Influence of bifidobacterium on NF-κB and IκB in murine peritoneal macrophages

AIM: To explore the regulatory effect of bifidobacteria adolescence on NF-κB in murine peritoneal macrophages (PMs). METHODS: Macrophages were collected and were divided into tow groups, normal control group and bifidobacterium stimulation groups. The cells were fixed at 60 min after stimulated with bifidobacterium at 106, 107, 108 and 109 cells/L, or fixed respectively at 15, 30, 60, 120 and 180 min after stimulated with bifidobacterium at 108 cells/L, then the total protein and nucleoprotein were extracted. The activities of NF-κB and IκBα were measured with the methods of MSA and Western blotting. RESULTS: NF-κB activity markedly increased and reached the peak at 0.5 h after stimulation, while IκBα markedly decreased at same time. CONCLUSION: NF-κB activity is markedly activated by bifidobacterium. NF-κB pathway participates in the regulation of peritoneal macrophage in this process.     

Effect of microwave ablation of liver cancer on cellular immunity in mice

AIM:To investigate the effect of microwave ablation of liver cancer on the cellular immunity in mice.METHODS:A C57BL/6J mouse model of liver cancer was established by subcutaneous injection of Hepa 1 - 6 cells.The tumors were subjected to micr owave ablation under the ablation condition of 45 ℃,50 ℃,55 ℃ or 60 ℃ fo r 180 s.The CD4＋ T cells,CD8＋ T cells and natural killer cells (NK) in p eripheral blood were detected by FACS.The cytotoxicity of splenic NK and splen ic cytotoxic T lymphocytes (CTL) activated by inactivated Hepa 1-6 cells was ass ayed by LDH method.RESULTS:The proportions of CD4＋ T cells,CD8＋ T cells and NK cells in peripheral blood in 50 ℃ and 55 ℃ group at 21 d after ablation we re significantly increased and that of NK cells in 60 ℃ group was significantly in creased.There was no significant difference between those in group 42 d after ablation and control.The cytotoxicities of splenic CTL and NK cells in 50 ℃ an d 55 ℃ groups at 21 d or 42 d after ablation were significantly increased,and they were much higher than those in 45 ℃ group at the same time.The cytotoxicities of splenic CTL in 50 ℃ and 55 ℃ groups at 21 d after ablation were much highe r than that in 60 ℃ group at the same time.CONCLUSION:Under a certain ablation temperature,microwave abla tion of liver cancer promotes the cellular immunity.     

Expression of hypoxia-inducible factor 1 and neuroglobin in piglet cortex during deep hypothermic circulatory arrest

AIM: To observe the expression of hypoxia-inducible factor 1 (HIF-1) and neuroglobin (NGB) in piglet cortex during deep hypothermic circulatory arrest. METHODS: Wuzhishan piglets were randomly assigned to cardiopulmonary bypass group (CPB group), 40 min of circulatory arrest (CA) at 18 ℃ without cerebral perfusion (DHCA group) or with selective antegrade cerebral perfusion (SACP group). After 180 min of reperfusion, cortical tissue was harvested for determining HIF-1α and NGB expression by HE staining, Western blot and real-time PCR. RESULTS: Severer cerebral injury was observed in DHCA group than that in SACP group. After 180 min of reperfusion, HIF-1α protein and mRNA levels were significantly higher in DHCA group than those in CPB group (P<0.05). Accordingly, SACP animal had higher levels of HIF-1α protein and mRNA than those in DHCA group (P<0.05). Simultaneously, higher NGB protein and mRNA levels were found in DHCA group than those in CPB group after 180 min of reperfusion (P<0.05). The SACP animal had higher levels of NGB protein and mRNA than those in DHCA group (P<0.05). CONCLUSION: Up-regulation of HIF-1 and NGB are involved in the mechanism against cerebral injury resulting from DHCA in the cortex and possibly a part of cerebral protective effect of SACP.     

