Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury.

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Non-invasive Visualization and Characterization of Bile Canaliculus Formation Using Refractive Index Tomography.

The vital role of bile canaliculus (BC) in liver function is closely related to its morphology. Electron microscopy has contributed to understanding BC morphology; however, its invasiveness limits its use in living specimens. Here, we report non-invasive characterization of BC formation using refractive index (RI) tomography. First, we investigated and characterized the RI distribution of BCs in two-dimensional (2D) cultured HepG2 cells. BCs were identified based on their distinct morphology and functionality, as confirmed using a fluorescence-labeled bile acid analog. The RI distribution of BCs exhibited three common features: (1) luminal spaces with a low RI between adjacent hepatocytes; (2) luminal spaces surrounded by a membranous structure with a high RI; and (3) multiple microvillus structures with a high RI within the lumen. Second, we demonstrated the characterization of BC structures in a three-dimensional (3D) culture model, which is more relevant to the in vivo environment but more difficult to evaluate than 2D cultures. Various BC structures were identified inside HepG2 spheroids with the three features of RI distribution. Third, we conducted comparative analyses and found that the BC lumina of spheroids had higher circularity and lower RI standard deviation than 2D cultures. We also addressed comparison of BC and intracellular lumen-like structures within a HepG2 spheroid, and found that the BC lumina had higher RI and longer perimeter than intracellular lumen-like structures. Our demonstration of the non-destructive, label-free visualization and quantitative characterization of living BC structures will be a basis for various hepatological and pharmaceutical applications.

Microscopic Analysis of Nuclear Speckles in a Viviparous Reptile.

Nuclear speckles are compartments enriched in splicing factors present in the nucleoplasm of eucaryote cells. Speckles have been studied in mammalian culture and tissue cells, as well as in some non-mammalian vertebrate cells and invertebrate oocytes. In mammals, their morphology is linked to the transcriptional and splicing activities of the cell through a recruitment mechanism. In rats, speckle morphology depends on the hormonal cycle. In the present work, we explore whether a similar situation is also present in non-mammalian cells during the reproductive cycle. We studied the speckled pattern in several tissues of a viviparous reptile, the lizard Sceloporus torquatus, during two different stages of reproduction. We used immunofluorescence staining against splicing factors in hepatocytes and oviduct epithelium cells and fluorescence and confocal microscopy, as well as ultrastructural immunolocalization and EDTA contrast in Transmission Electron Microscopy. The distribution of splicing factors in the nucleoplasm of oviductal cells and hepatocytes coincides with the nuclear-speckled pattern described in mammals. Ultrastructurally, those cell types display Interchromatin Granule Clusters and Perichromatin Fibers. In addition, the morphology of speckles varies in oviduct cells at the two stages of the reproductive cycle analyzed, paralleling the phenomenon observed in the rat. The results show that the morphology of speckles in reptile cells depends upon the reproductive stage as it occurs in mammals.

Ultrastructural evidence of four types of lipofuscins in the melanomacrophagic centers in hepatocytes of zebrafish (Denio rerio).

Melanomacrophagic centers (MMCs) were studied in the hepatocytes of zebrafish using transmission electron microscope (TEM). The MMCs with irregular or amoeboid nucleus were located in the hepatocytes adjacent to the bile canaliculi. Several engulfed structures were present in the cytoplasm of MMCs. The most frequent observation was the presence of mitochondria, ranging in size from small to giant, with distorted shape and inconspicuous cristae. Occasionally the fragments of erythrocytes were found. The rough endoplasmic reticulum (rER) showed whirling around the mitochondria and lipid droplets, forming membrane-like structures. The damaged mitochondria were invaded by the lysosomes, and this was covered by a membrane led to the formation of lipofuscin. Four different types of lipofuscins were observed; namely, (1) granular with/without vacuoles of high electron-density, (2) homogenous surrounded by indistinct limiting membrane, (3) lamellated structures similar to inner matrix and cristae of mitochondria, and, (4) compound structure made by the combinations of first 3 types, (granular and homogenous, granular and lamellated, homogenous and lamellated). The present evidence suggests that MMCs in the hepatocytes of zebrafish perform continuous functions of removal of the damaged cellular organelles. The lipofuscin formation work in coordination with the cellular players of immune system and remove pathogens and maintain the internal homeostasis of cells.

