 0.34±0.02)(P＜0.01). EGFR expression increased significantly on the 28th day than on the 14th day in the Iressa group(P＜0.01). Conclusion The expression of C-erbB2 and EGFR are enhanced in different stages of alveolitis and pulmonary fibrosis, c-erbB2 and EGFR may be participated in different stages of pulmonary fibrosis.     

硝酸甘油对大鼠重症急性胰腺炎ET/NO、TXA2/PGI2的影响

Objective To explore the effect of nitroglycerin on ET/NO, TXA2/PGI2 and pancreas pathomorphology changes in severe acute pancreatitis (SAP) rats. Methods Sixty SD rats were random divided into five groups, including control group (A group, n = 12) and experimental group(B,C,D and E group, n = 12). The SAP was induced by injection of 5% sodium taurocholate through retrograde common biliopancreatic ducts via duodenal papilla with epidural catheter. Group C, D and E were intravenously injected with nitroglycerin 0.5μg/kg/min,1μg/kg/min and 2μg/min in 30 min respectively, and group A and B was injected with Sodium Chloride 0.5ml. The indexes of changed pathomorphology and ET/NO, TXB2/6-keto-PGF1a, were determined at the 6th and 12th hour after operations, respectively. Results The specimen data of the 6th and 12th hour displayed that the indexes of changed pathomorphology, ET, ET/NO, TXB2, and TXB2/6-keto-PGF1a of the group C,D and E degraded respectively, compared to group B(P < 0.05). Conclusion The nitroglycerin could degrade ET, ET/NO,TXA2 and TXA2/PGI2, improve the microcirculation of pancreas, and delay the pathological inflammation change in SAP rats.     

Blepharis persica increases testosterone biosynthesis by modulating StAR and 3β-HSD expression in rat testicular tissues

Objective:To evaluate the effect of methanolic extract and ethyl acetate fraction of methanol extract prepared from the seeds of Blepharis(B.)persica on testosterone biosynthesis and also to elucidate the underlying mechanism.Methods:Forty-eight male Wistar rats were divided into eight groups(n=6 per group).GroupⅠreceived 0.3%w/w gum acacia suspension p.o.and served as the normal control group.GroupⅡwas administered testosterone propionate in arachis oil i.m.as the positive control group.GroupⅢtoⅣreceived B.persica methanolic extract p.o.at doses of 50,100 and 200 mg/kg body weight.GroupⅥtoⅦreceived B.persica ethyl acetate fraction p.o.at doses of 50,100 and 200 mg/kg body weight.The testis was used for biochemical estimation and histological studies.The effects of methanolic extract and ethyl acetate fraction of B.persica on testicular testosterone,mRNA expression corresponding to steroidogenic acute regulatory protein(StAR)and 3β-hydroxysteroid dehydrogenase(3β-HSD)along with 3β-HSD enzyme assay were evaluated in testicular tissues and sperm concentration.Ethyl acetate fraction of B.persica was subjected to column chromatography.Invitro studies were performed using TM3 cell line at three dose levels(50,100,200μg/mL),each for methanolic extract,ethyl acetate fraction and 2-benzoxazolinone for evaluation of their comparative effect on testosterone production.Results:Ethyl acetate fraction and methanolic extract of B.persica could elevate the testicular testosterone content compared to the normal control group.The treatment with methanolic extract and ethyl acetate fraction of B.persica increased the expression of mRNA corresponding to StAR by 6.7 fold and 10.6 fold,respectively,whereas the mRNA expression of 3β-HSD increased by 5.7 fold and 7.3 fold,respectively.Moreover,fraction and extract treatment exhibited increased 3β-HSD activity in the testicular tissues and were found to elevate sperm concentration in seminal fluid.The spermatogenic potential was further ensured by histological observations.2-benzoxazolinone was isolated from ethyl acetate fraction and identified using spectral studies.It showed the ability to increase the testosterone content in the TM3 Leydig cells.Conclusions:Methanolic extract and ethyl acetate fraction of B.persica are able to increase the testicular testosterone in rats by elevating mRNA expression of StAR and 3β-HSD in testicular tissues,leading to increase the sperm concentration.     

