Objective:To investigate the effect of hypoxia on vascular endothelial growth factor expression in breast carcinoma cell line(MD-MBA-435S) and its mechanism.Methods:Hypoxia culture model in vitro was established to test the expression of VEGF and HIF-1? and their correlation in MD-MBA-435S cell line.After cultured in hypoxia circumstance for a given period of time(4 hours,8 hours,12 hours,16 hours,20 hours),VEGF expression was tested in mRNA level and protein level by RT-PCR and Western blot analysis,HIF-1 ? expression tested in protein level.Cell viability in twenty hours was tested by trypan blue exclusion assay.Results:Cell survival was affected a little within 20 hours under hypoxia.Two isoforms of VEGFmRNA,VEGF-121 and VEGF165,were identified by RT-PCR,and their expressions increased after treatment of hypoxia for 4 hours.VEGF and HIF-1 ? protein expressions increased equally with VEGFmRNA confirmed by immunocytochemistry and Western blot and its positive correlation was found by Pearson product-moment correlation analysis.Conclusion:VEGF gene and protein can be induced remarkably by hypoxia in breast carcinoma cell line,which is regulated mostly by transcription factor HIF-1 ?.This mechanism may be involved in the malignant proliferation and transformation of breast neoplasm.

Objective To investigate the effect of vaccum-assisted closure(VAC)on the expression of C-ski and Smad3 in the human wound margin tissue and to explore their significance in triggering the wound healing.MethodsFourteen patients,9 males and 5 females with chronic wound admitted in our department from April 2007 to April 2009 were enrolled into the study after signed an informed consent,and the full-thickness skin defects were collected before treatment,and 1,4 or 7 d after treatment.Immunohistochemistry(IHC)was carried out to detect the expressions of C-ski and Smad3,and RT-PCR was used to determine their mRNA expressions of C-ski and Smad3.ResultsIHC results showed that there existed little C-ski in the wound margin tissue before treatment,and after treatment,it were gradually increased.Compared with before treatment group,the differences in 4 and 7 d after treatment were markedly significant(P0.05).Compared with above groups,the differences in 4 and 7 d after treatment groups were markedly significant(P

Objective:To investigate the effect of recombinant Elafin on A549 apoptosis induced by paraquat and the underlying mechanism.Methods:pEGFP-C1-Elafin was transformed into A549 competent cells by electroporation.Transformed A549 was cultured for additional 24 h before Elafin mRNA was detected by RT-PCR and cocultured with or without various concentration of paraquat( PQ ) for different duration.A549 apoptosis percentage and reactive oxygen species ( ROS ) content were tested by flow cytometry .Nuclear factor erythroid like-2(Nrf2) heme oxygenase-1(HO-1) were examed by Western blot .Results:Elafin expressed successfully in A549 after transformation.PQ caused A549 apoptosis in a concentration dependent manner and leaded to ROS content increasing and Nrf2 and HO-1 decreasing.However,elafin could inhibit ROS production and A549 apoptosis,upregulate Nrf2 and HO-1.Conclusion:Elafin could restrict A549 apoptosis to some extent and the possible mechanism lied in its ability to upregulate Nrf2 ex-pression.

The purpose of this study was to determine whether the Ca2+ signaling pathway is involved in the ability of osteoprotegerin (OPG) to inhibit osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-kappaB ligand (RANKL) to stimulate osteoclastogenesis and then treated with different concentrations of OPG, an inhibitor of osteoclast differentiation. The intracellular Ca2+ concentration [Ca2+]i and phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the different treatment groups were measured by flow cytometry and Western blotting, respectively. The results confirmed that M-CSF + RANKL significantly increased [Ca2+]i and CaMKII phosphorylation in osteoclasts (p < 0.01), and that these effects were subsequently decreased by OPG treatment. Exposure to specific inhibitors of the Ca2+ signaling pathway revealed that these changes varied between the different OPG treatment groups. Findings from the present study indicated that the Ca2+ signaling pathway is involved in both the regulation of osteoclastogenesis as well as inhibition of osteoclast differentiation and activation by OPG.
Animals ; Calcium/*metabolism ; *Calcium Signaling ; *Cell Differentiation/drug effects ; Cell Line ; Cell Survival/drug effects ; Gene Expression Regulation/drug effects ; Macrophage Colony-Stimulating Factor/metabolism ; Mice ; Osteoclasts/*cytology/*drug effects/*metabolism ; Osteoprotegerin/*pharmacology ; RANK Ligand/metabolism