The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ß-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of ß-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected versus transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is relocalized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M, and N are required for optimal production of virus-like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.

Mesenchymal stromal cells protect hepatocytes from lipotoxicity through alleviation of endoplasmic reticulum stress by restoring SERCA activity.

The aim of this study was to investigate how mesenchymal stromal cells (MSCs) modulate metabolic balance and attenuate hepatic lipotoxicity in the context of non-alcoholic fatty liver disease (NAFLD). In vivo, male SD rats were fed with high-fat diet (HFD) to develop NAFLD; then, they were treated twice by intravenous injections of rat bone marrow MSCs. In vitro, HepG2 cells were cocultured with MSCs by transwell and exposed to palmitic acid (PA) for 24 hours. The endoplasmic reticulum (ER) stressor thapsigargin and sarco/ER Ca2+ -ATPase (SERCA2)-specific siRNA were used to explore the regulation of ER stress by MSCs. We found that MSC administration improved hepatic steatosis, restored systemic hepatic lipid and glucose homeostasis, and inhibited hepatic ER stress in HFD-fed rats. In hepatocytes, MSCs effectively alleviated the cellular lipotoxicity. Particularly, MSCs remarkably ameliorated the ER stress and intracellular calcium homeostasis induced by either PA or thapsigargin in HepG2 cells. Additionally, long-term HFD or PA stimulation would activate pyroptosis in hepatocytes, which may contribute to the cell death and liver dysfunction during the process of NAFLD, and MSC treatment effectively ameliorates these deleterious effects. SERCA2 silencing obviously abolished the ability of MSCs against the PA-induced lipotoxicity. Conclusively, our study demonstrated that MSCs were able to ameliorate liver lipotoxicity and metabolic disturbance in the context of NAFLD, in which the regulation of ER stress and the calcium homeostasis via SERCA has played a key role.

A postprandial FGF19-SHP-LSD1 regulatory axis mediates epigenetic repression of hepatic autophagy.

Lysosome-mediated autophagy is essential for cellular survival and homeostasis upon nutrient deprivation, but is repressed after feeding. Despite the emerging importance of transcriptional regulation of autophagy by nutrient-sensing factors, the role for epigenetic control is largely unexplored. Here, we show that Small Heterodimer Partner (SHP) mediates postprandial epigenetic repression of hepatic autophagy by recruiting histone demethylase LSD1 in response to a late fed-state hormone, FGF19 (hFGF19, mFGF15). FGF19 treatment or feeding inhibits macroautophagy, including lipophagy, but these effects are blunted in SHP-null mice or LSD1-depleted mice. In addition, feeding-mediated autophagy inhibition is attenuated in FGF15-null mice. Upon FGF19 treatment or feeding, SHP recruits LSD1 to CREB-bound autophagy genes, including Tfeb, resulting in dissociation of CRTC2, LSD1-mediated demethylation of gene-activation histone marks H3K4-me2/3, and subsequent accumulation of repressive histone modifications. Both FXR and SHP inhibit hepatic autophagy interdependently, but while FXR acts early, SHP acts relatively late after feeding, which effectively sustains postprandial inhibition of autophagy. This study demonstrates that the FGF19-SHP-LSD1 axis maintains homeostasis by suppressing unnecessary autophagic breakdown of cellular components, including lipids, under nutrient-rich postprandial conditions.

Maturation of Lipophagic Organelles in Hepatocytes Is Dependent Upon a Rab10/Dynamin-2 Complex.

