High fat diet- induced obesity decreases ErbB receptors expression in liver and adipose tissue in C57BL/6 mice

Neuregulins (NRGs) are a family of signaling proteins that bind to receptor tyrosine kinases of the ErbB family (ErbB2 to ErbB4), which can homo- or heterodimerize depending on their structural features and cell type. Many studies have proposed that decreased NRG levels are a common characteristic of obesity. In liver and adipose tissue, the increase in NRG expression has protective effects against obesity. However, it is still unknown whether ErbBs expression is altered in this pathology. We hypothesized that high fat diet-induced obesity downregulates ErbB receptors expression in obese mice compared to normal weight mice. Males C57BL/6 mice (n=6-7 for each group) were fed for 12 weeks and divided into: (i) control diet (CD; 10%-kcal fat, 20%-kcal protein, 70%-kcal carbohydrates), and (ii) high fat diet (HFD; 60%-kcal fat, 20%-kcal protein, 20%-kcal carbohydrates). General parameters and ErbBs expression (qPCR, immunohistochemistry and Western blot) were evaluated. We observed a significant increase in final body weight (47%), adipose tissue to body weight ratio (244%) and HOMA-IR (69%), among other parameters, in obese mice. In HFD group significantly decreased ErbB2 (48%) and ErbB3 (66%) mRNA levels in liver (no change in ErbB4), and ErbB2 (43%), ErbB3 (76%) and ErbB4 (35%) in adipose tissue, compared to CD. Furthermore, ErbB2 and ErbB3 protein levels decreased significantly in HFD group compared to the CD in liver. Therefore, our results suggest that HFD-induced obesity significantly decreases ErbBs expression in liver and adipose tissue in this murine model, that may be associated with alterations in the NRG pathway in obese mice.

Las neuregulinas (NRGs) son una familia de proteínas de señalización que se unen a receptores tirosina quinasas de la familia ErbB (ErbB2 a ErbB4), que pueden homo- o heterodimerizar dependiendo de sus características estructurales y del tipo celular. Estudios han propuesto que la disminución de los niveles de NRG es una característica común de la obesidad. En el hígado y el tejido adiposo (TA), el aumento de la expresión de NRG tiene efectos protectores contra la obesidad. Sin embargo, aún se desconoce si la expresión de ErbBs está alterada en esta patología. Nuestra hipótesis es que la obesidad inducida por una dieta alta en grasas (DAG) disminuye la expresión de los ErbB en ratones obesos. Ratones machos C57BL/6 (n=6-7 para c/grupo) fueron alimentados durante 12 semanas y divididos en: (i) dieta control (DC; 10%-kcal grasa, 20%-kcal proteína, 70%-kcal carbohidratos), y (ii) DAG (60%-kcal grasa, 20%-kcal proteína, 20%-kcal carbohidratos). Se evaluaron los parámetros generales y la expresión de ErbBs (qPCR, inmunohistoquímica y Western blot). Observamos un aumento significativo del peso corporal final (47%), de la relación tejido adiposo/peso corporal (244%) y del HOMA-IR (69%), entre otros parámetros, en ratones obesos. En este grupo disminuyó significativamente los niveles de ARNm de ErbB2 (48%) y ErbB3 (66%) en el hígado (sin cambios en ErbB4), y de ErbB2 (43%), ErbB3 (76%) y ErbB4 (35%) en el TA. Además, los niveles de proteína ErbB2 y ErbB3 disminuyeron significativamente, en comparación con el grupo DC en el hígado. Nuestros resultados sugieren que la obesidad inducida por DAG disminuye significativamente la expresión de ErbBs en el hígado y el TA, que puede estar asociado con alteraciones en la vía NRG en ratones obesos.

Phosphatidic acid-enabled MKL1 contributes to liver regeneration: Translational implication in liver failure

Liver regeneration following injury aids the restoration of liver mass and the recovery of liver function. In the present study we investigated the contribution of megakaryocytic leukemia 1 (MKL1), a transcriptional modulator, to liver regeneration. We report that both MKL1 expression and its nuclear translocation correlated with hepatocyte proliferation in cell and animal models of liver regeneration and in liver failure patients. Mice with MKL1 deletion exhibited defective regenerative response in the liver. Transcriptomic analysis revealed that MKL1 interacted with E2F1 to program pro-regenerative transcription. MAPKAPK2 mediated phosphorylation primed MKL1 for its interaction with E2F1. Of interest, phospholipase d2 promoted MKL1 nuclear accumulation and liver regeneration by catalyzing production of phosphatidic acid (PA). PA administration stimulated hepatocyte proliferation and enhanced survival in a MKL1-dependent manner in a pre-clinical model of liver failure. Finally, PA levels was detected to be positively correlated with expression of pro-regenerative genes and inversely correlated with liver injury in liver failure patients. In conclusion, our data reveal a novel mechanism whereby MKL1 contributes to liver regeneration. Screening for small-molecule compounds boosting MKL1 activity may be considered as a reasonable approach to treat acute liver failure.

