Lbp On The Mechanism Of Diabetic Rats Blood - Retinal Barrier Function And Rock Pathway Expression

Object:To investigate the protection effects of lycium bararum polysaccharides (LBP) on blood-retinal barrier (BRB) in diabetic rats, and the mechanism of improving the leakage of BRB via ROCK signaling pathway and induced expression of P-MLC.Materials and methods:Experiment One:Fifty four male SD rats were randomly divided into 3 groups. CON group:normal control rats; DM group: diabetes model group; LBP group:diabetes model rats were intragastric administrated with LBP(250mg/Kg·d). DM group were intragastric administration with normal sodium. Blood sample were extracted at 4w, 8w,12w as well as eye globe. General condition, the blood glucose, the body weight, triglyceride and cholesterinwere determined on on 4w,8w and 12w; caudal vein injection evans blue (EB) 45 mg·kg-1, EB content were detected in retina. Experiment Two:Fifty four male SD rats(grouped and admisnistrated in the same way as experiment one). Cell amount and morphologic changes in each layer retina were observed by hematine and eosine(HE) stain with commonmicroscope, Retinal vascular preparations were performed by using trypsin digestion, morphology changes was reviewed, such as shape and calibre of arteriole and veinlet in rats retina and the quantity and appearance of vascular endothelial cells and pericytes.By immunohistochemistry assessment the VEGF expression of retinal vessel net and semiquantitative analysis with cell image analysis system, ultrastructural organization of micrangium endothelial cell, perithelial cell and photosensory cell were observed under transmission electron microscope. Experiment There:healthy adult masculinity SD rats 44 were divided 5 groups, medication and the time of making sample in CON group, DM group, LBP group were same as experiment two. F group: intraperitoneal injection fasudil (15mg/kg) after induction of diabetic model; L+F group:intraperitoneal injection fasudil (15mg/kg) at same as intragastric administration with LBP(250mg/Kg·d), to make sample at 8w. Immunohistochemistry assessment the Occludin,ROCK1 expression and semiquantitative analysis. Western blot was used to determine the expression of Occludin, ROCK1 and substrate P-MLC and were quantitative analysised by gelatum imaging system. Experiment Four:macaque choroids-retinal endothelial cells (RF/6As), The third generation of cells were treated with 5.5 mmol/L isoosmia glucose (normal control group),30 mmol/L glucose(high-glucose 1 group),40 mmol/L glucose (high-glucose 2 group),30 mmol/L glucose+1g/L LBP (LBP+H-G 1 group)and 40 mmol/L glucose+1g/L LBP (LBP+H-G 2 group) respectively. Morphologic changes of RF/6As in different groups were observed under the inverted microscope at the 1st,3rd,5th and 7th day after culture. Horseradish peroxidase (HRP) was used as a tracer agent to detect the permeability of monolaye RF/6As in transwell chamber. Cytoskeleton distribution in different groups was determined by using immumof luorescence, Western blot was used to determine the expression of ROCK1, Occludin and P-MLC at 1st,3rd,5th and 7th day. Results:1. The effect of LBP on the blood glucose, the blood fat and BRB leakage of diabetes rats(1)The changes of the blood glucose in different groups:The blood glucose of each LBP group decreased obviously than the DM group at the same time, the blood glucose decreased as 57% in 4w, decreased as 40% in 8w, decreased as 36% in 12w. The best outstanding effective in 4w. Accompaning the advancemen of desease, LBP's the effective of declining the blood glucose weakened and lasted between 35%-40%(2)The changes of body weight in different groups:DM group rats'average body weight declined insteaded of increasing, but increased slowly in fllowing two months and that obvious lower than the other group's. but LBP group rats'body weight decreased lightly, that were most better than DM group's.(3) The changes of body-fat in different groups:DM group's TGL is higher 2 time than the normal value, but LBP group's increased lightly in 12w, while the density of cholesterol total in blood of DM group was 1.4 time than the normal control group's. As the seam time density of cholesterol total in blood of LBP group was not difference P>0.05(4) The leakage of EB in different groups:The standard curve formula was calculated:y=0.0818x+0.0023. the amount of EB leakage DM group rats were higher 2.1,2.3,2.7times than the control group rats in 4w,8w,12w respectively. But the amount of LBP group's were decreased 50%,40%,27%, comparison among groups were significance P＜0.01. The protection were significant at first 4w.2 The morphology changes of LBP on BRB in diabetes rats.(1) The samples were stained with (HE) to examine the morphology changes: 12w DRgroup:nerve fiber layer rupture obviously;ganglion cells and internal granular layer cells disorded, micrangium obviously expand, vessel wall became thicker, the amount of micrangium increased. Every layer morphous were same as normal tissue in 4wLBP,8wLBP groups, only inner ane externa granular layer cells became anomalism lightly, and micrangium expanded lightly.