Specialized Pro-resolving Mediators Delay The Progression Of Retinal Degeneration

Background:Retinal degeneration?RD?is a series of blinding eye diseases,including retinitis pigmentosa?RP?,age-related macular degeneration?AMD?,diabetic retinopathy?DR?,and glaucoma.RD is the leading cause of irreversible blindness worldwide,making it the focus of WHO's current prevention of blindness.Its common pathophysiological mechanism is the gradual apoptosis of nerve cells such as photoreceptors and/or ganglion cells?RGC?,accompanying with the progressive loss of retinal photoreceptor function and visual signaling impairment.Due to the complexity and heterogeneity of the pathogenic mechanism,there is currently no effective treatment for such diseases.Whether different pathogenic factors,such as genetic mutations or damage stress in the retina,ischemia,hypoxia,etc.,can trigger the same mechanism of apoptosis is a critical question remained to be answered.If this question was addressed,curing such disease by regulating this apoptotic pathway will be promising and meaningful.Former work has found that the progressive apoptosis of photoreceptors and RGC in RD is mainly attributed to two aspects:1)Photoreceptors or RGC undergo apoptosis due to inner defects caused by gene mutation.Previous studies have shown that calpains are activated when abnormal proteins are synthesized under genetic defects and accumulate in retinal cells.By comparison with the classical caspase-dependent apoptotic pathway,the apoptotic pathway dependent on calpains in degenerative retinas plays a more important role;2)The imbalance of the retinal microenvironmental homeostasis exacerbates non-cell autonomous apoptosis of photoreceptors and RGC.The excessive glutamate levels in the retinas of glaucoma,DR and RP induce calpains activation to involve in cell apoptosis.In addition,microglia,the resident immune cell in the retina,is activated during the process of RD and acts as a double-edged sword.On the one hand,activated microglia can phagocytose apoptotic cells,clear inflammatory substances,and limit inflammation.On the other hand,hyperactivated microglia release excessive pro-inflammatory factors and worsen the retinal microenvironment,aggravating the apoptosis of photoreceptors and RGC.Therefore,inhibition of calpains-dependent apoptotic responses in the retina or regulation of microglia-mediated retinal inflammatory responses is expected to be a new strategy for the treatment of RD.Specialized pro-resolving mediators?SPM?are a class of lipid mediators with anti-inflammatory,pro-resolving and regenerative properties,which were produced from omega-6/3 polyunsaturated fatty acids in vivo.The most abundant omega-6 and omega-3PUFAs in the body are arachidonic acid?AA?and docosahexaenoic acid?DHA?.Under the action of 15-lipoxygenase?15-LOX?,DHA and AA are metabolized to produce lipoxin A4?LXA4?and Neuroprotectin D1?NPD1?,respectively.The former mainly exhibit strong anti-inflammatory effects,while the latter has more significant neuroprotective properties.Previous study suggests that the dysregulations of endogenous SPM can occur during neurodegenerative diseases such as Alzheimer's disease,stroke,AMD,and glaucoma.Besides,15-LOX also modulates the retinal inflammatory response in DR and the protective effects of RGC in glaucoma.Those suggest that SPM potentially are crucial targets for the treatment of neurodegenerative diseases.Therefore,we hypothesize that SPM may have important regulatory effects in the progression of RD.On the one hand,SPM protect photoreceptors,RGC and other retinal cells by directly ameliorating the calpains-involved neuronal apoptosis;on the other hand,SPM exert indirect neuroprotective effects by regulating the activation of microglia and improving the retinal inflammatory microenvironment.Purposes:This study mainly explored whether LXA4 and NPD1 are significant