To Investigate The Protective Effect Of Salidroside On DR Based On COX2/PGE₂ Signaling Pathway

Purpose:Salidroside(Sal),a major component isolated from the medicinal plant Rhodiola rosea L.,has strong antioxidant,anti-inflammatory,hypoglycemic and other protective effects.In vivo experiments were conducted to observe the protective effect of Sal on diabetic retinopathy(DR)and its inhibitory effect on COX2/PGE₂pathway.In vitro experiments were conducted to analyze the effect of SAL on COX2/PGE₂ signaling pathway in Retinal Muller cell line(rMC-1)reduced by high glucose in rats,and to explore the protective effect and mechanism of sal on diabetic retinopathy,so as to provide experimental basis for the research and treatment of sal in diabetic retinopathy.Material and method:Part 1:75 rats of 8-week-old healthy SPF grade SD(Sprague-Dawley)male,weighed(230±20)g.The rats were randomly divided into 5 groups,with a total of 15 rats in each group:control group,diabetes group,diabetes+Sal low dose(50mg·kg-1·d-1)group,diabetes+Sal medium dose(100mg·kg-1·d-1),diabetes+Sal high dose (200mg·kg-1·d-1)group,respectively.Rats in the diabetic model group were intraperitoneally injected with a dose of 65 mg·kg^-1streptozotocin(STZ).The rats were measured on the 3rd and 5th day.Rats with blood glucose?16.7mmol/L were considered diabetic.Diabetes+Sal low dose(50mg·kg^-1·d^-1)group,diabetes+Sal medium dose(100mg·kg^-1·d^-1),diabetes+Sal high dose(200mg·kg^-1·d^-1)group were given the corresponding dose of Sal.And the same amount of normal saline was given to control group and diabetic group.The above group were injected continuously for 12 weeks.After 12 weeks,the blood glucose and body weigh were measured.HE staining was used to detect the effect of Sal on Retinal ganglion cells(RGC)staining.The expression of glial fibrillary acidic protein(GFAP)in each group was detected by Immunofluorescence assay.Part 2:The expression of cyclooxygenase-2(COX2)was detected by immunohistochemistry of the retinal tissues.The expression levels of brain derived neurotrophic factor(BDNF),prostaglandin E2(PGE₂),NF-?B,Tumor necrosis factor-?(TNF-?)and interleukin-6(IL-6)were detected by Western blot.The activities of Superoxide Dismutase(SOD)and cata-lase(CAT)in retinal tissues of rats were detected by kit.Part 3:The rMC-1 cells were cultured in DMEM containing 10%fetal bovine serum(FBS)at 37°C in a 5%CO₂,95%humidified air atmosphere.RMC-1 cells in logarithmic growth phase were digested with trypsin and inoculated in 96-well plates at the cell density of 1×10⁴/well.The cells were cultured in normal medium until they were completely adherant to the wall,and the growth status was good under the microscope.The rMC-1 cells were treated with 0?0.125?0.25?0.5?1?mol/L SAL for24,48 and 72hr each.Then CCK-8 was added to each well for 1.5hr.Subsequently,the absorbance was assessed with an ELISA plate reader.And the optimal concentration and action time of SAL were selected for subsequent cell experiments.The experimental settings were:normal control group(NC,5.5 mmol/L),some cells were incubated under normal culture medium;High glucose group(HG,35.5 mmol/L),some cells were induced by high glucose;SAL(0.5 mmol/L)plus HG(35.5mmol/L)medium group(HG+SAL),cells were treated with SAL in HG medium;COX2 inhibitor(NS398,10?mol/L)group(HG+NS398),cells were treated with COX2 inhibitor in HG medium.The level of PGE₂in rMC-1 cells induced by HG was measured by ELISA.The expression of BDNF in rMC-1 cells induced by high glucose was detected by immunofluorescence assay,and the effect of SAL on the apoptosis of rMC-1 cells induced by high glucose was detected by flow cytometry.The expressions of COX2,NF-?B,TNF-?,IL-6 and proapoptotic proteins of Caspase-1,Caspase-3,Bcl-2 and Bax were used by Western blot.The effect of salidroside on reactive oxygen species(ROS)levels in RMC-1 cells induced by high glucose was detected by fluorescence probe.The activity of SOD and CAT in RMC-1cells induced by high glucose was detected by salidroside with enzyme label analyzer.Results:Part 11.Effects of Sal on blood glucose and body weight of rats in each group.Compared with the control group,the blood glucose of the diabetic model group (diabetes group and diabetes+Sal low,medium and high dose group)increased significantly and the weigh reduced significantly after 