| Objective: The chronic vascular complications of diabetes (including macrovascular and microvascular diseases, such as atherosclerosis and diabetic nephropathy) are a major cause of morbidity and premature mortality of diabetes. However, the pathogenesis of diabetic angiopathy is not fully known yet, inflammation and oxidative stress having been widely accepted to play an important role.In diabetes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) can be induced and actived, leading to damages. It is reported that there exists interaction between iNOS and COX-2 in vitro. However, whether there exists the interaction between iNOS and COX-2 in vivo, and its relationship with the pathogenesis and progression of diabetic angiopathy are not reported.In diabetes, once diffusing NO meets excessive superoxide, peroxynitrite (ONOO-) will be formed at a diffusion coefficient limited rate. ONOO- can cause conspicuous nitration to tyrosine residue of proteins. When proteins are nitrated by ONOO-, their structure and functions will be altered. The effect of ONOO- caused nitration of iNOS and COX-2 on their interaction in vivo has not been reported, especially in diabetic angiopathy.Urate is a specific endogenous ONOO- scavenger of ONOO-. Nourishing Yin and Promoting Blood flow Recipe (NYPBR) has been the core recipe to prevent and cure diabetic complications in previous serial studies.In this experiment, healthy male Wistar rats were used, and diabetic model was induced with streptozotocin (STZ). The rats were randomly divided into 4 groups: Control group, Diabetes group, Urate group and NYPBR group. The protein contents of iNOS and COX-2, their co-localization and interaction, and the nitration levels of iNOS and COX-2 were evaluated to clarify whether there exists interaction between iNOS and COX-2 in the diabetic rats; the relationship of interaction between iNOS and COX-2 with pathogenesis and progression of diabetic angiopathy, including macrovascular and microvascular diseases; the effect of nitration of iNOS and COX-2 on their interaction in vivo; whether the attenuated mechanism of NYPBR on diabeitc angiopathy is decreasing the nitration level of iNOS and COX-2 and then their interaction.Method:1 grouping and material36 healthy male Wistar rats (407.05±59.51g) were divided randomly into 4 groups: Control group (n=8), Diabetes group (n=10), Urate group (n=9), and NYPBR group (n=9). The latter 3 groups were injected intraperitoneally with STZ (40mg/kg, dissolved in 0.1mol/L citrate buffer, pH4.4, freshly made, 10mg/ml), to induce diabetes. Control group were injected with equivalent saline. 3 days after injection, diabetes was confirmed by the concentration of blood glucose higher than 13mmol/L and urine glucose (+++) or above. Rats in the 4 groups were all fed normal diet. Rats in Urate group were fed urate solution (160 mg/kg/day), rats in NYPBR group were fed NYPBR solution (1g/mL, 3mL/d with equivalent dose to people) and others equivalent water. After rats were raised for 13 weeks at room temperature (20-25°C), blood glucose and body weight were detected. Rats were killed by bloodletting from femoral artery, with aorta and renal cortex taken out quickly. Pieces of aorta and renal cortex were used for morphological observations and laser confocal microscopy analysis, and the rest pieces were put into liquid nitrogen and preserved at -70°C for future molecular biological measurement, such as RNA extraction, Western blotting and co-immunoprecipitation.2 Morphological observationsRat aorta and renal cortex were observed under optical microscope and transmission electron microscope.3 Detection of the transcriptional levels of iNOS and COX-2 in aorta and renal cortexTotal RNA was extracted from aorta and renal cortex using Trizol kit, and the transcriptional levels of iNOS and COX-2 were detected by reverse transcription-polymerase chain reaction (RT-PCR), withβ-actin as a"housekeeping gene".4 Detection of the protein contents and nitration levels of iNOS and COX-2 in aorta and renal cortexAfter aorta and renal cortex were homogenized, protein concentration was determined by Lowry method. Western-blotting was used to detect the protein contents and nitration levels (NT content) of iNOS and COX-2 in aorta and renal cortex.5 Co-localization of iNOS and COX-2 in aorta and renal cortexConfocal laser scanning microscope was used to detect the protein contents and co-localization of iNOS and COX-2 in aorta and renal cortex.6 Analysis of nitration levels and6.1 After aorta and renal cortex were homogenized, protein concentration was determined by Lowry method. Interaction between iNOS and COX-2 was analysed by co-immunoprecipitation.6.2 Western-blotting detected the immunoprecipitated iNOS and COX-2, and