Objective:To investigate the effect of microbubbles combined with gentamicin on the clearance of bacterial biofilms and the healing of diabetic foot ulcers under low-frequency ultrasound.Methods:From July 2021 to June 2022, 27 patients with chronic diabetic foot ulcers complicated with infection were prospectively selected from the Trauma Center of the Second Affiliated Hospital of Chongqing Medical University. The patients were divided into low-frequency ultrasound + microbubbles + gentamicin ointment group, low-frequency ultrasound + microbubbles group, and gentamicin ointment group by using a random number table, with 9 patients in each group. The three groups were all treated with simple debridement by the same surgeon.Afterward, in the low-frequency ultrasound+ microbubbles+ gentamicin ointment group, the wounds covered by 4% microbubble suspension were firstly irradiated with low-intensity focused ultrasound for 5 min, and then evenly applied with gentamicin ointment. In the low-frequency ultrasound + microbubbles group, the wounds covered by 4% microbubble suspension were irradiated with low-intensity focused ultrasound for 5 min. The gentamicin ointment group was treated with gentamicin ointment evenly. The treatment lasted for 2 weeks, and secretions and tissue specimens were collected during and 2 weeks after the treatment, respectively. The general indexes of wound surface (including ulcer depth score, secretion exudation score, fresh granulation tissue growth score, and total index score), ulcer area, ulcer healing rate, as well as negative rate of secretion culture were compared among the three groups after treatment. Additionally, the structural changes in bacterial biofilms under a scanning electron microscope and colony count under a laser confocal scanning microscope were compared among the three groups after treatment.Results:No significant differences were found in the general datas among the three groups (all P>0.05). After treatment for 2 weeks, the overall general indexes showed statistically and significant differences among the three groups (all P<0.05). Each index score in the low-frequency ultrasound + microbubbles + gentamicin ointment group was lower than that in the low-frequency ultrasound + microbubbles group and the gentamicin ointment group (all P<0.05). There were no significant differences in overall ulcer area among the three groups ( P>0.05). The overall ulcer healing rate presented significant differences among the three groups ( P<0.05). The healing rate in the low-frequency ultrasound + microbubbles + gentamicin ointment group was higher than that in the low-frequency ultrasound + microbubbles group and the gentamicin ointment group (all P<0.05). The overall negative rates of secretion culture among the three groups were significantly different ( P<0.05), the negative rate in the low-frequency ultrasound + microbubbles + gentamicin ointment group was higher than that in the low-frequency ultrasound + microbubbles group and the gentamicin ointment group (all P<0.05). Scanning electron microscopy confirmed bacterial biofilm infection in the three groups before treatment. After treatment for 2 weeks, the biofilm formation in the low-frequency ultrasound + microbubbles + gentamicin ointment group reduced significantly, while the low-frequency ultrasound + microbubbles group and the gentamicin ointment group had little change compared with that before treatment. Significant differences were detected in total colony count among the three groups under the confocal microscope ( P<0.05). The colony count in the low-frequency ultrasound + microbubbles + gentamicin ointment group was lower than that in the low-frequency ultrasound + microbubbles group and the gentamicin ointment group (both P<0.05). Conclusions:Ultrasound microbubbles combined with gentamicin can clear bacterial biofilms and promote the healing of diabetic foot ulcers.

Exposure to cadmium (Cd) induces apoptosis in osteoblasts (OBs); however, little information is available regarding the specific mechanisms of Cd-induced primary rat OB apoptosis. In this study, Cd reduced cell viability, damaged cell membranes and induced apoptosis in OBs. We observed decreased mitochondrial transmembrane potentials, ultrastructure collapse, enhanced caspase-3 activity, and increased concentrations of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 following Cd treatment. Cd also increased the phosphorylation of p38-mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK)1/2 and c-jun N-terminal kinase (JNK) in OBs. Pretreatment with the caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, ERK1/2 inhibitor (U0126), p38 inhibitor (SB203580) and JNK inhibitor (SP600125) abrogated Cd-induced cell apoptosis. Furthermore, Cd-treated OBs exhibited signs of oxidative stress protection, including increased antioxidant enzymes superoxide dismutase and glutathione reductase levels and decreased formation of reactive oxygen species. Taken together, the results of our study clarified that Cd has direct cytotoxic effects on OBs, which are mediated by caspase- and MAPK pathways in Cd-induced apoptosis of OBs.
Animals ; Apoptosis/*drug effects ; Cadmium/*toxicity ; Caspases/metabolism ; Environmental Pollutants/*toxicity ; Osteoblasts/*drug effects/metabolism ; Oxidative Stress/drug effects ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases/metabolism