12W(P<0.01).Compared with the diabetes group,the blood glucose of the diabetes+Sal low dose group(P<0.05),diabetes+Sal medium dose group and diabetes+Sal high dose group(P<0.01)decreased significantly,and the weigh increased significantly.2.Effect of salidroside on RGC of rats in each groupAfter 12 weeks of intragastric administration,compared with the control group,the density of RGC in diabetic group was significantly decreased(P<0.01).Compared with diabetic group,there was no significant difference in RGC density in diabetic+Sal low-dose group(P>0.05).With the increase of Sal dose,compared with the diabetes group,the density of RGC in diabetes+Sal medium dose group and diabetes+Sal high dose group increased significantly(P<0.05).But there were no statistical differences in the density of RGC between diabetes+Sal medium dose group and diabetes+Sal high dose group(P>0.05).3.Effect of salidroside on GFAP expressionAfter 12 weeks of intragastric administration,GFAP expression was significantly increased and fluorescence intensity was significantly enhanced in diabetic group,even in almost all retinal layers(P<0.01)compared with the control group.Compared with the diabetic group,the expression level of GFAP in the diabetic+Sal low-dose group,diabetic+Sal medium-dose group(P<0.05)and diabetic+Sal high-dose group(P<0.01)was decreased,and the decrease was most obvious in the diabetic+Salhigh-dose group.4.Effect of salidroside on GS expression Compared with the control group,the expression of GS in diabetic group was significantly decreased(P<0.01).There was no significant difference in GS expression in diabetic+Sal low-dose group compared with diabetic group(P>0.05).With the increase of Sal dose,GS expression level in diabetic+Sal medium dose group and diabetic+Sal high dose group was significantly increased(P<0.05).Part 21.Effect of salidroside on the expression of COX2 and PGE₂in retinal tissues of rats in each groupAfter 12 weeks of intragastric administration,immunofluorescence results showed that COX2 fluorescence intensity in retinal tissue of diabetic rats was significantly increased(P<0.01).Compared with the diabetes group,the fluorescence intensity of COX2 decreased to varying degrees in diabetes+Sal low dose group,diabetes+Sal medium dose group(P<0.05)and diabetes+Sal high dose group,and diabetes+Sal high dose group weakened the most obvious(P<0.01).The expression of PGE₂was detected by ELISA.Compared with the control group,the expression of PGE₂in the diabetes group were significantly increased(P<0.01).Compared with the diabetes group,the expression of PGE₂in diabetic+Sal low-dose group,diabetic+Sal medium-dose group(P<0.05)and diabetic+Sal high-dose group were significantly decreased,and diabetes+Sal high dose group,and diabetes+Sal high dose group weakened the most obvious(P<0.01).2.Effects of Sal on the expression of BDNF in retinal tissues.The expression of BDNF was detected by Western blot.Compared with the control group,the expression of BDNF in the diabetes group were significantly decreased(P<0.05).Compared with the diabetes group,there were no statistical differences in the expression of BDNF between diabetes group and diabetes+Sal low dose group(P>0.05).With the increase of Sal dose,compared with the diabetes group,the expression of BDNF in diabetes+Sal medium dose group and diabetes+Sal medium dose group increased significantly(P<0.05).3.Effects of Sal on the expression of NF-?B,TNF-?and IL-6 in retinal tissues.The expression of NF-?B,TNF-?and IL-6 was detected by Western blot.Compared with the control group,the expression of NF-?B,TNF-?and IL-6 in the diabetes group were significantly increased(P<0.01).Compared with the diabetes group,there were no statistical differences in the expression of NF-?B,TNF-?and IL-6between diabetes group and diabetes+Sal low dose group(P>0.05).With the increase of Sal dose,compared with the diabetes group,the expression of NF-?B,TNF-?and IL-6 in diabetes+Sal medium dose group and diabetes+Sal medium dose group increased significantly(P<0.05).4.Effects of Sal on the activities of SOD and CAT in retinal tissues.The activities of SOD and CAT in diabetic group were significantly decreased compared with the control group(P<0.01)after 12 weeks of intragastric administration.Compared with diabetic group,there were no significant differences in SOD and CAT