Histomorphological, Histochemical and Ultrastructural Studies on the Healthy Liver of Yemen Veiled Chameleon (Chamaeleo calyptratus) in Southern Saudi Arabia / Estudios Histomorfológicos, Histoquímicos y Ultraestructurales en el Hígado Sano del Camaleón Velado de Yemen (Chamaeleo calyptratus) en el Sur de Arabia Saudita

SUMMARY: The livers of reptiles are being studied as a model for the link between the environment and hepatic tissue. There have been few investigations on the histology of reptile livers, and very few or no studies have examined the histology of liver of veiled chameleon (Chamaeleo calyptratus). This paper describes the histomorphological, histochemical and ultrastructural characterization of the liver of veiled chameleons in southern Saudi Arabia. Seven Chamaeleo calyptratus were captured in the summer season in Abha City, Aseer region, southern Saudi Arabia. Chamaeleon liver samples were processed for histomorphology, histochemistry and ultrastructure analyses. Morphologically liver of Chamaeleo calyptratus was observed as a large dark brown organ with lighter speckles, which represent melanin deposits. It located at the ventral part of abdominal cavity forward of the stomach. Its dimensions approximately were 3.7 x 2 cm. The liver was a bilobed organ divided into two lobes, right and left lobes. The right one was bigger than the others. The gallbladder was well developed and had an elongated shape, situated between the two lobes and contained the bile for the digestion. Microscopically, the liver was found to be covered by a thick layer of connective tissue, which formed the hepatic capsule. Hepatic parenchyma probably appeared in cross sections as hepatic glandular-like alveoli "acini" or follicular structures with various diameters, each acinus contains approximately four to six hepatocytes, surrounded by sinusoidal capillaries filled with abundant melanomacrophages, which are absent in birds and mammals. Melanomacrophages are common in the hepatic parenchyma's perisinusoidal areas, particularly near portal spaces. Hepatocytes are polyhedral or pyramidal with and mostly contained large, rounded nuclei mostly peripherally located, with prominent dark oval nucleoli. Some of nuclei are eccentric or central position. The cytoplasm appeared spongy or vacuolated and more eosinophilic when stained by hematoxylin-eosin and strongly reactive to PAS staining technique, indicating abundant glycogen content. The reticular fibers that surround hepatocytes, blood arteries, and sinusoids supported the hepatic parenchyma. The blood sinusoids are seen interspersed among hepatocytes of varying sizes. The sinusoidal lumen was bordered by flattened endothelial cells and includes elliptical nucleated erythrocytes and liver macrophages as phagocytes, which are also known as Kupffer cells. Branches of the portal vein, hepatic artery, small bile duct, and lymph vessels were detected in the hepatic portal area "tract" or triad which made up of connective. Hematopoietic tissue was observed in subcapsular region and portal triads. Ultrastructurally, the hepatocyte appeared polyhedric containing a single large rounded basal or eccentric vesicular nucleus with prominent nucleolus. Extensive network of rough endoplasmic reticulum (RER) often arranged in an array parallel to the nuclear membrane with many mitochondria, and Golgi apparatus were described. The cytoplasm contained glycogen granules, vesicles or vacuoles scattered throughout the cytoplasm especially at the apical region were reported. The bile canaliculi and the hepatic "Kupffer" cells were also discussed. This is the first study on the histological characterization of the healthy liver of Yemen veiled chameleon in southern Saudi Arabia. The findings reported here should be used as a reference to compare with the pathological abnormalities of the liver in this animal.

está disponible en el texto completo

Mitochondrial protection effect of sirtuin2 inhibitor AGK2 on thioacetamide-induced L02 cells / 中国医师杂志

Objective:To explore the protective effect of AGK2, a selective inhibitor of sirtuin 2 (SIRT2), on the mitochondria of L02 hepatocytes induced by thioacetamide (TAA) and its related mechanism.Methods:Human-derived hepatocyte line L02 cells were cultured in vitro. Different concentrations of SIRT2 inhibitor AGK2 were used as intervention drugs. Cell counting kit-8 (CCK8) was used to detect the effects of different concentrations of AGK2 on the activity of L02 cells, and the appropriate concentration was selected as the AGK2 intervention group. The normal group was not given any drug intervention. The model group was given 90 mmol/L TAA for modeling. Low, medium and high dose AGK2 groups were added with 1, 2 and 4 μmol/L AGK2, respectively 2 h before modeling. CCK8 was used to detect cell activity in each group. Morphological changes of cells were observed under inverted light microscope. The relative protein expression levels of isocitrate dehydrogenase (IDH1), malate dehydrogenase (MDH1), SIRT2 and fission protein 1 homologue (FIS1) were detected by Western blot. The expression of SIRT2 in cells of each group was observed by confocal laser scanning microscope. The mitochondrial membrane potential of cells in each group was observed under a fluorescence microscope. Results:When AGK2 concentration was 1, 2 and 4 μmol/L, the survival rate of cells were 98.05%, 95.76% and 91.65%, respectively, with no statistical significance compared with normal group (all P>0.05). When AGK2 concentration was 8, 16, 32, 64, 128 μmol/L, the cell survival rate was significantly decreased compared with normal group (all P<0.05). Compared with the model group, the L02 cells in low, medium and high AGK2 groups had better activity and adherence, and the floating cells were significantly reduced. The higher the concentration of AGK2, the better the cell activity and adherence, and the less floating cells. Compared with the model group, the red fluorescence of L02 cells in AGK2 group was enhanced, while the green fluorescence was weakened. The higher the AGK2 concentration was, the stronger the red fluorescence was, and the weaker the green fluorescence was. Compared with the model group, the fluorescence of SIRT2 in L02 cells of low, medium and high AGK2 groups was weakened, and the higher the concentration of AGK2, the weaker the fluorescence of SIRT2. The protein expressions of IDH1 and MDH1 in L02 cells of low, medium and high AGK2 groups were significantly higher than those of model group (all P<0.05), and were positively correlated with the concentration of AGK2 ( r=0.818, P<0.05; r=0.960, P<0.05); the protein expressions of SIRT2 and FIS1 were significantly lower than those of the model group (all P<0.05), and were negatively correlated with the concentration of AGK2 ( r=-0.992, P<0.05; r=-0.998, P<0.05). Conclusions:AGK2 can reduce the mitochondrial membrane potential stimulated by TAA in L02 cells, increase the protein expression of IDH1 and MDH1, and inhibit the protein expression of SIRT2 and FIS1 in L02 cells in a dose-dependent manner.

