Liver mRNA Expression Research Articles

P06-19 Murine liver mRNA expression analysis of repeated exposure to chemicals by Percellome Project protocols

P06-19 Murine liver mRNA expression analysis of repeated exposure to chemicals by Percellome Project protocols

Transcriptome analysis reveals hepatic disordered lipid metabolism, lipotoxic injury, and abnormal development in IUGR sheep fetuses due to maternal undernutrition during late pregnancy

Although lipid metabolism in fetal livers under intrauterine growth restriction (IUGR) conditions has been widely studied, the implications of maternal undernutrition on fetal hepatic lipid metabolism, lipotoxic injury, and abnormal development remain largely unknown. Therefore, this study investigated the effects of maternal undernutrition on disordered hepatic lipid metabolism, lipotoxic injury, and abnormal development in IUGR sheep fetuses using transcriptome analysis. Seventeen singleton ewes were randomly divided into three groups on day 90 of pregnancy: a control group (CG; 0.63 MJ metabolic energy/body weight (ME/BW)0.75/day, n = 5), maternal undernutrition group 1 (MU1; 0.33 MJ ME/BW0.75/day, n = 6), and maternal undernutrition group 2 (MU2; 0.20 MJ ME/BW0.75/day, n = 6). The fetuses were euthanized and recovered on day 130 of pregnancy. The levels of free fatty acids (FFA) in maternal blood (P < 0.01), fetal blood (P < 0.01), and fetal livers (P < 0.05) were increased in the MU1 and MU2 groups, but fetal hepatic triglyceride (TG) levels in the MU2 group (P < 0.01) and β-hydroxybutyrate levels in the MU1 and MU2 groups (P < 0.01) were decreased compared to the CG. Severe inflammatory cell infiltration and increased non-alcoholic fatty liver disease activity scores were observed in MU1 and MU2 fetuses (P < 0.01). Progressive deposition of fetal hepatic reticular fibers and collagen fibers in the fetal livers of the MU1 and MU2 groups and significant hepatic fibrosis were observed in the MU2 fetuses (P < 0.05). Gene set enrichment analysis showed that genes involved in lipid accumulation and FFA beta oxidation were downregulated in both MU groups compared to those in the controls. The fetal liver mRNA expression of the β-oxidation regulator, acetyl-CoA acetyltransferase 1, and the TCA regulator, isocitrate dehydrogenase were reduced in MU1 (P < 0.05) and MU2 (P < 0.01) fetuses, and downregulated mRNA expression of long chain fatty acid CoA ligase 1 (P < 0.05) and glycerol-3-phosphate acyltransferase (P < 0.01) was observed in MU2 fetuses. Differentially expressed genes (DEGs) in MU1 versus CG (360 DEGs) and MU2 versus CG (746 DEGs) were identified using RNA sequencing. Bioinformatics analyses of the 231 intersecting DEGs between MU1 versus CG and MU2 versus CG indicated that neutrophil extracellular traps (NETs) were induced and played a central role in fetal hepatic injury in IUGR sheep. Increased maternal blood myeloperoxidase (MPO) levels (P < 0.01), NE (Elane)-positive areas in fetal liver sections (P < 0.05), and fetal liver MPO protein expression (P < 0.01) were found in the MU1 and MU2 groups; however, MPO levels were reduced in the fetal membrane (P < 0.01) and fetal blood (P < 0.05) in the MU1 group, and in the maternal-fetal placenta and fetal blood in the MU2 group (P < 0.01). Analysis of gene expression trends in the intersecting DEGs between MU1 versus CG (129 DEGs) and MU2 versus CG (515 DEGs) further revealed that 30 hub genes were essential regulators of the G2/M cell cycle, all of which were associated with hepatocellular carcinoma. G0/G1 phase cells of the fetal liver were reduced in the MU1 (P < 0.05) and MU2 (P < 0.01) groups, whereas G2/M phase cells were elevated in the MU1 and MU2 groups (P < 0.01). The representatives of upregulated hub genes and fetal liver protein expression of maternal embryonic leucine zipper kinase and protein regulator of cytokinesis 1 were progressively enhanced in the MU1 and MU2 groups (P < 0.01), and topoisomerase II alpha protein expression in the MU2 group (P < 0.05), as expected. These results indicate that FFA overload, severe lipotoxic injury, and NETs were induced, and disease-promoting regulators of the G2/M cell cycle were upregulated in the fetal liver of IUGR sheep. These findings provide new insights into the pathogenesis of impaired hepatic lipid metabolism and abnormal development and the molecular origin of post-natal liver disease in IUGR due to maternal undernutrition. This information can support the development of new therapeutic strategies.

