Post-ischemic administration of 5-methoxyindole-2-carboxylic acid at the onset of reperfusion affords neuroprotection against stroke injury by preserving mitochondrial function and attenuating oxidative stress

Abstract

We previously reported that 5-methoxyindole-2-carboxylic acid (MICA) could induce preconditioning effect in the ischemic brain of rat. In the present study, we addressed the question of whether MICA could also trigger a postconditioning effect in ischemic stroke. To this end, MICA (100 mg/kg body weight) was injected intraperitoneally at the onset of 24 h reperfusion following 1 h ischemia in rat brain. Results indicate that stroked animals treated with MICA showed less brain infarction volume than that of vehicle-treated animals. Further experiments revealed that brain mitochondrial complexes I and IV showed elevated enzymatic activities in MICA treated group and the elevation in complex I activity was likely contributed by seemingly enhanced expression of many complex I subunits, which was determined by mass spectral peptide sequencing. When compared with vehicle-treated rats, the preservation of complexes I and IV activities was shown to be accompanied by enhanced mitochondrial membrane potential, increased ATP production, and decreased caspase-3 activity. Additional studies also indicate the involvement of NQO1 upregulation by the Nrf2 signaling pathway in this MICA postconditioning paradigm. Consequently, attenuated oxidative stress in the MICA treated group reflected by decrease in H₂O₂ production and protein carbonylation and lipid peroxidation was detected. Taken together, the present study demonstrates that MICA can also induce a postconditioning effect in the ischemic brain of rat and the underlying mechanism likely involves preservation of mitochondrial function, upregulation of cellular antioxidative capacity, and attenuation of oxidative stress.

Keywords: 5-Methoxyindole-2-carboxylic acid; Mitochondria; Neuroprotection; Oxidative stress; Postconditioning; Stroke.

Fig. 1

A) Chemical structure of MICA. B) Timeline of MICA injection (IP) during the ischemia reperfusion procedure; MICA was administered at the onset of 24 h reperfusion following 1 h ischemia (MCAO). C) TTC staining of brain slices after 24 h reperfusion between control and MICA groups (N=3 for each group). D) Densitometric quantitation of the infarction size shown in C.

Fig. 2

A) BN-PAGE analysis of mitochondrial complex I and DLDH. B) Densitometric quantitation of complex I gel band intensity shown in A. C) BN-PAGE analysis of complex IV between control and MICA. D) Densitometric quantitation of complex IV gel band intensity shown in C. E) Comparison of protein expression of detected complex I subunits between control and MICA. Complex I band from each group were excised for mass spectral peptide sequencing and the number of peptides for each subunit were shown. The number of peptide for each subunit was the sum of three blue native gel bands for each group

Fig. 3

A) Western blot determination of complex I subunits NDUFS1 and NDUFS8, indicating a higher expression of the two subunits than that in the control. B and C) show densitometric quantitation of the two subunits, respectively. D) Comparison of mitochondrial membrane potential between control and MICA. E) Comparison of cellular ATP content between control and MICA. F) Comparison of caspase-3 activity between control and MICA.

Fig. 4

A) NQO1 expression determined by Western blot assay (N=3 for each group). B) Comparison of densitometric quantitation of gel bands between control and MICA shown in A. C) Comparison of electro-mobility of Nrf2 transcription factor between control and MICA. D) Comparison of H₂O₂ production between control and MICA. E) Comparison of protein carbonyl content between control and MICA. F) Comparison of lipid peroxidation (as TBARS) between control and MICA.