Research progress in lineage tracing to explore hepatic parenchymal cell regeneration and repair mechanisms / 中华肝脏病杂志

Hepatic parenchymal cells are a type of liver cells that performs important functions such as metabolism and detoxification. The contribution of hepatic parenchymal cells, bile duct cells, and hepatic stem/progenitor cells to new hepatic parenchymal cells in the process of liver injury repair has become a controversial issue due to their strong proliferation ability. Lineage tracing technology, which has emerged in the past decade as a new method for exploring the origin of cells, can trace specific type of cells and their daughter cells by labeling cells that express the specific gene and their progeny. The article reviews the current literature on the origin and contribution of hepatic parenchymal cells by this technique. About 98% of new hepatic parenchymal cells originate from the existing hepatic parenchymal cells during liver homeostasis and after acute injury. However, under conditions of severe liver injury, such as inhibition of hepatic parenchymal cell proliferation, bile duct cells (mainly liver stem/progenitor cells) become the predominant source of hepatic parenchymal cells, contributing a steady increased hepatocyte regeneration with the extension of time.

Research progress of the regulatory role of autophagy in metabolic liver diseases / 中华肝脏病杂志

Autophagy is one of several hepatic metabolic processes in which starved cells are supplied with glucose, free fatty acids, and amino acids to produce energy and synthesize new macromolecules. Moreover, it regulates the quantity and quality of mitochondria and other organelles. As the liver is a vital metabolic organ, specific forms of autophagy are necessary for maintaining liver homeostasis. Protein, fat, and sugar are the three primary nutrients that can be altered by different metabolic liver diseases. Drugs that have an effect on autophagy can either promote or inhibit autophagy, and as a result, it can either increase or inhibit the three major nutritional metabolisms that are affected by liver disease. Thus, this opens up a novel therapeutic option for liver disease.

Nonalcoholic fatty liver disease and bilirubin: correlation, mechanism, and therapeutic perspectives / 中华肝脏病杂志

Non-alcoholic fatty liver disease (NAFLD) is a metabolic-related disorder induced by multiple factors and mainly characterized by excessive fat buildup in hepatocytes. With the consumption of a Western-style diet and obesity prevalence in recent years, the incidence of NAFLD has gradually increased, becoming an increasingly serious public health problem. Bilirubin is a heme metabolite and a potent antioxidant. Studies have demonstrated that bilirubin levels have an inverse correlation with the incidence rate of NAFLD; however, which form of bilirubin plays the main protective role is still controversial. It is considered that the main protective mechanisms for NAFLD are bilirubin antioxidant properties, insulin resistance reduction, and mitochondrial function. This article summarizes the correlation, protective mechanism, and possible clinical application of NAFLD and bilirubin.

The role of Keap1/Nrf2/HO-1 signal pathway in liver injury induced by rare earth neodymium oxide in mice / 中华劳动卫生职业病杂志

Objective: To investigate the role of Keap1/Nrf2/HO-1 signaling pathway in liver injury induced by neodymium oxide (Nd(2)O(3)) in mice. Methods: In March 2021, forty-eight SPF grade healthy male C57BL/6J mice were randomly divided into control group (0.9% NaCl), low dose group (62.5 mg/ml Nd(2)O(3)), medium dose group (125.0 mg/ml Nd(2)O(3)), and high dose group (250.0 mg/ml Nd(2)O(3)), each group consisted of 12 animals. The infected groups were treated with Nd(2)O(3) suspension by non-exposed tracheal drip and were killed 35 days after dust exposure. The liver weight of each group was weighed and the organ coefficient was calculated. The content of Nd(3+) in liver tissue was detected by inductively coupled plasma mass spectrometry (ICP-MS). HE staining and immunofluorescence was used to observe the changes of inflammation and nuclear entry. The mRNA expression levels of Keap1, Nrf2 and HO-1 in mice liver tissue were detected by qRT-PCR. Western blotting was used to detect the protein expression levels of Keap1 and HO-1. The contents of catalase (CAT), glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD) were detected by colorimetric method. The contents of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were determined by ELISA. The data was expressed in Mean±SD. Two-independent sample t-test was used for inter-group comparison, and one-way analysis of variance was used for multi-group comparison. Results: Compared with the control group, the liver organ coefficient of mice in medium and high dose groups were increased, and the Nd(3+) accumulation in liver of mice in all dose groups were significantly increased (P<0.05). Pathology showed that the structure of liver lobules in the high dose group was slightly disordered, the liver cells showed balloon-like lesions, the arrangement of liver cell cords was disordered, and the inflammatory exudation was obvious. Compared with the control group, the levels of IL-1β and IL-6 in liver tissue of mice in all dose groups were increased, and the levels of TNF-α in liver tissue of mice in high dose group were increased (P<0.05). Compared with the control group, the mRNA and protein expression levels of Keap1 in high dose group were significantly decreased, while the mRNA expression level of Nrf2, the mRNA and protein expression levels of HO-1 were significantly increased (P<0.05), and Nrf2 was successfully activated into the nucleus. Compared with the control group, the activities of CAT, GSH-Px and T-SOD in high dose group were significantly decreased (P<0.05) . Conclusion: A large amount of Nd(2)O(3) accumulates in the liver of male mice, which may lead to oxidative stress and inflammatory response through activation of Keap1/Nrf2/HO-1 signal pathway. It is suggested that Keap1/Nrf2/HO-1 signal pathway may be one of the mechanisms of Nd(2)O(3) expose-induced liver injury in mice.