for the regulation of neuronal apoptosis and microglia activation during RD.Methods:Part ?:LXA4 restores the visual function of RD1 mice.1.Based on the correlation between RD1 mouse development and the progression of retinal degeneration,we chose RD1 mice aged 7,14 and 21 days to perform RT-PCR.PN7,PN14 and PN21 are considered as the pre-denaturing stage,the peaked stage,and the late stage of RD,respectively.The mRNA expression levels of LXA4 receptor and LXA4 key metabolic enzymes in the retinas of three-stage RD1 mice were evaluated.2.RD1 mice were treated with LXA4 or PBS by intravitreal injection three times at PN6,PN9 and PN12,respectively.And then we proceeded as follows:1)ERG and visual behavioral tests were used to evaluate the visual function of RD1mice;2)Immunostaining of retinal frozen sections were performed to analyze the apoptosis of photoreceptors in RD1 mice.Meanwhile,RT-PCR and Western blotting were used to detect the expression level of photoreceptor-specific marker,Rhodopsin;3)Using immunostaining,RT-PCR and Western blotting to assess the morphology of microglia in the RD1 retinas and the expression levels of microglia-related inflammatory factors,respectively;3.The intact retinas of PN13 RD1 mice were isolated and cultured in vitro under the LXA4 treatment.The mouse cytokine assays were performed to analyze the expression level of microglia-related factors in retinal explants and supernatants during RD.Part ?:NPD1 protects RGC in NMDA-injured retinal rat model.To investigate the calpains-dependent apoptotic response,we used a NMDA-injured retinal rat model.Firstly,in order to clarify whether NPD1 has a protective effect on NMDA-damaged RGC,we carried out:1.Long Evans?LE?rats aged 21 days?PN21?were randomly divided into three groups to received three treatments,intravitreal injection of NMDA,NMDA+NPD1,PBS,respectively.Visual electrophysiological examinations and visual behavioral tests were performed successively to evaluate visual function.At last,retinal frozen sections were prepared for immunostaining to assess RGC apoptosis;2.The PN21 LE rats were randomly divided into groups and fed with feeds rich in different levels of DHA for one month,and then ELISA was used to detect whether the level of endogenous NPD1 was increased.Subsequently,NMDA,or NMDA combined with NPD1was injected intravitreally followed by visual electrophysiological tests and visual behavioral tests to analyze the visual function.Immunostaining was used to evaluate the RGC apoptosis.Next,in order to explore the mechanism underlying the protection of NPD1 for RGC against NMDA damage,we carried out:3.The transcriptome sequencing and proteomic detection technique was used to analyze the changes of total RNA and protein expression profile between the retinas treated with co-injection of NMDA+NPD1 and injection alone of NMDA,then RT-PCR and Western blotting were used to verify our findings;4.The PN21 LE rats were randomly divided into three groups to received the intravitreal injection of PBS,calpain 1,or calpain 1 combined with NPD1,respectively.And then immunostaining was performed to evaluate RGC apoptosis and RT-PCR was used to analyze the expression levels of RGC-specific genes and apoptotic genes involved in calpains-dependent apoptotic pathway;5.Retinal frozen sections of NMDA injected and NMDA+NPD1 co-injected intravitreally rats were prepared for immunostaining with iba1 to evaluate the morphology of microglia.Meanwhile,Western blotting was used to detect the expression changes of CCL-2and ED-1 proteins,which are both biomarkers related to microglia.RT-PCR was conducted to assess the mRNA levels of inflammatory factor in NMDA-injured retinas of both two groups.Results:Part ?:LXA4 restores the visual function of RD1 mice.1.LXA4 maintains the visual function of RD1 mice.?1?Changes in mRNA levels of 15-LOX and FPR2 during the progression of RD.At