Inhibitory Effect Of Triglyceride Saponins On Apoptosis Of Retinal Ganglion Cells In Diabetic Rats And Its Mechanism

Objuctive: To reveal the benifical effect of panax notoginseng (PTS) ofdiabetes-induced apoptosis of retinal ganglion cell (RGC), and to explore underlyingmechanisms by detecting the alterations of Nogo receptor (NgR) siganaling pathway.Materials and methods: Part Ⅰ: Anti-apoptosis effect of PTS on diabeticRGC. SD rats were intraperitoneally injected with streptozotocin (STZ) at the dose of60mg/kg, rats with tail vein blood glucose concentration more than16.7mmol/L72hafter STZ injection were considered diabetic. Rats were randomly assigned into3groups: control group, diabetic group and PTS-treated group (diabetic rats intragastricadministration with PTS50mg·kg-1·d-1, twice a day). One month later, we detectedthe blood glucose concentration and body weight, reveald the coexistence of NgR andBrn3a (a specific marker of RGC) in retina by immunofluorescence histochemistrystaining, observed the expression of retinal NgR, and exhibited the level of retinalmalonaldehyde (MDA) and glutathione (GSH) in addition to show the number ofRGC by hematoxylin and eosin stain. Part Ⅱ: Effect of NgR on the apoptosis ofdiabetic RGC. STZ-induced diabetic rats just as we described in Part Ⅰ. Rats wererandomly divided into control group, diabetic group, siNgR group and siRNA controlgroup. Diabetic rats in siNgR group were intravitreally injected withlentivirus-transported NgR antisense nucleotide, the anti-NgR nucleotide sequence is5’-AATGACTCTCCATTTGGGACT-3’, while, diabetic rats in siRNA control group wereintravitreally injected with lentivirus transfected with negative nucleotide. One monthafter diabetes onset, rat blood glucose concentration and body weight were ditected,the apoptosis of RGC were revealed by terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling (TUNEL) staining, the lever of retinal MDAand GSH were ovseved by kits, and the expression of NgR and Caspase-3in retina were detected by Western blot. Part Ⅲ: mechanisms of NgR signaling pathwayparticipate in glucose-induced apoptosis of RGC. RGC were cultured in DMEMdedium plus10%FBS, RGCs were cultured in5groups for3days as follows: controlgroup, high glucose group (together with glucose15mmol/L), siRNA control group(15mmol/L glucose+negative nucleotide sepuence), siNgR group (15mmol/Lglucose+anti-NgR nucleotide sepuence), and PTS group (15mmol/L glucose+PTS25mg/L). RGC viabilities were detected by methyl thiazolyl tetrazolium (MTT),oxidative stress were reflected by MDA and GSH detection, the expression of NgRand apoptosis-related gene caspase-3were obserbed by Western blot.Results:1.Anti-apoptosis effect of PTS on diabetic RGC1.1Blood glucose concentration and body weightBlood glucose concentration: STZ-administered rats showed increased bloodglucose concentration>16.7mmol/L, and maintained throughout the experiments,showing significant difference to control group (P<0.05). Diabetic rats treated withPTS showd no alterations of blood glucose concentration, still>16.7mmol/L,exhibited no difference between PTS-treated group and control group (P>0.05).Body weight: rats in control group showed obviously body weight gaining, whilediabetic rats showed slightly body weight gaining, there is obvious difference betweencontrol group and diabetic group (P<0.05). While, PTS showd no effects on bodyweight gaining, no difference of bode weight could be detected1month after diabetesonset between PTS-treated group and diabetic group (P>0.05).1.2Double-immunofluorescence for NgR and Brn3a in retinaHere with Brn3a, the marker of RGC, we found that NgR is exlusively expressedin Brn3a-positive cells. Therefore, NgR expresses in RGC.1.3Effects of PTS on the expression of retinal NgRThe expression of NgR was increased in diabetic group compared with controlgroup (P<0.05). PTS-treated diabetic rats showed down-regulation of NgR inPTS-treated group than diabetic group (P<0.05). Thus, PTS inhibited the expression of NgR in diabetic retina.1.4Effects of PTS on the level of retinal MDAand