In this study, we demonstrated that CDDO-Im, a synthetic triterpenoid and the strongest inducer of the Nrf2 signaling pathway known so far, induced robust HO-1 expression in neuronal cultures and brains. Using three ischemia models, one in vitro and two in vivo, we found that CDDO-Im treatment attenuated ischemic neuronal injury at extremely low doses. The protective effect of CDDO-Im was blocked when HO-1 activity was inhibited by Sn-PPIX or when Nrf2 was knocked down with specific shRNA, indicating that HO-1 upregulation and Nrf2 activation played important roles in the neuroprotective effects of CDDO-Im.
Triterpenoids belong to a group of five-ring compounds that are produced by many plants, including ginsengs. Natural triterpenoids have been used as alternative medicines for centuries for their mild anti-oxidative, anti-inflammatory and anti-carcinogenic effects. In an effort to enhance their potency, oleanolic acid, a natural triterpenoid, has been further modified by extensive synthetic steps, and a new set of synthetic triterpenoids has been generated 12
. Among them, CDDO-Im displays the strongest bioactivities, including anti-cancer, anti-inflammatory and anti-oxidative effects 13–15, 19
The anti-oxidative role of CDDO-Im is dependent on its ability to activate Nrf2, a master transcription factor that governs phase-2 enzyme expression 14, 15
. Under normal conditions, Nrf2 is not active because it binds Keap1, which facilities their proteosomal degradation, resulting in a short half-life and a low basal level of Nrf2 20
. Keap1 is rich in cysteine, and Cys-151 and Cys-275 are important for Nrf2 degradation 21
. A recent study shows that dihydro-CDDO-trifluoroethyl amide, another derivative of CDDO, dissociates Keap1 from Nrf2 by interacting with Cys-151 of Keap1 via Michael addition 22
, leading to upregulation of phase-2 enzymes. CDDO-Im may function in a similar manner to upregulate phase-2 enzymes, though direct evidence for this is currently not available.
As a phase-2 enzyme, HO-1 is neuroprotective against stroke, as HO-1 knockout worsens infarcts 8
and HO-1 overexpression reduces infarcts in mice 2
. Our data support this notion. In addition to an increase in levels, HO-1 location is also likely to contribute to its neuroprotective capacity. HO-1 was primarily expressed in neurons after CDDO-Im treatment, as shown in & and in a previous report 17
. This is likely to be important for its neuroprotective role in our hands. HO-1 was upregulated in CA1 neurons during the early stage (<24 hr) after ischemia, further supporting the notion that HO-1 may contribute to protection against ischemic neuronal injury. In the late stage, HO-1 was also strongly expressed in astrocytes and microglia. The role of glial HO-1 remains unclear, though previous reports showed that astrocytic Nrf2 and HO-1 also offered neuronal protection against oxidative stress 4, 23
. A prior report also showed that HO-1 could be expressed in cultured astrocytes after CDDO treatment 17
; however, we did not notice strong astrocytic HO-1 in vivo
, probably because astrocytes were not activated after CDDO-Im injection.
The potential benefits of CDDO-Im in the nervous system are not limited to stroke. For example, CDDO-Im reduces retinal injury from photooxidation 24
. Other CDDO derivatives protect against Alzheimer’s disease 25
, Huntington’s and Parkinson’s disease 26
, and amyotrophic lateral sclerosis 27
. In future studies, the neuroprotective resume for CDDO-Im could be extended to other forms of acute brain injuries such as hemorrhagic stroke and traumatic brain injury. Compared to several Nrf2/HO-1 inducers such as sulforaphane 4, 5
and Gingko biloba 7
that have previously shown neuroprotective effects, a clear advantage of using CDDO-Im is its low doses required to achieve neuroprotection. Thus, the strong potency of CDDO-Im with no apparent toxicity makes this drug a potential candidate for clinical neuroprotection. A caveat for the clinical application of CDDO-Im though is its relatively mild capability to across BBB. However, since BBB is likely compromised after stroke, it is possible to systemically administrate CDDO-Im shortly after stroke and achieve neuroprotection, especially in conjunction with tPA treatment that would re-establish blood flow in ischemic regions. Alternatively, a CDDO-Im derivative with increased BBB penetrating properties may be designed and tested in future studies.
In summary, our data reveal for the first time that a low dose of a potent compound CDDO-Im upregulates the inducible phase-2 enzyme HO-1 primarily in neurons and protects neurons and brain from ischemic injury. The protection was apparent in three different models and was blocked with a competitive inhibitor of HO-1. Therefore, CDDO-Im is a promising candidate for protecting brain against human stroke in an Nrf2/HO-1 dependent manner. Nonetheless, several issues still need to be addressed in the future. Examples include whether other phase-2 enzymes or other signaling pathways are involved in the neuroprotective effects of CDDO-Im, as the nuclear factor kappa B, phosphatase and tensin homolog, and mammalian target of rapamycin appear to also mediate the effects of CDDO-Im in non-neuronal cells 12, 28, 29
. Clarification of these issues may help develop new strategies for stroke treatment.