their nitation levels (NT content).Result:1 General condition of rats and the effects of Urate and NYPBR on blood glucose and body weight of rats 3 days after diabetic model was made, the blood glucose of rats in Diabetes group (21.68±2.95), Urate group (23.28±3.98) and NYPBR group (21.93±3.93) increased significantly, compared with Control group (P<0.001). Rats in Diabetes group were gradually thinner, with rare hair without politure, slow reaction, polydipsia, polyuria, polyphagia and growth retardation, which were better in Urate group and NYPBR group. After 13 weeks, the blood glucose of rats in Diabetes group (22.20±5.57) was higher than Control group (6.05±0.94) and body weight (347.30±43.15) lighter than Control group (521.2±81.74), with statistical significance (P<0.001). Neither blood glucose (18.88±3.21) nor body weight (320.44±61.78) in Urate group had statistical difference from Diabetes group. The blood glucose (14.79±3.07) of rats in NYPBR group was significantly lower than Diabetes group (P<0.05), but body weight (340.89±51.24) had no statistical difference.2 Morphological changes of rat aortaObservation of rat aorta under optical microscopy (×400): The aortic endothelium was intact in Control group and disappeared extensively in Diabetes group. The morphological changes of aortic endothelium in Urate group and NYPBR group were between Control group and Diabetes group, with damage in NYPBR group lighter than Urate group.Observation of rat aorta under transmission electron microscope (×20K or×40K): The endothelial cells with numerous pinocytosis vesicles and intact mitochondria were seen in Control group. However, most mitochondrial membrane, crests and cell junction fused or disappeared in Diabetes group. In Urate group and NYPBR group, the morphological changes were between Control group and Diabetes group, with mitochondrial damages attenuated, and only parts of cell junction fused. Damage in NYPBR group was lighter than Urate group.3 Morphological changes of glomerulusObservation of glomerulus under optical microscopy (×400): The pathological changes were not found, and basement membrane was thin and clear with PAS staining in Control group. Glomerular sclerosis, broader capsular space and thickened basement membrane with PAS staining were seen in Diabetes group. The morphological changes of glomerulus in Urate group and NYPBR group were significantly lighter than that of Diabetes group. Damage in NYPBR group was lighter than Urate group.Observation of glomerulus under transmission electron microscope (×10K,×15K or×20K): The pathological changes were not found in Control group. Intercapillary cells and foot process fused mostly, basement membrane thickened and mesangial matrix increased were observed in Diabetes group. The morphological changes of glomerulus in Urate group and NYPBR group were significantly lighter than Diabetes group. Damage in NYPBR group was lighter than Urate group.4 The changes of the mRNA expressions of iNOS and COX-2 in rat aortaThe mRNA expressions of iNOS and COX-2 in rat aorta were not detected in Control group, but the mRNA expressions of iNOS (0.57±0.05) and COX-2 (0.49±0.06) were high in Diabetes group. The mRNA expressions of iNOS (0.28±0.01) and COX-2 (0.29±0.02) in Urate group were significantly lower than that in Diabetes group (P<0.01). The mRNA expressions of iNOS (0.22±0.03) and COX-2 (0.29±0.03) in NYPBR group were significantly lower than that in Diabetes group (P<0.01).5 The changes of the mRNA expressions of iNOS and COX-2 in rat renal cortexThe mRNA expressions of iNOS and COX-2 in rat aorta was not detected in Control group, but the mRNA expressions of iNOS (0.43±0.07) and COX-2 (0.56±0.05) were high in Diabetes group. The mRNA expressions of iNOS (0.22±0.03) and COX-2 (0.22±0.03) in Urate group were significantly lower than that in Diabetes group (P<0.01). The mRNA expressions of iNOS (0.11±0.03) and COX-2 (0.33±0.07) in NYPBR group were significantly lower than that in Diabetes group (P<0.01 or P<0.05).6 The protein contents and nitration levels of iNOS and COX-2 in rat aortaThe protein contents of iNOS and COX-2 couldn't be detected in Control group. The protein content (60.67±6.66) and nitration level (0.83±0.14) of iNOS, and the protein content (204.33±6.66) and nitration level (0.43±0.02) of COX-2 increased obviously in Diabetes group. The protein content (44±5.29) and nitration level (0.44±0.04) of iNOS, and the protein content (158.67±19.5) and nitration level (0.37±0.03) of COX-2 in Urate group lowered significantly than Diabetes group (P<0.05). The protein content (47±2.65) and nitration level (0.36±0.06) of iNOS, and the protein content (161.67±12.1) and nitration level (0.3±0.05) of COX-2 in NYPBR group lowered