Sodium butyrate activates HMGCS2 to promote ketone body production through SIRT5-mediated desuccinylation / 医学前沿

Ketone bodies have beneficial metabolic activities, and the induction of plasma ketone bodies is a health promotion strategy. Dietary supplementation of sodium butyrate (SB) is an effective approach in the induction of plasma ketone bodies. However, the cellular and molecular mechanisms are unknown. In this study, SB was found to enhance the catalytic activity of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting enzyme in ketogenesis, to promote ketone body production in hepatocytes. SB administrated by gavage or intraperitoneal injection significantly induced blood ß-hydroxybutyrate (BHB) in mice. BHB production was induced in the primary hepatocytes by SB. Protein succinylation was altered by SB in the liver tissues with down-regulation in 58 proteins and up-regulation in 26 proteins in the proteomics analysis. However, the alteration was mostly observed in mitochondrial proteins with 41% down- and 65% up-regulation, respectively. Succinylation status of HMGCS2 protein was altered by a reduction at two sites (K221 and K358) without a change in the protein level. The SB effect was significantly reduced by a SIRT5 inhibitor and in Sirt5-KO mice. The data suggests that SB activated HMGCS2 through SIRT5-mediated desuccinylation for ketone body production by the liver. The effect was not associated with an elevation in NAD+/NADH ratio according to our metabolomics analysis. The data provide a novel molecular mechanism for SB activity in the induction of ketone body production.

Blueberry attenuates liver injury in metabolic dysfunction-associated liver disease by promoting the expression of mitofilin/Mic60 in human hepatocytes and inhibiting the production of superoxide / 细胞与分子免疫学杂志

Objective To study the effect and mechanism of blueberry on regulating the mitochondrial inner membrane protein mitofilin/Mic60 in an in vitro model of metabolic dysfunction-associated liver disease (MAFLD). Methods L02 human hepatocytes were induced by free fatty acids (FFA) to establish MAFLD cell model. A normal group, a model group, an 80 μg/mL blueberry treatment group, a Mic60 short hairpin RNA (Mic60 shRNA) transfection group, and Mic60 knockdown combined with an 80 μg/mL blueberry treatment group were established. The intracellular lipid deposition was observed by oil red O staining, and the effect of different concentrations of blueberry pulp on the survival rate of L02 cells treated with FFA was measured by MTT assay. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), superoxide dismutase (SOD) activity, glutathione (GSH) and malondialdehyde (MDA) contents were measured by visible spectrophotometry. The expression of reactive oxygen species (ROS) in hepatocytes was observed by fluorescence microscopy, and the mRNA and protein expression of Mic60 were detected by real-time quantitative PCR and Western blot analysis, respectively. Results After 24 hours of FFA stimulation, a large number of red lipid droplets in the cytoplasm of L02 cells was observed, and the survival rate of L02 cells treated with 80 μg/mL blueberry was higher. The results of ALT, AST, TG, TC, MDA and the fluorescence intensity of ROS in blueberry treated group were lower than those in model group, while the levels of SOD, GSH, Mic60 mRNA and protein in blueberry treated group were higher than those in model group. Conclusion Blueberry promotes the expression of Mic60, increases the levels of SOD and GSH in hepatocytes, and reduces the production of ROS, thus alleviating the injury of MAFLD hepatocytes and regulating the disorder of lipid metabolism.

