Uric acid preconditioning alleviated doxorubicin induced JNK activation and Cx43 phosphorylation associated cardiotoxicity via activation of AMPK-SHP2 signaling pathway

Figure 1. Uric acid preconditioning significantly increased the survival rate and body weight of doxorubicin-treated mice. (A) Kaplan Meier survival analysis showed that uric acid preconditioning increased the survival rate of mice compared to doxorubicin treatment. The Log rank (Mantel-Cox) test was used to determine significance (n=10 of each group). (B) Body weight was increased in the uric acid-treatment group. Data are presented as the mean ± SEM. Significant difference was detected by one-way or two-way analysis of variance (***, P<0.001 vs. control group; #, P<0.05, ##, P<0.01, and ###, P<0.001 vs. doxorubicin-treatment alone group, Bonferroni post hoc tests). Allo, allopurinol; Dox, doxorubicin; UA, uric acid.

Figure 2. Uric acid preconditioning alleviated doxorubicin-induced cardiac abnormal conduction in mice. (A) Representative images of ECG indicated that doxorubicin induced conduction abnormalities, including slowed heart rate (B) and prolongation of QT intervals (C) and QT(c) (D), whereas preconditioning with uric acid (125 mg/kg) significantly alleviated doxorubicin-induced abnormal conduction in mice. ELISA kits were used to detect the levels of cardiomyocyte injury markers, including AST (E), LDH (F), CK (G), CK-MB (H), and UA (I). Data are presented as the mean ± SEM. n=6 in each group. Significant difference was observed by one-way or two-way analysis of variance (*, P<0.05, **, P<0.01, and ***, P<0.001 vs. control group; #, P<0.05, and ###, P<0.001 vs. doxorubicin-treatment alone group, Bonferroni post hoc tests). Dox, doxorubicin; UA, uric acid.

Figure 3. Uric acid preconditioning significantly induced activation of AMPK and SHP2 both in vivo and in vitro. (A) Uric acid treatment for 2 h significantly increased the levels of p-AMPK/AMPK and p-SHP2/SHP2 in H9C2 cells in vitro. (B) Uric acid treatment (5 mg/dL) time-dependently increased he levels of p-AMPK/AMPK and p-SHP2/SHP2 in H9C2 cells in vitro. (C) Consecutive administration of uric acid (125 and 250 mg/kg, i.g.) for 2 days significantly up-regulated the level of p-AMPK/AMPK and p-SHP2/SHP2 in heart tissue in vivo. (D) Intraperitoneal injection of a single dose of doxorubicin (20 mg/kg) markedly elevated the expression levels of p-JNK/JNK and p-Cx43 but reduced the total Cx43 level on day 5 in vivo. Data are presented as the mean ± SEM of three individual experiments. Significant difference by one-way or two-way analysis of variance (*, P<0.05, **, P<0.01, and ***, P<0.001 vs. control group; Bonferroni post hoc tests).

Figure 4. Uric acid preconditioning alleviated doxorubicin-induced cardiotoxicity via the AMPK-SHP2-JNK-Cx43 signaling axis. (A) Representative western blot images showing the levels of p-AMPK, p-SHP2, p-JNK/JNK, p-Cx43, and total Cx43 in heart tissue in vivo. (B) H9C2 cells were pretreated with uric acid (1.25, 2.5, 5 mg/dL) for 2 h, and then treated with doxorubicin (1 µM) for 12 h. Western blot images showing the levels p-AMPK, p-SHP2, p-JNK, and p-Cx43 in H9C2 cells in vitro. (C) H9C2 cells were treated with compound C (10 µM) and Shp099 (10 µM) 30 min before uric acid (5 mg/dL) administration, and then after 12 h of doxorubicin (1 µM) treatment. Western blot samples (n=3 of each group) were collected, and the images show the levels p-AMPK, p-SHP2, p-JNK, and p-Cx43 in H9C2 cells in vitro. (D) Real-time cell analyzer results showed that preconditioning with uric acid (5 mg/dL) or AICAR (10 µM) for 2 h significantly alleviated doxorubicin-induced cardiotoxicity in vitro, whereas these effects were abolished by compound C (10 µM). Data are presented as the mean ± SEM of three individual experiments. Significant difference by one-way or two-way analysis of variance (*, P<0.05, **, P<0.01…

Figure 5. Schematic indicated that uric acid preconditioning could inhibit doxorubicin-induced the changes of JNK-Cx43 gap junctions and conduction abnormalities via AMPK-SHP2 signaling pathway. Doxorubicin could significantly induce the activation of JNK-Cx43 signaling pathway, and lead to gap junctions changes and conduction abnormalities, and then mediate the production of cardiac toxicity. However, uric acid preconditioning could significantly activate AMPK and induce the upregulation of SHP2, which could in turn suppress the change of Cx43 gap junctions and conduction abnormalities, and alleviate doxorubicin-mediated cardiotoxicity.