Effect of MIF antisense oligonucleotides on expression of MIF in macrophages

AIM: To investigate the effect of antisense oligonucleotides on expression of macrophage migration inhibitory factor (MIF) in macrophages. METHODS: MIF phosphorothioate oligonucleotides was designed and synthesized. The phosphorothioate antisense, sense and missense oligonucleotides of mouse MIF was transfected into macrophages, separately. After that, macrophages were incubated with LPS. Cell culture medium was collected for MIF protein detection by EIA. Cellular RNA was extracted and the expression of MIF mRNA was examined by RT-PCR analysis. RESULTS: LPS stimulation resulted in a specific time-dependent expression of MIF derived from macrophages. MIF mRNA and MIF protein level increased at 6 h and reached a plateau at 9-12 h after LPS stimulation. The macrophages treated with antisense oligonucleotides showed a significant decrease in MIF mRNA and MIF protein after LPS stimulation than those with LPS stimulation only and LPS plus sense or missense oligonucleotides (P<0.05). CONCLUSION: Antisense oligonucleotides of MIF inhibit the expression of MIF mRNA and MIF protein in macrophages treated with LPS.     

Overexpression of Smad7 inhibits TGF-β-induced Smad2 mRNA and protein expression in peritoneal mesothelial cells

AIM: To investigate the role of Smad7 in the Smad2 expression induced by transforming growth factor-β₁ (TGF-β₁) in rat peritoneal mesothelial cells (PMCs).METHODS: Rat PMCs were cultured at different doses of TGF-β₁ (0，1.25，2.5，10 μg/L) for different time (0，5，15，30，60，120 min).PCDNA3-Smad7 was then transfected into cultured rat PMCs by lipofectamine, and the cells were stimulated like the above.Endogenous Smad2 and Smad7 expression was evaluated by RT-PCR and Western blotting.RESULTS: TGF-β₁ induced increase in Smad2 mRNA and protein expression at 5 min, peaked at 30 min, and declined to baseline levels at 120 min, which was in a time-dependent manner.TGF-β₁ also induced Smad7 mRNA expression at 5 min, and then declined, down to the lowest at 30 min, but at 60 min it increased again.Smad2, Smad7 mRNA and protein expression induced by TGF-β₁ were also dose-dependent.After transfection, overexpressions of Smad7 mRNA and protein in rat PMCs were observed, which did not decline with time.The expression of Smad2 mRNA significantly decreased by 33%, 56%, 67%, 71%, 63% and 57% (P<0.05), the expression of Smad2 protein declined by 78%，89%，89%，88% and 76% (P<0.05) respectively at 0, 5, 15, 30, 60 and 120 min.CONCLUSION: Overexpression of Smad7 inhibits Smad2 gene and protein expression in peritoneal mesothelial cells.Smad7 may be a negative regulator of TGF-β₁ signaling.     

通过玻璃化超低温处理脱除草莓轻型黄边病毒(SMYEV)研究

以草莓品种明宝为材料,取茎尖做初代培养,继代扩繁5次后,取2mm左右的茎尖,利用玻璃化超低温技术对SMYEV进行脱除,并利用结合内标的多重RT-PCR技术对再生植株进行病毒检测。结果表明,在病毒脱除过程中,预培养蔗糖浓度为0.5mol/L,处理3d;装载处理为25℃,60min;玻璃化处理为0℃,120min;液氮处理60min后,进行40℃水浴2min,草莓茎尖的存活率为76%,而草莓轻型黄边病毒的脱除率为95%。只有通过液氮处理才可以脱除该病毒,液氮处理前的玻璃化处理脱毒率为0。利用超低温脱毒法可以简便有效地脱除SMYEV。     

Effects of cerebral ischemia and postconditioning on signaling molecules PERK and GRP78 in hippocampus tissue of tree shrew during endoplasmic reticulum stress

