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.

AIMTo investigate the effects of c-myb on progesterone-induced mouse germinal vesicle(GV) stage denuded oocyte (DO) maturation in vitro.

METHODSWe used mouse GV stage oocyte cultured with special concentration progesterone, or/and antisense c-myb ODN, or/and db-cAMP, or/and heparin for 24 h, and observed oocyte maturation and analysed the relationship among them.

RESULTSWe cultured DO in the medium 199 for 24 h, and found 10 micromol/L progesterone had more significant effect than 5 micromol/L progesterone (2 h GVBD% P < 0.05, 8 h PB 1% P < 0.05), but had not more significant effect than 20 micromol/L progesterone. We found that 16 micromol/L antisense c-myb ODN significantly inhibited progesterone (10 micromol/L)-induced mouse germinal vesicle stage oocyte maturation in vitro (2 h GVBD% P < 0.05, 8 h PBI% P < 0.01). 1 x 10(-4) micromol/L dbcAMP, 100 microg/ml heparin could single significantly inhibited progesterone-induced mouse GV stage oocyte maturation in vitro (2 h PBI% all P < 0.01, 8 h PBI% all P < 0.01), and could enhanced the inhibition of 16 micromol/L antisense c-myb ODN (2 h GVBD% all P < 0.01, 8h PBI% all P < 0.01).

CONCLUSIONProgesterone, protooncogene c-myb,cAMP and calcium all pay important role in regulating oocyte maturation and the mechanism of progesterone, cAMP and calcium in regulating oocyte maturation may be through the expression of protooncogene c-myb.

Animals ; Cells, Cultured ; Genes, myb ; Meiosis ; Mice ; Mice, Inbred Strains ; Oocytes ; cytology ; drug effects ; Oogenesis ; Progesterone ; pharmacology

Child ; China ; Communicable Diseases ; Communicable Diseases, Emerging ; Humans ; Multicenter Studies as Topic

ObjectiveTo investigate the effect of cholinesterase inhibitor on endotoxin-induced brain injury in rabbits.Methods Twenty-one healthy male rabbits were randomly assigned into three groups ( n ＝ 7each):group sham operation (group S),lipopolysaccharide (LPS) group and cholinesterase inhibitor (tacrine hydrochloride,THA) group.LPS 200 μg/kg was intracerebroventricularly injected in LPS group,LPS 200μg/kgand tacrine hydrochloride 150 μg/kg were injected in THA group,while same volume of normal saline was injected in S group.Then blood and tissue samples were collected in different groups after 4 hours.Nuclear factor-kappa B (NF-κB) p65 activity of brain tissues was determined by using Western blot analysis.Tumor necrosis factor-alpha (TNF-α) levels in plasma,cerebrospinal fluid and brain tissues were measured using enzyme linked immunosorbent assay.The brain tissue's myeloperoxidase (MPO) activity and the ratio of wet to dry weight (W/D) were also analyzed.ResultsAs compared with S group,TNF-α level in plasma,cerebrospinal fluid and brain tissues,NF-κB p65 level,MPO activity and W/D ratio increased in LPS and THA groups (P < 0.05).When compared with LPS group,TNF-α level in plasma,cerebrospinal fluid and brain tissues,NF-κB p65 level,MPO activity and W/D ratio decreased in THA group ( P < 0.05 ).ConclusionCholinesterase inhibitor can attenuate the endotoxin-induced brain injury through inhibiting local inflammatory responses.

10.3969/j.issn.2095-4344.2013.23.026

AIMTo investigate the distribution of c-myb, an oncoprotein, in mouse oocytes-cumulus cell complex and sperm immunohistochemically.

METHODSTo study the effect of c-myb on mouse fertilization in vitro, various concentration of c-myb antisense-oligodeoxynucleotides (c-myb ASODNs) were incubated with sperms and oocytes during fertilization. To explore the possible mechanism involved in fertilization, the relationship between c-myb ASODNs and GABA or dbcAMP or Verapamil or Progesterone in fertilization was also observed by immunohistochemical methods.

