Background and purpose:Stanniocalcin 1 (STC1) has been reported to be up-regulated in various cancer tissues, and related to malignancy degree of cancer. However, the molecular mechanism of STC1 in lung cancer cells is still not clear. This experiment aimed to investigate the effects of STC1 on cell cycle and apoptosis of lung cancer A549 cells.Methods:A549 cells were transfected with validated siRNA for STC1 A549-STC1-siRNA and a negative control vector RNA A549-Vector. The gene and protein expression of cell cycle-related genes, including CyclinA, CyclinB1, CyclinD1, CyclinE, CDK2 and CDK4, as well as apoptosis-inhibiting genes Bcl-2, Bcl-xl and apoptosis-inducing genes Caspase-3, Bax, Bak and Bid, were detected by real-time lfuorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot. The cell cycle distribution was determined with lfow cytometry. Terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) was used to detect cell apoptosis.Results:After transfection with STC1-siRNA, the gene and protein expression of CyclinA, CyclinB1, CyclinD1, CyclinE, CDK2 and CDK4 decreased signiifcantly in A549 cells (P<0.05). The proportion of cells in G0/G1 phase signiifcantly increased,
whereas the proportion of cells in S phase and G2/M phase decreased (P<0.05). The cell cycle was blocked at G0/G1 phase. Furthermore, compared with that in A549-Vector, the gene and protein expression of Bcl-2 and Bcl-xl in A549-STC1-siRNA was reduced signiifcantly (P<0.05), while the expression of apoptosis-inducing genes Caspase-3, Bax, Bak and Bid increased obviously (P<0.05). In addition, the percentage of apoptotic cells significantly increased in A549-STC1-siRNA compared with that in A549-Vector detected by TUNEL method.Conclusion:Down-regulation of STC1 by RNAi can block the cell cycle of A549 cells, inhibit cell proliferation, and promote cell apoptosis.

Objective To analyze the effects of dengue virus 2 ( DENV-2 ) infection on the ex-pression of IL-29 in primary human umbilical vein endothelial cells ( HUVECs) cultured on hydrogel sub-strates .Methods Primary HUVECs were isolated and cultured on hydrogel substrates .DENV-2 stains were used to infect the primary HUVECs at a multiplicity of infection( MOI) of 10.Flow cytometry analysis was performed to detect the apoptosis and infection rate of HUVECs after 48 hours of culturing .The gene chip profiling was performed to analyze mRNA expression .The expression of IL-29 at mRNA and protein levels were measured by real-time fluorescent quantitative PCR analysis and double antibody sandwich ELISA as -say, respectively.Results Compared with 96.36%of baby hamster kidney (BHK) cells that were infected with DENV-2 stains, only 4.71%primary HUVECs cultured on hydrogel substrates were infected .The pri-mary HUVECs cultured on hydrogel substrates with or without DENV-2 infection showed no significant differ-ences with the rates of cell apoptosis and infection (P>0.05).A significant difference was observed with the expression of IL-29 at mRNA level between primary HUVECs cultured on hydrogel substrates and the cells cultured in plastic bottles (P<0.05).The results of the real-time quantitative PCR analysis and ELISA as-say showed that IL-29 was highly expressed in DENV-2 infected primary HUVECs cultured on hydrogel sub-strates as compared with those in control groups (P<0.05).Conclusion The expression of IL-29 was de-tected in DENV-2 infected primary HUVECs cultured on hydrogel substrates , which was significantly differ-ent from that in DENV-2 infected primary HUVECs cultured in plastic bottles .The successful establishment of hydrogel substrates as the model of vascular basement membranes might provide a new way for the investi -gation of the pathogenesis of DENV infection .

