枸杞多糖对糖尿病大鼠血—视网膜屏障的保护作用及ROCK通路表达的机理研究
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摘要
目的：明确枸杞多糖(LBP)对糖尿病大鼠血—视网膜屏障的保护作用,进一步探讨枸杞多糖通过调控ROCK信号通路及其底物P-MLC的表达,减少血—视网膜屏障渗漏的作用机制。
材料与方法：实验一.取健康雄性SD大鼠60只,随机分为3组：CON组：正常对照组；DM组：STZ造模；LBP组：STZ造模成模后予以LBP(250mg/Kg·d)灌胃,DM组予以等量的生理盐水灌胃。分别于4w、8w、12w抽取静脉血,并取眼球。观察大鼠的一般情况；记录各时间点各组大鼠体重变化、血糖值及甘油三酯和胆固醇的浓度；伊文思蓝(EB)45 mg/kg尾静脉注入,分别检测视网膜中EB含量。实验二.健康成年雄性(SD)大鼠54只,分组及给药方法取材时间同实验一,采用HE染色光镜观察各组大鼠视网膜各层组织细胞数量及形态变化。血管铺片形态学观察：视网膜动静脉的形态、管径,同时采取免疫组化方法,检测视网膜血管网VEGF的表达及灰度值半定量分析,采用透射电镜技术观察视网膜毛细血管内皮细胞、周细胞及感光细胞超微结构。实验三.健康成年雄性(SD)大鼠44只,随机分为5组,CON组、DM组、LBP组给药方法取材时间同实验一,F组：成模后法舒地尔(Fasudil)腹腔注射(15mg/kg),L+F组：成模后LBP灌胃+Fasudil腹腔注射,各组于8w取材。分别用免疫组织化学方法检测Occludin、ROCK1表达情况进行半定量分析。免疫印迹方法检测Occludin、ROCK及其底物P-MLC蛋白表达的量,并利用凝胶成像系统对其进行定量分析。实验四.实验细胞RF/6A,MEM培养基继续培养48h,按照1：2-1：4进行传代。分组检测：共分5组,正常对照组：含葡萄糖5.5mmol/L;高糖1组：含葡萄糖30mmol/L；高糖2组：含葡萄糖40mmol/L;LBP+高糖1组：含葡萄糖30mmol/L+LBP(1g/L)；LBP+高糖2组：含葡萄糖40mmol/L+LBP(1g/L)；于1d、3d、5d、7d进行指标检测。倒置显微镜下观察细胞形态的变化；以辣根过氧化酶(HRP)作示踪剂用二室弥散系统检测RF/6A单层通透性,免疫荧光法观察细胞骨架的变化、Western blot检测ROCK1、Occludin和P-MLC的蛋白表达量,并进行半定量分析。
结果：
1.LBP对DM大鼠血糖、血脂及血—视网膜屏障渗漏的影响：
(1)血糖变化：LBP干预后的各组大鼠对应同时期的DM大鼠的血糖均有明显下降,4w时血糖下降幅度为57%,8w时下降幅度为40%,12w血糖下降幅度为36%,其降血糖效果以4w最为明显,随着病情的进展LBP的降糖效果有所减弱,下降率基本稳定在35%～40%之间。
(2)体重的变化：DM组成模后,开始出现消瘦,体重下降或增加缓慢,明显低于相同时间段的其他两组大鼠的体重。而LBP组则体重与正常对照组比较略有下降,明显好于DM组。
(3)血脂的变化：DM组12w时甘油三酯基本达到正常值的3倍,而LBP组12w时甘油三酯的浓度有轻度上升。12w时DM组总胆固醇浓度是正常对照组的1.4倍；而LBP组胆固醇浓度与正常组基本无差异p>0.05。
(4)EB在血—视网膜屏障渗漏的变化：DM组大鼠EB渗漏量4w、8 w、12 w分别增加了2.1倍、2.3倍、2.7倍。而枸杞多糖组较糖尿病组以上三个时间点EB渗透量降低了50%、40%、27%,组间比较p<0.01。前4w保护作用表现就的最明显。
2.LBP对DM大鼠血—视网膜屏障形态学变化的影响：
(1)HE染色光镜观察：
12wDM组神经纤维层断裂明显；节细胞、内颗粒层细胞排列明显紊乱；毛细血管明显扩张,数量增加。4wLBP组、8wLBP组各层组织形态与正常组织基本无明显差别,12wLBP组仅出现内、外颗粒层排列稍有不规则,毛细血管的轻度扩张。
