Alcohol induced epigenetic perturbations during the inflammatory stage of fracture healing

Abstract

It is well recognized by orthopedic surgeons that fractures of alcoholics are more difficult to heal successfully and have a higher incidence of non-union, but the mechanism of alcohol's effect on fracture healing is unknown. In order to give direction for the study of the effects of alcohol on fracture healing, we propose to identify gene expression and microRNA changes during the early stages of fracture healing that might be attributable to alcohol consumption. As the inflammatory stage appears to be the most critical for successful fracture healing, this paper focuses on the events at day three following fracture or the stage of inflammation. Sprague-Dawley rats were placed on an ethanol-containing or pair-fed Lieber and DeCarli diet for four weeks prior to surgical fracture. Following insertion of a medullary pin, a closed mid-diaphyseal fracture was induced using a Bonnarens and Einhorn fracture device. At three days' post-fracture, the region of the fracture calluses was harvested from the right hind-limb. RNA was extracted and microarray analysis was conducted against the entire rat genome. There were 35 genes that demonstrated significant increased expression due to alcohol consumption and 20 that decreased due to alcohol. In addition, the expression of 20 microRNAs was increased and six decreased. In summary, while it is recognized that mRNA levels may or may not represent protein levels successfully produced by the cell, these studies reveal changes in gene expression that support the hypothesis that alcohol consumption affects events involved with inflammation. MicroRNAs are known to modulate mRNA and these findings were consistent with much of what was seen with mRNA microarray analysis, especially the involvement of smad4 which was demonstrated by mRNA microarray, microRNA and polymerase chain reaction.

Epigenetics in the perioperative period

“…Similarly, the proliferation of keratinocytes, key to re-epithelialization of wound surfaces, is regulated by epigenetic factors (Rafehi et al, 2011). Moreover, alcoholics have a higher risk of nonunion after bone fractures, and this has been linked to epigenetic changes affecting cell migration, proliferation and differentiation (Sampson et al, 2011). Zeybel and co-workers investigated whether these epigenetic traits might be handed down through generations, and in a rat model of liver damage, they found that the fibrogenic component of liver damage was still alleviated in the first-and secondgeneration offspring after ancestral chronic liver injury induced by carbon tetrachloride (Zeybel et al, 2012).…”

MicroRNAs and Ethanol Toxicity

“…Outside the brain, alcohol exposure also alters miRNA expression and function associated with gut leakiness and liver damage (Tang et al, 2008), steato-hepatitis (Dolganiuc et al, 2009), inflammation (Bala & Szabo, 2012), fibrosis (Meng et al, 2012), and bone remodeling (Sampson et al, 2011). In an early study exemplifying the promise of miRNAs for understanding alcoholism-associated disease, Tang and colleagues showed, in a gut epithelial monolayer model, that alcohol induced miR-212 expression while decreasing expression of the target tight-junction complex protein ZO-1.…”

Plasma miRNA Profiles in Pregnant Women Predict Infant Outcomes following Prenatal Alcohol Exposure

“…In 2007, we showed that miRNAs were sensitive to alcohol [19] and mediated alcohol effects on fetal neural [19, 20] and cranial development [21] in animal models. MiRNAs play an important role in alcohol addiction [22–26] and neurotoxicity [27], alcohol-associated alterations in intestinal and hepatic integrity [28, 29], inflammation [30], fibrosis [31], and bone remodeling [32]. Thus, miRNAs are not only sensitive to alcohol exposure, but also mediate many alcohol effects.…”