Effect Of Huoxue Jiedu Decoction On Retinopathy Of Diabetic Rats

ObjectiveTo examine the effect of Huoxue Jiedu Formula on the retina of diabetic rat; to research on its mechanism of improving the retinopathy of diabetic rat from the perspective that TGF-β /Smads signal transduction pathway regulates and adjusts matrix metabolism; to further clarify the pathogenesis of DR; and to provide effective methods and thoughts in clinical prevention and cure of DR.Methods1. A single intraperitoneal injection of STZ was given to induce models of diabetic rats. These rats were divided into five groups:model group (Group B), low dose group (of rats that have taken low dose of Huoxue Jiedu) (Group C), middle dose group (of rats that have taken middle dose of Huoxue Jiedu) (Group D), high dose group (of rats that have taken high dose of Huoxue Jiedu) (Group E) and calcium dobesilate group (Group F). There were 20 rats in each group. Besides, a normal group of 15 rats was set to make comparisons (Group A). In the 19 week, electron microscopy examinations were done on the rats to confirm retinopathy. Then they were dosed and lavaged once a day, which lasted 12 weeks. The conditions of the rats were observed generally, weights examined every week, and blood glucose examined every 4 weeks.2. In the 32nd week, the hemodynamic parameters of the central retinal artery and vein of rats’ eyes were examined by using high resolution small animal ultrasound imaging system to evaluate whether Huoxue Jiedu Formula had protective effect on the retinopathy of diabetic rats.3. In the 32nd week, intraperitoneal injections of sodium pentobarbital were given to anesthetize rats. Blood was taken from abdominal aorta and rats were killed. After the right eyeballs removed, retinas were stripped out and cutted into three sections, then fixed separately in 4% paraformaldehyde and 2.5% buffer solution of glutaraldehyde phosphate. Retinal vascular digestion and paraffin section HE dyeing were used to observe the changes of the retinal pathological tissue structure. Transmission electron microscope was used to observe the changes of the retinal ultrastructure.4. In the 32nd week, intraperitoneal injections of sodium pentobarbital were given to anesthetize rats. Blood was taken from abdominal aorta and rats were killed. After the left eyeballs removed, retinas were stripped out and cryopreserved in temperature of-80 ℃. The Real-time PCR method was used to examine the expressions of TGF-β2 mRNA、Smad-2 mRNA and Smad-7 mRNA in the retina of rats in all groups.5. Western blot method was used to examine the expression of MMP-9 and MMP-2 protein in the retina of rats in all groups after cryopreserved retina of-80 ℃ taken.6. Immunohistochemical method was used to examine the expression of collagen I and collagen IV in the retina of rats in all groups after the paraffin section taken.Results1. General conditions:The model group of diabetic rats had more food, water and polyuria and had gradual emaciation, which was a typical symptom of "three more and one less". They had other features like paleness in auricle and onyx stool, thin and putrid excrement, body hair, dry and disorderly hair, poor mental state, laziness, restlessness etc. The different dose groups of rats that had taken Huoxue Jiedu were in better conditions state of existence of each dose group of rats obviously improved compared with the model group in the same period. For Hair loss, quantity of drinking water and the volume of urine were all reduced. But the differences between the dose groups were not obvious.2. Changes in weight:Weights of the normal group of rats increased steadily and kept comparatively high while weights of the model group and different dose groups of rats increased slowly and were obviously low compared with the normal group in the same period. The difference between the dose groups and the normal group was of statistical significance (P<0.05). There was no difference between different dose groups (P>0.05).3. Changes in blood glucose:Blood glucose of the normal group of rats kept stable at a low level and there were no expressions of high blood glucose while blood glucose of the model group and different dose groups of rats kept at a higher level and was obviously high compared with the normal group in the same period. The difference between the dose groups and the normal group was of statistical significance (P<0.05). There was no difference between different dose groups (P>0.05).4. Changes in hemodynamic parameters:The fundus high frequency ultrasonic examination on all groups of rats showed:compared with the normal group of rats, pulse index (PI) and resistance index (RI) of the model group increased while systolic peak velocity (PSV), late diastolic velocity (EDV), mean blood flow velocity (MV) and central venous blood flow velocity (CRV) decreased. And the difference was of statistical significance (P<0.05). Compared with the model group, pulsation index (PI) of the different dose groups