(2)视网膜血管铺片：12w DM组毛细血管网迂曲,扭结,某些部位出现节段性膨大,管腔狭窄甚至闭塞。周细胞和内皮细胞有所减少；12wCON DM、LBP组VEGF灰度值216.13±2.71、105.30±4.25、176.72±4.31。组间有明显差异p<0.05,DM组并随着病程的进展,VEGF表达的量不断增高。
(3)电镜下超微结构的改变：12w DM组大鼠视网膜基底膜进一步增厚,血管内皮细胞及周细胞肿胀,胞质内线粒体肿胀变性,管腔可出现扩张。视杆细胞膜盘模糊,出现溶解、断裂,间隙进一步扩大。12w LBP胞质内部分线粒体轻度肿胀变性,基底膜增厚不明显,毛细血管壁完整,管腔无扩张。视杆细胞,膜盘结构略有些模糊,结构开始出现紊乱,但纹理尚清。
3.枸杞多糖对糖尿病大鼠血—视网膜屏障保护作用机理的研究：
(1)免疫组化法检测的Occludin表达：主要分布在视网膜内五层上。灰度值12w时,CON组143.70±2.29；DM组173.18±3.78；LBP组153.02±3.59,DM组表达较正常对照组显著减少。随着糖尿病视网膜病变时间的延长而逐渐下降。8w各治疗组与DM组比较,Occludin表达量明显增加。F+L组效果最好(p<0.05),LBP与Fasudil组之间无明显差异(p>0.01)
(2)免疫组化法检测的ROCK1表达：主要分布在神经节细胞层和内颗粒层,灰度值12w时,CON组178.32±5.92；DM组155.91±2.76；LBP组178.82±4.33,DM组从4w开始减弱,并随着病程的延长,ROCK1表达的量逐渐增加,而LBP组ROCK表达量并没有明显的增加。各治疗组间比较,F+L组与正常组无明显差异(p>0.05),治疗效果最好,Fasudil组的作用要比LBP的效果明显一些(p<0.05)。
(3)免疫印迹法检测Occludin蛋白表达：DM组随着病程的延长,Occludin蛋白的量逐渐下降,组间有明显差异p<0.01,而LBP组3.57±0.58；F组3.65±0.71；F+L组4.48±0.53,LBP组与F组比较LBP组表达略低,但无明显统计学意义P>0.05。F+L组表达量高于其他两治疗组p<0.01,与CON组相接近。
(4)免疫印迹法检测ROCK1蛋白表达：各组DM组大鼠ROCK蛋白表达随着病程的延长量逐渐上升,组间有明显差异p<0.01；而LBP组3.14±0.51；F组3.05±0.42；F+L组2.62±0.51,LBP组与F组比较LBP组表达略高,有统计学意义p<0.05。F+L组表达量高于其他两治疗组p<0.01,与CON组相接近。
(5)免疫印迹法检测P-MLC蛋白表达：各组DM组大鼠ROCK蛋白表达随着病程的延长量逐渐上升,组间有明显差异p<0.01,而LBP组2.97±0.42；F组2.78±0.53；F+L组2.44±0.47,LBP组与F组比较LBP组表达略高,有统计学意义p>0.05。F+L组表达量高于其他两治疗组p<0.01,与CON组相接近。
4.枸杞多糖对高糖培养下视网膜血管皮细胞Rho/ROCK信号传导通路的影响：(1)正常RF/6A呈单层扁平上皮状,高糖2组7d组内皮细胞收缩明显,有的伸出长长的伪足,边界模糊,细胞间形成明显的裂隙,细胞生长数量明显较对照组少。高糖1组则于5d后形态开始出现变化。而LBP+高糖1组、LBP+高糖2组,仅于7d时,细胞略有些变圆,细胞数量略少。