RESULTSc-myb oncoprotein was observed on the nucleus of cumulus cell and head of sperm. c-myb ASODNs inhibited the rate of fertilization in vitro in a dose-dependent way. The fertilization rates of the control group, low (5 micromol/L), medium (10 micromol/L), high (20 micromol/L) concentration c-myb ASODNs groups and nonsense tat oligodeoxynucleotides (20 micromol/L) group were 34.97%, 30.89%, 20.14%, 16.68%, 34.47%, respectively. All of GABA, Progesterone and dbcAMP inversed the c-myb ASODNs inhibition effects on fertilization rate, but neither of them showed significant effect on the percentages of immunohistochemical stain of Myb on sperm and cumulus cells. By contrast, Verapamil inhibited the fertilization rate. Co-treated with c-myb ASODNs, Verapamil showed synergic inhibiting effects on the fertilization with c-myb ASODNs. Verapamil also inhibited the expression of Myb on head of sperm. The fertilization rates of the control group, medium (10 micromol/L) concentration c-myb ASODNs group, GABA group, P4 group, Verapamil group, dbcAMP group were 34.81%, 22.96%, 40.83%, 39.12%, 7.46%, 40.61%, respectively.

CONCLUSIONc-myb ASODNs is closely correlated with fertilization. Verapamil can inhibit fertilization in vitro through regulating Myb expression of sperm, while GABA, dbcAMP and Verapamil may affect the process of fertilization through the way other than Myb expression.

Animals ; Bucladesine ; pharmacology ; Female ; Fertilization ; physiology ; Fertilization in Vitro ; Male ; Mice ; Mice, Inbred Strains ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Oocytes ; physiology ; Proto-Oncogene Proteins c-myb ; metabolism ; Spermatozoa ; physiology ; Verapamil ; pharmacology ; gamma-Aminobutyric Acid ; pharmacology

BACKGROUND: The dynamic changes of pneumocyte apoptosis and aspartate-specific cysteine proteases-3 (caspase-3) expression in lung tissue of rats during the process of lung ischemia/reperfusion (I/R) injury and the possible action mechanisms remain unclear.OBJECTIVE: This study was to observe the dynamic changes of pneumocyte apoptosis and caspase-3 expression in the rat lung tissue during the process of lung I/R injury, and to analyze the role of pneumocyte apoptosis and the possible action mechanism.DESIGN: A randomized controlled animal experiment.SETTING: Emergency Center, First Hospital, Nanjing Medical University.MATERIALS: This study was carried out in the Animal Laboratory of the First Hospital of Nanjing Medcial University and Nanjing Center for Radioimmunity between April 2006 and September 2006. Twenty-eight male healthy SD rats of clean grade, with body weight of 250 to 350 g, aged 49 to 76 days, were provided by the Experimental Animal Center of Nanjing Medical University. The involved rats were randomized into experimental group and control group, with 14 rats in each.METHODS: ①Experimental intervention: Rats in the experimental group were created into models of lung I/R injury according to the method of Eppinger et al. They were occluded for 45 minutes at the porta of lung (no systolic and diastolic reactions in lung tissue being considered as successful occlusion), and then they were reperfused (recovery of systolic and diastolic function being considered as successful reperfusion); After that, lung tissues were harvested at 3 and 6 hours after lung I/R injury, 7 rats at each time point. Each rat in the control group was subjected to a thoracotony only, but lung tissues were isolated at the same time point by the same method. ②Experimental evaluation: Apoptotic cells in the lung tissue were detected with a flow cytometer by Annexin-V-PI staining, and apoptosis rate was calculated. Caspase-3 expression in the lung tissue was observed by immunohistochemical method and image analysis. Wet to dry weight ratio(W/D) of lung tissue of rats in the two groups was calculated; the number of injured pulmonary alveoli at I/R 3 hours/that at I/R 6 hours was calculated for quantitative evaluation of injured lung tissue; Patho-morphological changes of lung tissue were observed by haematoxylin & eosin staining under an optical microscope.MAIN OUTCOME MEASURES: ①Pneumocyte apoptosis rate and caspase-3 expression in the lung tissue. ②W/D of lung tissue and quantitative evaluation of injured lung tissue. ③Patho-morphological changes of lung tissue.RESULTS: Twenty-eight rats were involved in the final analysis, without deletion. ①Pneumocyte apoptosis rates in the experimental group at I/R 3 and 6 hours were significantly increased as compared with control group (P＜0.01). In the experimental group, pneumocyte apoptosis rate was decreased a little at I/R 6 hours than at I/R 3 hours (P＜0.05). ②Caspase-3 expression in the lung tissue of rats of experimental group reached its top at I/R 3 hours, and was decreased a little at I/R 6 hours. At each time point, caspase-3 expression in the experimental group was increased as compared with control group (P＜0.01). ③In the experimental group, the number of injured pulmonary alveoli at I/R 3 hours/that at I/R 6 hours and W/D ratios of lung tissues were significantly increased as compared with control group (P＜0.01). In the experimental group, two ratios at I/R 6 hours were higher than those at I/R 3 hours (P＜0.05).④In the experimental group, the structure of pulmonary alveoli was destructed, collapsed and disappeared; lots of inflammatory cell infiltration was found; Patho-morphological changes of injured lung tissue at I/R 6 hours were severer than those at I/R 3 hours. No obvious changes were found in the control group.CONCLUSION: At the early stage of lung I/R injury, the alteration of caspase-3 maybe activate pneumocyte apoptosis and induce the apoptosis of lung tissue, and thereby leads to lung injury.

