Reducing DEN-evoked apoptosis and autophagy by AC and ACW in the livers

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We compared several treatments on DEN-induced apoptosis and autophagy in the livers. Figure 11 indicated the effect of silymarin, AC, ACW co-treatment or post-treatment PACW on DEN-induced Beclin-1-mediated autophagy (Figure 11A) and apoptosis-TUNEL responses (Figure 11B) by immunohistochemistry. These data showed that DEN toxicity significantly increased Beclin-1-positive stain and TUNEL-positive stain in 4WDEN and 8WDEN livers. Co-treatment of silymarin, AC, ACW or PACW significantly depressed DEN-enhanced Beclin-1 and TUNEL positive stain after 4 or 8 weeks of DEN injury when compared to their respective 4WDEN or 8WDEN livers. 8WDEN+AC, 8WDEN+ACW and 8WDEN+PACW significantly reduced DEN-evoked apoptosis and autophagy when compared to 8WDEN group.

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Figure 1. The grouping and experimental design.of Antrodia cinnamomea

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Figure 2. HPLC analysis of several components of extracts from the fruiting bodies of A. cinnamomea. Within 110 min, seven components are identified by the HPLC

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fingerprint analysis and the respective structure of antcin K (AC, ST14), Antcin C (ST4), Antcin H (ST12), Dehydrosulphurenic acid (TR15), Antcin B (ST3), Antcin A and Dehydroeburicoic acid (TR2) is indicated. Within 80 min, five major components are identified. Note that the highest content is Antcin K.

Figure 3. Different concentration of water extract (ACW) (A) and Antctin K (AC) (B) of A. cinnamomea on scavenging O2-., H2O2 and NO activity in vitro. These data show that the concentration from ACW of A. cinnamomea at a dose larger than 0.25 mg/mL significantly reduced the oxidative stress in O2-., H2O2 and NO. In addition, AC at a dose larger than 1 µg/mL displayed a similar response in significant inhibition of O2-., H2O2 and NO activity like ACW. All the experiments were repeated six times. * P <

0.05 when compared to control value.

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Figure 4. Effect of concomitant administration of Antcin K (AC) and water extract (ACW) of Antrodia cinnamomea on inflammation by H&E stain (A-D), oxidative stress by 3-NT (E-H) and 4-HNE (I-L) stain and ED-1 infiltration (M-P) after 2 weeks of DEN-induced liver injury (2WDEN). There are significantly increases in inflammation (Q), 3-NT (R), 4-HNE (S) and ED-1 stain (T) in 2WDEN livers as compared to Con rats. Co-treatment of AC and ACW significantly reduced these parameters in 2WDEN+AC and 2WDEN+ACW rats. The scale bar is 50 µm. * P <

0.05 when compared to Con value. # P <0.05 when compared to 2WDEN group.

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Figure 5. Effect of concomitant administration of Antcin K (AC) and water extract (ACW) of Antrodia cinnamomea on inflammation by H&E stain (A-D), oxidative stress by 3-NT (E-H) and 4 HNE (I-L) stain and ED-1 infiltration (M-P) after 4 weeks of DEN-induced liver injury (4WDEN). There are significantly increases in inflammation (Q), 3-NT (R), 4-HNE (S) and ED-1 stain (T) in 4WDEN livers.

Co-treatment of AC and ACW significantly reduced these parameters in 4WDEN+AC and 4WDEN+ACW rats. The scale bar is 50 µm. * P < 0.05 when compared to Con value. # P <0.05 when compared to 4WDEN group.

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Figure 6. Effect of concomitant administration of Antcin K (AC) and water extract (ACW) of Antrodia cinnamomea on inflammation and tumor formation by H&E stain (A-D), oxidative stress by 3-NT (E-H) and 4 HNE (I-L) stain and ED-1 infiltration (M-P) after 8 weeks of DEN-induced liver injury (8WDEN). There are significantly increases in ratio of liver weight/body weight (Q), 3-NT (R), 4-HNE (S) and ED-1 stain (T) in 8WDEN livers. Co-treatment of AC and ACW significantly reduced these parameters in 8WDEN+AC and 8WDEN+ACW rats. The scale bar is 50 µm. * P <

0.05 when compared to Con value. # P <0.05 when compared to 8WDEN group.

