The Mechanism Of Acteoside Regulating Autophagy And Apoptosis Of RGC-5 Cells Through MiR-342-3p/OPTN Molecular Axis And The Downstream PI3K/AKT Signaling Pathway

BackgroundGlaucoma is a group of diseases characterized by characteristic optic nerve atrophy and visual field defects.Increased intraocular pressure is a risk factor of glaucoma optic neuropathy.As the second leading cause of blindness after cataract,glaucoma is a serious threat to human visual health.Due to the induction or stimulation of some adverse factors in vivo or in vitro,the intraocular pressure increases or fluctuates greatly.When the intraocular pressure changes beyond the capacity of the intraocular tissue,especially the retinal optic nerve,damage will be caused to the intraocular tissue,especially the optic nerve and its visual pathway and visual function.The most typical and prominent manifestations are pitted atrophy of the optic disc and characteristic narrowing of the visual field.Without prompt and effective treatment,the field of vision can be completely lost and eventually lead to blindness.Glaucoma optic nerve damage is characterized by optic nerve atrophy,which is a direct manifestation of ganglion cell axonal degeneration.The primary pathological feature of all types of glaucoma is the death of retinal ganglion cells(RGCs).For a long time,the pathogenesis of glaucoma can be classified into two categories:mechanical stress theory and vascular ischemia theory.The theory of mechanical pressure emphasizes the role of intraocular pressure.It is believed that the increase of intraocular pressure causes the deformation and displacement of each layer of the sieve plate to produce shear force,which blocks the axial flow of retinal ganglion cells(RGCs)in the sieve plate area.The reduction of axon protein production and transport impedes the acquisition of brain-derived neurotrophic factors,which ultimately leads to the apoptosis of RGCs.According to the theory of vascular ischemia,due to various reasons,the visual papilla microcirculation is blocked,This leads to decreased nutrient supply to the optic papilla and surrounding tissues,resulting in hypoxia and eventual apoptosis of the RGCs axons,or optic nerve fibers.Since RGCs cannot regenerate after destruction,the death of RGCs will result in permanent loss of vision.Clinically,glaucoma is mainly treated with intraocular pressure lowering drugs or surgery,whose main purpose is to control intraocular pressure.However,although some patients have good intraocular pressure control,progressive loss of visual field still occurs and eventually leads to blindness.Therefore,the reduction of intraocular pressure alone cannot completely control the loss of RGCs in patients with glaucoma.With glaucoma optic nerve injury mechanism and pathophysiological process of in-depth study,to suppress RGCs death as soon as possible,promote nerve repair and the treatment of optic nerve protection measures,is important in the clinical treatment of improve vision in patients with optic neuropathy in way,got attaches great importance to clinical doctors,has become a critical and difficult point in the study of optic nerve protection.Acteoside(C29H36015)is the main component of phenylpropanin,which comes from yunnan kuting tea.Our previous study found that Acteoside had a protective effect on the damage model RGCs.In the model of chronic intraocular hypertension and the model of optic nerve clamp clipping in rats,the number of RGCs after treatment with Acteoside was significantly increased compared with the control group.At the same time,Thy-1,a specific RGCs marker,was significantly higher than that of the control group,indicating that Acteoside could reduce the apoptosis of RGCs.In the rat model of retinal reperfusion injury,Acteoside can promote the recovery of cell morphology,reduce the apoptosis of RGCs,and reduce the retinal ischemia reperfusion injury.However,the mechanism of Acteoside regulating RGCs apoptosis is still unclear,and further studies are needed.ObjectiveTo explore explore the protective effect of Acteoside on retinal ganglion cells RGC-5 through regulating autophagy and its molecular mechanism.Method1.RGC-5 cells were cultured in vitro and treated