Rat Liver Research Articles

Polydatin Prevents Electron Transport Chain Dysfunction and ROS Overproduction Paralleled by an Improvement in Lipid Peroxidation and Cardiolipin Levels in Iron-Overloaded Rat Liver Mitochondria.

Increased intramitochondrial free iron is a key feature of various liver diseases, leading to oxidative stress, mitochondrial dysfunction, and liver damage. Polydatin is a polyphenol with a hepatoprotective effect, which has been attributed to its ability to enhance mitochondrial oxidative metabolism and antioxidant defenses, thereby inhibiting reactive oxygen species (ROS) dependent cellular damage processes and liver diseases. However, it has not been explored whether polydatin is able to exert its effects by protecting the phospholipid cardiolipin against damage from excess iron. Cardiolipin maintains the integrity and function of electron transport chain (ETC) complexes and keeps cytochrome c bound to mitochondria, avoiding uncontrolled apoptosis. Therefore, the effect of polydatin on oxidative lipid damage, ETC activity, cytochrome levels, and ROS production was explored in iron-exposed rat liver mitochondria. Fe2+ increased lipid peroxidation, decreased cardiolipin and cytochromes c + c1 and aa3 levels, inhibited ETC complex activities, and dramatically increased ROS production. Preincubation with polydatin prevented all these effects to a variable degree. These results suggest that the hepatoprotective mechanism of polydatin involves the attenuation of free radical production by iron, which enhances cardiolipin levels by counteracting membrane lipid peroxidation. This prevents the loss of cytochromes, improves ETC function, and decreases mitochondrial ROS production.

Sub-Acute Toxicity Assay of the smoke of Seeds of Azadirachta indica and Peels of Citrus sinensis used as natural insecticides on the Liver, Kidney, Lungs and Heart of Albino Rats

Abstract Natural plant parts such as seeds of Azadirachta indica (neem seeds) and peels of Citrus sinensis (sweet orange) have been used as house-hold remedies against various ailments and bio-pesticides for agricultural purpose due to their repellent, growth regulatory and anti-feedant properties against insects. Despite their beneficial role, these natural insecticides could also have toxic effects on some vital organs of human. Therefore, this study aimed to investigate the sub-acute toxicity of smoke of the Seeds of Azadirachta indica and Peels of Citrus sinensis on the liver, kidney, lungs and heart of swiss albino rats. In this study, three groups of white swiss albino rats were exposed to the smoke of A indica seeds, peels of C sinensis and their combination at different duration of one hour and two hours respectively. The Control group were not exposed to any of the natural insecticides and were fed normal diet and water. After four weeks of the treatment, the animals were all sacrificed and their liver, kidneys, lung and heart were evaluated using standard biochemical and histopathological techniques. The levels of serum biochemical indices for liver (aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), total bilirubin and protein), kidneys (urea, creatinine and electrolytes), and heart (creatine kinase and lactate dehydrogenase) were found to be significantly higher at (p<0.05) for all the groups treated with the two natural insecticides when compared to control group. Histopathological changes including fibrosis, hemorrhages, vascular congestion and necrosis were observed in the liver, kidneys and lungs tissues of the rats exposed to the smoke of the seeds of A indica. However, no histopathological changes were observed in all organs of rats exposed to the smoke of the peels of C sinensis compared to control group. Moreover, the severity of the effects of smoke of these natural insecticides on both biochemical and histopathological indices increases with increase in duration of exposure. The results of this study revealed that seeds of A indica have significant toxic effects on both the tissue and biochemical markers of all the major organs of the Swiss albino rats, while peels of C sinensis shows effect on only the biochemical indices at the specific duration of time studied.

