Fasudil Alleviates Lipopolysaccharide-Mediated Injury in Rat Cardiomyocyte H9c2 Cells by Inducing Autophagy.

OBJECTIVE

By establishing an in vitro model of septic cardiomyopathy (SIC), we aimed to investigate whether fasudil exerts its cardioprotective effects by inducing autophagy in cardiomyocytes.

METHODS

Rat cardiomyocyte H9c2 cells were cultured in vitro, and lipopolysaccharide (LPS) was added to induce inflammation, simulating an in vitro model of SIC. The cells were divided into five groups, including control group, LPS group, fasudil group, intervention group (fasudil+LPS) and hydroxychloroquine intervention group (fasudil+LPS+hydroxychloroquine). Cell apoptosis was measured using Hoechst 33342/PI fluorescence staining. Changes in mitochondrial membrane potential were measured using JC-1 fluorescence staining. Autophagy and autophagic flux were evaluated using MDC staining and mCherry-EGFP-LC3B assays. Western blotting was conducted to measure the expression of apoptosis-related proteins (Bcl2, Bax, and Cleaved-Caspase3), autophagy-related proteins (LC3B and p62), and the phosphorylation levels of the Akt protein.

RESULTS

In the LPS group, H9c2 cells showed significant apoptosis and decreased mitochondrial membrane potential, which were markedly alleviated after treatment with fasudil. Western blotting revealed increased expression of the pro-apoptotic protein Bax in the LPS group, and treatment with fasudil inhibited LPS-induced up-regulation of Bax expression (P<0.05). Autophagic flux analysis showed that fasudil increased autophagic activity and reduced LC3B accumulation. Western blotting indicated that fasudil up-regulated the protein levels of the autophagy marker LC3B (P<0.05), inducing autophagy in H9c2 cells. However, the protein levels of both LC3B and the autophagosome adaptor protein p62 were significantly down-regulated in the intervention group (P<0.01), suggesting that fasudil promotes autophagic flux in H9c2 cells. Hydroxychloroquine potently inhibited autolysosome-dependent degradation of LC3B and p62 proteins, resulting in their accumulation. The phosphorylation level of the Akt protein was markedly up-regulated in the LPS group but was significantly down-regulated in the fasudil group. An even greater decrease in Akt phosphorylation was observed in the intervention group (P<0.01).

CONCLUSION

Fasudil induces autophagy in H9c2 cells. Treatment with LPS promotes autophagic flux and inhibits apoptosis, thereby exerting protective effects on the cells.