Objective To observe the difference of lung injury between toll-like receptor 4 (TLR4) mutant mice and wild type mice in a model of lipopolysaccharide (LPS) induced acute lung injury (ALI),discuss the role of TLR4 in ALI.Methods Different doses of LPS solution (1,5mg?kg~(-1)) were injected in vein tail to reproduce ALI model in both TLR4 mutant (TLR4~(-/-)) and wild type (WT,TLR4~+(/+)) mice.Lung tissues were collected for gross and micrographic histological injury analysis and for assessment of lung edema.Meanwhile,the levels of myeloperoxidase (MPO) in lung tissues in both strains were assessed to evaluate the extent of polymorphological neutrophils (PMN) infiltration.Results The gross and micrographic injury of lung was milder in TLR4 mutant mice than that in wild type mice.The extent of lung edema (W/D) was also reduced compared with wild type mice,especially in 5 mg?kg~(-1) group [(4.08?0.1)vs.(4.55+0.2),n=10,t=12.71,P＜0.01].With high dosage of LPS,the value of W/D in both mice strains was higher than that in sham operation group (P＜0.01).The extent of PMN infiltration in lung tissues in TLR4 mutant mice was reduced compared with wild type mice.But they were higher than sham operated mice (P＜0.01).Conclusion TLR4 May involve in the development of ALI,by sequestration of PMN into lung tissues.

Objective Calcium-sensing receptor(CaSR)belongs to the family C of G-protein coupled receptors.This study was carried out to observe the influence and mechanism of CaSR on anoxia/reoxygenation(A/R)-induced cardiomyocytes apoptosis.Methods The model of A/R injury was established through anoxia for 2 hours and reoxygenation for 24 hours in cultured cardiomyocytes of neonatal rats.Cardiomyocytes were randomly divided into three groups:control group,A/R group and GdCl3 group(300μmol/L GdCl3 was added to the culture medium at the beginning of reoxygenation).Apoptosis of cardiomyocytes was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL).The expression of CaSR,cysteine-requiring aspartate protease(caspase)-3,9 and cytochrom c (Cytc)were analyzed by Western blot.Results The TUNEL showed that cardiomyocytes apoptosis was 17%±3% in A/R group,and was higher than that in the control group.At the same time,expression of CaSR in A/R group was markedly increased in response to control group.Compared with A/R group,GdCl3,a specific activator of CaSR,further enhanced cardiomyocytes apoptosis to (28±4)% and decreased the ability of cardiomyocytes to (51.2±6.8)%,along with an increment in CaSR,caspase-3,caspase-9 and Cytc expressions.Conclusion CaSR is involved in anoxia/reoxygenation-induced apoptosis of neonatal rat cardiomyocytes through Cytc-caspase-3 pathway.

The relationship between M3 cholinergic receptor agonist (carbachol) hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen activation or NF-kappaB activation in rats was studied in vitro. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc), and NF-kappaB inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The cellular injury was evaluated by measuring the leakage of LDH from pancreatic acinar cells. The results showed that as compared with control group, 10(-3) mol/L carbachol induced a significant increase of the intracellular trypsin activity and the leakage of LDH from pancreatic acinar cells. Pretreatment with 2 mmol/L pefabloc could significantly decrease the activity of trypsin and the leakage of LDH from pancreatic acinar cells (P < 0.01) following the treatment with a high concentration of carbachol (10(-3) mol/L) in vitro. The addition of 10(-2) mol/L PDTC didn't result in a significant decrease in the activity of trypsin and the leakage of LDH from pancreatic acinar cells treated with a high concentration of carbachol (10(-3) mol/L) in vitro (P > 0.05). It was concluded that intracellular trypsinogen activation is likely involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro. NF-kappaB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro.
Carbachol/*pharmacology ; Cholinergic Agonists/pharmacology ; NF-kappa B/*metabolism ; Pancreas/metabolism ; Pancreas/*pathology ; Rats, Wistar ; Receptor, Muscarinic M3/agonists ; Trypsinogen/*metabolism