Effects of lactose content in milk replacer on apparent digestibility, growth, liver mRNA expression, and blood parameters related to metabolism of dairy calves

Even with the same energy supply, differences in energy sources may affect calf growth and metabolism. In this study, we evaluated the effects of feeding 3 different milk replacers (MRs) with different lactose supplies under isoenergetic conditions on calf digestibility, growth, and metabolism-related markers. Fifteen Holstein bull calves were randomly assigned to one of 3 MR-feeding treatments: low-lactose (L:38%), medium-lactose (M:41%), or high-lactose (H:46%). After birth, calves were provided colostrum and treatment MRs were fed from 1 d of age and gradually increased to a maximum feeding rate at 20 d of age (L:1.16 kg/d, M:1.21 kg/d, H:1.26 kg/d DM) without feeding solid feeds during the experimental period. Blood samples were temporarily collected weekly to assess blood concentrations of metabolites and hormones. From 30 to 36 d of age, the calves were euthanized and liver samples were collected to determine growth-related mRNA expression. L calves showed a greater body length than H calves and the highest GHR mRNA expression. Plasma concentrations of total cholesterol, urea nitrogen, total protein, albumin, insulin, and insulin-growth factor-1 were not different, but plasma concentrations of triglycerides were higher in order H, M, and L. These results showed that the difference in lactose content in the MR affected calf metabolism, and the L-MR was suggested to be more likely to enhance growth into the peripheral tissues.

Abstract P488: Diet Significantly Alters Metabolic Gene Expression Associated With Probiotic Administration in Mice

The microbial flora in the gastrointestinal tract is known to be mediated by diet and is thus one potential mechanism by which diet influences health. Furthermore, probiotic supplementation also affects various biological markers, however it is unclear if a diet and probiotic interaction exists. Therefore, the goal of our study was to assess how diet and probiotic administration influenced metabolic health in mice. Weanling, male C57Bl/6 mice were randomly assigned to receive one of three diets: chow, American Institute of Nutrition 93 formulation (AIN93), or our novel Americanized diet (AD) formulated to match the 50 th percentile of nutrient intake in Americans. Mice were then administered a commercially-available probiotic for 2-weeks and then euthanized. Insulin sensitivity was determined by performing an insulin-tolerance test (ITT) in a separate cohort. Blood was collected for serum analysis of circulating metabolic markers. Liver and adipose tissue were collected and processed to quantify the mRNA expression of key metabolic genes by real-time RT-PCR. All data were analyzed using General Linear Model Procedures in SPSS with significance determined at P&lt;0.05. Diet (P=0.001), but not probiotic supplementation (P=0.27), significantly affected ITT with the AD group having the lowest responsiveness to insulin ( P ≤0.001). Chow fed mice tended ( P =0.09) to have lower HDL than the AIN-93 and AD fed mice. Probiotic administration alone resulted in an upregulation of liver mRNA expression of SREBF2, particularly the AIN-93 diet group (P=0.024). Probiotic supplementation also modulated liver transcription of NF-κB1 with an increase in chow-fed mice, but decreased in those fed AD (P=0.016). Increased transcription of the key carbohydrate metabolism regulating gene, Mlxipl , was found to occur in liver of the chow and AIN-93 and also tended to differ in mice fed the AD (P=0.06). In the adipose tissue, diet (P&lt;0.001), probiotic (P&lt;0.001), and their interaction (P=0.01) influenced transcription of the androgen receptor ( Ar ) gene. The chow group had the most Ar transcription, while the AD had the lowest transcription in both the diet alone and probiotic treated group. Taken together, our data highlight the significance of diet in mediating the beneficial metabolic effects of probiotic administration. Diet appears to play a greater role in dictating the expression of key metabolic enzymes and transcription factors and future studies are needed to validate these findings and determine their physiological significance.

A Proof-of-Concept for a Hypolipidemic Brown Trout Model.

The impacts of hypolipidemic pharmaceuticals on fish lipid metabolism remain unexplored. However, data points to similar effects and mechanisms of action between fish and humans. Therefore, fish may be a strong model for screening hypolipidemic drug candidates and water pollution by lipid-modulating agents. This study aimed to test a new hypolipidemic model assay with juvenile brown trout using atorvastatin (ATV)-a hypolipidemic chemical. We selected 17α-ethinylestradiol (EE2), known to cause hyperlipidemia in fish, to ensure model functionality. Fish received intramuscular injections of 4 μL/g for two weeks under the following experimental conditions: control-C (0.7% NaCl), solvent control-SC (0.7% NaCl, 0.9% ethanol, 0.1% dimethyl sulfoxide), ATV (0.3 μg/g), EE2 (2 μg/g), and a mixture of both compounds-MIX (0.3 μg/g ATV and 2 μg/g EE2). Endpoints included blood lipid biochemistry, hepatic lipid droplet quantification, and liver mRNA expression of lipid-related target genes (related to lipogenesis, lipid transport, and β-oxidation pathways). ATV lowered blood total cholesterol, high-density lipoproteins (HDL), and low-density lipoproteins (LDL) levels, whilst triglycerides and very-low-density lipoproteins (VLDL) were highest under EE2. Hepatic lipid droplet deposition significantly increased in the ATV, EE2, and MIX groups. ATV and MIX caused a significant downregulation of the peroxisome proliferator-activated receptor γ (pparγ) and acetyl Co-A oxidase 3 (acox3). EE2 upregulated acyl-CoA long-chain synthetase 1 (acsl1) and downregulated both fatty acid binding protein 1 (fabp1) and acetyl Co-A oxidase 1-3I (acox1-3I). ATV caused hypolipidemic effects in juvenile brown trout and could even counteract EE2-stimulated hyperlipidemia, reinforcing the potential of fish hypo- and hyperlipidemic models.

