| ObjectiveTo investigate the protective effects and its mechanism of Chinese medicinal Formula shihusan on retinitis pigmentosa (RP) through study of retinal degeneration animal models and retinal neural cell culture, and further to study its effect positions and degree during the process of photoreceptor apoptosis, thereby to identify Chinese medicinal herbs which may be valuable as therapeutic agents for RP, and also to provide new thoughts and methods for future study of Traditional Chinese Medicine's effect on RP.Methods1 Animal model study: Mutant mice with naturally occurring inherited retinal degeneration, retinal degeneration slow (rds), had been treated with shihusan compounded by Dendrobium ,Herba Epimedii and Atractylodes Rhizome , from the time when male and female mice were fed together to 56 days postnatal. The retinal pathologic and ultrastructure changes were observed by light and electron microscopy, and the apoptosis rates of photoreceptor cells were determined through TUNEL (tdt-mediated biotin-dUTP nick-end labeling) technique.The retinal function changes were detected by Electroretinogram (ERG). The mRNA expressions of bax, bcl-2, caspase-3 and c-fos were determined by real-time fluorescent quantitative (FQ)-PCR method in rds mice treated with shihusan.2 Cell culture in vitro study: The cultured retinal cells of human were treated with formula and single herbs. The mitochondrial metabolic ability of the cells was examined by MTT test. The apoptosis cells were accounted by flow cytometry. The mitochondrial membrane potensial (MMP) and intracellular Ca2+ of cultured retinal neural cells were determined by laser scan confocal microscopy using rhodamine 123 and Furo 3/AM as indicator.Results1 Study on the effect of shihusan on the animal model of RP(D The effects of shihusan on pathologic changes of rds mice: At 14 days postnatal (P14) of rds mice, there was no difference in retinal structure and thickness of outer nuclear layer (ONL) between shihusan group and control group. Photoreceptors aligned in order. At P28, ONL and inner nuclear layers (INL) of control mice became obviously thinner than that of shihusan mice, hi addition, the decrease of photoreceptors in control mice was more significantly than shihusan mice (P<0.05). At P56, there were only 6 layers of photoreceptor cells which were less orderred in the control group than in the shihusan group. The changes in shihusan group were equivalent with the control group at its P28. At PI 12, there were only 3-4 layers of photoreceptors aligning in serious disorders. INL also became thinner. During all observing period, outer segments development was found neither in the mice of control group nor shihusan group.(2) The effects of shihusan on ultrastructure changes of rds mice: At PI4, under TEM observation, we found no outer segment structure was noticed, and few cells with apoptosis nucleus changes in rds mice. At P28 of control mice, TEM showed there were some disorganization in segment layer such as disaggregation of some inner segment and cilia, still no outer segment structure. More apoptosis nucleus changes in ONL were found than that of the mice at PI4. Moreover, there was some degeneration of mitochondrium in ONL. hi the control mice at P56, it was found by TEM there were less cells layers and more disorganization in inner segments and cilia, apoptosis nucleus changes were found in most cells, with more mitochondrium degeneration. Some apoptosis changes were also found in INL. Compare with the control group, no more degenerating cells were found in shihusan group. However, the degree of apoptosis at P56 of shihusan group was more serious than that at P28.(3) The effects of shihusan on photoreceptor apoptosis of rds mice: TUNEL was used during the study. At 14 days postnatal both control and shihusan group showed few positive stained cells (brownish-black) in ONL. There was no significant difference between the two groups compared with their apoptosis rate. .At P28, the number of positive stainedphotoreceptor cells were obviously increased in two groups. The apoptosis rates of photoreceptor cells were 6.27% and 3.46% respectively, and showed significant difference (P<0.05). At P56, the control group demonstrated conspicuous thinning of the ONL with decreased number of the cells and more positive stained cells in both ONL and INL compare with the shihusan group. The apoptosis rates were 15.8% and 8.53% respectively, and was statistically significant (P<0.05).? The effects of shihusan on retinal function of rds mice: There were different changes ofMax-ERG in different ages. In general, P56 mice were considered of mature mice due to high wave amplitude and stable electrophysiological response. The electrophysiological response of rds mice was much lower than that of normal mice (C57 mice) because of retinal degeneration. The results of Max-ERG response showed a-wave and b-wave amplitude was higher at P28, attenuated at P56 and PI4. The results were related with more serious degeneration at P56, and younger stage and other effect factors at PI4. The cone electrical response couldn't be recorded in two groups. The ERG response of shihusan mice was similar to the control mice, and no significant differences were observed.