| Diabetic retinopathy(DR)is one of the most common and serious complications of microvascular complications in diabetes(DM).Early interventions can prevent or delay the incidence or progression of microvascular complications,and reduce the mortality and disability and huge medical burden.At present,the pathogenesis of DR is not clear,and epigenetic regulation has become a hot topic in recent years.Hyperglycemia can activate epigenetic regulation,which leads to the onset of DR,and intervention on target genes through epigenetic modification at early stages is expected to reverse the progression of DR."Dredging collaterals in early treatment,and dredging collaterals in the whole process of diabetes" is our original theory in preventing diabetic vascular complications,on account of this,the principle of supplementing qi,warming yang and dredging collaterals has been applied in the prevention of DR for many years.The composition of Yiqi Wenyang Tongluo foumula(YWTF)is based on this principle above.Previous studies of our research group have shown that preventive treatment by YWTF before the onset of DR can alleviate or delay the process of DR in diabetic rats.YWTF formula can also ameliorate oxygen free radical injury,improve endothelial cell function and retinal vascular microcirculation.But the precise mechanism remains to be further studied.Objective:(1)To investigate whether preventive treatment by YWTF before the onset of DR can delay the occurrence of DR in diabetic rats,and improve vascular endothelial function.(2)To investigate whether preventive treatment by YWTF can reduce retinal vascular permeability and protect the blood retinal barrier through decreasing the Evans blue leakage and the expression of tight junction protein.(3)To investigate whether reversing the expression of microRNA-200b,then down-regulating the ratio of VEGF and PEDF,and up-regulating the expression of targeted genes Ang-1 and Tie-2 to protect endothelial cell function is probably the mechanism of YWTF to prevent the occurrence of DR.(4)To investigate whether reversing the expression of microRNA-146a,then regulating NF-κB and P38-MAPK pathway,and down-regulating the expression of ICAM-1 and TNF-a to inhibit the inflammation state is probably the mechanism of YWTF to prevent the occurrence of DR.(5)To investigate whether reversing the expression of microRNA-146a,then regulating NF-κB and P38-MAPK pathways,and down-regulating the expression of ICAM-1 and TNF-a to inhibit the inflammation state is probably the mechanism of YWTF to prevent the occurrence of DR.(6)To investigate whether improving the pathological damage of pancreas,increase the insulin expression of islet beta cell,down-regulate the expression of glucagon in islet alpha cells,and improve the expression of insulin receptor substrate-1 is probably the mechanism of YWTF to protect the function of pancreatic islet.Method:One hundred and sixty Sprague Dawley(SD)rats were divided into:1)Normal control(CON)group(40 rats);2)Diabetic model group(40 rats);3)DM rats treated with YWTF group(40 rats);4)DM rats treated with Calcium Dobesilate(CaD)group(40 rats).Diabetes was induced by injection of streptozotocin(2%STZ,60 mg/kg,Sigma Chemical Co.,USA)via intraperitoneal injection in SD rats.YWTF was given 42 mg/kg/day,and CaD was given 104 mg/kg/day dissolved in water after injection of STZ,and another two groups was given equal dose of distilled water.The intervention duration was 3 months.Following DM model establishment,body weight and fast blood glucose of all rats were measured at 4w,8w,12w,16w,20w and 24w,respectively;the rats in each group were sacrified at 12w and 24w.Hematoxylin and eosin(HE)staining was used to analyze pathological changes in retinas.Retinal digest preparations and periodic acid-schiff(PAS)staining was used to analyze pathological changes in retinal capillary.Additionally,six SD rats and six Brown Norway(BN)rats were divided into:1)Normal control(CON)group(6 rats);2)Diabetic model group(6 rats).Fundus photography and fluorography examination via Micron-III retina imaging system was used to analyze pathological changes in retinas.(2)Evens Blue(EB)perfusion were used to analyze retinal vascular permeability.Immunochemistry,Western blot and quantitative real time-polymerase chain reaction(RT-PCR)were used to analyze retinal expression of ZO-1,Occludin and Claudin-5.(3)TUNEL staining was used to analyze apoptosis of retinal endothelial cells in rats.Immunochemistry was used to analyze retinal expression of VEGF,PEDF,Tie-2,ICAM-1.Immunochemistry was used to analyze retinal expression of VEGF,PEDF,Tie-2,ICAM-1.Western blot was used to analyze retinal protein expression of VEGF,VEGFR,PEDF,Ang-1,Tie-2,TNF-a,ICAM-1,NF-κB,p38-MAPK.RT-PCR was used to analyze retinal gene expression of VEGF,p38-MAPK,MMP-9,miR-200b,miR-146a.