Distinct bile acid alterations in response to a single administration of PFOA and PFDA in mice

Per- and polyfluoroalkyl substances (PFASs), a group of synthetic chemicals that were once widely used for industrial purposes and in consumer products, are widely found in the environment and in human blood due to their extraordinary resistance to degradation. Once inside the body, PFASs can activate nuclear receptors such as PPARα and CAR. The present study aimed to investigate the impact of perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) on liver structure and functions, as well as bile acid homeostasis in mice. A single administration of 0.1 mmole/kg of PFDA, not PFOA, elevated serum ALT and bilirubin levels and caused cholestasis in WT mice. PFDA increased total and various bile acid species in serum but decreased them in the liver. Furthermore, in mouse livers, PFDA, not PFOA, down-regulated mRNA expression of uptake transporters (Ntcp, Oatp1a1, 1a4, 1b2, and 2b1) but induced efflux transporters (Bcrp, Mdr2, and Mrp2–4). In addition, PFDA, not PFOA, decreased Cyp7a1, 7b1, 8b1, and 27a1 mRNA expression in mouse livers with concomitant hepatic accumulation of cholesterol. In contrast, in PPARα-null mice, PFDA did not increase serum ALT, bilirubin, or total bile acids, but produced prominent hepatosteatosis; and the observed PFDA-induced expression changes of transporters and Cyps in WT mice were largely attenuated or abolished. In CAR-null mice, the observed PFDA-induced bile acid alterations in WT mice were mostly sustained. These results indicate that, at the dose employed, PFDA has more negative effects than PFOA on liver function. PPARα appears to play a major role in mediating most of PFDA-induced effects, which were absent or attenuated in PPARα-null mice. Lack of PPARα, however, exacerbated hepatic steatosis. Our findings indicate separated roles of PPARα in mediating the adaptive responses to PFDA: protective against hepatosteatosis but exacerbating cholestasis.

Effects of PEG antibodies on in vivo performance of LNP-mRNA vaccines

Polyethylene glycol (PEG) plays important roles in stabilizing and lengthening circulation time of lipid nanoparticle (LNP) vaccines. Nowadays various levels of PEG antibodies have been detected in human blood, but the impact and mechanism of PEG antibodies on the in vivo performance of LNP vaccines has not been clarified thoroughly. By illustrating the distribution characteristics of PEG antibodies in human, the present study focused on the influence of PEG antibodies on the safety and efficacy of LNP-mRNA vaccine against COVID-19 in animal models. It was found that PEG antibodies led to shortened blood circulation duration, elevated accumulation and mRNA expression in liver and spleen, enhanced expression in macrophage and dendritic cells, while without affecting the production of anti-Spike protein antibodies of COVID-19 LNP vaccine. Noteworthily, PEG antibodies binding on the LNP vaccine increased probability of complement activation in animal as well as in human serum and led to lethal side effect in large dosage via intravenous injection of mice. Our data suggested that PEG antibodies in human was a risky factor of LNP-based vaccines for biosafety concerns but not efficacy.

Targeting liver angiotensinogen using a GalNAc-siRNA improves cardiac remodeling in spontaneously hypertensive rats

Abstract Background Dysregulation of the Renin-Angiotensin-Aldosterone System (RAAS) leads to hypertension and cardiac hypertrophy. Angiotensinogen (AGT) is the only precursor of all angiotensin peptide products. Liver-specific AGT silencing has been proven to be a promising treatment option in lowering blood pressure (BP) in patients, characterized by full RAAS inhibition and little RAAS escape phenomena. However, the long-term effects of AGT knockdown on cardiac remodeling is still unclear. Purpose To explore the long-term effects of a GalNAc-conjugated siRNA drug R0797070 on AGT inhibition, BP and cardiac functions in Spontaneously Hypertensive Rats (SHRs). Methods In vitro inhibitory activities of siRNAs were assessed in primary rat hepatocytes. The selected siRNA was conjugated with GalNAc based on RIBO-GALSTAR liver targeting system and named R0797070. In vivo effects of repeated doses of R0797070 were evaluated in SHR model. The animals were treated with R0797070 (3 mg/kg, every 8 weeks; subcutaneous injection) or PBS (5 ml/kg, every 8 weeks; subcutaneous injection) for 7 months with the first dose (day1) at the 3 months of age. Captopril (100 mg/kg per day; oral administration) was taken as a positive drug control and Wistar rats were used as normotensive control group. Blood pressure was monitored by tail-cuff method after an initial acclimatization training period. Serum AGT protein was measured by ELISA and liver mRNA expression was detected by qPCR. Echocardiography was used to determine the changes of cardiac morphology and functions，and wheat germ agglutinin (WGA) staining was used to measure myocyte cross-sectional size. Results R0797070 showed 90-99% inhibitory activities on hepatic AGT mRNA at different doses. In SHRs, a single dose of R0797070 at 3 mg/kg resulted in a maximum inhibition of 97% for serum AGT protein with 27 mmHg mean blood pressure (MBP) reduction on Day 15. Daily administration of Captopril could reduce MBP by 33 mmHg on average, while the duration of drug efficacy of R0797070 could reach 2 months with the average 25 mmHg MBP reduction. At the end of study, the echocardiographic results showed that R0797070 significantly reduced left ventricular hypertrophy in the 10-month-old SHRs. Consistently, R0797070 treatment reduced the heart weight by 10%，and LV myocyte size by 17%. Conclusion We showed the long-term effects of a GalNAc-siRNA R0797070 on silencing angiotensinogen expression, reducing blood pressure and left ventricular hypertrophy. Compared to daily oral administration of Captopril and its effect on blood pressure R0797070 demonstrated significant effect on cardiac hypertrophy.Figure 1Figure 2

