| BackgroundChromium is a silvery-white metal, which has widely existed in the nature environment. The stable valence state includes trivalent chromium, Cr(Ⅲ) and hexavalent chromium, Cr(Ⅵ). Hexavalent chromium has been widely used in occupational production, such as welding, metallurgy, chroming, leather-tanning technology. It could cause adverse health damage, including nasal perforation and bronchogenic cancer and so on. In1990, the international agency for research on cancer (IARC) has confirmed hexavalent chromium as human carcinogen, according to the research that the accident of lung cancer in human and animals increased as a result of the exposure to Cr(Ⅵ). Recently, with the exposure of water pollution and "chromium containing poison capsule" event, how to control health damage caused by Cr(Ⅵ) through digestive system has become an urgent public health issue.Various studies have shown that Cr(Ⅵ) could induce mitochondria-dependent apoptosis; however, the corresponding measures to prevent Cr(Ⅵ)-induced liver injury have not yet been elaborately discussed. After entering into hepatocytes, Cr(Ⅵ) attacks mitochondrial respiratory chain and increases electron leakage and ROS formation. Excess ROS induces mitochondrial membrane oxidative damage and mitochondria permeability transition (MPT), which leads to apoptosis-inducing factors release and p53-caspase cascade effect, and finally mitochondria-dependent apoptosis. The present study explored whether inhibiting MPT or alleviating oxidative damage has protective effects on Cr(Ⅵ)-induced hepatocellular apoptosis. Autophagy, as a hotspot in recent years, could timely degrade injured mitochondria to diminish further damage. And meanwhile, the degradation production could be used as cellular energy material for the regeneration of damaged organelle. Hence, the study focused on the protective effects on Cr(Ⅵ)-induced mitochondria-dependent apoptosis by inhibiting MPT, diminishing oxidative damage, and increasing autophagy activity, which has preventive medical significance for the protection of Cr(Ⅵ)-exposed population health.Methods1. Cr(Ⅵ) induced mitochondria-dependent apoptosis in L-02hepatocytesAfter L-02hepatocytes treated with different concentrations of Cr(Ⅵ), cell viabilities were detected by MTT assay and the appropriate concentrations for subsequent experiments were chosen. Mitochondria membrane potentials were tested with specific fluorescent dye JC-1staining and flow cytometry. ATP productions were assessed by phosphorus determination method and visible spectrophotometer. By western blotting assay, the protein expressions of VDAC1and p53were detected and caspase-3activities were measured by microplate reader. And apoptosis rates were tested with fluorescent dye Annexin V-FITC/PI by flow cytometry.2. The effects of MPT inhibitor CsA on mitochondria-dependent hepatocyte apoptosis induced by Cr(Ⅵ)Firstly, the quality of mitochondria isolated from L-02hepatocytes was appraised. Mitochondrial ultrastructure was observed by TEM and mitochondrial respiratory function was measured by Clark oxygen electrode. Cell viabilities were assessed by MTT assay to select an appropriate concentration of CsA for subsequent experiments. The effects of CsA on mitochondrial membrane potential and the protein expression of VDAC1were tested. Mitochondrial respiratory function and oxidative phosphorylation efficiency were measured by Clark oxygen electrode. Other apoptosis related indicators, such as ATP production, protein expression of p53, caspase-3activity and apoptosis rate, were detected using the methods described as above.3. The effects of ROS scavenger NAC on mitochondria-dependent hepatocyte apoptosis induced by Cr(Ⅵ)Cell viabilities were assessed by MTT assay to choose an appropriate concentration of antioxidant N-acetylcysteine (NAC) for subsequent experiments. After hepatocytes were treated with Cr(Ⅵ) and/or NAC, superoxide anion radical and ROS contents were tested by fluorescence spectrophotometer. Ca2+contents were detected with Ca2+specific probe Fluo3-AM and fluorescent microplate. And Ca2+release-activated Ca2+channel (CRAC) activity were measured by MultiClamp700B patch clamp recording system. Moreover, the effects of NAC on hepatocellular mitochondria membrane function and respiratory function were measured. Finally, several energy metabolism and apoptosis related indicators were detected using the methods described as above.4. The effects of autophagy inducer Rap on mitochondria-dependent hepatocyte apoptosis induced by Cr(Ⅵ)Cell viabilities were detected by MTT assay to choose appropriate concentrations of autophagy inducer Rapamycin (Rap) and autophagy inhibitor3-Methyladenine (3-MA) for subsequent experiments. The occurrence of autophagy in Cr(Ⅵ)-treated hepatocytes was verified by the observation of autophagosomes by