Effects of Salicylic and Acetylsalicylic Acids in Mitochondrial and Erythrocyte Membranes

For further clarification of the mechanisms of pharmacological effects of salicylic and acetylsalicylic acids, the interactions of these acids with mitochondrial and erythrocyte membranes were studied and the role of calcium ions in the effects of salicylic and acetylsalicylic acids was examined. Salicylic acid and to a lesser extent acetylsalicylic acid at 0.5−2.0 mM concentration effectively inhibited the respiratory activity of isolated rat liver mitochondria, by uncoupling respiration and phosphorylation processes, induced depolarization of the mitochondrial membrane and potentiated Ca2+-stimulated formation of mitochondrial permeability transition pores in EGTA-free media. Cyclosporine A and ruthenium red partially inhibited the mitochondrial pore opening process induced by salicylic and acetylsalicylic acids both in the absence and presence of Ca2+ ions. Salicylic acid (180–360 µM) markedly accelerated proton-induced lysis of human erythrocytes (at pH 3.2) and caused hyperpolarization of erythrocyte membranes (at pH 5.5, but not at pH 7.4), probably as a result of proton transfer to the cytoplasm of the cell. Thus, salicylic and acetylsalicylic acids interact with mitochondrial and plasma membranes, act as effective proton/Ca2+ ionophores and stimulate the mitochondrial calcium uniporter.

Activity Variations of CYP2B6 Determine the Metabolic Stratification of Efavirenz.

To investigate the effects of hepatic enzyme activity variations and CYP2B6 gene polymorphisms on the in vivo and in vitro metabolism of efavirenz. In vitro enzyme systems using rat and human liver microsomes (RLM/HLM) were established, with in vivo studies conducted on Sprague-Dawley rats. Metabolite detection was performed via LC-MS/MS. Human recombinant CYP2B6 microsomes were prepared using a baculovirus-insect cell system and ultracentrifugation, with efavirenz serving as the substrate to study enzyme kinetics. Isavuconazole exhibited an IC50 of 21.14 ± 0.57 μM in RLM, indicating a mixed competitive and noncompetitive mechanism, and an IC50 of 40.44 ± 4.23 μM in HLM, suggesting an anticompetitive mechanism. In rats, coadministration of efavirenz and isavuconazole significantly increased the AUC, Tmax, and Cmax of efavirenz. Co-administration of efavirenz and rifampicin significantly elevated the AUC, Tmax, and Cmax of 8-OH-efavirenz. The activity of CYP2B6.4, 6, and 7 increased significantly compared to CYP2B6.1, with relative clearance ranging from 158.34% to 212.72%. Conversely, the activity of CYP2B6.3, 8, 10, 11, 13-15, 18-21, 23-27, 31-33, and 37 was markedly reduced, ranging from 4.30% to 79.89%. Variations in liver enzyme activity and CYP2B6 genetic polymorphisms can significantly alter the metabolism of efavirenz. It provides laboratory-based data for the precise application of efavirenz and other CYP2B6 substrate drugs.

Pharmacokinetic predictions of ROS-mediated targets and genotoxin combinations via multiple ligand simultaneous docking and ROS evaluation in vitro using HepG2 cell lines.

Although combination therapy is known for its high efficacy, reduced side effects and drug resistance, toxicity remains a major drawback. Some of the genes are likely to induce hepatotoxicity through ROS-mediated mechanisms when a drug is metabolized alone or in combination in the liver. To address this, we have developed a scientific approach to predict the toxicity of different genotoxin combinations and validate their interactions with various targets. The current study is an extensive study of our previous set of in vivo rat liver microarray data processed using R studio for their functional analysis. About five combinations of genotoxins such as CPT/ETP, CPT/CPL, ETP/CPL, CP/CPT and EES/CP along with their differential gene expression targeting Chemical carcinogenesis-ROS are chosen for this study. We aim to examine the binding affinity of different genotoxin combinations using in silico multiple ligand simultaneous docking (MLSD) and are then bio-evaluated for cytotoxicity in vitro using human hepatocellular carcinoma cell lines (HepG2) with the MTT assay. As a result, dose-response cytotoxicity with its strength of interactions and a significant variance in ROS levels in the treated cells is observed compared to their IC50 values. Out of 5 combinations such as CPT/CPL, ETP/CPL and EES/CP are found not only to be significantly cytotoxic but also induce oxidative stress specifically above their IC50 values with good and moderate binding interactions ensuring their toxicity. On the contrary, the safe combinations are found to be CTP/ETP and CP/CPT possibly with no and tolerable adverse effects standing as preliminary information for researchers in drug design and development.

