Curcumin Attenuates Iron‐Dysregulation in Human Hepatocytes Exposed to Oxidative Stress

Chronic inflammation is associated with low‐dose oxidative stress. This exposure has previously been shown to disrupt iron regulation by downregulating iron storage and increasing iron import. As a result, the increased unbound intracellular iron is able to generate damaging free radicals via Fenton reaction chemistry. Using Glucose Oxidase and Catalase, we produced a steady‐state, low‐dose hydrogen peroxide exposure for human hepatocytes in the HepG2 cell line to simulate this dysregulation caused by chronic inflammation. We have previously shown that curcumin is able to attenuate the effects of this oxidative stress in mouse C2C12 myoblasts. Because of the liver's key role in human iron homeostasis, we investigated curcumin's ability to produce the same effect in HepG2 cells. Purpose To determine if curcumin can adequately prevent oxidative‐stress induced iron dysregulation in human hepatocytes. Methods Cell viability was measured using MTT assays. Protein content was qualitatively compared between treatment groups using standard western blotting protocol. HepG2 cells were grown to 80% confluency and then subsequently treated with combinations of FeCl 3 , H 2 O 2 , and Curcumin. FeCl 3 was added directly into culture media. Except during H 2 O 2 treatments (Dulbecco's high‐glucose medium, Sigma D6429), Eagle's Minimal Essential Minimum from ATCC(30‐2003) was used for culture. Curcumin was also added as a bolus into the culture medium prior to treatment. Combined, 36‐hour treatments were carried out as described here: Iron (0‐12 hours); Iron + H 2 O 2 (12‐24 hours); Curcumin alone (24‐36 hours); followed by a 2x rinse with PBS and harvesting in RIPA buffer (Millipore 20‐188). Results MTT assays showed no significant cell death. Transferrin receptor (TfR) protein content is significantly decreased following 24‐hour iron exposure (Control=1±0.1 SEM vs. 0.71±0.1; p<0.01; n=12). Interestingly, The addition of H 2 O 2 to that treatment (Fe + H 2 O 2 ) resulted in TfR content that showed no significant difference compared to control–likely indicating that the oxidative stress had impaired the normal cellular response to iron exposure (Control=1±0.09 SEM vs. 0.88±0.04 SEM; p=0.8; n=4). Our most recent findings showed that when these two treatments were followed by a 12‐hour curcumin exposure, doses of 20 and 30 µM were able to significantly reduce TfR content when compared to control (Control=1±0.12 SEM vs. 30 µM 0.49±0.09 SEM; p=0.01; n=6). When all three treatments were blotted simultaneously for comparison, Ferritin light chain, a protein responsible for storing intracellular iron in hepatocytes, increased 13‐fold (Control=1±0.21 SEM vs. 13.1±2.4 SEM; p=0.01; n=4). compared to control in the Fe + H 2 O 2 + Curc group. This was a slight decrease from the Fe + H 2 O 2 group (data not shown). Conclusion Our results indicate that curcumin may effectively mitigate oxidative stress on hepatocytes and subsequent iron dysregulation through altering the expression of crucial iron regulatory proteins such as TfR and FLC. We are continuing this work to determine curcumin's effects on modulating the intracellular iron concentrations as a result of this protein regulation. Importantly, our results suggest that curcumin could reduce the effects of chronic inflammation on human hepatocytes.