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Figure 7. Effect of post-treatment of water extract (PACW) of Antrodia cinnamomea on inflammation by H&E stain (A-D), oxidative stress by 3-NT (E-H) and 4 HNE (I-L) stain and ED-1 infiltration (M-P) in the 2WDEN+PACW, 4WDEN+PACW and 8WDEN+PACW livers. The degree of inflammation (Q), 3-NT (R), 4-HNE (S), ED-1 stain (T) and plasma level of γ-GT (U) is indicated respectively in these DEN-treated livers. The scale bar is 50 µm. * P < 0.05 when compared to Con value. # P <0.05 when compared to 2WDEN+PACW group.

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Figure 8. Effect of co-treatment of Antcin K (AC) and water extract (ACW) of A.

cinnamomea on fibrosis by Masson stain (A, B) and collagen content (C) in the DEN-treated livers. There are significantly blue Masson stains and hepatic collagen contents in DEN-treated livers in 2WDEN, 4WDEN and 8WDEN groups.

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Co-treatment of AC or ACW significantly reduced these parameters in 2WDEN+ACW, 2WDEN+AC, 4WDEN+ACW, 4WDEN+AC, 8WDEN+ACW and 8WDEN+AC livers. The scale bar is 50 µm. * P < 0.05 when compared to Control value. a P < 0.05 when compared to 2WDEN. b P < 0.05 when compared to 4WDEN.

c P < 0.05 when compared to 8WDEN. The level of collagen was determined with the Sircol collagen kit according to the manufacture’s instruction.

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Figure 9. Effect of concomitant administration of water extract of A. cinnamomea (ACW) and AC on DEN-induced inflammation by EMSA assay. Gel shift of nuclear transcription factor NF-κB by EMSA found NF-κB transcription factor increased by DEN stimulation for 2-8 weeks (A). ACW or AC treatment markedly reduced DEN-induced NF-κB transcription factor shift. NC, negative control. The statistic data from triple experiments are demonstrated in (B). * P < 0.05 compared to Con group. # P < 0.05 compared to respectively time-matched DEN group.

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Figure 10. Effect of concomitant administration of Antcin K (AC) or water extract of A. cinnamomea (ACW), and post-treatment ACW (PACW) on phosphorelated p85, phosphorylated MAPK and CYP2E1 expression by western blot (A). DEN injury significantly enhanced p85 (B), MAPK (C) and CYP2E1 protein expression (D) in the

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2WDEN, 4WDEN and 8WDEN livers compared to Con group. Co-treatment of ACW in 2DEN+ACW, 4DEN+ACW and 8WDEN+ACW groups significantly depressed DEN-enhanced hepatic phosphorylated p85, phosphorylated MAPK and CYP2E1 expression compared to respective DEN group. Co-treatment of AC in 2DEN+AC, 4DEN+AC and 8WDEN+AC groups also significantly decreased DEN-enhanced hepatic p85, MAPK and CYP2E1 expression compared to respective DEN group.

After 2 weeks of DEN induction, PACW treatment significantly depressed DEN-enhanced hepatic p85, MAPK and CYP2E1 expression compared to respective 4WDEN+PACW and 8WDEN+PACW groups. * P < 0.05 compared to Con group. # P < 0.05 compared to respectively time-matched DEN group. a P < 0.05 compared to respective DEN+ACW group. b P < 0.05 compared to respective DEN+AC group.

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Figure 11. Effect of silymarin, antcin K (AC), co-treatment of water extract of A.

cinnamomea pretreatment (ACW) or post-treatment of water extract of A.

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cinnamomea (PACW) 2 weeks after DEN injury on Beclin-1-mediated autophagy (A) and apoptosis-TUNEL (B) responses by immunohistochemistry. DEN injury significantly enhanced Beclin-1-positive stain and TUNEL-positive stain at 4WDEN and 8WDEN livers. Co-treatment of silymarin, AC, ACW or PACW significantly depressed DEN-enhanced Beclin-1 and TUNEL positive stains compared to respective 4WDEN and 8WDEN livers. 8WDEN+AC and 8WDEN+ACW displayed a stronger effect in decreasing Beclin-1-autophagy and TUNEL-apoptosis than the positive group of 8WDEN+silymarin. * P < 0.05 compared to 2WDEN group. # P <

0.05 compared to 4WDEN group. a P < 0.05 compared to respectively time-matched DEN group. b P < 0.05 compared to time-matched silymarin group. c P < 0.05 compared to time-matched AC group. d P < 0.05 compared to time-matched ACW group.

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