Discovery of biomarkers related to abnormal lipid metabolism in liver and serum and intervention mechanism of ginsenoside Rb_1 in hyperlipidemia rats based on non-targeted metabolomics / 中国中药杂志

Through the non-targeted metabolomics study of endogenous substances in the liver and serum of hyperlipidemia rats, the biomarkers related to abnormal lipid metabolism in hyperlipidemia rats were found, and the target of ginsenoside Rb_1 in improving hyperlipidemia was explored and its mechanism was elucidated. The content of serum biochemical indexes of rats in each group was detected by the automatic biochemical analyzer. The metabolite profiles of liver tissues and serum of rats were analyzed by HPLC-MS. Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to compare and analyze the metabolic data in the normal group, the hyperlipidemia group, and the ginsenoside Rb_1 group, and screen potential biomar-kers. The related metabolic pathways were further constructed by KEGG database analysis. The results showed that hyperlipemia induced dyslipidemia in rats, which was alleviated by ginsenoside Rb_1. The non-targeted metabolomics results showed that there were 297 differential metabolites in the liver tissues of hyperlipidemia rats, 294 differential metabolites in the serum samples, and 560 diffe-rential metabolites in the hyperlipidemia rats treated by ginsenoside Rb_1. Perillic acid and N-ornithyl-L-taurine were common metabolites in the liver and serum samples, which could be used as potential biomarkers for ginsenoside Rb_1 in the improvement of hyperlipidemia. As revealed by pathway enrichment in the liver and serum, ginsenoside Rb_1 could participate in the metabolic pathway of choline in both the liver and serum. In addition, ginsenoside Rb_1 also participated in the ABC transporter, alanine, aspartic acid, and glutamate metabolism, protein digestion and absorption, β-alanine metabolism, taurine and hypotaurine metabolism, caffeine metabolism, valine, leucine, and isoleucine biosynthesis, arachidonic acid metabolism, and methionine and cysteine metabolism to improve dyslipidemia in rats.

Punicalagin inhibits hepatic lipid deposition in obese mice via AMPK/ACC pathway / 中国中药杂志

Hepatic lipid deposition is one of the basic manifestations of obesity, and nowadays pharmacological treatment is the most important tool. Punicalagin(PU), a polyphenol derived from pomegranate peel, is a potential anti-obesity substance. In this study, 60 C57BL/6J mice were randomly divided into a normal group and a model group. After establishing a model of simple obesity with a high-fat diet for 12 weeks, the successfully established rat models of obesity were then regrouped into a model group, an orlistat group, a PU low-dose group, a PU medium-dose group, and a PU high-dose group. The normal group was kept on routine diet and other groups continued to feed the high-fat diet. The body weight and food intake were measured and recorded weekly. After 8 weeks, the levels of the four lipids in the serum of each group of mice were determined by an automatic biochemical instrument. Oral glucose tole-rance and intraperitoneal insulin sensitivity were tested. Hemoxylin-eosin(HE) staining was applied to observe the hepatic and adipose tissues. The mRNA expression levels of peroxisome proliferators-activated receptor γ(PPARγ) and C/EBPα were determined by real-time quantitative polymerase chain reaction(Q-PCR), and the mRNA and protein expression levels of adenosine 5'-monophosphate-activated protein kinase(AMPK), anterior cingulate cortex(ACC), and carnitine palmitoyltransferase 1A(CPT1A) were determined by Western blot. Finally, the body mass, Lee's index, serum total glyceride(TG), serum total cholesterol(TC), and low-density lipoprotein cholesterol(LDL-C) levels were significantly higher and high-density lipoprotein cholesterol(HDL-C) levels were significantly lower in the model group as compared with the normal group. The fat deposition in the liver was significantly increased. The mRNA expression levels of hepatic PPARγ and C/EBPα and the protein expression level of ACC were increased, while the mRNA and protein expression levels of CPT-1α(CPT1A) and AMPK were decreased. After PU treatment, the above indexes of obese mice were reversed. In conclusion, PU can decrease the body weight of obese mice and control their food intake. It also plays a role in the regulation of lipid metabolism and glycometabolism metabolism, which can significantly improve hepatic fat deposition. Mechanistically, PU may regulate liver lipid deposition in obese mice by down-regulating lipid synthesis and up-regulating lipolysis through activation of the AMPK/ACC pathway.

Perfluorooctanoic acid-induced lipid metabolism disorder in SD rat liver and its effect on the expression of fatty acid metabolism-related proteins / 中南大学学报(医学版)