Characteristics and clinical significance of mitochondrial injury of T lymphocyte subsets in patients with autoimmune hepatitis / 中国医师进修杂志

Objective:To study the characteristics and clinical significance of mitochondrial injury of T lymphocyte subsets in patients with autoimmune hepatitis (AIH).Methods:The clinical data of 57 patients with AIH (AIH group) from June to December 2021 in Hangzhou Xixi Hospital were retrospectively analyzed, while 60 healthy physical examiners were included as healthy group. The peripheral blood T lymphocyte subsets (CD 8+ T lymphocyte count and CD 4+ T lymphocyte count) were detected by flow cytometry, and matched mitochondrial staining value according to certain algorithm was used to determine the mitochondrial damage of helper T lymphocyte (Th cell) and suppressor T lymphocyte (Ts cell). The levels of IgG, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using a Roche E170 automatic electrochemiluminescence immunoassay. Anti-nuclear antibody (ANA) titer was measured by immunofluorescence. Multivariate Logistic regression was used to analyze the independent risk factors of mitochondrial damage of Th cell and Ts cell in patients with AIH. Results:The ALT, AST, IgG, positive rate of ANA titer, CD 4+ T lymphocyte count, CD 8+ T lymphocyte count, rate of Th cell mitochondrial injury and rate of Ts cell mitochondrial injury in AIH group were significantly higher than those in healthy group: (118.90 ± 37.61) U/L vs. (30.96 ± 14.37) U/L, (102.40 ± 36.51) U/L vs. (31.12 ± 14.06) U/L, (18.40 ± 3.71) g/L vs. (13.89 ± 1.98) g/L, 96.49% (55/57) vs. 16.67% (10/60), 438 (323, 637) × 10 6/L vs. 398 (272, 469) × 10 6/L, 296 (211, 296) × 10 6/L vs. 270 (193, 322) × 10 6/L, 61.40% (35/57) vs. 8.33% (5/60) and 82.46% (47/57) vs. 11.67% (7/60), and there were statistical differences ( P<0.01 or <0.05). Multivariate Logistic regression analysis result showed that the AST elevated and CD 8+ T lymphocyte count reduced were the independent risk factors of Ts cell mitochondrial injury in patients with AIH ( OR = 1.06 and 0.99, 95% CI 1.01 to 1.10 and 0.99 to 1.00, P<0.05); the ALT elevated and IgG elevated were the independent risk factors of Th cell mitochondrial injury in patients with AIH ( OR = 1.08 and 1.66, 95% CI 1.02 to 1.14 and 1.11 to 2.48, P<0.05). Conclusions:It is of positive clinical significance to measure the T lymphocyte subtype mitochondrial injury in patients with AIH. The probability of mitochondrial injury of T lymphocyte subtype can be predicted by biochemical indexes such as ALT, AST and IgG, so as to indirectly evaluate the liver cell necrosis.

The protective effect of trigonelline on H / 中国药理学通报

Aim To study the protective effect of trigonelline on H

Liver cell therapies: cellular sources and grafting strategies / 医学前沿

The liver has a complex cellular composition and a remarkable regenerative capacity. The primary cell types in the liver are two parenchymal cell populations, hepatocytes and cholangiocytes, that perform most of the functions of the liver and that are helped through interactions with non-parenchymal cell types comprising stellate cells, endothelia and various hemopoietic cell populations. The regulation of the cells in the liver is mediated by an insoluble complex of proteins and carbohydrates, the extracellular matrix, working synergistically with soluble paracrine and systemic signals. In recent years, with the rapid development of genetic sequencing technologies, research on the liver's cellular composition and its regulatory mechanisms during various conditions has been extensively explored. Meanwhile breakthroughs in strategies for cell transplantation are enabling a future in which there can be a rescue of patients with end-stage liver diseases, offering potential solutions to the chronic shortage of livers and alternatives to liver transplantation. This review will focus on the cellular mechanisms of liver homeostasis and how to select ideal sources of cells to be transplanted to achieve liver regeneration and repair. Recent advances are summarized for promoting the treatment of end-stage liver diseases by forms of cell transplantation that now include grafting strategies.

Synchronization isolation method for multiple types of cells from mouse liver / 中华肝脏病杂志

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.

Efecto del extracto hidroalcohólico de la planta Lampaya medicinalis Phil . sobre marcadores inflamación inducidos por ácidos grasos en hepatocitos humanos HepG2

RESUMEN Introducción: la enfermedad de hígado graso no alcohólico (EHGNA) se caracteriza por la acumulación de gotas lipídicas (GL) y sobre expresión de la proteína de GL Perilipina 1 (PLIN1) en los hepatocitos. En su patogénesis y progresión participan NF-ĸB, caspasa-1 y citoquinas proinflamatorias como IL-1β. La medicina popular del norte de Chile utiliza la planta Lampaya medicinalis Phil. (Verbenaceae) contra enfermedades. Objetivo: evaluar el efecto de un extracto hidroalcohólico de lampaya (EHL) sobre la expresión de marcadores inflamatorios y proteínas asociadas a las GL en hepatocitos tratados con ácidos grasos. Métodos: se incubó hepatocitos HepG2 con 0,66 mM de ácido oleico (AO) y 0,33 mM de ácido palmítico (AP) por 24 o 48 horas en presencia o no de EHL. Se evaluó la expresión proteica de NF-ĸB, PLIN1 y caspasa-1 por Western blot y la expresión de ARNm de IL-1β por qPCR. Resultados: los hepatocitos tratados por 48 h con AO/AP mostraron un aumento en la expresión de IL-1β que fue revertido por la co-incubación con EHL. Conclusión: estos antecedentes aportan nueva evidencia respecto a la actividad biológica del EHL en un modelo de alteraciones metabólicas e inflamatorias, asociadas a la EHGNA, inducidas por AO/AP en hepatocitos humanos.

