Study Of Mitochondrial Energy Metabolism Impairment In Hepatocytes Induced By SiO₂-NPs And HA-NPs

Objective:As the biosafety of nanotechnology becomes a hot field of nanotechnology research, more and more attention has been paid to the toxicity of nanoparticles(NPs). It has been proved that as the vital organ of a body, the liver is one of the primary target of NPs, thus the toxic effects of NPs on liver have become a research focus. The liver is the main organ for the body metabolism, and mitochondria which are most populous in hepatocytes, provide critical location in converting saccharides, lipids and amino acids to ATP, supply life activities about 95% of the energy, and play important roles in energy metabolism. However, until now, research focusing on the potential toxic effects of NPs on mitochondria-associated energy metabolism in hepatocytes has been lacking. In this project, two kind of inorganic NPs—silica nanoparticles(Si O2-NPs) and hydroxyapatite nanoparticles(HA-NPs) were chosen as the research materials, and corresponding micron-sized particles were utilized as control materials. Attempts were made to elucidate the impact of Si O2-NPs and HA-NPs on mitochondrial energy metabolism and related mechanism by exploring their toxic effects on hepatocyte, hepatic mitochondria and function of mitochondria, providing a scientific basis for applications of NPs in the future.Materials and Methods:(1) Si O2-NPs/ μm-Si O2 or HA-NPs/ μm-HA were incubated directly with normal rat liver cells(BRL) cells in vitro, then, BRL cytotoxicity, mitochondrial dehydrogenase activity, NP distribution in mitochondria after entering the BRL cells and mitochondrial membrane potential(MMP) were measured and further, changes of enzymatic expression in the Krebs cycle and oxidative phosphorylation pathway were detected;(2) Si O2-NPs/ μm-Si O2 or HA-NPs/ μm-HA were incubated directly with Kupffer cells(KC), then the culture medium was transferred to stimulate BRL cells in vitro. Changes of oxidative phosphorylation in the mitochondrial energy metabolism in affected by NPs via a KC-mediated pathway was observed and the mechanisms behind were investigated, through observing KC activation and a series of proinflammatory fectors [tumor necrosis factor-α(TNF-α), nitric oxide(NO), reactive oxygen species,(ROS)] released from KCs.Results:(1) Direct impact of Si O2-NPs and HA-NPs on BRL cells. 1) With the treatment dose increased, BRL cell viability decreased, injury enhanced gradually, activity of mitochondrial dehydrogenase decreased and MMP decreased in both NPs. Meanwhile, Si O2-NPs could enter the mitochondria, resulting in damage to the structure of the mitochondria. 2) Both Si O2-NPs and HA-NPs induced the decline of enzymatic expression in the Krebs cycle and activity of the mitochondrial respiratory chain complexes at different levels(Si O2-NPs and HA-NPs induced dysfunction of complex I、III、IV and complex I、II、III, respectively.) Besides, HA-NPs could reduce α-KGDH expression in the Krebs cycle.(2) Indirect effects of Si O2-NPs and HA-NPs on BRL cells. 1) Both Si O2-NPs and HA-NPs could induce dysfunction of mitochondrial respiratory chain complexes at different levels via a KC-mediated pathway(Si O2-NPs and HA-NPs induced dysfunction of complex I and complex I、II、III, respectively.). 2) Both Si O2-NPs and HA-NPs induced KC activation and the subsequent release of proinflammatory factors such as TNF-α, NO and ROS from the KCs, at different levels, while these proinflammatory factors might be the mechanism of NP indirectly-induced mitochondrial dysfunctionin in energy metabolism.Conclusions:(1) Both Si O2-NPs and HA-NPs can directly induce hepatic cytotoxicity, mitochondrial activity decrease and mitochondrial integrity interruption, at different levels;(2) Both Si O2-NPs and HA-NPs can directly cause series of mitochondrial dysfunction including the Krebs cycle and the oxidative phosphorylation pathway in energy metabolism, at different levels;(3) Both Si O2-NPs and HA-NPs can activate KCs, resulting in the release of proinflammatory factors, and subsequently indirectly cause hepatic mitochondrial oxidative phosphorylation dysfunction in energy metabolism.(4) Compared with micron-sized particles, Si O2-NPs and HA-NPs can cause mitochondrial energy metabolism disorder in hepatocyte either directly or through a KC-mediated pathway, more obviously.