PN14,Alox15 and Fpr2 mRNA expression levels in RD1 retinas were significantly higher than PN7?p<0.0001?,but comparing to that of PN14,their expressions were significantly decreased in the late-stage degeneration?PN21??p<0.0001?.?2?LXA4 delays the loss of visual function in RD1 mice.LXA4-treated mice continuously exhibited higher b-wave amplitudes in the ERG responses at PN15,PN18?p<0.01?and PN21?p<0.05?than those of PBS-treated mice.In the open field visual behavioral tests,LXA4-treated RD1 mice of PN18?p<0.05?and PN21?p<0.01?stayed in the dark box for significantly longer than PBS-treated RD1 mice,suggesting LXA4 restores the visual function in late-stage RD1 mice.2.Mechanisms underlying the restoration of visual function in LXA4-treated RD1 mice.?1?LXA4 reduces photoreceptors apoptosis in RD1 retinas.Immunostaining showed that the number of Rhodopsin-positive cells in the LXA4-treated retinas was significantly higher than that in the PBS-treated ones.RT-PCR?p<0.01?and Western blotting results?p<0.001?both confirmed this result.TUNEL staining showed that LXA4 significantly reduced photoreceptor apoptosis?p<0.05?and maintained the thickness of outer nuclear layer?p<0.01?.?2?LXA4 suppresses overactivation of microglia in RD1 retinas.1)LXA4 reduces retinal inflammatory response in RD1 mice.LXA4 treatment remarkably reduced Alox5 mRNA expression levels?p<0.05?in late-stage RD1 retinas.2)LXA4 regulates the expression of inflammatory cytokines associated with activated microglia in RD1 retinas.The iba1-positive microglia in the PN21 RD1 retinas of both LXA4-treated and PBS-treated group exhibited an amoebic-like activated state.Western blotting results indicated that LXA4 significantly decreased the expression of iba1?p<0.01?.LXA4significantly reduced the mRNA levels of cytokines,such as CCL2,iNOS,TNF-?,IFN-?,IL-1?and GM-CSF?p<0.05?,but increased the mRNA levels of IL-10?p<0.0001?,suggesting LXA4 reduces activated microglia-involved inflammation in RD1 retinas.3)LXA4 suppresses the activation of microglia in cultured RD1 retinas.Cytokine protein assays showed that all the detectable factors in the LXA4-treated RD1retinas and their supernatants were significantly reduced compared to the levels of PBS-treated ones.Abundant CCL-2 was detected both in the retinal explants and supernatants?PN21?,and LXA4 significantly reduced the production and secretion of CCL-2 compared to the PBS-treated group?p<0.05?.Part ?:NPD1 protects RGC in NMDA-injured retinal rat model.1.NPD1 protects RGC in NMDA-injured retinal rat model.?1?Intravitreal NPD1 protects NMDA-injured RGC in rats.1)Intravitreal NPD1 reduces NMDA-induced RGC apoptosis in rats.RGC underwent a rapid apoptosis after intravitreal injection of NMDA within 72h.Immunostaining showed that the TUNEL-and Brn3a-positive cells in the retina of rats receiving the co-injection of NMDA+NPD1 decreased from 60±4.8%to 41±6.3%?p<0.05?,55±7.5%to 31±4.3%?p<0.01?,and 50±3.7%to 27±3.1%?p<0.001?at 24h,48h and 72h,respectively,compared with those of NMDA-injected rats,suggesting that NPD1suppresses NMDA-induced RGC apoptosis.2)Intravitreal NPD1 maintains RGC electrophysiological function in rats.LE rats received the intravitreal injection of NMDA or co-injection of NMDA+NPD1were examined.ERG data showed that the PhNR amplitudes of the rats in latter group decreased from 102±11?V to 63±1 3?V in a steady trend,while the PhNR amplitudes of the former ones dropped sharply from 103±14?V to 24±8?V.fVEP data showed that rats of latter group maintained higher P₂-wave amplitudes?p<0.05?.Those data suggest that NPD1maintains the NMDA-injured RGC function.3)Intravitreal NPD1 restores the visual acuity of NMDA-injured rats.The visual acuity of rats challenged by NMDA alone decreased from 0.76±0.05 c/d to0.27±0.19 c/d,but NPD1 co-injected with NMDA significantly increased the visual acuity to0.56±0.05 c/d?p<0.001?.?2?Endogenous