Hepatic Cell Damage Research Articles

Progression of DNA damage induced by a glyphosate-based herbicide in fish (Anguilla anguilla) upon exposure and post-exposure periods — Insights into the mechanisms of genotoxicity and DNA repair

Roundup® is a glyphosate-based herbicide widely used with both agricultural and non-agricultural purposes, which has been demonstrated to represent a risk to non-target aquatic organisms, namely fish. Among the described effects to fish, genotoxicity has been pointed out as one of the most hazardous. However, the genotoxic mechanisms of Roundup® as well as the involvement of the oxidative DNA damage repair system are not entirely understood. Hence, this work aimed to improve the knowledge on the progression of DNA damage upon short-term exposure (3days) and post-exposure (1–14days) periods in association with DNA repair processes in Anguilla anguilla exposed to Roundup® (58 and 116μgL−1). DNA damage in hepatic cells was evaluated by the comet assay improved with the DNA-lesion specific endonucleases FPG and EndoIII. In order to evaluate the oxidative DNA damage repair ability, an in vitro base excision repair (BER) assay was performed, testing hepatic subcellular extracts. Besides the confirmation of the genotoxic potential of this herbicide, oxidative damage was implicit as an important mechanism of genetic damage, which showed to be transient, since DNA integrity returned to the control levels on the first day after cessation of exposure. An increased capacity to repair oxidative DNA damage emerging in the post-exposure period revealed to be a crucial pathway for the A. anguilla recovery; nevertheless, DNA repair machinery showed to be susceptible to inhibitory actions during the exposure period, disclosing another facet of the risk associated with the tested agrochemical.

Experimenting Liver Fibrosis Diagnostic by Two Photon Excitation Microscopy and Bag-of-Features Image Classification

The accurate staging of liver fibrosis is of paramount importance to determine the state of disease progression, therapy responses, and to optimize disease treatment strategies. Non-linear optical microscopy techniques such as two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) can image the endogenous signals of tissue structures and can be used for fibrosis assessment on non-stained tissue samples. While image analysis of collagen in SHG images was consistently addressed until now, cellular and tissue information included in TPEF images, such as inflammatory and hepatic cell damage, equally important as collagen deposition imaged by SHG, remain poorly exploited to date. We address this situation by experimenting liver fibrosis quantification and scoring using a combined approach based on TPEF liver surface imaging on a Thioacetamide-induced rat model and a gradient based Bag-of-Features (BoF) image classification strategy. We report the assessed performance results and discuss the influence of specific BoF parameters to the performance of the fibrosis scoring framework.

In vivo restoration of hepatic and nephro protective potential of hesperidin and ellagic acid against mercuric chloride intoxicated rats

The present experimental study is to investigate the efficacy of some phytochemicals on heavy metal intoxicated animals. Commonly phytochemicals have played a vital role for protective effect of oxidative stress, which is induced by heavymetals in animals. At sub-lethal dose of mercuric chloride (1.23mg/kg body weight) treated rat liver tissue shows hepatic cell damage and alteration of its metabolic activities by the way of liver marker enzymes. The hepato-protective effect of Hesperidin and ellagic acid was tested against mercuric chloride induced hepato-toxicity in rats. In the present study, drastically altered in the level of Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Lactic dehyrogenase (LDH), bilirubin, albumin, cholesterol, urea and creatinine levels were observed in the blood serum of mercury intoxicated rats. The activity of liver marker enzymes such as ALT AST, ALP and LDH were significantly increased and albumin was simultaneously decreased in mercuric chloride intoxicated rats. Administration of Hesperidin and Ellagic acid (5mg/kg body weight) on mercuric chloride intoxicated rats not only reduced the liver markers enzymes and bilirubin and cholesterol levels and also maintain their level to near normal condition. Hesperidin and ellagic acid alone treated animals did not alter the ALT, AST, ALP, LDH, bilirubin, albumin, cholesterol urea and creatinine levels in serum. Our results indicate that treatment for hesperidin and ellagic acid exhibited the strong hepatoprotective activity against mercuric chloride induced hepatotoxicity.