ABSTRACT Introduction: Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipid droplets (LD) and overexpression of the LD-associated protein Perilipin 1 (PLIN1). NF-ĸB, caspase-1 and proinflammatory cytokine such as IL-1β participate in the pathogenesis and progression of NAFLD. Traditional medicine in northern Chile uses the plant Lampaya medicinalis Phil. (Verbenaceae) against diseases. Objective: To evaluate the effect of a hydroalcoholic extract of lampaya (HEL) on the expression of inflammatory markers and LD-associated proteins in hepatocytes treated with fatty acids. Methods: HepG2 hepatocytes were incubated with 0.66 mM oleic acid (OA) and 0.33 mM palmitic acid (PA) for 24 or 48 h in the presence or not of HEL. The protein expression of NF-ĸB, PLIN1 and caspase-1 was evaluated by Western blot while the mRNA expression of IL-1β was assessed by qPCR. Results: hepatocytes treated for 48 h with OA/AP showed an increase in IL-1β expression that was reversed by co-incubation with HEL. Conclusion: These antecedents provide new evidence regarding the biological activity of HEL in a model of metabolic and inflammatory alterations, associated with NAFLD, induced by OA/PA in human hepatocytes.

Hepatotoxic Effect of Aqueous Extract of Parkia biglobosa Seeds on Adult Albino Wistar Rats

Background: Parkia biglobosa belongs to the plant family Fabaceae and is popularly known as the African locust bean tree is gotten from medium-sized, tree high (20-20 cm), whose leaves are edible and are used in many African local dishes. The phytochemical screening of the methanolic extracts of P. biglobosa revealed the presence of saponins, tannins, terpenes, and phenols, reducing sugars, sterols, flavonoids. Methods: 21 adult Wistar rats (100-120 g) were distributed into 3 groups (A, B and C) consisting of 7 in each. Group B and C were administered orally with aqueous seed extract of P. biglobosa at a dose of 300 mg/kgB wt and 500 mg/kg B wt, respectively for 30 days. Group A was normal control and received 300 mg/kgB wt of normal saline. After 30 days, the weights were recorded and the animals were sacrificed using cervical dislocation. The changes in body weight, liver histology and enzymes were evaluated. Results: This study shows a significant difference (p<0.01) in the body weight gain between animals in the low, high and control groups, respectively. Photomicrograph of the liver tissue from animals in low dose reveals a liver cytoarchitecture with mildly dilated sinusoids, while the liver tissue from animals high dose group revealed a portal tract with dilated sinusoids. Results from histochemical observation of the liver of the control group showed marked periodic Acid Schiff (PAS) staining on predominant hepatocytes but little or no staining of cytoplasm, the low dose reveals a mild PAS staining while that of high dose shows moderate staining on tissue degeneration. Serum chemistry revealed a significant increase (p<0.05) AST and ALT in the test groups when compared to control group. Conclusion: Results from this study shows that the aqueous extract of P. biglobosa at a dose of 500 mg/kgB wt over 30 days may have adversely affected the morphology of the liver with the increase in serum levels.

The effect of heme oxygenase-1 on liver sinusoidal endothelial cells proliferation and pro-regeneration / 中华肝胆外科杂志

Objective:To investigate the effects of heme oxygenase-1 (HO-1) on hepatic sinusoidal endothelial cells (LSECs) proliferation, migration, and hepatocyte proliferation.Methods:Eighteen male C57BL/6 mouse aged 6-8 weeks old were underwent partial hepatectomy. Cell proliferation and HO-1 expression in residual liver tissue were detected by immunofluorescence histochemistry at 0 d, 2 d and 4 d after operation. In vitro, LSECs were transfected with adenovirus carrying HO-1 gene (HO-1 group), and the cells were transfected with empty vector adenovirus and the non-transfected cells were used as control. In addition, LSECs from different transfection groups were co-cultured with hepatocyte without contact to evaluate the effect of HO-1 expression on promoting hepatocyte proliferation. Western Blotting and RT-PCR were used to detect the protein and mRNA expression of HO-1, inhibitor of DNA binding and or differentiation (Id1), hepatocyte growth factor (HGF) and Wnt2. Cell proliferation was detected by EdU. The ability of cell migration was detected by Transwell migration assay.Results:Compared with 0 d after hepatectomy, LSECs proliferation and HO-1 expression within LSECs were increased significantly at 4 d after surgery. EdU positive rate of LSECs in HO-1 group (27.20±4.80)% was higher than that in empty vector group (12.47±3.30)% and non-transfected group (15.97±2.50)%. The number of LSECs migration in HO-1 group (258.70±36.56) was higher than that in empty vector group (122.00±38.16) and non-transfected group (107.70±30.01). The protein and mRNA expression level of HO-1, Id1, HGF and Wnt2 in HO-1 group were higher than that in empty vector group and non-transfected group. EdU positive rate of hepatocytes that co-cultured with LSECs in HO-1 group (18.33±2.52) % was higher than that in empty vector group (11.33±1.53)% and non-transfected group (11.7±2.08)%. The differences were statistically significant (all P<0.05). Conclusion:Up-regulation of HO-1 promoted LSECs proliferation and migration of, as well as up-regulation of HO-1 in LSECs enhanced the capacity of LSECs to promote hepatocyte proliferation.

