OA-induced steatosis in HepG2 cells may serve as an in vitro
model for studying fatty liver disease. Consistent with previous reports [30
], we revealed OA induces steatosis in HepG2 cells in a dose-dependent manner that can be assessed by ORO biochemical staining. However, this method is neither convenient, nor accurate in quantification, therefore, cannot be used as a routine steatosis assay, especially for the study of disease mechanisms and development of new therapy to NAFLD.
In the present study, we utilized the feature that after biochemical staining of steatosis, ORO can be released from the stained cells that could be further quantitatively measurable. Using this concept, we were able to develop an ORO-based colorimetric quantification assay to measure OA-induced steatosis in HepG2 cells. Our further analysis indicated that this novel method is not only convenient, and highly reproducible, but also accurate with a very high correlation between the dose of OA and the degree of steatosis as expressed as absorbance of optical density and histological ORO digital pixel measurement. Thus, the cell model of OA-induced steatosis, together with this novel ORO-based colorimetric quantitative assay, will provide valuable tools to study the pathogenesis and develop new treatment for NAFLD.
It is well known that TNF-α plays important pathogenic roles in both ALD and NAFLD [16
]. For instance, TNF-α has been associated with insulin resistance and induce inflammatory cytokines formation. The mean plasma level of TNF-α was significantly higher in NAFLD patients with abnormal ALT than controls [31
]. A key question is whether increased TNF-α in NAFLD is from hepatocytes and/or from other inflammatory cells. A recent study demonstrated that FFA treatment induces TNF-α mRNA in HepG2 cells [19
], but it remains unknown if FFA-induced steatosis also promotes translation of TNF-α mRNA. In the present study, we demonstrated that OA-induced steatosis significantly increased TNF-α production and secretion from HepG2 cells. These in vitro results indicate that OA-induced steatosis promotes translation of TNF-α mRNA. Our data further support the pathogenic role of hepa-tocyte-derived TNF-α in NAFLD. Studies reported that TNF-α also stimulates ROS generation and induces lipid peroxidation [34
]. We demonstrated that OA-induced steatosis is associated with increased lipid peroxides in HepaG2 cells. However, the pathogenic role of increased TNF-α in up regulation of lipid peroxides in OA-induced steatosis remains to be determined.
As HepG2 cells accumulate intracellular lipids, there is a significant rise in lipid peroxides. In previous studies, unsaturated fatty acids have been shown to induce the cytochrome P450 2E1 (CYP2E1) enzyme pathway and stimulate lipid peroxidation, which subsequently promotes apoptosis and cell toxicity [35
]. Consistent with these findings, we found that OA-induced steatosis was associated with a significant rise in lipid peroxide formation in the OA-treated HepG2 cells. The pathogenic role of increased lipid peroxides in the cell injury of OA-induced steatosis in HepG2 cells is further supported by our findings that OA-induced steatosis in these cells was associated with a significantly decreased SOD-1, a free radical scavenger enzyme that protects against cellular membrane injury mediated by lipid peroxidation. These finding also indicated a potential therapeutic role of anti-peroxidation agents for NAFLD.
PPARα activates expression of a series of target genes involved in the uptake, transportation, and β-oxidation of fatty acids [36
]. Several studies have shown that PPARα increases fatty acid catabolism, and therefore, may prevent hepatic fat deposition [38
]. Our findings that OA-induced steatosis resulted in reduced PPARα expression in HepG2 cells indicated its possible pathogenic role in NAFLD. Taken together, our results suggested that the association of OA-induced steatosis with increased lipid peroxidation may be mediated by decreased PPARα expression in these cells. This speculation is supported by the clinical and experimental evidence that PPARα antagonists may improve steatosis in patients and animal model [43
]. Further determining this association will be help in developing novel therapeutic approach to this disease.
Hepatocyte apoptosis is a feature of fatty liver disease [45
], which may reduce cell regeneration or proliferation. We found that OA, at 0.1-lmM dose range, leads to increased apoptosis of HepG2 cells with OA-induced steatosis. This was associated with increased production of activated caspase-9 of the apoptosis cascade. Although apoptosis may be associated with decreased proliferation of the liver cells with steotosis, it may also contribute to the pathogenesis of the liver injury in non-alcoholic fatty liver disease. We could not demonstrate any significant effects of OA-treatment on Bcl-2 and Bax expression, the two other apoptotic modulators, indicating that they are unlikely regulating apoptosis in OA-induced steatosis in HepG2 cells.
The effects of steatosis on hepatocyte proliferation are unknown. Using MTT assay, we demonstrated that OA-induced steatosis in HepG2 cells was associated with inhibition of cell proliferation. These results are supported by a recent report of microarray analysis [27
]. Since OA-increased apoptosis was saturated at the dose of 1 mM, the decreased HepG2 proliferation could not be solely explained by apoptosis. On the other hand, we demonstrated its association with increased p27 expression. Since p27 functions as a cyclin-dependent kinase (CDK) inhibitor involved in suppression of the cell cycle at the G1
-S checkpoint, our results indicated that OA-induced steatosis decreases cell proliferation by inhibiting p27 expression, therefore, G1
The clinical presentation of NAFLD could be very variable from persistently normal to significantly increased ALT. It is not known if steatosis results in direct liver injury and elevated ALT. Using HepG2 in vitro model, we demonstrated that baseline level of ALT was comparable in the culture medium of both untreated and OA-treated HepG2 cells. At a wide range of concentration, OA treatment does not alter the ALT level in the culture medium. These findings suggest that OA-induced steatosis itself does not result in liver cell injury. Instead, it is likely that hepatocytes’ direct response to steatosis results in generation of a series of inflammatory mediators that may cause liver cell injury. Further studies will be needed to detail theses underlying mechanisms.
In conclusion, the present study developed an ORO-based colorimetric assay to quantify lipid accumulation in OA-induced HepG2 cells, which provides a convenient tool for studying the pathogenesis and therapy for NAFLD. We also assessed the effects of OA-induced steatosis on HepG2 cell proliferation, apoptosis, and lipid peroxidation through a complicated signaling. These data are very valuable in understanding the pathogenesis of NAFLD.