BACKGROUND AND AIMS: Hepatocytes play a central role in storage and utilization of fat by the liver. Selective breakdown of lipid droplets (LDs) by autophagy (also called lipophagy) is a key process utilized to catabolize these lipids as an energy source. How the autophagic machinery is selectively targeted to LDs, where it mediates membrane engulfment and subsequent degradation, is unclear. Recently, we have reported that two distinct GTPases, the mechanoenzyme, dynamin2 (Dyn2), and the small regulatory Rab GTPase, Rab10, work independently at distinct steps of lipophagy in hepatocytes. APPROACH AND RESULTS: In an attempt to understand how these proteins are regulated and recruited to autophagic organelles, we performed a nonbiased biochemical screen for Dyn2-binding partners and found that Dyn2 actually binds Rab10 directly through a defined effector domain of Rab10 and the middle domain of Dyn2. These two GTPases can be observed to interact transiently on membrane tubules in hepatoma cells and along LD-centric autophagic membranes. Most important, we found that a targeted disruption of this interaction leads to an inability of cells to trim tubulated cytoplasmic membranes, some of which extend from lipophagic organelles, resulting in LD accumulation. CONCLUSIONS: This study identifies a functional, and direct, interaction between Dyn2 and a regulatory Rab GTPase that may play an important role in hepatocellular metabolism.

Ground-glass hepatocellular inclusions are associated with polypharmacy.

Ground-glass (GG) hepatocytes are classically associated with chronic hepatitis B (HBV) infection, storage disorders, or cyanamide therapy. In a subset of cases, an exact etiology cannot be identified. In this study, we sought to characterize the clinical, histological, and ultrastructural findings associated with HBV-negative GG hepatocytes. Our institutional laboratory information system was searched from 2000 to 2019 for all cases of ground-glass hepatocytes. Ten liver biopsies with GG hepatocellular inclusions and negative HBV serology, no known history of storage disorders, or cyanamide therapy were reviewed. Half of the patients had history of organ transplantation and/or malignancy. These patients took on average 8.1 medications (range: 3-14) with the most common medications being immunosuppressive and health supplements. Histologically, GG hepatocytes show either peri-portal or centrizonal distribution. The inclusions are PAS-positive and diastase sensitive. Electron microscopy showed intracytoplasmic granular inclusions with low electron density, consistent with unstructured glycogen. In summary, GG hepatocytes are a rare finding in liver biopsies, but are more common in patients with hepatitis B. They can also be seen in HBV-negative patients who have polypharmacy. In these cases, they are the result of unstructured glycogen accumulation putatively due to altered cell metabolism.

Hepatic and Extrahepatic Sources and Manifestations in Endoplasmic Reticulum Storage Diseases.

Alpha-1-antitrypsin (AAT) and fibrinogen are secretory acute phase reactant proteins. Circulating AAT and fibrinogen are synthesized exclusively in the liver. Mutations in the encoding genes result in conformational abnormalities of the two molecules that aggregate within the rough endoplasmic reticulum (RER) instead of being regularly exported. That results in AAT-deficiency (AATD) and in hereditary hypofibrinogenemia with hepatic storage (HHHS). The association of plasma deficiency and liver storage identifies a new group of pathologies: endoplasmic reticulum storage disease (ERSD).

Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis.

