The Intervention Effect Of Traditional Chinese Medicine Purendan On C-Jun N-terminal Kinase And Cysteine Aspartic Acid Specific Protease-3 Expression Of Retina In OLETF Rat

Along with the extension of life expectancy and people lifestyle changes, the prevalence and blindness rates of diabetic retinopathy(DR) is rising year by year, has become our important cause of irreversible blindness.Therefore, how to defense and treatment of DR become an important topic in the research field of diabetes complications.In recent years, studies have shown that DR is not only a kind of microvascular lesion but also a kind of nerve degeneration, the nerve cells apoptosis of retinal is the main form of cases in the early of DR.JNK signaling pathway as a mediated apoptosis is one of the important pathways in DR,and plays an important role in the process of development, the c- Jun N- terminal kinase(JNK) and cysteine aspartic acid specific protease-3(Caspase-3) as JNK signaling pathways is the two important factors which closely associated with the diabetic retinopathy.Chinese materia medica(CMM) which has multi-targets and multi-pathways treatment effedt, has the unique advantages for the treatment of DR.Purendan superfine powder(PRD) has the functions of boosting the energy,clearing away heat,activating blood,removing obstructing in collaterals,which aims at the key of the nosogeny of diabetes mellitus(DM),heat, deficiency and stasis.In this study,diabetic rats models built up by the spontaneous diabetic rats model OLETF rats,are used to investigate the protective effects of PRD on retinopathy of diabetic rats and theoretical basis of the treatment and experimental of PRD.Objective:To investigate the effects of PRD on phosphorylation c-Jun N-terminal kinase and cysteine aspartic acid specific protease-3 in retina of spontaneous type 2 diabetic rat.Methods:With spontaneous type 2 male diabetes OLETF rats and homologous nodiabetesmale LETO rats as experimental object,with a peak level of fasting blood glucose>16.8mmol/L and a level of blood glucose at 120 min>11.2mmol/L as standard.Divided 24 mold male OLETF rats into diabetes model group and PRD treatment group,each group of 12,the same weeks male LETO rats as the normal control group.After the model was succssesfully established,the rats in PRD treatment group were lavaged with PRD(1.8g·kg-1·d-1) for 2 months.The morphological changes of rtina were observed by HE staining,neuron apoptosis in retina was detected by TUNEL technique,the expression of p-JNK and Caspase-3 protein in retina were detected by Immunohistochemical staining and Western blot.The Reverse reanscription-polymerase chaine reaction(RT-PCR) was employed to examine the expression of JNK and Caspase-3 m-RNA in retina.2 Fasting blood glucose meter was used to detect the blood glucose of all groups.3 The morphological changes of rtina were observed by HE staining.4 Neuron apoptosis in retina was detected by TUNEL technique.5 The expression of p-JNK and Caspase-3 protein in retina were detected by Immunohistochemical staining and Western blot.6 RT-PCR was employed to examine the expression of JNK and Caspase-3 m-RNA in retina.Results:1.Before the DM models successfully established,the FBG of each group has no obvious difference, after the DM models successfully established in addition to the normal group, the FBG of other groups significantly increased, was statistically significant(P < 0.01).After treated with PRD,the FBG of rats in PRD treatment group were significantly reduced compared with diabetes model group(P < 0.01).2.Change of retinal histomorphology in each group: the retinal structure of normal group rats was complete, the surface of inner limiting membrane was smooth,the layers were clear.Ganglion cells were circle, elliptic, hypochromic and the arrangment was orderly. Inner plexiform layer thicker and loosen.The inner nuclear layer hypochromic, made up of 3 ~ 5 layers of cells.The outer plexiform layer is obviously thinner than inner plexiform layer.The outer nuclear layer dyeing deeply, consisted of 8 ~ 10 layer cells and the arrangement was closely.The outside limiting membrane boundary clear and the arrangment was orderly.In diabetic model group,inner limiting menbrane became edema and incrassation, part of the inner limiting membrane rupture and the boundary is not clear, some cells vacuole-like transformed,karyopycnosis,partial hypochromatosis.In PRD treatment group,the layere clearer,inner limiting membrane became edema lightly and some cells vacuole-like transformed.3.Neurons apoptosis in retina:There were no obviousiy TUNEL positive cells in normal control group rats retina and the positive cells in diabetes model group and PRD treatment group expanded distributed.The TUNEL positive products were brown,granules,located in nucleus and were seen in inner nuclear layer and RGCs layer.For the three groups of rats retina,the comparison of AI were statistically significant.AI of retina was obviously higher in diabetes model group rats than in normal control group rats(P < 0.01),AI of retina was obviously iower in PRD treatment group rats than in diabetes model group rats(P < 0.01).4.p- JNK expression in retina of rats: immunohistochemical staining showed,p- JNK immunopositive products were buffy granules and located in nucleus and cytoplasm, p- JNK immunopositive cells were seen in RGCs layer and inner nuclear layer.Western blot showed,the p-JNK protein band located at 42 kDa and β-actin protein band located at 43 kDa.RT-PCR showed,the JNK mRNA band located at 422 bp and β-actin mRNA band located at 425 bp.The expression of p- JNK protein and mRNA in retina were obviously higher in diabetes model group rats than in normal control group rats(P < 0.01),those were obviously lower in PRD treatment group rats than in diabetes model group rats(P < 0.01).5.Caspase-3 expression in retina of rats: immunohistochemical staining showed,Caspase-3 immunopositive products were buffy granules and located in nucleus, Caspase-3 immunopositive cells were seen in RGCs layer and inner nuclear layer.Western blot showed,the Caspase-3 protein band located at 34 kDa and β-actin protein band located at 43 kDa.RT-PCR showed,the Caspase-3 mRNA band located at 329 bp and β-actin mRNA band located at 425 bpThe expression of Caspase-3 protein and mRNA in retina were obviously higher in diabetes model group rats than in normal control group rats(P< 0.01),those were obviously lower in PRD treatment group rats than in diabetes model group rats(P< 0.01).Conclusion:PRD can improve nerve tissue damage in diabetic retinopathy, it can be manifestedby the reduce of nervecell apoptosis, this may be related to inhibiting the expression of p-JNK and Caspase-3.