ABSTRACT
@#AIM:To investigate the role of cellular autophagy in the expression of IL-1β and IL-6 by retinal pigment epithelium(RPE)cells under hypoxia.<p>METHODS: A cultured human RPE cell line was randomly divided into the hypoxia group, 3-methyladenine(3-MA)+ hypoxia group, chloroquine(CQ)+ hypoxia group and the control group. Cells incubated in a hypoxic incubator were set as the hypoxia group. After culture for 24h, western blot was used to analyze the relative expression of autophagy-associated key proteins in each group, including microtubule associated protein 1 light chain 3B(LC3B), Beclin-1 and p62. Then, ELISA was applied to detect the concentration of IL-1β and IL-6 in the cell culture supernatants.<p>RESULTS: The concentration of IL-1β and IL-6 in the hypoxia group was significantly higher than that in the control group 3-MA+hypoxia group and CQ+ hypoxia group(all <i>P</i><0.01). The relative expression of LC3B-II/I and Beclin-1 in the hypoxia group was significantly higher than that in the control group and 3-MA+hypoxia group. The relative expression of LC3B-II/I in the hypoxia group was significantly lower than that in the CQ+ hypoxia group(all <i>P</i><0.01). The relative expression of p62 in the hypoxia group was significantly lower than that in the control group, 3-MA+hypoxia group and CQ+ hypoxia group(all <i>P</i><0.01).<p>CONCLUSION: Hypoxia can enhance the expression of LC3B-II/I and Beclin-1 in RPE cells, weaken the expression of p62, and promote the secretion of IL-1β and IL-6. Autophagy inhibitors 3-MA and CQ can reduce the expression of IL-1βand IL-6 in RPE cells.
ABSTRACT
Objective To investigate the role of IL-17 in immune inflammatory reaction after spinal cord injury, and provide more evidence for clinical treatment of spinal cord injury on cytokine levels. Methods Male C57BL/6 mice were randomly divided into 4 groups: in the spinal cord injury group, mice were made into spinal cord clamp model. In the sham surgery group, the dura was cut without injuring the spinal cord. The IL-17 neutralizing antibody group received IL-17 neutralizing antibody injection through the cadual vein at 1 h after the spinal cord clamp. The solvent control group received the sterile PBS (0.01 μmol/L) through the cadual vein at 1 h after the spinal cord clamp. Mouse scale for locomotion(BMS) was applied to evaluate the mice's behavior change of hindlimb in 1-7 d. The real time fluorescent quantitative PCR was used to detect the expression changes of IL-1β、IL-6 and TNF-αmRNA, and the immunohistochemistry technique was conducted to observe the morphological changes of neurons of NeuN on the 7th day after spinal cord injury respectivly. Results The behavior score of mice after spinal cord injury indicates: the BMS scores were all 9 on the 1st to the 7th day in the sham surgery group, but were 0 on the 1st day in the model group, the IL-17 neutralizing antibody group and the solvent control group. With time extension, the motor function of hindlimbs of mice in each group were improved, but improved even better in the IL-17 neutralizing antibody group than in the model group and the solvent control group. Immunohistochemistry staining showed that after spinal cord injury, there were much complete structure of NeuN positive staining cells in the gray matter in the sham surgery group, which were obviously shrinking and protrusions disappearing in the model group and the solvent control group, while large number of NeuN neurons vacuolated and reduced significantly. It could be seen that part of neurons morphology was normal and with complete NeuN neuronal cell bodies and branches of the synapse, and the amount of NeuN neuron staining positive cells rebounded in the IL-17 neutralizing antibody group. The results of RT-qPCR on the 7th day after spinal cord injury indicated that compared with sham surgery group, IL-1β mRNA increased significantly in the model group and the solvent control group(P<0.01); compared with the model group and the solvent control group, IL-1β mRMA decreased significantly in the IL-17 neutralizing antibody group(P<0.05); compared with sham surgery group , TNF-α mRNA increased significantly in the model group (P<0.01); compared with the sham surgery group, TNF-α mRNA increased significantly in the solvent control group (P<0.05); compared with the model group , TNF-α mRNA decreased significantly in the IL-17 neutralizing antibody group (P< 0.05). IL-6 mRNA expression was on the decline in the IL-17 neutralizing antibody group, but without statistically significant difference with other groups. Conclusion Combined action of IL-17, IL-1β, IL-6 and TNF-α deteriorates the immune inflammatory of spinal cord injury, and it might relieve spinal cord injury in mice by inhibition of IL-17.