孕哺期铝暴露对仔鼠海马 N-甲基-D-天门冬氨酸受体表达的影响

目的 研究孕哺期染铝对仔鼠学习记忆和仔鼠海马组织中N-甲基-D-天门冬氨酸受体(NMDAR)表达的影响,以探讨铝对发育中的中枢神经系统的毒性作用及机制.方法 Wistar大鼠60只,雌雄各半,饲养1周后按1∶1雌雄合笼,发现阴栓即可认为雌鼠怀孕,按体重将孕鼠随机分为3组:对照组(饮用蒸馏水)、低剂量组(饮用0.2%AlCl3蒸馏水溶液)、高剂量组(饮用0.4%AlCl3蒸馏水溶液),每组10只,母鼠从怀孕开始染毒,至仔鼠哺乳期(出生后21 d)结束,共染毒6周.原子吸收石墨炉法测 定血铝和脑铝含量;跳台试验法观察大鼠学习、记忆行为学的改变;用免疫印迹(Western blot)法测定海马NMDA受体表达水平.结果 随着染铝剂量的增加,仔鼠血铝、脑铝含量明显增加,与对照组比较,差异有统计学意义(P＜0.05).低、高剂量染铝组仔鼠跳台试验的潜伏期逐渐缩短[分别为(202.71±81.99)、(19.67±8.44)s],与对照组[(300.00±0.00)s]比较,差异有统计学意义(P＜0.01),而错误次数逐渐增加(低、高剂量染铝组分别为1.43±0.85、2.47±0.99),与对照组(0.00±0.00)比较,差异有统计学意义(P＜0.01).铝暴露也可导致NMDAR各亚型的比例发生变化,低、高剂量染铝组仔鼠海马组织中NR1和NR2B含量下降(低剂量染铝组NR1和NR2B灰度值为25.22±0.68、81.23±15.37,高剂量染铝组NR1和NR2B灰度值为24.75±0.71、56.63±7.82,与对照组(NR1和NR2B灰度值分别为31.69±3.44、107.61±9.05)比较,差异均有统计学意义(P＜0.05).结论 孕哺期铝暴露可以引起发育中大鼠学习记忆能力下降,导致NMDAR各亚型的比例发生变化,NR1和NR2B含量下降可能是母体铝暴露影响子代大鼠学习记忆能力的重要分子机制之一.

Abstract：

Objective To investigate the effects of aluminum on learning and memory and the expression of N-methyl-D-aspartic acid receptor (NMDAR) of hippocampus in offspring from female rats exposed to Al in the pregnancy or lactation, and to explore the mechanism of toxic effects of Al on central nervous system (CNS) during development. Methods The pregnant Wistar rats were randomly divided into 3 groups based on their body weight, I.e. Control group was exposed to distilled water, low exposure group (0.2 %AlCl3) and high exposure group (0.4 %AlCl3) were exposed orally to AlCl3 in pregnancy and lactation for 6 weeks, 10 rats each group. Aluminum content in blood and brains was determined by atomic absorption spectrophotometry (AAS). Platform experiment was used to detect the abilities of learning and memory. The expression levels of NMDARs were detected by western blot assay. Results The Al content in blood and brains of rats in exposure groups increased significantly with Al dose, as compared with the control group ( P＜0.05 ). In platform experiment, the incubation periods of rats in low and high exposure groups were (202.71±81.99 ) and ( 19.67±8.44 )s respectively, which were significantly lower than that [( 300.00±0.00 )s] in control group (P＜0.01), but the mistake times of rats in low and high exposure groups were 1.43±0.85 and 2.47±0.99 respectively, which were significantly higher than that (0.00±0.00) in control group (P＜0.01). The Al exposure could change the proportion of NMDAR subtypes, the expression levels of NR1 and NR2B in hippocampus of newborn rats in low and high exposure groups were 25.22±0.68, 81.23±15.37 and 24.75±0.71, 56.63 ±7.82, respectively, which were significantly lower than those (31.69±3.44, 107.61±9.05 ) in control group (P＜0.05). Conclusion Aluminum exposure in pregnancy and lactation could reduce theabilities of learning and memory in newborn rats, and change the proportion of NMDAR subtypes. The reduced NR1and NR2B expression levels may be one of important mechanisms to influence the abilities of learning and memory in offspring.     