TAK1 Binding Protein 2 Is Essential for Liver Protection from Stressors

The liver is the first line of defense from environmental stressors in that hepatocytes respond to and metabolize them. Hence, hepatocytes can be damaged by stressors. Protection against hepatic cell damage and cell death is important for liver function and homeostasis. TAK1 (MAP3K7) is an intermediate of stressors such as bacterial moieties–induced signal transduction pathways in several cell types. Tak1 deficiency has been reported to induce spontaneous hepatocellular carcinoma. However, the regulatory mechanism of TAK1 activity in liver stress response has not yet been defined. Here we report that activation of TAK1 through TAK1 binding protein 2 (TAB2) is required for liver protection from stressors. We found that a bacterial moiety, lipopolysaccharides (LPS), activated TAK1 in primary hepatocytes, which was diminished by deletion of TAB2. Mice having hepatocyte-specific deletion of the Tab2 gene exhibited only late-onset moderate liver lesions but were hypersensitive to LPS-induced liver injury. Furthermore, we show that a chemical stressor induced greatly exaggerated liver injury in hepatocyte-specific Tab2-deficient mice. These results demonstrate that TAB2 is a sensor of stress conditions in the liver and functions to protect the liver by activating the TAK1 pathway.

Histopathological Changes Associated with Exposure of Male Mice to Profenofos and Chlorpyrifos

Aims: The histopathological effects of Profenofos, and Chlorpyrifos, as synthetic organophosphorus pesticides, on the liver, kidney, brain and spleen tissues in mice (Mus musculus) were determined by light microscopy. Recently the toxic effects of pesticides have been of public interest. The usage of pesticides is still the most effective and accepted means to protect plants from the pests and to increases productivity. The misuse of pesticides is connected with serious problems of pollution and health hazards. Profenofos and Chlorpyrifos is used widely in Egypt and they play a vital role in controlling Lepidopteron pests of cotton and vegetables [1]. Study Design: Mice were treated with Profenofos, and Chlorpyrifos sub-lethal concentrations (1/10, 1/40 and ADI LD50) orally to twice a week for 30, 60, and 90 consecutive days. Place and Duration of Study: Department of chemistry Faculty of Agriculture, Cairo University, Egypt, between June 2012 and January 2013. Results: Histopathological examination revealed various abnormalities in liver tissues, such as congestion of blood vessels, vacuolar degeneration of hepatic cells, focal Original Research Article Annual Research & Review in Biology, 4(5): 766-777, 2014 767 infiltration and mononuclear cells, Moreover, all central veins and other hepatic blood vessels were dilated, some hepatic cells showed necrosis, disorganization with the formation of a denoid structure and some areas showed hepatocytomegaly with the increase of the number of cells showing double nuclei. Pathological finding in kidney showed perivascular edema with congestion of renal blood vessels, infiltration of mononuclear cells and around some of glomerular tubules, edema of Bowman's capsule and some renal tubules showed coagulation necrosis. Pathological finding in spleen showed disorganization of lymphocytes in lymphoid follicles and in white pulp, depletion of lymphocyts with sub capsular edema, and other cases showed increasing the number of megaterocytes with hemorrhages and haemosiderosis. Pathological finding in Brain showed menengial hemorrhages and congestion of blood vessels, with neuronophagia and satelletosis and sub meningial encephalomalacia, with neuronal degeneration of purkinjie cells were noticed and lesions, there was lyses of some neurons with demylenation of nerve fibers and privascular and pricellular edema. This investigation proves the toxic effects of Profenofos, and Chlorpyrifos at organ level. Conclusion: The histopathological data showed that profenofos exhibited histopathological changes in liver, kidney, spleen and brain. Liver showed hepatic cell damage with degenerative changes. Kidney showed heamorrhages, edema, necrosis and glomeruli shrinkage. The spleen showed slight deplesion of the lymphocytes of the white pulp. The brain showed interstitial edema and severe necrosis. From these results we concluded that liver is the most sensitive organ and profenofos damage the structure of liver cells more severely than chlorpyrifos on albino mice.

Amifostine enhances the antioxidant and hepatoprotective effects of UW and HTK preservation solutions.

