Study of rat hepatocytes in primary culture submitted to hypoxia and reoxygenation: action of the cytoprotectors prostaglandin E1, superoxide dismutase, allopurinol and verapamil

Abstract

-Context -Exposure of hepatocytes to pathological conditions in a microenvironment of hypoxia and reoxygenation is very frequent in hepatic diseases. Several substances present perspectives for cytoprotective action on hepatocyte submitted to reoxygenation after hypoxia and simple hypoxia. Objective -We research therapeutic options for hepatocytes submitted to hypoxia and hypoxia + reoxygenation injury. Methods -Primary culture of rat hepatocytes was submitted to hypoxia (2 hours) plus reoxygenation (2 hours) and simple hypoxia (4 hours) in the presence or the absence of cytoprotectors. The hepatocyte lesion was evaluated by functional criteria through percentage of lactate dehydrogenase released and cell viability. The effects of the cytoprotectors prostaglandin E1 3 ηg/mL, superoxide dismutase 80 μg/mL, allopurinol 20 μM and verapamil 10 -4 M were studied in this model of injury. Results -Reoxygenation after hypoxia induced more significant lesion in cultured hepatocytes compared to simple hypoxia, detected by analysis of functional criteria. There was a significant reduction of percentage of lactate dehydrogenase released and a significant increase of percentage of cell viability in the hypoxia + reoxygenation + cytoprotectors groups compared to hypoxia + reoxygenation groups. Prostaglandin E1, superoxide dismutase and verapamil also protected the group submitted to simple hypoxia, when evaluated by functional criteria. Conclusions -We conclude that reoxygenation after hypoxia significantly increased the lesion of cultured rat hepatocytes when compared to simple hypoxia. Prostaglandin E1, superoxide dismutase, allopurinol and verapamil acted as cytoprotectors to the rat cultured hepatocytes submitted to hypoxia + reoxygenation in vitro. The substances prostaglandin E1, superoxide dismutase and verapamil protected hepatocytes submitted to simple hypoxia on the basis of all the criteria studied in this experimental model.

Protective effects of fish oil, allopurinol, and verapamil on hepatic ischemia-reperfusion injury in rats

“…Allopurinol has a well-known action in the blockade of xanthine oxidase (XO) enzyme, and it has a cytoprotective potential against injury provoked by reoxygenation. [ 9 ] Because Kupffer cells contain XO, which is one potential source of free radicals, allopurinol has been suggested to ameliorate hepatic injury by inhibition of XO and reduction of oxidative stress.…”

Variable responses of small and large human hepatocytes to hypoxia and hypoxia/reoxygenation (H-R)

“…Death of hepatocytes by hypoxia and H–R injury is a multifactorial event including alteration of the plasma membrane, increase of autophagic vacuoles, rupture of blebs on the cell surface, calcium accumulation and mitochondrial damage [9]. The common mediator of these death pathways appears to be the accumulation of ROS [17]. Studies in rodent models suggest that large/PV hepatocytes are more vulnerable to damage during hepatic injury [8].…”

“…Therefore, an understanding of the differential vulnerability of hepatocytes to liver injury would provide an important basis for preventing loss of liver function and/or failure caused by cirrhosis, hepatic resection and liver transplantation [8]. Common factors to most liver injuries are hypoxia and the generation of ROS [14,17]. However, the susceptibility of hepatocytes, and in particular human hepatocytes, to oxidative stress has not been assessed directly.…”

Histidine‐Rich Glycoprotein Alleviates Liver Ischemia/Reperfusion Injury in Mice With Nonalcoholic Steatohepatitis

“…Then the cells were incubated at 1% O 2 at 37°C for 2 hours, and the cells were moved to normal conditions for 4 hours. ( 22 ) Treated cells were used for Western blot analysis of HRG, FXR, HIF1A, and HIF2A protein expressions.…”