(2)RF/6As单层通透性的变化：3、5、7 d高糖组HRP的光密度值明显高于正常对照组(p<0.01),尤其高糖2组渗漏更加明显。而LBP则能逆转这种因高糖造成的渗漏(p<0.01)。尤以LBP+高糖1组效果较理想,与对应的高糖1组通透性明显降低(p<0.01)(3) F-actin形态及分布的变化：对照组F-actin分布均匀,呈网状有序排列,细胞相互融合,连接紧密。高糖2组7d外周致密带明显断裂,胞质内应力纤维断裂,细胞周边出现“绒毛状”短的指状突起,“空洞状”裂隙增大,细胞近似脱落状态。LBP+高糖2组7d外周致密带进一步变细,并且部分出现断裂,细胞间开始出现裂隙,但细胞形态明显好于高糖7d组。(4)ROCK1蛋白表达量：随葡萄糖浓度的增加而升高,与β-actin的光密度比值,正常组0.54±0.25、高糖1组0.85±0.31、高糖2组1.321±0.38,高糖1组和高糖2组与对照组比较p<0.01,而这种增高趋势能被LBP所逆转,在葡萄糖浓度为30mmol/L的条件下LBP抑制ROCK1表达的作用更明显。(5)P-MLC蛋白表达量：表达量随葡萄糖浓度的增加而升高,与β-actin的光密度比值,正常组1.34±0.25、高糖1组2.45±0.31、高糖2组3.32±0.38,高糖1组和高糖2组与对照组比较p<0.01,LBP抑制P-MLC表达的升高,LBP+高糖1组1.38±0.27,LBP+高糖2组1.87±0.51。(6) Occludin蛋白表达量：随着葡萄糖浓度的增加Occludin蛋白表达量逐渐下降,LBP—高糖1组0.90±0.16,LBP—高糖2组0.69±0.14,各治疗组均可明显提升其相对应高糖组Occludin表达的量,有明显统计学意义p<0.01,尤其LBP-高糖1组可明显抑制Occludin表达的量的下降。
结论：
1.LBP可以显著减低糖尿病大鼠的血糖、血脂并能降低血-视网膜屏障的渗漏。
2.LBP可以减低糖尿病大鼠的视网膜血管上VEGF的表达,并能减轻血管内皮细胞和周细胞的水肿,减少基底膜的增厚,并减轻视细胞的损害。3.DR的过程中确实存在着ROCK通路的激活,ROCK及其底物P-MLC表达的上调而该过程可以被LBP所阻断,减低以上信号分子的表达,通过稳定Occludin的表达,起到保护血-视网膜屏障的作用。4.LBP可以明显减轻体外高糖环境培养下的单层EC的渗漏,通过抑制ROCK及其底物P-MLC该信号通路,而起到稳定细胞骨架,维持紧密连接的作用。
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.
材料与方法：实验一.取健康雄性SD大鼠60只,随机分为3组：CON组：正常对照组；DM组：STZ造模；LBP组：STZ造模成模后予以LBP(250mg/Kg·d)灌胃,DM组予以等量的生理盐水灌胃。分别于4w、8w、12w抽取静脉血,并取眼球。观察大鼠的一般情况；记录各时间点各组大鼠体重变化、血糖值及甘油三酯和胆固醇的浓度；伊文思蓝(EB)45 mg/kg尾静脉注入,分别检测视网膜中EB含量。实验二.健康成年雄性(SD)大鼠54只,分组及给药方法取材时间同实验一,采用HE染色光镜观察各组大鼠视网膜各层组织细胞数量及形态变化。血管铺片形态学观察：视网膜动静脉的形态、管径,同时采取免疫组化方法,检测视网膜血管网VEGF的表达及灰度值半定量分析,采用透射电镜技术观察视网膜毛细血管内皮细胞、周细胞及感光细胞超微结构。实验三.健康成年雄性(SD)大鼠44只,随机分为5组,CON组、DM组、LBP组给药方法取材时间同实验一,F组：成模后法舒地尔(Fasudil)腹腔注射(15mg/kg),L+F组：成模后LBP灌胃+Fasudil腹腔注射,各组于8w取材。分别用免疫组织化学方法检测Occludin、ROCK1表达情况进行半定量分析。免疫印迹方法检测Occludin、ROCK及其底物P-MLC蛋白表达的量,并利用凝胶成像系统对其进行定量分析。实验四.实验细胞RF/6A,MEM培养基继续培养48h,按照1：2-1：4进行传代。分组检测：共分5组,正常对照组：含葡萄糖5.5mmol/L;高糖1组：含葡萄糖30mmol/L；高糖2组：含葡萄糖40mmol/L;LBP+高糖1组：含葡萄糖30mmol/L+LBP(1g/L)；LBP+高糖2组：含葡萄糖40mmol/L+LBP(1g/L)；于1d、3d、5d、7d进行指标检测。倒置显微镜下观察细胞形态的变化；以辣根过氧化酶(HRP)作示踪剂用二室弥散系统检测RF/6A单层通透性,免疫荧光法观察细胞骨架的变化、Western blot检测ROCK1、Occludin和P-MLC的蛋白表达量,并进行半定量分析。