all decreased and the difference was of statistical significance (P<0.05). The change was especially obvious in group E. The resistance index (RI) decreased and systolic peak velocity (PSV), late diastolic velocity (EDV), mean blood flow velocity (MV) all increased. And the difference was of statistical significance (P<0.05).5. Changes in retinal vascular digest preparations morphologically:The retinal vascular network of the model group distorted, had a disordered arrangement, and was uneven; the size of capillary diameter was not the same and capillary hemangioma was visible; pericytes reduced significantly, endothelial cells increased diffusely; capillary area density, endothelial cells and pericytes proportion increased significantly compared with the normal group, the difference was of statistical significance (P<0.001). Capillaries of the Huoxue Jiedu Formula groups was distributed evenly and vascular morphology was more regular and orderly, endothelial cell proliferation was not obvious, no plexiform, capillary hemangioma or apoptotic bodies; capillary area density, endothelial cells and pericytes decreased compared with the model group, the difference was of statistical significance (P<0.05), especially in the Group D.6. Changes in HE staining morphological retina:The retinal layers of the model group all had an edema and loose organizational structures. Nerve fiber layer was lost, the number of cells in the ganglion cell layer significantly reduced, inner plexiform layer and outer plexiform layer both had an edema, inner nuclear layer cell disordered, and the number of cells reduced; visible dot-like or flame-like bleeding in the retina, with blurred boundaries. Huoxue Jiedu Formula group improved significantly compared with the model group, retinal layers evenly arranged, some levels had a slight edema, occasional minor bleeding, the emergence of new blood vessels was not observed.7. Changes in retinal ultrastructure under electron microscope:The retinal capillary endothelial cells of the model group swelled and deformated, nucleus were irregular, endothelial cells protruded into vascular lumen, making stenosis even occlusion, capillary basement membrane thickening was especially obvious. Retinal vascular basement membrane of the Huoxue Jiedu Formula group periodically thickened but not obvious, endothelial cells connected tightly, and lumen relatively smooth with unobvious deformation.8. Results of Real-time PCR method:TGF-β2 mRNA and Smad-2 mRNA expression of the model group were higher than the normal group, the difference was of statistical significance (P<0.05), Smad-7 mRNA expression was lower than the normal group, but the difference was of no statistical significance (P>0.05); TGF-β2 mRNA and Smad-2 mRNA expression of Huoxue Jiedu Formula group were lower than the model group, the difference was of statistical significance (P<0.05), Smad-7 mRNA expression higher than the model group, but the difference was of no statistical significance (P>0.05).9. Results of Western blot method:MMP-9, MMP-2 protein expression of model group were higher than the normal group, the difference was of statistical significance (P<0.05); MMP-9, MMP-2 protein expression of Huoxue Jiedu Formula group was lower than the model group, the difference was of statistical significance (P<0.05).10. Results of immunohistochemical method:collagen Ⅰ and collagen Ⅳ positive cells showed brown cytoplasm, visible under microscope:collagen Ⅰ and collagen IV expression of normal group is lower and showed pale brown; collagen Ⅰ and collagen Ⅳ expression of model group increased and showed dark brown; collagen Ⅰand collagen Ⅳ expression Huoxue Jiedu Formula group showed light brown, and lower than model group.Conclusion1. Huoxue Jiedu Formula can improve the survival state of STZ diabetic rats, relieve some symptoms of diabetes, but has no significant effects on weight and blood glucose in rats.2. Huoxue Jiedu Formula can reduce peripheral vascular resistance in rats’ retina, accelerate blood velocity, increase retinal blood perfusion, so as to improve microcirculation resistance, protect retinal vessels, and postpone the further development of DR.3. Huoxue Jiedu Formula can inhibit proliferation of endothelial cells, reduce angiogenesis; improve edema in retinal layers, alleviate the pathological damage in retina of diabetic rats; and inhibit increase of the basement membrane collagen synthesis, reduce matrix deposition, inhibite constant thickening of the basement membrane, which suggests that Huoxue Jiedu Formula can improve diabetic rats’ retinopathy.4. Huoxue Jiedu Formula can decrease TGF-β2 and Smad-2 gene expression, intervene TGF-β2/Smads signal transduction pathways to regulate matrix metabolism, reduce the expression of MMP-9, MMP-2, collagen Ⅰ, collagenⅣ, thereby improving DR basement membrane thickening, neovascular proliferation, protecting the retina tissue, which may be Huoxue Jiedu Formula’ mechanism of improving diabetic retinopathy.