Advances in research based on primary human hepatocytes in vitro / 中华肝胆外科杂志

Due to the special functions of liver, the topic of liver physiological function and pathophysiological changes has always been the main issue in life science area. Human liver is composed of multiple types of cells, among which the hepatocyte is the most essential one. Primary human hepatocytes are considered as the gold standard model in vitro for the liver study and the ideal cell for hepatocyte transplantation and bioartificial liver. Nowadays, primary human hepatocytes play a vital role in the field of basic science research and applied research. Therefore, we presented recent advances in research based on primary human hepatocytes in vitro.

M1 macrophages promote non-alcoholic steatohepatitis / 中华消化杂志

Objective:To investigate the function, mechanism and therapeutic potential of macrophages in non-alcoholic steatohepatitis (NASH).Methods:Eight-week-old male foz/ foz (Alms mutant) mice were fed with a high fat diet (HFD) for 6, 8 and 10 weeks and 8-week-old male C57BL/6 mice were fed with a methionine and choline-deficient (MCD) diet for 7 d, 3 weeks and 4 weeks to establish NASH models. The mice of control group were fed with normal diet or MCD control diet. The expression of F4/80 mRNA level in the livers of mice of NASH model group and control group was detected by fluorescence quantitative polymerase chain reaction. Macrophages in the livers of mice of NASH group and control group were determined by immunofluorescence staining. After transgenic lysM-Cre/DTR mice were fed with MCD diet for 5 weeks, they were divided into transgenic experimental group (ablation of macrophages induced by diphtheria-toxin (DTox) injection) and transgenic control group (phosphate buffer saline injection). The levels of triglyceride and lipid peroxide in the livers of transgenic experimental group and transgenic control group were detected, and the inflammation of the livers of the mice was scored. The mechanism of macrophages regulating inflammation in NASH was investigated by cytokine profiliny analysis and Western blotting. The interaction between hepatocytes and macrophages were determined by co-culturing the conditional medium of hepatocytes AML-12 and macrophages RAW264.7. Macrophages of mice of control group and NASH model group were depleted by liposomal clodronate to confirm its value in NASH prevention. Independent sample t-test was used for statistical analysis. Results:F4/80 mRNA level in the livers of NASH model foz/ foz mice fed with HFD for 6 weeks, 8 weeks and 10 weeks was higher than that of control group (1.49±0.19, 1.70±0.15 and 1.93±0.04 vs.1.05±0.22), and the differences were statistically significant ( t=3.06, 4.92 and 7.92, all P<0.05). The expression of F4/80 mRNA level of the livers of NASH model mice fed with MCD for 7 d and 3 weeks was higher than that of control group (2.70±0.99 and 3.08±1.71 vs.1.00±0.83), and the differences were statistically significant ( t=3.43 and 3.54, both P<0.01). The results of immunofluorescence demonstrated that compared with that of control group, the number of F4/80 + inducible nitric oxide synthase (iNOS) + M1 macrophages were significantly increased, while F4/80 + CD206 + M2 macrophages were significantly decreased in the livers of NASH model mice fed with MCD for 4 weeks. After macrophages depletion, the inflammation score, the levels of triglyceride and lipid peroxide in the liver of transgenic experimental mice were all lower than those of transgenic control mice (0.69±0.32 vs. 1.95±0.74, (43.97±13.24) g/mg vs. (63.09±14.85) g/mg, (24.84±6.21) nmol/mg vs.(37.91±8.91) nmol/mg), and the differences were statistically significant ( t =3.14, 2.72 and 2.41, all P<0.05). The results of cytokine profiling analysis showed that macrophage depletion could lower the levels of interleukin (IL)-12 and macrophages inflammatory protein-1α (the difference between multiples: -3.98, -2.74, both P<0.05). CCAAT/enhancer binding protein β was defected in the nuclear of transgenetic experimental mice. In vitro study showed that RAW264.7 macrophages conditional medium could promote lipid accumulation in AML-12 hepatocytes, while conditional medium from MCD medium-treated AML-12 hepatocytes could promote RAW264.7 macrophages to M1 polarization. After treated with liposomal clodronate, the levels of triglyceride and lipid peroxidation in the liver of control mice were both lower than those of MCD-induced NASH model mice((45.33±14.59) g/mg vs. (63.10±16.02) g/mg, (2.11±0.48) nmol/mg vs. (2.73±0.17) nmol/mg), and the differences were statistically significant ( t=2.84 and 2.73, both P<0.05). The results of Western blotting indicated that after treating with liposomal clodronate, the relative content of phosphorylated protein kinase R-like endoplasmic reticulum kinase, inositol requiring enzyme-1α, protein disulfide isomerase, glucose regulatory protein 78, phosphorylated eukaryotic initiation factor 2α in the liver of NASH model mice were all lower than those of NASH model mice without liposomal clodronate treatment (1.84±0.36 vs. 3.05±0.83, 1.50±0.84 vs. 6.65±1.47, 0.87±0.12 vs. 2.28±0.52, 1.68±0.43 vs. 4.76±1.13, 1.42±0.19 vs. 2.75±0.79), and the differences were statistically significant( t=2.32, 5.28, 4.56, 4.41 and 2.85, all P<0.05). Conclusions:Macrophages are polarized into M1 phenotype in NASH. M1 macrophages contributed to NASH progression by interacting with hepatocyets to promote the secretion of inflammatory cytokines, up-regulation of lipogenic factors, oxidative stress and endoplasmic reticulum stress, resulting in the progression of NASH. Macrophages depletion by liposomal clodronate is a potential noval approach for NASH prevention.