elevation of NPD1 in rats protects RGC from NMDA injury.1)Dietary supplementation with DHA improves endogenous NPD1 content in rats.ELISA analysis showed that dietary supplementation with DHA increased the level of endogenous NPD1 in retinal tissue from 36.2±2.8 to 48.8±0.9 pg/mg compared to the DHA-deficiency diet.And the NPD1 content in plasma significantly increased from 6.0±0.03 to 8.5±0.3 pg/ml?p<0.001?.2)Endogenous elevation of NPD1 in rats reduces NMDA-induced RGC apoptosis.Immunostaining showed that the number of TUNEL-and Brn3a-positive cells accounted for about 89.93±2.73%of GCL in the NMDA-injured retina of rats intaking DHA-deficiency diet,while the DHA-enriched diet group only reached 21.33±2.36%?p<0.001?,but when the NMDA-injured retinas of rats consuming the DHA-deficiency diet were treated with exogenous NPD1,the proportion of double positive cells was significantly reduced to about 11.58±3.13%?p<0.001?,suggesting that the lack of DHA in the diet makes RGC more susceptible to NMDA-induced damage,but both elevation of endogenous and exogenous NPD1 can diminish the apoptosis of RGC.Rats fed DHA-enriched diet maintained the PhNR amplitude?44.23±4.66?V?after the intravitreal injection of NMDA,while the PhNR amplitude of the rats fed DHA-deficiency diet?22.78±1.93?V?was significantly lower?p<0.05?.However,the PhNR amplitude was significantly maintained?43.40±2.82?V?in the rats fed DHA-deficiency diet received the co-injection of NMDA and NPD1?p<0.05?,suggesting that both elevation of endogenous and exogenous NPD1 can restore the function of RGC.2.Mechanism underlying the protective effect of NPD1 on NMDA-injured RGC in rats.?1?NPD1 suppresses neuronal apoptosis by downregulating calpain 1 in NMDA-injured rat retinas.1)NPD1 regulates neuronal death-related pathways in the NMDA-injured rat retinas.Functional clustering of differentially expressed genes and proteins between NMDA injected and NMDA+NPD1 co-injected intravitreally rat retinas suggested that NPD1regulated neuronal death and development as well as inflammatory responses.2)NPD1 negatively regulates the neuronal apoptosis in NMDA-injured rat retinas.The heatmap of differentially expressed genes suggested that NPD1 downregulated the process of apoptosis.Calpain 1 ranked the top among 281 downregulated differentially expressed genes in the volcano map.3)NPD1 reduces the expression of calpain 1 to ameliorate neuronal apoptosis in NMDA-injured rat retinas.Western blotting showed that intravitreal injection of NMDA induced a remarkable upregulation of calpain 1?p<0.05?,but NPD1 significantly decreaesed its expression?p<0.05?when injected simultaneously.The quantitative analysis of calpain 1 mRNA came to the consistent results.The mRNA levels of the four genes involved in the calpain 1-dependent apoptotic pathway,including Bax,Bid,AIF and caspase 3,were also significantly reduced in the retinas subjected to NMDA combined with NPD1 by comparison with NMDA alone.Those data suggest that NPD1 downregulates calpain-1 in the retinas of rat with NMDA injury.4)NPD1 protects RGC from calpain 1-induced apoptosis in NMDA-injured rat retinas.Immunostaining revealed that calpain 1 colocalized with NeuN/Brn3a in NMDA-injured rat retinas,indicating that calpain 1 was expressed in RGC.Intravitreal calpain 1 significantly induced RGC apoptosis compared to that of the PBS-treated retinas in rats?p<0.001?,and RGC apoptosis increased from 1.96±0.51%to 19.08±1.58%.But Intravitreal calpain 1 and NPD1 simultaneously significantly decreased RGC apoptosis to 8.90±1.10%?p<0.001?.Meanwhile,NPD1 significantly maintained the expression levels of genes which expressed in RGC specifically,including Brn3a,Thy-1 and RBPMs.RT-PCR results indicated that intravitreal calpain 1 induced a significant increase in the expression of four apoptotic genes,namely Bax,Bid,AIF?p<0.001?and caspase 3?p<0.01?in rat