An Experimental Study Of QiMing Granule On Shh Signaling Pathway Of Radiation Retinopathy

Part Ⅰ The establishment of rat model with radiation retinopathyObjective:Established the rats models on radiation retinopathy which eyes were irradiated by the linear accelerator. Observed the changes of the retinal morphology of rats eyeballs and defined the intuitive experimental evidence of RR.Method:30 SD rats (aged 2 months) were randomly divided into 15 in normal group and 15 in model control group. The model control group rats’eyes were radiated by the linear accelerator after anesthetized. The irradiation single dose was 10 Gy. They were radiated once a week for 3 weeks, so the total dose was 30 Gy in all. The 5 rats of each group were killed randomly at 4 weeks and 8 weeks and enucleated the eyeballs under the electron microscope. All the left eyeballs were sectioned after fixed. Retinal morphology was observed by hematoxylin-eosin staining. Compared with each retinal layers of cells and tissues to analyze their changes.Result:Compared with the normal group, the cell morphology is abnormal in rats retina which were radiated after 4 weeks and 8 weeks. The ganglion cell layer and inner nuclear layer cell morphology were abnormal especially. Most ganglion cells were hydropic degeneration, and the nuclear lied in the side of cytoplasmic. The cell size of inner nuclear layer were smaller and the chromosomes of nucleus were pyknosis. The retinal ganglion cell layer, inner nuclear layer and outer nuclear layer were edema, the cells were disorder.Conclusions:The retinal cells morphology and tissue structure were obviously abnormal in rats eyes in the early after radiation.Part II Expression of Shh signaling pathway in the retina of rats with radiation retinopathyObjective:To explore the mechanism of radiation retinopathy based on the Shh signaling pathway and provide new research ideas and direction for preventing and treating RR.Method:30 SD rats (aged 2 months) were randomly divided into 15 in normal group and 15 in model control group. The model control group rats’eyes were radiated by the linear accelerator after anesthetized. The irradiation single dose was 10 Gy. They were radiated once a week for 3 weeks, so the total dose was 30 Gy in all. The 5 rats of each group were killed randomly at 4 weeks and 8 weeks and enucleated the eyeballs under the electron microscope. All the left eyeballs were sectioned after fixed. Retinal morphology was observed by hematoxylin-eosin staining. VEGF and HIF-1α were detected by immunohistochemistry. The right eyeballs were opened along corneoscleral limbus. The retinas were separated from the balls. The protein content of PEDF, Shh, Patched, Gli1 were detected by western blot. The gene content of Shh, Patched, Gli1 were detected by RT-PCR. All data were analyzed by SPSS 17.0. The figures were done by medical professional graphics software Graphpad Prism 6.Result:1.VEGF detection:Compared with the normal group, the retinal tissue VEGF protein expression was significantly increased in rats after their eyes radiated 4 weeks and 8 weeks(P<0.01), and it had a trend of rising continuously as time passed.2.PEDF detection:Compared with the normal group, the retinal tissue PEDF protein expression was significantly lower in rats which eyes radiated after 4 weeks and 8 weeks(P<0.01), and it had a trend of decreasing as time passed.3. HIF-la detection:Compared with the normal group, the retinal tissue HIF-la protein expression was significantly higher in rats which eyes radiated after 4 weeks (P<0.01). And there was no significant difference in rats which eyes radiated after 8 weeks (P> 0.05), but there was till a trend of decreasing.4.Shh signaling pathway detection:The Shh, Patched and Gli1 proteins were hardly expressed in normal mature retina of rats. Compared with the normal group, the Shh, Patched, Glil protein expression and Shh、Patched、Gli1 mRNA level in retinal tissue were significantly increased in the model control group in 4 weeks and 8 weeks(all P<0.01), the protein expression and gene levels tended to increase as time goes on.Conclusions:At the early of radiation, the retinal PEDF expression decreasing, Shh signaling pathway activity and HIF-1α expression increasing, these were caused a high degree of expression of VEGF and induced retinal angiogenesis.Part Ⅲ Regulation mechanism of QiMing granule on Shh signaling pathway in radiation retinopathyObjective:Based on the therapeutic effects on retinal vascular lesions of QiMing granule. Observed the changes of pathological and Shh signaling pathway in the retina, studied the effect of Qi Ming granule on the early radiation retinopathy (RR) in rats by taking Cyclopamine as positive control drug.Method:60 SD rats (aged 2 months) were randomly divided into 4 groups, included 15 in normal group,15 in model control group,15 in positive drug group and 15 in Qiming granule group. In addition to the normal group, rats’eyes in other groups were radiated by the linear accelerator after anesthetized. The irradiation single dose was 10 Gy. They were radiated once a week for 3 weeks, so the total dose was 30 Gy in all. From the second day, the normal group and the model control group rats were administered lml saline once per day. The positive drug group rats were injected with 2ul Cyclopamine with the concentration of 2.5mg·ml-1 twice per week. The Qiming granule group rats