activities in diabetic+Sal low-dose group(P>0.05).With the increase of sal dose,SOD and CAT activity forces in diabetic+Sal medium dose group and diabetic+Sal high dose group were significantly increased(P<0.05).Part 31.The effects of SAL on HG-induced cell viability.To assess the biological role of SAL on rMC-1cells,cells were subcultured in different doses of SAL(0,0.125,0.25,0.5,1?M)for 24 hr,48 hr and 72hr separately.When treated for 24 h,there was no significant effect of SAL on cell viability in the rMC-1 cells compared with that in NC conditions(P>0.05).Next,the cells were treated with different doses of SAL for 48hr and 72hr in HG culture conditions.SAL(0.5 and 1?M)markedly protected rMC-1 cells from HG-induced inhibition(P<0.05).Thus,SAL at 0.5?M and 48hr time points was used for further study.2.The effects of SAL on HG-induced cell morphology.Compared with the NC group,the growth of cells in the HG group was slower,the cell body was smaller.Compared with the HG group,the cell morphology of HG+SAL group and HG+NS398 group was improved,the cell body was enlarged.3.The effects of SAL on the expression of COX2 and PGE₂in rMC-1 cells under HG conditions.Compared with the NC group,the expression of COX2 and PGE₂in rMC-1 cells of the HG group were significantly increased(P<0.01).Compared with the HG group,the expression of COX2 and PGE₂in rMC-1 cells of the HG+SAL group and HG+NS398 group were significantly decreased(P<0.05).4.The effects of SAL on the expression of BDNF in rMC-1 cells under HG conditions.Compared with the NC group,the expression of BDNF in rMC-1 cells of the HG group were significantly decreased(P<0.01).Compared with the HG group,the expression of BDNF in rMC-1 cells of the HG+SAL group and HG+NS398 group were significantly increased(P<0.05).5.The effects of SAL on the expression of NF-?B,TNF-?and IL-6 in rMC-1 cells under HG conditions.Compared with the NC group,the expression of NF-?B,TNF-?and IL-6 in rMC-1 cells of the HG group were significantly increased(P<0.01).Compared with the HG group,the expression of NF-?B,TNF-?(P<0.01)and IL-6(P<0.05)in rMC-1 cells of the HG+SAL group and HG+NS398 group were significantly decreased.6.Effects of Sal on the expression of ROS,SOD and CAT in RMC-1 cellsCompared with the control group,the ROS level in RMC-1 cells of high glucose group was increased,and the activities of SOD and CAT were significantly decreased(P<0.01).Compared with high glucose group,ROS level in SAL intervention group and COX2 inhibitor group was significantly decreased,while SOD and CAT activities were significantly increased(P<0.05).7.The effects of SAL on apoptosis of rMC-1 cells under HG conditions.Compared with the control group,the expressions of Caspase-1,Caspase-3 and Bax in RMC-1 cells of high glucose group were significantly increased(P<0.01),while the expression of Bcl-2 was significantly decreased(P<0.01).Compared with the high glucose group,the expressions of Caspase-1,Caspase-3 and Bax in Sal treatment group and COX2 inhibitor group were significantly decreased(P<0.05),while the expression of Bcl-2 was significantly higher(P<0.05).In addition,Annexin-V and PI staining of flow cytometry analysis also showed that SAL could protect rMC-1 cells from HG-induced apoptosis.Compared with the NC group,the apoptosis rate of rMC-1 cells in HG group was significantly increased(P<0.05).Compared with HG group,apoptosis rate of cells in the HG+SAL group and HG+NS398 group was significantly decreased(P<0.05).Conclusions:1.Sal had no toxic effects on blood glucose,body weight and RGC cell morphology of normal rats.However,it could protect RGC,reduce the blood glucose and prevent weight gain.2.Sal could decrease the expression of NF-?B,TNF-?and IL-6 in retinal tissues,increase the level of neurotrophic factor BDNF,and the activities of SOD and CAT were up-regulated,which may be related to the inhibition of COX2/PGE₂signaling pathway.3.Sal can reduce the expression of NF-?B,TNF-?,IL-6,Caspase-1 and Caspase-3,up-regulate the expression of BDNF and GS,increase the Bcl-2/Bax ratio,and down-regulate the apoptosis rate of rMC-1 cells induced by high glucose.The activity of antioxidant enzymes SOD and CAT is up-regulated,and the ROS level is down-regulated.The mechanism of which is related to the protective effect of Sal on rMC-1 cells through COX2/PGE₂signaling pathway.