RNA干扰caspase-3基因对染铝小鼠神经行为的影响

目的 研究RNA干扰caspase-3基因对染铝小鼠神经行为的影响.方法 取健康3月龄雄性昆明种小鼠,按体重随机分为4组:空白对照组(给予生理盐水4 μl)、染铝组(给予0.5%AlCl3·6H2O4μ1)、Al+空载体组(给予0.5%AlCl3·6H2O 3 μl+对照siRNA表达载体1 μl)、Al+RNAi组(给予0.5%AlCl3·6H2O 3μl+目的 siRNA表达载体1 μl),侧脑室注射连续染毒5 d,采用Morris水迷宫试验、旷场试验、跳台试验检测小鼠神经行为改变,电子显微镜观察小鼠海马病理改变,反转录-聚合酶链反应(RT-PCR)法检测caspase-3基因表达量.结果 与空白对照组[分别为(279.00±17.17)s、1.13±0.35]相比,染铝组的潜伏期(LT)[(44.67±10.60)s]明显缩短,错误次数(3.63±0.52)明显增加,Al+空载体组LT[(68.00±14.70)s]明显缩短,差异均有统计学意义(P<0.05);Al+RNAi组LT[(239.50±19.36)s]比染铝组明显延长,错误次数明显减少,差异均有统计学意义(P<0.05).与空白对照组相比,染铝组逃避潜伏期明显延长,原平台象限停留时间明显缩短,Al+空载体组逃避潜伏期明显延长,Al+RNAi组逃避潜伏期较染铝组明显延长,差异均有统计学意义(P<0.05).与空白对照组相比,染铝组和Al+空载体组小鼠中央格停留时间明显延长,直立次数、修饰次数明显减少,差异均有统计学意义(P<0.05);Al+RNAi组小鼠中央格停留时间较染铝组明显缩短,跨格次数、直立次数、修饰次数较染铝组明显增加,差异均有统计学意义(P＜0.05).空白对照组海马细胞出现轻微改变,染铝组和Al+空载体组海马细胞改变明显,Al+RNAi组海马细胞的病理形态变化减少.空白对照组海马CA3区神经细胞层次较为清楚,基本无变性损伤现象发生;染铝组和Al+空载体组小鼠CA3区细胞数量明显减少,排列不规则,神经元尼氏体着色较空白对照组浅;RNA干扰后,CA3区神经细胞数量明显增加,排列较规则,神经元尼氏体着色逐渐加深.染铝组和Al+空载体组的caspase表达量(分别为2.24±0.57、2.28±0.33)较空白对照组(1.00±0.00)明显增加,Al+RNAi组的的caspase-3表达量(0.44±0.08)较空白对照组和染铝组明显降低,差异均有统计学意议(P＜0.05).结论 铝可以引起小鼠学习和记忆能力障碍,活跃程度及探索能力下降,而RNAi技术能够在一定程度上减轻该毒性作用.

