OBJECTIVE Autosomal dominant polycystic kidney disease (ADPKD) is a common-monogenetic disease characterized by progressive development of renal cysts. Thereis still further need for effective therapy.Based on our precious study that Ganoderma triterpenes(GT),which is the major secondary metabolites of Ganoderma lucidum,is able to attenuate renal cyst development.The aim of this study was to investigate the effect of a monomer,Ganoderic acid A(GA-A)that was purified from the GT,which has been reported to exhibit antinociceptive,antioxidative,hepatoproctive and anti-cancer activities,to have a potent anti-cyst effect in ADPKD. METHODS We first evaluated the potential cytotoxicity of GA-A on MDCK cells using a CCK-8 assay.Then we used MDCK cyst model,cultivated MDCK cells in vitro to form fluid-filled cysts surrounded by monolayer cells.MDCK cells were co-incu-bated with 10 μmol·L-1FSK with or without GA-A(25 μg·mL-1)and equal concentration GT as positive control from day 0 to day 6 to investigate the inhibitory effect of GA-A on cyst formation.And to further investigate the inhibitory effect of GA-A on cyst enlargement, MDCK cysts were treated with different concentration of GA-A(6.25,25 and 100 μg·mL-1)from day 5 to day 12.Next we used an embryonic kidney cyst model, wile-type mice kidneys were taken out on embryonic day 13.5 to form renal cysts stimulated with 8-Br-cAMP to prove the renal cyst inhibition at organ level.Meanwhile,we explored the possible mechanisms underlying GA-A inhibition on renal cyst development using MDCK cells treated with 10 μmol·L-1FSK co-incubated with GA-A(25 μg·mL-1)and equal concentration GT.Several key components of Ras/MAPK pathway was evaluated by Western blot,the protein expression of H-ras,B-raf, p-ERK, Egr-1 and c-fos was evaluated. RESULTS MDCK cell viability was not affected by GA-A that were used ofincreasing concentrations up to 200 μg·mL-1. GA-A had no significant influence on cyst formation,but inhibited cyst enlargement dose-dependently and the inhibitory effect is significantly better than that of the same concentration of GT which proved that GA-A may be an effective monomer from GT.And after washing out GA-A on day 8,MDCK cysts regrew to a large size,suggesting that the inhibitory effect of GA-A on MDCK cyst enlargement was reversible. GA-A inhibited embryonic kidney cyst development significantly in a dose-dependent and reversible manner proving GA-A cyst inhibition at organ level,which is also more effective than equal concentration GT.Treatment of MDCK cells with FSK caused a significant elevation of H-ras,B-raf,p-ERK,Egr-1 and c-fos signaling molecules,while treatment with GA-A reduced the level of H-ras,B-raf,p-ERK,Egr-1 and c-fos expression.GA-A down-regulated Ras/MAPK signaling pathway could contribute to its inhibitory effect on cyst development. CONCLUSION Ganoderic acid A from Ganoderma lucidum retard ADPKD renal cyst development via down-regulating Ras/MAPK signaling pathway.

[Objective] To explore the cytoprotecfion of hydrogen sulfide (H_2S) against cobalt chloride (CoCl_2)-induced apeptosis in PC12 cells and the underlying mechanisms. [Methods] CoCl_2 (a chemical hypoxia-mimetic agent) was used to establish the chemical hypoxia-induced PC12 cell injuries model. Sodium hydrosulfide (NaHS) was used as a H_2S donor. The viability of PC12 cells was measured by CCK-8 assay. The percentage of apeptotic cells was assessed by propidium iodide stain flow cytometry (FCM). The morphological change of apeptotic cells was tested by using the chromatin dye Hoechst 33258. The mitochondrial membrane potential (MMP) was analyzed by rhodamine 123 staining and photofluorography. The level of reactive oxygen species (ROS) in PC12 cells was measured by DCFH-DA staining and photofluorography. [Results] CoCl_2 induced a decrease in cell viability and an increase in percentage of apeptosis in PC12 cells along with dissipation of MMP as well as overproduction of ROS. When PC12 cells were treated with Naris 30 min before CoCl_2 treatment a decrease in viability of PC12 cells induced by 600 μmol/L CoCl_2 was concentration-dependently blocked by NaHs (100, 200, and 400 μmol/L). Pretreatment with NaHS at 200 and 400 μmol/L obviously reduced the apepetotic percentage of PC12 cells induced by 600 μmol/L CoCl_2 and inhibited the dissipation of MMP and overproduction of ROS. [Conclusion] H_2S protected PC12 cells against CoCl_2-induced apeptosis, which may be associated with the inhibition of H_2S on the dissipation of MMP and overproduction of ROS induced by CoCl_2.