Objective To investigate the effects of substrate stiffness on the proliferation of human umbilical vein endothelial cells ( HUVEC) during dengue virus infection.Methods Polyacrylamide gels were prepared for cell culture [(0±4) kPa].The proliferation of HUVEC cultured on substrates with differ-ent stiffness was determined by using 3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-etrazolium,inner salt ( MTS) assay.The cycle and apoptosis of HUVEC were determined by flow cytometry analysis.Dengue virus serotype 2 (DENV-2) strains were propagated and identified by con-ventional assays.The HUVEC were infected with DENV-2 strains at a MOI of 10 and cultured on traditional plastic and hydrogel substrates, respectively.The levels of nitric oxide (NO) and endothelin-1 (ET-1) were detected by nitric acid reductase assay and double antibody sandwich ELISA.Results Young′s modulus E value of the hydrogels was (3030 ±0.44) Pa.The proliferation of HUVEC and the expression of NO and ET-1 were enhanced along the increased substrate stiffness.However, no significant differences with the cell cycle and apoptosis were observed between cells cultured on different substrates.Conclusion The stiffness of substrates affected not only the proliferation of HUVEC, but also the release of cytokines during DENV-2 infection.The development of dengue fever was associated with the decreased secretion of vascular active substances as a result of blood vessel injury.The establishment of hydrogel substrates as the model of vascu-lar basement membranes might provide a new way for the in vitro investigation of the pathogenesis of DENV infection.

Child ; Female ; Gastroscopy ; Humans ; Pylorus ; abnormalities

Objective: To predict the action targets of anti-acute lung injury active ingredients of Xuebijing Injection, and investigate the “multi-components, multi-targets, and multi-pathways” mechanism. Methods: Reverse molecular docking was used to forecast its targets of 38 main active components of Xuebijing Injection. The relevant targets of acute lung injury were searched through literature mining and multiple databases and compared with the predicted component targets. The protein interaction network was constructed by Cytoscape software, and topological analysis was performed to screen the key targets of anti-acute lung injury of Xuebijing Injection. And GO enrichment analysis and KEGG analysis were carried out on the key targets through DAVID database. Results: The network analysis indicated that 38 active ingredients affected 143 key target proteins directly or indirectly, involved in 71 biological processes, 29 cellular components, 40 molecular function, and 25 KEGG pathways. These active compounds might participate in regulating the processes of gene, protein, external stimulus, and interfering the mitogen-activated protein kinase (MAPK), phosphatidyl inositol 3-kinase (PI3K)-Akt, and other signaling pathways to play a role in resisting to acute lung injury. Conclusion: The anti-acute lung injury effect of Xuebijing Injection showed the characteristics of traditional Chinese medicine in multi-components, multi-targets, and multi-pathways. This research provides a scientific basis for elucidation of the anti-acute lung injury pharmacological mechanism of Xuebijing Injection.

Objective: To clarify the chemical constituents of Tiandan Tongluo Capsule and establish a precise and effective identification method for the complex composition of TCM. Methods: This objective was achieved mainly depending on the information of the accurate mass and the multistage fragment ions obtained by ultra-high performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS), comparing the relative retention time and the mass spectrometric data of the standard substance and consulting the reference literature. Results: Forty compounds were identified in this study, including the phenoliacids, anthraquinones, flavones, phthalides, fatty acids, and the others. Conclusion: This study can identify various chemical constituents of Tiandan Tongluo Capsule systematically, accurately, and rapidly. Moreover, the scientific theory basis will be provided for the pharmacodyamic material basis and the quality control of this drug at the same time.

OBJECTIVETo observe the effect of Zhibai Dihuang Decoction (ZDD) on mRNA and protein expressions of transient receptor potential family vanilloid subtype 1 (TRPV1) and transient receptor potential family vanilloid subtype 5 (TRPV5) in Ureaplasma urealyticum (UU)-infected rat semens and spermatogenic cells, and to explore the pathomechanism of UU-infected infertility and the intervention of ZDD.