AIM: To investigate the effects of cerebral ischemia and postconditioning on protein kinase R-like endoplasmic reticulum kinase (PERK) and glucose-regulated protein 78 (GRP78) in the hippocampus tissue of tree shrew during endoplasmic reticulum stress and the mechanism of post-conditioning protecting the brain from damage. METHODS: The focal cerebral ischemic model was duplicated by photochemical reaction in tree shrew and the postconditioning was induced by alternatively occluding and opening the carotid artery of ischemic side for 3 cycles (5 min each cycle) at 3.5 h after ischemia. The damage and ultrastructural changes of the hippocampal neurons were observed by HE staining. The expression of PERK and GRP78 at mRNA and protein levels in the hippocampal tissue at different time points after cerebral ischemia and postconditioning was determined by RT-PCR, immunohistochemistry and Western blot. RESULTS: The injuries of hippocampal neurons were aggravated with prolonged cerebral ischemia, which was most severe at 24 h after ischemia while the postconditioning alleviated these damages correspondingly. The expression of PERK at mRNA and protein levels was upregulated at 4 h, 24 h and 72 h after ischemia (P<0.05), while postconditioning downregulated the expressions of PERK at ischemia and postconditioning 4 h (IP4 h) gruop and IP24 h group (P<0.05). The expression of GRP78 at mRNA and protein levels was not changed at 4 h, 24 h and 72 h after ischemia, while postconditioning upregulated the expressions of GRP78 at IP24 h group (P<0.05). CONCLUSION: The focal thrombotic cerebral ischemia activates the endoplasmic reticulum stress in ischemic hippocampus of tree shrews, leading to the changes in mRNA and protein expression of PERK in the PERK/eIF2α signal transduction pathway. The postconditioning treatment alleviates endoplasmic reticulum stress and neuronal damages by downregulating PERK and upregulating GRP78, thereby protecting the brain from injury.     

香蕉果皮2个HSP70基因片段的克隆及其在热激和冷藏过程中的表达

温度逆境胁迫可以诱导热激蛋白的表达,而热激处理能够减轻果实冷害。为了探讨温度逆境对香蕉热激蛋白基因表达的调控、辨析香蕉热激蛋白基因的表达与果实冷害的关系,采用同源序列法分离了香蕉果皮HSP70基因序列-根据已经报道的HSP70基因的氨基酸保守序列设计引物、以香蕉果皮总RNA为模板,用RT-PCR方法克隆香蕉的HSP70 cDNA,Northern杂交分析该基因在不同贮藏温度下的表达特征。结果得到2个序列不同的HSP70基因片段,分别命名为Ma-HSP70-1和Ma-HSP70-2,Ma-HSP70-1和Ma-HSP70-2之间的碱基同源性为86.9%,氨基酸同源性为97.1%。Northern杂交的结果表明,38℃短期热激处理诱导了Ma-HSP70-2基因的表达,低温冷藏能使2个基因的表达增强。并初步认为Ma-HSP70可能与香蕉果实耐冷性有关。     

Expression of dopamine receptor in rat anoxia-reoxygenation injured cardiac tissue

AIM: To investigate the expression of dopamine receptor (DR) in rat cardiac tissue and the relationship with the anoxia-reoxygenation injury (ARI).METHODS: The rat cardiac ARI model in vitro was rebuilt on Langendorf device.RT-PCR and Western blotting were used to detect the mRNA and protein expression of DR1and DR2.The activity of LDH in coronary effluent volume was assayed with ultraviolet spectrophotometer.The ultrastructure of cardiac tissue was determined by transmission electron microscope.RESULTS: The mRNA and protein expressions of DR1and DR2 in rat cardiac tissue increased at anoxia 40 min (P<0.05)，markedly increased at reperfusion 1 h (P<0.01),reached the highest level at 2 h,but lightly decreased at 3 h and 4 h.The activity of LDH increased and the myocardial ultrastructure injured seriously after reperfusion.CONCLUSION: The expressions of DR1 and DR2 in rat cardiac tissue increased firstly then decreased during anoxia-reoxygenation processes.The two kinds of receptors may take part in the cardiac anoxia-reoxygenation injury.     