A chitosan macroporous hydrogel integrating enrichment, adsorption and delivery of blood clotting components for rapid hemostasis

Traditional hemostatic hydrogels face considerable limitations in achieving rapid control of severe bleedings, a crucial factor in reducing casualties in both military and civilian settings. This study presents a chitosan-based hemostatic hydrogel with interconnected secondary macropores designed to enhance interactions with blood clotting components by reducing diffusion resistance and increasing contact area. The macropores were created using a straightforward process involving NaOH-mediated SiO2 template dissolution and NH3 generation. The resulting macroporous structure increased the hydrogel's overall porosity without compromising its viscoelasticity. Functional studies demonstrated that the macroporous hydrogel effectively concentrated and adsorbed blood clotting components, while also facilitating the delivery of artificially embedded clotting factor to further expedite clot formation. These combined actions resulted in improve hemostatic efficacy, reducing whole blood clotting time by over 94 % in vitro. Furthermore, in vivo studies using rat tail amputation and liver injury models showed a reduction in blood loss by over 65 % and a decrease in bleeding time by over 70 %. Additionally, the porous chitosan hydrogel exhibited minimal biotoxicity and promoted biodegradability in vivo. In conclusion, this work introduces a macroporous chitosan-based hemostatic hydrogel with great potential for rapid hemorrhage control.

Immunoexpression of CD34, CD68 and CD3 in Cadmium-Induced Liver Damage and Protective Effectiveness of Bee Bread (Perga)

Cadmium (Cd) is one of the potent environmental toxicants that causes oxidative stress in many organs of the body, including the liver. Perga (bee bread) is used for apitherapeutic purposes due to its medicinal properties. This study was conducted to investigate the effectiveness of perga on endothelial damage and inflammatory cell activation in the liver as a result of exposure to Cd. For this purpose, 32 male Wistar rats (8 rats/group) were randomly divided into 4 groups, as the control, perga (0.5 g/kg of perga), Cd (5 mg/kg of CdCl2), and Cd + perga (0.5 g/kg of perga + 5 mg/kg of CdCl2) groups. Daily intragastric Cd and/or perga was administered for 4 weeks. At the end of the study, the rats were euthanized and liver tissue sections were taken and stained with hematoxylin-eosin and Masson’s Trichrome. Immunohistochemically, the reactivity of the liver sinusoidal endothelium was determined using CD34, the reactivity of the Kupffer cells was determined using CD68, and the levels of T-lymphocyte cells were determined using CD3 antibodies. Exposure to Cd caused significant histological changes in the liver. Immunohistochemically, exposure to Cd caused an increase in the expressions of CD34, CD68, and CD3. On the other hand, the cotreatment of Cd and perga caused partial improvement in some histopathological changes. Compared to the Cd group, there was a decrease in CD34 and CD68 positivity in the Cd + perga group, while no significant difference was detected in the number of CD3-positive cells between the groups. The results revealed that the histopathological changes and inflammation in the rat liver could partially improve with perga supplementation.

Metabolic dysfunction-associated steatotic liver disease exhibits sex-specific microbial heterogeneity within intestinal compartments.

Evidence suggests that the gastrointestinal microbiome plays a significant role in the biology of metabolic dysfunction-associated steatotic liver disease (MASLD). However, it remains unclear whether disparities in the gut microbiome across intestinal tissular compartments between the sexes lead to MASLD pathogenesis. Sex-specific analyses of microbiome composition in two anatomically distinct regions of the gut, the small intestine and colon, were performed using an experimental model of MASLD. The study involved male and female spontaneously hypertensive rats and the Wistar-Kyoto control rat strain, which were fed either a standard chow diet or a high-fat diet for 12 weeks to induce MASLD (12 rats per group). High-throughput 16S sequencing was used for microbiome analysis. There were significant differences in the overall microbiome composition of male and female rats with MASLD, including variations in topographical gut regions. The beta diversity of the jejunal and colon microbiomes was higher in female rats than in male rats (PERMANOVA p-value=0.001). Sex-specific analysis and discriminant features using LEfSe showed considerable variation in bacterial abundance, along with distinct functional properties, in the jejunum and colon of animals with MASLD. Significantly elevated levels of lipopolysaccharide and protein expression of Toll-like receptor 4 were observed in the livers of male rats with MASLD compared with their female counterparts. This study uncovered sexual dimorphism in the gut microbiome of MASLD and identified microbial heterogeneity within intestinal compartments. Insights into sex-specific variations in gut microbiome composition could facilitate customised treatment strategies.

