| Object: Renal interstitial fibrosis may result from the excessive deposition of extracellular matrix components caused by many fac- tors. It was the common pathway to kidney structure abandoned. It was a common pathological basis of the end-stage renal disease and the development. At present the exact pathogenesis has not been f- ully elucidated. Oxidative stresses promote the process, while anti-oxidant postpone it. The Keap1-Nrf2-ARE path way can active a variety of anti- oxidant genes and phase II detoxification enzyme gene and relieve the injury result from ROS cells and electrophilic material. In recent years, the pathway mediated by the Keap1-Nrf2-ARE signaling was founded to play an important role in antioxidant, antitumor, anti-aging and protecting the ischemic reperfusion. It was seldom reported that the rule of the Keap1-Nrf2-ARE in Renal interstitial fibrosis. Most stu- dies revealed that Butylphthalide widely used for the treatment of ischemic brain injury resisted oxidative stress. But it was not reported that the role of Butylphthal ide in treating mouse model of renal interstitial fibrosis induced by ureteral obstruction.The mou- se model of renal interstitial fibrosis induced by unilateral ureteral obstruction was established. Observed the expression of Nrf-2, ga- mma-GCS, type I collagen in mice’s kidney. Polymerase Chain Rea- ction and Immunoh-istochemical laboratory check were used. Exp- lored the mechanism of Butylphthalide against oxidative stress and the renal protective effect in obstructive nephropathy in mice mod- el and treated it with Benazepril.Methods: A total of 72 subjects(CD-1mice of clean grade, male) were included. These subjects were divided into 4 groups at rand- om, Sham group, UUO group, NBP group, ACEI group. The Sham group and UUO group were gavaged with physiological saline. The NBP group was gavaged with Butylphthalide. The ACEI group was gavaged with Benazepril. At 3, 7, 14 days, 6 mice were sacrificed after surgery in every group. The kidney in side of the surgery was divided into three parts. One was fixed by 10% neutral formalin an- d was detected by immunohistochemisty. One was placed in alumini- um foil which was stripped with DEPC and was reserved under-70℃, so as to be used for Real-time PCR. One was reserved under-70℃, so as to be used for western blot. The correlation of TNF-α and Hb A1 c was assessmented by Linear regression. The experimental data was analyzed by SPSS 16.0 statistical software. ANOVA test and SNK-q test were used for data analysis.Results: Part one, the Pathology of mice kidney. The Sham group was normal. The other three group had significant figures, especially in the UUO group. The renal size was hypertrophy and the color was light. The renal cortex was thinning, cystic and rough. The demarcation between the renal cortex and the renal medulla was quite fuzzy. The renal capsule was not bounded firmly with nephridial tissue. The renal pelvis and renal calyces dilated obviously filled with deep brown urine. The damage in NBP group was less than other grouops. With HE and Masson observation, The Sham group was found normal. In UUO group, the renal tubular epithelial cells were shedding and vacuolar degeneration. In UUO group, the renal interstitium filled with inflammatory cells. Renal interstitial fibrosis, renal tubular dilatation, renal tubular atrophy and renal tubular collapse were found in seriously damaged mice in UUO group. The longer the obstructed time was, the seriously expression was. The damage in NBP group was less than UUO grouops. The damage of ACEI group was between NBP group and UUO grouop. Part two, the results of immunohistochemisty. The expression of Nrf2 was weakly positive in Sham group. It was more positive in other three groups. The longer the obstructed time was, the more positive expression was. Compared with the UUO group, the expression of Nrf2 in NBP group was more positive(7d, 0.1973±0.0294 vs 0.1579±0.0076; 14 d, 0.2722±0.0338 vs 0.1152±0.0128; P<0.05) Compared with the ACEI group, the expression of Nrf2 in NBP group was more positive(14d, 0.2722±0.0338 Vs 0.2104±0.0358, P<0.05). The expression of γ-GCS was weakly positive in Sham group. Compared with the UUO group, the expression of γ-GCS in NBP group was more positive(7d, 0.1487±0.0207 vs. 0.1035±0.0021; 14 d, 0.3009±0.0366 vs. 0.0729±0.0039; P<0.05). Compared with the ACEI group, the expression of γ-GCS in NBP group was more positive(14d, 0.3009±0.0366 Vs 0.2255±0.0436, P<0.05). The express of Type I collagen in Sham group was normal. The express of Type I collagen was enhanced 3 days after surgery. Compared with the UUO group, the expression of Type I collagen in NBP group was more(14d, 0.1089±0.0055 vs. 0.1527±0.0339, P<0.05). Part three, the result of RT-PCR. Compared with Sham group, the expression of m RNA of Nrf-2, γ-GCS and Type I collagen was all increased(3, 7, 14 days after surgery). Compared with UUO group, the production of Nrf-2 m RNA were apparently increased in NBP group(7d, 0.4917±0.0129 vs. 0.3554±0.0274; 14 d, 0.5205±0.0165 vs. 0.3648±0.0101; P<0.05). Compared with the ACEI group, the expression of Nrf2 in NBP group was more positive(14d, 0.5205±0.0165 vs. 0.4246±0.0097, P<0.05) Compared with UUO group, the production of m RNA γ-GCS were apparently increased in NBP group(7d, 0.5731±0.0205 vs. 0.4519± 0.0149; 14 d, 0.7186±0.0342 vs. 0.4855±0.0117; P<0.05). Compared with the ACEI group, the expression of γ-GCS in NBP group was more positive(14d, 0.7186±0.0342 vs. 0.5721±0.0293, P<0.05). But compared with the ACEI group, the expression of Type I collagen in NBP group was decreased after treatment with Bu tylphthalide. Part four, the result of Western Blot. Compared with UUO group, the production of Nrf-2 were apparently increased in NBP group(7d, 0.6143±0.0118 vs. 0.3522±0.0065; 14 d, 0.7027± 0.0218 vs. 0.3356±0.0030; P<0.05). Compared with the ACEI group, the expression of Nrf2 in NBP group was more positive(7d, 0.6143±0.0118 vs. 0.4450±0.0107; 14 d, 0.7027±0.0218 vs. 0.4531±0.0054, P<0.05) Compared with UUO group, the production of γ-GCS were apparently increased in NBP group(7d, 0.6843±0.0147 vs 0.3268±0.0099;14d, 1.0053±0.0113 vs. 0.4589±0.0145; P<0.05). Compared with the ACEI group, the expression of γ-GCS in NBP group was more positive(7d, 0.6843±0.0147 vs. 0.3863±0.0097;14d,1.0053±0.0113 vs.0.7232±0.0063;P<0.05).But compared with the ACEI group,the expression of Type I collagen in NBP group was decreased after treatment with Butylphthalide(7d,0.5649±0.0064 vs.0.9681±0.0109;14d,0.4392±0.0077 vs.1.1199±0.0902).Conclusion: The Renal Protective Effect of Butylphthalide in the Renal Interstitial Fibrosis was more predominant than Benazepril. The roles maybe occurred through increased the expression of Nrf-2 and γ-GCS and alleviated the expression of Type I collagen in nephridial tissue. The Keap1-Nrf2-ARE sign aling played an important role in Renal interstitial fibrosis. |
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