ABSTRACT
Objective:To evaluate the effect of lycopene preconditioning on Toll-like receptors (TLRs)/nuclear factor kappa B (NF-κB) signaling pathway during hypoxia-reoxygenation (H/R) injury to rat cardiomyocytes.Methods:The rat H9C2 cardiomyocytes were cultured in vitro, inoculated in a petri dish at a density of 8×10 4 cells/ml and divided into 3 groups( n=30 each) using a random number table method: control group (C group), H/R group and lycopene preconditioning group (LP group). The H9C2 cardiomyocytes were reoxygenated for 6 h after hypoxia to establish a model of H/R injury in H/R group and LP group.The cells were preconditioned with lycopene 20 μmol/L at 12 h before establishing the model in LP group.The cell viability was detected by CCK-8 assay.The cell apoptosis rate was detected by flow cytometry (Annexin V and PI double staining). The levels of lactic dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) and reactive oxygen species (ROS) were detected by microplate method.The expression of TLR2, TLR3 and NF-κB was detected by Western blot. Results:Compared with group C, the cell viability and SOD level were significantly decreased, the apoptosis rate and levels of LDH, MDA and ROS were increased, and the expression of TLR2, TLR3 and NF-κB was up-regulated in H/R and LP groups ( P<0.05). Compared with group H/R, the cell viability and SOD level were significantly increased, the apoptosis rate and levels of LDH, MDA and ROS were decreased, and the expression of TLR2, TLR3 and NF-κB was down-regulated in group LP ( P<0.05). Conclusion:The mechanism by which lycopene preconditioning attenuates H/R injury may be related to inhibiting activation of TLRs/NF-κB signaling pathway and inhibiting oxidative stress response in rat cardiomyocytes.
ABSTRACT
Objective To explore the effects of Compound Antler capsule on the NO secretion of macrophages, cell cycle and[Ca2 +]i of mouse T lymphocytes.Methods ICR mice were randomly divided into 4 groups:experimental groups were respectively given Compound Antler capsule 233, 467, 1400 mg/kg via intragastric administration once a day and the control group were given the same volume of water for 30 days.NO concentration of mouse peritoneal macrophages was measured by Griess assay.The cell cycle distribution of activated mouse spleen lymphocytes was measured by flow cytometry.Fluorescent probe Fluo 4-AM was used to mark Ca2 + in lymphocytes, and the changes of its fluorescence intensity were observed with the multiscan spectrum.Results The result showed that NO concentration in experimental groups was higher than that in control group (P<0.01).More activated spleen lymphocytes of 467, 1400 mg/kg dose groups were entried into S and G2/M phase than control group (P<0.05).After activated by ConA for 8 min, the intracellular[Ca2 +]i in mouse spleen lymphocytes of 233, 1400 mg/kg dose group was higher than that of control group, respectively (P<0.05).After activated by LPS for 1, 4, 8 min, the[Ca2 +]iin mouse spleen lymphocytes of 233 mg/kg dose group was higher than that of control group, especially at 1 min(P<0.01).Conclusion Compound Antler capsule can improve NO secretion of macrophages and facilitate the entry of mouse spleen lymphocytes from the G0/G1 into the S phase.It also can increase the [ Ca2 +] i of activated lymphocytes to promote their proliferation.Thus Compound Antler capsule can improve the immune regulating ability.
ABSTRACT
To explore a sensitive , stable and handleable method for evaluating phagocytosis of mouse peritoneal macrophages by flow cytometry , and get a set of optimized solutions.Methods: The peritoneal macrophages obtained from ICR mice were divided into two part.One part was used directly ,and another part was 1∶1 diluted.Three fluorescent microsphere concentrations were used (5×106/well,1×107/well and 1.5×107/well).Incubation time were respective 1 h,1.5 h and 2 h.The adherent cells were digested by enzyme or cell scraper.The percentage of phagocytic cells ( PP) and the phagocytic index ( PI) were determined by flow cy-tometry.To verify and confirm the reliability of experiment conditions , effect of JKS on phagocytosis of mouse macrophages were evaluated with flow cytometric assays and chicken red blood-cell method.Results:The higher concentration of fluorescent microspheres meant PP and PI were higher.When cell concentration was 1×105-2×105 ml-1 ,incubation time was 1.5 h,concentration of fluorescent microspheres was 1.5 ×107/well,the PP and PI were the highest (89.87%,1.54).When incubation time was 2 h,the PP and PI declined(57.71%,1.51).Effect of cell concentration on the PP and PI were negatively correlated with fluorescent microspheres .After adherent macrophages were digested by trypsin+EDTA,the PP and PI were 44.51%,0.68.The PP and PI were 37.92%,0.57 after di-gestion by EDTA.The results were lower than using cell scraper.The PP(1 485 mg/kg group) of JKS were higher than control group that were evaluated with flow cytometric assays and chicken red blood-cell method.The difference was statistically significant ( P<0.05 ).Conclusion: These are the optimized solutions for the experiment such as the concentration of peritoneal macrophaes is (1-2)×105,the incubation time is 1 h and the concentration of fluorescent microspheres is 1×107/well.