Effects Of Lycium Barbarum Extracts On Microglia/Macrophages And Autophagy After Partial Optic Nerve Transection In Rats

Aim: Lycium barbarum is a traditional Chinese herbal medicine,which is believed to have the effects of clearing the liver and clarifying the eyes.Lycium barbarum extracts(LBE)are extracted from Lycium barbarum fruit.Existing researches had shown that LBE had good therapeutic effects on visual diseases and some other neurological diseases,but the mechanisms were still not fully understood.It was known that LBE could regulate the function of microglia/macrophages and autophagy,but further investigatio n was needed to explore the details of these effects.The microglia/macrophages have different phenotypes,namely M1 type which aggravates tissue damage and M2 type which are favorable for tissue repair.The aim of this study was to explore whether LBE co uld regulate the polarization of microglia/macrophages and whether LBE had effects on autophagy after partial optic nerve transection(PONT).Methods: The adult female SD rats were used for three parts of study: I.The rats(n=6)were administrated with PONT from the temporal sides of the optic nerves using diamond knife and several crystals of DiI were put into the cut site of each rat immediately after PONT to label the cell bodies of retinal ganglion cells(RGCs)whose axons were transected during the surgery and then the rats were sacrificed 4 days after PONT;II.Nine groups of total 45 rats(n=4 to 9 in each group)were used for determine the percentages RGC death and the expression levels of some proteins after PONT: three normal groups;three 1-week PONT groups and three 4-week PONT groups.Three groups from each treatment were used for RGC counting which were retrogradely labeled by fluoro-gold(FG),Western blotting analysis and immunohistochemical staining respectively.Immunohistochemical staining was used to detect the number of microglia/macrophages(CD68 staining)and western blotting was used to detect the expression of CD68(activated microglia/macrophages),iNOS(expressed by M1 microglia/macrophages),Arginase-1(expressed by M2 microglia/macrophages),LC3(the marker of autophagy).III.There were nine groups of total 49 rats in this part(n=4 to 9 in each group): one normal group for RGC counting after FG labeling;four PONT + PBS groups and four PONT + LBE groups(oral administrated with 0.01 mol/L PBS or 1 mg/kg of LBE in 0.01 mol/L PBS from 1 week before PONT and until 1 week or 4 weeks after PONT).In either four PONT + PBS or LBE groups: the first group was used for RGC counting 1 week after PONT;the second for RGC counting 4 weeks after PONT;the third for CD68 immunohistochemical staining 1 week after PONT and the last for Western blotting analysis 1 week after PONT.Results: ?.There were much more DiI-labeled RGCs in the temporal retinas than the nasal sides 4 days after PONT and the ratio was about 4.9:1 between temporal and nasal retinas.II.There were more surviving FG labeled RGCs in the nasal retinas than in the temporal sides both 1 week and 4 weeks after PONT and the expression levels of CD68,iNOS,Arginase-1 and LC3 II increased in the optic nerves 1 week after PONT compared with normal retinas.III.The number of activated microglia/macrophages in the rats fed with LBE one week before PONT and four week after PONT was greater than that of the rats fed with PBS.In addition,LBE could significantly increase the expression levels of CD68 and Arginase1;and decreased the expression of LC3 II.Conclusion: ?.After PONT,the primary degeneration mainly occurred in the temporal retinas and in the nasal retinas most RGCs would die from secondary degeneration based on the DiI results.II.There were more surviving RGCs in the nasal retinas than in the temporal sides both 1 week and 4 weeks after PONT.There were more activated total microglia/macrophages,M1 type microglia/macrop hages and M2 type microglia/macrophages 1 week after PONT in the optic nerves;and the level of autophagy increased 1 week after PONT.III.LBE could delay the death of RGCs 4 weeks after PONT;increase the number of total activated microglia/macrophages a nd the number of M2 type microglia/macrophages and decrease the level of autophagy 1 week after PONT in the optic nerve.