(4)Hematoxylin and eosin(HE)staining was used to analyze pathological changes in pancreatic islet.Immunochemistry was used to analyze pancreatic expression of insulin and glucagon.Western blot was used to analyze pancreatic protein expression of IRS-1.’Results:1.Effects on general status,body weight,blood glucose and retinal pathology:(1)Four weeks after STZ injection,body weight of rats in DM group were significantly decreased compared with normal control group,the difference was statistically significant(P<0.05);body weight of rats in YWTF group and CaD group were significantly increased compared with DM group,the difference was statistically significant(P<0.05).At each time point,fast blood glucose levels of rats in DM group were significantly increased compared with normal control group,the difference was statistically significant(P<0.05);fast blood glucose levels of rats in YWTF group and CaD group were not significantly decreased compared with DM group,the difference was not statistically significant(P<0.05).Cataract rate in DM rats after 12 weeks:bilateral turbidity rate(96%),no turbidity(4%),and no turbidity in normal rats.(2)Retinal HE staining for 3 months of diabetes:Non-diabetic animal showed a normal retina,all cell layers of retina were clear and neatly arranged;In the DM group,the retinal tissue exhibited slightly edema,the cell boundaries of each layer were vague,and the ganglion cells were reduced;In the YWTF group and CaD group,each layer of the retina was arranged neatly and exhibited slight tissue edema,no telangiectasia was seen.Retinal HE staining for 6 months of diabetes:In the DM group,all cell layers of retina were clear and neatly arranged,and the ganglion cells were reduced with vacuoles;the cells in the core layer are obviously disorganized,surrounding edema and the capillary dilatation was obvious;In the YWTF group and CaD group,the retina exhibited slightly edema,the cells were disorganized,the endothelial cells are seen to proliferate.The distribution of endothelial cells and pericytes was relatively normal.(3)Retina digest preparations for 3 months of diabetes:capillary vessel network of the normal group arranged in order and go straight,and the vascular diameter was uniform;In the DM group,capillary vessel network arranged in disorder,the basement membrane was thicken slightly,and pericytes was decreased and the endothelial cells began to proliferate;In the YWTF group and CaD group,the manifestation of retinal digest preparations was close to the normal group;the distribution of capillaries was normal,and the distribution of pericytes and endothelial cells was normal.Retina digest preparations for 6 months of diabetes:In the DM group,capillary vessel network arranged in disorder,the distortion and aggregation existed.Some capillaries were dilated,and the.microangioma,the pericellal cells and the acellular capillaries were scattered;In the YWTF group and CaD group,retinal microvasculature was obviously protected.Distortion and aggregation was not seen and the capillary dilated slightly;The protective effect of CaD on retina is not as good as that of YWTF.(4)Fundus photography and angiofluorography:none of the SD diabetic rats had distinct photos because rapid interference of choroid fluorescent light,capillaries of normal BN rats were in normal shape,while all of the 6 BN diabetic rats showed evident vascular disorder as background diabetic retinopathy,such as dilated and tortuous veins,microaneurysm,pathological manifestations of fluorescein leakage.2.Effect of protecting the blood retinal barrier:(1)EB leakage of rats in DM group were significantly increased compared with normal control group,the difference was statistically significant(P<0.05);EB leakage of rats of rats in YWTF group and CaD group were significantly decreased compared with DM group,the difference was statistically significant(P<0.05).(2)The retinal expression of ZO-1,Occludin and Claudin-5 in the DM group were significantly decreased compared with normal control group,the difference was statistically significant(P<0.05);the retinal expression of ZO-1 in the YWTF group were significantly increased compared with DM group,the difference was statistically significant(P<0.05),while the retinal expression of ZO-1 in the CaD group was elevated,but there was no significant difference(P>0.05);the retinal expression of Occludin in the YWTF group and CaD group were significantly increased compared with DM group,the difference was statistically significant(P<0.05);the retinal expression of Claudin-5 in the CaD group were significantly increased compared with DM group,the difference was statistically significant(P<0.05),while the retinal expression of Claudin-5 in the YWTF group was elevated,but there was no significant difference(P>0.05).3.The mechanism of improving vascular endothelial function:(1)There were significant increased the numbers of TUNEL(+)cells in the ganglion cell layer(GCL)of the diabetic retina compared with to normal control group(P<0.05).Treatment with YWTF was able to significantly decrease retinal cell apoptosis compared with that of the diabetic group(P<0.05).(2)The retinal expression of VEGF and VEGFR in the DM group were significantly decreased compared with normal control group,the difference was statistically significant(P<0.05);the retinal expression of VEGF in the YWTF group and CaD group were significantly increased compared with DM group,the difference was statistically significant(P<0.05),while the retinal expression of VEGFR in the YWTF group and CaD group was decreased,but there was no significant difference(P>0.05).