Date Published: January 29, 2019
Publisher: Public Library of Science
Author(s): Youn Ju Lee, Mi-Sun Shu, Jong-Yeon Kim, Yun-Hye Kim, Kyeong Hwa Sim, Woo Jung Sung, Jong Ryeol Eun, Vladimir Trajkovic.
Alcoholic liver disease (ALD) is a worldwide health problem and hepatocyte apoptosis has been associated with the development/progression of ALD. However, no definite effective pharmacotherapy for ALD is currently available. Cilostazol, a selective type III phosphodiesterase inhibitor has been shown to protect hepatocytes from ethanol-induced apoptosis. In the present study, the underlying mechanisms for the protective effects of cilostazol were examined. Primary rat hepatocytes were treated with ethanol in the presence or absence of cilostazol. Cell viability and intracellular cAMP were measured. Apoptosis was detected by Hoechst staining, TUNEL assay, and caspase-3 activity assay. The roles of cAMP and AMP-activated protein kinase (AMPK) pathways in the action of CTZ were explored using pharmacological inhibitors and siRNAs. Liver from mice received ethanol (5 g/kg body weight) by oral gavage following cilostazol treatment intraperitoneally was obtained for measurement of apoptosis and activation of AMPK pathway. Cilostazol inhibited ethanol-induced hepatocyte apoptosis and potentiated the increases in cAMP level induced by forskolin. However, the anti-apoptotic effect of cilostazol was not reversed by an inhibitor of adenylyl cyclase. Interestingly, cilostazol activated AMPK and increased the level of LC3-II, a marker of autophagy. The inhibition of AMPK abolished the effects of cilostazol on LC3-II expression and apoptosis. Moreover, the inhibition of LKB1 and CaMKK2, upstream kinases of AMPK, dampened cilostazol-inhibited apoptosis as well as AMPK activation. In conclusion, cilostazol protected hepatocytes from apoptosis induced by ethanol mainly via AMPK pathway which is regulated by both LKB1 and CaMKK2. Our results suggest that cilostazol may have potential as a promising therapeutic drug for treatment of ALD.
Alcohol is an important risk factor for development of liver disease. Alcoholic liver disease (ALD) represents a spectrum of pathological conditions ranging from simple hepatic steatosis to alcoholic hepatitis, fibrosis and eventually to cirrhosis [1, 2]. Among cellular pathogenesis of ALD, hepatocyte apoptosis is a prominent feature of alcoholic hepatitis and hepatic fibrosis [3, 4]. The inhibition of hepatocellular apoptosis in various liver injury models has been shown to reduce liver damage and progression of liver diseases [5, 6]. Therefore, apoptosis has been considered as a target for therapeutic management of ALD.
AMPK has recently emerged as an important regulator of various liver functions. Therefore, AMPK has been suggested to be a crucial therapeutic target to treat liver injury including steatosis, inflammation and apoptosis . Cilostazol has been reported to have protective effects on diverse disease models including liver damage. The beneficial effects of cilostazol are mediated by multiple signaling pathways. However, the involvement of AMPK in the effects of cilostazol on hepatocyte apoptosis induced by ethanol has not been determined. In the present study, we have shown that cilostazol suppressed ethanol-induced hepatocyte apoptosis in in vitro primary hepatocyte and in vivo alcohol drinking mice. The pharmacological and gene knockdown approaches clearly demonstrated that the anti-apoptotic effect of cilostazol was mediated mainly through AMPK-dependent pathway. Of interest, the protective effect of cilostazol was cAMP/PKA independent although cAMP has shown an anti-apoptotic role. The activation of AMPK by cilostazol was down regulated by both LKB1- and CaMKK2- siRNA, which abrogated the anti-apoptotic effect of cilostazol. In addition, pharmacological inhibitor of CaMKK2, STO-609, but not inhibitors of cAMP/PKA pathway blunts AMPK activation, supporting the anti-apoptotic role of cilostazol via LKB1-CaMKK2/AMPK pathway. The protective effect of cilostazol on hepatocyte apoptosis is consistent with a recent report, in which cilostazol prevented hepatocyte apoptosis induced by ethanol by ameliorating ROS generation .