结果：
1.LBP对DM大鼠血糖、血脂及血—视网膜屏障渗漏的影响：
(1)血糖变化：LBP干预后的各组大鼠对应同时期的DM大鼠的血糖均有明显下降,4w时血糖下降幅度为57%,8w时下降幅度为40%,12w血糖下降幅度为36%,其降血糖效果以4w最为明显,随着病情的进展LBP的降糖效果有所减弱,下降率基本稳定在35%～40%之间。
(2)体重的变化：DM组成模后,开始出现消瘦,体重下降或增加缓慢,明显低于相同时间段的其他两组大鼠的体重。而LBP组则体重与正常对照组比较略有下降,明显好于DM组。
(3)血脂的变化：DM组12w时甘油三酯基本达到正常值的3倍,而LBP组12w时甘油三酯的浓度有轻度上升。12w时DM组总胆固醇浓度是正常对照组的1.4倍；而LBP组胆固醇浓度与正常组基本无差异p>0.05。
(4)EB在血—视网膜屏障渗漏的变化：DM组大鼠EB渗漏量4w、8 w、12 w分别增加了2.1倍、2.3倍、2.7倍。而枸杞多糖组较糖尿病组以上三个时间点EB渗透量降低了50%、40%、27%,组间比较p<0.01。前4w保护作用表现就的最明显。
2.LBP对DM大鼠血—视网膜屏障形态学变化的影响：
(1)HE染色光镜观察：
12wDM组神经纤维层断裂明显；节细胞、内颗粒层细胞排列明显紊乱；毛细血管明显扩张,数量增加。4wLBP组、8wLBP组各层组织形态与正常组织基本无明显差别,12wLBP组仅出现内、外颗粒层排列稍有不规则,毛细血管的轻度扩张。
(2)视网膜血管铺片：12w DM组毛细血管网迂曲,扭结,某些部位出现节段性膨大,管腔狭窄甚至闭塞。周细胞和内皮细胞有所减少；12wCON DM、LBP组VEGF灰度值216.13±2.71、105.30±4.25、176.72±4.31。组间有明显差异p<0.05,DM组并随着病程的进展,VEGF表达的量不断增高。
(3)电镜下超微结构的改变：12w DM组大鼠视网膜基底膜进一步增厚,血管内皮细胞及周细胞肿胀,胞质内线粒体肿胀变性,管腔可出现扩张。视杆细胞膜盘模糊,出现溶解、断裂,间隙进一步扩大。12w LBP胞质内部分线粒体轻度肿胀变性,基底膜增厚不明显,毛细血管壁完整,管腔无扩张。视杆细胞,膜盘结构略有些模糊,结构开始出现紊乱,但纹理尚清。
3.枸杞多糖对糖尿病大鼠血—视网膜屏障保护作用机理的研究：
(1)免疫组化法检测的Occludin表达：主要分布在视网膜内五层上。灰度值12w时,CON组143.70±2.29；DM组173.18±3.78；LBP组153.02±3.59,DM组表达较正常对照组显著减少。随着糖尿病视网膜病变时间的延长而逐渐下降。8w各治疗组与DM组比较,Occludin表达量明显增加。F+L组效果最好(p<0.05),LBP与Fasudil组之间无明显差异(p>0.01)
(2)免疫组化法检测的ROCK1表达：主要分布在神经节细胞层和内颗粒层,灰度值12w时,CON组178.32±5.92；DM组155.91±2.76；LBP组178.82±4.33,DM组从4w开始减弱,并随着病程的延长,ROCK1表达的量逐渐增加,而LBP组ROCK表达量并没有明显的增加。各治疗组间比较,F+L组与正常组无明显差异(p>0.05),治疗效果最好,Fasudil组的作用要比LBP的效果明显一些(p<0.05)。
(3)免疫印迹法检测Occludin蛋白表达：DM组随着病程的延长,Occludin蛋白的量逐渐下降,组间有明显差异p<0.01,而LBP组3.57±0.58；F组3.65±0.71；F+L组4.48±0.53,LBP组与F组比较LBP组表达略低,但无明显统计学意义P>0.05。F+L组表达量高于其他两治疗组p<0.01,与CON组相接近。
