ObjectiveTo investigate the expression of heme oxygenase-1 (HO-1) in lung tissue of mice with acute paraquat poisoning, and discuss its pathological mechanism.Methods Fifty-eight healthy male mice were randomly divided into control group (n = 8) and poisoned group (n = 50). The mice in poisoned group were lavaged with 20% paraquat (50 mg/kg), and those in control group with equal amount of normal saline. The mice were sacrificed on the day of experiment in control group, and those in poisoned group at 6 hours and 1, 3, 7, 14 days after poisoning. The lung tissue was harvested to observe the changes in pathology of lung with hematoxylin and eosin (HE) staining. The positive expression of HO-1 was determined with immunohistochemistry, and the protein expression of HO-1 was determined with Western Blot. The contents of superoxide dismutase (SOD) and malonaldehyde (MDA) were determined.Results The mice showed shortness of breath and signs of exhaustion 1 hour after poisoning, getting worse on 3-5 days, but returned to normal 14 days after poisoning. Under the light microscope, it showed that the control group had no significant pathological changes in lung tissue. One day after the ingestion, pulmonary alveolar structure disorder, obvious hemorrhage, edema and infiltration of inflammatory cells were found. At 3 days, the pathological changes in the lung tissue were more pronounced. They were less pronounced on 7 days, and inflammatory changes disappeared on 14th day, but alveolar structure disorder remained. Immunohistochemical test showed that HO-1 was seldom expressed in the lung tissue, and a little amount was expressed in the mucosal epithelial cells of the airway in control group. It was shown that inflammatory cell and endothelial were mainly distributed in the mucosal epithelial cells of airway 1 day after poisoning followed by a gradually decrease tendence, and came to normal level of control group 7 days after poisoning. It was shown by Western Blot that HO-1 (gray value) in lung tissue increased 6 hours after poisoning (2.438±0.467 vs. 0.475±0.167,P< 0.01), peaked at 1 day (9.200±0.940 vs. 0.475±0.167,P< 0.01), continued to increase till 7 days after poisoning, and it lowered to normal level thereafter (0.825±0.260 vs. 0.475±0.167,P> 0.05). The SOD activity (μU/L) in lung tissue was lowered 6 hours after poisoning, and it was significantly lower than that of control group (649.681±13.951 vs. 1 167.051±15.744,P< 0.01), and it continued to decrease up to 14 days after poisoning (859.733±121.079 vs. 1 167.051±14.744,P< 0.01). MDA content (μmol/L) in the lung tissue homogenate was elevated 6 hours after poisoning with significant difference compared with that of the control group (4.542±0.266 vs. 3.705±0.176,P< 0.01). It peaked on day 1 (5.956±0.281 vs. 3.705±0.176,P< 0.01), then it declined and reached normal level 3 days after poisoning (4.134±0.168 vs. 3.705±0.176,P> 0.05).Conclusion HO-1 expression was increased significantly in lung tissue of mice with acute paraquat poisoning, which may be considered as an important protection mechanism against paraquat poisoning.

Objective To construct eukaryotic expression plasmid of hCD137 Fc gene and express hCD137 Fc fusion protein with high biological activity. Methods PCR technique was employed to clone the human CD137 cDNA from a normal human activated T cells cDNA library, and then clone its extramembrane encoding region. The extramembrane sequence together with human IgG1 Fc cDNA were inserted into the eukaryotic expression plasmid pcDNA3. CD137 Fc gene was expressed transiently in 293T cells, and then CD137 Fc protein was purified by recombinated protein A affinity chromatography column. At last, the MW, purity and antigenicity of CD137 Fc were identified by sandwich ELISA, SDS PAGE and Western blotting, respectively. Results The ORF of CD137 Fc gene was coincident with what we expected. ELISA and SDS PAGE confirmed protein expression in 293T cells. Western blotting proved the antigenicity of the purified CD137 Fc protein. Conclusion We obtained a purified recombinated CD137 Fc protein with biological activity and the expected MW. This lays the foundation for further studies of CD137 such as its role in immune homeostasis and other biological functions.

