| Background:Age-related macular degeneration(AMD),is characterized by degenerative changes of macular choroidal capillaries,vitreous membrane and pigment epithelium.This disease often leads to severe visual impairment and even blindness.AMD can be divided into exudative and non-exudative,characterized by choroidal neovascularization(CNV)and choroidal pattern atrophy(GA),respectively.Nonexudative macular degeneration,also known as dry or atrophic age-related macular degeneration,is characterized by progressive atrophy of retinal pigment epithelial cells,resulting in photosensitive cell degeneration and central vision loss.Dry AMD accounts for almost 90%of AMD cases,and it is even considered that dry AMD may be a risk factor or precursor state for wet AMD,as most AMD cases usually start with dry AMD and progress to wet AMD.However,there is still no effective treatment for dry AMD.According to previous studies,dry AMD is characterized by the formation of glass membrane warts due to the accumulation of toxic substances in retinal pigment epithelial(RPE)cells or at the interface of Bruch membrane.Oxidative stress and other stresses can lead to the death of RPE and photoreceptor(PR)cells,the accumulation of toxicity and the deposition of RPE glass membrane warts.Since vitreous membrane warts cannot be removed once they are formed,one of the main goals of AMD therapy is to prevent the formation of vitreous membrane warts on the retina as soon as possible after the diagnosis of the disease,and to avoid oxidative stress and apoptosis of RPE cells,which is also a key and difficult point in the prevention and treatment of AMD at present.Klotho is an anti-aging protein that has many health benefits for animals and can prolong life.The overexpression of Klotho gene delayed the aging of mice and prolonged the life span of mice.Interestingly,inhibition of Klotho accelerates the development of aging-related diseases,including atherosclerosis,osteoporosis,infertility and cognitive decline.Klotho can enhance the resistance to oxidative stress and inflammation.The protein is mainly expressed in the kidney and brain,but it can also be present in cerebrospinal fluid,urine and blood in the form of soluble protein.Recent studies have shown that Klotho can regulate the differentiation and apoptosis of ocular lens epithelial cells.In addition,Klotho gene can also affect retinal health.Objectives:AMD is one of the common diseases leading to vision loss in the elderly,and oxidative stress has been considered to be the main pathogenic factor of AMD.Klotho is an anti-aging protein that can enhance resistance to oxidative stress and inflammation.However,the effect of Klotho on AMD is not clear.Molecular,cellular and pathological experimental techniques,focusing on Klotho,will be used in this study to reveal the mechanism of Klotho protecting retinal pigment epithelial cells and retina from oxidative stress of cellular,animal and clinical aspects,so as to provide theoretical basis for clinical prevention and treatment of AMD.Methods:(1)Clinical data analysis:20 patients with simple cataract,15 patients with untreated exudative AMD and 18 patients with treated exudative AMD were collected from the Department of Ophthalmology of the first affiliated Hospital of Kunming Medical University.the blood samples were centrifuged at 4℃ with 3000 rmp 10min,and the supernatants were collected,and the aqueous humor samples were collected to detect the expression of α-klotho in peripheral blood and the expression of α-klotho,SOD and MDA in aqueous humor.The linear relationships between α-klotho and SOD,α-klotho and MDA,SOD and MDA were analyzed.(2)In vitro experiment:ARPE-19 cells in logarithmic phase were treated with H2O2,Klotho,NAC,LY294002 and knockdown of Nrf2 genes,respectively.The proliferation of ARPE-19 cells was detected by CCK-8 and 5-ethynyl-2’-deoxyuridine(EdU)incorporation;apoptosis of ARPE-19 cells was detected by flow cytometry,TUNEL apoptosis and mitochondrial membrane potential;ROS level in ARPE-19 cells was detected by DCFH-DA staining;the expression of MDA in ARPE-19 cells was detected by malondialdehyde(MDA)kit;the expression and localization of Nrf2 in ARPE-19 cells were detected by immunofluorescence staining and RT-qPCR was used to detect the expression of Klotho,SOD2,GPX and CAT in ARPE-19 cells,and Western blot was used to detect the expression of Bax,Bcl-2,cleaved-caspase-3,p-Akt,Akt,Nrf2(cytoplasm/nucleus)and HO-1 in ARPE-19 cells.(3)In vivo experiment:the AMD mouse model was established by injecting sodium iodate(NaIO3)into the tail vein of C57BL/6J mice,and the corresponding intraperitoneal injection of Klotho and vitreous injection of LY294002 were given.After the establishment of the model,the mice were examined by optical correlation tomography(OCT)and electroretinogram(ERG).Then the mice were killed,and the right eyes were removed.After fixed and embedded in paraffin,histological analysis was performed by HE staining.The retina of the left eye was removed and extracted.ROS level was detected by flow cytometry,retinal apoptosis was detected by TUNEL,MDA level was detected by MDA kit,and the protein expression of Bax,Bcl-2,p-Akt,Akt,Nrf2 and HO-1 was detected by Western blot.The localization and expression of Nrf2 and HO-1 were detected by retinal immunofluorescence staining.Results:(1)The results of clinical sample analysis showed that:① The expression of α-klotho and SOD in aqueous humor of patients with AMD untreated group was lower than that of normal control group and AMD treatment group,while the expression of MDA in aqueous humor of AMD untreated group and AMD treated group was higher than that of normal control group.The expression of α-klotho and SOD in aqueous humor of patients with normal control group and AMD treatment group had no difference,and the expression of MDA in aqueous humor of patients with AMD untreated group and AMD treatment group had no difference.