To investigate whether amifostine contributes to the antioxidant and cytoprotective effects of histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin (UW) preservation solutions. Forty-eight Sprague Dawley male rats were equally divided into six groups: (1) ringer Lactate (RL) group; (2) RL + amifostine (RL + A) group; (3) HTK group; (4) HTK + A group; (5) UW group; and (6) UW + A group. Rats in the RL + A, HTK + A and UW + A groups were administered amifostine intraperitoneally at a dose of 200 mg/kg prior to laparotomy. The RL group was perfused with RL into the portal vein. The RL + A group were perfused with RL into the portal vein after amifostine administration. The HTK group received an HTK perfusion while the HTK + A group received an HTK perfusion after administration of amifostine. The UW group received a perfusion of UW, while the UW + A group received a UW perfusion after amifostine administration. Liver biopsy was performed to investigate histopathological, immunochemical [transferase mediated dUTP nick end labeling (TUNEL), inducible nitric oxide syntetase (iNOS)] and ultrastructural alterations. Biochemical alterations were determined by examining levels of alanine aminotransferase, alkaline phosphatase and nitric oxide in the perfusion fluid. Pathological sinusoidal dilatation and centrilobular hydropic alteration were significantly lower in the groups that received amifostine prior to preservation solution perfusion. Although the best results were obtained in the UW + A group, we did not observe a statistically significant difference between the UW + A and HTK + A groups. iNOS grades were significantly lower in the amifostine groups 12 h after treatment. When the amifostine groups were compared against each other, the iNOS grades obtained from the UW + A and HTK + A groups were similar while the RL + A group had a much poorer score. TUNEL assays demonstrated a lower apoptosis ratio in the amifostine groups than in the non-amifostine groups 12 h after treatment. No statistically significant difference was observed between the UW + A and HTK + A groups for apoptosis. Cellular ultrastructure was best preserved in the UW + A and HTK + A groups. Here, we show that preoperative administration of a single dose of amifostine is sufficient to minimize the preservation damage in hepatic cells.

Protection of hepatic cell damage and antimicrobial evaluation of chitosan-catechin conjugate

The chitosan-catechin conjugate was developed by free radical-induced conjugating reaction, and its protection ability against hydrogen peroxide-induced hepatic damage in human normal Chang liver cells and antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and foodborne pathogens were investigated. Treatment of hydrogen peroxide (650 μM) on Chang liver cells decreased cell viability up to 59.38% compared to the non-treatment group; however, cotreatment of the chitosan-catechin conjugate increased cell viability up to 76.90% at 200 μg/mL, and the protection ability was significantly higher than the unmodified chitosan (p <0.05). The chitosan-catechin conjugate significantly (p <0.05) inhibited the formation of intracellular reactive oxygen species and lipid peroxidation in Chang liver cells. Moreover, the chitosan-catechin conjugate increased glutathione levels in normal condition as well as under oxidative stress by hydrogen peroxide. Additionally, the chitosan-catechin conjugate showed increased antimicrobial activity against MRSA and foodborne pathogens as compared to those of the unmodified chitosan.

Gymnaster koraiensis and its major components, 3,5-di-O-caffeoylquinic acid and gymnasterkoreayne B, reduce oxidative damage induced by tert-butyl hydroperoxide or acetaminophen in HepG2 cells

We investigated the protective effects of Gymnaster koraiensis against oxidative stress-induced hepatic cell damage. We used two different cytotoxicity models, i.e., the administration of tert-butyl hydroperoxide (t-BHP) and acetaminophen, in HepG2 cells to evaluate the protective effects of G. koraiensis. The ethyl acetate (EA) fraction of G. koraiensis and its major compound, 3,5-di-O-caffeoylquinic acid (DCQA), exerted protective effects in the t-BHP-induced liver cytotoxicity model. The EA fraction and DCQA ameliorated t-BHP-induced reductions in GSH levels and exhibited free radical scavenging activity. The EA fraction and DCQA also significantly reduced t-BHP-induced DNA damage in HepG2 cells. Furthermore, the hexane fraction of G. koraiensis and its major compound, gymnasterkoreayne B (GKB), exerted strong hepatoprotection in the acetaminopheninduced cytotoxicity model. CYP 3A4 enzyme activity was strongly inhibited by the extract, hexane fraction, and GKB. The hexane fraction and GKB ameliorated acetaminophen-induced reductions in GSH levels and protected against cell death. [BMB Reports 2013; 46(10): 513-518]

