Effect Of LXRs On The Retinal Degeneration Of RD1 Mice And Its Protective Effect On Implanted Neural Stem Cells

Background and purposeRetinal degeneration(RD)including retinitis pigmentosa(RP)is a kind of hereditary eye disease characterized by progressive photoreceptor loss[1,2],is one of the most common blinding eye disease in developed countries,there is no effective treatment.Recent studies have shown that retinal microglia activation mediated immune inflammatory response [3-5]and Müller cell glial activation[6,7] constitute retinal major microenvironment changes,and play a key role in retinal degeneration which are expected to be a new target of treatment[8].At present,Stem cell transplantation is the most promising strategy of treating blind ophthalmopathy such as RD.The nutritional effect and immunomodulatory effect of implanted cells are considered to be important mechanisms for short-term therapeutic effect,and cell replacement is considered its long-term mechanism[9].It is well known that the fate of transplanted cells is affected by its microenvironment.Recent studies have found that inhibition of the immune inflammatory response[10] or Müller cell activation formed glia scar[11],can improve the survival and functional integration of transplanted cells,and conducive to long-term cell replacement effect.Liver X receptors(LXRs)are one of the nuclear receptor superfamily members and play an important role in regulating lipid and glucose metabolism[12,13].Recent studies have reported that LXRs also play an important regulatory role in immune inflammatory responses in neurodegenerative diseases [13]and are closely related to glial cell activation[14].Lei et al.found that LXRs receptor agonist T0901317 can protect N-methyl-D-aspartate(NMDA)-induced retinal damage and inhibit ocular inflammation caused by experimental uveitis[15,16].No relevant research has been reported that whether LXRs activation influenced the degenerated retina or its microenvironment for the implanted stem cells.In this study,we investigated the effect of T0901317-mediated LXRs activation on the gliosis of Müller cells and the immunoinflammatory response of microglia in the retina degeneration mouse model Rd1.Whether the effect could delay the degeneration process of binocular photoreceptor cells in Rd1 mice,save the visual function and affect the fate and treatment of implanted mouse neural stem cells C17.2.And preliminary discuss the effect and mechanism of LXRs activation on degeneration microenvironment and implanted stem cells.Method:Part I: Effects of LXRs Activation on Degenerated Retina in Rd1 Mice1.Taking postnatal 7 days(P7)Rd1 mice,the same age C57 / BL6 mice as a control.LXRs agonist T0901317 or 2% dimethylforma(DMSO)were injected intraperitoneally for 7 days.The retinal specimens of mice were taken from P15 and the activation of LXRs in the retina was detected by RT-qPRC.2.Immunohistochemistry and Western blotting were performed to compare the effects of LXRs activation on photoreceptor apoptosis,Müller cell and microglia activation in mouse retina.3.The effect of LXRs activation on the retinal function of Rd1 mice was observed by retinal electroretinogram(ERG)test in retinal function of P15 mice.Part II: Mechanism of LXRs activation in the treatment of Rd1 mouse retinal degeneration1.At p15,the expression of inflammatory-related genes,such as interleukin-6(IL-6),cyclooxygenase-2(COX-2)and nitric oxide synthase(iNOS),in mice retina were detected by RT-PCR.2.Gene expression patterns of T0-or vehicle-treated Rd1 mice retina were detected by microarray technique.RT-qPCR was used to validate the results.Part III: Effects of LXRs Activation on the biological characteristics of Neural Stem Cells and Implanted with Rd1 Mice1.Different concentrations of T0 were used to treat the rat neural stem cells in vitro.After 48 h,the proliferation of the L2.3 was observed and the apoptosis was detected by flow cytometry.2.Rd1 mice at P11 were injected intraperitoneally with T0 for 3 days.Neural stem cells(NSCs)C17.2 were transplanted into subretinal space of Rd1 mice at P14.After 1 week,immunofluorescence was performed to observe the changes of photoreceptor cells,glial cells and NSCs in mouse retina.3.ERG was performed to detect the visual function of degenerated mice at 1 week after transplantation.Result:Part I: Effects of LXRs Activation on Denoted Retina in Rd1 Mice1.RT-PCR results showed that LXR? ABCA1 and ABCG1 in the T0-treated mice were up-regulated compared with the vehicle-treated mice,but LXR? expression was decreased,suggesting that T0 could mainly activate LXR? subtype in Rd1 mice retina.2.T0-mediated LXRs activation significantly inhibited the expression of GFAP in Müller cells,reduced the number of activated microglia and apoptotic photoreceptor cells in Rd1 mice,suggesting that activation of LXRs could delay retinal degeneration by inhibiting the activation of retinal glial cells.3.ERG results showed a significant increase in b-wave amplitude of T0-treated Rd1 mice under dark adaptation compared with the control,suggesting that T0-mediated LXRs activation can partially save the visual function during the denaturation of Rd1 mice.Part II: Mechanism of LXRs Activation in Rd1 Mouse Retina1.RT-qPCR results showed that activation of LXRs significantly inhibited the expression of IL-6,COX-2 and i NOS,which indicated that LXRs were involved in the regulation of inflammatory reaction in degenerative retina.2.Microarray and RT-qPCR results showed that T0 regulated the microenvironment of degenerative retina and delayed the process of retinal degeneration through the JAK3-STAT pathway.Part III: Effects of LXRs Activation on the biological characteristics of Neural Stem Cells and Implanted with Rd1 Mice1.LXRs activation can increase the proliferation of L2.3,which has no effect on its apoptosis.2.The survival rate of neural stem cells C17.2 transplanted into vitreous cavity of Rd1 mice treated with T0 was higher than that in the control group,and the thickness of the outer nuclear layer of the retina was thicker than that of the control group.3.T0 pretreatment combined with C17.2 transplantation had no obvious protective effect on the visual function of Rd1 mice at P21.Conclusion:1.By activating the LXR? receptor in the degenerated retina of Rd1 mice,thereby modulating the JAK3-STAT signaling pathway,T0 inhibited the hyperactivity of Müller cells,microglia-induced immunoinflammatory responses,reduced apoptosis of photoreceptor cells and saved visual function of Rd1 mice.2.T0 pretreatment can improve the retinal degeneration microenvironment of Rd1 mice and improve the survival rate of mouse neural stem cells C17.2 transplanted into Rd1 mouse retina.T0 pretreatment and C17.2 transplantation have synergistic effect on the protection of the outer nuclear layer of the retina of Rd1 mice.