Investigation of ponatinib metabolism and drug-drug interactions with lycopene and shikonin in vitro and invivo

Ponatinib is approved for use in patients with chronic myeloid leukemia (CML) who are resistant to or intolerant to prior tyrosine kinase inhibitor (TKI) therapy. Given that ponatinib can induce significant cardiotoxicity when taken, and that most Chinese medicines have cardioprotective effects, it is possible to administer them in combination in clinic to alleviate adverse effects. The quantitative determination of ponatinib and its metabolite N-desmethyl ponatinib was optimized and fully verified by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). And the drug-drug interactions (DDI) of ponatinib with lycopene and shikonin, both in vivo and in vitro, were studied. The results of bioanalytical methodology showed that ponatinib and N-desmethyl ponatinib had good linearity in plasma samples, and their selectivity, accuracy, precision, stability, matrix effect and recovery were all satisfied with the need of quantitative analysis of samples. In animal experiments, compared with the control group, lycopene and shikonin significantly changed the pharmacokinetic parameters of ponatinib, including AUC(0-t), AUC(0-∞) and CLz/F, while having no effect on the pharmacokinetic parameters of N-desmethyl ponatinib. In vitro interaction studies indicated that lycopene showed mixed inhibition mechanism on ponatinib metabolism in both rat liver microsomes (RLM) and human liver microsomes (HLM). And, shikonin displayed mixed inhibition mechanism in RLM and competitive inhibition mechanism in HLM, respectively. In summary, the UPLC-MS/MS method can accurately and sensitively quantify ponatinib and N-desmethyl ponatinib, and provide further reference for clinical drug combination between ponatinib and lycopene or shikonin.

Енергетичний обмін у печінці щурів із паркінсонічним синдромом, індукованим ротеноном, при дії метаніндіазенону

Parkinson’s disease (PD) is symptomatically characterized by motor disorders in the human body, which are associated with the degeneration of dopamine neurons in the substantia nigra of the midbrain. It is widely recognized that mitochondrial dysfunction occurs in dopaminergic neurons, leading to their death and the development of this pathology. Recently, benzodiazepine derivatives have been found to have a neuroprotective effect against damage of dopaminergic neurons in an experimental model of Рarkinsonian syndrome (PS). The aim of this study was to investigate the effect of a novel molecule, named methanіdiazenone, on energy metabolism in the liver of rats induced by rotenone. To achieve the set goal, the concentration of ATP, ADP, AMP, hypoxanthine аnd xanthine were determined in the bile samples of rats with PS by the method of thin-layer chromatography. The obtained data indicate that under the influence of rotenone compared to the control values the content of ATP in bile decreased by 40%, while the levels of AMP, xanthine and hypoxanthine, on the contrary, increased by 87,5%, 55,6%, and 25%, respectively. Our findings suggested that the ratio of AMP/ATP, which is an important indicator of the functional state of mitochondria, under the influence of rotenone increased by 200% compared to the control. Metanindiazenone in a dose of 1,0 and 2,0 mg/kg normalized all studied parameters of purine metabolism. The presented results show that methanindiazenone significantly improves purine metabolism in the liver of rats with PS induced by rotenone, indicating the normalization of mitochondrial function in hepatocytes. Thus, methanindiazenone may be recommended for clinical trials regarding its use in combination with other drugs to treat Parkinson’s disease and possibly other neurodegenerative diseases, in which mitochondrial dysfunction occurs.

Phase-change ultrasound contrast agents for proton range verification: towards an in vivo application