OBJECTIVES@#Perfluorooctanoic acid (PFOA) can cause lipid metabolism disorders in animal body and affect the lipolysis and synthesis of fatty acids. Peroxisome proliferators-activated receptor (PPAR) plays an extremely important role in this process. This study aims to explore the effects of PFOA on liver lipid metabolism disorders in Sprague Dewley (SD) rats and the expression of PPAR.@*METHODS@#A total of 40 male SD rats were randomly divided into 4 groups (n=10 in each group): a control group (ddH2O), a low-dose PFOA group [PFOA 1.25 mg/(kg·d)], a middle-dose PFOA group [PFOA 5.00 mg/(kg·d)], and a high-dose PFOA group [PFOA 20.00 mg/(kg·d)]. The rats were fed with normal diet, and PFOA exposure were performed by oral gavage for 14 days, and the rats were observed, weighted and recorded every day during the exposure. After the exposure, the blood was collected, and the livers were quickly stripped after the rats were killed. Part of the liver tissues were fixed in 4% paraformaldehyde for periodic acid-schiff (PAS) staining; the contents of HDLC, LDLC, TG, TC in serum and liver tissues, as well as the activities of their related enzymes were assayed; The expression levels of cyclic adenosine monophosphate-response element binding protein (Cbp), general control of amino acid synthesis 5-like 2 (Gcn5L2), peroxidation peroxisome proliferation factor activated receptor γ (PPAR), silent information regulator 1 (Sirt1) and human retinoid X receptor alpha 2 (Rxrα2) ) were detected by Western blotting.@*RESULTS@#After 14 days of PFOA exposure, the PAS staining positive particles in the cytoplasm and nucleus of SD rats in the medium and high dose groups were significantly reduced compared with the control group. The serum levels of LDLC and TC in the low-dose and middle-dose groups were significantly reduced compared with the control group (all P<0.05), while the high-dose group showed an increasing tendency, without siginificant difference (P>0.05), there was no significant difference in HDLC and TG (both P>0.05). The activities of alkaline phosphatase (AKP) and alanine aminotransferase (ALT) were increased significantly (both P<0.05) compared with control group; the ratio of ALT/aspartate aminotransferase (AST) in the high-dose group was increased significantly (P<0.05), there was no significant difference in LDH and TG (both P>0.05); the HDLC content in the liver tissues in the high-dose group was significantly reduced, compared with the control group (P<0.05); the TC contents in the liver tissues in the low, medium and high-dose groups were significantly increased (all P<0.05), there was no significant difference in LDLC and TG (both P>0.05); the AKP activity in the livers in the medium and high-dose groups was significantly increased (both P<0.05), there was no siginificant difference in LDH, ALT, and the ratio of ALT/AST (all P>0.05); the protein expression levels of Ppar γ, Cbp and Rxrα2 in the liver in the high dose groups were significantly down-regulated compared with the control group (all P<0.05), while the protein expression levels of Sirt1 were significantly up-regulated (all P<0.05).@*CONCLUSIONS@#PFOA exposure can cause lipid metabolism disorder and glycogen reduction in SD rat livers, which may be related to the activation of Sirt1 and inhibition of Ppar γ expression, leading to affecting the normal metabolism of fatty acids and promoting glycolysis.

Deficiency in glutathione synthesis and reduction contributes to the pathogenesis of colitis-related liver injury / 中南大学学报(医学版)

OBJECTIVES@#Liver disease is the most common extra-intestinal manifestation of ulcerative colitis (UC), but the underlying pathogenesis is still not clarified. It is well accepted that the occurrence of UC-related liver disease has close correlation with immune activation, intestinal bacterial liver translocation, inflammatory cytokine storm, and the disturbance of bile acid circulation. The occurrence of UC-related liver disease makes the therapy difficult, therefor study on the pathogenesis of UC-related liver injury is of great significance for its prevention and treatment. Glutathione (GSH) shows multiple physiological activities, such as free radical scavenging, detoxification metabolism and immune defense. The synthesis and the oxidation-reduction all contribute to GSH antioxidant function. It is reported that the deficiency in hepatic GSH antioxidant function participates in multiple liver diseases, but whether it participates in the pathogenesis of UC-related liver injury is still not clear. This study aims to investigate the feature and underlying mechanism of GSH synthesis and oxidation-reduction function during the development of UC, which will provide useful information for the pathogenesis study on UC-related liver injury.@*METHODS@#UC model was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS)-ethanol solution (5 mg/0.8 mL per rat, 50% ethanol) via intra-colonic administration in rats, and the samples of serum, liver, and colon tissue of rats were collected at the 3rd, 5th, and 7th days post TNBS. The severity degree of colitis was evaluated by measuring the disease activity index, colonic myeloperoxidase activity, and histopathological score, and the degree of liver injury was evaluated by histopathological score and the serum content of alanine aminotransferase. Spearman correlation analysis was also conducted between the degree of colonic lesions and index of hepatic histopathological score as well as serum aspartate aminotransferase level to clarify the correlation between liver injury and colitis. To evaluate the hepatic antioxidant function of GSH in UC rats, hepatic GSH content, enzyme activity of GSH peroxidase (GSH-Px), and GSH reductase (GR) were determined in rats at the 3rd, 5th, and 7th days post TNBS, and the protein expressions of glutamine cysteine ligase (GCL), GSH synthase, GSH-Px, and GR in the liver of UC rats were also examined by Western blotting.@*RESULTS@#Compared with the control, the disease activity index, colonic myeloperoxidase activity, and histopathological score were all significantly increased at the 3rd, 5th, and 7th days post TNBS (all P<0.01), the serum aspartate aminotransferase level and hepatic histopathologic score were also obviously elevated at the 7th day post TNBS (all P<0.05). There was a significant positive correlation between the degree of liver injury and the severity of colonic lesions (P=0.000 1). Moreover, compared with the control, hepatic GSH content and the activity of GSH-Px and GR were all significantly decreased at the 3rd and 5th days post TNBS (P<0.05 or P<0.01), and the protein expressions of GCL, GSH-Px, and GR were all obviously down-regulated at the 3rd, 5th, and 7th days post TNBS (P<0.05 or P<0.01).@*CONCLUSIONS@#There is a significant positive correlation between the degree of liver injury and the severity of colonic lesions, and the occurrence of reduced hepatic GSH synthesis and decreased GSH reduction function is obviously earlier than that of the liver injury in UC rats. The reduced hepatic expression of enzymes that responsible for GSH synthesis and reduction may contribute to the deficiency of GSH synthesis and oxidation-reduction function, indicating that the deficiency in GSH antioxidant function may participate in the pathogenesis of UC related liver injury.