Abstract：

Objective To investigate the effects of caspase-3 siRNA on the neurobehavior of mice exposed to aluminum. Methods Male KunMing mice ( 3 months old ) were randomly divided into 4 groups by weight:blank control group(4 μl normal saline), Al group(4 μl 0.5%AlCl3), Al plus empty vector group(3 μl 0.5% AlCl3 plus control siRNA expression vector)and Al plus RNAi group (3μl 0.5% AlCl3 plus targeted siRNA expression vector). All groups were treated by lateral cerebral ventricle micro-injection for 5 days. The neurobehavior was tested by the Morris water maze test, Open-field and Step-down tests for all treated mice. Pathological changes in hippocampus was observed by electron microscopy, the caspase-3 gene expression levels were detected using RT-PCR. Results The results of Step-down test indicated that as compared with control group, the latent time [LT, (44.67±10.60) s] in Al group decreased significantly, the error number (3.63±0.52) in Al group increased significantly and the LT [(68.00±14.70) s] in Al plus empty vector group decreased significantly(P<0.05). the LT [(239.50±19.36) s] in Al plus RNAi group increased significantly and the error number in Al plus RNAi group decreased significantly, as compared with Al group (P<0.05). The results of Morris water maze test showed that as compared with control group, the LT in Al group increased significantly, and residence time in the former platform quadrant decreased significantly and the LT in Al plus empty vector group increased significantly (P<0.05). The LT in Al plus RNAi group was significantly longer than that in Al group(P<0.05). The results of open-field test demonstrated that as compared with control group, the time in the central grid in Al group and Al plus empty vector group increased significantly, the rearing number and the modification number in Al group and Al plus empty vector group decreased significantly (P< 0.05). As compared with Al group, the time in the central grid in Al plus RNAi group decreased, the inter-cell number, the rearing number and the modification number increased significantly (P<0.05). The results of electron microscopic examination exhibited that a slight change of hippocampal cells appeared in control group, the obvious pathological changes of hippocampal cells appeared in Al group and Al plus empty vector group, but the pathological changes of hippocampal cells in Al plus RNAi group significantly reduced as compared with Al group. The results of thionin staining indicared that the layers of neural cells of hippocampal CA3 were more clear and there was not obvious denatured injury of neural cells of hippocampal CA3 in control group. The number and Nissl body color of neural cells of hippocampal CA3 in Al group and Al plus empty vector group decreased significantly. After RNA interference, the number and Nissl body color of neural cells of hippocampal CA3 increased obviously. The expression levels of caspase-3 gene in Al group and Al plus empty vector group were 2.24±0.57 and 2.28±0.33, respectively, which were significantly higher than that (1.00±0.00) in control group (P<0.05). The expression level of caspase-3 gene in Al plus RNAi group was 0.44 ±0.08, which was significantly lower than those in Al group and control group (P<0.05). Conclusion Aluminum can decrease the learning and memorizing ability, and inhibited the activity or exploration function of mice. It is suggested that Caspase-3 siRNA may reduce the neurotoxicity induced by aluminum to a certain extent.     

雷米芬太尼对全麻患者气管插管期平均动脉压、心率和QTc间期延长的影响

Objective To evaluate the effect of remifentanil on mean arterial pressure (MAP), heart rate (HR) and QTc interval during tracheal intubation of general anesthesia patients. Methods Seventy-five ASA Ⅰ -Ⅱ grade patients were selected and allocated to receive either saline (group C), remifentanil 0.50 μg/kg (group R1) or remifentanil 0.75 μg/kg(group R2) by random digits table with 25 cases in each, they were administrated as a bolus intravenous, followed by a continuous infusion at 0.10 μg/ (kg·min), 1 min before laryngoscopy. All patients received fentanyl 3 μg/kg,propofol 1.0 - 1.5 mg/kg and vecuronium 0.1 mg/kg. The ECG.MAP and HR were recorded prior to induction of anesthesia (T0), 2 min following the start of drug intravenous of fentanyl and propofol with vecuronium (T1), 1 min following remifentanil or saline (T2), before laryngoscopy(T3), 30 s (T4), 2 min (T5) and 4 min (T6) after intubation. Results The QTc interval was significantly prolonged immediately following intubation in group C and group R1, but it remained stable in group R2, compared with the QTc interval just before laryngoscopy. In group R2, QTc interval was significantly shorter at T4-T6 compared to group C(P＜ 0.05 or ＜ 0.01). QTc interval significantly increased from baseline at T4 in group R1 and T4-T6 in group C (P＜ 0.05 or ＜ 0.01). The number of patients with QTc interval ＞ 440 ms were significantly greater immediately following tracheal intubation in group C than that in group R2 [44% (11/25) vs. 12% (3/25)] (P ＜ 0.05). Conclusions QTc interval increases following tracheal intubation during induction of anesthesia using fentanyl and propofol. Intravenous of remifentanil attenuates the QTc interval prolongation associated with tracheal intubation. In addition, remifentanil decreases the hemodynamic responses to tracheal intubation.     