with CoCl2 with concentration gradient to establish an oxidative stress injury model of RGC-5 cells.CoCl2 of 0,10,20,40,80,160 and 320 μmol/L were used to treat the cells and explore the appropriate concentration for constructing the model of damaged cells.RGC-5 cells damaged by oxidative stress was treated with three different concentrations of Acteoside at high,medium and low levels was conducted to detect the protective effect of Acteoside on CoCl2 damaged RGC-5 cells.2.CCK-8 experiment was used to detect the effect of Acteoside on RGC-5 cell proliferation.Transmission electron microscope and flow cytometry were used to detect autophagy and apoptosis of RGC-5 cells.3.The Affymetrix microRNA expression profile microarray was used to detect the differential expression of miRNA in RGC-5 cells during injury and repair.After removing the cryopreserved cells from the liquid nitrogen,the cells were continuously shaken in a 37℃ water bath to promote their melting.After centrifugation,cleaning,inoculation and culture in the incubator.When the subculture was used,the cells were sucked up and down several times with a straw after cleaning to break up the cell mass.After mixing evenly,the ml medium(MEM)of 3n(n is the number of subculture bottles)was supplemented.In accordance with the dilution ratio,it was transferred to a new culture bottle and cultured in a CO2 incubator.According to the Hybridization operation procedure provided by the Affymetrix microRNA expression profile Chip,the corresponding Gene Chip Hybridization Wash and Stain Kit was used to hybridize RNA samples and the Chip.According to the kit operation instructions;The hybrid reaction solution was prepared and added to the above biotin-labeled samples.Results were scanned using Gene chip-scanner 3000 and read by GeneChip Command Console Software5.0.4.Detection of the relationship between the differential miRNA(miR-342-3p)and its target gene OPTN.The targeted effects of miR-342-3p and OPTN were detected by dual luciferase reporter gene.The recombinant plasmid of wild type and mutant OPTN(pmirglo-optn-wt/MUT)was constructed and co-transfected with miR-342-3p mimics into HEK293T cells.The control group was cotransfected with wild or mutant recombinant plasmids of OPTN using miR-342-3p scramble negative control.The relative fluorescence intensity was detected according to the instructions of luciferase activity detection kit(Promega)5.Overexpression or knockdown transfection of miR-342-3p and OPTN.miR-342-3p mimic or inhibitor was used to overexpress or knock down miR-342-3p.OPTN was over-expressed or down-transfected with pcdna3.1-optn recombinant plasmid or OPTN knockdown sequence RNA.Among them,miR-342-3p overexpression or knockdown transfection was controlled by mir-scramble.Overexpression of OPTN was transfected with no-load plasmid as control,knockdown transfection with nonsence shRNA as control.NC control was set for each transfection group,and 3 multiple Wells were set for each group.6.qRT-PCR and Western blotting were used to detect the influence of miR-342-3p and OPTN expression in RGC-5 cells.After centrifugation,cells in each group were collected,and the total RNA of rRGC-5 cells in each group was frayed and extracted by Trizol method.Reverse transcription kit was used for reverse transcription,and SYBR Green Master Mix real-time fluorescence quantitative PCR kit was used for relative expression detection.The expression of OPTN in RGC-5 cells was detected by Western blotting.7.Western blot and immunofluorescence were used to detect the influence of PI3K/AKT/mTOR pathway and autophag-related protein expression in RGC-5 cells.Extraction of total cell protein,sds-page gel electrophoresis,Western blot reaction detection in the RGC-5 PI3K/AKT signaling pathway and autophagy related protein(Beclin 1,Soluble p62,Insoluble p62,LC3-Ⅰ/Ⅱ),cell apoptosis related proteins(such as caspase3 and caspase7)expression.Prepare cell slide,4%paraformaldehyde fixed,immunofluorescence was used to test the expression of autophagy protein Beclin 1 and LC3 Ⅱ.Results1.The protective effect of Acteoside on RGC-5 cell damage.Using 30 μ mol/L CoCl2 to intervene RGC-5 cells for 24h to construct the RGC cell damage model significantly