Study On The Preventive And Therapeutic Effects And Mechanism Of Tangwangmingmu Granule On Diabetic Retinopath

Diabetic retinopathy(DR)is a kind of complications of diabetes,mainly reflects in the aspect of microvascular pathological change,with the characteristics of high incidence and easy to cause blindness.At present,some drugs are used to treat DR by protecting blood vessels and improving retinal microcirculation,such as calcium dobesilate,Difrarel 100,methycobal,Qiming Granule and so on.All of these drugs have a certain effect on the prevention and treatment of DR,but the lasting effect is not ideal.In recent years,traditional Chinese medicine has showed great advantage in the treatment of DR.Tang Wang Ming Mu Granule(TWMM)comes from "Mimeng Flower prescription",which is the clinical experience prescription of Jiansheng Gao professor,a famous specialist in ophthalmology of traditional Chinese medicine."Mimeng Flower prescription" is effective and safe,which has been used for many years in clinical.If it could be developed into a newly safe and effective drug preparation with clear clinical location and relatively clear action mechanism,that will have important medical and social value.Objective:This study aimed to research the efficacy of TWMM in the prevention and control of DR,by researching the protective effect of TWMM on the diabetic retinopathy of type 2 diabetic rats,the effect of TWMM on the retinal permeability of type 1 diabetic rats and on the new blood vessels in mice.The other purpose is to explore the mechanisms of TWMM,by researching the effect of TWMM on JAK2/STAT3/VEGF pathway and SOCS3 expression,the effect of the representative components of TWMM on VEGF induced proliferation,migration and tube formation of EA.hy 926 cells.Methods:(1)Effects of TWMM on rats of type 2 diabetic retinopathy1.The rat model of type 2 diabetic retinopathy was established by using the high fat diet combined with low dose of STZ.The rats were divided into seven groups:normal control,diabetes model(DM),diabetes with TWMM treatment,the positive control treatment groups of Qi Ming granules and Calcium dobesilate capsules.All rats were treated for 8 weeks.2.During the experiment,the hair color,skin,urine,excrement,activity degree,death rate and lens turbidity rate of the rats were observed every day.The body weight and blood glucose levels were recorded every week.3.At the end of the experiment,HbAlc values were determined by biochemical method.Retinal digest stretched preparation was used to observe retinal capillary morphology changes.HE staining was used to observe the pathological changes.Transmission electron microscopy was used to observe the changes of ultrastructure.ELISA method was used to determine the changes of MDA,sICAM-1 and VEGF in serum of rats.(2)Effectof TWMM on retinal permeability of ratsUsing the STZ-induced type 1 diabetic rat model and with Evans blue(EB)as the tracer,the effect of TWMM on retinal permeability was observed by quantitative detection of EB content in the retina.(3)Effectof TWMM on angiogenesisThe effect of TWMM on angiogenesis was evaluated by using matrigel plug assay.HE staining was used to observe the morphology of matrix gel and the number of microvessels was observed by CD31 staining.(4)Effects of TWMM on JAK2/STAT3/VEGF pathway and SOCS3 expressionUsing type 2 diabetic rat model,immunohistochemical method was used to determine the changes of VEGF,JAK2,P-JAK2,STAT3,P-STAT3,and SOCS3 in rat retinas.