| Background: Retinal edema is an important cause of impaired visual function in diabetic retinopathy.Breakdown of the inner blood-retinal barrier(iBRB)and the uncoupling state between the aquaporin-4 and Kir4.l expressioned on Müller cells are important pathological mechanisms of diabetic retinal edema.While the retinal vascular endothelial cells are the main target in most of the traditional Chinese medicine researches about the mechanism of diabetic retinal edema.Up to now,there are no paper reports the coupling of aquaporin-4 and Kir4.1 and the mechanism of iBRB injury and protection how to intervention the retinal edema.Objiective: To investigate the interventional effect of the bushenhuoxue method on the formation of early diabetic retinopathy and its related pathways from multiple angles,such as iBRB injury and protection mechanism and the association between retinal AQP4 and Kir4.1.To uncovering the possible interventions of bushenhuoxue method in prevent ing and treating DR.Methods: Diabetic rats were induced by high-sugar and high-fat diet combined with STZ intraperitoneal injection.Diabetic rats were randomly divided into 5 groups: model group(negative control group),positive control group(diabetic plus calcium dobesilate also called calcium dobesilate group),Bushenhuoxue compound groups(divided into high,medium and low dose groups).The same age normal SD rats as normal control group.So,experimental animals were divided into 6 groups.The indexes of diabetes were detected in the 1st,2nd,4th,8th week respectively.The distributions of Occludin,ZO-1,AQP4 and Kir4.1 proteins were detected by immunohistochemistry.The expressions of Occludin,ZO-1,IL-1?,ICAM-1,VEGF,PEDF,AQP4 and Kir4.1 proteins were detected by Western blot.The expression of AQP4 mRNA and Kir4.1mRNA proteins were detected by RT-PCR.The blood-retinal barrier leakage was detected by Rhodamine isothiocyanate(RHIC).Results:1.Using the way that high-sugar and high-fat diet for 1 week combined withintraperitoneal injection of medium-dose STZ(40mg/Kg)could successfully induce the type 2 diabetes animal model in SD rats with low mortality and high rate of molding.2.No retinal RHIC leakage was detected in normal control rats during each period of the experiment,the retina of the model group showed significant RHIC leakage in the nerve fiber layer,ganglion cell layer,inner and outer network layers,and inner nuclear layer.The RHIC leakage in the outer network layer was more serious than that in other layers,and the leakage gradually increased with the course of disease.3.(1)The expression of retinal tight junction protein ZO-1,0ccludin in the normal control group was stable.(2)The expression of retinal tight junction protein ZO-1,0ccludin in the negative control group was significantly lower than that of the normal control group from the 1st week of diabetes course(P <0.05),and the decrease of ZO-1 was more obvious than that of 0ccludin.The expression of 0ccludin protein showed a tendency of decreasing gradually with time.(3)The expression of ZO-1 and0 ccludin proteins in the retina of calcium dobesilate group and Bushenhuoxue Compound high,medium and low dose groups was significantly higher than that of the negative control group(P <0.05).(4)Comparison between the calcium dobesilate group and the Bushenhuoxue Compound groups: the expression of Occludin protein in middle dose group,high dose group at the 1st week and high dose group at the 4th week was higher(P<0.05).There was no significant difference in the expression of Occludin between the other TCM groups and the calcium dobesilate group(P> 0.05).At 1st week,the expression of ZO-1 protein in low and high dose Chinese medicine groups was lower than that in the calcium dobesilate group(P<0.05),this condition was also existd in low dose group at the 4th week.The expression of ZO-1 protein in high dose group at the 4th week and middle dose group at the 8th week was higher than that in calcium dobesilate group(P <0.05).There was no significant difference at various points in the expression of ZO-1 between other Chinese medicine groups and calcium dobesilate groups(P> 0.05).4.(1)The expression of IL-1? and ICAM-1,the retinal inflammation-relatedfactors in the normal control rats,were stable at each stage of the experiment.(2)The expression of IL-1? and ICAM-1 in the retina of the negative control group was significantly higher than that of the normal control group at the1 st,2nd,4th and 8th weeks(P<0.05).The expression of IL-1? has been significantly increased at the 1st week,the expression of ICAM-1 showed a gradual increase with the course of disease.(3)The protein expression levels of IL-1? and ICAM-1 in the retinas of calcium dobesilate groups and Bushenhuoxue Compound groups were significantly lower than those in the negative control group(P<0.05).