inhibited the proliferation activity of RGC cells and promoted the level of apoptosis.The results of transmission electron microscope showed that the autophagy level of RGC-5 cells in the CoCl2 group was significantly increased,and the content of autophagy bodies was significantly higher than that in the normal cell group.Westernblot and immunofluorescence detection results showed that beclin-1 expression,soluble p62 protein and insoluble p62 were significantly up-regulated in the injured group,and the conversion of LC3 protein type II was also significantly higher than that in the control group.Acteoside treatment significantly promoted the proliferation activity of RGC-5 cells and reduced the levels of autophagy and apoptosis after cell damage.2.Acteoside significantly up-regulated the expression of miR-342-3p in RGC-5 cells.MiRNAs expression analysis of RGC-5 cells in the normal control group,the CoCl2 injury group and the ak-treated group showed that,64 miRNAs were differentially expressed in RGC-5 cells in the Acteoside group,and 41 miRNAs were upregulated.The largest upregulated multiple was miR-342-3p,with a upregulated multiple of 4.653.Knockdown of miR-342-3p significantly reduced the protective effect of Acteoside on RGC-5 cells.Compared with the Acteoside group,Acteoside+knockdown miR-342-3p group significantly reduced the proliferation activity of RGC-5 cells and promoted the autophagy and apoptosis levels of RGC-5 cells.Immunofluorescence and Western Blot detection of autophagy-related proteins showed that beclin-1 expression in RGC cells in the miR-342-3p group was significantly up-regulated,soluble p62 protein was significantly increased,insoluble p62 was significantly decreased,and the conversion level of type Ⅱ LC3 protein was increased.4.OPTN was negatively regulated by MiR-342-3p.The prediction results from starBase database indicated that OPTN might be the target gene of miR-342-3p.The results of the dual-luciferase reporter gene experiment showed that the luciferase activity of RGC-5 cells in the control group and the miR-342-3p mimics group was 10.044±0.462 and 4.820±0.284 respectively(t=5.685,P=0.008).In the mutant vector of OPTN,luciferase activity of RGC-5 cells in the control group and the mimics group was 10.015±0.439 and 9.957±0.514 respectively(t=0.402,P=0.859).5.Acteoside regulates proliferation,autophagy and apoptosis of RGC-5 cells through miR-342-3p/OPTN molecular axis.Compared with the RGC-5 cells in the injury model group(NC group),the proliferation activity of the RGC-5 cells in the over-expressed OPTN group was significantly inhibited,and the apoptosis and autophagy levels of the cells were further significantly increased.Compared with the group of Acteoside,the group of Acteoside+over-expressing OPTN significantly reduced the protective effect of Acteoside on damaged cells.There was no significant difference in cell proliferation,apoptosis and autophagy between the Acteoside+overexpression of miR-342-3p+overexpression of OPTN group and the Acteoside group.6.Acteoside inhibits autophagy of RGC-5 cells by activating the PI3K/AKT/mTOR pathway.Western blot results showed that both Acteoside and 3-MA could independently activate PI3K and AKT at the same time,resulting in inhibition of autophagy in RGC-5 cells.Treatment with Acteoside significantly reduced rapamycin-induced autophagy.Moreover,Acteoside combined with the autophagy inhibitor 3-ma had a stronger effect on the inhibition of autophagy in cells than the single one.7.Acteoside inhibits autophagy of RGC-5 cells through the synergistic effect of OPTN and PI3K/AKT/mTOR pathway and reduces the apoptosis level of RGC-5 cells.Conclusion1.Acteoside promotes the proliferation of damaged RGC-5 cells and inhibits the autophagy and apoptosis of cells caused by RGC-5 damage2.Acteoside significantly promoted the expression of miR-342-3p in damaged RGC-5.3.Acteoside promoted RGC-5 cell proliferation and inhibited autophagy and apoptosis by upregulating miR-342-3p/OPTN molecular axis.4.Acteoside reduces autophagy and apoptosis of RGC-5 cells by activating PI3K/AKT signaling pathway.5.Acteoside protects RGC-5 cells by promoting the synergistic effect of OPTN and PI3K/AKT signaling pathways.