Complement C3 Upregulation Via RAGE Is Involved In The Mechanisms Of Diabetic Encephalopathy

Objectives:1.To investigate whether the C3 and RAGE alteration exists in the hippocampus of diabetic mice;2.To investigate whether C3 upregulation can be modulated by RAGE under high glucose condition and elucidate the molecular mechanisms;3.To investigate the effect of RAGE and C3 upregulation on the structure and cognitive function of diabetic mice.Methods:1.In vivo:(1)C57BL/6J mice were randomly divided into four groups: Control group,DM group,DM+FPS-ZM1 group and DM+C3aRA group.Type I diabetic mice were induced by a single 150 mg/kg intraperitoneal injection of STZ.Three months after diabetic model induction,mice in the DM+FPS-ZM1 group and in the DM+C3aRA separately received 1mg/kg/d intraperitoneal injection of FPS-ZM1(0.1 mg/mL)and C3aRA(60mg/kg)for 4 weeks.(2)Assay the learning and memory by Morris water maze.(3)Double immunofluorescence staining GFAP with RAGE and C3respectively;immunofluorescence staining SYP to determine the expression of SYP in each group.(4)Measurement of C3,RAGE,p38 MAPK,NF-?B,SYP and PSD-95 protein levels by Western blot.(5)To observe the number of neuronal cells and apoptosis by Nissl and TUNEL staining.2.In vitro:(1)Astrocytes were randomly divided into 8 groups:Normal glucose(NG)group;High glucose(HG)15 mM group;HG30mM group;NG+30 mM mannitol group;HG15 mM+15 mM mannitol group;HG30mM+FPS-ZM1 group;HG30mM+SB203580 group;HG30mM+ PDTC group.(2)Measurement of C3 mRNA by qRT-PCR.(3)Measurement of protein level of C3 and RAGE related signaling molecules.(4)To measure ROS contents in astrocytes by flow cytometry.(5)To determine C3 secreted level from astrocytes by ELISA.Results:1.In vivo:(1)In the Morris water maze,the escape latency of diabetic mice was longer than control mice,which was improved by the FPS-ZM1 and C3 aRA treatment.The number of crossing the platform and the time spent in the target region in the diabetes group was more than the control group.However,FPS-ZM1 group and C3 aRA group had the less crossing frequency of platform and time spent in the target region.(2)Double label immunofluorescence staining with C3 and GFAP showed that C3 immunoreactivity increased in the astrocytes of the diabetic brain as compared to the control group.However,the FPS-ZM1 treatment significantly reduced the C3 immunoreactivity in the diabetic brain.In addition,enhancement of RAGE immunoreactivity in the astrocytes stained by GFAP in the CA1 region of diabetic hippocampus compared to the control group,which were significantly attenuated by the FPS-ZM1 treatment.Moreover,the density of SYP immunoreactivity decreased in the diabetic hippocampus as compared to the control group,which were improved by FPS-ZM1 and C3 aRA treatment separately.(3)By western blot,we observed upregulated RAGE/p38MAPK/NF-?B and C3 protein expression and downregulated SYP and PSD-95 expression in the diabetic mice as compared to the control group,which were attenuated in the diabetic mice treated with FPS-ZM1.(4)Nissl staining showed that neuronal cell density decreased in diabetic mice as compared to the control mice.However,FPS-ZM1 andC3 aRA treatment significantly increased the neuronal cell density in diabetic mice,respectively.TUNEL staining showed that apoptosis index increased in hippocampus of diabetic mice as compared to the control mice,which was ameliorated by FPS-ZM1 and C3 aRA treatment.2.In vitro:(1)Western blot and ELISA results showed that C3 protein in cell extracts and secreted level in medium elevated significantly in the HG15 mM and HG30 mM groups.In addition,there was no additional effect of mannitol on both intra-and extra-cellular C3 expression protein.Moreover,by Western blot,HG30 mM induced increasing RAGE protein level and phosphorylated level of p38 MAPK and NF-?B in astrocyte as compared to the NG group.While inhibition of RAGE,p-p38 MAPK and p-NF-?B respectively can reverse the intra-and extra-cellular C3 upregulation caused by high glucose.(2)Results from qRT-PCR showed that C3 mRNA in astrocytes of HG30 mM group was higher than that of NG,which can be reduced by the pretreatment of RAGE,p38 MAPK and NF-?B inhibitors separately.(3)The ROS contents elevated and Crry expression decreased in astrocytes in the HG30 mM group as compared to the NG group,which can be attenuated by the RAGE inhibitor FPS-ZM1.Conclusion:1.C3 upregulated in astrocytes of hippocampus in diabetic mice;2.Astrocytic RAGE upregulation mediated the activation of p38 MAPK and NF-?B,which modulated the transcription and secretion of C3;3.High glucose induced ROS accumulation in astrocytes by RAGE activation,resulting in downregulation of Crry and C3 activation;4.C3 upregulation via RAGE signaling mediated the neurodegeneration and cognitive dysfunction in diabetic mice.