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OBJECTIVES@#The proliferation, migration capacity, and expression of activation-related proteins of NHGFs+Cal27-exo were determined by coculturing Cal27 exosome (Cal27-exo) with normal human gingival fibroblasts (NHGFs) to explore the effects of Cal27-exo on the activation and biological behavior of NHGFs.@*METHODS@#Cal27-exo was extracted using supercentrifugation, and exosomes were identified using Western blot, transmission electron microscopy (TEM), and particle size detection. Cal27-exo was cocultured with NHGFs to detect the uptake of Cal27-exo by NHGFs, and the proliferation and migration capacity of NHGFs+Cal27-exo were detected using CCK8 and wound healing tests, respectively. The expression levels of NHGF activation-related proteins, i.e., matrix metalloproteinase-9 (MMP-9), fibroblast-activating protein (FAP), alpha smooth muscle actin (αSMA), and transforming growth factor-β (TGF-β), were detected using real-time quantitative polymerase chain reaction (qRT-PCR).@*RESULTS@#Cal27-exo was extracted u-sing supercentrifugation, and Western blot showed the positive expression levels of Alix and CD63. TEM showed that Cal27-exo had a circular double-layer vesicle. The particle size was between 30 and 150 nm. Cal27-exo labeled with PKH67 entered NHGFs after the coculture method. The wound healing test showed that the migration capacity of NHGFs+Cal27-exo was stronger after the scratch compared with that of NHGFs. CCK8 results showed that the proliferation activity of NHGFs+Cal27-exo was enhanced. qRT-PCR results showed that the MMP-9 levels of NHGFs+Cal27-exo were upregulated, whereas the TGF-β and αSMA mRNA levels of NHGFs+Cal27-exo were downregulated (@*CONCLUSIONS@#The proliferation and migration ability of NHGFs+Cal27-exo are enhanced, and the mRNA expression of related proteins is changed. Cal27-exo can activate NHGFs, which suggests that Cal27-exo has potential significance in tumor invasion and metastasis.
Subject(s)
Humans , Cell Proliferation , Exosomes , Fibroblasts , Gingiva , Matrix Metalloproteinase 9ABSTRACT
The epigenetic changes of clear cell renal cell carcinoma(ccRCC )are considered to be the main molecular mechanisms of its pathogenesis ,including DNA methylation ,histone modification ,microRNA change ,and so on.DNA methyltransferases(DNMTs)catalyze the occurrence of DNA methylation.DNA methylation changes are mani-fested in the overall low methylation of the genome and the high methylation of specific sites ,which were involved in the de-velopment of ccRCC by affecting the expression of tumor suppressor genes.Due to histone-modified enzyme involvement ,histone modification is shown as possible genetic reversal.MicroRNA plays an important role in the abnormal expression of ccRCC genes.With the studies of epigenetic mechanism and molecular pathology ,it is important to explore the mechanisms and to seek effective early diagnosis ,treatment and prognosis intervention of ccRCC.
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Objective To evaluate preventive effect of polysaccharides from Lycium ruthenicum Murray against photodamage in HaCaT cells,and to explore its possible mechanism.Methods Ultrasoundassisted extraction was used to extract polysaccharides from Lycium ruthenicum Murray in Qaidam Basin.In vitro cultured HaCaT cells were randomly divided into 3 groups:control group receiving no treatment,ultraviolet B (UVB) group irradiated with 30 nJ/cm2 UVB alone for 1 hour,experimental group pretreated with 2 g/L Lyeium ruthenicum polysaccharide solution followed 6 hours later by 30 mJ/cm2 UVB radiation for 1 hour.At twelve hours after UVB radiation,an inverted microscope was used to observe cell morphology.Then,MTS assay was performed to estimate cell proliferation,flow cytometry to detect cell apoptosis,an enzyme-labeled antigen method to detect levels of malondialdehyde (MDA) and glutathione peroxidase (GSH-Px),as well as to evaluate the activity of superoxide dismutase (SOD) and catalase (CAT),and enzyme-linked immunosorbent assay (ELISA) to measure levels of intedeukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) in HaCaT cells and their culture supernatant.Results Compared with the control group,the UVB group showed obscure cell morphology,cell death and floating phenomenon,while cells became swollen but renained morphologically distinct in the experimental group.MTS assay revealed that the cell proliferative activity significantly differed among the above 3 groups (F =48.88,P ＜ 0.01),and the cell proliferative activity was significantly lower in the UVB group (1.72 ± 0.12) than in the control group (2.34 ± 0.11,P ＜ 0.05) and experimental group (2.11 ± 0.10,P ＜ 0.05).Moreover,the apoptosis rate was significantly higher in the UVB group (82.41％ ± 2.49％) than in the control group (3.98％ ± 0.19％,P ＜ 0.05) and experimental group (22.79％ ± 0.97％,P ＜ 0.05),as well as higher in the experimental group than in the control group (P ＜ 0.05).Compared with the control group,the UVB group showed significantly higher levels of MDA,supernatant levels of IL-1 and TNF-α,and intracellular levels of TNF-α,but significantly lower GSH-Px levels and activity of SOl and CAT (all P ＜ 0.05).However,there was no signifi-cant difference in the intracellular level of TNF-α between the UVB group and control group (P ＞ 0.05).Conclusion Lycium ruthenicum polysaccharide has protective effects against photodamage in HaCaT cells,likely by reducing the synthesis and secretion of inflammatory substances as well as free radicals.
