Exploring the Effects of Astragalus Polysaccharide on High Glucose-induced RGC Apoptosis via the miR-148a-3p/SMAD2 Axis
OBJECTIVE To investigate the effects of astragalus polysaccharide(APS)on high glucose-induced retinal ganglion cell(RGC)apoptosis and its mechanism via the microRNA-148a-3p/SMAD family member 2(miR-148a-3p/SMAD2)axis.METHODS RGC was transfected with miR-148a-3p mimics,miR-148a-3p inhibitors,and their respective controls(mimics-NC,miR-NC),and divided into the control group(CG),model group(MG),APS low-dose group(APS-L),APS high-dose group(APS-H),APS-H+miR-148a-3p control group(APS+miR-NC),and APS-H+miR-148a-3p inhibitor group(miR-148a-3p inhibitor).Treatments were administered with corresponding high glucose and APS.Expression levels of miR-148a-3p and SMAD2 mRNA were measured by real-time fluorescence quantitative PCR(qRT-PCR).Cell viability,apoptosis,and oxidative stress indicators were assessed.SMAD2 protein expression was detected by Western Blot,and the targeting relationship between miR-148a-3p and SMAD2 was verified using dual-luciferase reporter assays.RESULTS(1)Expression of miR-148a-3p/SMAD2:The APS-L,APS-H,and APS-H+miR-NC groups showed higher miR-148a-3p expression level compared to the MG group(tAPS-L=6.564,tAPS-H=10.542,tAPS-H+miR-NC=9.945,all P=0.000),and lower SMAD2 mRNA expression level(tAPS-L=4.147,P=0.004;tAPS-H=6.464,tAPS-H+miR-NC=6.586,both P=0.000).The miR-148a-3p inhibitor group showed lower miR-148a-3p expression level compared to the APS-H+miR-NC group(t=7.558,P=0.000),and higher SMAD2 mRNA expression level(t=4.513,P=0.001),with statistically significant differences.(2)Cell viability and apoptosis rate:The APS-L,APS-H,and APS-H+miR-NC groups had higher cell viability than the MG group(tAPS-L=8.105,tAPS-H=11.509,tAPS-H+miR-NC=11.996,all P=0.000),and lower apoptosis rates(tAPS-L=8.729,tAPS-H=15.690,tAPS-H+miR-NC=14.709,all P=0.000).The miR-148a-3p inhibitor group showed lower cell viability(t=10.212,P=0.000)and higher apoptosis rates(t=12.247,P=0.000)than the APS-H+miR-NC group,with statistically significant differences.(3)Oxidative stress:The APS-L,APS-H,and APS-H+miR-NC groups had lower levels of lactate dehydrogenase(LDH)and malondialdehyde(MDA)compared to the MG group(LDH:tAPS-L=11.257,tAPS-H=18.770,tAPS-H+miR-NC=18.364,all P=0.000.MDA:tAPS-L=11.121,tAPS-H=17.139,tAPS-H+miR-NC=17.768,all P=0.000),and higher levels of superoxide dismutase(SOD)and glutathione(GSH)(SOD:tAPS-L=9.408,tAPS-H=14.833,tAPS-H+miR-NC=13.240,all P=0.000.GSH:tAPS-L=5.616,tAPS-H=12.080,tAPS-H+miR-NC=12.642,all P=0.000).The miR-148a-3p inhibitor group had higher levels of LDH and MDA(tLDH=15.121,tMDA=14.613,all P=0.000),and lower levels of SOD and GSH(tLDH=15.121,tMDA=14.613,both P=0.000)than the APS-H+miR-NC group,with statistically significant differences.(4)Apoptotic proteins:The APS-L,APS-H,and APS-H+miR-NC groups had lower expression levels of SMAD2,Bcl-2-associated X protein(Bax),and Caspase-3 compared to the MG group(SMAD2:tAPS-L=4.565,tAPS-H=8.042,tAPS-H+miR-NC=7.390,all P=0.000.Bax:tAPS-L=4.916,tAPS-H=8.763,tAPS-H+miR-NC=8.336,all P=0.000.Caspase-3:tAPS-L=4.214,tAPS-H=10.201,tAPS-H+miR-NC=9.536,all P=0.000),and higher expression of B-cell lymphoma 2(Bcl-2)(tAPS-L=3.713,P=0.001;tAPS-H=10.108,tAPS-H+miR-NC=10.314,both P=0.000).The miR-148a-3p inhibitor group showed higher expression of SMAD2,Bax,and Caspase-3(tSMAD2=5.651,tBax=6.840,tCaspase-3=8.205,all P=0.000),and lower expression of Bcl-2(t=9.283,P=0.000)compared to the APS-H+miR-NC group,with statistically significant differences.(5)Targeting relationship:miR-148a-3p has binding sites at the 3'-UTR of SMAD2.Co-transfection with miR-148a-3p mimics and wild-type SMAD2 resulted in lower luciferase activity than co-transfection with mimics-NC and wild-type SMAD2,with statistically significant differences(t=12.781,P=0.000).CONCLUSIONS Astragalus polysaccharide may improve HG-induced RGC apoptosis and oxidative stress damage by up-regulating miR-148a-3p and down-regulating SMAD2.
astragalus polysaccharideretinal ganglion cellmicroRNA-148a-3pSmad fam-ily member 2
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