Chlorogenic acid ameliorates intestinal mitochondrial injury by increasing antioxidant effects and activity of respiratory complexes

Figures & data

Fig. 1. Effects of different concentrations of H₂O₂ on mitochondrial function. Intestinal mitochondria were treated with 0.125, 0.25, 0.5, or 1 M H₂O₂ for 50 min. Mitochondrial swelling (A) was measured by monitoring the absorbance at 540 nm. Mitochondrial membrane potential changes (B) were indicated by Rhodamine 123 fluorescence after staining mitochondria with Rhodamine 123. Mitochondrial ROS production (C) was measured following incubation of mitochondria with DHA with the use of Rhodamine 123 fluorescence.

Notes: Data are presented as the mean ± SE, n = 6. Values with different lowercase superscript letters are significantly different (p < 0.05), as analyzed by one-way ANOVA and Tukey’s test.

Fig. 2. CGA alleviated H₂O₂-induced injury to isolated intestinal mitochondria. Intestinal mitochondria were treated with 0.5 mM H₂O₂ or a combination of H₂O₂ and CGA (CGA + H₂O₂). CGA was added 5 min prior to treatment. Mitochondrial swelling (A), mitochondrial membrane-potential change (B), mitochondrial ROS production (C), content of 8-OHdG in mitochondrial fractions (D), content of cytochrome c in mitochondrial fractions (E), and content of cytochrome c in mitochondrial supernatant (F) are shown.

Notes: Data are presented as the mean ± SE, n = 6. Values with different lowercase superscript letters are significantly different (p < 0.05), values with different uppercase superscript letters are significantly different (p < 0.01), as analyzed by one-way ANOVA and the Tukey’s test.

Table 1. Effects of CGA on activities of T-AOC, SOD, and GSH-Px in mitochondrial fractions.

	SOD (U/mg prot)	GSH-Px (U/mg prot)	T-AOC (U/mg prot)
Control	227.95 ± 4.85^A	139.92 ± 6.00^A	8.65 ± 0.27^A
CGA	210.16 ± 4.84^A	140.48 ± 5.56^A	8.72 ± 0.47^A
H2O2	126.16 ± 1.11^C	71.92 ± 3.32^B	5.67 ± 0.24^B
CGA+H2O2	149.75 ± 3.45^B	136.25 ± 2.34^A	8.27 ± 0.28^A

Notes: Intestinal mitochondria were treated with 160 μM CGA (CGA), 0.5 mM H₂O₂ (H₂O₂), or a combination of 160 μM CGA and 0.5 mM H₂O₂ (CGA + H₂O₂). Data are presented as the mean ± SE, n = 6. Values with different uppercase superscript letters are significantly different (p < 0.01), as analyzed by one-way ANOVA and the Tukey’s test.

Table 2. Effects of CGA on intestinal mitochondrial respiratory chain complexes.

	Complexes I（U/mg protein）	Complexes IV（U/mg protein）	Complexes V（U/mg protein）
Control	162.18 ± 4.96^AB	64.28 ± 1.98^A	66.77 ± 0.97^a
CGA	175.47 ± 11.04^A	68.85 ± 2.75^A	68.97 ± 4.12^a
H2O2	117.51 ± 3.07^C	35.03 ± 1.48^C	47.64 ± 1.48^c
CGA+H2O2	146.95 ± 5.60^B	48.40 ± 3.40^B	52.91 ± 2.14^b

Notes: Intestinal mitochondria were treated with 160 µM CGA (CGA), or 0.5 mM H₂O₂ (H₂O₂), or combination of 160 µM CGA and 0.5 mM H₂O₂ (CGA+H₂O₂). Data are presented as the mean ± SE, n = 6. Values with different lowercase superscript letters (a, b, c) are significantly different (P < 0.05), values with different uppercase superscript letters (A, B, C) are significantly different (P < 0.01), as analyzed by one-way ANOVA and the Tukey’s test.

Fig. 3. CGA ameliorates colonic injury in TNBS-induced colitis rat. Histological findings of proximal colon samples stained with hematoxylin and eosin (A). Arrows indicate inflammatory cells. Histological injury scoring (B) indicated a grading scale for histological assessment of inflammation in colitis. The length of colon (C), weight of colon (D), activity of MPO (E), and ALP (F).

Notes: Data are presented as the mean ± SE, n = 8. Values with different uppercase letters are significantly different (p < 0.01), as analyzed by one-way ANOVA and the Tukey’s test.

Fig. 4. CGA alleviated TNBS-induced mitochondrial injury and ultrastructural changes. Ultrastructure of mitochondria (A), arrows in control and CGA groups show the normal mitochondrial ultrastructure. Arrows in the TNBS group show mitochondrial ultrastructural changes, such as swelling and lost cristae. Arrows in the CGA + TNBS group indicate amelioration of mitochondrial ultrastructural changes. Mitochondrial damage score (B), the content of 8-OHdG (C) and cytochrome c (D) in mitochondria, and cytochrome c in cytoplasm (E) are shown.

Notes: Data are presented as the mean ± SE, n = 8. Values with different lowercase superscript letters are significantly different (p < 0.05), as analyzed by one-way ANOVA and the Tukey’s test.