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ObjectiveTo investigate the effects of autophagy on exocrine function of pancreas in rats with acute sepsis, and to determine whether the mitochondrial coenzyme Q (Mito Q) can prevent exocrine dysfunction of pancreas mediated by autophagy.Methods ExperimentⅠ: 30 Sprague-Dawley (SD) rats were randomly divided into three groups, with 10 rats in each group. All the rats were given lipopolysaccharide (LPS, 10 mg/kg) intraperitoneally, and Wortmannin (2 mg/kg), the specific inhibitor of autophagy (LPS+ Wortmannin group), Mito Q (6.5μmol/kg, LPS+Mito Q group), or the same volume of normal saline (LPS group) was respectively injected via the tail vein 1 hour later. Survival rate was assessed within 12 hours after LPS injection. ExperimentⅡ: another 100 male SD rats were randomly divided into ten groups with 10 rats in each group: namely control 4, 6 and 12 hours groups, LPS 4, 6 and 12 hours groups, and LPS+ Wortmannin 4 hours group, Wortmannin 4 hours group, LPS+ Mito Q 6 hours group, and Mito Q 6 hours group. The protocols of model reproduction and drug administration were the same as in the experimentⅠ. Blood samples were collected at each time point, and the amylase content was determined with the velocity method. The levels of reactive oxygen species (ROS) in the pancreases were measured with enzyme-linked immunosorbent assay (ELISA). The expression of the autophagy-related protein LC3 was determined with Western Blot. The pathological changes in the pancreas were observed with microscopy.Results① The survival time in the LPS+ Wortmannin group was significantly shorter than that in the LPS group (hours: 7.50±0.64 vs. 11.90±0.13,χ2= 19.847,P= 0.001). There was no significant difference in the survival time between LPS+ Mito Q and LPS groups (hours: 11.60±0.24 vs. 11.90±0.13,χ2= 1.055,P= 0.137).② The serum amylase in the LPS 6 hours, LPS+ Wortmannin 4 hours, and LPS+ Mito Q 6 hours groups were significantly higher than those in the control group at the same time points (U/L:2 881.00±550.12 vs. 2 099.20±249.57, 3 672.00±779.24 vs. 2 081.36±245.18, 2 975.20±687.03 vs. 2 099.20± 249.57, allP 0.05). Light microscopy showed that obvious pathological changes were found in the pancreas in the LPS 6 hours and 12 hours groups, LPS+Wortmannin 4 hours group, and LPS+ Mito Q 6 hours group. Electron microscopy showed that the number of autophagic vacuoles increased 6 hours after LPS administration. There was no difference at any time point in the number of autophagic vacuoles between LPS+ Mito Q 6 hours group and LPS 6 hours group, and the autophagic vacuoles were not found after Wortmannin intervention. It was demonstrated by Western Blot that the levels of LC3 protein in the LPS 6 hours and 12 hours groups, and LPS+ Mito Q 6 hours group were significantly higher than those of the control group at the same time points (A value: 0.34±0.02 vs. 0.17±0.02, 0.37±0.03 vs. 0.18±0.04, 0.36±0.02 vs. 0.17±0.02, allP 0.05).Conclusions Autophagy prevents exocrine dysfunction of pancreas in septic rats, and the autophagic capacity or autophagosome-formation rate may determine the development of exocrine pancreatic dysfunction. The mitochondria-targeted antioxidant Mito Q does not prevent exocrine dysfunction of pancreas.