METHODSTotally 45 were randomly selected from 60 4-5 months old SD rats. UU testicular infected animal models were set up after bladder inoculation of UU suspension. The remaining 15 rats were simultaneously injected with normal saline as a normal control group. After a successful modeling, UU infected model rats were randomly divided into the model group, the azithromycin group, and the ZDD group, 15 in each group. Rats in the ZDD group were administered with ZDD at the daily dose of 1 g/kg by gastrogavage. Rats in the azithromycin group were administered with azithromycin suspension at the daily dose of 0. 105 mg/kg by gastrogavage. Equal volume of normal saline was administered to rats in the normal control group and the model group. All medication was performed once daily for 21 successive days. Testes and epididymis were extracted after rats were killed and UU positive rates were compared among all groups. Sperm cells were separated using a mechanical separation technique. Sperm motility parameters were detected using color sperm motion detection system. mRNA and protein expressions of TRPV1 and TRPV5 in spermatogenic cells were determined by real-time quantitative PCR and Western blot.

RESULTSThe UU positive rate was obviously higher in the model group than in the normal control group [(86.7% (13/15 cases) vs. 0] P < 0.05). It was lower in the ZDD group [33.3% (5/15 cases)] and the azithromycin group [26.7% (4/15 cases)] than in the model group (P < 0.05). Compared with the normal control group, class A and B sperms were reduced, the linear velocity and the average velocity were significantly lowered, mRNA and protein expressions of TRPV1 and TRPV5 in spermated genic cells significantly decreased in the model group (P < 0.05). Compared with the model group, class A and B sperms were increased, linear and curve velocities and the average velocity were significantly elevated, mRNA and protein expressions of TRPV1 and TRPV5 significantly increased in the ZDD group and the azithromycin group (P < 0.05, P < 0.01). Compared with azithromycin group, class A and B sperms were increased, the linear velocity and the average velocity were significantly elevated, mRNA and protein expressions of TRPV1 and TRPV5 significantly increased in the ZDD group (P < 0.05, P < 0.01).

CONCLUSIONZDD could fight against UU infection and elevate semen quality, which might be associated with up-regulating mRNA and protein expressions of TRPV1 and TRPV5 in spermatogenic cells.

Animals ; Calcium Channels ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Infertility ; Male ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Semen Analysis ; Sperm Motility ; Spermatozoa ; TRPV Cation Channels ; metabolism ; Testis ; Ureaplasma Infections ; Ureaplasma urealyticum

OBJECTIVEThis study aimed to induce carcinogenesis of lingual mucosa in C57BL/6 mice by feeding them 4-nitroquinoline 1-oxide (4NQO) solution.

METHODSA total of 85 C57BL/6 mice were randomly divided into distilled water control group (DD group, n=5), 1,2-propylene glycol control group (PG group, n=5), and experimental group (EP group, n= 75). The mice in the experimental group were medially fed in 15 cages. By contrast, the mice in DD, EP, and PG groups were watered with distilled water, 50 mg.L-1 4NQO solution, and 1,2-propylene glycol solution. The mice in EP group were executed every two weeks from week 0, and the mice in the control groups were sacrificed at the 28th week. The mice were weighed. Mucosal lesions were measured by macroscopic observation and histopathologic detection.

RESULTSOne mouse in EP group died of unknown reason. The weight of the mice in EP group presented weight loss compared with the mice in DD and PG groups after the 24th week. Seventy-nine macroscopic lesions were observed in the lingual mucosa, oral floor, and upper palatal and buccal mucosa. A total of 70 macroscopic lesions (88.6%) were located in the lingual mucosa. Mucosal lesions changed from simple hyperplasia to squamous cell carcinomas. Well-differentiated squamous cell carcinomas were observed in all mice of EP group by pathological section at the 28th week. No lesion was found in the mice of DD and PG groups.

CONCLUSIONThe animal model of lingual squamous cell carcinomas was successfully established. The periods from 12th to 16th week and 20th to 28th week were the ideal times for the research on pathogenesis of early and medial-advanced stage during carcinogenesis of squamous cell carcinomas.