The effects of WNT1 inducible signaling pathway protein 2 on hepatocyte lipid metabolism and associated mechanisms / 中华老年医学杂志

Objective:To investigate the effects of various concentrations of recombinant human WISP2 protein(WISP2)on lipid metabolism in HepG2 cells.Methods:HepG2 cells were treated with different concentrations(0, 0.4, 1 and 2 μg/L)of recombinant human WISP2 for 48 hours.Cell viability was detected by Cell-Titer, and enzymatic hydrolysis methods were used to measure intracellular triacylglycerol(TG)and total cholesterol(TC)levels.The mRNA expression was detected by quantitative real-time reverse transcription-PCR(RT-qPCR)and protein expression in HepG2 cells was detected by western blot.Results:Compared with the control group, the WISP2 groups treated with various concentration did not significantly reduce the viability of HepG2 cells.TG and TC in HepG2 cells were significantly increased by recombinant human WISP2 treatment(all P<0.05).The concentrations of TG in the 0.4, 1 and 2 μg/L recombinant human WISP2-treated groups were 1.254±0.039, 1.216±0.028 and 1.174±0.014)times the concentration in the untreated group, respectively( F=6.791, P=0.006).The concentration of TC in the untreated group was 1.264±0.057, 1.394±0.101 and 1.392±0.077), respectively, times the concentration in each of the treated groups( F=7.045, P=0.005).Further experiments found that the mRNA expression of sterol regulatory element binding protein 1(SREBP1), 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGCR), acetyl-CoA carboxylase(ACC), type 2 diacylglycerol acyltransferase(DGAT2)and the protein expression of SREBP1, ACC and fatty acid synthase(FAS)were significantly increased in the recombinant human WISP2-treated groups, compared with the control group(all P<0.05).However, the expression of lipid transporters such as the low-density lipoprotein receptor(LDLR), ApoB and ApoE and adipose triglyceride lipase(ATGL), a key lipolysis protein, was not significantly affected. Conclusions:Human recombinant WISP2 protein increases lipid levels in hepatocytes and the key underlying mechanisms may be through promoting lipid synthesis.

Inhibitory effect of flufenidone on TGF-β1/Smads pathway in hepatocytes of rats with diethylnitrosamine (DEN)-induced liver injury / 中国临床药理学与治疗学

AIM: To explore the protective effect of fluorofenidone (AKF-PD) on diethylnitrosamine-induced liver injury in rats and its inhibition of the TGF-β1/Smads pathway in hepatocytes. METHODS: Fifty-five male Sprague Dawley (SD) rats were randomly divided into three groups: model group (DEN group, n=20) were gavaged with DEN (10 mg/kg), 5 times for 14 weeks; control group (n=20) were gavaged with saline with the same volume of the model group; treatment group (DEN+AKF-PD Group, n=15), after 4 weeks of modeling, they were gavaged with AKF-PD (500 mg/kg) daily, and stopped at 14 weeks. At the end of experiment, the rats were killed by anesthesia and spinal dislocation. Masson staining was used to observe collagen deposition; primary hepatocytes were extracted and identified, and the levels of α-smooth muscle actin (α-SMA), TGF-β1, Smad3, and Smad7 mRNA, and the expression of Smad3 and Smad7 proteins in hepatocytes were detected. RESULTS: Compared with the control group, Masson staining showed that collagen deposition increased in the DEN group; AKF-PD treatment could significantly improve liver pathological damage and reduce collagen deposition. In addition, compared with the DEN group, the α-SMA, TGF-β1, and Smad3 mRNA levels of the AKF-PD group were significantly reduced, and the Smad7 mRNA level was increased. Moreover, AKF-PD treatment could dependably reduce the expression of Smad3 and increase Smad7. CONCLUSION: AKF-PD can significantly improve liver injury and fibrosis in rats caused by DEN. This effect may be related to the down-regulation of α-SMA, TGF-β1, and Smad3 mRNA levels in hepatocytes and the increase of Smad7 mRNA levels.