Objective To observe the effects and mechanism of lung inflation with carbon monoxide (CO) during the cold ischemia phase on lung ischemia-reperfusion injury (IRI) after rat lung transplantation.Method Twenty-four pairs of SD rats were selected to establish the model of lung transplantation,and random number method was used to divide 24 donors into 3 groups with 8 rats in each group.(1) CO inflation group (CO group):During the cold ischemia phase,500 ppm CO +volume fraction 40％ O2 + N2 was used for lung inflation,and the volume was 5 mL/kg;(2) O2 inflation group (O2 group):During the cold ischemia phase,volume fraction 40％ O2 + volume fraction 60％ N2 was used for lung inflation;(3) Control group:The lung was deflated during the cold ischemia phase.The gas was replaced every 30 min in the CO and O2 groups,and the lung transplantations were performed after 180 min of cold ischemia.The arterial blood gas analysis was performed at baseline,3 min after reperfusion,and 60,120,and 180 min after reperfusion.The recipient serum levels of relative inflammatory factors,lung tissue cell apoptosis and nuclear factor kappa B (NF-κB) protein expression were detected after 180 min of reperfusion.Result As compared with the control group (238 ± 61 mm Hg),the oxygenation index in the O2 group (293 ± 78 mm Hg) and CO group (361 ± 48 mm Hg) was increased (P＜0.05),and as compared with the O2 group,that in the CO group was increased (P＜0.05).Furthermore,as compared with the control group,the interleukin (IL)-8,tumor necrosis factor (TNF)-α,and cell apoptosis in the O2 group and CO group were decreased significantly,and as compared with the O2 group,those in the CO group and NF-κB protein expression were significantly decreased (P＜0.05).Conclusion Lung inflation with CO during the cold ischemia phase ameliorated the rat lung IRI via reducing the inflammatory response and cell apoptosis mediated by the NF-κB pathway.

Objective: To optimize the determination of pentachlorophenol in wooden chopping boards through pretreatment of miniaturized samples. Methods: The pretreated wooden chopping board samples were subjected to ultrasound extraction (1 mL of 0.5 mol/L K2CO3 added in 5 mL extraction solution) in 8 mL acetone and 2 mL water, followed by derivatization with 0.3 mL acetic anhydride, extraction with n-hexane and separation with DB-5ms column (30 m×0.25 mm, 0.25 μm). Gas chromatography tandem mass spectrometry (GC-MS/MS) was performed in multiple reaction monitoring (MRM) mode with quantitative analysis using the internal standard method. Results: The GC-MS/MS assay showed a good linear relationship within the range of 0.01 to 0.2 µg/mL (R2>0.999), with a 0.003 mg/kg limit of detection and 0.01 mg/kg limit of quantitation. The mean recovery rates were 84.2% to 96.7% at spiked concentrations of 0.003, 0.01 and 0.03 mg/kg, with relative standard deviation of 2.2% to 6.1%. Conclusions The established GC-MS/MS assay is easy to perform, environment-friendly, highly accurate and sensitivity, which is feasible for determination of pentachlorophenol in wooden chopping boards.