Featuring molecular regulation of bile acid homeostasis in pediatric short bowel syndrome.

Disturbed bile acid homeostasis may foster development of short bowel syndrome (SBS) associated liver disease during and after weaning off parenteral nutrition (PN). Our aim was to study hepatic molecular regulation of bile acid homeostasis in relation to serum and fecal bile acid profiles in pediatric SBS. Liver histopathology and mRNA expression of genes regulating synthesis, uptake and export of bile acids, and cellular receptors involved in bile acid signaling were measured in SBS patients (n=33, median age 3.2 years). Simultaneously, serum (n=24) and fecal (n=10) bile acid profiles were assessed. Sixteen patients were currently on PN. Results of patients were compared to healthy control subjects. Nine of ten (90%) patients with histological cholestasis received current PN, while portal inflammation was present in 60% (6/10) of patients with cholestasis compared to 13% (3/23) without cholestasis (P=0.01). In all SBS patients, hepatic synthesis and uptake of bile acids was increased. Patients on current PN showed widespread repression of hepatic FXR target genes, including downregulated canalicular (BSEP, MDR3) and basolateral (MRP3) bile acid exporters. Serum and fecal primary bile acids were increased both during and after weaning off PN. Bile acid homeostasis in SBS is characterized by interrupted enterohepatic circulation promoting increased hepatic synthesis and conservation of bile acids. In PN dependent SBS patients with hepatic cholestasis and inflammation, the molecular fingerprint of downregulated hepatocyte canalicular and basolateral bile acid export with simultaneously increased synthesis and uptake of bile acids could favor their accumulation in hepatocytes and predispose to liver disease.

Feeding strategies for rearing replacement beef heifers in a grazing system under tropical conditions

Our objective was to evaluate the effects of pre- and post-weaning protein supplement on the productive response and nutritional and metabolic status of replacement beef heifers grazed on tropical pasture. Fifty-six Nellore heifers (7.7 ± 0.1 months age) with an average initial body weight (BW) of 237 ± 3.5 kg were distributed in a completely randomized design in 2 × 2 factorial arrangement. The four strategies were: US-ME, unsupplemented heifers at pre-weaning and supplemented with 5 g/kg BW at post-weaning; US-HI, unsupplemented heifers at pre-weaning and supplemented with 7.5 g/kg BW at post-weaning; ME-ME, heifers supplemented with 5 g/kg BW at pre-weaning and post-weaning; and ME-HI, heifers supplemented with 5 g/kg BW at pre-weaning and 7.5 g/kg BW at post-weaning. On average, crude protein (CP) intake and CP: digested organic matter (DOM) ratio were influenced (P < 0.05) by protein supplement in the post-weaning period, with a higher intake of CP and CP:DOM ratio in HI heifers than in ME heifers (average CP of 0.812 vs 0.665 kg/day and average DOM of 204 vs 174 g CP/kg). Nonetheless, the post-weaning protein supplement did not affect the intake of total dry matter (DM), pasture DM, organic matter (OM), neutral detergent fiber corrected for ash and protein residue (apNDF), non-fibrous carbohydrates, and DOM. The CP digestibility was highest (P = 0.005) for HI heifers than for ME heifers (average of 0.732 vs 0.645 g/g DM). The pre-weaning and post-weaning protein supplement did not affect the concentration of insulin-like growth factor–1 (IGF-1), progesterone, albumin, globulins, glucose, and triglycerides, and liver mRNA expression of carbamoyl phosphate synthetase-1 (CPS-1) and phosphoenolpyruvate carboxykinase (PEPCK). Finally, the pre-weaning and post-weaning protein supplement did not affect growth performance, carcass traits, and body growth in the post-weaning. Supplementation during pre-weaning does not bring any carry-over effect on the productive performance and metabolic status of heifers supplemented during post-weaning. However, supplementation in post-weaning with 5 or 7.5 g/kg BW allows a compensatory growth of heifers that are unsupplemented at pre-weaning. Therefore, the supply of 7.5 g/kg BW of protein supplement for beef heifers only during the post-weaning period is recommended.