Establishment of 3D hepatocyte genotoxicity testing assay / 中国药理学通报

Aim To establish the 3D hepatocyte model by selecting the humanized hepatocyte HepG2 cells and 3D cell culture methods, and to establish the 3D hepatocyte cytokinesis-block micronucleus cytome(CBMN-cyt)assay and 3D hepatocyte comet assay by using chemicals of different mode of action.Methods In this study, a scaffold-free culture method was used to successfully establish a 3D HepG2 hepatocyte spheroid model.The appearance of the sphere, the survival rate of cells inside the sphere, the gene expression of phase I and II metabolic enzymes, and the expression of liver-specific biomarkers were selected as the observation indicators to obtain the best culture conditions for the 3D hepatocyte model.The 3D hepatocyte model was combined with in vitro micronucleomics test and in vitro comet test to explore its applicability for genotoxicity test.Results The best culture conditions for the 3D hepatocyte model was 5×103 cells/20 μL /drop inoculation, cultivating for seven days.A 3D hepatocyte CBMN-cyt assay was established using mitomycin C(MMC), a micronucleus positive compound, and the results showed that it could successfully detect the genotoxicity and cytotoxicity of MMC.Compared with the CBMN-cyt results of 2D hepatocyte model, 3D hepatocyte model had higher sensitivity in detecting MN and Nbud.The 3D hepatocyte comet assay methods were established using the known in vivo and in vitro comet assay positive compound methyl methanesulfonate(MMS), and the results showed that MMS could significantly increase the tail DNA% of 3D hepatocytes with low cytotoxicity.The sensitivity of 3D hepatocyte model to MMS genotoxicity detection was higher than that of 2D cells.Conclusions The 3D hepatocyte model established in this study is easy to use and low in cost, and shows good sensitivity and specificity in the in vitro micronucleus test and comet test, suggesting that the 3D hepatocyte genotoxicity test method is used in early drug genotoxicity screening.It has good application prospects in additional experimental research.

Nicotinamide mononucleotide alleviates ethanol-induced hepatic insulin resistance / 中国药理学通报

Aim To explore the potential protective effects of nicotinamide mononucleotide(NMN)on ethanol-induced hepatic insulin resistance. Methods Primary rat hepatocytes were divided into groups of different concentrations(0, 0.1, 0.25, 0.5, 1 mmol·L-1). The effects on cell viability of hepatocytes by different concentrations of NMN were assessed by CCK-8 method. Next, the hepatocytes were incubated with ethanol(25 mmol·L-1)to establish insulin resistance model, and incubated with different concentrations of NMN(0.1, 0.5 mmol·L-1)with or without Ex527(an inhibitor of SIRT1, 10 μmol·L-1). Glucose oxidase and anthrone methods were used to measure the glucose utilization and glycogen content respectively. Western blot was used to detect the ratios of p-PI3K/PI3K and p-Akt/Akt and the expression of SIRT1. Fluorimetric NAD+ Assay Kit was used to measure the NAD+ level in hepatocytes. Moreover, the effects of NMN on the ethanol-induced hepatic insulin resistance were explored. Results Compared with ethanol group, NMN treatment not only increased the glucose utilization and glycogen content, but also up-regulated the ratios of p-PI3K/PI3K and p-Akt/Akt at both concentrations. Meanwhile, NMN effectively recovered the NAD+ level and SIRT1 expression in hepatocytes. Furthermore, the protective effects of NMN on ethanol-induced hepatic insulin resistance was blocked by Ex527. Conclusions NMN could alleviate ethanol-induced hepatic insulin resistance by up-regulating NAD+/SIRT1 pathway and further recovering PI3K/Akt insulin signaling pathway.

Sodium butyrate affected the proliferation and apoptosis of steatosis HepG2 cells via G protein-coupled receptor 43/109a-phosphorylated protein kinase B-mammalian target of rapamycin signaling pathway / 中华消化杂志

Objective:To investigate the effects and mechanism of gut metabolite sodium butyrate on the proliferation and apoptosis of steatosis HepG2 cells in vitro. Methods:The in vitro steatosis hepatocyte model was established with human liver cell line HepG2 and free fatty acid (FFA; the concentration ratio of oleic acid to palmitic acid was 2∶1). Normal control group, model group and intervention groups with different concentration (1, 2, 5, 10, 20 and 50 mmol/L) of sodium butyrate were set up. The inhibition of sodium butyrate on the proliferation of steatosis HepG2 cells was detected by cell counting kit (CCK-8). The proportion of apoptotic cells of normal control group, model group and sodium butyrate 5 mmol/L (sodium butyrate intervention) group was detected by flow cytometry. Normal control group, model group, intervention group with different concentration (1, 2, 5 and 10 mmol/L) of sodium butyrate, negative small interfering RNA (siRNA) control group, G protein-coupled receptor (GPR) 43-siRNA group, GPR109a-siRNA group, GPR43+ GPR109 a double knockout group were set up. The change of the levels of phosphorylated protein kinase B (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR) before and after transfection were detected by Western blotting. One-way analysis of varivance, SNK- q test and logistic regression analysis were used for statistical analysis. Results:The results of CCK-8 test indicated that sodium butyrate inhibited the proliferation of steatosis HepG2 cells in a dose-dependent and time-dependent manner. The results of flow cytometry showed that the proportion of apoptotic cells of the sodium butyrate intervention group was higher than that of the model group and normal control group ((3.400±0.100)% vs. (1.800±0.400)% and(1.067±0.451)%), and the differences were statistically significant ( t=6.721 and 8.705, both P<0.01). There was no significant difference in the proportion of apoptotic cells between the model group and the normal control group ( P>0.05). Before transfection, the expressions of p-AKT and p-mTOR at protein level of the model group were both higher than those of the normal control group (2.300±0.058 vs. 1.000±0.012, 2.160±0.125 vs. 1.000±0.052), and the differences were statistically significant ( t=22.080 and 8.575, both P<0.05). The expressions of p-AKT and p-mTOR at protein level of sodium butyrate intervention groups at 1, 2, 5 and 10 mmol/L were all lower than those of the model group (1.530±0.085, 1.407±0.096, 1.032±0.035 and 1.036±0.099 vs. 2.300±0.058; 1.483±0.073, 1.297±0.048, 1.067±0.035 and 0.970±0.072 vs. 2.160±0.125), and the differences were statistically significant ( t=7.491, 7.997, 19.790, and 11.020; 4.683, 6.445, 8.424, and 8.245; all P<0.05). After transfection, the expressions of p-AKT and p-mTOR at protein level of GPR43-siRNA group, GPR109a-siRNA group and GPR43/ GPR109 a double knockout group were all higher than those of the negative siRNA control group and 5 mmol/L sodium butyrate group (1.474±0.045, 1.471±0.058 and 2.067±0.120 vs. 1.158±0.030 and 1.139±0.031; 1.850±0.082, 1.683±0.058 and 2.160±0.091 vs. 1.469±0.037 and 1.490±0.116), and the differences were statistically significant ( tp-AKT=5.807, 4.816, 7.322, 6.109, 5.080 and 7.463; tp-mTOR=4.235, 3.113, 7.044, 2.542, 1.497 and 4.562; all P<0.05). Conclusions:The effect of sodium butyrate on the proliferation and apoptosis of steatosis HepG2 cells is associated with the GPR43/GPR109a-pAKT-mTOR signaling pathway.

