Pro-Inflammatory CXCR3 Impairs Mitochondrial Function in Experimental Non-Alcoholic Steatohepatitis

Abstract

Mitochondrial dysfunction plays a crucial role in the development of non-alcoholic steatohepatitis (NASH). However, the regulator of mitochondrial dysfunction in the pathogenesis of NASH is still largely unclear. CXCR3 is an essential pro-inflammatory factor in chronic liver diseases. We explored the significance of CXCR3 in regulating mitochondrial function during NASH development in animal models and cultured hepatocytes.Methods: The effects of CXCR3 on mitochondrial function were evaluated by genetic knockout or pharmacological inhibition in mouse models and in vitro. The ultrastructural changes of mitochondria were assessed by transmission electron microscopy (TEM). Hepatic levels of mitochondrial reactive oxygen species (ROS), DNA damage, membrane potential and ATP were examined.Results: CXCR3 ablation by genetic knockout or pharmacological inhibition in mice protected against NASH development by influencing mitochondrial function. Similarly, depletion of CXCR3 reduced steatohepatitis injury in cultured hepatocytes. TEM analysis revealed that liver mitochondrial integrity was much improved in CXCR3 knockout (CXCR3-/-) compared to wildtype (WT) mice. In agreement with this, impaired mitochondrial function was pronounced in WT mice compared to CXCR3-/- mice, evidenced by increased protein expression of dynamic-related protein-1 (DRP1) and fission-1 (FIS1) and decreased protein expression of mitofusin-1 (MFN1). Mitochondrial dysfunction was induced in AML-12 hepatocytes by methionine and choline deficient medium and in HepG2 cells by palmitic acid. The impaired mitochondrial function in both cell lines was evidenced by reduced membrane potential and ATP content, and by increased mitochondrial ROS accumulation and DNA damage. However, CXCR3 knockdown by siCXCR3 significantly diminished the mitochondrial dysfunction in both AML-12 and HepG2 hepatocytes. In addition, inhibition of CXCR3 by CXCR3 specific antagonists SCH546738 and AMG487 restored mitochondrial function and inhibited mitochondrial-dependent apoptosis in the liver of WT mice fed with methionine and choline deficient diet.Conclusion: CXCR3 induces mitochondrial dysfunction, which contributes to the pathogenesis of steatohepatitis. Pharmacologic blockade of CXCR3 prevents mitochondrial dysfunction and restores the severity of steatohepatitis, indicating a potential clinical impact for controlling the disease.

Corilagin Alleviates Nonalcoholic Fatty Liver Disease in High-Fat Diet-Induced C57BL/6 Mice by Ameliorating Oxidative Stress and Restoring Autophagic Flux

“…However, intracellular ROS levels were significantly decreased after Cori treatment ( Figure 5A). Recent reports have revealed that increased proinflammatory cytokines levels were a pivotal factor triggering ROS formation as well as inducing oxidative stress, which was related to HFD-induced lipid droplet accumulation (Du et al, 2017). Our results also showed that Cori treatment significantly decreased the expression of proinflammatory cytokines in livers of HFD-fed mice, including TNF-a, IL-6 ( Figure 1N).…”

“…ROS-mediated mitochondrial dysfunction can cause ineffective capacity of mitochondrial fatty acid oxidation, which inversely aggravates excessive ROS production, thus constituting a vicious cycle in the progression of NAFLD (Wang et al, 2015). To evaluate the degrees of mitochondrial DNA damage caused by ROS, we next measured 8-OHdG (a marker of oxidative DNA damage) levels in hepatocytes (Du et al, 2017). In comparison FIGURE 4 | Cori ameliorated nonalcoholic fatty liver disease (NAFLD) by restoring autophagic flux in vitro.…”

“…The extracted mtDNA was digested with DNAse I and alkaline phosphatase (TaKaRa, Otsu, Japan).Then, the levels of 8hydroxydeoxyguanosine (8-OHdG) in hepatocytes and liver tissues were measured by a commercial ELISA kit (Cayman Chemical) according to the standard protocol (Du et al, 2017). Intracellular ROS Measurement AML12 cells were seeded in 12-well plates and cultured for 24 h. Cells were stimulated with PA/OA (200 mM) in the absence or presence of tested concentration of Cori (10, 20 mM) for 24 h. The culture medium was removed, washed three times with PBS buffer and then incubated with 10 mM of DCFH-DA (S0033, Beyotime, China) at 37°C for 20 min.…”

Transmission electron microscopy of the benzbromarone-induced change in mitochondrial morphology in HepG2 cells

“…2A and B). These results are consistent with previous reports that alteration of mitochondrial morphology impairs mitochondrial function, activates caspase pathways, and ultimately induces cell death in a mouse model of non-alcoholic steatohepatitis (Du et al, 2017). Based on these findings, we thought that our TEM assay was a suitable method for detecting of mitochondria-related cell death.…”

Polyphenols in Liubao Tea Can Prevent CCl4-Induced Hepatic Damage in Mice through Its Antioxidant Capacities

“…The mitochondrial respiratory chain complex uses electron transfer to produce ATP, which is the main source of ROS. The liver is rich in mitochondria, and is, therefore, the main organ susceptible to ROS attack, and oxidative stress has a close relationship with most liver damage [ 12 ]. ROS can also initiate a variety of cytokines such as transforming growth factor-beta (TGF-β), interleukin-8 (IL-8), and NF-κB.…”