Effect of supplementing rumen-protected fat during the second half of gestation on maternal performance and metabolism in ewes during pregnancy and subsequent lactation

This study aimed to evaluate the effects of supplementing ewes' diet with calcium salts of soybean oil (CSSO) during the second half of gestation on their nutritional, metabolic, and physiological parameters. Additionally, it also aimed to assess the their offspring's sex effect and its interaction with rumen-protected fat. To achieve this, 30 multiparous hair ewes pregnant from a single fetus and weighing 44.1 ± 6.9 kg were randomly divided into two feeding regimens at 75 days of gestation: (1) Control (CON; n = 16), which received a basal diet that met the basic nutritional requirements of ewes gestating a single fetus, and (2) CSSO (n = 14), which received the basal diet plus calcium salts of soybean oil at a level of 1.5 g per kg of body weight (5 % inclusion based on dry matter). The ewes and their offspring were kept together after birth, and lambs were weaned once they reached 20 kg of body weight. Results showed that CSSO ewes were heavier during late gestation and had higher body weight at lactation than CON ewes (P ≤ 0.05). However, CSSO ewes showed an earlier decline in dry matter intake during late gestation (P ≤ 0.05). CSSO ewes also had greater apparent total tract digestibility of ether extract (P < 0.01) and tended to have lower fat milk concentration by approximately 17 % (P = 0.08). In terms of gene expression, CSSO ewes had greater CPT2 expression and ACACA mRNA levels in the subcutaneous adipose tissue (P = 0.08 and P = 0.04, respectively). ACACA mRNA expressions in the maternal liver tissue were also greater (P ≤ 0.01) for CSSO ewes gestating female offspring. However, liver mRNA expressions of IGF1, PC, PCK1, GK, and PPARy were similar between feeding regimens (P > 0.05). Lambs from CSSO ewes were born 0.57 kg heavier than those from CON ewes (P = 0.01) and tended to be weaned at an younger age (P = 0.06). Additionally, CSSO lambs had greater MyoG expression than CON lambs (P = 0.05). In summary, the supplementation of ewes' diet with CSSO during the second half of gestation shows promise as a technology to enhance fetal development, as well as improve long-term maternal and offspring performance.

Changes in cortisol and corticosteroid receptors during dynamic salinity challenges in Mozambique tilapia

In estuarine environments, euryhaline fish maintain a narrow range of internal osmolality despite daily changes in environmental salinity that can range from fresh water (FW) to seawater (SW). The capacity of euryhaline fish to maintain homeostasis in a range of environmental salinities is primarily facilitated by the neuroendocrine system. One such system, the hypothalamic-pituitary-interrenal (HPI) axis, culminates in the release of corticosteroids such as cortisol into circulation. Cortisol functions as both a mineralocorticoid and glucocorticoid in fish because of its roles in osmoregulation and metabolism, respectively. The gill, a key site for osmoregulation, and the liver, the primary storage site for glucose, are known targets of cortisol’s actions during salinity stress. While cortisol facilitates acclimation to SW environments, less is known on its role during FW adaptation. In this study, we characterized the responses of plasma cortisol, mRNA expression of pituitary pro-opiomelanocortin (pomc), and mRNA expression of liver and gill corticosteroid receptors (gr1, gr2, and mr) in the euryhaline Mozambique tilapia (Oreochromis mossambicus) under salinity challenges. Specifically, tilapia were subjected to salinity transfer regimes from steady-state FW to SW, SW to FW (experiment 1) or steady state FW or SW to tidal regimen (TR, experiment 2). In experiment 1, fish were sampled at 0 h, 6 h, 1, 2, and 7 d post transfer; while in experiment 2, fish were sampled at day 0 and day 15. We found a rise in pituitary pomc expression and plasma cortisol following transfer to SW while branchial corticosteroid receptors were immediately downregulated after transfer to FW. Moreover, branchial expression of corticosteroid receptors changed with each salinity phase of the TR, suggesting rapid environmental modulation of corticosteorid action. Together, these results support the role of the HPI-axis in promoting salinity acclimation, including in dynamically-changing environments.