Objective:To investigate the effects of methylprednisolone(MP)on pneumocyte apoptosis during lung ischemia/reperfusion injury in rats and to study the possible role of MP in pneumocyte apoptosis.Methods:Forty-two male Sprague-Dawley rats used for unilateral lung ischemia/reperfusion model were randomly divided into three groups:sham operation group(Sh group),ischemia/reperfusion group(I/R group),and methylprednisolone group(MP group).Each group has two subgroups of three hours and six hours.Apoptosis rate in lung tissue was detected by the way of Annexin-V-PI in flow cytometer.Expression of I?B-? in lung was observed by immunohistochemical stain.The index of quantitative assessment of histological lung injury(IQA),the wet to dry weight ratio(W/D),the pathological and ultrastructure changes of lung tissue were measured.Results:Apoptosis rate,W/D,IQA of lung tissue were significantly higher in I/R group than which in Sh group(P

Objective To observe the protective effect of silybin-phosphatidylcholine compound(SPC) on acute liver damage induced by alpha-naphthylisothiocyanate(ANIT) in mice.Methods Forty mice were randomly divided into 4 groups:normal group,model group,SPCⅠgroup,SPCⅡgroup.Intragastric administration of 0.5% carboxymethylcelluloes-Na(CMC-Na) suspension were given to the normal and model group,and 2 quantities of SPC suspension(150 and 300 mg/kg) were respectively given to SPCⅠ and SPC Ⅱ groups for 1 week.At 12 h after last administration of SPC,all of groups besides normal were given ANIT(dissolved in peanut oil,(60 mg/kg)) by lavage and the normal group just only were given peanute oil by the same volume and same way.After 16 h of ANIT administration,the levels of serum alanine transferase(ALT),aspartate aminotransferase(AST),serum total bilirubin(TB),malondialdehyde(MDA) and superoxide dismutase(SOD) were detected by biochemical method.Results Serum levels of ALT,AST,TB and MDA markedly increased after ANIT was administered via intragastric tube,and the level of serum SOD also decrease.SPC could obviously inhibit the alteration of the activities of serum ALT,AST,TB,MDA and SOD(P

Objective To establish an experimental subarachnoid hemorrhage(SAH)model with endo- cerebrovascular peroration.Method The right external carotid artery of SD rats were isolated,leaving a stump of approximately 3 to 4 mm.A-3-O monofilament nylon suture was inserted up through the stump of external carotid artery to the internal carotid artery for about 18～19 mm.A small resistance was usually felt,and the suture was then advanced 2 mm further and the suture was immediately withdrawn.Two hours or two days after SAH induction,SAH extension was observed.Two days after SAH induction,the diameter of the basilar artery was measured.Results SAH extends from the ipsilateral artery to the eontralateral artery after SAH induction.The diameters of basilar arteries in SAH animals were smaller than those of control rats,indicating the present of cerebrovascular spasm in SAH animals.Conclusions The endo-cerebrovascular perforation technique for establishing a non-craniotomy SAH model is reliable.