| BackgroundThe diabetic renal injury is a specific complication of diabetic microangiopathy, which is the pathophysiological foundation of renal failures in perioperative period among diabetic patients. One of the most common pathophysiological mechanisms accompanying clinical trauma and shock is renal ischemia/reperfusion injury. Moreover, oxidative stress and perioperative diabetic renal injury are closely related to renal ischemia/reperfusion injury, and remain a research hot spot and important project of disciplines related. It has been shown in latest researches that, DJ-1, a key protein found taking part in Parkinson's disease, is related to oxidative stress. DJ-1 can regulate and control the expressions of many endogenous antioxidant genes, including the nuclear transcription factor Nrf2, and therefore enhances the capacity of anti-oxidative stress. Nrf2 is a key transcription factor that can regulate and control cells, and that participates the endogenous protection of the oxidative stress process of cells. Nrf2 can induce the cells to withstand oxidative stress injuries effectively, which is an important endogenous protective mechanism of human beings. However, few researches have been found related to the mechanism of the protective actions of DJ-1 on renal cells against high glucose-induced ischemia/reperfusion injuries. Therefore, as a highly important endogenous protective mechanism, the mechanism of the protective actions of DJ-1 and Nrf2 against renal ischemia/reperfusion injuries in diabetic renal injuries calls for intensive researches.ObjectiveTo study the influences of DJ-1 and Nrf2 in rat renal proximal tubular epithelial cells induced by high glucose-induced ischemia/reperfusion injuries and the interactive relation in renal cell expression between them and the protective mechanism in endogenous anti-oxidative stress injuries.Methods1. The NRK-52E cells were divided into four groups randomly:(1)the low glucose group (LG group); (2)the low glucose+hypoxia/reoxygenation group (LG+HR group); (3)the high glucose group (HG group); (4)the high glucose+hypoxia/reoxygenation group (HG+HR group). The cells were placed in the environment of high glucose for 72 hours, then in hypoxia for 4 hours, and in reoxygenation for 2 hours in sequence. Therefore, the high glucose hypoxia/reoxyg enation model was established of renal proximal tubular epithelial cells of rats. The CCK-8 and LDH cytotoxicity test was adopted to detect the activity of cells. Then, the content of MDA and the alteration of SOD activity were measured. The Western blot was adopted to detect the expression of DJ-1, Nrf2 and HO-1 proteins.2. The NRK-52E cells were divided into nine groups randomly:(1)the low glucose group (LG group); (2)the low glucose+DJ-1 over expression group (LG+DJ-1 group); (3)the low glucose+antioxidant N-acetylcysteine group (LG+NAC group); (4)the high glucose group (HG group); (5)the high glucose+DJ-1 over expression group (HG+ DJ-1 group); (6)the high glucose+antioxidant N-acetylcysteine group (HG+NAC group); (7)the high glucose hypoxia/reoxygenation group (HG+HR group); (8)the high glucose+hypoxia/reoxygenation+DJ-1 over expression group (HG+HR+DJ-1 group) (9)the high glucose+hypoxia/reoxygenation+antioxidant N-acetylcysteine group (HG+HR+NAC group). The CCK-8 and LDH cytotoxicity test was adopted to detect the activity of cells. Then, the content of MDA and the alteration of SOD activity were measured. The Western blot was adopted to detect the expression of DJ-1, Nrf2 and HO-1 proteins.Results1. Compared with the low glucose group, the cell activity of those in the 72-hour high glucose group declined sharply, the activity of LDH grew sharply, and the difference was statistically significant (P<0.05). Compared with the high glucose group, the cell activity of those in the HG+HR group dropped more sharply, while the activity of LDH grew more rapidly, and the difference was also statistically significant (P<0.05). As with the oxidative stress level, compared with the low glucose group, content of MDA grew much higher, and the activity of SOD was much lower in cells of the high glucose group, and the difference was statistically significant (P<0.05). Compared with the high glucose group, in cells of the HG+HR group, content of MDA grew much higher, and the activity of SOD was much lower, and the difference was statistically significant (P<0.05). After the 72-hour stimulation by high glucose, the expressions of the DJ-1, Nrf2 and HO-1 protein sharply increased, and the difference was statistically significant (P<0.05). After the hypoxia/reoxygenation stimulation on the low glucose group, the expressions of the DJ-1 and Nrf2 protein sharply increased, and the difference was statistically significant (P<0.05) compared with the low glucose group, while there was a tend of growing in the expression of the protein HO-1, yet the difference was not statistically significant. After the hypoxia/reoxygenation stimulation on the high glucose group, the expressions of the DJ-1, Nrf2 and HO-1 protein sharply declined, and the difference was statistically significant (P<0.05) compared with the high glucose group. 2. The over expression of the protein DJ-1 and the treatment of antioxidant NAC could inverse the cell activity detected by CCK-8 and LDH obviously. Compared with the HG+HR group, the activity of NRK-52E cells in the HG+HR+DJ-1 group and HG+HR+NAC group ascended sharply, while the activity of LDH dropped sharply, and the difference was statistically significant (P<0.05). Besides, both of the over expression of the protein DJ-1 and the treatment of antioxidant NAC could strongly inverse the content of MDA and the activity of SOD. Compared with the HG+HR group, the content of MDA of NRK-52E cells in the HG+HR+DJ-1 group and HG+HR+NAC group dropped, while the activity of SOD grew, and the difference was statistically significant (P<0.05). After the pEX-2-EGFP-DJ-1 transfection of NRK-52E cells, compared with the corresponding comparison group, the expressions of the DJ-1, Nrf2 and HO-1 protein sharply increased, and the difference was statistically significant (P<0.05). The comparison between the HG+NAC group and the HG group showed that the expressions of the DJ-1, Nrf2 and HO-1 protein dropped sharply, and the difference was statistically significant (P<0.05). The comparison between the HG+HR+NAC group and the HG+HR group showed that the expressions of the DJ-1 and Nrf2 protein grew sharply, and the difference was statistically significant (P<0.05), while there was a tend of growing in the expression of the protein HO-1, yet the difference was not statistically significant.ConclusionThe stimulation of high glucose and hypoxia/reoxygenation in the rat renal proximal tubular epithelial cells would induce the acute injuries of the renal cells, while the DJ-1 protein could effectively alleviate the descend of cell activity and the oxidative stress induced by high glucose and hypoxia/reoxygenation, of which the mechanism might by related with the activation of the signal pathway of Nrf2/HO-1. |
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