| Purpose:Diabetic retinopathy(DR)is the primary cause of blindness in adults.There are significant neuropathy in the early stage.Salidroside(Sal)has neuroprotective effect,and has obvious prevention and treatment effect on many complications of diabetes,but the effect of Sal on DR neuropathy is not clear.The purpose of this study is to explore the neuroprotective effect of Sal on diabetic retinopathy in rats,and to explore the mechanism of action through PI3K/Akt signaling pathway,hoping that this study can further reveal the pathogenesis of DR,and provide theoretical basis for the clinical prevention and treatment of DR by Sal.Material and methods:Part?: SD rats were intraperitoneally(IP)injected with 1% Streptozotocin(STZ)at the dose of 50 mg/kg.After 72 hours,tail vein blood sugar concentration more than16.7 mmol/L after STZ injection were considered diabetic.Rats were randomly divided into control group,diabetes group,Sal low-dose group,Sal medium-dose group,Sal high-dose group and metformin group(positive control).Sal low-dose group was given 50 mg·kg-1·d-1,Sal medium-dose group was given 100 mg·kg-1·d-1,Sal high-dose group was given 200 mg·kg-1·d-1,metformin group was given 20mg·kg-1·d-1,control group and diabetic group were given the same dose of saline.After 12 weeks,blood glucose and body weight were measured.The density of retinal inner nuclear layer(INL)cells and retinal ganglion cells(RGC)were measured by HE staining.Part?: STZ-induced diabetic rats just as we described in Part?.Rats were randomly divided into control group,diabetes group,Sal treatment group and Sal+LY294002 group.The diabetic rats in Sal treatment group were given 100mg·kg-1·d-1 by gastric perfusion.In Sal+LY294002 group,except 100 mg·kg-1·d-1 Sal by gastric perfusion,PI3K/Akt pathway inhibitor LY294002 was injected into vitreous once at a dose of 5 ?l,the concentration was 80 ng·m L-1,once every 4 w,three times in total.The control group and diabetic group were given equal dose of PBS.After 12 weeks,blood sugar and body weight of rats were measured,the density INL and RGC were detected by HE staining,the expression of p-Akt was detected by immunohistochemical staining,and the expression of glial fibrillary acidic protein(GFAP)and glutamic acid/aspartate transporter(GLAST)were detected by immunofluorescence.Western blot was used to detect the relative expression of glutamine synthetase(GS),GFAP,GLAST,Akt and p-Akt in retina,and high performance liquid chromatography was used to detect the content of glutamate(Glu)in retina.Part ?: Müller cells were cultured with DMEM+10% FBS.After 24 hours of cell culture,Müller cells were treated with different treatment factors.The cells were divided into control group,high glucose group(30 mmol/L glucose),Sal treatment group(30 mmol/L glucose+120 ug/m L Sal)and Sal+LY294002 group(30mmol/L glucose+120 ug/m L Sal+25?mol/L LY294002).The indexes were detected on the third day of culture.Methyl thiazolyl tetrazolium(MTT)colorimetry was used to detect the viability of Müller cells,the expression of GLAST and GS were detected by immunofluorescence,the apoptosis of Müller cells were reflected by TUNEL staining,the expression of GLAST,GS,Akt,p-Akt,Bcl-2 and Bax in Müller cells were obserbed by Western blot.Results:Part?1.Effects of Sal on blood sugar and body weight in diabetic rats: Rats in control group showed obviously body weight gaining.Compared with the control group,the body weight of diabetes group,Sal low-dose group,Sal medium-dose group,Sal high-dose group and metformin group decreased significantly(P < 0.01).Compared with the control group,the blood sugar of diabetes group,Sal low-dose group,Sal medium-dose group and Sal high-dose group were more than 16.7 mmol/L(P < 0.01),while the blood sugar of metformin group had no significant change compared with the control group(P > 0.05).Compared with the diabetic group,the blood glucose were decreased in the low dose Sal group(P < 0.05),the middle dose Sal group and the high dose Sal group(P < 0.01),but all of them were higher than the critical value of 16.7 mmol/L of diabetic rats.2.Effect of Sal on the density of INL and RGC: Compared with the control group,the density of INL and RGC in the diabetic group decreased significantly(P < 0.01).Compared with the diabetes group,the cell density of Sal middle dose group,Sal high dose group and metformin group increased(P < 0.01),but there was no significant change between Sal middle dose group and Sal high dose group(P > 0.05).Part?1.Effects of Sal on blood sugar and body weight in diabetic rats: Compared with the control group,the body weight of diabetes group,Sal treatment group and Sal+LY294002 group decreased significantly(P < 0.01).There was no significant change in body weight among diabetes group,Sal treatment group and Sal+LY294002 group(P > 0.05).Compared with the control group,the blood sugar in diabetic group was more than 16.7 mmol/L(P < 0.01).Compared with the diabetic group,the blood sugar of Sal treatment group and Sal+LY294002 group decreased but were more than the critical value of 16.7 mmol/L of diabetic rat(P < 0.01).2.Effect of Sal on the density of INL and