Objective.In proton therapy, range uncertainties prevent optimal benefit from the superior depth-dose characteristics of proton beams over conventional photon-based radiotherapy. To reduce these uncertainties we recently proposed the use of phase-change ultrasound contrast agents as an affordable and effective range verification tool. In particular, superheated nanodroplets can convert into echogenic microbubbles upon proton irradiation, whereby the resulting ultrasound contrast relates to the proton range with high reproducibility. Here, we provide a firstin vivoproof-of-concept of this technology.Approach.First, thein vitrobiocompatibility of radiation-sensitive poly(vinyl alcohol) perfluorobutane nanodroplets was investigated using several colorimetric assays. Then,in vivoultrasound contrast was characterized using acoustic droplet vaporization (ADV) and later using proton beam irradiations at varying energies (49.7 MeV and 62 MeV) in healthy Sprague Dawley rats. A preliminary evaluation of thein vivobiocompatibility was performed using ADV and a combination of physiology monitoring and histology.Main results.Nanodroplets were non-toxic over a wide concentration range (<1 mM). In healthy rats, intravenously injected nanodroplets primarily accumulated in the organs of the reticuloendothelial system, where the lifetime of the generated ultrasound contrast (<30 min) was compatible with a typical radiotherapy fraction (<5 min). Spontaneous droplet vaporization did not result in significant background signals. Online ultrasound imaging of the liver of droplet-injected rats demonstrated an energy-dependent proton response, which can be tuned by varying the nanodroplet concentration. However, caution is warranted when deciding on the exact nanodroplet dose regimen as a mild physiological response (drop in cardiac rate, granuloma formation) was observed after ADV.Significance.These findings underline the potential of phase-change ultrasound contrast agents forin vivoproton range verification and provide the next step towards eventual clinical applications.

Discovery of Novel 5-(Pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione Derivatives as Potent and Orally Bioavailable Inhibitors Targeting Ecto-5'-nucleotidase.

Ecto-5-nucleotidase (CD73) is overexpressed in a variety of cancers and associated with the immunosuppressive tumor microenvironment, making it an attractive target for cancer immunotherapy. Herein, we designed and synthesized a series of novel (pyridazine-3-yl)pyrimidine-2,4(1H,3H)-dione derivatives as CD73 inhibitors. These compounds exhibited remarkable inhibitory activity against CD73 in both enzymatic biochemical and cellular assays. Among them, compound 35j proved to be one of the most potent inhibitors and an uncompetitive inhibitor with no obvious cytotoxicity. This compound showed high metabolic stability in rat liver microsomes and favorable pharmacokinetic profiles in rats (T1/2 = 3.37 h, F = 50.24%). Importantly, orally administered 35j significantly inhibited tumor growth in the triple-negative breast cancer 4T1 mouse model (TGI = 73.6%, 50 mg/kg). Immunoassays suggested that 35j remarkably increased the infiltration of positive immune cells, thereby reinvigorating antitumor immunity. These results demonstrate that 35j is a potent CD73 inhibitor worthy of further development.

Oxidative Stress, Inflammation, and Altered Lymphocyte E-NTPDase Are Implicated in Acute Dyslipidemia in Rats: Protective Role of Arbutin

Background/Objectives: Dyslipidemia is frequently linked to various disorders, and its clinical relevance is now recognized. The role of inflammation and oxidative stress (OS) in dyslipidemia has been acknowledged. This study assessed the potential of arbutin (ARB) to prevent dyslipidemia and its associated OS and inflammation in rats with acute hyperlipidemia. Methods: Rats received ARB orally for 14 days and a single intraperitoneal injection of poloxamer-407 on day 15. Results: Poloxamer-407 elevated circulating cholesterol (CHOL), triglycerides (TG), very low-density lipoprotein (vLDL), and LDL, and reduced high-density lipoprotein (HDL)-C and lipoprotein lipase (LPL). ARB ameliorated the circulating lipids and LPL, and suppressed 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) in rat liver and in vitro. Fatty acid synthase (FAS) in rat liver and its in vitro activity were suppressed by ARB, which also upregulated the LDL receptor (LDL-R) and ABCA1, and had no effect on ABCG5 and ABCG8 mRNA. ARB ameliorated liver malondialdehyde and nitric oxide and enhanced antioxidants in rats with dyslipidemia. Liver NF-κB p65 and blood inflammatory cytokines were increased in dyslipidemic rats, effects that were reversed by ARB. Moreover, ARB effectively suppressed lymphocyte E-NTPDase and E-ADA activities in dyslipidemic rats. The biochemical findings were supported by in silico data showing the affinity of ARB to bind LDL-R PCSK9 binding domain, HMGCR, FAS, and E-NTPDase. Conclusions: ARB possessed anti-dyslipidemia, anti-inflammatory, and antioxidant effects mediated via the modulation of CHOL and TG synthesis, LPL, lymphocyte E-NTPDase and E-ADA, and cytokine release in rats. Thus, ARB could be an effective agent to attenuate dyslipidemia and its associated OS and inflammation, pending further studies as well as clinical trials.