BACKGROUND & AIMS: Wilson disease (WD) is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in the liver and brain. It is caused by mutations in the adenosine triphosphatase copper transporting ß gene (ATP7B), which encodes a protein that transports copper from hepatocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to decrease copper toxicity in patients with WD. METHODS: We used RNA-seq to compare gene expression patterns between wild-type and ATP7B-knockout HepG2 cells exposed to copper. We collected blood and liver tissues from Atp7b-/- and Atp7b+/- (control) rats (LPP) and mice; some mice were given 5 daily injections of an autophagy inhibitor (spautin-1) or vehicle. We obtained liver biopsies from 2 patients with WD in Italy and liver tissues from patients without WD (control). Liver tissues were analyzed by immunohistochemistry, immunofluorescence, cell viability, apoptosis assays, and electron and confocal microscopy. Proteins were knocked down in cell lines using small interfering RNAs. Levels of copper were measured in cell lysates, blood samples, liver homogenates, and subcellular fractions by spectroscopy. RESULTS: After exposure to copper, ATP7B-knockout cells had significant increases in the expression of 103 genes that regulate autophagy (including MAP1LC3A, known as LC3) compared with wild-type cells. Electron and confocal microscopy visualized more autophagic structures in the cytoplasm of ATP7B-knockout cells than wild-type cells after copper exposure. Hepatocytes in liver tissues from patients with WD and from Atp7b-/- mice and rats (but not controls) had multiple autophagosomes. In ATP7B-knockout cells, mammalian target of rapamycin (mTOR) had decreased activity and was dissociated from lysosomes; this resulted in translocation of the mTOR substrate transcription factor EB to the nucleus and activation of autophagy-related genes. In wild-type HepG2 cells (but not ATP7B-knockout cells), exposure to copper and amino acids induced recruitment of mTOR to lysosomes. Pharmacologic inhibitors of autophagy or knockdown of autophagy proteins ATG7 and ATG13 induced and accelerated the death of ATP7B-knockout HepG2 cells compared with wild-type cells. Autophagy protected ATP7B-knockout cells from copper-induced death. CONCLUSION: ATP7B-deficient hepatocytes, such as in those in patients with WD, activate autophagy in response to copper overload to prevent copper-induced apoptosis. Agents designed to activate this autophagic pathway might decrease copper toxicity in patients with WD.

Effect of lithium carbonate on autophagy and proliferative activity of isolated hepatocytes.

The hepatocytes were cultivated in the presence of lithium carbonate (LC) for drugs testing or possible source for transplantation in the treatment of hereditary or terminal liver diseases. The LC, as an inducer of autophagy, is a promising drug for maintaining cell homeostasis and has a significant effect on the ultrastructural organization of hepatocyte cells. Within current investigation, new mechanisms of the biological effects of lithium and the ultrastructural analysis of the primary culture of hepatocytes were studied via flow cytofluorometry, light, and electron microscopy methods. Obtained results demonstrate the absence of the toxic effect of 5 mM of LC on the primary hepatocyte culture. In addition, LC does not block the cell cycle at the G0/G1 stage after 24 h of hepatocyte cultivation and promotes the preservation of their viability by 48 h of the experiment. Moreover, LC does not stimulate hepatocyte apoptosis, induces autophagy and the preserves the proliferative activity of hepatocytes.

Paracrine signals regulate human liver organoid maturation from induced pluripotent stem cells.

A self-organizing organoid model provides a new approach to study the mechanism of human liver organogenesis. Previous animal models documented that simultaneous paracrine signaling and cell-to-cell surface contact regulate hepatocyte differentiation. To dissect the relative contributions of the paracrine effects, we first established a liver organoid using human induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as previously reported. Time-lapse imaging showed that hepatic-specified endoderm iPSCs (HE-iPSCs) self-assembled into three-dimensional organoids, resulting in hepatic gene induction. Progressive differentiation was demonstrated by hepatic protein production after in vivo organoid transplantation. To assess the paracrine contributions, we employed a Transwell system in which HE-iPSCs were separately co-cultured with MSCs and/or HUVECs. Although the three-dimensional structure did not form, their soluble factors induced a hepatocyte-like phenotype in HE-iPSCs, resulting in the expression of bile salt export pump. In conclusion, the mesoderm-derived paracrine signals promote hepatocyte maturation in liver organoids, but organoid self-organization requires cell-to-cell surface contact. Our in vitro model demonstrates a novel approach to identify developmental paracrine signals regulating the differentiation of human hepatocytes.

Exosomal Transport of Hepatocyte-Derived Drug-Modified Proteins to the Immune System.