Effect of heat stress and recovery on viability, oxidative damage, and heat shock protein expression in hepatic cells of grass carp (Ctenopharyngodon idellus)

In this study, we investigated the effects of hyperthermia and recovery on cell viability, lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, malondialdehyde (MDA), total antioxidant capacity (T-AOC), and heat shock protein (HSP60, 70, and 90) mRNA expression in the hepatic cells of the grass carp, Ctenopharyngodon idellus. Triplicate groups of cultured cells were exposed to 30, 32, or 34°C for 0.5h and then immediately incubated at 27°C in 5% CO2 for 6, 12, 24, or 48h. Hyperthermia stress greatly reduced cell viability and increased LDH release. Cell damage declined after recovery. Hyperthermia stress increased the lipid peroxide levels and reduced the antioxidant capacity (e.g., reduced SOD and T-AOC) of the cells. However, oxidative damage declined as the recovery period increased, and the levels of MDA, SOD, and T-AOC were restored. After cells were exposed to 32°C, the expression of HSP60 after recovery for 1, 2, and 4h (P<0.05), the expression of HSP70 after recovery for 0.5 and 1h (P<0.01), and the expression of HSP90 throughout recovery were significantly higher (P<0.01) than the prestress levels. During the recovery period, the variations in HSP gene expression reflected the transition period from a state of cellular growth to one of the cellular repairs. In conclusion, hyperthermia depresses cell viability, induces oxidative damage, and increases HSP expression, which plays an important role during hyperthermic stress in grass carp hepatic cells.

Anticarcinogenic effect of brucine in diethylnitrosamine initiated and phenobarbital-promoted hepatocarcinogenesis in rats

We evaluated the effects of brucine on N-nitrosodiethylamine (DENA)-induced hepatocarcinogenesis in rats. Initiation of hepatocarcinogenesis was done by intraperitoneal injection of diethylnitrosamine (DENA) followed by promotion with phenobarbital. The rats were exposed to dietary brucine for 4weeks prior to initiation, and the treatment was continued for 22 consecutive weeks. Brucine decreased the incidence, total number, multiplicity, size and volume of preneoplastic hepatic nodules in a dose-dependent manner. Administration of DENA induced hepatocellular carcinoma (HCC), as evidenced by changes in histopathological architecture, increased activity of cytochrome P450, decreased activity of glutathione Stransferase (GST) as well as decreased antioxidant status, enhanced lipid peroxidation, increased liver marker enzymes. Western blot analysis showed decreased expression of cyclin D1 and Bcl-2 with activation of caspase-3 and increased expression of Bax. Immunohistochemical demonstrated the decreased expression of the PCNA and VEGF. These results indicate that brucine prevents lipid peroxidation and hepatic cell damage and also protects the antioxidant system in DENA-induced hepatocarcinogenesis.

Specific Inhibition of the Redox Activity of Ape1/Ref-1 by E3330 Blocks Tnf-Α-Induced Activation of Il-8 Production in Liver Cancer Cell Lines

APE1/Ref-1 is a main regulator of cellular response to oxidative stress via DNA-repair function and co-activating activity on the NF-κB transcription factor. APE1 is central in controlling the oxidative stress-based inflammatory processes through modulation of cytokines expression and its overexpression is responsible for the onset of chemoresistance in different tumors including hepatic cancer. We examined the functional role of APE1 overexpression during hepatic cell damage related to fatty acid accumulation and the role of the redox function of APE1 in the inflammatory process. HepG2 cells were stably transfected with functional and non-functional APE1 encoding plasmids and the protective effect of APE1 overexpression toward genotoxic compounds or FAs accumulation, was tested. JHH6 cells were stimulated with TNF-α in the presence or absence of E3330, an APE1 redox inhibitor. IL-8 promoter activity was assessed by a luciferase reporter assay, gene expression by Real-Time PCR and cytokines (IL-6, IL-8, IL-12) levels measured by ELISA. APE1 over-expression did not prevent cytotoxicity induced by lipid accumulation. E3330 treatment prevented the functional activation of NF-κB via the alteration of APE1 subcellular trafficking and reduced IL-6 and IL-8 expression induced by TNF-α and FAs accumulation through blockage of the redox-mediated activation of NF-κB. APE1 overexpression observed in hepatic cancer cells may reflect an adaptive response to cell damage and may be responsible for further cell resistance to chemotherapy and for the onset of inflammatory response. The efficacy of the inhibition of APE1 redox activity in blocking TNF-α and FAs induced inflammatory response opens new perspectives for treatment of inflammatory-based liver diseases.

