Restoration of liver sinusoidal cell phenotypes by statins improves portal hypertension and histology in rats with NASH

Non-alcoholic steatohepatitis (NASH) is a common chronic liver disorder in developed countries, with the associated clinical complications driven by portal hypertension (PH). PH may precede fibrosis development, probably due to endothelial dysfunction at early stages of the disease. Our aim was to characterize liver sinusoidal endothelial cell (LSEC) dedifferentiation/capillarization and its contribution to PH in NASH, together with assessing statins capability to revert endothelial function improving early NASH stages. Sprague-Dawley rats were fed with high fat glucose-fructose diet (HFGFD), or control diet (CD) for 8 weeks and then treated with simvastatin (sim) (10 mg·kg−1·day−1), atorvastatin (ato) (10 mg·kg−1·day−1) or vehicle during 2 weeks. Biochemical, histological and hemodynamic determinations were carried out. Sinusoidal endothelial dysfunction was assessed in individualized sorted LSEC and hepatic stellate cells (HSC) from animal groups and in whole liver samples. HFGFD rats showed full NASH features without fibrosis but with significantly increased portal pressure compared with CD rats (10.47 ± 0.37 mmHg vs 8.30 ± 0.22 mmHg; p < 0.001). Moreover, HFGFD rats showed a higher percentage of capillarized (CD32b−/CD11b−) LSEC (8% vs 1%, p = 0.005) showing a contractile phenotype associated to HSC activation. Statin treatments caused a significant portal pressure reduction (sim: 9.29 ± 0.25 mmHg, p < 0.01; ato: 8.85 ± 0.30 mmHg, p < 0.001), NASH histology reversion, along with significant recovery of LSEC differentiation and a regression of HSC activation to a more quiescent phenotype. In an early NASH model without fibrosis with PH, LSEC transition to capillarization and HSC activation are reverted by statin treatment inducing portal pressure decrease and NASH features improvement.

Effect of melatonin on cellular composition of the liver in wistar rats with alimentary obesity