Objective:To analyze the effects of espO gene knockout on the biological characteristics of enterhemorrhagic Escherichia coli (EHEC). Methods:Two-step methods mediated by the suicide plasmid pCVD442-Δ espO and plasmid pTrc99a were used to construct the espO gene-deleted strain (Δ espO) and the complemented mutant (CΔ espO), respectively. HeLa cells were infected with different EHEC strains to analyze the biological functions and lethal effects of espO gene during infection. Results:PCR, electrophoresis and gene sequencing showed that the Δ espO and CΔ espO mutants were successfully constructed. Compared with the wild-type strain, neither the Δ espO nor CΔ espO mutant showed significant difference in growth rate, indicating that the espO gene had no influence on the growth and replication of EHEC. Furthermore, EspO could activate the tumor necrosis factor receptor (TNF)-induced NF-κB signaling pathway, while the effector protein NleB could inhibit the process. EspO could not inhibit the death of HeLa cells induced by TNF or TNF-related apoptosis-inducing ligand (TRAIL) after EHEC infection. Conclusions:In this study, we successfully constructed the espO gene-deleted and complemented mutants of EHEC and preliminarily analyzed the interaction between espO gene and host cells and the effects of espO gene on cell apoptosis during infection, which provided reference for further research on the in vitro biochemical activity and in vivo pathogenic roles of EspO.

Objective To detect the expressions of nm23-H1 and heat shock protein 27 (HSP27) and their clinical significance on development and metastasis in non-small cell lung carcinoma (NSCLC).Methods 75 tumor tissues from patients with NSCLC were included as experimental group and 28 pulmonary benign lesion tissues were as control group.The expressions of nm23-H1 and HSP27 in patients with different clinical and pathological characters were detected by immunohistochemistry.Results nm23-H1 and HSP27 were mainly expressed in cytoplasm,the positive rates of nm23-H1 and HSP27 were significantly higher in the experimental group than that in control group [41.3 ％ (31/75) vs 7.1％ (2/28),x2 =10.946,P =0.001,80.0 ％ (60/75) vs 46.4 ％ (13/28),x2 =11.131,P =0.001].Compared with control group,the positive rate of HSP27 was correlated with the degree of tumor differentiation (x2 =4.191,P =0.041).nm23-H1 was related with HSP27 in lung cancer (r =0.284,P =0.013).Conclusion nm23-H1 and HSP27 are related to the occurrence and development of NSCLC.The joint detection of nm23-H1 and HSP27 should be helpful to the diagnosis and judge the biological behavior of NSCLC.

Objective To valuate the left ventricular function and myocardial fibrosis using Dopple tissue imaging(DTI) and integrated backscatter(IBS) in diabetic cardiomyopathy(DCM) rats. Methods Diabetes mellitus(DM) in healthy male SD rats was induced by single injection of streptozotocin (STZ,Sigma) into intraperitoneal at a dose of 65mg/kg body weight. Left ventricular fractional shortening (LVFS),ratio of peak early to late diastolic mitral inflow velocity(E/A) by color Doppler flow imaging-pulsed wave (CDFI-PW), ratio of peak early to late diastolic mitral annulus velocity (Ea/Aa) by DTI,myocardial calibrated IBS (IB%), cyclic variation of IBS(CVIB) by IBS, were determined in rats of 4,12 and 24 weeks after DM was induced. Heart weight index(HWI), Masson's trichrome staining, expression of connective tissue growth factor(CTGF) and activity of sarcoplasmic reticulum Ca2+ -ATPase(SERCA2a) in heart were measured, while the age-matched health rats served as control group. Results ① Diastolic dysfunction of left ventricular was found in diabetic rats detected by CDFI-PW(E/A<1) at the 4th week,and developed gradually. Pseudonormal filling(E/A>1) was found in major diabetic rats at the 24th week,which could be identified by DTI(Ea/Aa<1). The IB% of left ventricular posterior wall was significantly higher in diabetic rats than those in the control group( P<0.05), but the CVIB of the 24th week diabetic rats was lower(P<0.05). The LVFS had no great alteration in six groups(P>0.05).②Compared with the control group, the activity of SERCA2a was reduced, while HWI, collagen volume fraction(CVF),perivascular collagen area(PVCA) and the expression of connective tissue growth factor(CTGF) protein in heart were increased in diabetic rats (P<0.05), and correlation between IB% and HWI, CVF, PVCA,CTGF was significant (P<0.05). Conclusions DTI and IBS can assess early left ventrieular diastolic function and fibrosis of diabetic cardiomypathy in STZ-induced diabetic rats. DTI echoeardiography can identify "pseudonormal" mitral inflow patterns. DCM may be relatived with overexpression of CTGF and decreasion of SERCA2a activity.