(4)Comparison between the calcium dobesilate group and the Bushenhuoxue Compound groups: the expression of IL-1?protein in the traditional Chinese medicine high-dose group was lower than that of the periodontal calcium dobesilate group at the 4th week(P<0.05);The expression of IL-1? protein in the low-dose group at the1 st week,4th week,8th week,and the high-dose group at the 1st week was higher than that in the periodontal calcium dobesilate group(P<0.05).There was no significant difference at various points in the expression of IL-1? between other Chinese medicine groups and calcium dobesilate groups(P> 0.05).At the 1th week,the expression of ICAM-1 protein in middle-dose group was lower than that in calcium dobesilate group(P<0.05).The expression of ICAM-1 protein in low-dose group at the 4th week and high-dose group at the 8th week was higher than that in calcium dobesilate group(P<0.05).There was no significant difference at various points in the expression of ICAM-1 between the Chinese medicine groups and calcium dobesilate groups(P> 0.05).5.(1)The expression of VEGF in normal control group rats retina was stable during each period of the experiment.(2)The expression of VEGF protein in the retina of the negative control group was significantly higher than that of the normal control group at the 1st,2nd,4th and 8th week(P<0.05),and the expression gradually increased with the course of disease.The expression of VEGF in calcium dobesilate group and Bushenhuoxue Compound high,medium and low dose groups was significantly lower than that in negative control group(P<0.05).(4)Comparison between the calcium dobesilate group and the Bushenhuoxue Compound groups:the expression of VEGF in Bushenhuoxue Compound groups at the 1st week and themiddle dose of traditional Chinese medicine at the 2nd week was lower than that of calcium dobesilate(P<0.05).In the 2nd week,the expression of VEGF in high dose group was higher than that in calcium dobesilate group(P<0.05).There was no significant difference at various points in the expression of VEGF between the Chinese medicine groups and calcium dobesilate groups(P> 0.05).6.(1)The expression of PEDF in normal control group rats retina was stable.(2)There was no significant difference in the expression of PEDF protein between the negative control group and the normal control group at the 1st week(P> 0.05).At the2nd?4th and 8th week,the expression of PEDF protein in negative control group was significantly higher than that of normal control group(P<0.05).(3)The expression of PEDF in the calcium dobesilate group rats retina was stable.(4)The expression of PEDF in the Bushenhuoxue compound groups was raher than the normal control group at the 1st and 2nd weeks,and obviously higher than the normal control group at the 4th and 8th weeks(P<0.05).(5)Comparison between the calcium dobesilate group and the Bushenhuoxue Compound groups: there was no significant difference in the expression of PEDF protein between the two groups at the 1st and 2nd week(P> 0.05).The expression of PEDF in the middle dose group and high dose group at the 4th week and the Bushenhuoxue Compound groups at the 8th week was significantly higher than the calcium dobesilate group(P<0.05).7.The balance of VEGF and PEDF:(1)In the negative control group,the ratio of VEGF / PEDF increased at the 1st week,and the balance of VEGF and PEDF was destroyed.It was similar to the normal control group at the 2nd and 4th week.At the8 th week,it increased again,and the balance of VEGF and PEDF was broken again.(2)The ratio of VEGF / PEDF in the calcium dobesilate group was similar to that of the normal group at the first,second and fourth weeks.At the eighth week,it increased and the balance of VEGF and PEDF was broken.(3)The ratio of VEGF /PEDF in all the Chinese medicine groups was close to that of the normal control group expect the ratio of high dose group significantly increased at the second week.8.(1)The expression of AQP4 and AQP4 mRNA proteins in normal control group rats retina was stable at the 1st,2nd,4th and 8th week.(2)The expression ofAQP4 and AQP4 mRNA of the negative control group was significantly higher than that of the normal control group at the 1st,2nd,4th and 8th week.((P<0.05),and gradually increased with the course of disease.Their expression trends were similar.(3)The expression of AQP4 and AQP4 mRNA of Calcium dobesilate group and Bushenhuoxue compound groups were obviously lower than that of the negative control group(P <0.05).9.(1)The expression of Kir4.1 and Kir4.1mRNA proteins in normal control group rats retina was stable at the 1st,2nd,4th and 8th week.(2)The expression of Kir4.1 and Kir4.1mRNA in the negative control group at the 1st,2nd,4th and 8th week was significantly lower than that in the normal control group(P <0.05).