Hgsd Prevents Diabetic Retinopathy Through The AMPK Signaling Pathway

Diabetic retinopathy(DR)is a common chronic complication of diabetes which could frequently lead to blindness in working-age people in the world.In recent years,the studies have found that DR is not only the microvascular disease,but also the neurodegenerative disease.Before any microcirculatory abnormalities occur,retinal cells have been injured and could been be detected by electroretinogram(ERG).However,the pathogenesis of DR neurodegeneration has not been known clearly.Müller cells are the principal glial cells in retinas and play a key role in the progress of DR.Müller cells could support and nourish the retinal neuron cells,regulate the signaling transportations and maintain the integrity of the blood-retinal barrier and homeostasis.While apoptotic death of Müller cells could result in the abnormalities of glutamate cycle,increase of vascular endothelial growth factor(VEGF)and is an important cause of the pathogenesis of DR.Studies have shown that defect or abnormality of autophagy could lead to apoptosis and AMPK/m TOR signaling pathway is related to the modulation of autophagy.Berberine is a kind of isoquinoline alkaloid which is extracted from rhizomacoptidis and cortex phellodendri and is a traditional therapy for diarrhea and digestive tract infections.Recently,more and more studies have found berberine exerts positive protect effects on regulating blood pressure and blood cholesterol and lowering blood glucose.Furthermore,many effects of berberine could be mediated through activation of AMPK pathway.Nevertheless,the oral bioavailability of berberine is so poor that has been limited.To promote the oral bioavailability of BBR,our laboratory has developed an amorphous solid dispersion called Huang-Gui solid dispersion(HGSD)that could protect against neuroapoptosis induced by diabetes without any gastrointestinal damage.Experimental evidence has shown that HGSD could effectively improve the bioavailability of BBR and protect against neurotoxicity induced by diabetes without any gastrointestinal damage.However,the effect of HGSD in DR has not been reported.Aim:In vivo,type 2 diabetic mice were induced through injection of STZ and high fat diet to explore the protective activity of HGSD in modulating AMPK in DR.In vitro,Müller cells were induced by high glucose and given AICAR and Compound C to further explore the protective mechanism of HGSD through AMPK/m TOR signaling pathway.Method:1.In vivo,the protective activity of HGSD in modulating AMPK in DRType 2 diabetic mice were induced through injection of STZ and high fat diet.Retinal function was assessed through electroretinogram,apoptosis was detected through TUNEL assay and the levels of anti-apoptotic protein Bcl-2,pro-apoptotic protein Bax,autophagy-related proteins Beclin-1 and LC3-II and phosphorylated AMPK and phosphorylated m TOR were detected through immunohistochemistry.2.In vitro,the mechanism of the protective effect of HGSD via AMPK/m TOR signaling pathwayApoptosis model of Müller cells was established by using high glucose.The level of autophagy was detected by MDC;the changed of nucleus were assayed through Hoechst33258.Western blot was used to detect the expressions of apoptosis related proteins,autophagy related proteins,p-AMPK/AMPK and p-m TOR/m TOR.Müller cells were treated with compound C and AICAR,staining was used to assess the changed of nucleus and MDC staining was used to assess the autophagy of Müller cells.The levels of anti-apoptotic protein Bcl-2,pro-apoptotic protein Bax,autophagy-related proteins Beclin-1 and LC3 and phosphorylated AMPK and phosphorylated m TOR were assayed through Western Blot.Results:In vivo,ERG revealed a significant decrease in the amplitude and increase in the implicit time of b-wave.Furthermore,the number of TUNEL-positive cells and the level of pro-apoptotic protein Bax were significantly increased,and the level of anti-apoptotic protein Bcl-2 was significantly decreased.Furthermore,there was a marked reduction of autophagy-related proteins Beclin-1 and LC3-II.There was a significant tendency toward decrease of phosphorylated AMPK and increase of phosphorylated m TOR in retina of diabetic mice.Treatment with HGSD effectively restored all of the changes mentioned above in diabetic mice.In vitro,compared with the control group,the condensation and fragmentation of nuclear were increased and the green fluorescent particles of cytoplasm were decreased in HG group.Furthermore,the level of pro-apoptotic protein Bax was significantly increased,and the level of anti-apoptotic protein Bcl-2 was significantly decreased in HG group as compared with the control group.There was a marked reduction of autophagy-related proteins Beclin-1 and LC3-II and there was a significant tendency toward decrease of phosphorylated AMPK and increase of phosphorylated m TOR in the Müller cells induced by HG.Treatment with BBR effectively restored all of the changes mentioned above in HG group.The above-mentioned effects of BBR were diminished after treatment with Compound C and were increased through treatment with AICAR.Conclusion: HGSD could treat DR and it possible mechanisms may be related with downregulation of Müller cells apoptosis through improvement of autophagy via activating AMPK/m TOR signaling pathway.