The effect of hyperthermia on cell viability, oxidative damage, and heat shock protein expression in hepatic cells of grass carp (Ctenopharyngodon idellus)

The purpose of the study was to investigate the effects of mild hyperthermia on cell viability, release of lactate dehydrogenase (LDH), superoxide dismutase (SOD) activity, malondialdehyde (MDA) formation, total antioxidant capacity (T-AOC), and the relative mRNA levels of heat shock protein (HSP60, 70, and 90) in hepatic cells of grass carp (Ctenopharyngodon idellus) before and after temperature stress. Cultured cells were exposed to thermal stress (32°C) for 0.5, 1, 2, 4, and 8h. The results showed that hyperthermia stress significantly reduced cell viability (P<0.01) and increased LDH release at 0.5 and 1h (P<0.05). Additionally, hyperthermia stress led to oxidative stress as evidenced by significantly decreased T-AOC after treating cells for 0.5 and 8h (P<0.05). SOD activity also significantly decreased after 1h of stress (P<0.05), but MDA formation increased after 8h of stress (P<0.05). This may be partly responsible for the lower cell viability and higher LDH release we observed. The differences between SOD activity, MDA formation, and T-AOC between the 2h treatment group and the control were smaller than that of other groups. This indicated that cellular antioxidant enzyme systems play an important role in the defense against oxidative stress. Further tests showed that the expression of HSP60 at 1, 2, and 4h (P<0.05), HSP70 at 0.5 and 1h (P<0.01), and HSP90 at all time points after stress were higher (P<0.01) than pre-stress levels. This suggested that HSPs possess the ability to modulate cellular anti-stress responses and play key roles in protecting organisms from heat stress. In conclusion, hyperthermia inhibits cell proliferation, induces cell oxidative stress, and enhances HSP expression in hepatic cells of grass carp.

Lipopolysaccharide blocks induction of unfolded protein response in human hepatoma cell lines

In the present study, we examined whether unfolded protein response (UPR) determined the hepatic cell damage induced by an innate immune response including TLR signaling pathways. We observed that lipopolysaccharide (LPS) transcriptionally downregulates 78-kDa glucose-regulated protein/immunoglobulin heavy-chain binding protein (GRP78/Bip), known to confer resistance to apoptosis. We also observed that LPS blocked the induction of UPR and led to poly(ADP-ribose) polymerase (PARP) cleavage in hepatocytes. We also demonstrated that overexpression of GRP78 rescued HepG2 cells treated with LPS from PARP cleavage. These data suggest that UPR downregulation could be a collateral effect of the LPS treatment. We speculate that UPR is an important factor of hepatic cell damage induced by an innate immune response.

Function of the liver and bile ducts in humans exposed to lead

Lead is very common in the environment, and it is therefore important to characterize its possible adverse health effects. The aim of this study was to evaluate the impact of lead exposure on selected functions of the liver and bile ducts in people who are chronically exposed to the metal because of their occupations. To provide this information, the activity of specific enzymes and the bilirubin concentration were determined in blood serum, and morphological parameters of the liver and bile ducts were evaluated using the ultrasonic imaging method. Healthy male employees of a lead-zinc processing facility (n=145) who were occupationally exposed to lead were divided into two subgroups as a function of the lead concentrations in blood (PbB): low lead exposure (PbB=20-35μg/dl; n=57) and high lead exposure (PbB=35-60μg/dl; n = 88). Human exposure to lead compounds was found to cause liver enlargement and to activate inflammatory reactions with the characteristics of moderate cholestasis within the bile ducts, while no characteristics of necrotic damage of hepatic cells were noted. It seems that lipid peroxidation can be one of the toxic mechanisms of lead which induce moderate cholestasis. The effects depend on the extent of the lead exposure and were greater in subjects with higher exposure levels, particularly subjects with PbB values greater than 35μg/dl.