Pro-inflammatory cytokines gene expression in liver and kidneys of rats exposed to a sub-lethal dose of Bitis arietans snake venom.

Bitis arietans (Puff adder) is a poisonous snake and its bite causes pain, edema, blistering, tissue damage and neutrophilia. There are limited studies on inflammatory process involved in Bitis arietans envenomation. We therefore investigated the role of proinflammatory cytokines in Bitis arietans venom (BAV)-induced liver and kidney toxicities in rats. Adult male Sprague Dawley rats were treated with BAV (0.5 mg/kg) and were sacrificed after specific time intervals (2 h, 24 h, 1 week). Blood samples were collected for liver and renal function tests and tissues were collected for histopathology and gene expression analysis of IL-1β, IL-6, and TNF-α in liver and kidneys. There was no significant difference in serum ALT activities among different treatment groups. Serum AST was significantly increased at 24 h following BAV injection. In both organs, injection of BAV resulted in mild inflammatory cell infiltration at 2 h post-dosing which normalized after 1 week. In liver, there was a significant increase in IL-1β expression in BAV-treated rats at 2 and 24 h post-dosing that reduced after one week. Significant increases in IL-6 and TNF-α were observed at 24 h and 1 week after BAV exposure. In kidneys, there were significant increases in IL-1β and TNF-α expression at 24 h that subsided after 1 week. In conclusion, a single sub-lethal dose of BAV caused an acute phase inflammation in liver and kidneys. It is most probable that a higher dose of BAV may result in greater and irreversible damage to these organs.

Protective effects of lemon nanovesicles: evidence of the Nrf2/HO-1 pathway contribution from in vitro hepatocytes and in vivo high-fat diet-fed rats

The cross-talk between plant-derived nanovesicles (PDNVs) and mammalian cells has been explored by several investigations, underlining the capability of these natural nanovesicles to regulate several molecular pathways. Additionally, PDNVs possess biological proprieties that make them applicable against pathological conditions, such as hepatic diseases. In this study we explored the antioxidant properties of lemon-derived nanovesicles, isolated at laboratory (LNVs) and industrial scale (iLNVs) in human healthy hepatocytes (THLE-2) and in metabolic syndrome induced by a high-fat diet (HFD) in the rat. Our findings demonstrate that in THLE-2 cells, LNVs and iLNVs decrease ROS production and upregulate the expression of antioxidant mediators, Nrf2 and HO-1. Furthermore, the in vivo assessment reveals that the oral administration of iLNVs improves glucose tolerance and lipid dysmetabolism, ameliorates biometric parameters and systemic redox homeostasis, and upregulates Nrf2/HO-1 signaling in HFD rat liver. Consequently, we believe LNVs/iLNVs might be a promising approach for managing hepatic and dysmetabolic disorders.

Ameliorative Effect of Curcuma longa Ethanolic Extract on the Histology, Hepatic Glycogen Content and Some Biochemical Parameters of the Liver in Streptozotocin-Induced Hyperglycemic Wistar Rats