The effects and mechanisms of exogenous phosphatase and tensin homolog deleted from chromosome ten (PTEN) gene on phosphatase activity-dependent apoptosis of breast cancer cell line MDA468 were investigated. PTEN gene packaged with lipofectin was transferred into breast cancer cell line MDA468 and parental MDA468 cells served as controls. RT-PCR and Western blot were done to detect the expression of target genes. The expression of phosphospecific protein kinase B (PKB/Akt) and focal adhesion kinase (FAK) protein stimulated by epidermal growth factor (EGF) was also detected. Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI. MDA468 cells transfected with PTEN gene could express PTEN mRNA and protein. PTEN decreased the phosphorylation level of AKT protein and down-regulated FAK protein expression in MDA468 stimulated by EGF. The apoptosis rate was 21.68%. PTEN induced breast cancer apoptosis phosphatase activity-dependently. The mechanism is possibly related with phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB)/AKT signaling pathway. Those results may provide new clues on the gene therapy in breast cancer.

Over-expression of P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, represents one of the major mechanisms that contribute to multidrug resistance (MDR) in cancer cells. This study examined the effects of troglitazone, a ligand of peroxisome proliferator-activated receptor gamma (PPARgamma), on P-gp-mediated MDR in SGC7901/VCR cells (a vincristine-resistant human gastric cancer cell line). The expression of P-gp was detected by RT-PCR and Western blotting, respectively. The SGC7901/VCR cells were treated with 0.1 mg/L vincristine (VCR) alone or in combination with 1, 5, 10 mumol/L troglitazone for 24 h. PPARgamma was measured by electrophoretic mobility shift assay (EMSA). The intracellular concentration of Rhodamine123 (Rh123, a fluorescent P-gp substrate) was assayed to evaluate the activity of P-gp. The cell cycle and apoptosis were measured by flow cytometry. The results showed that the P-gp was increasingly expressed in SGC7901, BGC823 and SGC7901/VCR cells in turn, suggesting that MDR in the SGC7901/VCR cells was mediated by the increased expression of P-gp. In the SGC7901/VCR cells, the expression level of total PPARgamma was increased, however, the protein level and activity of PPARgamma in the nuclei of cells decreased significantly. Troglitazone elevated the PPARgamma activity in SGC7901/VCR cells in a dose-dependent manner. Troglitazone decreased the P-gp expression and markedly enhanced the accumulation of Rh123 in SGC7901/VCR cells in a dose-dependent manner. We also found that troglitazone significantly increased the percentage of SGC7901/VCR cells in the G(2)/M phase and decreased the cell percentage in G(1) and S phase in a dose-dependent manner. Troglitazone significantly increased the apoptotic rate of SGC7901/VCR cells treated by VCR or ADR in a dose-dependent manner. It was concluded that P-gp-overexpressed SGC7901/VCR cells have minor endogenous PPARgamma activity. Elevation of the PPARgamma activity by troglitazone can reverse P-gp-mediated MDR via down-regulating the expression and activity of P-gp in SGC7901/VCR cells. It was suggested that troglitazone can dramatically enhance the sensitivity of P-gp-mediated MDR cancer cells to chemotherapeutic agents.

OBJECTIVE:To observe clinical efficacy and safety of Mailuoning injection combined with benazepril in the treat-ment of chronic glomerulonephritis. METHODS:130 patients with chronic glomerulonephritis were randomly divided into observa-tion group and control group,with 65 cases in each group. Both groups received rountine treatment as low salt and fat diet. Control group was additionally given Benazepril tablet 10 mg,qd;observation group was additionally given Mailuoning injection 20 ml added into 5% Sodium chloride injection 250 ml,ivgtt,qd,on the basis of control group,20 days of drug withdrawal every 10 days. Both group received 4 months of treatment. Clinical efficacy of 2 groups were observed as well as the levels of BUN,UCr, SCr and 24 h urine protein quantitative (UPE) before and after the treatment. The incidence of ADR was compared between 2 groups. RESULTS:Total effective rate of observation group was 83.1%,which was significantly higher than 67.7% of control group,with statistical significance (P<0.05). There was no statistical significance in BUN,UCr,SCr and 24 h UPE between 2 groups before treatment (P>0.05);those indexes of 2 groups decreased significantly after treatment,and the observation group were lower than the control group,with statistical significance(P<0.05). There was no statistical significance in the incidence of ADR between 2 groups (P>0.05). CONCLUSIONS:Mailuoning injection combined with benazepril is effective for chronic glo-merulonephritis,and can effectively improve renal function with good safety.

