The Intervention Effect Of The Chinese Medicine Purendan On OLETF Rats Retinal Glucose Regulated Protein 78 And C/EBP Homologous Protein Expression

Objective:Diabetic retinopathy(DR) is the most serious and common in the diabetic microvascular(DM)complications. Statistics showed that the prevalence of DR of diabetics was about 25%. DR may still continue to develop even blood glucose was controlled well.And it will reduce the quality of life of patients and their families by reduce one’s visual acuity severely. There is a variety of theories about the pathogenesis of DR. And there is no satisfactory treatment for advanced DR. Therefore it is significant to find the pathomechanism of DR and new methods for treating it. According to the research, retinal microvascular disease and associated neurodegenerative diseases, which is most occurred by apoptosis, play a major role in the pathogenesis of DR. Apoptosis including death receptor pathway, the mitochondrial pathway and endoplasmic reticulum stress pathway(ER stress), and ER stress is a hotspot in resent studies. As endoplasmic reticulum stress pathway iconic factors, glucose-regulated protein78(GRP78) and C / EBP homologous protein(CHOP)plays an important role in apoptosis. Traditional Chinese Medicine considers that the “heat, deficiency, stasis” is relative to pathomechanism of diabetic. Purendan superfine powder(PRD), which is made of bitter melon, ginseng, salvia, polygonum, leeches, kudzu, is a traditional Chinese medicine with efficacy of clearing away heat, benefiting lung and removing blood stasis. This study aim to investigate protective effect of PRD on the retina of type 2 diabetic rats through TUNEL, HE, IHC, PCR, Western boltting. In order to provide references for the clinical application value of PRD on the treatment of retinal nerve tissue damage in DR.To explore the protective effects of PRD on diabetic retinopathy by observing the intervention effect of PRD on the factor GRP78 and CHOP expressions of OLETF rats retina.Methods:A.experimental subjects are male OLETF rats which are the spontaneous type 2 diabetic rats model and its homologues in non-diabetic control LETO rats.We let the rats which meet the diabetic standard that the peak glucose is higher than 16.7 mmolL-1 or 120 min after glucose load is higher than 11.1 mmol L-1 as the model,choose 24 of them,and randomly divided into PRD treatment group, diabetic model group.each group had 12 rats, with 12 of the same age male LETO rats as normal control group.After the model was successfully established, the PRD treatment group rats continuous intragastric administrate PRD(1.8g.kg-1.d-1)2 months.B. Fasting blood glucose meter was used to detect the blood glucose of all groups and living state was observed at regular intervals.C. After administration, all rats received intraperitoneal anesthesia, removed the eye balls for text specimen processing.D. HE staining was used to observe the pathologic changes of retinal tissue.E. TUNEL was used to detect the apoptosis of retinal nerve cells of OLETF ratsF. SP immunohistochemical staining was used to observe the GRP78 and CHOP protein expression of retinal tissue.G. Western blotting was used to detect the GRP78 and CHOP protein expression and quantitative analysis.H. RT-PCR was used to detect the GRP78 and CHOP mRNA expression and quantitative analysis.Result:Living state of rats in each group: diabetes model group rats were more irritable, with poor quality of hair, shedding, lost weight, drinking and urinating more than before. After treated with PRD lavage, the hair of rats were more shining, gained weight, less drinking and urinating than DM groups’. During the whole process, the normal control group rats has no obvious change. Blood glucose monitoring result showed that the blood glucose of rats in diabetes model group increased obviously compared with that in PRD treatment group(P<0.01);the blood glucose of rats in normal control group decreased significantly compared with that in diabetes model group(P<0.01). The HE staining changes showed as follows. Normal control group: the retinal structure was complete and stratified clearly, cells morphology were in good condition and the inner limiting membrane was smooth, no changes of swelling and thickening. Diabetes model group: inner limiting membrane was markedly swelling and thickening, the retinal layers owed clear, some cells vacuolar degeneration and karyopyknosis, cell distribution disorder and sparse. Some fragmentation of inner limiting membrane causing membrane surface rough, hyperplasia of capillaries endothelial cells out from the membrane. PRD treatment group: stratified clear, inner limiting membrane mild swelling and thickening. Compared with diabetes model group,the appearance of decreasing in the cell number and cell disorder were less. Immunohistochemical staining in combination with Western blotting and RT-PCR results shows that compared with normal control group, the expression of GRP78,CHOP protein and mRNA increased in diabetes model group(P<0.01);compared with diabetes model group, the expression of GRP78,CHOP protein and mRNA decreased significantly in PRD treatment group(P<0.01).Conclusion:PRD treatment may have diabetic rat’s retina neuroprotective effect, its mechanism may be mediated by an inhibition of ER stress and apoptosis in diabetic retinal neurons.