Introduction: Diabetes mellitus is a life threatening disease that requires immediate medical treatment. Numerous medicinal plants have been known for their anti-diabetic effects and Curcuma longa (turmeric) is one of them. This attribute is possible due to the pharmacological properties of its bio-constituents. In this study, the ameliorative potential of ethanolic extract of Curcuma longa was investigated on the liver of hyperglycemic adult Wistar rats. The objectives were to determine its effects on liver histology, hepatic glycogen content and some liver biochemical parameters. Methods: Forty (40) rats weighing between 120-180g were used for the research. Sixteen (16) rats were used for its acute toxicity study, while twenty-four (24) rats were grouped into four. The groups were labelled A to D and contained six rats each. Group A (normal control) rats were given food pellets and distilled water only. Group B (diabetic control) rats were given 65mg/kg BW of streptozotocin IP. Group C served as the standard group and was given 65mg/kg of streptozotocin IP + 250mg/kg of metformin IP, while Group D (test group) rats were given 65mg/kg BW of streptozotocin IP + 500mg/kg of Curcuma longa extract. Curcuma longa extract was administered through the oral route with the aid of an oral gavage. The administration lasted for twenty-eight (28) days, after which the rats were made to fast overnight with access to only water. The rats were then anaesthetized and whole blood obtained through cardiac puncture for biochemical analysis. Liver tissue was obtained for histological and glycogen content analyses. Results: The results showed severe destruction of hepatocytes with widely spread cytoplasmic vacuolation, loss of hepatic lobules and hepatic inflammation in the diabetic group, whereas it showed prominent nuclei, hepatocytes and distinct sinusoidal spaces in the normal control group. The metformin group showed an intact architecture of the liver tissue with indicating regeneration in the liver, while the Curcuma longa extract group showed a preserved liver cytoarchitecture. There was a significant decrease (p&lt;0.05) in the fasting blood glucose level and liver enzyme activities in the standard and test groups compared to the diabetic group (p&lt;0.05). There was also improvement in glycogen content of the standard and test groups in comparison with the diabetic group (p&lt;0.05). Conclusion: Curcuma longa possesses hepato-protective properties and a strong anti-diabetic potential, therefore it can be considered as an alternative drug in the management of diabetes mellitus and its complications.

A compact machine perfusion device for whole blood perfusion in isolated rat liver.

We established a compact machine perfusion system for whole blood perfusion of rat liver by making use of oxygenation filters as an artificial lung. Livers removed from rats were divided into Krebs-Henseleit (control), 50% blood (hemoglobin: 7g/dL), and whole blood (hemoglobin: 14g/dL) groups, then perfused (total perfusate volume: 25ml) with a small oxygenation filter at 37°C for 120min. Blood or perfusate was collected over time, and blood gas and blood cell were measured. In addition, bile volume and portal venous pressure measurements were taken. In all groups, the partial pressure of oxygen was controlled to approximately 400mmHg. Flow rates were maintained at approximately about 20-30ml/min according to liver size. Portal venous pressure was normal in the 50% blood and whole blood groups, while lower than the reference value in the Krebs-Henseleit group. Twice as much bile was produced in the 50% blood and whole blood groups relative with the Krebs-Henseleit group. We observed no differences in hemoglobin and red blood cell levels. Lactate levels were normal in the 50% blood and whole blood groups, but were elevated in the Krebs-Henseleit group. Our compact perfusion system using oxygenation filters was able to maintain rat liver function by perfusing a small amount of extracorporeal blood. This system is simple and stable, and may contribute to the future development of machine perfusion systems.

Identification of the metabolites of nimbolide in rat by liquid chromatography combined with quadrupole/orbitrap mass spectrometry.

Nimbolide is a major furanoid compound isolated from Azadirachta indica. The aim of this study was to characterize the metabolites of nimbolide in rats and to propose the metabolic pathways. The metabolites were generated by incubating nimbolide (10μM) with rat liver microsomes, nicotinamide adenine dinucleotide phosphate (NADPH), and nucleophiles (glutathione [GSH] or N-acetyl-lysine [NAL]) at 37°C for 60 min. For the in vivo study, nimbolide was intravenously administered to rats at a single dose of 10mg/kg, and the bile and urine were collected. The metabolites were identified by ultra-high-performance liquid chromatography-quadrupole/orbitrap mass spectrometry (UPLC-Q/Orbitrap-MS) using electrospray ionization in positive ion mode. Totally, nine metabolites were detected, and their identities were characterized by accurate MS and MS/MS data. In GSH-supplemented liver microsomes, GSH conjugation was the primary elimination pathway. The furan ring was bioactivated into cis-butene-1,4-dial that can be trapped by GSH. In NAL-supplemented liver microsomes, two NAL conjugates (M4 and M5) derived from cis-butene-1,4-dial were observed. In rat bile and urine, N-acetyl-cysteine, cysteine-glycine, and GSH conjugate were also found. The current study provides an overview of the metabolism and the bioactivation profiles of nimbolide in rats, which aids in understanding its safety and activity.