significantly than Diabetes group (P<0.01 or P<0.05).7 The protein contents and nitration levels of iNOS and COX-2 in rat renal cortexThe protein contents of iNOS and COX-2 couldn't be detected in Control group. The protein content (205.67±26.76) and nitration level (0.54±0.02) of iNOS, and the protein content (281.67±28.75) and nitration level (1.64±0.12) of COX-2 increased obviously in Diabetes group. The protein content (142±10.54) and nitration level (0.39±0.06) of iNOS, and the protein content (182±8.62) and nitration level (0.95±0.1) of COX-2 in Urate group lowered significantly than Diabetes group (P<0.01 or P<0.05). The protein content (131.67±11.02) and nitration level (0.45±0.04) of iNOS, and the protein content (152.33±9.07) and nitration level (0.85±0.05) of COX-2 in NYPBR group lowered significantly than Diabetes group (P<0.01 or P<0.05).8 The protein contents and co-localization of iNOS and COX-2 in rat aortaThe protein contents of iNOS and COX-2 were not detected in Control group. The protein contents of iNOS (61.6±9.95) and COX-2 (60.7±8.96) increased sharply, with obvious yellow (co-localization of iNOS and COX-2) in Diabetes group. The protein contents of iNOS (23.24±4.15) and COX-2 (42.38±4.2) lowered significantly (P<0.01 or P<0.05), with weakened yellow in Urate group than Diabetes group. The protein contents of iNOS (17.65±4.34) and COX-2 (25.18±3.69) lowered significantly (P<0.01), with weakened yellow in Urate group than Diabetes group.9 The protein contents and co-localization of iNOS and COX-2 in rat renal cortexThe protein contents of iNOS and COX-2 were not detected in Control group. The protein contents of iNOS (20.52±2.73) and COX-2 (16±1.59) increased sharply, with obvious yellow (co-localization of iNOS and COX-2) in Diabetes group. The protein contents of iNOS (2.7±0.6) and COX-2 (9.95±2.23) lowered significantly (P<0.01 or P<0.05), with weakened yellow in Urate group than Diabetes group. The protein contents of iNOS (4.75±2.28) and COX-2 (10.91±2.05) lowered significantly (P<0.01 or P<0.05), with weakened yellow in Urate group than Diabetes group.10 The nitration levels and interaction between iNOS and COX-2 in rat aortaThe interaction between iNOS and COX-2 couldn't be detected in Control group. The interacted iNOS (0.92±0.12) and its nitration level (0.94±0.03), and interacted COX-2 (0.83±0.09) and its nitration level (1.3±0.11) increased sharply in Diabetes group. The interacted iNOS (0.65±0.09) and its nitration level (0.75±0.07), and interacted COX-2 (0.64±0.05) and its nitration level (1.08±0.07) lowered significantly in Urate group than Diabetes group (P<0.05). The interacted iNOS (0.71±0.03) and its nitration level (0.85±0.03), and interacted COX-2 (0.55±0.12) and its nitration level (1.05±0.09) lowered significantly in NYPBR group than Diabetes group (P<0.05).11 The nitration levels and interaction between iNOS and COX-2 in rat renal cortexThe interaction between iNOS and COX-2 couldn't be detected in Control group. The interacted iNOS (0.8±0.03) and its nitration level (1.14±0.07), and interacted COX-2 (0.57±0.05) and its nitration level (1.21±0.09) increased sharply in Diabetes group. The interacted iNOS (0.6±0.07) and its nitration level (1.01±0.02), and interacted COX-2 (0.47±0.02) and its nitration level (1.02±0.06) lowered significantly in Urate group than Diabetes group (P<0.05). The interacted iNOS (0.7±0.04) and its nitration level (0.84±0.09), and interacted COX-2 (0.44±0.05) and its nitration level (1.02±0.06) lowered significantly in NYPBR group than Diabetes group (P<0.05).Conclusion:1 In aorta and renal cortex of diabetic rats, the mRNA expressions and protein contents of iNOS and COX-2 increased significantly, and then it was found obviously co-localization, indicating that there might exist interaction between iNOS and COX-2 in diabetic angiopathy. 2 The alterations of interaction between iNOS and COX-2 were consistent with the pathological changes of aorta and glomerulus, suggesting that the interaction between iNOS and COX-2 might play important roles in the pathogenesis of diabetic macrovascular and microvascular diseases.3 In aorta and glomerulus of diabetic rats, iNOS and COX-2 were nitrated obviously and their interaction enhanced. Inhibition of nitration by urate could attenuate the interaction between iNOS and COX-2, and the pathological changes, showing that nitration of iNOS and COX-2 could promote their interaction, and inhibition of nitration may play a key role in prevention and cure of diabetic angiopathy.4 NYPBR could inhibit nitration of iNOS and COX-2 and then weaken their interaction in aorta and glomerulus of diabetic rats, protecting diabetic angiopathy from injury.
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