GSHCompared with control group, the level of retinal MDA in diabetic group wassignificantly increased (P<0.05), showing no difference to PTS-treated group(P>0.05). Compared with control group, the level of retinal GSH in diabetic groupwas significantly decreased (P<0.05), and showing no difference to PTS-treated group(P>0.05)1.5Effects of PTS on the number of diabetic RGCHematoxylin and eosin stain showed that the number of RGC in diabetic group isdecreased compared with control group (P<0.05). While, the number of RGC inPTS-treated group showed no difference to control group (P>0.05).2. Effect of NgR on the apoptosis of diabetic RGC2.1Rat blood glucose concentration and body weight.Blood glucose concentration: STZ-induced diabetic rats showed blood glucoseconcentration>16.7mmol/L, and maintained to the end of experiments, showingsignificant difference to control group (P<0.01). Diabetic rats intravitreally injectedwith lentivirus-transported NgR antisense nucleotide in siNgR showd no alterations ofblood glucose concentration, exhibited no difference between to control group(P>0.05).Body weight: rats in control group exhibited obviously body weight gaining,diabetic rats showed slightly body weight gaining, the body weight in diabetic groupwas lower than control group (P<0.05). While, siNgR showd no effects on bodyweight gaining, there was no difference of bode weight between siNgR group anddiabetic group (P>0.05).2.2Expressions of NgR and Caspase-3in retinaCompared with control group, both the expressions of NgR and Caspase-3wereincreased in diabetic group and siRNA group (P<0.05), showing no difference tosiNgR group (P>0.05).2.3Effect of NgR on retinal oxidative stress in diabetic ratsThe level of retinal MDA was increased in diabetic group than control group (P<0.05), the level of retinal MDA showed no difference between siNgR group andcontrol group (P>0.05); The level of retinal GSH was decreased in diabetic groupthan control group (P<0.05), while, the level of MDA in retina showed no differencebetween siNgR and control groups (P>0.05).2.4Effect of NgR on RGC apoptosisTUNEL staining revealed that the number of apoptosis RGC were significantlyincreased than control group (P<0.05), while, the number of apoptosis RGC weredecreased in siNgR group compared with diabetic group (P<0.05).3. The mechanisms of NgR signaling pathway contributing to glucose-inducedapoptosis of RGC3.1Effect of high glucose concentration on RGC viabilitiesCompared with control group, cell viabilities in high glucose group and siRNAgroups were decreased (P<0.05); compared with diabetic group, cell viabilities in PTSgroup and siNgR groups were increased (P<0.05).3.2Investigation of oxidative stress by detection of the level of MDA and GSHCompared with control group, the level of MDA in high glucose group andsiRNA control group were increased (P<0.05), while, in siNgR group and PTS groupshowing no difference to control group (P>0.05). The level of MDA in high glucoseand siRNA control groups were increased compared with control group (P<0.05),while, in PTS and siNgR groups showing no difference to control group (P>0.05).3.3Expressions of NgR and Caspase-3Compared with control group, the expressions of NgR and Caspase-3wereincreased in high glucose group and siRNA group (P<0.05), while, showing nodifference to PTS group and siNgR group (P>0.05).Conclusion:Based on STZ-induced diabetic rats, we explored the effects and mechanisms ofPTS underlying RGC apoptosis in diabetic retina. Breifly, here we conclude that:1. PTS inhibites diabetic RGC apoptosis in a glycemia indepanded manner.2. Up-regulation of NgR plays a vital role in the apoptosis of diabetic RGC. In addition, PTS ihhibites diabetic RGC apoptosis, least in part, via revesing theup-regulation of NgR.3. Oxidative stress, up-regulation of apoptosis-releated gene caspase-3contribute toNgR-induced apoptosis of RGC.4. PTS inhibites the expression of retinal NgR, and then inhibites the oxidative stressand expression of caspase-3, thus, PTS shows the effect of inhibites the apoptosisof RGC in diabetic rats.