The Mechanism Study Of Protection Effect Of Melatonin In Cadmium Induced Hepatotoxicity

BackgroundMelatonin is an indolamine synthesized in the pineal gland that has a wide range of physiological functions, including sleep timing, blood pressure regulation, seasonal reproduction and many others, and it has been under clinical investigation for expanded applications. Cadmium is an extremely toxic metal commonly found in industrial workplaces and environment. It can cause occupational-exposure in workplaces and nonoccupational-exposure in population through transmiting in food chain. After been absorbed by the body, cadmium will combine with the albumin in blood, after that they were transported to the liver, which is the biggest detoxification organ. It was cleared that the liver was supposed to be one of most sensitive targets of Cd. Increasing evidence demonstrates that melatonin can ameliorate cadmium-induced hepatotoxicity. However, the potentially protective effects of melatonin against cadmium-induced hepatotoxicity and the underlying mechanisms of this protection remain unclear. The critical targets of Cd binding are the thiol groups of proteins, including the uclei, mitochondria and endoplasmic reticulum. Thus mitochondria dysfunction may play a crucial role in the hepatotoxicity of Cd. Mitochondrial biogenesis is responsible for mitochondria quality control, and PGC-1α has been extensively described as a master regulator of mitochondrial biogenesis. One of its upstream regulator, SIRT1, can regulate the activity of PGC-1α through its acetylation status. Therefore, the purpose of this study is to investigate the protective effects of melatonin pretreatment on cadmium-induced hepatotoxicity and elucidates the potential mechanism of melatonin-mediated protection.Methods① The Hep G2 cells were exposure to different concentrations of cadmium chloride(2.5, 5, and 10μM) for 12 h. Parallel, the cell viability, ROS generation level, mitochondrial membrane potential, mitochondria DNA copy number and mitochondrial mass were evaluated immediately after the exposure ended for identification the cadmium-induced hepatotoxicity in cytotoxicity, mitochondrial dysfunction and impairment of mitochondrial biogenesis.② On the basis of exposure model established before, the pretreatment of mitochondrial protective agent, melatonin, before cadmium exposure was used to elucidate whether melotonin could protest against the cadmium-induced cytotoxicity, mitochondrial dysfunction and impairment of mitochondrial biogenesis. In addition, whether melatonin interferes the SIRT1/PGC-1αsignaling during the protection effect was also been assayed.③ On the basis of exposure model established before, the pretreatment of SIRT1 inhibitor agent, Sirtinol and SIRT1 siRNA, before cadmium exposure was used to evaluate whether melatonin is involved in Cd-induced mitochondrial dysfunction and the disruption of SIRT1-regulated mitochondrial biogenesis. Finally, the expression of MT1 and MT2, were investigated in HepG2 cells, and the pretreatment of melatonin receptor antagonist, Luzindole, was added to investigate whether it could attenuate the protection effect of melatonin on cadmium-induced hepatotoxicity.Results① 2.5μM CdCl2 exposure could not cause cytotoxicity in HepG2 cells. But higher concentration Cd Cl2 exposure significantly stimulated cytotoxicity, disrupted the mitochondrial membrane potential, increased reactive oxygen species production, and decreased mitochondrial mass and mitochondrial DNA content. All of the effects were in a dose-dependent manner.② The pretreatment of 0.5μM melatonin before exposure of 5μM CdCl2 for 12 h could markedly decreased ROS production, preserved the mitochondrial membrane potential, inhibit Cd-stimulated cytotoxicity, and maintained mtDNA content and mitochondrial mass. Also the treatment of cadmium could inhibit the expression and activity of SIRT1, promoting the expression of acetylated-PGC-1 alpha. In addition, melatonin stimulated Cd-suppressed SIRT1 protein expression and activity, and thus inhibited acetylated-PGC-1 alpha expression.③ With the pretreatment of Sirtinol and SIRT1 siRNA before cadmium exposure, we observed a decrease of melatonin enhanced SIRT1 protein activity or expression. Also, they inhibit the protective effects of melatonin in ROS generation, mitochondrial membrane potential, Cd-stimulated cytotoxicity, mtDNA content and mitochondrial mass. Importantly, melatonin-induced decreases in acetylated-PGC-1αexpression were significantly attenuated by Sirtinol and SIRT1 si RNA. After Luzindole, a melatonin receptor antagonist, was added alongside melatonin, the melatonin-induced enhancement of SIRT1 activity but not expression level was partially reversed by luzindole. And as expected, the melatonininduced decrease in the expression of acetylated-PGC-1α was also blocked by luzindole.ConclusionOverall, we proposed an intriguing mechanism whereby cadmium induces hepatotoxicity by disrupting mitochondrial biogenesis and function. Most importantly, the protective effect of melatonin is associated with enhanced mitochondrial biogenesis through the SIRT1-dependent PGC-1α pathway, and this effect appears to be mediated partially through the binding of melatonin to the melatonin receptor MT1 to activate signaling to mitochondria. Taken together, our data illustrate a new molecular mechanism underlying the ability of melatonin to be explored for future clinical treatment of cadmium-induced hepatotoxicity.