镉对HepG2细胞DNA损伤作用以及对gadd基因表达的影响

目的 探讨氯化镉对HepG2细胞DNA损伤作用以及对gadd153和gadd45β启动子和mRNA表达的影响.方法 应用彗星试验检测氯化镉对HepG2细胞的DNA损伤作用;分别构建含有gadd153和gadd45β启动子和荧光素酶报告基因的载体pGADD153-Luc和pG45-Luc,荧光素酶活性检测反映gadd153和gadd45β启动子的活性,生物发光检测荧光素酶活性;反转录-聚合酶链反应(RT-PCR)检测gadd153和gadd45β基因mRNA的表达.结果 彗星试验结果显示,在100、300 μmol/L氯化镉处理细胞24 h后,Olive尾矩(分别为0.78±0.06、1.10±0.12)明显高于对照组(0.53±0.08),差异有统计学意义(P<0.05),并有良好的剂量-效应关系(r=0.9761,P<0.05);报告基因分析显示,1、5、10 μmol/L氯化镉处理组gadd153启动子表达[分别为(9.45±1.26)、(11.76±1.12)、(21.14±1.47)RIU/μg Pro]均明显高于对照组[(7.02±0.82)RIU/μg Pro]差异均有统计学意义(P<0.05),并有良好的剂量-效应关系(r=0.8755,P<0.05);5、10μmol/L氯化镉处理组gadd45β启动子表达明显高于对照组,差异有统计学意义(P<0.05),并有良好的剂量-效应关系(r=0.8856,P<0.05);RT-PCR结果显示,1、5、10μmol/L氯化镉处理组gadd153 mRNA表达均明显高于对照组,5、10 μmol/L氯化镉处理组gadd45β mRNA表达明显高于对照组,差异有统计学意义(P<0.05).结论 氯化镉可诱导HepG2细胞DNA损伤,较低剂量氯化镉即使未引起明显的DNA损伤,亦可促进HepG2细胞gadd153和gadd45β启动子和mRNA的表达.

Abstract：

Objective To investigate the effects of the cadmium chloride on the DNA damage and the expression of the gadd153 and gadd45β promoter and mRNA in HepG2 cells.Methods DNA damage induced by cadmium chloride was detected by comet assay.The plasmids (pGADD153-Luc and pG45-Luc ) containing DNA damage and repair inducible gene 153 and 45 (gadd153 and gadd45β) promoter and luciferase and gadd45β reporter gene were constructed.The activity of gadd 153 and gadd45β promoter were represented by the luciferase activity,the inducible luciferase activities was detected by bioluminescence.The expression of gaddl53 and gadd45β mRNA was detected by RT-PCR.Results The results of comet assay indicated that Olive Tail Moment induced by the cadmium chloride increased significantly at the dose of 100,300 μmol/L,compared with the control (P<0.05).The luciferase activity analysis showed that the expression levels of gaddl53 promoter increased significantly in 1,5,10 μmol/L treatment group,compared with the control (P<0.05).The expression levels of gadd45β promoter in 5,10μmol/L treatment group were significantly higher than that in control group (P<0.05).The expression levels of gaddl53 mRNA induced by cadmium chloride at the doses of 1,5,10 μ-mol/L and the expression levels of gadd45β mRNA induced at the doses of 5,10μmol/L were significantly higher than thoae in control group (P<0.05).Conclusion The cadmium chloride can induce the DNA damage and increase the expression levels of the gaddl53 and gadd45β promoters in HepG2 cells.     