Research progress on the relationship between liver cirrhosis and thyroid hormones / 中华肝脏病杂志

There exists a complex relationship between liver and thyroid hormones. Liver plays an important role in the activation, inactivation, transportation, and metabolism of thyroid hormones. At the same time, thyroid hormones also affect hepatocytes activity and liver metabolism, such as lipid and bilirubin metabolism. Importantly, thyroid hormone levels often change abnormally in patients with liver cirrhosis. Therefore, studying the change of thyroid hormone levels in patients with liver cirrhosis has a certain clinical value for assessing the severity, prognosis, diagnosis and treatment. This paper reviews the research progress on the relationship between liver cirrhosis and thyroid hormone.

Correlation of Lipin gene expression with hepatic fat content in rats with intrauterine growth retardation / 中国当代儿科杂志

OBJECTIVES@#To study the correlation of the expression of Lipin1 in visceral adipose tissue and Lipin2 in liver tissue with hepatic fat content in rats with intrauterine growth retardation (IUGR).@*METHODS@#Pregnant rats were given a low-protein (10% protein) diet during pregnancy to establish a model of IUGR in neonatal rats. The pregnant rats in the control group were given a normal-protein (21% protein) diet during pregnancy. The neonatal rats were weighed and liver tissue was collected on day 1 and at weeks 3, 8, and 12 after birth, and visceral adipose tissue was collected at weeks 3, 8, and 12 after birth. The 3.0T 1H-magnetic resonance spectroscopy was used to measure hepatic fat content at weeks 3, 8, and 12 after birth. Real-time PCR was used to measure mRNA expression levels of Lipin2 in liver tissue and Lipin1 in visceral adipose tissue. Western blot was used to measure protein levels of Lipin2 in liver tissue and Lipin1 in visceral adipose tissue. A Pearson correlation analysis was performed to investigate the correlation of mRNA and protein expression of Lipin with hepatic fat content.@*RESULTS@#The IUGR group had significantly higher mRNA and protein expression levels of Lipin1 in visceral adipose tissue than the control group at weeks 3, 8, and 12 after birth (P<0.05). Compared with the control group, the IUGR group had significantly lower mRNA and protein expression levels of Lipin2 in liver tissue on day 1 after birth and significantly higher mRNA and protein expression levels of Lipin2 at weeks 1, 3, 8, and 12 after birth (P<0.05). At week 3 after birth, there was no significant difference in hepatic fat content between the IUGR and control groups (P>0.05), while at weeks 8 and 12 after birth, the IUGR group had a significantly higher hepatic fat content than the control group (P<0.05). The protein and mRNA expression levels of Lipin1 were positively correlated with hepatic fat content (r=0.628 and 0.521 respectively; P<0.05), and the protein and mRNA expression levels of Lipin2 were also positively correlated with hepatic fat content (r=0.601 and 0.524 respectively; P<0.05).@*CONCLUSIONS@#Upregulation of the mRNA and protein expression levels of Lipin1 in visceral adipose tissue and Lipin2 in liver tissue can increase hepatic fat content in rats with IUGR and may be associated with obesity in adulthood.