高温胁迫对设施番茄和黄瓜光合特性及抗氧化酶活性的影响

以黄瓜品种"津优35"和番茄品种"金粉2号"为试材,于2011年1～6月在南京信息工程大学人工气候箱进行环境控制试验,系统研究高温32～40℃处理不同时间对黄瓜、番茄叶片光合特性及抗氧化酶活性的影响。结果表明:高温胁迫处理明显抑制了黄瓜和番茄光合速率,处理时间越长,光合速率下降越快,当38～40℃高温持续一段时间后,黄瓜、番茄的最大光合速率为负值;随高温胁迫程度的增加和胁迫时间的延长,黄瓜和番茄的PSII潜在光化学效率(Fv/Fm)、光化学淬灭(qP)均呈现下降趋势,40℃高温胁迫5d后再经过恢复,黄瓜和番茄的Fv/Fm及qP无法恢复到对照水平;当温度达到38℃以上时,黄瓜和番茄的SOD、POD、CAT、MDA酶随着胁迫温度的升高和持续时间的增加而呈现与38℃以下时相反的情况,根据高温对黄瓜和番茄叶片光合参数的影响将高温胁迫划分为轻度胁迫、中度胁迫、重度胁迫和极重度胁迫4个等级,并确定气象指标。     

Effect of L-arginine on expression of bcl-2 and bax mRNA in pulmonary injury induced by ischemia-reperfusion in rabbits

AIM:To investigate the effect of L-arginine (L-Arg) on expression of bcl-2, bax mRNA during pulmonary ischemia and reperfusion injury (PIRI) in rabbits.METHODS:Single lung ischemia and reperfusion animal model was used in vivo. The rabbits were randomly divided into three groups: sham operated group (sham, n=12), ischemia-reperfusion group (I/R, n=12) and I/R+ L-arginine group (L-Arg, n=12). Changes of several parameters, which included apoptotic index (AI), wet to dry ratio of lung tissue weight (W/D) and index of quantitative assessment of histologic lung injury (IQA), were measured at 300 min after reperfusion in lung tissue. Meanwhile the location and expression of bcl-2, bax mRNA as well as the ratio of bcl-2 mRNA/bax mRNA were observed. The lung tissue was prepared for light microscopic and electron microscopic observation at 60, 180 and 300 min after reperfusion. RESULTS:As compared with I/R group, in intima and extima of small pulmonary artery, alveoli, and bronchiole epithelia, the expression of bcl-2 mRNA and the ratio of bcl-2 mRNA/bax mRNA were increased, and the expression of bax mRNA was decreased in L-Arg treatment group. The values of AI, W/D and IQA showed significantly lower than that in I/R group at 180 minutes after reperfusion in lung tissue (P<0.01 and P<0.05). Meanwhile, abnormal changes of the lung tissue in morphologically were markedly lessened in L-Arg treatment group.CONCLUSION:L-arginine produces a notable protective effect on PIRI in rabbits by up-regulating bcl-2 mRNA expression, down-regulating bax mRNA expression in lung tissue and regulating the balance of bcl-2 mRNA and bax mRNA to decrease apoptosis.     

Model construction of rat coronary artery smooth muscle cell endoplasmic reticulum stress induced by thapsigargin

AIM: To investigate the primary culture method for coronary artery smooth muscle cells (CASMCs), and to establish the endoplasmic reticulum stress (ERS) model in CASMCs of SD rats. METHODS: CASMCs were cultured by tissue explant method. The morphological characteristics were observed under optical microscope. The marker proteins of CASMCs, including α-SMA and SM-MHC, were identified by immunofluorescence technique. The protein expression levels of BiP and CHOP, the marker molecules of ERS, were determined by Western blot. RESULTS: The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d, and formed the typical "hill and valley" growth pattern of CASMCs at 9~10 d. The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed. The results of Western blot showed that the protein expression of BiP and CHOP in TG (1 and 2 μmol/L) treatment groups was increased compared with control group. Compared with control group, the protein expression of BiP and CHOP was significantly increased after 1 μmol/L TG treatment for 24 and 48 h. CONCLUSION: CASMCs can be successfully cultured by tissue explant method. ERS model of CASMCs was established by 1 μmol/L TG treatment for 24 h.     