(3)The retinal expression of PEDF in the DM group were significantly decreased compared with normal control group,the difference was statistically significant(P<0.05);the retinal expression of PEDF in the YWTF group were significantly increased compared with DM group,the difference was statistically significant(P<0.05),while the retinal expression of PEDF in the CaD group was elevated,but there was no significant difference(P>0.05).(4)The retinal expression of Ang-1 and Tie-2 in the DM group were significantly decreased compared with normal control group,the difference was statistically significant(P<0.05);the retinal expression of Ang-1 and Tie-2 in the YWTF group were significantly increased compared with DM group,the difference was statistically significant(P<0.05),while the retinal expression of Ang-1 and Tie-2 in the CaD group was elevated,but there was no significant difference(P>0.05).(5)The retinal expression of miR-200b in the DM group were significantly decreased compared with normal control group,the difference was statistically significant(P<0.05);the retinal expression of miR-200b in the YWTF group and CaD group were significantly increased compared with DM group,the difference was statistically significant(P<0.05).4.The mechanism of alleviating inflammation:(1)The retinal expression of TNF-a and NF-κB in the DM group were significantly increased compared with normal control group,the difference was statistically significant(P<0.05);the retinal expression of NF-κB in the YWTF group and CaD group were significantly decreased compared with DM group,the difference was statistically significant(P<0.05),while the retinal expression of TNF-a in the YWTF group and CaD group were decreased,but there was no statistically significance(P>0.05).(2)The retinal expression of ICAM-1 and p38-MAPK in the DM group were significantly increased compared with normal control group,the difference was statistically significant(P<0.05);the retinal expression of ICAM-1 and p38-MAPK in the YWTF group and CaD group were significantly decreased compared with DM group,the difference was statistically significant(P<0.05).(3)The retinal expression of miR-146a in the DM group were significantly decreased compared with normal control group,the difference was statistically significant(P<0.05);the retinal expression of miR-146a in the YWTF group and CaD group were increased,but there was no significant difference(P>0.05).5.Effects on pancreatic pathology:(1)Pancreatic HE staining for 3 months of diabetes:In the normal group,the morphology of the islets was round or oval cell mass,there was the high number of islet cells,and the boundary was clear,while the morphology of the islets in the DM group was irregular,and the number of islet cells decreased and the distribution was sparse;In the YWTF and CaD group,the morphology of the islets was less regular,the number of islet cells decreased,and the boundaries of the exocrine glands were relatively clear,and the arrangement of the islet was more regular compared with DM group.Pancreatic HE staining for 6 months of diabetes:In the DM model group,the islets were severely atrophied,the number of islet cells was reduced and distributed sparsely,the exocrine glands invaded the islets,the islet cells swelled,and some nuclei was contracted and split;In the YWTF and CaD group,the morphology of the islets was irregular,the islets were slightly atrophied,and the number of islet cell was decreased.(2)There was less number of insulin positive β cells and glucagons positive a cells in the DM group compared with normal control group(P<0.05);Treatment with YWTF increased the number of insulin positive β cells and positive reactions while glucagons positive a cells decrease in number and positive reactions were less compared with DM group(P<0.05).The protective effect of CaD Group on islet cells is not as good as that of YWTF group.(3)The pancreatic expression of IRS-1 in the DM group were significantly decreased compared with normal control group,the difference was statistically significant(P<0.05);the pancreatic expression of IRS-1 in the YWTF group and CaD group were increased,but there was no significant difference(P>0.05).Conclusion:1.Twelve weeks after STZ injection,SD rats appeared the typical pathological manifestations in the early stage of DR,with the duration of diabetes prolonged,the pathological manifestations of the retina are gradually aggravated.2.Preventive treatment by YWTF before the onset of DR can alleviate pathological state,and reduce the retinal vascular leakage,and increase the expression of tight junction protein ZO-1 and Occludin,thus protecting the blood-retinal barrier.3.The mechanism of YWTF may be "reversing the expression of microRNA-200b,then down-regulating the ratio of VEGF and PEDF,and up-regulating the expression of targeted genes Ang-1 and Tie-2 to protect endothelial cell function",thus preventing the occurrence of DR.4.The mechanism of YWTF also may be "down-regulating the expression of ICAM-1 and TNF-a through NF-κB and P38-MAPK pathways to inhibit the inflammation state",thus preventing the occurrence of DR.5.The mechanism of YWTF may be "improving the pathological damage of pancreas,increase the insulin expression of islet beta cell,down-regulate the expression of glucagon in islet alpha cells,and improve the expression of insulin receptor substrate-1",thus protecting the function of pancreatic islet. |
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