Dietary zinc supplementation in breeding pigeons improves the carcass traits of squabs through regulating antioxidant capacity and myogenic regulatory factor expression

The purpose of this experiment was to explore the effects of zinc supplementation in breeding pigeons diet on carcass traits, meat quality, antioxidant capacity and mRNA expressions of myogenic regulatory factors of squabs. A total of 120 healthy White King pigeons were randomly assigned to 5 treatments, each involving 8 replicates. The experiment lasted for 46 d (18-d incubation period of eggs and 28-d growth period of squabs). The 5 groups were 0, 30, 60, 90, and 120 mg/kg zinc addition. Results showed that the 28-d body weight, breast muscle yield, zinc content in crop milk and myogenic factor 6 (MyF6) abundance of breast muscle were linearly increased (P < 0.050), but the abdominal fat yield linearly decreased (P=0.040) with increasing dietary zinc supplementation. Both the linear (P < 0.050) and quadratic responses (P < 0.001) were observed in copper zinc superoxide dismutase (Cu-Zn SOD), total antioxidant capacity (T-AOC) and malondialdehyde (MDA) contents in liver and breast muscle. The 28-d body weight was increased by 90 mg/kg zinc supplementation (P < 0.05), and there is no significant difference between 90 and 120 mg/kg zinc addition. The breast muscle yield, Cu-Zn SOD and T-AOC contents in breast muscle and liver, zinc contents in crop milk and breast muscle, MyF6 mRNA expression in breast muscle were higher (P < 0.05) in the group supplemented with 120 mg/kg zinc than the control. The abdominal fat yield was numerically lowest, and MDA contents in breast muscle and liver were significantly lowest in the group fed 120 mg/kg zinc (P < 0.05). However, the meat quality traits were not affected (P > 0.05) by zinc supplementation, except for shear force. It should be stated dietary zinc supplementation at the level of 120 mg/kg for breeding pigeons increased body weight and breast muscle yield of squabs, which may be associated with the up-regulating MyF6 mRNA expression and antioxidant capacity in liver and breast muscle.

In vivo assessment of the pharmacokinetic interactions between donafenib and dapagliflozin, donafenib and canagliflozin in rats

Donafenib (DONA), a deuterium derivative of sorafenib, is used for advanced hepatocellular carcinoma (HCC). Dapagliflozin (DAPA) and canagliflozin (CANA) are sodium–glucose co-transporter 2 (SGLT2) inhibitors used for T2DM, which is frequently comorbid with HCC. Three drugs are substrates of UGT1A9 isoenzyme. This study aimed to evaluate donafenib–dapagliflozin and donafenib–canagliflozin pharmacokinetic interactions and explore the potential mechanisms. Rats were divided into seven groups (n = 6) that received donafenib (1), dapagliflozin (2), canagliflozin (3), dapagliflozin and donafenib (4), canagliflozin and donafenib (5), donafenib and dapagliflozin (6), donafenib and canagliflozin (7). The concentrations of drugs were determined by an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. The messenger RNA (mRNA) expressions were measured by quantitative RT-PCR. Multiple doses of dapagliflozin caused donafenib maximum plasma concentration (Cmax) to increase 37.01%. Canagliflozin increased donafenib Cmax 1.77-fold and the area under the plasma concentration–time curves (AUC0−t and AUCinf) 1.39- and 1.41-fold, respectively, while reducing the apparent clearance (CLz) 28.38%. Multiple doses of donafenib increased dapagliflozin AUC0−t 1.61-fold, AUCinf 1.77-fold, whereas its CLz reduced 40.50%. Furthermore, donafenib caused similar changes in canagliflozin pharmacokinetics. The PCR results demonstrated that dapagliflozin inhibited the mRNA expression of Ugt1a7 in liver and donafenib decreased the expression of Ugt1a7 mRNA in liver and intestine. Increased exposure to these drugs may be due to their metabolism inhibition mediated by Ugt1a7. These pharmacokinetic interactions observed in this study may be of clinical significance, which may help adjust dose properly and avoid toxicity effects in patients with HCC and T2DM.

Liver insulin-like growth factor-1 mediates effects of low-intensity vibration on wound healing in diabetic mice.

Chronic wounds in diabetic patients are associated with significant morbidity and mortality; however, few therapies are available to improve healing of diabetic wounds. Our group previously reported that low-intensity vibration (LIV) could improve angiogenesis and wound healing in diabetic mice. The purpose of this study was to begin to elucidate the mechanisms underlying LIV-enhanced healing. We first demonstrate that LIV-enhanced wound healing in db/db mice is associated with increased IGF1 protein levels in liver, blood, and wounds. The increase in insulin-like growth factor (IGF) 1 protein in wounds is associated with increased Igf1 mRNA expression both in liver and wounds, but the increase in protein levels preceded the increase in mRNA expression in wounds. Since our previous study demonstrated that liver was a primary source of IGF1 in skin wounds, we used inducible ablation of IGF1 in the liver of high-fat diet (HFD)-fed mice to determine whether liver IGF1 mediated the effects of LIV on wound healing. We demonstrate that knockdown of IGF1 in liver blunts LIV-induced improvements in wound healing in HFD-fed mice, particularly increased angiogenesis and granulation tissue formation, and inhibits the resolution of inflammation. This and our previous studies indicate that LIV may promote skin wound healing at least in part via crosstalk between the liver and wound. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Effects of dietary iron sources on growth performance, iron status, Fe-containing enzyme activity and gene expression related to iron homeostasis in tissues of weaned pigs.