Mechanism of Zexie Decoction in improvement of nonalcoholic fatty liver disease based on LKB1/AMPK/PGC-1α pathway / 中国中药杂志

The present study investigated the pharmaceutical effect and underlying mechanism of Zexie Decoction(ZXD) on nonalcoholic fatty liver disease(NAFLD) in vitro and in vivo via the LKB1/AMPK/PGC-1α pathway based on palmitic acid(PA)-induced lipid accumulation model and high-fat diet(HFD)-induced NAFLD model in mice. As revealed by the MTT assay, ZXD had no effect on HepG2 activity, but dose-dependently down-regulated alanine aminotransferase(ALT) and aspartate aminotransferase(AST) in the liver cell medium induced by PA, and decreased the plasma levels of ALT and AST, and total cholesterol(TC) and triglyceride(TG) levels in the liver. Nile red staining showed PA-induced intracellular lipid accumulation, significantly increased lipid accumulation of hepatocytes induced by PA, suggesting that the lipid accumulation model in vitro was properly induced. ZXD could effectively improve the lipid accumulation of hepatocytes induced by PA. Oil red O staining also demonstrated that ZXD improved the lipid accumulation in the liver of HFD mice. JC-1 staining for mitochondrial membrane potential indicated that ZXD effectively reversed the decrease in mitochondrial membrane potential caused by hepatocyte injury induced by PA, activated PGC-1α, and up-regulated the expression of its target genes, such as ACADS, CPT-1α, CPT-1β, UCP-1, ACSL-1, and NRF-1. In addition, as revealed by the Western blot and immunohistochemistry, ZXD up-regulated the protein expression levels of LKB1, p-AMPK, p-ACC, and PGC-1α in vivo and in vitro. In conclusion, ZXD can improve NAFLD and its mechanism may be related to the regulation of the LKB1/AMPK/PGC-1α pathway.

Study on mechanism of curcumol against liver fibrosis based on autophagy and apoptosis of hepatic stellate cells / 中国中药杂志

The present study clarified the molecular mechanism of curcumol against liver fibrosis based on its effects on the autopha-gy and apoptosis of hepatic stellate cells. The hepatic stellate cells were divided into a blank control group, a transforming growth factor-β1(TGF-β1)(10 ng·mL~(-1)) group, and low-(12.5 mg·L~(-1)), medium-(25 mg·L~(-1)), and high-dose(50 mg·L~(-1)) curcumol groups. The effect of curcumol on the viability of hepatic stellate cells induced by TGF-β1 was detected by the MTT assay kit. The apo-ptosis in each group was determined by flow cytometry. Real-time fluorescence-based quantitative PCR(RT-PCR) was employed for the detection of mRNA expression of α-smooth muscle actin(α-SMA), type Ⅰ collagen(collagen Ⅰ), and type Ⅲ collagen(collagen Ⅲ). Western blot was used to detect the protein expression of p62, microtubule-associated protein 1 light chain 3(LC3), beclin1, B cell lymphoma 2(Bcl-2), and Bcl-2-associated X protein(Bax). Transmission electron microscopy(TEM) was used to observe cell morphology and autophagosome formation in each group. The autophagic flux was observed after cell infection with adenovirus under double fluorescence labeling. The cell viability assay revealed that compared with the TGF-β1 group, the curcumol groups showed significantly decreased cell viability. The apoptosis assay showed that the apoptosis rates of the curcumol groups were significantly higher than that of the TGF-β1 group. RT-PCR indicated that the mRNA expression of α-SMA, collagenⅠ, and collagen Ⅲ in the curcumol groups was significantly lower than that of the TGF-β1 group. Western blot showed that the expression of p62, LC3, beclin1, Bcl-2, and Bax in the curcumol groups was significantly different from that in the TGF-β1 group. As demonstrated by TEM, compared with the TGF-β1 group, the curcumol groups showed significantly increased autophagosomes. The detection of autophagic flow by the adenovirus under double fluorescence labeling showed that autolysosomes in the curcumol groups were significantly increased compared with those in the TGF-β1 group. Curcumol can induce the autophagy and apoptosis of hepatic stellate cells, which may be one of its anti-liver fibrosis mechanisms.

Influencing factors of iron metabolism assessment in patients with myelodysplastic syndrome: A retrospective study / 中华血液学杂志

