Objective: To study the effects of mitochondrial permeability transition (MPT) on hepatocyte apoptosis and mitochondrial damage, and investigate the effects of curcumin on MPT and the related mechanisms in septic rat.
Methods: Fifteen healthy male Sprague-Dawley (SD) rats were randomly divided into three groups: sham group, sepsis group and curcumin group, with 5 rats in each group. Septic rat model was reproduced by cecal ligation and puncture (CLP). The rats in sham group were flipped the cecum without perforation and ligation. The rats in the curcumin group were treated with curcumin 100 mg × kg⁻¹ × d⁻¹ (dissolved in saline to 10 mL/kg) by oral gavage for 7 days, while the other groups were treated with normal saline. Tissue samples were harvested in each group at 12 hours after operation. Pathological changes in hepatic mitochondria were observed under electron microscopy, concentration of free calcium was examined with confocal laser scanning microscope. After Fluo-3/AM staining, protein and mRNA expression of active caspase-3, Bcl-2 and Bax were examined by Western Blot and reverse transcription-polymerase chain reaction (RT-PCR).
Results: Under the transmission electron microscope, intact cell membrane, adqulis cytoplasm, and normal and clear mitochondrion was found in the sham group. Mitochondria in sepsis group swelled obviously with mitochondrial cristae broken or disappearance, unclear bilateral membrane structure, while the curcumin group showed much less pathological changes, with few mitochondria swell, and smear bilateral membrane structure. The fluorescence intensity index of sham group, sepsis group and curcumin group was raised successively (417.33 ± 15.88, 772.95 ± 42.37, 1 560.84 ± 160.78, respectively, F=184.149, P=0.000). The protein and mRNA expression of active caspase-3 and Bax had the highest level in sepsis group, followed by the curcumin group, and that in the sham group was the lowest [active caspase-3 protein (gray scale): 1.698 ± 0.061, 0.694 ± 0.045, 0.246 ± 0.027, F=1 289.667, P=0.000; active caspase-3 mRNA (2(-ΔΔCt)): 1.031 ± 0.135, 0.578 ± 0.144, 0.183 ± 0.036, F=66.958, P=0.000; Bax protein (gray scale): 1.826 ± 0.126, 1.254 ± 0.140, 0.623 ± 0.901, F=94.536, P=0.000; Bax mRNA (2(-ΔΔCt)): 2.774 ± 0.338, 1.661 ± 0.226, 0.656 ± 0.114, F=124.710, P=0.000], all of these values had statistical significance among the three groups (all P<0.01). While Bcl-2 protein and mRNA had the highest level in curcumin group and lowest level in the sham group [Bcl-2 protein (gray scale): 0.716 ± 0.091, 1.328 ± 0.147, 1.656 ± 0.104, F=84.918, P=0.000; Bcl-2 mRNA (2(-ΔΔCt)): 0.617 ± 0.118, 1.393 ± 0.096, 1.650 ± 0.167, F=83.846, P=0.000]. The protein and mRNA expressions of Bcl-2/Bax ratio were lowest in sepsis group, then sham group, and highest in curcumin group [Bcl-2/Bax protein (gray scale): 0.726 ± 0.055, 1.150 ± 0.043, 1.333 ± 0.163, F=46.265, P=0.000; Bcl-2/Bax mRNA (2(-ΔΔCt)): 0.505 ± 0.041, 0.944 ± 0.097, 1.006 ± 0.168, F=12.211, P=0.001].
Conclusions: MPT can lead to mitochondrial dysfunction and further cause hepatocyte apoptosis. Mechanism of effect of curcumin on MPT may be related to reduction of intracellular calcium concentration, promotion of anti-apoptotic Bcl-2 gene expression, inhibition of caspase-3 activation and Bax gene.