(5)Effects of the representative components of TWMM on VEGF induced proliferation,migration and tube formation of EA.hy 926 cells1.We selectd astragaloside in Radix Astragali(the monarch drug),berberine hydrochloride in Rhizoma Coptidis(the minister drug),cinnamaldehyde in Cinnamon(the minister drug)and linarin in Flos Buddlejae(the guide drug)as the representative components in TWMM.The MTT assay,scratch test and tube formation experiment were used to observe the effects of representative components in TWMM on the proliferation,migration and tube formation of EA.hy 926 cells induced by VEGF.2.Western Blot method was used to explore the effects of cinnamaldehyde on the JAK2/STAT3 signaling pathway and the expression of SOCS3 in EA.hy 926 cells induced by VEGF.Results:(1)Effects of TWMM on rats of type 2 diabetic retinopathy1.Effects of TWMM on the general condition of ratsIn DM group,the amount of food intake and drinking water,the blood glucose levels increased significantly(P<0.01),the body weight decreased obviously(P<0.01),the lens turbidity rate increased(P>0.05)and the survival rate reduced(P>0.05),compared with the normal group.L-TWMM,M-TWMM,H-TWMM,Qi Ming granules and Calcium dobesilate capsules treatments can reduce the amount of food intake and increased the survival rate;L-TWMM and Calcium dobesilate capsules treatments can decrease the amount of drinking water,but there were no differences compared with DM group(P>0.05).All treatments showed no effect on the the body weight,the blood glucose levels and the lens turbidity rate,compared with DM group(P>0.05).2.Effect of TWMM on serum HBA1C in ratsHbAlc levels of DM rats were significantly higher than normal rats(P<0.01).The HbA1c values of L-TWMM,M-TWMM,and H-TWMM groups revealed a significant decrease(P<0.05-0.01),compared with that of DM animals.Qi Ming and Calcium dobesilate groups can reduce the HbAlc levels,but there were no differences compared with DM group(P>0.05).3.Effects of TWMM on retinal capillary morphologyRetinal capillary network in the normal group was uniformand regular.However,retinal capillaries were disordered and the diameter of capillary tubes was not uniform in the DM group.At the same time,endothelial cells proliferated and pericytes decreased in the DM group.Compared with the DM group,the distribution of capillaries was even more uniform and regular,and the proliferation of endothelial cell was not obvious in all treatment groups.4.Effects of TWMM on Retinal HistologyThe retinal layers of control group had integrity structure,as well as normal cells.Moreover,retinal ILM remained tidy and smooth.However,hyperplasia,loose,and swelling of ILM were seen in DM group,as well as new or dilated vessels in RNFL and OPL.TWMM groups had an obvious suppression of retinal new and dilated vessels.There were obvious amelioration in Qi Ming granules and Calcium dobesilate capsules treatment groups.5.Effect of TWMM on Ultrastructure of Retinal VesselsThe retinal capillaries of control group were integrated,and the endothelial cells proliferated and pericytes were normal.In the DM group,the basement membranes were uneven,thickened or split.The endothelial cells disappeared and the peripheral cells edema.Compared with the DM group,the basement membranes were relatively complete,and there were a small number of endothelial cells and pericytes in all treatment groups.6.Effects of TWMM on serum MDA,sIC AM-1,and VEGF in ratsThe DM rats exhibited greatly higher MDA,ICAM-1,and VEGF levels than those determined in retinal tissue of the normal control rats(P<0.05-0.01).All treatment groups decreased the level of MDA significantly,compared with the DM group(P<0.05-0.01).L-TWMM,H-TWMM,and Calcium dobesilate capsules treatments could reduce sICAM-1 level significantly(P<0.05-0.01).L-TWMM,M-TWMM,H-TWMM and Qi Ming granules treatments showed significantly reduced VEGF level(P<0.05-0.01).(2)Effect of TWMM on retinal permeability of ratsRetinal levels of EB in the DM group were significantly higher than that in the control group(P<0.01).All treatments significantly reduced retinal levels of EB(P<0.05-0.01),compared with the DM group.(3)Effect of TWMM on angiogenesisCompared with the normal group,the microvascular number of implant in the model group increased significantly(P<0.01).Compared with the model group,H-TWMM could significantly reduce the microvascular number of implant(P<0.01),and there was no significant change in L-TWMM,M-TWMM,Qi Ming granules and Calcium dobesilate capsules treatments.