We have reported that sulforaphane (SFN) prevented diabetic cardiomyopathy in both type 1 and type 2 diabetes (T2DM) animal models via the upregulation of nuclear transcription factor erythroid 2–related factor 2 (Nrf2) and metallothionein (MT). In this study, we tested whether SFN protects the heart from T2DM directly through Nrf2, MT, or both. Using Nrf2-knockout (KO), MT-KO, and wild-type (WT) mice, T2DM was induced by feeding a high-fat diet for 3 months followed by a small dose of streptozotocin. Age-matched controls were given a normal diet. Both T2DM and control mice were then treated with or without SFN for 4 months by continually feeding a high-fat or normal diet. SFN prevented diabetes-induced cardiac dysfunction as well as diabetes-associated cardiac oxidative damage, inflammation, fibrosis, and hypertrophy, with increases in Nrf2 and MT expressions in the WT mice. Both Nrf2-KO and MT-KO diabetic mice exhibited greater cardiac damage than WT diabetic mice. SFN did not provide cardiac protection in Nrf2-KO mice, but partially or completely protected the heart from diabetes in MT-KO mice. SFN did not induce MT expression in Nrf2-KO mice, but stimulated Nrf2 function in MT-KO mice. These results suggest that Nrf2 plays the indispensable role for SFN cardiac protection from T2DM with significant induction of MT and other antioxidants. MT expression induced by SFN is Nrf2 dependent, but is not indispensable for SFN-induced cardiac protection from T2DM.

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Oxidative stress contributes to the pathogenesis of diabetic nephropathy (DN). Nuclear factor erythroid 2-related factor 2 (NRF2) plays a key role in cellular defense against oxidative stress. NRF2 activators have shown promising preventive effects on DN. Sodium butyrate (NaB) is a known activator of NRF2. However, it is unknown whether NRF2 is required for NaB protection against DN. Therefore, streptozotocin-induced diabetic C57BL/6 Nrf2 knockout and their wild-type mice were treated in the presence or absence of NaB for 20 weeks. Diabetic mice, but not NaB-treated diabetic mice, developed significant renal oxidative damage, inflammation, apoptosis, fibrosis, pathological changes and albuminuria. NaB inhibited histone deacetylase (HDAC) activity and elevated the expression of Nrf2 and its downstream targets heme oxygenase 1 and NAD(P)H dehydrogenase quinone 1. Notably, deletion of the Nrf2 gene completely abolished NaB activation of NRF2 signaling and protection against diabetes-induced renal injury. Interestingly, the expression of Kelch-like ECH-associated protein 1, the negative regulator of NRF2, was not altered by NaB under both diabetic and non-diabetic conditions. Moreover, NRF2 nuclear translocation was not promoted by NaB. Therefore, the present study indicates, for the first time, that NRF2 plays a key role in NaB protection against DN. Other findings suggest that NaB may activate Nrf2 at the transcriptional level, possibly by the inhibition of HDAC activity.

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Recent studies show that biomaterials are capable of regulating immune responses to induce a favorable osteogenic microenvironment and promote osteogenesis and angiogenesis. In this study, we investigated the effects of zinc silicate/nanohydroxyapatite/collagen (ZS/HA/Col) scaffolds on bone regeneration and angiogenesis and explored the related mechanism. We demonstrate that 10ZS/HA/Col scaffolds significantly enhanced bone regeneration and angiogenesis in vivo compared with HA/Col scaffolds. ZS/HA/Col scaffolds increased tartrate-resistant acid phosphatase (TRAP)-positive cells, nestin-positive bone marrow stromal cells (BMSCs) and CD31-positive neovessels, and expression of osteogenesis (Bmp-2 and Osterix) and angiogenesis-related (Vegf-α and Cd31) genes increased in nascent bone. ZS/HA/Col scaffolds with 10 wt % ZS activated the p38 signaling pathway in monocytes. The monocytes subsequently differentiated into TRAP+ cells and expressed higher levels of the cytokines SDF-1, TGF-β1, VEGF-α, and PDGF-BB, which recruited BMSCs and endothelial cells (ECs) to the defect areas. Blocking the p38 pathway in monocytes reduced TRAP+ differentiation and cytokine secretion and resulted in a decrease in BMSC and EC homing and angiogenesis. Overall, these findings demonstrate that 10ZS/HA/Col scaffolds modulate monocytes and, thereby, create a favorable osteogenic microenvironment that promotes BMSC migration and differentiation and vessel formation by activating the p38 signaling pathway.

