The Role And Mechanism Of Sonic Hedgehog Regulating PI3K/AKT Pathway In From-deprivation Myopic Development Of Guinea Pigs

PrefaceDespite many years of intensive research, the mechanism of myopic occurence and development still remains unclear. One commonly held belief is that the abnormal visual stimuli including diffused or defocus image can act on the retina. This effect can transmit to the sclera by choroid and induce some relative changes of metalloproteinases (MMPs), which can finally act on the extracellular matrix (ECM) and cause scleral degradation and development of myopia. Our previous studies have indicated that Sonic hedgehog (Shh) play a key role in myopic occurrence and development, which can also regulate the levels of MMPs and transforming growth factor-β (TGF-β). Nevertheless, the specific pathway of this role and regulating is still unknown. Many studies in tumor have found out that Shh pathway can modulate the expression of MMPs by regulating PI3K/AKT signal, which may influence the invasion and metastasis of the tumor. This shows us the possible intermediate pathways with Shh in myopia. Thus, we also use form-deprived myopic (FDM) animal model by covering method in guinea pigs. For on hand, we observe the changes of PI3K/AKT signals in FDM and other corresponding changes after inhibiting PI3K/AKT pathway with LY294002. For another, we observe some relative changes including MMP-2 and bFGF by upregulating Shh pathway with exogenous Shh and downregulating with cyclopamine. Through this study, we aimed to figure out the exact acts of Shh on myopic development and its complete regulating pathway, providing basis for myopic prevention and therapeutics.PART Ⅰ The expression of retinal PI3K/AKT signaling and scleral MMP-2/bFGF in guinea pigs with form deprivation myopiaPurpose:To observe the expression of retinal PI3K/AKT signaling and scleral MMP-2/bFGF in guinea pigs with form deprivation myopia.Methods:Seventy-two healthy guinea pigs of 2 to 3-week-old were randomly assigned to the FDM group (n=48) and the control group (n=24). The right eyes of guinea pigs in the FDM group were wore a translucent diffuser. Six guinea pigs were separately choosen from the FDM group and the control group for retinoscopic refraction and A-scan measurement at 0,1,2 an 4 weeks. Then, animals were killed and the eyeballs were enucleated for detection of retinal PI3K and AKT immunohistochemically, mRNA of retinal PI3K and AKT by real-time PCR and protein of retinal PI3K and AKT, scleral MMP-2/TGF-β with Western-blot.Results:The right eyes in the FDM group developed significant myopia of-1.74±1.23D、-4.82±1.65、-6.43±1.17D compared with the left eyes at 1,2 and 4 weeks. And the myopia progressed with the prolonging of diffuser wearing. The right eyes in the FDM group had an elongation of 0.09±0.03mm、0.12±0.04mm and 0.33±0.16mm in axial lengths compared with the contralateral eyes at 1.2 and 4 weeks, which were statistically significant. And The right eyes in the FDM group had an elongation of 0.06±0.03mm、0.11±0.05mm and 0.27±0.13mm in lengths of the vitreous compared with the contralateral eyes at 1,2 and 4 weeks, which were statistically significant. PI3K/AKT was immunohistochemically detedcted in retina and the induction of myopia caused significant increase in expression of PI3K mRNA (P=0.0052) and AKT mRNA (P<0.001) at week 4 compared with the contralateral eyes. Significant increase of retinal PI3K/AKT protein and scleral MMP-2, bFGF were seen in the right eyes of the FDM group since week 2 and continued to week 4.Conclusions:The changes in the expression of PI3K/AKT and the similar trend comparing with Shh, MMP-2 and bFGF in guinea pigs with form deprivation myopia suggests an involvement of PI3K/AKT signaling pathway in the development of FDM.PART II Effect of intravitreal injection of LY294002 on the myopic development in guinea pigs with form deprivation myopiaPurpose:To investigate the effect of intravitreal injection of LY294002 on the myopic development and its associated expression of retinal Shh, scleral MMP-2/bFGF in guinea pigs.Methods:Fifty four guinea pigs of 2 to 3-week-old were randomly assignedinto three groups (n=18, each). The right eyes of guinea pigs in each group were wore a translucent diffuser. Both eyes in all 3 groups were intravitreally injected with LY294002. The concentrations of LY294002 for the three groups were 10μM/L (group 1),20μM/L (group 2) and 40μM/L (group 3), separately. The injection was performed every 1 days for 3 times with a volume of 10μl for each eye. Retinoscopic refraction and A-scan measurements were performed at week 0,1,2,4 weeks. Then, animals were killed and the eyes were enucleated for scleral MMP-2/bFGF detection with Western-blot, Shh detection with RT-PCR as well. The results were also compared with those in group of part I.Results:Intravitreal injection of LY294002 in guinea pigs with FDM can induce significant development of myopia of -1.15±1.09D.