(4)Effects of TWMM on JAK2/STAT3/VEGF pathway and SOCS3 expression1.Effects of TWMM on JAK2/STAT3/VEGF pathwayRetinal expression of VEGF,JAK2,P-JAK2,STAT3,and P-STAT3 in the DM group were significantly higher than that in the control group(P<0.01).All treatments significantly reduced retinal levels of VEGF,JAK2,STAT3,and P-STAT3(P<0.01),compared with the DM group.2.Effect of TWMM on SOCS3 expressionRetinal expression of SOCS3 in the DM group were significantly higher than that in the control group(P<0.05).Compared with the DM group,the expression of SOCS3 was further increased by M-TWMM and H-TWMM treatment groups significantly(P<0.05).(5)Effects of the representative components in TWMM on VEGF induced proliferation,migration and tube formation of EA.hy 926 cells1.Effect on proliferation of VEGF-induced EA.hy 926 cells.Compared with the control group,VEGF(7 ng/mL)promoted the proliferation of EA.hy 926 cells obviously at 24 h(P<0.01),but there was no obvious effect at 48 h and 72 h.Compared with the VEGF group,astragaloside(62.5,125,187.5,250 μmol/L)at 24 h,astragaloside(62.5,125μmol/L)at 48 h,and astragaloside(125,187.5μmol/L)at 72 h was showed to suppress VEGF-induced proliferation of EA.hy 926 cells significantly(P<0.05-0.01),and there was no obvious dose-and time-effect relationship.At 24 h,48 h,and 72 h,berberine hydrochloride(12.5,25,37.5,50μmol/L)and cinnamaldehyde(60,90,120,150 μmol/L)suppressed VEGF-induced proliferation of EA.hy 926 cells significantly(P<0.01),which showed dose-and time-effect relationship.Linarin(30,45,60,120,180μmol/L)suppressed VEGF-induced proliferation of EA.hy 926 cells significantly(P<0.01),which showed dose-effect relationship,but no time-effect relationship.2.Effect on migration of VEGF-induced EA.hy 926 cells.Compared with the control group,VEGF(7 ng/mL)promoted the migration of EA.hy 926 cells obviously(P<0.05).Compared with the VEGF group,astragaloside(125,187.5,250 μmol/L)was shown to suppress VEGF-induced migration of EA.hy 926 cells,but there were no statistically differences(P>0.05).Berberine hydrochloride(12.5,25,37.5,50μmol/L)and cinnamaldehyde(60,90,120,150 μmol/L)suppressed VEGF-induced migration of EA.hy 926 cells significantly(P<0.01).Linarin(180 μmol/L)suppressed VEGF-induced migration of EA.hy 926 cells significantly(P<0.01),but which showed some cytotoxicity.3.Effect on tube formation of VEGF-induced EA.hy 926 cells.Compared with the control group,VEGF(7 ng/mL)could promote the tube formation of EA.hy 926 cells(P>0.05).Compared with the VEGF group,astragaloside(125,250μmol/L),berberine hydrochloride(25,50 μmol/L)and cinnamaldehyde(90,150 μmol/L)and linarin(45,60 μmol/L)was showed to suppress VEGF-induced tube formation of EA.hy 926.The action of cinnamaldehyde(150 μmol/L)was most significant,with almost no intact tubular structure formed.4.Effect of cinnamaldehyde on the JAK2/STAT3 Pathway and SOCS3 expression of VEGF-induced EA.hy 926 cells.Expression of P-JAK2,STAT3,P-STAT3 in the DM group were significantly higher than that in the control group(P<0.05-0.01),while the levels of JAK2 and SOCS3 showed a higher trend,but there was no statistical difference(P>0.05).Cinnamaldehyde significantly reduced the levels of P-JAK2,STAT3,P-STAT3(P<0.05-0.01),compared with the DM group,which reduced the levels of JAK2 and SOCS3,but it was not significant(P>0.05).Conclusion:TWMM had protective effect on retinopathy of type 2 diabetic rats,could improve diabetic retinal capillary permeability and inhibited the formation of new blood vessels.The protective effect of TWMM on diabetic retina might be related to anti-oxidative,anti-inflam-matory,upregulation of SOCS3 expression,inhibition of the JAK2/STAT3/VEGF signaling pathway.Astragaloside,berberine hydrochloride,cinnamaldehyde and linarinwas,the representative components in TWMM,showed inhibitory effect on the proliferation,migration and tube formation of VEGF-induced EA.hy 926 cells.Cinnamaldehyde showed inhibitory effect on the JAK2/STAT3 Pathway of VEGF-induced EA.hy 926 cells.