The aim of this study is to evaluate the effects of dietary iron sources on growth performance, iron status and activities of Fe-containing enzymes and gene expression related to iron homeostasis in tissues of weaned pigs. A total of 480 piglets at d 28 (Duroc X Landrace) were allotted to four groups as a factorial arrangement of treatments with 30 pigs/pen (male: female = 1:1) and 4 replicate pens/treatment. The treatments for iron in the diets were: control basal diet (Con); Con + 150 mg Fe/kg as inorganic Fe (iFe); Con + 75 mg Fe/kg as inorganic Fe + 75 mg Fe/kg as organic Fe-peptide complex (iFe+oFe) and Con + 150 mg of Fe/kg as organic Fe-peptide complex (oFe). The feeding trial lasted for 36 days. There were no significant differences in final body weight, ADG, ADFI, and G/F as well as blood hemoglobin and MCHC contents between piglets fed the control and iron-supplemented groups (P > 0.05). The iron supplemented groups exhibited increased iron content in the liver, kidney and spleen as well as the CAT and SDH activities in liver compared to the control group (P < 0.05), while piglets in oFe group experienced greater Fe accumulation and activities of CAT and SDH in the liver than piglets in the iFe group. Compared with the control group, dietary supplementation of iron increased the NCOA4 mRNA expression and decreased the TfR1 mRNA expression in liver of piglets. The TfR1, NCOA4 and Ferritin mRNA expressions of bone marrow in both iFe and iFe+oFe groups were greater than both in the Con and oFe groups. These results suggest that dietary supplementation of iron does not influence the growth performance and hematological parameters in weaned pigs fed a corn-soybean meal basal diet (75.8 mg/kg) from d 28 to d 70, but increased tissue iron status and activities of Fe-containing enzymes at d 70. The addition of organic Fe-peptide complexes presents greater beneficial effects on enhancing tissue Fe accumulation and Fe-containing enzyme activities, which may be involved in different gene expression patterns related to iron intake and transport in tissues of weaned pigs.

Preparation and characterization of the myricetin-loaded solid lipid nanoparticles decorated with folic acid-bound chitosan and evaluation of its antitumor and anti-angiogenic activities in vitro and in vivo in mice bearing tumor models

Myricetin is a flavonoid with anticancer properties. This study aimed to formulate myricetin in the form of solid lipid nanoparticles (SLN), decorated with chitosan (CS) and active-targeted with folic acid (FA). After characterization, the in vitro release, cytotoxicity, antioxidant, and ability of the formulation to induce apoptosis using flow cytometry, fluorescent microscopy, and real-time qPCR were examined. Then in vivo anti-angiogenesis on chick chorioallantoic membrane (CAM) and antitumor activities on mice bearing tumor models were investigated. The present study showed that the size of 310 nm and zeta potential of + 30 mV were acceptable for oral administration. The Michaelis–Menten model fitted the drug release pattern with lag during 144 h of the study. The cytotoxicity assay showed that myricetin-SLN-CS-FA significantly killed cancer cells at the concentrations of 6.25, 12.5, 25, 50 and 100 µg/mL (*p < 0.05, **p < 0.01, and ***p < 0.001). The highest level of apoptosis was shown at the concentration of 45 µg/ml in flow cytometry, and fluorescent studies. These results showed the anticancer properties of myricetin-SLN-CS-FA in a dose-dependent manner. The real-time results also indicated that the formulation exerted its cytotoxic effect by activating apoptosis genes. The DPPH, ABTS, and FRAP studies also demonstrated the significant antioxidant properties of the myricetin-SLN-CS-FA (*p < 0.05, **p < 0.01, and ***p < 0.001). The anti-angiogenic activities of the formulations depicted in the CAM assay significantly decrease the number and length of the vessels (*p < 0.05, **p < 0.01, and ***p < 0.001), and also affect VEGF and VEGFR, genes involved in angiogenesis (**p < 0.01, and ***p < 0.001). The antitumor studies indicated the statistically significant effects of myricetin-SLN-CS-FA on reducing tumor volume (*p < 0.05 and ***p < 0.001). The H&E staining of the liver and monitoring of the animal weights also indicated the safety of the formulation. The analysis of mRNA expression in liver and tumor demonstrated that myricetin-SLN-CS-FA exerts its antitumor activities by modulating the inflammatory and oxidative responses in the tissues.

Scytosiphon lomentaria Extract Ameliorates Obesity and Modulates Gut Microbiota in High-Fat-Diet-Fed Mice.