RGC:Compared with the control group,the density of INL and RGC in the diabetic group were significantly decreased(P < 0.01).Compared with the diabetic group,the density of INL and RGC in Sal treatment group and Sal+LY294002 group increased(P < 0.01),and the Sal treatment group increased more significantly(P > 0.05).3.Effect of Sal on the expression of p-Akt in retina: Compared with the control group,the expression of p-Akt in the diabetic group was significantly lower(P < 0.01),while the expression of p-Akt in the Sal treatment group was significantly higher(P < 0.01)than that in the diabetic group,and the expression of p-Akt in the Sal + LY294002 group was decreased(P < 0.05).4.Effect of Sal on the expression of retinal GFAP in diabetic rats: Compared with the control group,the expression of GFAP in diabetes group was high and penetrated the whole retina(P < 0.01).Compared with diabetes group,the expression of GFAP in Sal treatment group and Sal+LY294002 group decreased,especially in Sal treatment group(P < 0.01).5.Effect of Sal on the expression of retinal GLAST in diabetic rats: Compared with the control group,the expression of GLAST in the diabetic group decreased significantly(P < 0.01),while the expression of GLAST in Sal treatment group and Sal+LY294002 group increased,and the Sal treatment group increased more significantly(P < 0.01).6.Effect of Sal on relative expressions of GS,GFAP,GLAST,Akt and p-Akt in retina of diabetic rats: Compared with the control group,the expression of GFAP increased significantly(P < 0.01),GS,GLAST,p-Akt decreased significantly(P<0.01),and Akt expression did not change significantly(P > 0.05).Compared with diabetes group,the expression of GFAP decreased significantly in Sal treatment group(P < 0.01),while the expression of GS,GLAST and p-Akt increased significantly(P< 0.01),while the expression of Akt did not change significantly(P > 0.05),while LY294002 could reverse the therapeutic effect of Sal to some extent.7.Effect of Sal on Glu content in retina of diabetic rats: Compared with the control group,the Glu content in diabetic group increased significantly(P < 0.01).Compared with diabetes group,the Glu content in Sal treatment group and Sal+LY294002 group decreased,and the Sal treatment group decreased more significantly(P < 0.01).Part ?1.Effect of Sal on morphology and cell growth of Müller cells in different groups:Microscopic observation showed that compared with the control group,the growth of cells in the high glucose group was slower,the cell body was swollen,the refractive index of the cell edge was enhanced,the number of cell processes was less and the processes atrophied.Compared with the high glucose group,the Sal treatment group and Sal+LY294002 group improved the above-mentioned state,and the Sal treatment group improved more significantly.2.Effect of Sal on the viability of retinal Müller cells: Compared with the control group,the cell viability in the high glucose group was significantly decreased(P <0.01).Compared with diabetes group,cell viability of Sal treatment group and Sal+LY294002 group increased,and the Sal treatment group increased more significantly(P < 0.01).3.Effect of Sal on the expression of GLAST and GS in retinal Müller cells: The expressions of GLAST and GS in diabetic group were significantly lower than in control group(P < 0.01).Compared with diabetes group,the expression of GLAST and GS in Sal treatment group and Sal+LY294002 group increased,and the Sal treatment group increased more significantly(P < 0.01).4.Effect of Sal on the expression of GLAST,GS,Akt,p-Akt,Bcl-2 and Bax in retinal Müller cells: Compared with the control group,the expression of Bax increased significantly(P < 0.01),while the expression of GS,GLAST,p-Akt and Bcl-2decreased significantly(P < 0.01),while the expression of Akt did not change significantly(P > 0.05).Compared with diabetes group,the expression of Bax in Sal treatment group was significantly decreased(P < 0.01),the expression of GS,GLAST,p-Akt,Bcl-2 were significantly increased(P < 0.01),the expression of Akt was not significantly changed(P > 0.05),while LY294002 could reverse the therapeutic effect of Sal to some extent.5.Effect of Sal on apoptosis of retinal Müller cells: Compared with the control group,the apoptotic rate of the diabetic group increased significantly(P < 0.01),while the apoptotic rate of Sal treatment group and Sal+LY294002 group decreased significantly,and the Sal treatment group decreased more significantly(P < 0.01).Conclusions:1.Sal could reduce the blood glucose of diabetic rats,and prevent the decrease of the number of retinal inner layer cells and RGC.2.Sal could up-regulate the expression of GLAST and GS,down-regulate the expression of GFAP and down-regulate the content of Glu in the retina of diabetic rats,which may be related to the activation of PI3K/Akt signaling pathway.3.Sal could up-regulate the expression of GLAST and GS,increase the ratio of Bcl-2/Bax,and reduce the apoptosis of Müller cells induced by high glucose.The mechanism was related to the activation of PI3 K /Akt signaling pathway. |
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