Protective effect of bioactive compounds from Lonicera japonica Thunb. against H2O2-induced cytotoxicity using neonatal rat cardiomyocytes

Abstract

Objectives: Pharmacological studies showed that the extracts of Jin Yin Hua and its active constituents have lipid lowering, antipyretic, hepatoprotective, cytoprotective, antimicrobial, antibiotic, antioxidative, antiviral, and anti-inflammatory effects. The purpose of the present study was to investigate the protective effects of caffeoylquinic acids (CQAs) from Jin Yin Hua against hydrogen peroxide (H2O2)-induced and hypoxia-induced cytotoxicity using neonatal rat cardiomyocytes.

Materials and methods: Seven CQAs (C1 to C7) isolated and identified from Jin Yin Hua were used to examine the effects of H2O2-induced and hypoxia-induced cytotoxicity. We studied C4 and C6 as preventative bioactive compounds of the reactive oxygen species (ROS) production, apoptotic pathway, and apoptosis-related gene expression.

Results: C4 and C6 were screened as bioactive compounds to exert a cytoprotective effect against oxidative injury. Pretreatment with C4 and C6, dose-dependently attenuated hypoxia-induced ROS production and reduced the ratio of GSSG/GStotal. Western blot data revealed that the inhibitory effect of C4 on H2O2-induced up and down-regulation of Bcl-2, Bax, caspase-3, and cleaved caspase-3. Apoptosis was evaluated by detection of DNA fragmentation using TUNEL assay, and quantified with Annexin V/PI staining.

Conclusion: In vitro experiments revealed that both C4 and C6 protect cardiomyocytes from necrosis and apoptosis during H2O2-induced injury, via inhibiting the generation of ROS and activation of caspase-3 apoptotic pathway. These results demonstrated that CQAs might be a class of compounds which possess potent myocardial protective activity against the ischemic heart diseases related to oxidative stress.

Keywords: Anti-apoptosis; Caffeoylquinic acids; Cardiomyocytes; Lonicera japonica Thunb; Oxidative stress.

Figure 1

Protective effect against H₂O₂-induced and hypoxia-induced cytotoxicity in caffeoylquinic acids-treated primary cultured cardiomyocytes. A, structural identification of CQAs (1-7). B, the cells viability was measured following H₂O₂-induced myocardial injury. CQAs increased the cell viability, n=3. C, the cells viability was measured following hypoxia-induced myocardial injury. CQAs increased the cell viability, (n=3;, *P<0.05). Data are means±SEM of tree independent experiments

Figure 2

Anti-apoptotic effects of C4 and C6 in cardiomyocytes following H₂O₂ stimulation. A, TUNEL-positive cardiomyocytes were significantly more than that treated with C4 and C6. B, quantitative analysis of apoptotic cardiomyocytes by flowcytometry. Treatment of cardiomyocytes with C4 and C6 resulted in anti-apoptotic effect, (n=3; **, P<0.01). Data are mean±SEM

Figure 3

C4 and C6 enhanced the cellular adaptation to hypoxia. A, the fluorescent probe DCFH-DA indicated the intracellular ROS level; the green fluorescence intensity was proportional to the production of ROS. Treatment of cardiomyocytes with C4 and C6 decreased the production of hypoxia-induced intracellular ROS. B, bar chart shows the GS total and GSSG levels; C4 and C6 inhibited the production of hypoxia-induced intracellular ROS by increasing the GS total and decreasing the ratio of GSSG/GStotal (n=3; *, P<0.05). Data are mean±SEM

Figure 4

Inactivation of caspase-3 cell apoptosis pathway is involved in C4 treated cardiomyocytes following H2O2 stimulation. A, Western blot analysis of the expression and phosphorylation levels of caspase-3 cell apoptosis pathway. B, bar chart shows the effect of C4 on expression of apoptotic proteins (n=3; *, P<0.05). Data are mean±SEM

Figure 5

Quantitative real-time PCR of apoptotic genes, caspase-3 and caspase-9, in C4 treated cardiomyocytes following H₂O₂ stimulation (n=3; *, P <0.05). Data are mean ± SEM