(2) Retinal vascular preparation to observe:Capillary network became winding and kink, caliber became disparity. Some intumescentias shaw in some parts, even lumens were narrow or blocked up. pericytes and endothelial cells reduced in the sample of 12w DM group; gray scale of VEGF in every groups were 216.132.71,105.30±4.25,176.72±4.31 in 12th weeks. Obviously difference were among groups P＜0.05. Expression of VEGF continual increase, comparing LBP group with control group had obviously statistics difference P<0.01 (3) Ultramicrostructure changes under transmission electron microscop In 12w, the nuclear metachromatin in the pericytes and endothelial cells clumped together in a dense mass at the side, the capillaries expanded and the basementmembrane increased. The membrane disk space swere distinctly enlarged, dividing and dissolving locally. Rod cell nuclei showed pyknosis and chromatin condensation. The condrocyte in the inner segments of the rods swelled and even denatured in the DM group. while, the cytochondriome swelled slightly. Basement membrane were thicker slightly. Membrane disks in rods were unclear and spaces between them were slightly enlarged but still maintain the texture.3. Mechanism of the protection of LBP on BRB in diabetic rats(1) Occludin expression determined by Immunohistochemistry:Occludin expressed at inner four layers of retina and shew yellow or brown. In 12w, gray scale of CON group, DM group, LBP group were 143.70±2.29, 173.18±3.78,153.02±3.59. The value of DM group decreased significantly than CON group's, and following the time prolong, the expression decreased. In 8w, every therapy group expressed Occludin highly than DM group, Effective of F+L group was best. There was no manifest difference between LBP group and Fasudil group.(2) Rock1 expression:Rock1 expressed main at retinal ganglial cells layer and internal granular layer, In 12w, gray scale of CON group, DM group, LBP group were178.32±5.92,155.91±2.76,178.82±4.33. The value of DM group began falling from 4 week, and following the time prolong, the expression increased, In 8w, every therapy group expressed Rock1 less than DM group, Effective of F+L group was best. Effective of Fasudil group was better than that of LBP group (p＜0.05).(3)The expression of Occludin:Accompaning the prolong course of disease, the expression of Occludin decreased gradually. There were obvious difference among groups p＜0.01, The value were 3.57±0.58,3.65±0.71, 4.48±0.53 in LBPgroup, F group, F+L group. The expression of LBP group was litter less than F group of, but there wasn't statistical significance p>0.05.That of F+L group was the highest, nearly CON group's.(4) The expression of Rockl by Western blot to test:Accompaning the prolong course of disease, the expression of Rockl increased gradually.There were obvious difference among groups p＜0.01, The value were 3.14±0.51,3.05±0.42,2.62±0.51 in LBPgroup,F group, F+L group. The expression of LBP group was litter more than F group of, there was statistical significance p>0.05. That of F+L group was the best, nearly CON group's.(5) The expression of P-MLC by Western blot to test Accompaning the prolong course of disease, the expression of Rockl increased gradually, The expression of Rockl by Western blot to test, There were obvious difference among groups p＜0.01, The value were 2.97±0.42,2.78±0.53, 2.44±0.47. The expression of LBP group was litter more than F group of, there was statistical significance.p>0.05. That of F+L group was the best, nearly CON group's.4. Effects of LBP on the Rho/ROCK signal pathway of retinal vascular endothelial cell in high glucose condition:(1)Normal RF/6As are adherent monolayer simple squamous epithelium, Deformed RF/6As were gradually increased with the prolong of time in glucose group in, but the cells shape was near normal in LBP+hing glucose groupone and LBP+hing glucose group two. Only cell became round and the amout of cells were reduced lightly.(2)Monolayer RF/6As permeability:The A values of HRP were significantly enhanced in 3,5,7 days after treated in glucose group compared with isoosmia glucose group (P＜0.01), especially high glucose group two were Severity. And those in high glucose+LBP group were considerablly declined in comparison with high glucose group in various time points (P＜0.01). (3)Morphous and distribution changes of F-actin:The rupture of stree fibers contructed by F-actin, deformation and digitation of RF/6As were exhibited in 40 mmol/L glucose group, in 7days,but no similar findings were seen in isoosmia glucose and high glucose+LBP group under the fluorescence microscope.(4)Expression of ROCK1:Expression of ROCK1 was gradually arised with the add of glucose concentration comparison with isoosmia glucose group (P＜0.01), Expression of isoosmia glucose group, high glucose group one, high glucose group two were 0.54±0.25,0.85±0.31,1.32±0.38, and that in high glucose+LBP group was significantly lower than glucose group (P＜0.05). The effect was best in 30 mmol/L glucose+LBP group.(5)Expression of P-MLC:Expression of ROCK1 was gradually arised with the add of glucose concentration comparison with isoosmia glucose group (P＜0.01), Isoosmia glucose group expressed 1.34±0.25, high glucose group one expressed 2.45±0.31,high glucose group two expressed 3.32±0.38, LBP canrefrain the increase of P-MLC.(6)Expression of Occludin by Western blot:Expression of ROCK1 was gradually declined with the add of glucose concentration comparison with isoosmia glucose group (P＜0.01),30 mmol/L glucose+LBP group expressed 0.90±0.16,40 mmol/L glucose+LBP group expressed 0.69±0.14, LBP can enhance obviously the expression of Occludin in high glucose environment Conclusion:1. LBP significantly decreases blood glucose, blood-fat and BRB leakage in diabetes Rats.2. LBP decreases the expression of VEGF on retina micrangium in diabetes Rats, as well as improves the edema of vascular endothelial cells, and pericytes, ameliorate basement membrane thickening, prevent the damage of visual cells.3. ROCK pathway is activated in the pathology of DR. The expression of ROCK and its substrate P-MLC increase, which is blocked by LBP and leads to the decreased expression of the molecular signal, stabilizes the expression of occluding, protects BRB.4. LBP significantly decreases the leakage of monolayer permeability of vascular endothelial cell under the condition of high glucose, which maintains the tight junction via inhibiting ROCK and its substrate P-MLC signal pathway and stabilizes the cytoskeleton.