Aims/hypothesis Diabetic nephropathy is the leading cause of end-stage renal disease. Previously we reported that C66, a novel analogue of curcumin with a very high bioavailability, ameliorated diabetic nephropathy in mice, with little known about the mechanism. The present study aimed to define the mechanism by which C66 ameliorates diabetic nephropathy. Methods Our aim was to discover whether C66 acts through the activation of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2), which governs the antioxidant response. Streptozotocin-induced Nrf2 (also known as Nfe2l2)-knockout and wild-type (WT) diabetic mice were treated with C66. To determine whether the actions of C66 on NRF2 are mediated by microRNA (miR)-200a, WT diabetic mice were treated with C66 in the presence or absence of an in vivo miR-200a inhibitor (locked nucleic acid-modified anti-miR-200a [LNA-200a]) for 6 months. To determine whether miR-21 downregulation provided an NRF2-independent basis for C66 protection, Nrf2-knockout diabetic mice were treated with either C66 or an inhibitor of miR-21 (locked nucleic acid-modified anti-miR-21 [LNA-21]). Results Deletion of Nrf2 partially abolished diabetic nephropathy protection by C66, confirming the requirement of NRF2 for this protection. Diabetic mice, but not C66-treated diabetic mice, developed significant albuminuria, renal oxidative damage and fibrosis. C66 upregulated renal miR-200a, inhibited kelch-like ECH-associated protein 1 and induced NRF2 function, effects that were prevented by LNA-200a. However, LNA-200a only partially reduced the protection afforded by C66, suggesting the existence of miR-200a/NRF2-independent mechanisms for C66 protection. C66 was also found to inhibit diabetes induction of miR-21. Both C66 and LNA-21 produced similar reductions in miR-21, albuminuria and renal fibrosis. Conclusions/interpretation The present study indicates that in addition to upregulating NRF2 by increasing miR-200a, C66 also protects against diabetic nephropathy by inhibiting miR-21.

Sulforaphane (SFN) prevents diabetic nephropathy (DN) in type 1 diabetes via up-regulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, it has not been addressed whether SFN also prevents DN from type 2 diabetes or which Nrf2 downstream gene(s) play(s) the key role in SFN renal protection. Here we investigated whether Nrf2 is required for SFN protection against type 2 diabetes-induced DN and whether metallothionein (MT) is an Nrf2 downstream antioxidant using Nrf2 knockout (Nrf2-null) mice. In addition, MT knockout mice were used to further verify if MT is indispensable for SFN protection against DN. Diabetes-increased albuminuria, renal fibrosis, and inflammation were significantly prevented by SFN, and Nrf2 and MT expression was increased. However, SFN renal protection was completely lost in Nrf2-null diabetic mice, confirming the pivotal role of Nrf2 in SFN protection from type 2 diabetes-induced DN. Moreover, SFN failed to up-regulate MT in the absence of Nrf2, suggesting that MT is an Nrf2 downstream antioxidant. MT deletion resulted in a partial, but significant attenuation of SFN renal protection from type 2 diabetes, demonstrating a partial requirement for MT for SFN renal protection. Therefore, the present study demonstrates for the first time that as an Nrf2 downstream antioxidant, MT plays an important, though partial, role in mediating SFN renal protection from type 2 diabetes.

L. Sulforaphane reduction of testicular apoptotic cell death in diabetic mice is associated with the upregulation of Nrf2 expression and function. Am J Physiol Endocrinol Metab 307: E14 -E23, 2014. First published May 6, 2014 doi:10.1152/ajpendo.00702.2013.-Diabetes-induced testicular cell death is due predominantly to oxidative stress. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an important transcription factor in controlling the antioxidative system and is inducible by sulforaphane (SFN). To test whether SFN prevents diabetes-induced testicular cell death, an insulin-defective stage of type 2 diabetes (IDS-T2DM) was induced in mice. This was accomplished by feeding them a high-fat diet (HFD) for 3 mo to induce insulin resistance and then giving one intraperitoneal injection of streptozotocin to induce hyperglycemia while age-matched control mice were fed a normal diet (ND). IDS-T2DM and ND-fed control mice were then further subdivided into those with or without 4-mo SFN treatment. IDS-T2DM induced significant increases in testicular cell death presumably through receptor and mitochondrial pathways, shown by increased ratio of Bax/Bcl2 expression and cleavage of caspase-3 and caspase-8 without significant change of endoplasmic reticulum stress. Diabetes also significantly increased testicular oxidative damage and inflammation. All of these diabetic effects were significantly prevented by SFN treatment with upregulated Nrf2 expression. These results suggest that IDS-T2DM induces testicular cell death presumably through caspase-8 activation and mitochondria-mediated cell death pathways and also by significantly downregulating testicular Nrf2 expression and function. SFN upregulates testicular Nrf2 expression and its target antioxidant expression, which was associated with significant protection of the testis from IDS-T2DM-induced germ cell death. male germ cell death; sulforaphane; nuclear factor (erythroid-derived 2)-like 2; type 2 diabetes; high-fat diet