摘要
Objective:To investigate the effect of macrophage polarization in the pathogenesis of necrotizing enterocolitis(NEC).Methods:C57BL/6 mice were chose to construct the NEC model.The preterm pups were randomly assigned into the control group( n=10) and the NEC group( n=19). The pups in the control group were breastfed by mothers while the NEC group were treated with hypoxia, hypothermia, hypertonic feeding and lipopolysaccharide treatment.The intestinal tissues from the lower part of duodenum to the colon were collected after the pups were born after 96 hours.HE staining was used to observe the intestinal histological structure.Intestinal mucosal permeability was detected by the measurement of concentration of FD70 in plasma after gastric gavage.The expression of Pan-keratin of intestinal epithelium was detected by immunofluorescence histochemistry.Enterocyte apoptosis was detected by TUNEL staining.The expression of CD86 and CD206 protein were determined by western blotting and the percentage of M1 and M2 macrophages was calculated by flow cytometry.RT-PCR was used to detect the expression of mRNA levels of granulocyte-macrophage colony stimulating factor, interleukin(IL)-6, IL-10 and IL-12. Results:Compared with the control group, the pups in the NEC group had low survival rate(100.0% vs.36.8%), different level of intestinal injury, incomplete integrity of intestinal epithelium, increased mucosal permeability(1.53±0.80 vs.14.32±1.27, P<0.05)and enterocyte apoptosis(1.9%±1.1% vs.7.6%±2.6%, P<0.05). Western blotting showed that the expression of CD86 protein(1.00±0.01 vs.1.50±0.10, P<0.05) increased while CD206 protein decreased(1.00±0.01 vs.0.60±0.05, P<0.05) in the NEC group.Flow cytometry showed that the percentage of CD68 + CD86 + M1 macrophages increased(1.90%±0.19% vs.10.20%±0.38%, P<0.05) while the CD68 + CD206 + M2 macrophages decreased(5.8%±0.33% vs.3.7%±0.56%, P<0.05) in the NEC group.The expression of the mRNA levels of pro-inflammatory cytokines, granulocyte-macrophage colony stimulating factor(1.00±0.05 vs.1.83±0.17, P<0.05), IL-6 (1.00±0.13 vs.2.00±0.58, P<0.05) and IL-12(1.00±0.05 vs.1.49±0.22, P<0.05) increased and the anti-inflammatory cytokine IL-10(1.00±0.22 vs.0.09±0.01, P<0.05) decreased. Conclusion:Polarization of macrophages towards the pro-inflammatory M1 subtype plays an important role in the pathogenesis of NEC.
摘要
Objective To study the activity of macroautophagy and chaperone-medicated antophagy (CMA) in dopaminergic cells under oxidative stress and their possible roles in neurodegeneration.Methods PC 12 cells were treated with various concentrations of rotenone (0,20,100 and 500 nmol/L).The morphological characteristics of autophagosomes were observed by transmission electron microscopy (TEM),and macroautophagic activity was determined by detecting light chain (LC3) expression of microtubule-associated protein by Western blotting and monodansylcadaverine (MDC) immunofluorescence.Molecular chaperone mediated autophagy was estimated by the expressions of lysosomal-associated membrane protein 2α (Lamp-2α),heat shock cognate 70 (Hsc70) and heat shock protein 90 (Hsp90),which were detected by Western blotting.Results In control group (0 nmol/L rotenone),TEM showed that the cells presented a low autophagic activity with less autophagic vacuoles; in rotenone treatment groups,the autophagosomes with different forms were significantly increased,and the phenomenon ofautophagic mitochondria was observed.A large of vacuole-like bodies were found in the cytoplasm of the cells treated with high concentration of rotenone.The MDC immunofluorescence showed that after the treatment of rotenone,the red fluorescence showed punctate distribution,which was also significantly enhanced with the increasing of the concentrations of rotenone.Western blotting showed dose-dependent expressions of LC3-Ⅰ and LC3-Ⅱ.As compared with control cells,the expressions ofLamp-2α,Hsc70 and Hsp90 in rotenone treatment groups were also increased significantly (P＜0.05),but didn't show statistical difference between each two rotenone treatment groups (P＞0.05).Conclusions The macroautophagy and autophagy mediated by molecular chaperone are both activated in PC12 cells under oxidative stress conditions.And the increased oxidative stress results in an enhanced activity of macroautophagy,while the activity of CMA is saturated.