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Objective To investigate the effects of autophagy on cardiac function and to determine whether the mitochondrial coenzyme Q (MitoQ) prevents cardiac dysfunction,mediated by autophagy,in rats with acute sepsis.Methods Forty-five Sprague Dawley (SD) rats were randomly divided into 9 groups (n =5,each group):control group,4 h lipopolysaccharide(LPS) group,6 h LPS group,12 h LPS group,4 h LPS + Wortmannin group,4 h LPS + MitoQ group,6 h LPS + MitoQ group,MitoQ group and Wortmannin group.Rats in LPS + Wortmannin group and LPS + MitoQ group were intraperitoneally given LPS(10 mg/kg) and followed by an injection of Wortmannin(2 mg/kg) and MitoQ (6.5 μmol/kg) via tail vein 1 hour later,respectively.Rats in each group were given the same amount of normal sodium in addition to different intervention drugs.The cardiac function parameters were measured by a BL-420E + biosignal collection system.Blood samples from abdominal aorta were taken at each time point,and creatine kinase MB isoenzyme (CK-MB) content was detected by using the velocity method.The content of reactive oxygen species (ROS) in isolated myocardial tissues in rats was measured by enzyme-linked immunoadsorbent assay(ELISA).The protein expression of microtubule-associated protein 1 light chain 3 (LC3) was detected by Western blot method.The pathological changes of myocardial tissue were observed by light and electronic microscopy.Results Compared with the control group,the left ventricular systolic pressure(LVSP),the rate of the rise in left ventricular pressure (± dp/dt max) were significantly decreased in 6 h LPS group,6 h LPS + MitoQ group and 4 h LPS + Wortmannin group(P ＜0.05),left ventricular end-diastolic pressure(LVEDP) was significantly increased in these 3 groups(P ＜0.05).The contents of CKMB and ROS in 6 h LPS group,6 h LPS =MitoQ group and 4 h LPS + Wortmannin group were higher than those in the control group(P ＜ 0.05).Electron microscopy showed that the number of autophagic vacuoles increased 6 h after LPS was administered,but did not increase significantly thereafter to 12 h.There was no difference at any time point in the number of autophagic vacuoles in the group given MitoQ and LPS.Immunoblotting demonstrated that the levels of LC3Ⅱ protein in the LPS 6 h group and LPS + MitoQ 6 h group were higher than those in the control group(P ＜0.05),but there was no difference between the LPS 12 h and LPS 6 h groups (P ＞ 0.05).Conclusions The mitochondria-targeted antioxidant MitoQ does not prevent cardiac dysfunction.However,autophagy prevents cardiac dysfunction,and the autophagic capacity or autophagosome-formation rate may determine whether cardiac dysfunction develops.
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<p><b>OBJECTIVE</b>To discuss the rationale, hypothesis, modality of extracorporeal blood purification (EBP) techniques for the critically ill animal models or patients, and to summarize the experimental and clinical studies with inconsistent data which explored the EBP's efficacy in the areas of critical care medicine.</p><p><b>DATA SOURCES</b>Articles referred in this review were collected from the database of PubMed published in English up to June 2014.</p><p><b>STUDY SELECTION</b>We had done a literature search by using the term "(sepsis OR acute lung injury OR acute respiratory distress syndrome) AND (extracorporeal blood purification OR hemofiltration OR hemoperfusion OR plasma exchange OR plasmapheresis OR adsorpiton)". Related original or review articles were included and carefully analyzed.</p><p><b>RESULTS</b>Acute cellular and humoral immune disturbances occur in both sepsis and acute respiratory distress syndrome (ARDS). Treatments aimed at targeting one single pro-/anti-inflammatory mediator have largely failed with no proven clinical benefits. Such failure shifts the therapeutic rationale to the nonspecific, broad-spectrum methods for modulating the over-activated inflammatory and anti-inflammatory response. Therefore, EBP techniques have become the potential weapons with high promise for removing the circulating pro-/anti-inflammatory mediators and promoting immune reconstitution. Over the years, multiple extracorporeal techniques for the critically ill animal models or patients have been developed, including hemofiltration (HF), high-volume hemofiltration (HVHF), high-cutoff hemofiltration (HCO-HF), hemo-perfusion or -adsorption (HP/HA), coupled plasma filtration adsorption (CPFA), and plasma exchange (PE). These previous studies showed that EBP therapy was feasible and safe for the critically ill animal models or patients. However, data on their efficacy (especially on the clinical benefits, such as mortality) were inconsistent.</p><p><b>CONCLUSIONS</b>It is not now to conclude that EBP intervention can purify septic or ARDS patients with high clinical efficacy from current experimental and clinical practice. Prospective, randomized controlled, and well-designed clinical or experimental studies and most suitable EBP modalities should be further developed.</p>
Subject(s)
Humans , Hemofiltration , Respiratory Distress Syndrome , Therapeutics , Sepsis , TherapeuticsABSTRACT
OBJECTIVE: To study the changes of growth and biofilm formation capability of Enterococcus faecalis (Ef) in different stress conditions. METHODS: The changes of growth of Ef in stress conditions were observed by measuring the A600 value with ultraviolet spectrophotometer. Ef was incubated on glass slide in stress conditions, biofilm formation capability of cells was investigated by colony-forming unit (CFU) counting of the culturable bacteria and fluorescence confocal laser scanning microscopy. RESULTS: Ef couldn't growth under the conditions of 2%, 5%NaClO, pH = 11 and 12, the A600 value was unchanged in 96 hours. But the growth curve changed at different levels in other stress conditions: under 1%NaClO, the A600 value peaked at 1.461 at 16 hour (the peaked level was 1.238 at 6 hours in control group) ; under 0,0.05%,0.15% glucose, it peaked at 0.645,0.890, 1.173, respectively, at 6 hour (it was maximized to 1.195 at 6 hours in control group); the A600 value peaked at 1.704 at 6 hours at pH = 9 and 1.225 at 10 hours at pH = 10 (the peak level was 1.732 at 6 hours at pH = 7) . Biofilm assay showed that Ef were able to form biofilm in these stress conditions except 5%NaClO and pH = 12. CONCLUSIONS: Ef could growth and form biofilms in energy starvation, low concentrations of sodium hypochlorite and weak alkaline stress.