Scytosiphon lomentaria (SL) is a brown seaweed with antioxidant and anti-inflammatory properties; however, its effects on obesity are unknown. In this research, we investigated the anti-obesity properties and underlying mechanisms of the SL extract in vitro and in vivo. In 3T3-L1 preadipocytes, SL extract inhibited lipid accumulation, decreased the expression of Acc1, C/ebpa, Pparg mRNA and p-ACC1, and increased the expression of Ucp1 mRNA, UCP1 and p-AMPK. In animal experiments, mice were fed a chow diet, a high-fat diet (HF; 60% of calories as fat), and high-fat diet with SL extract (150 and 300 mg/kg body weight) for eight weeks (n = 10/group). SL extract reduced HF-induced weight gain, epididymal fat weight, fat cell size, LDL-C, leptin, fasting glucose, and glucose tolerance. In addition, SL extract had comparable effects on mRNA expression in WAT and liver to those observed in vitro, thereby inhibiting p-ACC1/ACC1 and increasing p-AMPK/AMPK and UCP1 expression. Furthermore, SL extract decreased HF-induced Firmicutes/Bacteroidetes ratio and reversed HF-reduced Bacteroides spp., Bacteroides vulgatus, and Faecalibacterium prausnitzii. These findings suggest that SL extract can aid in weight loss in mice fed a high-fat diet by altering adipogenic and thermogenic pathways, as well as gut microbiota composition.

Ewe early gestation supplementation with eicosapentaenoic and docosahexaenoic acids affects the liver, muscle, and adipose tissue fatty acid profile and liver mRNA expression in the offspring.

Our objectives were to assess the effects of eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) supplementation to pregnant ewes during the first third of gestation on their offspring's liver, adipose, and muscle tissues fatty acid (FA) profile and liver mRNA expression after a finishing period receiving diets with different FA profiles. Twenty-four post-weaning lambs, blocked by sex and body weight, were used in a 2 × 2 factorial arrangement of treatments. The first factor was dam supplementation (DS) in the first third of gestation with 1.61% of Ca salts of palm fatty acid distillate (PFAD) or Ca salts enriched with EPA-DHA. Ewes were exposed to rams with marking paint harnesses during the breeding. Ewes started DS at the day of mating, considered day 1 of conception. Twenty-eight days after mating, ultrasonography was used to confirm pregnancy, and nonpregnant ewes were removed from the groups. After weaning, the offspring lambs were supplemented (LS, second main factor) with two different FA sources (1.48% of PFAD or 1.48% of EPA-DHA) during the growing and fattening phase. Lambs were fed the LS diet for 56 d and sent to slaughter, where the liver, muscle, and adipose tissue samples were collected for FA analysis. Liver samples were collected for relative mRNA expression for genes associated with FA transport and metabolism. The data were analyzed as a mixed model in SAS (9.4). In the liver, the amount of C20:5 and C22:6 (P < 0.01) increased in lambs with LS-EPA-DHA, while some C18:1 cis FA isomers were greater in the lambs from DS-PFAD. In muscle, amounts of C22:1, C20:5, and C22:5 increased (P < 0.05) in lambs born from DS-EPA-DHA. The adipose tissue amounts of C20:5, C22:5, and C22:6 were greater (P < 0.01) in lambs from LS-EPA-DHA. Interactions (DS × LS; P < 0.05) were observed for DNMT3β, FABP-1, FABP-5, SCD, and SREBP-1; having greater mRNA expression in liver tissue of LS-EPA-DHA, DS-PFAD and LS-PFAD, DS-EPA-DHA lambs compared with the lambs in the other two treatments. Liver ELOVL2 mRNA relative expression (P < 0.03) was greater in the offspring of DS-PFAD. Relative mRNA expression (P < 0.05) of GLUT1, IGF-1, LPL, and PPARγ increased in the liver from LS-EPA-DHA lambs. Dam supplementation during early gestation using with different FA sources changed the lipid FA profile in MT, LT, and SAT during the finishing period depending on the tissue and type of FA source administered during the growing phase.

Serum FGF19 predicts outcomes of Kasai portoenterostomy in biliary atresia.

Outcomes after Kasai portoenterostomy (KPE) for biliary atresia remain highly variable for unclear reasons. As reliable early biomarkers predicting KPE outcomes are lacking, we studied the prognostic value of FGF19. Serum and liver specimens, obtained from biliary atresia patients (N=87) at KPE or age-matched cholestatic controls (N=26) were included. Serum concentration of FGF19 and bile acids, liver mRNA expression of FGF19 , and key regulators of bile acid synthesis were related to KPE outcomes and liver histopathology. Immunohistochemistry and in situ hybridization were used for the localization of liver FGF19 expression. Serum levels (223 vs. 61pg/mL, p <0.001) and liver mRNA expression of FGF19 were significantly increased in biliary atresia. Patients with unsuccessful KPE (419 vs. 145pg/mL, p =0.047), and those subsequently underwent liver transplantation (410 vs. 99pg/mL, p =0.007) had significantly increased serum, but not liver, FGF19, which localized mainly in hepatocytes. In Cox hazard modeling serum FGF19 <109pg/mL predicted native liver survival (HR: 4.31, p <0.001) also among patients operated <60 days of age (HR: 8.77, p =0.004) or after successful KPE (HR: 6.76, p =0.01). Serum FGF19 correlated positively with increased serum primary bile acids ( R =0.41, p =0.004) and ductular reaction ( R =0.39, p =0.004). Increased serum FGF19 at KPE predicted inferior long-term native liver survival in biliary atresia and was associated with unsuccessful KPE, elevated serum primary bile acids, and ductular reaction.