Effects Of Tongluo Recipe On Rats With Diabetic Retinopathy And The Study Of Mechanism

Diabetic Retinopathy is one of the most commonly chronic complications in Diabetes Mellitus.It is the leading cause of acquired blindness among young adults in devepoment countries.Hyperglycemia resulting from uncontrolled glucose regulation is widely recognized as the causal link between diabetes and diabetic complications.The diabetes mellitus microvasclular complication including retinopathy,nephropathy.The retinopathy have many pathological changes:reduction of pericytes around capillary;thickeness of basement membrance;stenosis of capillary lument,;structure disorder of membranous disc in photoreceptor cell,even broken or lysis;swelling and degeneration of cellular organ,suh as mitochondria;karyopyknosis and so on .Four major molecular mechanisms have been implicated in hyperglycemia-induced tissue damage: increased polyol pathway flux, increased hexosamine pathway flux, activation of protein kinase C (PKC) isoforms via de novo synthesis of the lipid second messenger diacylglycerol (DAG) and increased advanced glycation end product (AGE) formation.But treatments according to different pathways did not have significant effects. There was also no unifying hypothesis linking these four mechanisms. Experiments recently have shown that oxidative stress induced by hyperglycemia plays a key role in the diabetic chronic complications. Oxidative stress is regarded as the trigger that drives each of these pathways and/or common downstream mechanism of these pathways,activation signal transduction system,induing apoptosis;injury mitochondrial inner membrane,producing more ROS,lead energy dysbolism,decreasing the ATP synthesis.Tongluo Recipe(TLR) consists of ginseng,leech, scorpio, centipede,steleophaga, paeoniae rubra and so on.TLR have many effections about expand blood vessel,anticoagulation,degrade blood viscosity,improve microcirculatory. TLR can remove the ROS to therapy the microvascular complications with DM. Experiments showed that melatonin had protective effects in diabetes mellitus and diabetic chronic complications through antioxidation.It has been difficult to recognize which abnormalities are critical,thus making the rational for possible therapies limited. This research aimed to observe the antioxidation of TLR and MEL in rats with diabetic retinopathy. There were three parts in this research: first,to observe the effection of TLR on metabolism about antioxidation;second, to observe the effection of TLR on retinal apoptosis; third,to observe the effection of TLR on structure of retina. PartⅠPreventive effects of TLR on oxidative stress in rats with diabetic retinopathyObjective: To investigate the effect of TLR on ATP syntesis,oxidative stress,PAI-1 and othe materials changes in diabetic rats.Methods: Models of diabetic rat were induced by streptozocin(60mg/kg) and randomly assigned to 5 groups according to different blood glucose and body weights: (1) DM (Untreated); (2) TLR1, TLR 0.5g·kg-1·d-1 by gavage;(3)TLR2, 0.5g·kg-1·d-1 by gavage;(4) MEL1, melatonin 0.5mg·kg-1·d-1 by gavage;(5)MEL2, melatonin 10mg·kg-1·d-1 by gavage. Seven rats were in each group. Seven normal rats were assigned to normal control (NC) group with equivalent ages and body weights before experiment. TLR was given after diabetic models were induced by streptozotocin. Twelve weeks later, blood glucose and body weights,concentration of blood fat,insulin,antioxidant enzyme superoxide dismutase, glutathion peroxidase activities, lipid peroxide product malonaldehyde levels in serum and retina and concentration of ATP/ADP/AMP were evaluated.Results: Blood glucose was much higher and body weights were much lower in diabetic rats than in normal rats(P<0.01). The concentration of HDL was higher,LDL was lower;PAI-1was higher(P<0.05) in TLR group than in DM group. The level of insulin had no significant difference in TLR group and DM group.Superoxide dismutase and glutathione peroxidase activities were lower and malonaldehyde levels were higher in plasma and retina of DM group than those in NC group(P<0.05). TLR remarkably increased superoxide dismutase and glutathione peroxidase activities and reduced malonaldehyde levels in retina and plasma(P<0.05).The content of ATP was much more in NC group than in DM group.TLR can heighten the content of ATP.Conclusion: TLR remarkably increased superoxide dismutase and glutathione peroxidase activities and reduced malonaldehyde levels in retina and plasma and can heighten the content