Inhibitory effect of naringenin (citrus flavonone) on N-nitrosodiethylamine induced hepatocarcinogenesis in rats

We evaluated the effects of naringenin on N-nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis in rats. Administration of NDEA induced hepatocellular carcinoma (HCC), as evidenced by changes in histopathological architecture, increased activity of cytochrome P450, decreased activity of glutathione S-transferase (GST) as well as decreased antioxidant status, enhanced lipid peroxidation and increased liver marker enzymes. Pre- and post-treatment with naringenin effectively suppressed NDEA-initiated hepatocarcinoma and the associated preneoplastic lesions by modulating xenobiotic-metabolizing enzymes (XMEs), alleviating lipid peroxidation (through both free radical scavenging and the enhanced antioxidant status), and decreased levels of liver marker enzymes. These results indicate that naringenin prevents lipid peroxidation and hepatic cell damage and also protects the antioxidant system in N-nitrosdithylamine-induced hepatocarcinogenesis.

Expression of MMP-9 in hepatic sinusoidal obstruction syndrome induced by Gynura segetum

Hepatic sinusoidal obstruction syndrome (HSOS) is characterized by painful hepatomegaly, ascites, increased body weight, and jaundice. Gynura segetum (Compositae), a plant widely used in Chinese traditional medicine, often leads to the development of HSOS. However, the mechanism is unclear. The aim was to study the role of matrix metalloproteinase-9 (MMP-9) in the onset of HSOS induced by Gynura segetum. Twenty-five male Sprague-Dawley rats were randomly divided into two groups. Twenty were exposed to 600 mg/kg daily Gynura segetum extract solution for three weeks; five control rats were exposed to tap water alone. Liver sections were evaluated by light microscopy with a modified scoring system. Routine transmission electron microscopy (TEM) methods were used to evaluate the ultrastructual features of fixed liver tissue, and blood samples were collected to determine liver enzyme concentrations. MMP-9 expression was assessed by both immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) methods. A stable and reproducible rat model of HSOS was achieved by long-term exposure to Gynura segetum extract. The treated rats presented clinical symptoms and the histopathological manifestation of HSOS, including abnormal liver enzyme concentrations (alanine aminotransferase (ALT): (84.8±13.62) vs. (167.0±72.63) U/L, P<0.05; aspartate aminotransferase (AST): (27.6±6.31) vs. (232.8±108.58) U/L, P<0.05). Hematoxylin and eosin (H&E) staining and TEM together revealed deposition of red blood cells, the damage and destruction of hepatic sinusoidal endothelial cells, collapse of hepatic sinusoids, hemorrhage of subendothelial cells, atrophy and destruction of hepatocytes, etc. Compared with controls, the expression of MMP-9 in the blood sample, the lung and liver tissues of HSOS rats was increased. MMP-9 may have an important role in early pathological changes of HSOS, and thus the onset of the disease.

Evaluation of efficacious activities of aqueous extract of &lt;em&gt;Phyllanthus niruri&lt;/em&gt; against acetaminophen-induced hepatitis in rats

The efficacy of aqueous extract of Phyllanthus niruri against acetaminophen-induced hepatitis in rats was evaluated. The hepatic injury was induced with 200 mg/kg, p.o. of acetaminophen, which led to rise in serum levels of the biochemical parameters observed. These are the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) which were elevated by 22% compared to respective negative control. Treatment with the plant extract (500, 1000 and 2000 mg/kg, p.o.) lowered the biochemical parameters of the respective serum AST 31 to 38%, ALT 20 to 31%, Bilirubin -2 to 4%, protein 5 to 15%, cholesterol 0.2 to 0.5%, and albumin 5 to 12%. The histopathological results indicated that, the effect of the extracts on the condition of the liver as compared to the normal control ranges from mild to moderate. The biochemical variations were as a result of the different treatment involved in the study. The result therefore, shows lowering of the elevated parameters in the serum and possible reversal of hepatic cell damage with aqueous extract of Phyllanthus niruri. The trend of the study shows that, the longer the period of treatment at lower doses, the better the efficacy of the plant extract.Keywords: Phyllanthus niruri, hepatitis, acetaminophen, biochemical, histopathology, rats

Effect of Notoginseng Extracts and Its Components on Lipopolysaccharide and Galactosamine Mixture-induced Impaired Hepatic Function in Mice