Цель - морфофункциональная характеристика нарушений функции печени при алиментарном ожирении и их коррекция мелатонином. Методы. В эксперименте использовано 3 группы половозрелых (2 мес.) крыс-самок Wistar с исходной массой 180-200 г. 1-я группа - контроль (интактные крысы); 2-я - группа «ожирение» (модель алиментарного ожирения воспроизводилась путем добавления (без ограничения) к стандартному лабораторному рациону в течение 3 мес. пищевых жиров животного происхождения) и 3-я группа («ожирение + мелатонин») - животные с алиментарным ожирением, которым в течение 14 сут. per os через желудочный зонд 1 раз в сут. вводили водный раствор мелатонина (0,1 г на 100 г массы тела), животные жиры из рациона во время введения препарата не исключались. Крыс декапитировали под этаминаловым наркозом (40 мг на кг), извлекали печень для морфометрического и светооптического исследования (микроскоп LEICA DM 750, камера LEICA ICC 50 HD). Патогистологические препараты готовили по общепринятой методике. Исследование препаратов печени проводили при увеличении х1000 на срезах толщиной 5 мкм, окрашенных гематоксилином Майера и эозином, сульфатом нильского голубого. Для морфометрического анализа использовали метод наложения точечных морфометрических сеток (сетка 256 точек). Определяли относительную площадь сети синусоидов, ядер и цитоплазмы гепатоцитов, численную плотность синусоидных клеток и гепатоцитов и двуядерных паренхиматозных клеток; рассчитывали ядерно-цитоплазматическое соотношение, отношение численной плотности синусоидных клеток к численной плотности всех гепатоцитов, вычисляли долю диплокариоцитов от общего числа гепатоцитов, рассчитывали коэффициент Vizotto - отношение площади сети синусоидов к площади всех гепатоцитов. Результаты. Введение мелатонина нивелировало признаки нарушения кровообращения, крово- и лимфотока. Отмечалась сохранность сосудов портального тракта, восстановление архитектоники центральных вен. Большинство участков гемо- и лимфообращения не имело признаков грубых нарушений. Заключение. Ожирение приводит к значительным нарушениям в системе кровообращения и лимфотока в печени, развитию жировой дистрофии. Введение таким животным гормона эпифиза мелатонина способствует усилению репаративных процессов в печени, нормализации микроциркуляторных процессов, восстановлению микроструктурной и функциональной организации органа. Aim. To identify and assess morpho-functional changes in the liver of Wistar rats on a model of alimentary obesity and correct the changes with the pineal hormone, melatonin, a universal adaptogen, immune modulator, and potent antioxidant. Methods. Sexually mature female Wistar rats aged 2 months and weighing 180-200 g at baseline were used for the experiment. Rats were allocated to three groups, 1) control group (intact rats); 2) group with alimentary obesity modeled by adding animal fat to the ad libitum standard laboratory diet for 3 months (obesity group); and 3) obesity group treated with melatonin 0.1 g/100 g body weight in 200 µl of distilled water, orally through a gastric tube, once daily for 14 days; during the treatment, animal fat was not excluded from the diet (obesity + melatonin group). Rats were sacrificed under etaminal anesthesia (40 mg/kg body weight) by decapitation. For morphometric and optical studies (LEICA DM 750 microscope, LEICA ICC 50 HD camera), histopathological preparations were fixed in 10% buffered formalin and examined with a standard method. Morphometric studies of liver samples were performed at a x1000 magnification on 5 µm sections stained with Mayer’s hematoxylin and eosin stain and Nile blue sulfate using superposition of point morphometric grids (grid of 256 points). Relative areas of sinusoid network, hepatocyte nuclei and cytoplasm; numerical density of sinusoidal cells, hepatocytes, and dual-parenchymal cells were measured. The nuclear-cytoplasmic ratio; ratio of sinusoidal cell numerical density to the numerical density of all hepatocytes; and per cent of diplocaryocytes of the total number of hepatocytes were computed; the Vizotto coefficient was calculated as a ratio of the area of sinusoid network to the area of all parenchymal hepatocytes. Results. Administration of the animal hormone, melatonin, exerted a pronounced effect on the studied morphometric parameters and reversed signs of circulatory and lymph flow disorders. Blood vessels of the portal area were preserved, and the architectonics of central veins was recovered. Most parts of hemo- and lymph circulation had no abnormal features. Conclusion. Obesity leads to significant disorders of blood circulation and lymph flow in the liver and results in fatty degeneration of hepatic parenchyma. Administration of the pineal hormone, melatonin, to such animals enhances reparative processes in the liver, normalizes microcirculation, and restores the structural and functional organization of the body.

The Effects of Chronic Iron Overload in Rats with Acute Acetaminophen Overdose

Background and Aims: Rats are resistant to acetaminophen (APAP) hepatotoxicity. In this study, we evaluated whether by augmentation of the hepatic oxidative stress, through the induction of hepatic iron overload (IO), it will be feasible to overcome the resistance of rats to the toxic effects of APAP. Method: Rats with no or increased hepatic IO. Results: Providing iron by diet induced hepatocellular IO, while parenteral iron administration induced combined hepatocellular and sinusoidal cell IO. APAP administration to rats with no IO caused an increase in hepatic oxidative stress and a decrease in the hepatic antioxidative markers but no hepatic cell damage. APAP administration to rats with hepatocellular IO further amplified the hepatic oxidative stress but induced only hepatocyte feathery degeneration without any increase in serum aminotransaminases. APAP administration to rats with combined hepatocellular and sinusoidal cell IO caused an unexpected decrease in hepatic oxidative stress and increase in the hepatic antioxidative markers and no hepatic cell damage. No hepatic expression of activated c-jun-N-terminal kinase was detected in any of the rats. Conclusions: The hepatic distribution of iron may affect its oxidative/antioxidative milieu. Augmentation of hepatic oxidative stress did not increase the rats’ vulnerability to APAP.