CD137-CD137l Signaling Influences The Autophagy Via JNK Pathway In Mouse Vascular Smooth Muscle Cells

ObjectiveTo investigate whether CD137-CD137 L signaling could affect the autophagy of vascular mouse smooth muscle cells through c-Jun N-terminal kinase(JNK)signal pathway.MethodsPrimary culture of C57 BL / 6J mouse thoracic aorta vascular smooth muscle cells(VSMCs)was performed by tissue block adherence method.VSMCs were divided into four groups: control group,agonist-CD137 group,JNK inhibition group and DMSO group.Western blot was used to detect the protein expression of phosphorylate c-Jun N-terminal kinase(p-JNK),microtubule associated protein1 light chain3 II(LCII)and Sequestosome1(p62/SQSTMl)in each group.Fluorescence microscopy was used to track the changes of autophagy in cells which was infected tandem mRFP-GFP-LC3 adenovirus.Transmission electron microscope(TEM)was used to observe intracellular autophagosomes and autolysosomes.Results(1)Compared with the control group,stimulating CD137-CD137 L axis increased the expression of p-JNK,LCII and p62(P<0.05).These effects could be reduced by JNK inhibitor(P<0.05).The expression of these proteins in DMSO group did not change significantly compared with stimulate group(P>0.05).(2)The number of total fluorescent spots and yellow fluorescent spots in agonist-CD137 group was more than that in control group(P<0.05)and the yellow fluorescent spots was higher than the red in stimulate group(P<0.05).Compared with agonist-CD137 group,adding JNK inhibitor could decrease the number of total fluorescent spots and yellow fluorescent spots(P<0.05).The red fluorescent spots was higher than the yellow in JNK inhibition group(P<0.05).The condition of DMSO group was approximately same as stimulate group(P>0.05).(3)The number of intracellular autophagosomes and autolysosomes in agonist-CD137 group was higher than control group(P<0.05)and the number of autophagosomes was greater than that of autolysosomes in stimulate group(P<0.05).These situations were improved in JNK inhibition group(P<0.05).There were no significant difference in intracellular autophagosomes and autolysosomes between DMSO group and agonist-CD137 group(P>0.05).ConclusionCD137-CD137 L may influence autophagy of mouse VSMC by JNK signal pathway.