Effects of TGF-β1 and signal protein Smad3 on rat cardiac myocyte hypertrophy

AIM: To investigate the effects of TGF-β1 and signal protein Smad3 on rat cardiac myocyte hypertrophy. METHODS: The total protein was analyzed by flow cytometry and the ANF mRNA expression was measured by RT-PCR to judge the hypertrophy of cultured neonatal cardiac myocytes. Smad3 mRNA expression in cardiac myocytes was measured by RT-PCR, and the protein expression of Smad3 was analyzed by Western blotting. RESULTS: TGF-β1 significantly increased the total protein in cardiac myocytes and promoted ANF mRNA expression, compared with control group. In cultured neonatal myocytes, AS-ODN of Smad3 inhibited myocyte hypertrophy induced by TGF-β1. Smad3 mRNA and protein expression increased at 15 min after incubated with TGF-β1, reached the peak at 1 h, and declined at 4 h. CONCLUSION: TGF-β1 and signal protein Smad3 may participate in the progress of rat cardiac myocyte hypertrophy.     

Effect of PPAR γ-ACAT1 pathway on macrophage foam cell formation induced by Chlamydia pneumoniae

AIM: To investigate the expressions of peroxisome proliferator-activated receptor γ (PPAR γ) and acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT1), and to discuss the mechanisms and pathways of Chlamydia pneumoniae (C.pn)-induced macrophage foam cell formation. METHODS: THP-1-derived macrophages were incubated for 48 h with or without C.pn (1×105 to 1×106 IFU) and/or rosiglitazone (1 to 20 μmol/L), a specific PPAR γ agonist. Lipid droplets in cytoplasm were observed by oil red O staining. The contents of intracellular cholesterol ester were detected by enzyme-fluorescence. PPAR γ, ACAT1 mRNA and protein expressions were determined by RT-PCR and Western blotting, respectively. RESULTS: THP-1-derived macrophages infected with C.pn at concentration of 5×105 and 1×106 IFU resulted in the large accumulation of lipid droplets and the ratio of cholesteryl ester (CE) to total cholesterol (TC) was much higher than 50%when co-incubated with low density lipoprotein (LDL). C.pn up-regulated the expressions of ACAT1 mRNA and protein, and down-regulated the expressions of PPAR γ mRNA and protein in a concentration-dependent manner (P<0.05). Rosiglitazone (10, 20 μmol/L) markedly suppressed the accumulation of lipid droplets and CE by C.pn. Moreover, rosiglitazone inhibited the up-regulation of ACAT1 mRNA and protein expression by C.pn infection in a concentration-dependent manner (P<0.05). CONCLUSION: C.pn induces macrophage foam cell formation by up-regulating ACAT1 expression via PPARγ pathway, which may provide new evidences for the development and progression of atherosclerosis initiated by C.pn infection.     

巴西菇麦角甾醇抗肿瘤活性及作用机理初探

采用小鼠S180移植瘤实验研究了巴西菇子实体中麦角甾醇的体内抗肿瘤活性,并通过巨噬细胞吞噬功能测定(中性红法)、脾淋巴细胞增殖功能检测(MTT法)、体外抑杀肿瘤细胞(MTT法)以及鸡胚绒毛尿囊膜(CAM)等试验初步探讨了麦角甾醇抗肿瘤活性的作用机理.结果表明,巴西菇子实体中提取的麦角甾醇在剂量10 mg·kg-1·d-...     

Glucose-regulated protein 78 promote cardiomyocyte apoptosis in liver cirrhotic rats

AIM: To explore the cardiomyocyte apoptosis induced by glucose-regulated protein 78/immunoglobulin heavy chain-binding protein (GRP78/BiP) in liver cirrhotic rats and its mechanism. METHODS: The liver cirrhotic rat model was established with multiple pathogenic factors, and sampled at the time points of 4 weeks, 6 weeks and 8 weeks. In experiment 1, the heart was collected and weighed, the thickness of the left ventricular wall was measured, and the ratios of the left ventricular wall thickness to the heart weight, and the heart weight to the body weight were calculated. In experiment 2, TUNEL was used to detect the apoptotic cardiomyocytes,and the protein levels of GRP78/BiP, CCAAT/enhancer-binding protein homologous protein/growth arrest and DNA damage-inducible protein 153 (CHOP/GADD153), caspase-12, nuclear factor-κB p65 (NF-κB p65) and B-cell lymphoma/leukemia-2 (Bcl-2) in the myocar-dium were detected by the method of immunohistochemistry. RESULTS: Compared with the normal myocardium, significant larger ratios of the left ventricular wall thickness to the heart weight and the heart weight to the body weight, a significant increase in the cardiomyocyte apoptosis, and a significant larger positive expression index of GRP78/BiP in the hearts 8 weeks after modeling were observed. The protein levels of CHOP/GADD153 and caspase-12 were gradually increased during the development of liver cirrhosis and were significantly increased at 8 weeks. The positive expression of NF-κB p65 and Bcl-2 showed consistent changes, and were markedly higher at 4 weeks than those at the other time points. The positive expression index of GRP78/BiP was positively correlated with the apoptotic index, and the levels of CHOP/GADD153 and caspase-12. The positive expression index of CHOP/GADD153 was negatively correlated with NF-κB p65 and Bcl-2. CONCLUSION: Elevated expression of GRP78/BiP may play a crucial role in the pathogenesis of liver cirrhotic cardio-myopathy mediated by endoplasmic reticulum stress.     