② There was no difference in the expression of serum α-klotho among the groups.③ There was a positive correlation between α-klotho and SOD in aqueous humor,a negative correlation between α-klotho and MDA,and a negative correlation between SOD and MDA in aqueous humor of patients in each group.(2)In vitro experiment:① H2O2 significantly inhibited the proliferation of ARPE-19 cells in a concentration dependent manner,H2O2 could selectively down-regulate the expression of Klotho gene,while Klotho protein protected ARPE-19 cells from H2O2-induced injury in a concentration-dependent manner.② Klotho pretreatment could increase the expression of anti-apoptotic protein bcl-2 and decrease the expression of pro-apoptotic proteins Bax and cleaved-caspase-3,inhibit the apoptosis of ARPE-19 cells induced by H2O2,and promote cell proliferation.③ Klotho pretreatment inhibited the increase of ROS level and the decrease of mitochondrial membrane potential induced by H2O2 in ARPE-19 cells,and restored the changes of SOD2,GPX,CAT and MDA induced by H2O2 to nearly normal levels.④ NAC pretreatment could inhibit the increase of ROS level and apoptosis of ARPE19 cells induced by H2O2,and improve the proliferation activity of ARPE-19 cells inhibited by H2O2.⑤ H2O2 treatment could increase the expression of p-Akt/Akt,nuclear Nrf2 and HO-1 and decrease the expression of cytoplasmic Nrf2,while Klotho pretreatment could further increase the expression of these proteins and increase the nuclear translocation of Nrf2 induced by H2O2.⑥ LY294002 and knockdown of Nrf2 gene could block Klotho-mediated Nrf2 nuclear translocation,HO-1 expression and cytoprotective activity.(3)In vivo experiment:① After injection of sodium iodate,the retinal pigment epithelial(RPE)layer,rod cell layer(R&CL)and outer nuclear layer(ONL)were obviously damaged in HE staining,the cells in each layer were disordered,the cell density decreased,the normal monolayer continuous structure of retinal pigment epithelium(RPE)disappeared,delamination appeared,and protruding brown-black round deposits(glass membrane warts)were formed.The outer nuclear layer showed wavy changes,which became thinner as the outer plexiform layer and photoreceptor layer,and the boundary between the inner nuclear layer and the outer nuclear layer was not clear.Klotho treatment could reverse the retinal histomorphological changes induced by sodium iodate,increase the retinal thickness and reduce the number of vitreous membrane warts,however the pretreatment of LY294002 weakened the protective effect of Klotho.② OCT examination showed that abnormally high reflection turbidity appeared in the outer retina and vitreous cavity of mice,the retina lost its layered and clear structure,the outer nuclear layer also showed high reflection area,the thickness of the whole layer of retina became thinner,and several hyperreflective protuberances appeared in RPE layer.After Klotho treatment,the effect of NaIO3 on retina was alleviated,the thickness of retina was increased,and the delamination was clearer than that in NaIO3 group,and the high reflection process in RPE layer decreased.③ ERG examination showed that the amplitudes of a and b waves in mice were significantly lower than those in the control group,and the amplitudes of a and b waves were significantly increased after Klotho treatment.④The frozen sections of mouse retina were stained with TUNEL apoptosis.A large number of apoptotic positive cells appeared in the retina,mainly in the ganglion cell layer(GCL)and outer nuclear layer(ONL).After Klotho treatment,the apoptosis positive cells were significantly less,and the pretreatment of LY294002 weakened the inhibitory effect of Klotho on apoptosis.⑤ The levels of ROS and MDA in retina increased significantly,and the levels of ROS and MDA decreased after Klotho treatment.The pretreatment of LY294002 weakened the inhibitory effect of Klotho on oxidative stress.⑥ Western blot and immunofluorescence staining showed that the expression levels of p-Akt,Nrf2 and HO-1 were significantly up-regulated,and the expression levels of p-Akt,Nrf2 and HO-1 were further up-regulated after Klotho treatment.LY294002 could significantly inhibit the expression of the above proteins.Conclusions:(1)The abnormal low expression of α-klotho in aqueous humor of untreated AMD patients may be related to the increased oxidative stress of retina in AMD patients.(2)Klotho protein may play a cytoprotective role through PI3K/Akt-Nrf2/HO-1 pathway in H2O2-induced injury of human retinal pigment epithelial cells(ARPE19 cells).(3)The animal model further verified that Klotho could protect the retina from oxidative stress injury induced by sodium iodate through PI3K/Akt-Nrf2/HO-1 pathway. |
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