Objective To conduct a case-control study on the association of the nucleotide polymorphisms in the promoter region of the matrix metalloproteinase-9 (MMP-9) gene with phenotype of esophageal cancer. Methods All subjects were unrelated residents in northern regions of China. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used to determine the MMP-9 genotypes. Results The overall distribution of genotypes in the patients was not different from that in the controls (OR=0.77, 95% CI=0.45-1.34; P=0.36). There were no significant differences between the patients and the control subjects in terms of the distributions of sex and age, smoking status, alcohol dependence, pickled diet status, or history of environmental exposure. The patients were further examined with stratifications by age, sex, grade, depth of tumor invasion, lymphatic invasion, venous invasion and TNM staging. The results showed no pronounced association among the stratifications. Conclusion There is no significant association between the MMP-9 single nucleotide polymorphism genotypes and phenotype of esophageal cancer.

Objective To construct the prokaryotic recombinant expression vector of PTD4-Cu,Zn-SOD.Methods By using the techniques of gene recombination,the primers of Cu,Zn-SOD and the oligonucleotide sequences of PTD4 were designed,PCR amplification was performed for Cu,Zn-SOD genes,the PCR products were identified,reclaimed and purified,and pET16b served as carrier.The prokaryotic recombinant expression vector of pET16b-Cu,Zn-SOD was constructed using double digestion with Xho Ⅰ and BamH Ⅰ,ligated reaction and plasmid transformation.Then PTD4 gene and pET16b-Cu,Zn-SOD carrier were double digested with Nde Ⅰ and Xho Ⅰ and ligated,and the plasmid was transformed,and the prokaryotic recombinant expression vector of pET16b-PTD4-Cu,Zn-SOD was constructed.The reconstructed vector was analyzed by restriction mapping and was verified by gene sequencing.Results The prokaryotic recombinant expression vector of pET16b-PTD4-Cu,Zn-SOD with a length of 6 207 bp was constructed successfully.The carrier fragment about 5.7 kp and PTD4-Cu,Zn-SOD gene fragment about 510 bp were obtained by double digestion with Nde Ⅰ and BamH Ⅰ,which was consistent with the expected results.The results of gene sequencing showed that the base sequences of pET16b-PTD4-Cu,Zn-SOD were correct when compared with the expected gene sequences.Conclusion The prokaryotic recombinant expression vector of pET16b-PTD4-Cu,Zn-SOD is constructed successfully.

In order to enhance the specificity of TNF-α monoclonal antibody to inflamed site, a bispecific antibody BsDb that targets TNF-α and the extra-domain B (ED-B) of fibronectin (FN) was constructed by covalently linking the anti-TNF-α single chain Fv antibody (TNF-scFv) and the anti-ED-B scFv L19 via a flexible peptide linker deriving from human serum albumin (HSA). ED-B is an antigen specifically expressed at the inflamed site. BsDb is expressed in E. coli, identified by immunoblot, and purified with affinity chromatography. This was followed by further examination of its bioactivities and pharmacokinetics. We demonstrated that BsDb retained the immunoreactivity of its original antibodies as it could simultaneously bind to TNF-α and ED-B and neutralize the biological action of TNF-α. In the collagen-induced arthritis mice model, BsDb selectively accumulate in the inflamed joint with a maximal uptake of (12.2 ± 1.50)% ID/g in a single inflamed paw and retain in the inflamed paw for at least 72 h. In contrast, BsDb showed a short serum half-life of (0.50 ± 0.05) h and a rapid clearance from normal tissues. The findings reported herein indicate that BsDb has good specificity to the inflamed site and low toxicity to normal tissues. BsDb is therefore likely to have greater clinical applications in the treatment of rheumatoid arthritis and other autoimmune diseases. This laid a stable basis for its preclinical study.
Animals ; Antibodies, Bispecific ; chemistry ; Antibodies, Monoclonal ; chemistry ; Arthritis, Experimental ; Escherichia coli ; Fibronectins ; chemistry ; Half-Life ; Humans ; Mice ; Single-Chain Antibodies ; chemistry ; Tumor Necrosis Factor-alpha ; chemistry