Tanshinone â…  Pretreatment Protects Against Ischemia-reperfusion Induced Kidney Injury In Mice Via Nrf2-ARE Activation

[Abstract]Ischemia-reperfusion injury (IRI) is not only a major cause of acute kidney injury (AKI) in native kidneys,conferring a high mortality rate of up to 50%; But Renal IRI is also associated with an increased risk of death in transplant patients, and is the primary cause of delayed graft function in renal allograft recipients from deceased donors. Despite these risks, there is currently no specific therapy to treat AKI, and finding an effective treatment remains an urgent priority. Oxidative stress is a major known contributor to the pathogenesis of renal IRI, responsible for direct and progressive cell damage; Previous reports have demonstrated that deficiency of anti-oxidant could exacerbate IR injury and many studies have proved in renal ischemia reperfusion injury,catalase, superoxide dismutase, vitamin E had beneficial effects for IR injury, supporting oxidative stress as a major contributor in the development of IR-induced injury. Therefore, factors that control this pathway may be attractive drug targets.NF-erythroid-2-related factor 2 (Nrf2), a member of the Cap ’n’ collar (CNC)-bZIP transcription factor family, is responsible for regulating the cell’s antioxidant response via the antioxidant response element, and is thought to be involved in repair and recovery from AKI. Nrf2 and its downstream target genes were found to be upregulated in the kidney tissue of wild-type (WT) mice following induction of renal ischemia and reperfusion and deficiency of Nrf2 caused worsened ischemic and nephrotoxic AKI in mice. On the basis of these studies, we hypothesized that pharmacological upregulation of Nrf2 would confer protection against AKI.Tanshinones, also known as Total Ketone, one of the most well-known Chinese herbs, includes lipophilic diterpenoids, astanshinone Ⅰ (T-I), dihydro-tanshinone ⅡB ect [9], and found it anti-cancer, anti-inflammatory, anti-tumor, preventing dementia, cardiovascular disease effects. In addition, it has antioxidant activity, in particular its scavenging oxygen free radical function. It has been reported, tanshinone I has been identified as a novel class of Nrf2 strong activator andexperimental therapeutics for Nrf2-dependent cytoprotection in many diseases [9]. But the application in renal disease has not been reported.Objective:To explorethe protective effect of tanshinone I control Nrf2-ARE pathway in mice renal ischemia-reperfusion injury. It has opened a new way for the prevention and treatment of ischemia-reperfusion induced renal injury.Methods:Male ICR mice were in four different groups:sham group (Sham), Sham group treated with tanshinone I (Sham+T-1), ischemia-reperfusion group (IR), ischemia-reperfusion group treated with T-1 (IR+T-1). T-1(10 mg/kg) or corn oil was administrated through intraperitoneal (i.p.) injection every other day for 15 days. Renal ischemia-reperfusion injury was induced by clipping the left kidney for 45 minutes. Blood serum and kidney tissue specimens were collected 24 hours after IR procedure. Blood urea nitrogen (BUN) and serum creatinine (Cr) were detected to evaluate renal function. Renal histopathological grading was used to evaluate renal histology between each experimental group. We was used to assess Histologic examination in kidney by HE staining with a light microscopy; Determination of MDA content 50 mg of kidney tissue homogenates;By qPCR method to detect Nrf2, HO-1, Gsta2 related gene expression at the transcriptional level; Analysis of the expression of Nrf2 and antioxidant genes HO-1, Gsta2 by IHC methods.Using the Statistical analysis software SPSS 17.0 and using One-way ANOVA Compares diverse groups by a Bonferroni post-hoc correction. All relevant experimental data were presented as mean ± standard deviation (S.D.). Definition of P <0.05 was considered statistically different.Results:(1) Compared with Sham group, IR groups exhibited a obvious increase in both Cr and BUN and showed obvious features of severe acute tubular damage (p< 0.05); Compared with IR group,IR+T-1 groupsobvious decreased the level of Cr and BUN, attenuated the histologic injury by IR (p< 0.05). Compared withthe group, in IR+T-1 group the level of SOD increased significantly while the level of MDA decreasedsignificantly; and renal histopathological score in IR+T-1 group decreased significantly (P< 0.05).(2)Thetanshinone Ipromote the mRNA expression levels of Nrf2 andits downstream gene of HO-1 and Gsta2 increased in kidney tissue cells. Compared with the sham group, the mRNA expression levels of Nrf2, HO-1 and Gsta2elevatedSlightly in IR group, the difference was not statistically significant (p> 0.05), for a description of ischemia and reperfusion injury in kidney tissues of Nrf2, HO-1 and Gsta2 the mRNA expression levels had little effect.Compared with the IR group, the mRNA expression levels ofantioxidant gene of Nrf2, HO-1,Gsta2 increased (p<0.01), in the IR+T-1 group.(3) We use the method of immunohistochemical to detect the protein expression level of Nrf2 in the cytoplasm and the nucleus of kidney tissue. Experimental results show that tanshinone I pretreatment promote Nrf2 into the nucleus andthe up-regulated expressionof its protein expression.Conclusion:(1) Tanshinone I significantly improve IR-induced renal injury in mice with a lower level of renal impairment, attenuated the renal tubular damage and oxidative stress. Our results indicated that tanshinone I may be used for preventingrenal injury induced ischemia-reperfusion in rats.(2)Tanshinone Ⅰ is an Nrf2 activator, which increases the Nrf2 of transcription and translation level, while causing the transfer occurs Nrf2 from cytoplasm to the nucleus.(3)Tanshinone Ⅰ can upregulate and activate Nrf2, promoting its into the nucleus, activating its downstream Antioxidant regulation of gene expression. Results indicate that Nrf2/ARE pathway in IRI causing each endogenous oxidative stress mechanism plays a significant role within the species.