(3)The expression of Kir4.1protein in calcium dobesilate group and Bushenhuoxue compound groups were similar at the1 st,2nd week.But,it was obviously higher than the negative control group at the 4th and 8th week(P <0.05).The expression of Kir4.1mRNA of calcium dobesilate group and Bushenhuoxue compound groups were obviously higher than that of negative control group(P <0.05).10.AQP4 / Kir4.1 coupling:(1)In the negative control group,the ratio of AQP4 /Kir4.1 was significantly increased compared with the normal control group at the first week,the coupling of AQP4 and Kir4.1 was destroyed.The ratio decreased at the second week while it was significantly higher than the normal control group.The ratio was further increased at the 4th and 8th week.(2)The ratio of calcium dobesilate group and Bushenhuoxue compound groups at the first week was higher than that of the normal control group but obviously lower than that of the negative control group,the coupling of AQP4 and Kir4.1was destroyed.At the second week,The ratio was declining than that in the first week but higher than that in the normal control group.The ratio increased and became stable at the 4th and 8th week,which was obviously lower than that of the negative control group.Conclusions:1.High-sugar and high-fat diet for 1 week combined with medium dose of STZ(40mg / Kg)intraperitoneal injection can successfully induce type 2 diabetic SD rat model,taking into account the mortality and the molding rate at a good level.2.Type 2 diabetic rats with a course of 8 weeks can be used as a model of early diabetic retinal edema.The expression of Occludin and ZO-1 in the retina of diabetic rats decreased with the prolongation of diabetes,especially at 8 weeks,suggesting that the structural basis of the tight junction(TJ)of retinal vascular endothelial cells in rats with diabetes duration of 8 weeks has occurred Pathological changes.TJ injury is the main reason for the increase of retinal RHIC leakage in early diabetic rats and it is an important pathological change of iBRB damage in early diabetic retina.The Bushenhuoxue compound can significantly increase the expression of retinal Occludin and ZO-1 proteins in early diabetic rats,and it may be an important mechanism for the compound to reduce retinal edema in early diabetic rats.3.The expression of IL-1? and ICAM-1 protein in retina of early diabetic rats increased significantly,and the expression of IL-1? protein increased earlier than that of ICAM-1 protein,suggesting that the inflammatory factors such as IL-1?,ICAM-1may be an important reason of retinal iBRB damage in early diabetic rats.Bushenhuoxue Compound can significantly reduce the expression of IL-1?,ICAM-1in the retina of early diabetic rats,which may be an important intervention route to protect the retina iBRB in early diabetic rats.4.The expression of VEGF and PEDF protein in retina of early diabetic rats are higher than that of normal control group,and the increase of VEGF was earlier than that of PEDF,suggesting that the elevation of PEDF in retina of early diabetic rats is a kind of "compensatory increase phenomenon ".The break of dynamic balance of retinal PEDF and VEGF is an important reason for early diabetic retinal edema.Bushenhuoxue compound can partly restore the balance of VEGF / PEDF in retina of early diabetic rats.This may be an important mechanism for protecting the retinal iBRB of early diabetic rats and reducing the retinal edema.5.The expression of AQP4 and AQP4 mRNA protein in retina of diabetic rats increased gradually with the course of disease.The expression of Kir4.1 protein and Kir4.1mRNA protein decreased significantly in retina of diabetic rats.The coupling of retinal AQP4 and Kir4.l in diabetic rats was broken at the 1th week,that is,AQP4 and Kir4.1 were uncoupled at the 1th week and gradually aggravated with the courseof disease,resulting in dysfunction of water and K+ transport,which may be an important cause of early diabetic retinal edema.Bushenhuoxue compound can obviously inhibit the upregulation of AQP4 and AQP4 mRNA and the downregulation of Kir4.1 and Kir4.1mRNA in retina of early diabetic rats and regulate the expressions of AQP4 and Kir4.1 from the transcriptional level.This may be an important mechanism of Bushenhuoxue compound to relieve early diabetic retinal edema.6.Many reasons result the retinal edema in early diabetic retinopathy,such as the increase of cytokines IL-1??ICAM-1?VEGF and AQP4,the expression of Occludin,ZO-1,and Kir4.1 proteins decreased,the uncoupling of AQP4 and Kir4.1 and the imbalance of VEGF and PEDF.The damage of iBRB in retina of diabetic rats may be the result of multiple mechanisms.The Bushenhuoxue compound reduces retinal edema in early diabetic rats may be through multiple levels and multiple targets. |
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