The Effect Of Pancreatic Kininogenase On Non-Proliferative Retinopathy In Type 2 Diabetic Mice

Objective:Diabetic retinopathy(DR)is one of the most common microvascular complications of diabetes and also an important cause of non-traumatic blindness in adult.The early stage of DR is mainly manifested by the loss of vascular cells and leakage of blood vessels.Many studies have shown that oxidative stress,inflammation and apoptosis play an important role in this stage.Therefore,inhibition of oxidative stress,inflammation and apoptosis may prevent diabetic retinopathy.Kallikrein kinin system(KKS)is a complex and multifunctional endogenous peptidergic system consisting mainly of kininogen,serum or tissue kallikrein,bradykinin and kinin.Among them,kallikrein is a serine protease,which is a key enzyme of KKS system.Kininogenase could convert kininogen to bradykinin,which acts on the bradykinin B1 R and B2 R.Many studies have shown that tissue kallikrein has effects on diabetic complications such as nephropathy,cardiomyopathy and neuropathy,but its effects on diabetic retinopathy are not fully understood.Here,we discussed the retinal protective role of exogenous pancreatic kininogenase(PK)and studied potential mechanism in KKAy—a spontaneous type 2 diabetic mouse and HFD/STZ-induced type 2 diabetic mice.Methods:We used KKAy mice,spontaneous type 2 diabetic mice and HFD/STZ-induced type 2 diabetic mice as our models.After adapting for two weeks,KKAy mice were randomly divided into two groups.One group was injected with saline(0.1ml/10g/d,KKAy group)and the other group was injected with pancreatic kininogenase(40U/kg/d,KKAy +PK group)for 12 weeks.C57BL/6J mice were used as the control group(C57 group).For further validation,we selected another animal models —HFD/STZ-induced type 2 diabetic mice.C57BL/6 mice were randomly divided into two groups: normal control(NC group)and high fat diet(HFD group).After 12 weeks of HFD feeding,intraperitoneal glucose tolerance test(IPGTT)was used to measure insulin resistance.Then a small dose of streptozocin(STZ)was intraperitoneally injected for 7 consecutive days and mice with random blood glucose levels above 16.7 mmol/L were considered to be type 2 diabetic mice.After successful modeling,they were randomly separated into two groups.One group was intraperitoneally injected with saline(0.1 ml/10 g/d,STZ group)and the other group was intraperitoneally injected with pancreatic kininogenase(40U/kg/d,STZ+PK group).After 12 weeks of pancreatic kininogenase treatment,we used Optical Coherence Tomography(OCT)to observe the pathological changes of the retina in live mice.Blood samples were collected to detect liver function,renal function and blood lipid.H&E staining was used to observe the retinal thickness and number of cells in each layer;Retinal trypsin digestion preparation was performed to observe vascular lesions;Confocal was used to observe the changes of retinal vascular leakage;ROS was used to detect retinal oxidative stress;Immunohistochemistry and TUNEL was adopted to analyze the levels of apoptosis;Western blot was used to examine the protein expression of oxidative stress,inflammation,apoptosis and KKS system;RT-PCR was performed to detect the mRNA expression of relevant indicators of KKS system.Results:1.The results of both models showed that compared with the normal group,the body weight,blood glucose,kidney weight ratio,and alanine aminotransferase and triglyceride levels in diabetic group mice were significantly higher(P<0.05),while in PK treatment group these indicators did not change significantly except for a decrease in kidney weight/weight ratio(P>0.05).2.OCT results showed that compared with the C57 group,the blood vessels and the thickness of the retina was thinned in KKAy group,but was improved after PK treatment.3.The results of H&E staining in both animal models showed that compared with the normal group,the thickness of the retinal ganglion cell layer(GCL),the inner nuclear layer(INL)and the outer nuclear layer(ONL)of the diabetic group was significantly thinned.But that this effect was reversed by PK treatment.4.Retina trypsin digestion of two animal models showed that compared with the normal group,retinal acellular capillaries were significantly increased.However,PK treatment reduced this pathologic change.5.The confocal results of KKAy mice showed that the capillary density in the peripheral region of the retina was significantly lower in the KKAy group than in the C57 group,and that PK treatment attenuated this reduction in capillary density.In contrast,there was no significant change in the density of capillaries in the central region of the retina,but vascular leakage was observed in the KKAy group,and PK treatment improved the leakage.6.The confocal results of HFD/STZ model mice showed that there were no significant changes in the density of retinal capillaries in these three groups in either the peripheral or central regions,but vascular leakage was observed in the STZ group in both the peripheral and central regions,and pancreatic kallikrein improved this phenomenon.7.ROS,TUNEL and Western blot results in both animal models showed that compared with C57 group,KKAy group significantly increased oxidative stress,inflammation and apoptosis levels(P<0.05)and after PK treatment these indicators were decreased significantly(P<0.05).8.The results of Western blot and RT-PCR in both animal models showed that the KKS system related indicators were significantly increased after PK treatment(P<0.05)but there was no significant change in C57 and KKAy group(P>0.05).Conclusions:1.Pancreatic kininogenase could improve the pathological structure of retinal in KKAy and HFD/STZ-induced type 2 diabetic mice.2.Pancreatic kininogenase could relieve the oxidative stress,inflammation and apoptosis of the retina in KKAy and HFD/STZ-induced type 2 diabetic mice.3.Pancreatic kininogenase could increase the expression of KKS in KKAy and HFD/STZ-induced type 2 diabetic mice.