氯化镧对大鼠海马即刻早期基因表达的影响

目的 研究氯化镧(lanthanum chloride,LaCl3)对大鼠海马即刻早期原癌基因(c-jun)、早期生长反应基因1(early growth response gene 1,Egr1)和活性调节细胞骨架基因(activity-regulatedcytoskeletal gene,Arc)表达的影响,探讨LaCI3损害学习记忆能力的机制.方法 按完全随机化法将40只Wistar雌性成年大鼠分为对照组和低(0.25%)、中(0.50%)、高(1.00%)LaCl3染毒组,各组雌鼠10只,与5只雄鼠按2∶1比例交配.对照组和低、中、高剂量染毒组产仔鼠数分别是80、83、78、75只.各LaCl3染毒组仔鼠断乳前吸吮母乳染镧,断乳后饮用0.25%、0.50%和1.00%LaCl3溶液1个月.分别采用跳台试验测试仔鼠学习记忆能力,电感耦合等离子体质谱仪测定海马镧含量,RT-PCR法检测海马c-jun、Egr1和Arc mRNA表达,采用Western blotting法检测海马c-jun、Egr1和Arc蛋白表达.结果 低、中、高剂量组仔鼠在跳台测试时错误次数分别是(1.75±0.71)、(2.38±0.92)、(3.00±0.76)次,中、高剂量组均高于对照组的(1.25±0.46)次(q值分别为4.386、6.793,P值均＜0.05);潜伏期分别为(174.13±33.72)、(139.25±45.83)、(75.50±18.56)s,均低于对照组的(206.75±20.47)s(q值分别为2.958、6.121、11.902,P值均＜0.05).对照组和低、中、高剂量组仔鼠海马c-jun mRNA表达量分别为(0.89±0.08)、(0.77±0.12)、(0.58±0.14)、(0.29±0.11),c-jun蛋白表达量分别为(0.72±0.13)、(0.64±0.11)、(0.43±0.11)、(0.31±0.14),Egr1 mRNA表达量分别为(0.78±0.09)、(0.61±0.13)、(0.53±0.10)、(0.22±0.08),Egr1蛋白表达量分别为(0.65±0.18)、(0.40±0.15)、(0.32±0.13)、(0.14±0.09),均与染毒剂量具有剂量-反应关系,相关系数r值分别为-0.900(t=11.309,P=0.000)、-0.969(t=7.058,P=0.000)、-0.898(t=11.179,P=0.000)和-0.962(t=6.739,P=0.000).结论 LaCl3损害大鼠学习记忆能力的机制可能与其致海马c-jun和egr1基因和蛋白表达降低有关.

Abstract：

Objectiye To study influence of lanthanum chloride(LaCl3)on the expression of immediate early genes(IEGs)including c-jun,early growth response gene 1(Egr1)and activity-regulated cytoskeletal gene(Arc)in the hippocampus of rats,and discuss the mechanism of LaCl3 undermining learning and memory capability.Methods Forty female Wistar adult rats were divided into control group,low LaCl3-contaminated group(0.25%),medium LaCl3-contaminated group(0.50%),and high LaC13-contaminated group(1.00%)by randomized desigr.Each group had ten female rats along with five separately.The pups in respective group were La-dyed by lactation,and then the pups in LaCl3-contaminated groups drank 0.25%,0.50% and 1.00% LaCl3 separately for one month.Learning and memory capability of pups were measured in jumping stairs experiment.Hippocampal lanthanum content was determined by inductively coupled plasma mass spectrometry(ICP-MS).Hippocampal c-jun,Egr1 and Arc mRNA expression was detected by RT-PCR,and corresponding protein expression was measured by Western blotting method.Results In the jumping stairs experiment,pups in 0.25%,0.50% and 1.00%LaCl3-contaminated groups respectively made(1.75 ±0.71),(2.38 ±0.92)and(3.00 ±0.76)mistakes;significantly higher than control group(1.25 ±0.46)(q values were 4.386,6.793,P ＜0.05).However,the incubation period of 0.25%,0.50% and 1.00% LaCl3-contaminated groups were(174.13 ± 33.72),(139.25 ±45.83)and(75.50 ± 18.56)respectively,which were all significantly lower than that of control group(206.75 ± 20.47)(q values were 2.958,6.121,11.902,P ＜0.05).Hippocampal c-jun mRNA expression were(0.89 ±0.08),(0.77 ±0.12),(0.58 ±0.14)and(0.29 ±0.10); while the c-jun protein expression were(0.72 ± 0.13),(0.64 ± 0.11),(0.43 ± 0.11)and(0.31 ± 0.14),and the Egr1 mRNA expression were(0.78 ±0.09),(0.61 ±0.13),(0.53 ±0.10)and(0.22 ±0.08),Egr1 protein expression were(0.65 ± 0.18),(0.40 ± 0.15),(0.32 ± 0.13)and(0.14 ± 0.09)in 0.25%,0.50%and 1.00% LaCl3-contaminated groups; and all of which presented a dose-effect relationship that the correlation coefficients of these parameters with dose were-0.900(t = 11.309,P = 0.000),-0.969(t =7.058,P=0.000),-0.898(t=11.179,P=0.000)and-0.962(t=6.739,P=0.000).Conclusion LaCl3 undermines the learning and memory capability of rats,which is possibly related to lower expression of e-jun and Egr1 gene and protein induced by lanthanum in hippocampus.