retinas.However,the mRNA expression levels of the four apoptotic genes were significantly decreased in the retinas of rat received intravitreal calpain 1 combined with NPD1.The results suggest that NPD1ameliorates the intravitreal calpain-1 induced RGC apoptosis in rats.?2?NPD1 protects RGC by regulating microglia-mediated inflammation in NMDA-injured rat retinas.1)NPD1 regulates inflammatory response in NMDA-injured rat retinasFunctional clustering of differentially expressed genes and proteins between NMDA injected and NMDA+NPD1 co-injected intravitreally rat retinas suggested that NPD1regulated inflammatory responses,and the heatmaps suggested that NPD1 downregulated the expressions of differential expressed genes involved in inflammatory responses.2)NPD1 reduces the expression of inflammatory factors in NMDA-injured rat retinasComparing to the injection of NMDA intravitreally,co-injection of NMDA and NPD1 in rats significantly reduced the mRNA levels of cytokines in retinas,such as CCL-2,IL1?,IFN-?,TNF?,but increased the expression of anti-inflammatory factors TGF?and IL10,all of which had significant difference?p<0.001?,suggesting that NPD1 decreases the expression of inflammatory factors in NMDA-injured rat retinas.3)NPD1 attenuates activated microglia-mediated inflammation in NMDA-injured rat retinas.LE rats were divided into two groups received the intravitreal injection of NMDA combined with NPD1,or NMDA alone.Immunostaining revealed that iba1-positive microglia exhibited the morphology with more branches and longer processes in the former retinas?p<0.001?.Western blotting results showed that the former treatment decreased CCL-2expression?p<0.05?,whereas induced an 8.5-fold change of ED-1 expression compared with that of the latter ones?p<0.05?.Those data suggest that NPD1 suppresses the activation of microglia in NMDA-injured rat retinas and promotes its phagocytic clearance,thereby limiting the inflammatory response.Conclusions:1.This study revealed that in the RD1 mouse model of RD,the expression level of15-LOX,a key enzyme of SPM metabolism,increased first in the early stage of degeneration,and decreased significantly in the late stage,indicating endogenous SPM disorders can occur in RD.This provides a new idea for the application of SPM to regulate the progression of RD.2.This study found that SPM,including both LXA4 and NPD1,could regulate the activation and function of microglia in the retinas of RD1 mice and NMDA-induced retinal rat models,and limit activated microglia-mediated inflammation,thereby improving the degenerative retinal microenvironment to indirectly protect retinal cells.3.This study observed that SPM,including both LXA4 and NPD1,exhibit anti-apoptotic effects on degenerative neurons in RD.LXA4 reduced the apoptosis of photoreceptors in the late-stage RD1 retinas and maintains the thickness of the outer nuclear layer,saving the retinal electrophysiological function.NPD1 suppressed the apoptosis of RGCs in NMDA-induced retinal rat model and maintains its electrophysiological response.4.This project innovatively used retinal explant culture method to study the regulation of SPM on inflammatory response in degenerative retinas at the tissue level,and confirmed that LXA4 significantly reduced the production and secretion of CCL-2 in the late-stage degenerative retina of RD1 mice.This clarifys that the inhibitory effect of LXA4 on microglia activation is an important mechanism for limiting retinal inflammation in degenerative retinas.5.This study reported for the first time that calpains involved in the RGC apoptosis pathway in the retina of NMDA-induced retinal rat model,and found that exogenous NPD1exerted its anti-apoptotic effect on RGC by downregulating calpain 1 in the retina of NMDA-induced retinal rat model.This provides a new intervention target for delaying progressive neuronal degeneration and apoptosis in RD.