were administered 2.7g·kg-1 Qiming granule with the concentration of 0.81g·ml-1 once per day. The 5 rats were killed randomly at 4 weeks and 8 weeks and enucleated the eyeballs under the electron microscope. All the left eyeballs were sectioned after fixed. Retinal morphology was observed by hematoxylin-eosin staining. VEGF and HIF-la were detected by immunohistochemistry. The right eyeballs were opened along corneoscleral limbus. The retina was separated from the balls. The protein content of PEDF, Shh, Patched, Gli1 were detected by western blot. The gene content of Shh, Patched, Gli1 were detected by RT-PCR. All data were analyzed by SPSS 17.0. The figures were done by medical professional graphics software Graphpad Prism 6.Result:1.Retinal morphology:Treated for 4 weeks by Qiming granule:Compared with the model control group, the edema of inner nuclear layer and outer nuclear layer were better, the cell arrangement was more neatly and closely. Compared with the positive control group, the ganglion cell layer was more obvious edema. The inner nuclear layer and outer nuclear layer was slightly edema.The cells arrangement was slightly disordered. Compared with the normal group, the ganglion cells were hydropic degeneration, and the nuclear lied in the side of cytoplasmic. The cell size of inner nuclear layer was smaller and the chromosomes of nucleus were pyknosis. The cell arrangement of core layer and outer nuclear layer were disorder. Treated for 8 weeks by Qiming granule:compared with the model control group, the edema of the ganglion cell layer, inner nuclear layer and outer nuclear layer obviously improved, the cells arrangement were more neatly and closely. Compared with the positive control group, the cells morphology of inner nuclear layer were slightly abnormal. There’s no other significant differences. Compared with normal group, only the inner nuclear layer cell morphology is slightly abnormal, and there’s no other significant differences."2.VEGF detection:Treated for 4 weeks by Qiming granule:Compared with the model control group, VEGF protein expression was significantly reduced (P<0.01); VEGF protein levels decreased in the positive control group (p<0.05). Compared with the positive control group, VEGF protein expression difference was not significant(P> 0.05). Treated for 8 weeks by Qiming granule:Compared with model group, VEGF protein expression had a trend of decreasing(P> 0.05). Compared with the positive control group, VEGF protein expression difference was not significant (p> 0.05).3.PEDF detection:Treated for 4 weeks by Qiming granule:Compared with the model control group, PEDF protein expression level was all increased of the Qiming granule and positive control group(P<0.05). Compared with the positive control group, PEDF protein expression difference was not significant (P> 0.05). Treated for 8 weeks by Qiming granule:Compared with model group, PEDF protein level was all increased of the Qiming granule and positive control group (P<0.05). Compared with the positive control group, PEDF protein expression difference was not significant (P> 0.05).4. HIF-1α detection:Treated for 4 weeks by Qiming granule:Compared with the model control group, HIF-1α protein were significantly reduced(P<0.01), HIF-1α protein changes had a trend of decreasing in positive control group (p>0.05). Compared with the positive control group, HIF-1α protein expression had no significant change(P>0.05). Treated for 8 weeks by Qiming granule:Compared with the model control group, HIF-1α protein expression had a trend to be low in the Qiming granule and positive control group (P>0.05). Compared with the positive control group, HIF-1α protein level had no significantly change(P>0.05).5.Shh signaling pathway detection:Treated for 4 weeks by Qiming granule: Compared with the model control group, the Shh protein, Patched protein, Gli1 protein expression in retinal tissue were significantly reduced (all P<0.01). The Shh mRNA, Patched mRNA in retinal tissue reduced (P<0.05), Gli1 mRNA in retinal tissue was significantly reduced (P<0.01).Compared with the positive control group, the Shh protein expression in retinal tissue was reduced obviously(P<0.01), Patched protein, Glil protein expression in retinal tissue had no significant change (p>0.05). the Shh mRNA level reduced (P<0.05). Patched mRNA、Gli1 mRNA had no significant change (p>0.05). Treated for 8 weeks by Qiming granule:Compared with the model control group, the Shh protein, Patched protein, Gli1 protein expression in retinal tissue were reduced significantly (all P<0.01). The Shh mRNA, Gli1 mRNA level reduced (p<0.05), Patched mRNA level reduced significantly (P<0.01). Compared with the positive control group, the Shh protein expression in retinal tissue was reduced significantly (P 0.01). Shh mRNA level reduced (p<0.05). There were no difference in Patched protein, Gli1 protein and Patched mRNA、Gli1 mRNA level expression (P>0.05).Conclusions:The early intervention of QiMing granule could reduce retinal edema and protect retinal tissue structure of radiation-induced retinal damage. It maybe inhibit the occurrence and development of retinal neovascular diseases of radiation retinopathy by promoting the expression of PEDF, inhibition of Shh pathway activity,reducing HIF-la expression to some extent reduce the expression of VEGF. So QiMing granule had a preventive effect for radiation retinopathy.