Objective To investigate obstructive sleep apnea syndrome (OSAS) in patients with aortic dissection (AD). Methods Questionnaire analysis was applied to patients with or without AD according to Berlin questionnaire. Questionnaires were collected and common characters and related symptoms were compared between the two groups. Further comparison on related symptoms was made between the AD group and hypertensive patients in the control group. Results Totally 70 questionnaires were collected with 33 for the AD group (29 males and 4 females) and 37 for the control (29 males and 8 females). The average age (P ＜0.05) was 50.9 years for the AD group (range 32 to 70) and 53.4 years for the control (range 25 to 83). Snoring occurred in 29 AD patients (87. 88% ) and in 22 control patients (59. 46% ) (P ＜ 0. 05 ). Snoring everyday occurred in 19 AD patients (57. 58% ) and in 12 controls (32.43%) (P ＜0.05). Loud snoring was reported from 23 AD patients (69.70%) and 10 controls (27.03%) (P ＜0. 05). Apnea occurred in 15 AD patients (45. 45% ) and 8 controls (21.62%) ( P ＜0. 05). Apnea nearly everyday occurred in 9 AD patients ( 27.27% ) and 5 controls ( 13. 51% ) ( P ＜0. 05). Fatigue after sleep occurred in 23 AD patients (69. 70% ) and 15 controls (40. 54% ) (P ＜0. 05).Fatigue nearly everyday after sleep occurred in 10 AD patients (30. 30% ) and 6 controls ( 16. 22% ) ( P ＜0. 05). Hypertension was found in 28 AD patients ( 84. 85% ) and 20 controls ( 54. 05% ) ( P ＜ 0. 05 ).The average age of hypertensive control were 62 ± 16, greater than that of AD group (P ＜0. 05). In the 20 hypertensive control patients, loud snoring in 7 (35%), lessen than that of AD group (P ＜0.05).Compared with hypertensive controls, AD patients had greater body length ( P ＜ 0. 05 ) and lesser waist-tohip ration (P ＜ 0. 05 ). Conclusions Compared with normal control, OSAS is more common in AD patients.

The recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector Ad-PTEN containing green fluorescent protein (GFP) was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2. 5 X 10(10) pfu/mL, and about 70% breast cancer cells were infected with Ad-PTEN when multiplicity of infection (MOI) reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs I restrict endoenzyme site were cloned from plasmid psiRNA-hH1neo and reconstructed them into plasmid pEGFP-C1 in the Mlu I restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H1/siRNA forming plasmid pEGFP-H1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-alpha) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37+/-8.23) % and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-alpha released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-alpha release by RAW264.7 cells evoked by LPS.

Objective To explore the association of CD40 gene single nucleotide polymorphisms (SNPs) and haplotypes with the susceptibility to systemic lupus erythematosus (SLE),as well as the association of serum levels and genotypes of CD40 with the occurrence of SLE.Methods A multiplex PCR single-base extension assay (PCR-SBE) and DNA sequencing were performed to analyze 4 SNPs of the CD40 gene,including rs1883832 C/T,rs13040307 C/T,rs752118 C/T and rs3765459 G/A,in 205 patients with SLE (SLE group) and 220 healthy human controls (control group).Enzyme-linked immunosorbent assay (ELISA) was conducted to measure serum levels of CD40 in these subjects.Results Compared with the control group,the SLE group showed significantly increased serum levels of CD40 (P ＜ 0.05).There were significant differences in genotype and allele frequencies of the SNP rs1883832 C/T in the CD40 gene between the SLE group and control group (all P＜ 0.01).Relative risk analysis showed that the risk of developing SLE in rs1883832 T allele carriers was 1.517 times that in rs1883832 C allele carriers (OR =1.517,95％ CI:1.157-1.990,P=0.003).Moreover,serum levels of CD40 were significantly higher in rs1883832 T allele carriers than in rs1883832 C allele carriers (P ＜ 0.01).The risk of developing SLE was significantly increased in TCCA haplotype carriers compared with the healthy controls (OR =2.322,95％ CI:1.181-4.564,P=0.012).Conclusion The CD40 gene rs1883832 C/T polymorphism and its TCCA haplotype were both associated with the occurrence of SLE,and the rs1883832 T allele may be a gene predisposing to SLE.