Objective: To analyze the influencing factors of iron metabolism assessment in patients with myelodysplastic syndrome. Methods: MRI and/or DECT were used to detect liver and cardiac iron content in 181 patients with MDS, among whom, 41 received regular iron chelation therapy during two examinations. The adjusted ferritin (ASF) , erythropoietin (EPO) , cardiac function, liver transaminase, hepatitis antibody, and peripheral blood T cell polarization were detected and the results of myelofibrosis, splenomegaly, and cyclosporine were collected and comparative analyzed in patients. Results: We observed a positive correlation between liver iron concentration and ASF both in the MRI group and DECT groups (r=0.512 and 0.606, respectively, P<0.001) , only a weak correlation between the heart iron concentration and ASF in the MRI group (r=0.303, P<0.001) , and no significant correlation between cardiac iron concentration and ASF in the DECT group (r=0.231, P=0.053) . Moreover, transfusion dependence in liver and cardiac [MRI group was significantly associated with the concentration of iron in: LIC: (28.370±10.706) mg/g vs (7.593±3.508) mg/g, t=24.30, P<0.001; MIC: 1.81 vs 0.95, z=2.625, P<0.05; DECT group: liver VIC: (4.269±1.258) g/L vs (1.078±0.383) g/L, t=23.14, P<0.001: cardiac VIC: 1.69 vs 0.68, z=3.142, P<0.05]. The concentration of EPO in the severe iron overload group was significantly higher than that in the mild to moderate iron overload group and normal group (P<0.001) . Compared to the low-risk MDS group, the liver iron concentration in patients with MDS with cyclic sideroblasts (MDS-RS) was significantly elevated [DECT group: 3.80 (1.97, 5.51) g/L vs 1.66 (0.67, 2.94) g/L, P=0.004; MRI group: 13.7 (8.1,29.1) mg/g vs 11.6 (7.1,21.1) mg/g, P=0.032]. Factors including age, bone marrow fibrosis, splenomegaly, T cell polarization, use of cyclosporine A, liver aminotransferase, and hepatitis antibody positive had no obvious effect on iron metabolism. Conclusion: There was a positive correlation between liver iron concentration and ASF in patients with MDS, whereas there was no significant correlation between cardiac iron concentration and ASF. Iron metabolism was affected by transfusion dependence, EPO concentration, and RS.

Effective fraction from Simiao Wan prevents hepatic insulin resistant by inhibition of lipolysis via AMPK activation / 中国天然药物

Simiao Wan (SMW) is a traditional Chinese formula, including Atractylodis Rhizoma, Achyranthis Bidentatae Radix, Phellodendri Chinensis Cortex and Coicis Semen at the ratio of 1:1:2:2. It can be used to the treatment of diabetes. However, its bioactive compounds and underlying mechanism are unclear. This study aimed to screen the antilipolytic fraction from SMW and investigate its therapeutic mechanisms on hepatic insulin resistance. Different fractions of SMW were prepared by membrane separation combined with macroporous resin and their antilipolytic activities were screened in fasted mice. The effects of 60% ethanol elution (ESMW) on lipolysis were investigated in 3T3-L1 adipocytes stimulated by palmitic acid (PA) and high fat diet (HFD)-fed mice. In our study, ESMW is the bioactive fraction responsible for the antilipolytic activity of SMW and 13 compounds were characterized from ESMW by UHPLC-QTOF-MS/MS. ESMW suppressed protein kinase A (PKA)-hormone-sensitive lipase (HSL) related lipolysis and increased AMP-activated protein kinase (AMPK) phosphorylation in PA challenged 3T3-L1 adipocytes. AMPKα knockdown abolished the inhibitory effects of ESMW on IL-6 and HSL pSer-660, revealing that the antilipolytic and anti-inflammatory activities of ESMW are AMPK dependent. Furthermore, ESMW ameliorated insulin resistance and suppressed lipolysis in HFD-fed mice. It inhibited diacylglycerol accumulation in the liver and inhibited hepatic gluconeogenesis. Conditional medium collected from ESMW-treated 3T3-L1 cells ameliorated insulin action on hepatic gluconeogenesis in liver cells, demonstrating the antilipolytic activity contributed to ESMW beneficial effects on hepatic glucose production. In conclusion, ESMW, as the antilipolytic fraction of SMW, inhibited PKA-HSL related lipolysis by activating AMPK, thus inhibiting diacylglycerol (DAG) accumulation in the liver and thereby improving insulin resistance and hepatic gluconeogenesis.

Olanzapine causes non-alcoholic fatty liver disease via inhibiting the secretion of apolipoprotein A5 / 中南大学学报(医学版)