(D The effects of shihusan on apoptosis related genes of rds mice: The expression of bax gene was conspicuously higher than that of other apoptosis related genes, and increased with the age of rds mice. To bcl-2 gene, the expression was significantly higher at P28 and P56 than it was at PI4 (PO.05). The expression of caspase-3 gene has the similiar trend as bcl-2 gene;the value at P56 was the highest, about 0.72±0.17. The expression of c-fos gene was very low, and there was no significant difference among three age groups. Compared with the control mice, the expression of bax gene in shihusan mice was obviously less at P56 (P<0.05), the difference was not significant at P14 and P28. The expression of caspase-3 gene in the mice of shihusan group was obviously less than that of control mice at P28 and P56 (P<0.05).But there were no significantly difference of bcl-2 and c-fos expression between the control and shihusan group mice at the same age. 2 Study on the effect of shihusan on the cultured human retinal cells: ? The effect of shihusan on the human retinal neuron cells activity: The human retinal cells began to adhere after 12 hours, spread in 2-3 days, and grew well in 15 to 30 days.Approximately 65% of these cells reacted with NSE antibody, 30% with GFAP, which means neuron cells were the major cell type. After being cultured for 80 days, the retinal neuron cells died away gradually. MTT test: Shihusan groups (1.58mg/ml and 0.5mg/ml) and Dendrobium group (5mg/ml) showed promotional effect on the growth of human retinal cells. This change was significantly compare with the control group (P<0.05).Among all of these groups, Shihusan group (0.5mg/ml) was the most efficient one. It's found that other single herbs could not increase retinal neuron cells activity, hi addition, NGF groups (10-1000ng) showed obvious promotional effect of the retinal neural cells..(2) The effects of shihusan on apoptosis of cultured human retinal neural cells: Living cell rates were 61.32% in control group and 73.8% in shihusan group without adding Glu, the difference was significant (P<0.05). Dead cell and non-viable apoptotic cell rate was 32.40% in control group without adding Glu.After adding Glu in these groups, living cell rates were 44.23% and 58.91%, Dead cell and non-viable apoptotic cell rates were 46.53% and 24.39% respectively. The difference was significantly (P<0.01). But viable apoptotic cell rate was higher in shihusan group (16.7%) than that of control group.(3) The effects of shihusan on MMP of cultured human retinal neural cells: Shihusan could raise MMP of retinal cells slightly, and resist the decline of MMP by glutamic acid.? The effects of shihusan on intracellular Ca2+ of cultured human retinal neural cells:fluorescence intensity increased 88% in glutamic acid group,decreased 14.8% and 57.3% in shihusan group and verapamil group respectively. Shihusan couldn't decrease intracellular Ca2+ increased by glutamic acid, but could inhibit increasing tendency induced by glutamic acid. The difference is significent compared to control group (P<0.01).Conclusion1 Shihusan could protect the structures but function of rds mice by early intervention. The study indicated that shihusan could delay apoptosis of retinal neural cells by affecting the expression of bax and caspase-3 genes, not bcl-2 and c-fos genes.2 As a tonijying kidney and nourishing essence formula, shihusan could inhibit apoptosis of retinal neural cells, promote growth of the cells and protect the cells from death throughadjusting mitochondrium metabolism. The effect of shihusan was better than other singleherbs.3 The results suggest that shihusan could protect retinal neural cells and delay retinaldegeneration, in accordance with clinical dialectical feature of RP. Therefore, it might be agood basic formula to treat RP and associated diseases, and be benefit for the patients.
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