(282f) Recapitulating the Effects of Ethanol on an Inflamed Gut-Liver Axis in Vitro

Authors: 
Kothari, A., Virginia Tech
Rajagopalan, P., Virginia Tech
The gut and the liver together affect many physiological functions such as glucose homeostasis, inflammation and response to toxic xenobiotics. Chronic alcohol abuse, non-alcoholic fatty liver disease and inflammatory bowel diseases among others can lead to disruption of the gut barrier, bacterial dysbiosis, and subsequent translocation of bacteria and their metabolites to the liver. These phenomena can cause or exacerbate liver damage. Such an inflamed system can get further damaged when challenged with a toxic xenobiotic such as ethanol (EtOH) due to metabolism by the gut, the liver and the gut bacteria. We were thus motivated to develop an in vitro model to recapitulate the response of an inflamed integrated gut-liver system to assess EtOH toxicity.

Rat jejunum explants were inverted and cultured on PDMS rods in Transwell® inserts. Primary rat hepatocytes were isolated and cultured in a collagen sandwich (CS) model. CS models and jejunum explants were integrated at 4h and treated with 100mM and 200mM EtOH (LC50/2 and LC50 respectively for rats). At 24h, cultures were ended by lysing the cells and tissues for protein/DNA or embedding the explants in OCT compound for cryosectioning.

Jejunum explants when cultured alone showed an increase in intestinal alkaline phosphatase (ALP) activity over time, with the ALP activity being significantly higher by >2.5-fold after 24h of culture than at 0h or 4h. This indicates an increase in inflammation in these cultures. The effects of this inflammatory phenotype were further observed when explants were integrated with primary rat hepatocytes. Hepatocyte damage after integration with primary jejunum tissues was clear from the 39% decrease in hepatocyte protein and 76% increase in hepatic alkaline phosphatase (ALP) activity. Blebbing was observed in the membranes of hepatocytes integrated with jejunum explants for 20h, which could be caused by possible induction of apoptosis in these cells. These observations suggest the appearance of an inflammatory phenotype in integrated gut-liver cultures.

Next, we treated these cultures with EtOH to assess its toxic effects in an inflamed system. Upon EtOH treatment at LC50, hepatic alcohol dehydrogenase (ADH) activity was significantly reduced only in integrated cultures, which is consistent with in vivo data. Explants in integrated cultures treated with ethanol at both LC50/2 and LC50 showed a significant ~40% decrease in villus length. This observation concurs with in vivo studies in rats where a decrease in villus length has been reported in EtOH-fed rats. Mucus-forming mucins, as visualized through Alcian blue/Periodic Acid-Schiff’s base staining, were decreased dramatically when jejunum explants integrated with hepatocytes were treated with EtOH at both LC50/2 and LC50. However, these changes were not observed upon EtOH treatment of jejunum explants cultured alone. These results also correlated with significantly higher tumor necrosis factor-α (TNF-α) levels in integrated cultures treated with EtOH at LC50. This suggests the importance of gut-liver integration in understanding EtOH toxicity in inflamed tissues.

Taken together, our results underscore the importance of integration of organs to study ethanol toxicity as well as inflammation.