Effect of ferroptosis on hepatic ischemia reperfusion injury in diabetic rats model / 中华肝胆外科杂志

Objective:To investigate the effect of ferroptosis on hepatic ischemia reperfusion injury in diabetic rats model.Methods:Forty Sprague Dawley rats were randomly divided into four groups: sham operation group (Sham), hepatic ischemia reperfusion group (HIRI), diabetes mellitus group (DM), diabetes mellitus + hepatic ischemia reperfusion group (DM+ HIRI). The diabetic rat model was established by feeding the rats with high-fat and high-sugar feed for four consecutive weeks combined with intraperitoneal injection of 50 mg/kg 1% streptozotocin, and on this basis, the hepatic ischemia reperfusion injury model was established by local hepatic blood flow occlusion. ELISA assay was used to detect insulin content, liver function and serum lipid metabolism biomarkers. Chemiluminescence method was used to detect liver superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), polyunsaturated fatty acids (PUFA) and lipoxygenase (LOX-1) contents. HE staining was used to evaluate the pathological changes of liver tissue structure, and Western blotting was used to detect the ferroptosis-related protein expression of ACSL4 and GPX4.Results:Compared with Sham group, the level of fasting blood glucose, insulin, insulin resistance index, serum TC, TG and liver PUFA content of DM group were increased significantly ( P<0.05), HE staining showed there were a large number of fatty degeneration of hepatocytes in DM group, and extensive ballooning and necrosis of hepatocytes in DM+ HIRI group. Compared with HIRI group, level of serum TC, TG, ALT, AST and the liver PUFA and LOX-1 in DM+ HIRI group were significantly increased [TC (5.87±0.76) vs (1.34±0.2) mmol/L, TG (2.93±0.47) vs (0.71±0.34) mmol/L, ALT (339.5±40.09) vs (155.17±18.53) U/L, AST (325.50±37.52) vs (102.39±22.68) U/L, PUFA (21.58±3.01) vs (8.12±0.94) mg/g, LOX-1 (200.81±26.03) vs (73.34±10.66) U/m ] ( P<0.05). Compared with HIRI group, the protein expression of ACSL4 in DM+ HIRI group was up-regulated [(0.46±0.06) vs (1.02±0.11)], while the expression of GPX4 was down-regulated [(0.43±0.07) vs (0.14±0.02)] significantly ( P<0.05). Conclusion:The tolerance of DM rats to hepatic ischemia reperfusion injury was significantly reduced, which may be related to hepatic abnormal lipid metabolism and ferroptosis.

Catalpol inhibits cell apoptosis through alleviating endoplasmic reticulum stress in nonalcoholic fatty liver disease / 药学学报

This study was designed to explore the protective effect and underlying mechanism of catalpol on hepatocyte apoptosis in nonalcoholic fatty liver disease (NAFLD). High fat diet (HFD) was used to establish NAFLD model in the in vivo experiment, and the procedures of the experiments and animal care protocol were approved by the Animal Care and Use Committee of Jianghan University. Human liver cancer cell line HepG2 was treated with palmitate (PA) to establish a lipid toxicity model in the in vitro experiments. The results showed that catalpol significantly decreased the contents of serum total glyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate transaminase (AST) in HFD-fed mice. Results of TUNEL staining and flow cytometry analyses revealed that catalpol significantly inhibited hepatocytes apoptosis in HFD-fed mice and PA-treated HepG2 cells. Moreover, catalpol treatment significantly reduced the endoplasmic reticulum stress-related protein expression levels of binding immunoglobulin protein (BiP), phosphorylated PKR-like endoplasmic reticulum kinase (p-PERK), inositol-requiring kinase 1α (IRE1α), and transcriptional factor activating transcription factor 6 (ATF6), and apoptosis-related protein expression levels of C/EBP homology protein (CHOP), phosphorylated c-Jun N-terminal kinase (p-JNK), and cleaved cysteinyl aspartate specific proteinases (caspases)-12, -9, and -3 in HFD-fed mice and PA-treated HepG2 cells. Furthermore, endoplasmic reticulum stress agonist tunicamycin (TM) significantly reversed the inhibitory effect of catalpol on protein expression levels of BiP, p-PERK, IRE1α, and ATF6, subsequently the inhibitory effect of catalpol on expression levels of CHOP, p-JNK, Bcl-2, Bax, and cleaved caspases (-12, -9, and -3) was also attenuated in PA-treated HepG2 cells. Taken together, these findings demonstrated that catalpol could inhibit hepatocytes apoptosis and had a significant protective effect on liver injury, and its mechanism might be related to the relief of endoplasmic reticulum stress.