High levels of
homocysteine (Hcy) known as
hyperhomocysteinemia (HHcy), contribute to autophagy and
ischemia/reperfusion injury (I/R). Previous studies have shown that I/R injury and HHcy cause increased cerebrovascular permeability; however, the associated mechanism remains obscure. Interestingly, during HHcy, cytochome-c becomes homocysteinylated (Hcy-cyto-c).
Cytochrome-c (cyto-c) transports electrons and facilitates bioenergetics in the system. However, its role in autophagy during
ischemia/reperfusion injury is unclear.
Tetrahydrocurcumin (
THC) is a major herbal
antioxidant and
anti-inflammatory agent. Therefore, the objective of this study was to determine whether
THC ameliorates autophagy during
ischemia/reperfusion injury by reducing homocysteinylation of cyto-c in
hyperhomocysteinemia pathological condition. To test this hypothesis, we employed 8-10-week-old male
cystathionine-beta-synthase heterozygote knockout (CBS⁺/⁻) mice (genetically hyperhomocystemic mice). Experimental group was: CBS⁺/⁻, CBS⁺/⁻ + THC (25 mg/kg in 0.1%
DMSO dose); CBS ⁺/⁻/I/R, and CBS⁺/⁻/I/R + THC (25 mg/kg in 0.1%
DMSO dose).
Ischemia was performed for 30 min and reperfusion for 72 h.
THC was injected intra-peritoneally (I.P.) once daily for a period of 3 days after 30 min of
ischemia. The
infarct area was measured using
2,3,5-triphenyltetrazolium chloride staining. Permeability was determined by
brain edema and
Evans Blue extravasation. The brain tissues were analyzed for oxidative stress,
matrix metalloproteinase-9 (MMP-9), damage-regulated autophagy modulator (DRAM), and
microtubule-associated protein 1 light chain 3 (LC3) by Western blot. The
mRNA levels of S-adenosyl-L-
homocysteine hydrolases (SAHH) and
methylenetetrahydrofolate reductase (MTHFR) genes were measured by quantitative real-time polymerase chain reaction. Co-immunoprecipitation was used to determine the homocysteinylation of cyto-c. We found that
brain edema and
Evans Blue leakage were reduced in I/R + THC-treated groups as compared to
sham-operated groups along with reduced
brain infarct size.
THC also decreased oxidative damage and ameliorated the homocysteinylation of cyto-c in-part by MMP-9 activation which leads to autophagy in I/R groups as compared to
sham-operated groups. This study suggests a potential therapeutic role of dietary
THC in
cerebral ischemia.