The protective effects of notoginseng against hepatic damage were investigated in mice. To prepare a model animal of hepatitis, a mixture of lipopolysaccharide and galactosamine (LPS/GAlN) was administered intraperitoneally, leading to the impairment of hepatic function. Extracts of notoginseng or its components (ginsenoside Rb1 and ginsenoside Rg1) were orally administered 2 h before LPS/GalN injection. Eight hours after LPS/GalN injection, blood and liver tissue samples were collected. The levels of serum aspartate amino transferase (AST), alanine aminotransferase (ALT), tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were measured using commercial assay kits. Histologic changes in the tissue samples were also observed after hematoxylin-eosin staining. LPS/GalN administration increased the serum levels of AST and ALT, and histologic changes were noted, indicating hepatic cell damage. Prior to the increase in ALT, the serum levels of TNF-α and IFN-γ were elevated after LPS/GalN injection. Pretreatment of the mice with either notoginseng extract or gensenoside Rb1 and Rg1 attenuated the LPS/GalN-induced hepatic damage markedly. The protective effects of the components against hepatic damage appeared to be less potent than those of the crude extract and prescription of notoginseng. Notoginseng may be clinically useful in patients with hepatitis.

Transmission electron microscopy study on the effects of the ultrasound contrast agent Levovist on hepatic cells.

The Ultrasound Equipment and Safety Committee of The Japan Society of Ultrasonics in Medicine performed experiments to confirm whether contrast-enhanced ultrasonography damages liver cells. Rats were injected with 0.1ml of 300mg/ml ultrasound contrast agent (UCA). Diagnostic ultrasound pulses with a center frequency of 6MHz and a mechanical index of 1.9 were applied to rat livers with a water bag as a coupler to maintain a distance of 2-6cm between the ultrasound probe surface and the liver. Contrast-enhanced ultrasonography was carried out for 10s to visualize the entire liver. Then, specimens of liver tissue were fixed using two types of fixation: immersion and perfusion fixation. Although some variations were found in electron micrographs of liver tissue fixed using immersion fixation, none of three blinded readers found any significant differences between micrographs of liver tissue from rats receiving UCA with sonication and those from sham-treated control rats. Changes observed were not thought to be group-specific but instead due to differences between individual rats. When the livers were fixed using perfusion fixation and the hepatic vein was cut after injection of physiological saline for perfusion, a large number of vacuoles ≥2μm in diameter were observed. This finding suggested that hepatic cell damage observed in this study was caused by high perfusion pressure during the liver fixation process rather than by sonication with UCA. Blinded readings of electron micrographs showed no clear evidence that the use of Levovist in ADI mode ultrasonography causes significant damage to liver tissue.

Phenolics in mulberry leaves protect Hep G2 cells against hyperglycemia‐induced oxidative damage

Hyperglycemia is associated with increased risk of type 2 diabetes, partially due to increases in oxidative stress. Phenolics in mulberry leaves (MLP) may protect tissues from hyperglycemia‐induced oxidative damage. We examined the effect of MLP on hyperglycemia‐induced oxidative damage in hepatic HepG2 cells. HepG2 were treated with glucose (5.5 or 50 mM) with or without MLP at 10 or 100 μM GAE. Hyperglycemia‐induced oxidative damage in HepG2 was demonstrated by a 666% increase in O2.− production at 0.5 h, 400% increase in malondialdehyde (MDA) at 24 and 48 h, mitochondrial membrane hyperpolarization (MMHP) at 2 h followed by depolarization, and 474% increase in nucleus NFκB‐P65 at 4 h relative to the control cells (P ≤0.05). Both MLP doses inhibited hyperglycemia‐induced O2.− production by 40%. Hyperglycemia‐induced MDA production at 24 h was diminished by 34 and 54% by MLP at 10 and 100 μM, respectively. MLP treatment protected HepG2 cells from hyperglycemia‐induced MMHP in a dose‐dependent manner with the highest dose maintaining mitochondrial polarization status. MLP at 10 μM inhibited 35% hyperglycemia‐induced NFκB‐P65 translocation and at 100 μM completely prevented the NFκB activation. Our data show that MLP in vitro protects hepatoctyes from hyperglycemia‐induced oxidative damage.