Idiosyncratic drug-induced liver injury (DILI) is a rare, often difficult-to-predict adverse reaction with complex pathomechanisms. However, it is now evident that certain forms of DILI are immune-mediated and may involve the activation of drug-specific T cells. Exosomes are cell-derived vesicles that carry RNA, lipids, and protein cargo from their cell of origin to distant cells, and they may play a role in immune activation. Herein, primary human hepatocytes were treated with drugs associated with a high incidence of DILI (flucloxacillin, amoxicillin, isoniazid, and nitroso-sulfamethoxazole) to characterize the proteins packaged within exosomes that are subsequently transported to dendritic cells for processing. Exosomes measured between 50 and 100 nm and expressed enriched CD63. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) identified 2,109 proteins, with 608 proteins being quantified across all exosome samples. Data are available through ProteomeXchange with identifier PXD010760. Analysis of gene ontologies revealed that exosomes mirrored whole human liver tissue in terms of the families of proteins present, regardless of drug treatment. However, exosomes from nitroso-sulfamethoxazole-treated hepatocytes selectively packaged a specific subset of proteins. LC/MS-MS also revealed the presence of hepatocyte-derived exosomal proteins covalently modified with amoxicillin, flucloxacillin, and nitroso-sulfamethoxazole. Uptake of exosomes by monocyte-derived dendritic cells occurred silently, mainly through phagocytosis, and was inhibited by latrunculin A. An amoxicillin-modified 9-mer peptide derived from the exosomal transcription factor protein SRY (sex determining region Y)-box 30 activated naïve T cells from human leukocyte antigen A*02:01-positive human donors. Conclusion: This study shows that exosomes have the potential to transmit drug-specific hepatocyte-derived signals to the immune system and provide a pathway for the induction of drug hapten-specific T-cell responses.

A stereological study of developmental changes in hepatocyte ultrastructure of zebrafish (Danio rerio).

Histopathology can reveal toxicant-induced changes in the structure of a tissue or organ. A prerequisite for histopathological studies is a sound knowledge of the morphology of the anatomical structure in the normal or healthy state. Zebrafish larvae can provide a tool for studies focused on hepatotoxicity at early stages of development; therefore, the fine structure of the organ should be well characterised. To date, liver structure at 72 and 120 hr post-fertilisation (hpf) has not been reported in detail and this study aimed to fill this scientific gap. A stereological approach allowed for quantitative description of the liver and revealed ultrastructural alterations occurring with time of development. These included a significant increase in the absolute volume of hepatocytes, mitochondria and rough endoplasmic reticulum (rER) during the period of study. The surface area of rER, and of outer and inner mitochondrial membranes also increased. There was no change in the absolute volume of the nuclei. This study provides a quantitative spatial and temporal framework for future research aiming to detect early developmental changes in the liver.

Ultrastructure of Hepatocytes from Laboratory Mice Fed a Standard Dry Laboratory Animal Diet.

The significant destructive changes in ultrastructure of hepatocytes from laboratory mice kept in different vivariums in Moscow and fed with dry laboratory animal diets acquired from different domestic manufacturers that were not standardized for initial products were demonstrated using electron microscopy. Furthermore, disruption in the ultrastructure of liver parenchymal cells occurred regardless of the animal status (SPF or conventional), conditions of various vivariums, as well as the feed manufacturer. At the same time, studies on ultrastructure of liver hepatocytes from mice kept in the Charles River Laboratory facilities in Germany and fed with the Altromin Spezialfutter laboratory animal diet (GmbH & Co., Germany) that was produced using quality control of ingredients did not reveal destructive changes in the internal ultrastructure of hepatocytes. However, if these mice were later fed with the food produced in local manufactures, changes in the structure of liver cells developed after 2 months. Thus, feeding with dry diet from the domestic producers of an unspecified composition causes significant changes in the ultrastructure of hepatocytes in control animals, reflecting the development of some pathological processes in the body.

A fluorescence method to visualize the nuclear boundary by the lipophilic dye DiI.

Here, we describe a procedure to fluorescently contrast the nuclear boundary using the lipophilic carbocyanine dye DiI in cultured human cells. Our procedure is simple and is applicable to detect nuclear boundary defects, which may be relevant to studies on nuclear envelope dynamics, micronuclei formation and cancer biology. ABBREVIATIONS: DiI: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; DiO: 3,3'-dioctadecyloxacarbocyanine perchlorate; NE: nuclear envelope; RanBP2: Ran-binding protein 2/Nucleoporin 358.

