Specifically Sized Hyaluronan (35 kDa) Prevents Ethanol‐Induced Disruption of Epithelial Tight Junctions Through a layilin‐Dependent Mechanism in Caco‐2 Cells

Abstract

Background: Specific-sized species of the carbohydrate hyaluronan elicit a variety of cellular responses mediating tissue integrity and repair, as well as regulating inflammatory responses. Orally provided hyaluronan with an average molecular weight of 35 kDa (HA35) protects mice from shortterm ethanol (EtOH)-induced liver injury. This protection was associated with maintenance of the colocalization of zonula occludens-1 (ZO-1) and occludin at tight junctions in the proximal colon. However, it is not known whether HA35 also protects other regions of the intestine or whether protection is due to a direct and/or indirect interaction of HA35 with the intestinal epithelium.Methods: Female C57BL/6J mice were fed an EtOH containing diet or pair-fed control diet (4 days) and treated with or without HA35 via daily gavage during the last 3 days of EtOH feeding. Intestinal morphology and tight junction integrity were assessed. Differentiated Caco-2 cells were transfected or not with scrambled siRNA or siRNA targeting layilin, a hyaluronan receptor. Caco-2 cells were treated with or without HA35 prior to challenge with EtOH. Localization of tight junction proteins, fluorescein isothiocyanate (FITC)-dextran permeability, and transepithelial electrical resistance (TEER) were evaluated.Results: While short-term EtOH did not result in any apparent changes in the gross morphology of the intestine, colocalization of ZO-1 and occludin at tight junctions was decreased in the proximal and distal colon. HA35 prevented these effects of EtOH. In differentiated Caco-2 cells, EtOH decreased the localization of ZO-1 and occludin at tight junctions and increased permeability of FITC-dextran. At higher concentrations, EtOH also decreased TEER. Pretreatment with HA35 prevented these changes. When the hyaluronan receptor layilin was knocked down in Caco-2 cells, HA35 no longer protected cells from EtOH-induced loss of tight junctions.Conclusions: Taken together, these data indicate that HA35 interacts with layilin on intestinal epithelial cells and maintains intestinal tight junction integrity during short-term EtOH exposure.

Prophylactic Treatment of Probiotic and Metformin Mitigates Ethanol-Induced Intestinal Barrier Injury: In Vitro, In Vivo, and In Silico Approaches

“…Also, ZO-1 is a cytoskeletal linker protein that forms cross-links with other transmembrane proteins like occludins, necessary for the connection of other TJ proteins to the cytoskeleton [ 65 ]. The report demonstrated that the administration of 40 mM ethanol to Caco-2 monolayers disrupted the TJs, specifically occludins and ZO-1 [ 66 ]. Also, acute alcohol exposure disrupts the barrier function by downregulating the expressions of ZO-1 and occludin in colons of mice [ 18 ].…”

“…17 Mediators of Inflammation and ZO-1 [66]. Also, acute alcohol exposure disrupts the barrier function by downregulating the expressions of ZO-1 and occludin in colons of mice [18].…”

Immunological mechanisms and therapeutic targets of fatty liver diseases

“…Related strategies have included treatment with butyrate, an important fuel source for colonic enterocytes, as well as multiple molecules shown to improve tight junction integrity in the intestine, such as zinc, saturated fatty acids, glutamine, and hyaluronic acid with an average molecular weight of 35 kDa (HA35). [141][142][143][144][145] To date, only zinc supplementation has been tested in patients with AH, used in combination with anakinra and pentoxifylline as part of a large multicenter clinical trial. 146 Hepatocyte injury While PAMPs entering the portal circulation from the gut are one source of inflammatory signals contributing to ALD, DAMPs derived from injured or dead cells are other potential targets for therapeutics in ALD.…”

“…Related strategies have included treatment with butyrate, an important fuel source for colonic enterocytes, as well as multiple molecules shown to improve tight junction integrity in the intestine, such as zinc, saturated fatty acids, glutamine, and hyaluronic acid with an average molecular weight of 35 kDa (HA35). 141 – 145 To date, only zinc supplementation has been tested in patients with AH, used in combination with anakinra and pentoxifylline as part of a large multicenter clinical trial. 146 …”

Pathogenesis of Alcohol-Associated Liver Disease

“…266,267 Other investigations state that HA-35 treatment protects intestinal tight junctions from alcoholinduced barrier damage. 268 Given these findings that HA-35 regulates KC activation and protects gut barrier function, this HA fragment may have therapeutic value for ALD treatment.…”