TEM. Additionally, the protein expression of LC3-Ⅱ and the phosphorylation level of mTOR downstream protein4EBP1were assessed by western blot assay. The mRNA expression of autophagy related genes, Beclin-1and Atg7were detected by RT-PCR assay. Hepatocytes were treated with Cr(Ⅵ) and/or Rap and3-MA, then mitochondrial damage related indicator, such as mitochondria membrane potential, mitochondrial respiratory function and oxidative phosphorylation efficiency were tested. And apoptosis related indicators, such as ATP production, protein expression of p53, caspase-3activity and apoptosis rate, were detected using the methods described as above.Results1. Cr(Ⅵ) induced mitochondria-dependent apoptosis in L-02hepatocytesThe percentage of viable cells significantly diminished in a dose-dependent manner after treated with0,0.1,0.25,0.5,0.75,1,2,3,4,6,8μM Cr(Ⅵ) for24h. Then cells were exposed to Cr(Ⅵ) of increasing concentrations (1-4μM) for24h, and mitochondrial membrane potential and ATP production decreased. The protein expression of VDAC1also diminished. Moreover, Cr(Ⅵ) induced the increase of p53protein expression and caspase-3activities, then apoptosis rates increased significantly compared with control group in L-02hepatocytes.2. The effects of MPT inhibitor CsA on Cr(Ⅵ)-induced mitochondria-dependent hepatocyte apoptosisThe mitochondria isolated from L-02hepatocytes have intact structure and respiratory function.0.5μM CsA effectively inhibited the decrease of cell viability and mitochondrial membrane potential induced by4μM Cr(Ⅵ). However,0.5μM CsA did not show the ameliorative effect on the protein expression of VDAC1. In addition,0.5μM CsA protected mitochondrial respiratory function, oxidative phosphorylation efficiency and ATP production, and therefore decreased the activation of p53and casapse-3and the raise of apoptosis rates.3. The effects of ROS scavenger on Cr(Ⅵ)-induced mitochondria-dependent hepatocyte apoptosisROS scavenger,5mM NAC could effectively protect hepatocellular viability. Besides,5mM NAC also limited superoxide anion radical and ROS production in hepatocytes treated with4μM Cr(Ⅵ). Moreover, NAC protected the increase of Ca2+contents and the decrease of CARC channel activities induced by Cr(Ⅵ). After application of4μM Cr(Ⅵ) and5mM NAC for24h, mitochondria membrane potential and the protein expression of VDAC1increased compared to4μM Cr(Ⅵ) group. Finally,5mM NAC were beneficial for the protection of mitochondrial function and energy metabolism, and protected hepatocytes against Cr(Ⅵ)-induced mitochondria-dependent apoptosis.4. The effects of autophagy inducer Rap on Cr(Ⅵ)-induced mitochondria-dependent hepatocyte apoptosisCr(Ⅵ) induced the activation of autophagy in L-02hepatocytes, which is approved by the increase of endogenous LC3-Ⅱ levels, the decrease of phospho-4EBP1protein levels, the direct observation of double-membrane autophagosomes containing injured mitochondria, and the increase of Beclin1and Atg7mRNA expression. The process that4μM Cr(Ⅵ) induced apoptosis was intervened by autophagy inducer1.5μM Rap and autophagy inhibitor5mM3-MA. The results showed that1.5μM Rap ameliorated the decrease of mitochondria membrane potential, respiratory function, oxidative phosphorylation efficiency and ATP production caused by Cr(VI), which is deteriorated by autophagy inhibitor3-MA. And p53levels, caspase-3activities and apoptosis rates decreased in hepatocytes treated with4μM Cr(VI) and1.5μM Rap, and in contrast, apoptosis related indicators were further exacerbated in cells exposed to5mM3-MA and4μM Cr(Ⅵ).Conclusions1. Cr(Ⅵ) induced mitochondria-dependent apoptosis in L-02hepatocytes. The treatment of Cr(Ⅵ) caused mitochondria damage, energy metabolism disorder, p53protein activation and caspase activity increase, leading to hepatocellular apoptosis.2. The inhibitor of mitochondrial permeability transition, CsA, effectively inhibited mitochondrial PTP open, protected respiratory function, oxidative phosphorylation efficiency and ATP production, inhibited the activation of p53levels and capsase-3activities, and finally diminished hepatocyte apoptosis induced by Cr(Ⅵ).3. The ROS scavenger, NAC, decreased Cr(Ⅵ)-induced oxidative damage and calcium overload, and protected Ca2+channel activity by clearing over excess ROS. Therefore, NAC significantly diminished mitochondrial damage and protected hepatocytes against Cr(Ⅵ)-induced mitochondria-dependent apoptosis.4. Activation of autophagy was beneficial for the inhibition of hepatocyte apoptosis. The activation of autophagy timely cleared over damaged mitochondria and converted the degradation production into energy substances, which is beneficial for the regeneration of mitochondria and maintenance of mitochondrial function, and then inhibited mitochondria-dependent apoptosis. |
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