-4.06±1.38D、-6.11±1.24D at 1,2, 4 weeks in group 1. The refraction results in other two groups was -0.84±1.03D、-1.66±1.16D、-1.41±1.21D and -0.63±0.89D、-0.91±1.18D、-1.15±1.13D in group 2 and group 3. Meanwhile, it can induce significant elongation of axial lengths and vitreous lengths in all 3 groups, separately when compared with the left eyes and relevant results in part Ⅰ. Of which, the myopia progressed (P<0.001) and the axial lengths elongated (P<0.01) with the lower concentration of LY294002. Upregulation of Shh mRNA in retina was observed from RT-PCR in all 3 groups, but the uptrend decreased with the concerntration of LY294002. The same changes of Shh, MMP-2 protein were seen in Western-blot results. However, there was no significant difference in bFGF among 3 groups.Conclusions:Intravitreal injection of LY294002 can slow the development of myopia and the axial growth. Besides, it can inhibit the Shh and MMP-2 upregulating with FDM. PI3K/AKT signal may be the intermediate pathway of Shh in myopia regulating.PART III Effect of intravitreal injection of Shh-N in guinea pigs and Cyclopamine in guinea pigs with form deprivation myopiaPurpose:To investigate the effect of intravitreal injection of Shh-N in guenia pigs, cyclopamine in guinea pigs with form deprivation myopia and the associated expression of retinal PI3K/AKT and scleral MMP-2/bFGF in guinea pigs.Methods:Forty eight healthy guinea pigs of 2 to 3-week-old were randomly assigned into 2 groups (n=24, each) as section A. Seventy two ones were randomly assigned into 3 groups (n=24, each) as section B. The right eyes in each group of section A were intravitreally injected with Shh-N/0.1% BSA and the other eyes were intravitreally injected with 0.1% BSA. The injection was performed every 2 days for 4 times with a volume of 10μl for each eye. The concentrations of Shh-N for the two groups were 20μg/ml (the group of Shh20) and 50μg/ml (the group of Shh50), separately. In section B, both eyes in each group were intravitreally injected with cyclopamine at the same concentration while the right eyes in each group were wore a translucent diffuser. The injection was performed every 2 days for 4 times with a volume of 10μl for each eye. The concentrations of cyclopamine for the three groups were 0μg/ml (FDMO group),100μg/ml (FDM50 group) and 200μg/ml (FDM200 group), separately. Retinoscopic refraction and A-scan measurement were performed at week 2. Then, animals were killed and the eyes were enucleated for retinal PI3K/AKT and scleral MMP-2/bFGF detection with Western-blot. The results in section B were also compared with those in group of part ⅠResults:Section A:Intravitreal injection of Shh-N at concentration of 20μg/ml and 50μg/ml can induce significant development of myopia of -1.43±0.66D and-4.01±1.27D, elongation of axial lengths of 0.10±0.07mm and 0.13±0.06mm, elongation of vitreous lengths of 0.10±0.08mm、0.13±0.04mm, separately when compared with the left eyes (P<0.01). Of which, the myopia progressed (P<0.01) and the axial lengths elongated with the higher concentration of Shh-N. Upregulation of PI3K/AKT in retina and MMP-2 in sclera was observed with the Shh-N injection from the Western-blot results. The same results of PI3K/AKT mRNA were observed from RT-PCR.Section B:The right eyes in the group of FDM0、FDM50 and FDM200 induce relative myopia of -4.37±.52D、-2.81±0.73D2 及-0.66±0.92D, separately. Intravitreal injection of cyclopamine at 50 and 200 concentration can significantly slow the development of myopia (P<0.001). The right eyes in the group of FDM0、 FDM50 and FDM200 induce relative axial elongation of 0.13±0.06mm、 0.08±0.04mm、0.03±0.02mm, separately. Meanwhile, the relative elongations of vitreous lengths were 0.12±0.07mm、0.07±0.05mm、0.03±0.02mm for the FDMO、 FDM50 and FDM200 group, separately. The differences among 3 groups were all statistically significant (P<0.001). Upregulation of PI3K and AKT mRNA due to FDM was inhibited by cyclopamine, which was proportional to its concertration. Besides, Upregulation of PI3K protein in retina was inhibited by cyclopamine of different concertration. However, there was no significant changes in AKT, MMP-2 and beg protein.Conclusions:Upregulation and inhibiton of Shh signal can induce relevant changes of refraction status, PI3K/AKT pathway, MMP-2 and bFGF. In myopic occurrence and development, Shh may act on the MMPs by regulating PI3K/AKT pathway.PART Ⅳ Effect of intravitreal injection of Shh-N and LY294002 on the myopic development in guinea pigsPurpose:To investigate the effect of simultaneous intravitreal injection of Shh-N and LY294002 on the myopic development and its associated expression of scleral MMP-2/bFGF in guinea pigs.Methods:Forty eight guinea pigs of 2 to 3-week-old were randomly assignedinto two groups (n=24, each). The right eyes of guinea pigs in each group were intravitreally injected with Shh-N and LY294002. The left eyes in each group were intravitreally injected with Shh-N and BSA. The concertration of Shh-N in two groups was 25μg/ml and 50μg/ml while the concentrations of LY294002 were 40μM/L. The injection was performed every1 days for 4 times with a volume of 10μl for each eye. Retinoscopic refraction and A-scan measurements were performed at 14 days (2 weeks). Then, animals were killed and the eyes were enucleated for scleral MMP-2/bFGF detection with Western-blot. The results were also compared with those in group of part Ⅲ.Results:Intravitreal injection can induce -1.03±0.74D、-2.15±0.92D in left eyes comparing to the right eyes.The relative elongations of axial lengths and vitreous lengths and were 0.07±0.03mm、0.12±0.05mm and 0.06±0.02mm、0.11± 0.05mm.There was no significant statistical difference both in left eyes of two groups and relevant results in part Ⅲ(P>0.05). So were differences of the right eyes in two groups (P>0.05).Conclusions:Intravitreal injection of LY294002 can slow the development of myopia and the axial growth due to the Shh-N. Besides, the effect had no association with the concerntration of Shh-N. PI3K/AKT signal may be the unique intermediate pathway of Shh in myopia regulating.