Through Inhibition of HMGB1/TLR4/NF-κB Signaling Pathway, Piceatannol Attenuates the Damage Caused to H9c2 Cardiomyocytes by Doxorubicin

OBJECTIVE  To examine the potential protective effect and underlying mechanism of piceatannol against doxorubicin(DOX)-induced injury in H9c2 cardiomyocytes.

METHODS  H9c2 cardiomyocytes were cultured in groups and subsequently treated with doxorubicin at a final concentration of 1 μmol·L⁻¹ and piceatannol at various levels. The cells were incubated for 24 h, and the MTT method was employed to assess the cell survival rate and determine the most effective co-administration concentration. Hoechst 33342 staining and flow cytometry were utilized to detect apoptosis in H9c2 cardiomyocytes. ROS levels were assessed using DCFH-DA staining and fluorescence microscopy. Western blotting was employed to determine the expression levels of apoptotic proteins Bcl-2, Bax, and Cleaved caspase-3. The relationship between piceatannol and the HMGB1/TLR4/NF-κB signaling pathway was further confirmed by using the TLR4 agonist lipopolysaccharide.

RESULTS  DOX treatment resulted in a significant decrease in H9c2 cardiomyocytes activity, which was inhibited by piceatannol. Annexin V-FITC/PI staining and Hoechst 33342 staining demonstrated that piceatannol reduced the apoptotic rate of DOX-induced H9c2 cardiomyocytes. ROS staining results showed that piceatannol significantly decreased cellular ROS levels. Western blotting analysis revealed that piceatannol increased the expression level of the anti-apoptotic protein Bcl-2 and decreased the expression levels of the pro-apoptotic proteins(Bax, Cleaved caspase-3). Furthermore, it was found that piceatannol inhibited the increase of TLR4 protein level in H9c2 cardiomyocytes induced by DOX, and this effect was reversed by the TLR4 agonist lipopolysaccharide.

CONCLUSION  Piceatannol may ameliorate doxorubicin-induced H9c2 cardiomyocytes injury by inhibiting the HMGB1/TLR4/NF-κB pathway.