Objective To clone the full length cDNA of human LIGHT and to construct the recombinant eukaryotic expression plasmid pCI neo LIGHT for the stable expression on 293T cells. Methods Human LIGHT cDNA was cloned from a normal human activated T cell cDNA library phAD.CAD by PCR. After sequencing, LIGHT cDNA was inserted into plasmid pCI neo for the construction of the eukaryotic expression. The recombinant was transfected into 293T cells by electroporation. The expression of LIGHT on the surface of 293T cells was detected by flow cytometry after screening with G418. Results Sequencing confirmed that ORF of LIGHT gene was intact and right. Restrictive enzyme digestion proved that LIGHT gene was inserted into the recombinant plasmid of LIGHT pCI neo correctly. FACS analysis revealed that about 78.69% 293T cells expressed LIGHT protein on the cell surfaces at 3 months after screening with G418. Conclusion LIGHT gene has been cloned successfully, and a 293T cell line expressing LIGHT protein on its membrane surface has been obtained.

Objective To investigate the p53,Bax,bcl-2 gene in NaAsO2-induced human embryonic lung fibroblasts(HELF)apoptosis.Methods HELF was divided into HELF cells transfected with p53 plasmid(p53 group),HELF cells transfected with PC plasmid(PC group)and normal cultured HELF cells(normal group).The mRNA expression of p53,Bax and bcl-2 gene was detected by real-time PCR,the protein expression of p53,Bax and bcl-2 was assessed by immunohistochemical SABC and the cell apoptosis of HELF was detected by flow cytometry(FCM),in a 6-well plate and cultured for 48 hours,which was exposed to different doses(0,3,9,15mmol/L)NaAsO2 for 24 hours.Results The p53 gene mRNA expression level of p53 group(0.51±0.29)was lower than that of the normal group and PC group [ (1.00 ± 0.20), (1.32 ± 0.26), all P < 0.05 ]. The p53 protein expression level of p53 group(4.10 ± 1.20) was lower than the PC group and normal group[ (8.00 ± 1.63), (7.90 ± 1.79), allP < 0.05]. In p53 group, PC group, normal group exposed to 0,3,9,15 mmol/L NaAsO2 doses, the apoptotic rate [(0.57 ± 0.28)%, (22.91 ± 4.86)%, (40.05 ± 3.93)%, (44.87 ± 3.58)%; (0.65 ± 0.24)%, (14.09 ± 3.49)%,(20.31 ± 3.66)%, (32.42 ± 3.63)%; (0.56 ± 0.25)%, (12.14 ± 3.70)%, (19.61 ± 3.63)%, (30.43 ± 2.83)%], Bax mRNA expression level[(12.73 ± 3.96), (25.12 ± 6.42), (104.96 ± 26.77), (154.04 ± 30.52); (14.63 ± 3.57),(36.75 ± 3.67), (272.26 ± 66.11), (846.12 ± 243.36); (14.75 ± 5.65), (37.22 ± 11.27), (278.51 ± 37.42),(861.67 ± 369.29) ], Bax protein expression level [ ( 15.07 ± 0.83 ) %, ( 23.79 ± 3.99 ) %, (38.51 ± 1.58 ) %, (53.86 ±1.74)%;(15.43 ± 1.45)%,(36.11 ± 1.37)%, (56.86 ± 1.97)%, (76.09 ± 2.01)%; (15.20 ± 1.03)%,(35.25 ±1.09)%, (55.56 ± 2.17)%, (74.48 ± 2.85)% ] was respectively increased in a dose-dependent manner with the increased concentration of NaAsO2(all P < 0.05). The bel-2 mRNA expression level [ (443.00 ± 244.47), (156.79 ±53.18), (62.13 ± 13.66), (23.10 ± 6.44); (420.55 ± 110.77), (48.15 ± 10.02), (14.91 ± 6.53), (7.54 ± 2.62);(577.75 ± 123.22), (49.68 ± 10.11), (12.41 ± 1.28), (7.22 ± 1.89)], bcl-2 protein expression level[(47.20 ±3.77)%, (41.80 ± 2.94)%, (36.00 ± 2.36)%, (29.00 ± 2.91)%; (45.90 ± 4.15)%, (35.70 ± 2.77)%, (29.80 ±2.78)%, (24.80 ± 2.66)% ; (46.70 ± 3.47)%, (36.20 ± 2.90)%, (30.10 ± 3.21)%, (25.10 ± 2.28)% ] wasdecreased in a dose-dependent manner with the increased concentration of NaAsO2(all P < 0.05 ). In 3,9,15 mmol/L NaAsO2, apoptotic rate of p53 group, mRNA expression of bcl-2, protein expression of bcl-2 was higher than that ofnormal group and PC group, respectively (all P < 0.05), but mRNA expression of Bax, protein expression of Bax was respeetivelylower than that normal group and the PC group(P < 0.05 ). Conclusion p53 gene reduced the apoptosis induced by NaAsO2 in HELF, possibly by changing the apoptosis pathway.