(4)免疫印迹法检测ROCK1蛋白表达：各组DM组大鼠ROCK蛋白表达随着病程的延长量逐渐上升,组间有明显差异p<0.01；而LBP组3.14±0.51；F组3.05±0.42；F+L组2.62±0.51,LBP组与F组比较LBP组表达略高,有统计学意义p<0.05。F+L组表达量高于其他两治疗组p<0.01,与CON组相接近。
(5)免疫印迹法检测P-MLC蛋白表达：各组DM组大鼠ROCK蛋白表达随着病程的延长量逐渐上升,组间有明显差异p<0.01,而LBP组2.97±0.42；F组2.78±0.53；F+L组2.44±0.47,LBP组与F组比较LBP组表达略高,有统计学意义p>0.05。F+L组表达量高于其他两治疗组p<0.01,与CON组相接近。
4.枸杞多糖对高糖培养下视网膜血管皮细胞Rho/ROCK信号传导通路的影响：(1)正常RF/6A呈单层扁平上皮状,高糖2组7d组内皮细胞收缩明显,有的伸出长长的伪足,边界模糊,细胞间形成明显的裂隙,细胞生长数量明显较对照组少。高糖1组则于5d后形态开始出现变化。而LBP+高糖1组、LBP+高糖2组,仅于7d时,细胞略有些变圆,细胞数量略少。
(2)RF/6As单层通透性的变化：3、5、7 d高糖组HRP的光密度值明显高于正常对照组(p<0.01),尤其高糖2组渗漏更加明显。而LBP则能逆转这种因高糖造成的渗漏(p<0.01)。尤以LBP+高糖1组效果较理想,与对应的高糖1组通透性明显降低(p<0.01)(3) F-actin形态及分布的变化：对照组F-actin分布均匀,呈网状有序排列,细胞相互融合,连接紧密。高糖2组7d外周致密带明显断裂,胞质内应力纤维断裂,细胞周边出现“绒毛状”短的指状突起,“空洞状”裂隙增大,细胞近似脱落状态。LBP+高糖2组7d外周致密带进一步变细,并且部分出现断裂,细胞间开始出现裂隙,但细胞形态明显好于高糖7d组。(4)ROCK1蛋白表达量：随葡萄糖浓度的增加而升高,与β-actin的光密度比值,正常组0.54±0.25、高糖1组0.85±0.31、高糖2组1.321±0.38,高糖1组和高糖2组与对照组比较p<0.01,而这种增高趋势能被LBP所逆转,在葡萄糖浓度为30mmol/L的条件下LBP抑制ROCK1表达的作用更明显。(5)P-MLC蛋白表达量：表达量随葡萄糖浓度的增加而升高,与β-actin的光密度比值,正常组1.34±0.25、高糖1组2.45±0.31、高糖2组3.32±0.38,高糖1组和高糖2组与对照组比较p<0.01,LBP抑制P-MLC表达的升高,LBP+高糖1组1.38±0.27,LBP+高糖2组1.87±0.51。(6) Occludin蛋白表达量：随着葡萄糖浓度的增加Occludin蛋白表达量逐渐下降,LBP—高糖1组0.90±0.16,LBP—高糖2组0.69±0.14,各治疗组均可明显提升其相对应高糖组Occludin表达的量,有明显统计学意义p<0.01,尤其LBP-高糖1组可明显抑制Occludin表达的量的下降。
结论：
1.LBP可以显著减低糖尿病大鼠的血糖、血脂并能降低血-视网膜屏障的渗漏。
2.LBP可以减低糖尿病大鼠的视网膜血管上VEGF的表达,并能减轻血管内皮细胞和周细胞的水肿,减少基底膜的增厚,并减轻视细胞的损害。3.DR的过程中确实存在着ROCK通路的激活,ROCK及其底物P-MLC表达的上调而该过程可以被LBP所阻断,减低以上信号分子的表达,通过稳定Occludin的表达,起到保护血-视网膜屏障的作用。4.LBP可以明显减轻体外高糖环境培养下的单层EC的渗漏,通过抑制ROCK及其底物P-MLC该信号通路,而起到稳定细胞骨架,维持紧密连接的作用。
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.
引文
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