of ATP.TLR may adjust the function of endothelium and inhibit the produce of PAI-1,so it can be decrease the incidence of microthrombus.Overproduction of reactive oxygen species and insufficieny in antioxidant enzymes,microcirculation disturbance may contribute to the development of diabetic retinopathy. PartⅡEffects of TLR on apoptosis in rats with DRObjective: To investigate the effect of TLR on apoptosis of vascular cell in rats with diabetic retinopathy.Methods: Models of diabetic rat were induced by streptozocin(60mg/kg) and randomly assigned to 5 groups according to different blood glucose and body weights: (1) DM (Untreated); (2) TLR1, TLR 0.5g·kg-1·d-1 by gavage;(3)TLR2, 0.5g·kg-1·d-1 by gavage;(4) MEL1, melatonin 0.5mg·kg-1·d-1 by gavage;(5)MEL2, melatonin 10mg·kg-1·d-1 by gavage. Seven rats were in each group. Seven normal rats were assigned to normal control (NC) group with equivalent ages and body weights before experiment. TLR was given after diabetic models were induced by streptozotocin. Twelve weeks later, we utilized the immunohistochemistry method and RT-PCR to detect protein and mRNA expression of NF-kB and VEGF in retina.The quantity of expression using image analyzer were calculated.Results: The protein expression of NF-kB and VEGF can be detected in NC group and obviously enhanced in other groups(P<0.05). The protein expression of NF-kB were weaken compare with DM rats after drug treatment(P<0.05).The protein expression of VEGF were potentized in retinal of DM rats than normal rats (P<0.01) and it decreased after treatment(P<0.05). It can notable suppressed the protein expression of NF-kB and VEGF with large dose than low dose management(P<0.01).The mRNA expression of NF-kB,VEGF were significantly heighten in DM groups than NC rats(P<0.05).It is significantly difference between DM group and management group(P<0.05). It can visible suppressed the expression mRNA of NF-kB and VEGF with large dose than low dose management(P<0.05).Conclusion: TLR has the same effect in inhibited the protein and mRNA expression of NF-kB,VEGF in DM rats with MEL.So it can be relieve the development of diabetic retinopathy by decreasing the apoptosis of retina pericyte,inhibiting the generation of new vessels.The large dose may be have more therapeutic efficacy than low dosage. PartⅢPreventive effects of TLR on retina structure under light microscope and electron microscope in rats with DMObjective: To investigate the preventive effect of TLR on retinal structure under light microscope and electron microscope in diabetic rats.Methods: Models of diabetic rat were induced by streptozocin(60mg/kg) and randomly assigned to 5 groups according to different blood glucose and body weights: (1) DM (Untreated); (2) TLR1, TLR 0.5g·kg-1·d-1 by gavage;(3)TLR2, 0.5g·kg-1·d-1 by gavage;(4) MEL1, melatonin 0.5mg·kg-1·d-1 by gavage;(5)MEL2, melatonin 10mg·kg-1·d-1 by gavage. Seven rats were in each group. Seven normal rats were assigned to normal control (NC) group with equivalent ages and body weights before experiment. TLR was given after diabetic models were induced by streptozotocin. Twelve weeks later, the ultrastructural changes of retiana tissue in all groups under light microscope and electron microscope.Results: Light microscope:1.NC group:capillary vessels of retina walked regularly,caliber of vessels were uniform,they have no obviously abnormal changes;DM group:the vessel cells were apparent lost,vessels changed crooked and saccular dilatation,caliber of vessel were not coincidence.Electron microscope:NC group:the endochrome were well-distributed, the shape of cellular organ and karyon were normal, structure of basement membrane were distinctness and complete continuation.DM group:vascular endothelial cell was edema and even worse in the pericyte, basement membrane was thickness; mitochondrial was swelling to degeneration,endochrome was enrichment to the border. Caliber of vessels were manifest,some vessels even occluded.After TLR treatment,the pathological changes of diabetic retinopathy were taken a favorable turn under the light microscope and electron microscope.Conclusion: TLR can decrease the lost of endothelial cell and pericyte,inhibit thickness of basement membrane,reduce the neovacularization,protect structure and function of cellular organ.TLR may adjust the function of endothelium and improve hemodynamics in diabetic retinopathy. Microcirculation disturbance may contribute to the development of diabetic retinopathy.TLR may can inhibit the progress of diabetic retinopathy.