With homology cloning approaches coupling with RACE (rapid-amplification of cDNA ends) techniques, the full-length coding sequence of pathogenesis-related protein PR10-1 with differential expression was cloned from the total RNA of the root of Panax notoginseng, and its function was explored furtherly. As a result, the longest 465 bp ORF (named as PnPR10-1 with the Accession No. KJ741402 in GenBank) was detected from the cloned sequence with full-length of cDNA of 863 bp. The corresponding peptide encoded consisted of 155 amino acids, contained some domains such as Bet-v-I, and showed high similarity with that from Panax ginseng by analysis of phylogenetic trees created from the alignments. Real-time quantitative PCR showed that the expression of PnPR10-1 gene was constitutive in different tissues of 1-3 year old plant, suggesting that it might be involved in growth, development, and secondary metabolism; yet it was up-regulated significantly with the infection of Fusarium oxysporum in root, suggesting that it might be involved in defense against many diseases including root rot in P. notoginseng.
Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; Genes, Plant ; Glycoside Hydrolases ; genetics ; Molecular Sequence Data ; Open Reading Frames ; Panax notoginseng ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Plant Roots

Objective To develop an interlocking-style vascular shunt device for the treatment of distal limb ischemia resulting from vascular disconnection and defect.Methods A one-way interlocking buckle was designed with the space between the clamping teeth being 0.5 mm,which prevented the device from moving backwards and fixed the vessel and shunt tube conveniently.The interlocking buckle combined with silicone tube was used to connect the two ends of the defected vessel,which was compared with conventional method by suture ligation and silicone tube by the tests on vessel bursting pressure and tensile biomechanics.Results The vessel repaired with the developed device behaved better than that by the conventional method in the tests on vessel bursting pressure and tensile biomechanics (P＜0.05).Conclusion The vascular shunt device can be used for the treatment of distal limb ischemia resulting from vascular disconnection and defect,and thus facilitates the vascular graft in rear hospital after evacuation.

Aged ; Epidural Abscess ; complications ; Female ; Humans ; Methicillin-Resistant Staphylococcus aureus ; Osteomyelitis ; complications ; Paralysis ; etiology ; Spinal Diseases ; complications ; Staphylococcal Infections ; complications ; Thoracic Vertebrae

AIM:To observe the influence of erythropoietin (EPO) on eryptosis and production of reactive oxygen species (ROS) in erythrocytes under stimulation of hydrogen peroxide (H2O2),.and to explore its related mecha-nism. METHODS:The erythrocyte suspension (1%) was cultured in vitro and divided into 3 groups:control group (C group,the culture medium was PBS),H2O2group (H group,the culture medium was PBS containing H2O2at final con-centration of 100 μmol/L) and EPO group (E group,the culture medium was PBS containing H2O2at final concentration of 100 μmol/L and EPO at final concentration of 2×104U/L). The erythrocytes were collected at 24 h and 60 h. The eryptosis was detected by flow cytometry with Annexin V staining. The production of ROS and intracellular calcium ion con-centration (Ca2+]i) were also analyzed by flow cytometry. RESULTS:The eryptosis in C group was increased as the in-cubating time extended. The eryptosis in H group was higher than that in C group (P<0.01),while that in E group was lower than that in H group(P<0.01). Meanwhile,ROS production andCa2+]iwere higher in H group than those in C group (P<0.01), but those were lower in E group than those in H group (P<0.05 or P<0.01). CONCLUSION:EPO inhibits eryptosis induced by H2O2and its mechanism may be related to antioxidant effect and change of Ca2+]i.