4-Nitroquinoline-1-oxide ; Animals ; Cell Transformation, Neoplastic ; Disease Models, Animal ; Mice ; Mice, Inbred C57BL ; Mouth Mucosa ; Tongue

Objective:To reveal the primary mechanism of changing permeability in DENV-2 infected pHDMECs. Methods:pHDMECs was incubated by DENV-2 on the concentration of 103 TCID50 ,and the penetrability of the cell was detected by Transwell at 4,8,12,24,48 h,respectively. Then,the partial sequence of DENV-2 NS1 was analyzed by Real time-PCR,and NS1 protein was detected by immunofluorescence and flow cytometer (FCM). The apoptosis rate of pHDMECs was assayed by FCM. Finally,IL-6 and IL-8 secreted by pHDMECs were analyzed by Real time-PCR and double antibody sandwich ELISA. Results:The relative expression of NS1 gene was elevated but NS1 protein was not detected;the permeability of DENV-2 infected pHDMECs had dramatically increased both at 24,48 h,but the apoptosis rate has little changed even been influenced by DENV-2 at 72 h. However,the relative expression of IL-6/IL-8 mRNA was boosted at 8,24 h[(2. 49±0. 50) and (6. 82±1. 69) fold,respectively,P<0. 05]. In protein level,compared with control(869. 6±50. 70)pg/ml,IL-6 secreted by DENV-2 infected pHDMECs could reach by(1 248. 8±86. 9)pg/ml(P<0. 05),and IL-8 was(1 331. 0±86. 3)pg/ml(P<0. 05) while the control was (967. 6±156. 6)pg/ml. Conclusion:Indeed,pHDMECs can be infected by DENV-2;the increasing permeability of DENV-2 infected pHDMECs may not be caused by the pHDMECs′ apoptosis but the enhancing of pro-inflammatory cytokine IL-6 /IL-8.

Objective To investigate the effects of dengue type 2 virus(DENV-2)on the apopto-sis and autophagy of primary human hepatic sinusoidal endothelial cells(HHSECs)and the expression of ICAM-1 and Beclin-1 at mRNA level and to analyze the possible pathogenic mechanism of DENV-2. Meth-ods Immunohistochemistry(IHC)and flow cytometry analysis(FCM)were performed to identify HHSECs by detecting factor Ⅷ and CD31. The DENV-2 strain was identified by using PCR and HindⅢ. The 50%tissue culture infective dose(TCID50 )of DENV-2 was calculated after infecting C6 / 36 cells with DENV-2. Dynamic changes of DENV-2 NS1 were measured by real-time PCR after infecting HHSECs with DENV-2. CCK-8 was used to dynamically detect the cytotoxicity of DENV-2 to HHSECs. The transcriptional levers of Beclin-1 and ICAM-1 in DENV-2-infected HHSECs were detected by real-time PCR. FCM was performed to analyze the apoptosis of HHSECs and the expression of LC3B and ICAM-1. Results The cells in the exper-imental group were stained brown by DAB and the positive expression rate of CD31 reached 87. 1% . The TICD50 of DENV-2 to C6 / 36 cells was 10-6. 845 / 0. 1 ml. Compared with the uninfected cells,partial se-quences of NS1 gene were expressed in DENV-2-infected HHSECs. DENV-2 suppressed the cell activities of HHSECs. The suppression rates of DENV-2 to HHSECs at 12 h,24 h,36 h and 48 h were respectively (10. 90±1. 24)% ,(16. 40±0. 42)% ,(17. 00±0. 46)% and(29. 60±0. 26)%(P﹤0. 05). The tran-scriptional levels of Beclin-1 and ICAM-1 in HHSECs were significantly increased at the time point of 24 h after DENV-2 infection,the 2-△△Ct values of which were 46. 77±2. 55 and 40. 97±4. 91,respectively. The expression of LC3B and ICAM-1 in DENV-2-infected HHSECs were increased,the peaks of which were reached at 24 h(14. 7% )and 36 h(35. 5% ),respectively. The apoptosis of DENV-2-infected HHSECs was remarkably enhanced at 12 h with an apoptosis rate of 13. 17% . Conclusion HHSECs was susceptible to DENV-2. DENV-2 induced the upregulation of ICAM-1 and the activation of HHSECs. Moreover,autoph-agy and apoptosis of HHSECs could also be induced by DENV-2.