Pharmacokinetics and related gender difference studies of four active components of Codonopsis Pilosula by LC-MS/MS determination

Ethnopharmacological relevanceCodonopsis pilosula (C. pilosula), commonly known as Dangshen in Chinese, had been used to regulate the immune, digestive, and circulatory systems of human. The reported pharmacokinetic studies on C. pilosula are mainly limited to in vivo profile studies of a single component. It has not been detected simultaneously the in vivo pharmacokinetic profiles of multiple active components as well as related gender difference after oral dosing of the extraction of C. pilosula. Aim of the studyThis study aims to reveal the pharmacokinetic characteristics of the four main active components of C. pilosula after oral dosing of its extraction in rats, and to explain the gender differences in absorption and metabolism. Materials and methodsThe plasma pharmacokinetic characteristics of four main active components of C. pilosula was explored using the established LC-MS/MS method after oral dosing of the extraction of C. pilosula in male and female rats. In vitro intestinal pouch permeability and liver microsome metabolic stability were also observed to classify the possible mechanism of gender difference existed in the pharmacokinetic profiles of the four active components in rats. ResultsFour effective components were absorbed quickly in rats after oral administration of alcoholic extract of C. pilosula (1.36 g/mL, equivalent to 2 g/mL as crude drug), and their exposure order was as follows: Atractylenolide III > Lobetyolin > Tangshenoside I > Syringin. The exposure (AUC) and peak concentration (Cmax) of Atractylenolide III in female rats were much higher than those in male rats, indicating a significant gender difference in pharmacokinetics of Atractylenolide III between female and male animals. With the help of the rat model of intestinal sac in vitro, it was found that Lobetyolin was a hypertonic compound, and both Tangshenoside I and Syringin were compounds with medium permeabiltiy. Notably, the Papp of Atractylenolide III was 3.3 × 10−6 cm/s in male rat intestinal sac assay, while that was 10 × 10−6 cm/s in female rat intestinal sac model, showing a significant gender difference in intestinal permeability (P < 0.05). After the addition of NADPH, the four compounds were reduced in a time-dependent manner, suggesting that CYP450s could catalyze their metabolism. After incubation, the remaining content of Atractylenolide III in the liver microsomes of male and female rats was 27% and 57%, respectively, suggesting slower metabolic rate of in female rat liver microsomes. ConclusionA simple, efficient and reliable LC-MS/MS method for the simultaneous determination of four active index components of C. pilosula, Lobetyolin, Tangshenoside I, Atractylenolide III and Syringin, in rat plasma was established and verified. This method was successfully applied in the pharmacokinetic study after single oral administration of the alcoholic extract of C. pilosula in rats. Gender difference was observed in the pharmacokinetic profile of Atractylenolide III in rats. Intestinal absorption and liver metabolism might be two key factors that resulted in the gender difference in exposure and pharmacokinetics of Atractylenolide III in rats. This study provides supportive data for clinical rational application of C. pilosula in individualized medication therapy.

The effect of chronic predatory stress on the histostructure of the rat liver

One of the topical issues of modern circulatory physiology is the study of the peculiarities of the influence of chronic emotional stress on the histostructure of the liver.Aim: To study changes in the histostructure of the liver in the simulation of chronic predatory stress.Methodology and Research Methods. The study was conducted on 60 male Wistar rats weighing 220–370 g, divided into control and experimental groups of 30 rats each. Modeling of chronic predatory stress was performed according to the method of V.E. Zeilikman et al. (2021). Histological examination of the liver was performed in animals of the control and experimental groups. The preparations were stained with hematoxylin-eosin.Results. Under conditions of chronic predatory stress, stress-induced changes develop in the liver of rats. Transformation of hepatocytes occurs with the development of dystrophic changes, expansion and fullness of sinusoidal capillaries with compression of hepatic beams and destruction of hepatic lobules, a decrease in the parenchymal-stromal ratio. Foci of foamy accumulation of macrophages appear without signs of necrosis, single or group apoptotic hepatocytes. Necrotic foci are formed. The number of Kupffer cells and binucleated hepatocytes increases.Conclusion. With chronic predatory stress, the processes of dystrophy, necrosis and apoptosis of hepatocytes, signs of phagocytosis and regenerative processes, and decreased immune protection develop in the liver of rats.