Effects of Astragalus injection on neuronal apoptosis and expression of JNK3 in rat hippocampus after cerebral ischemia reperfusion

AIM:To investigate the effects of Astragalus injection on neuronal apoptosis and expression of c-Jun N-terminal kinase 3(JNK3) in the rat hippocampus after cerebral ischemia reperfusion. METHODS:The rat model of cerebral ischemia reperfusion was set up by a four-vessel occlusion method. The SD rats were randomly divided into 4 groups:sham operation group, cerebral ischemia reperfusion group(model group), cerebral ischemia reperfusion+Astragalus injection group(Astragalus injection group) and cerebral ischemia reperfusion+vehicle group(vehicle group). The rats in model group, Astragalus injection group and vehicle group after transient global cerebral ischemia(30 min) were then divided into 7 subgroups according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. The apoptosis of the neuron in the hippocampus was measured by the method of TUNEL staining. The expression of JNK3 at mRNA and protein levels was determined by real-time PCR and Western blotting,respectively. RESULTS:Compared with sham operation group, the number of apoptotic neurons increased in model group(P<0.05). Compared with model group, the number of apoptotic neurons decreased obviously in Astragalus injection group(P<0.05). Compared with sham operation group, the expression of JNK3 at mRNA and protein levels in the hippocampus increased obviously in model group at all time points except 120 h(P<0.05). Compared with model group, the expression of JNK3 at mRNA and protein levels in the hippocampus decreased obviously in Astragalus injection group at all time points except 120 h(P<0.05). CONCLUSION:Astragalus injection decreases neuronal apoptosis in rat hippocampus after cerebral ischemia reperfusion by inhibiting the expression of JNK3 at mRNA and protein levels.     

Effects of intratracheal administration of aflatoxin G1 on the expression of CC-10 in rat lung tissues

AIM: To explore the putative effects of single intratracheal administration of aflatoxin G1 (AFG1) on the expression of CC-10 of lung tissues in SD rats. METHODS: Male SD rats were intratracheally administrated with AFG1 (30 μg/kg body weight) and the animals were respectively sacrificed at 1, 3, 7 and 14 d after AFG1 treatment. Bronchial alveolar lavage fluid (BALF) was centrifuged and the supernatants were collected for LDH release assay using Detection Kit with biochemical method. The expression of clara cell 10 kD protein (CC-10 protein) in lung tissues was determined by FCM analysis and Western blotting respectively. The expression of CC-10 at mRNA level was analyzed by RT-PCR. RESULTS: LDH activity in BALF after AFG1 treatment for 1, 3 and 7 d was significantly increased as compared to that in their corresponding control group (P<0.01) and restored to control level at 14 d after AFG1 treatment. FCM, Western blotting and RT-PCR results showed that no significant changes in CC-10 expression at both protein and mRNA levels were found between AFG1 group and control group 1 day after AFG1 treatment. While the CC-10 expressions of lung tissues at both protein level and mRNA levels in AFG1 treated group were significantly decreased as compared to those in their corresponding control group at 3, 7 and 14 d after AFG1 treatment (all P<0.01). CONCLUSION: Intratracheal administration of AFG1 may cause injuries and decrease the expression of CC-10 in lung tissues of SD rats in vivo.