Effect of Betaine Supplementation on Liver Tissue and Ultrastructural Changes in Methionine-Choline-Deficient Diet-Induced NAFLD.

Nonalcoholic fatty liver disease (NAFLD) represents a hepatic manifestation of metabolic syndrome. The aim of this study was to examine the effect of betaine on ultrastructural changes in the mouse liver with methionine- and choline-deficient (MCD) diet-induced NAFLD. Male C57BL/6 mice were divided into groups: Control-fed with standard chow, BET-standard chow supplemented with betaine (1.5% w/v drinking water), MCD-fed with MCD diet, and MCD + BET-MCD diet with betaine supplementation for 6 weeks. Liver samples were taken for pathohistology and transmission electron microscopy. The MCD diet-induced steatosis, inflammation, and balloon-altered hepatocytes were alleviated by betaine. MCD diet induced an increase in mitochondrial size versus the control group (p < 0.01), which was decreased in the betaine-treated group. In the MCD diet-fed group, the total mitochondrial count decreased versus the control group (p < 0.01), while it increased in the MCD + BET group versus MCD (p < 0.01). Electron microscopy showed an increase in the number of autophagosomes in the MCD and MCD + BET group versus control, and a significant difference in autophagosomes number was detected in the MCD + BET group by comparison with the MCD diet-treated group (p < 0.05). Betaine decreases the number of enlarged mitochondria, alleviates steatosis, and increases the number of autophagosomes in the liver of mice with NAFLD.

Induction of hepatic portal fibrosis, mitochondria damage, and extracellular vesicle formation in Sprague-Dawley rats exposed to copper, manganese, and mercury, alone and in combination.

Increased anthropogenic activity and subsequent environmental exposure to heavy metals induce the production of reactive oxygen species (ROS), which increases oxidative stress and the risk of associated diseases. The aim of this study, in a subacute model of toxicity, was to investigate the effects of copper (Cu), manganese (Mn), and mercury (Hg) alone and in combination on the liver tissue of male Sprague-Dawley rats, exposed orally to 100 times the World Health Organization's acceptable water limits of each metal. General histological alterations as well as ultrastructural changes were investigated using light microscopy and transmission electron microscopy (TEM) respectively. Exposure to Cu, Mn, and Hg, alone and in combinations, caused hydropic swelling of the hepatocytes, dilation of the sinusoids, formation of binucleated hepatocytes with an increased inflammatory cell accumulation at the portal triad. Increased collagen deposition with associated fibrosis was also observed. Evaluation of hepatocyte ultrastructure revealed mitochondrial membrane damage and inner membrane swelling especially for hepatocytes exposed to Mn. Extracellular vesicle (EV) formation was observed in the liver tissue of all exposed rats. Furthermore, increased damage observed for metal combinations was possibly due to synergism. In conclusion, Cu, Mn, and Hg alone and as part of a mixture cause cellular damage, inflammation, and fibrosis increasing the risk of associated diseases.

Effect of MGHPPGP Peptide Administered during the Post-Traumatic Period on Morphometric Parameters of the Neocortex and Hippocampus, Hepatocytes, and Epithelium of the Tongue and Small Intestine in Rats.

The effect of glyproline-containing peptide MGHPPGP (Met-Glu-His-Phe-Pro-Gly-Pro) was studied in experiments on male Wistar rats with modeled traumatic brain injury. The peptide was administered intraperitoneally in a dose of 1 mg/kg in 3 h and on days 2, 3, 4, 5 after injury. We evaluated morphometric parameters of the epithelial cells of the tongue, small intestine, and liver cells (AgNOR staining), neuronal layers II and V of the neocortex of the parietal lobe and hippocampal CA1 field (staining with gallocyanin) were evaluated in the post-traumatic period. Traumatic brain injury (TBI) was induced in rats by using the impact model (WDM; weight drop method). MGHPPGP peptide corrected the activity indicators of the nuclear organizer regions in epitheliocytes of the tongue.