Objective To investigate the association of angiotensinogen(AGT)gene A-6G、T174M and G-217A polymorphisms with the risk of essential hypertension(EH)in the elderly of Han nationality.Methods Genotypes of AGT gene A-6G,T174M and G-217A polymorphisms in 177 aged EH patients and 86 sex and age-matched controls were analyzed with gene chip technology.Results The A-6G and T174M polymorphisms of AGT gene were significantly associated with EH.The numbers of the three genotypes of A-6G were 113,58 and 6 in the patient group and 70,15 and 1 in the control group(P= 0.014)and those of T174M were 94,77 and 6,60,25 and 1(P=0.031),respectively.G-217A polymorphism was not related to EH.Individuals carrying A-6G AA and T174M CC genotypes showed 57% and 56% lower risk of EH(OR=0.43;95%CI=0.23-0.82 and OR=0.44;95%CI=0.25-0.79, respectively).Conclusions The A-6G AA and the T174M CC genotype may be related with decreased risk of EH and G-217A polymorphism may have little role in the etiology of EH in Han nationality.

OBJECTIVETo investigate the possible protection provided by oral quercetin pretreatment against hepatic ischemia-reperfusion injury in rats.

METHODSThe quercetin (0.13 mmol/kg) was orally administrated in 50 min prior to hepatic ischemia-reperfusion injury. Ascorbic acid was also similarly administered. The hepatic content of quercetin was assayed by high performance liquid chromatography (HPLC). Plasma glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) activities and malondialdehyde (MDA) concentration were measured as markers of hepatic ischemia-reperfusion injury. Meanwhile, hepatic content of glutathione (GSH), activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and xanthine oxidase (XO), total antioxidant capacity (TAOC), contents of reactive oxygen species (ROS) and MDA, DNA fragmentation were also determined.

RESULTSHepatic content of quercetin after intragastric administration of quercetin was increased significantly. The increases in plasma GPT, GOT activities and MDA concentration after hepatic ischemia-reperfusion injury were reduced significantly by pretreatment with quercetin. Hepatic content of GSH and activities of SOD, GSH-Px and TAOC were restored remarkably while the ROS and MDA contents were significantly diminished by quercetin pretreatment after ischemia-reperfusion injury. However, quercetin pretreatment did not reduce significantly hepatic XO activity and DNA fragmentation. Ascorbic acid pretreatment had also protective effects against hepatic ischemia-reperfusion injury by restoring hepatic content of GSH, TAOC and diminishing ROS and MDA formation and DNA fragmentation.

CONCLUSIONIt is indicated that quercetin can protect the liver against ischemia-reperfusion injury after oral pretreatment and the underlying mechanism is associated with improved hepatic antioxidant capacity.

Administration, Oral ; Animals ; Antioxidants ; pharmacokinetics ; therapeutic use ; Ascorbic Acid ; pharmacokinetics ; therapeutic use ; Biological Availability ; Biomarkers ; blood ; DNA Fragmentation ; Glutathione Peroxidase ; metabolism ; Liver ; blood supply ; drug effects ; enzymology ; Male ; Malondialdehyde ; blood ; Quercetin ; pharmacokinetics ; therapeutic use ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Reperfusion Injury ; blood ; enzymology ; metabolism ; prevention & control ; Superoxide Dismutase ; metabolism ; Transaminases ; blood ; Xanthine Oxidase ; metabolism