OBJECTIVES@#Long-term treatment of olanzapine, the most widely-prescribed second-generation antipsychotic, remarkably increases the risk of non-alcoholic fatty liver disease (NAFLD), whereas the mechanism for olanzapine-induced NAFLD remains unknown. Excessive hepatic fat accumulation is the basis for the pathogenesis of NAFLD, which results from the disturbance of TG metabolism in the liver. Apolipoprotein A5 (ApoA5) is a key regulator for TG metabolism in vivo that promotes TG accumulation in hepatocytes, thereby resulting in the development of NAFLD. However, there are no data indicating the role of apoA5 in olanzapine-induced NAFLD. Therefore, this study aims to investigate the role of apoA5 in olanzapine-induced NAFLD.@*METHODS@#This study was carried out via animal studies, cell experiment, and ApoA5 gene knockdown experiment. Six-week-old male C57BL/6J mice were randomized into a control group, a low-dose group, and a high-dose group, which were treated by 10% DMSO, 3 mg/(kg·d) olanzapine, and 6 mg/(kg·d) olanzapine, respectively for 8 weeks. The lipid levels in plasma, liver function indexes, and expression levels of ApoA5 were detected. HepG2 cells were treated with 0.1% DMSO (control group), 25 μmol/L olanzapine (low-dose group), 50 μmol/L olanzapine (medium-dose group), and 100 μmol/L olanzapine (high-dose group) for 24 h. HepG2 cells pretreated with 100 μmol/L olanzapine were transfected with siRNA and scrambled siRNA (negative control), respectively. We observed the changes in lipid droplets within liver tissues and cells using oil red O staining and fat deposition in liver tissues using HE staining. The mRNA and protein levels of ApoA5 were determined by real-time PCR and Western blotting, respectively.@*RESULTS@#After intervention with 3 and 6 mg/(kg·d) olanzapine for 8 weeks, there was no significant difference in body weight among the 3 groups (P>0.05). Olanzapine dose-dependently increased the plasma TG, ALT and AST levels, and reduced plasma ApoA5 levels (all P<0.05), whereas there was no significant difference in plasma cholesterol (HDL-C, LDL-C, and TC) levels among the 3 groups (all P>0.05). Olanzapine dose-dependently up-regulated ApoA5 protein levels in liver tissues (all P<0.05), but there was no significant change in ApoA5 mRNA expression among groups (P>0.05). In the control group, the structure of liver tissues was intact, the morphology of liver cells was regular, and only a few scattered lipid droplets were found in the cells. In the olanzapine-treated group, there was a large amount of lipid deposition in hepatocytes, and cells were balloon-like and filled with lipid droplet vacuoles. The nucleus located at the edge of cell, and the number of lipid droplets was increased significantly, especially in the high-dose group. Likewise, when HepG2 cells were treated with olanzapine for 24 h, the number and size of lipid droplets were significantly elevated in a dose-dependent manner. Moreover, olanzapine dose-dependently up-regulated ApoA5 protein levels in HepG2 cells (all P<0.05), but there was no significant difference in ApoA5 mRNA expression among groups (P>0.05). Compared with the HepG2 cells transfected with scrambled siRNA, the number and size of lipid droplets in HepG2 cells transfected with ApoA5 siRNA were significantly reduced.@*CONCLUSIONS@#The short-term intervention of olanzapine does not significantly increase body weight of mice, but it can directly induce hypertriglyceridemia and NAFLD in mice. Olanzapine inhibits hepatic apoA5 secretion but does not affect hepatic apoA5 synthesis, resulting in the pathogenesis of NAFLD. Inhibition of apoA5 secretion plays a key role in the development of olanzapine-related NAFLD, which may serve as an intervention target for this disease.

TSPAN8 is involved in lipid metabolism in non-alcoholic fatty liver disease in mice / 南方医科大学学报

OBJECTIVE@#To investigate the changes of tetraspanin 8 (TSPAN8) expression levels and its role in lipid metabolism during the development of non-alcoholic fatty liver disease (NAFLD).@*METHODS@#Thirty male C57BL/6J mice were randomly divided into normal diet group and high-fat diet (HFD) group (n=15), and after feeding for 1, 3, and 6 months, the expression levels of TSPAN8 in the liver tissues of the mice were detected with Western blotting. In a HepG2 cell model of NAFLD induced by free fatty acids (FFA), the effect of TSPAN8 overexpression on lipid accumulation was examined using Oil Red O staining and an automated biochemical analyzer, and the mRNA expressions of the key genes involved in lipid metabolism were detected using qRT-PCR.@*RESULTS@#Western blotting showed that compared with that in mice with normal feeding, the expression of TSPAN8 was significantly decreased in the liver tissues of mice with HFD feeding for 3 and 6 months (P < 0.05). In HepG2 cells, treatment with FFA significantly decreased the expression of TSPAN8 at both the mRNA and protein levels (P < 0.01). TSPAN8 overexpression in FFA-treated cells showed significantly lowered intracellular triglyceride levels (P < 0.001) and obviously reduced mRNA expression of fatty acid transport protein 5 (FATP5) (P < 0.01). The expression of FATP5 was significantly increased in FFA-treated cells as compared with the control cells (P < 0.001).@*CONCLUSION@#TSPAN8 is involved in lipid metabolism in NAFLD, and overexpression of TSPAN8 may inhibit cellular lipid deposition by reducing the expression of FATP5.

Hepatic mitochondria in diet-induced obese mice (stereology and molecular analysis) / Mitocondrias hepáticas en ratones obesos inducidos por la dieta (estereología y análisis molecular)

SUMMARY: The world population is going through an obesity epidemic that has severe consequences for the health system. This study focused on studying hepatic mitochondria in obese animals induced by a high-fat (HF) diet and used the model-based stereology in electron micrographs for the quantitative study. Besides, the gene expressions of molecular markers of mitochondrial biogenesis carnitine palmitoyltransferase 1a (Cpt 1α), mitochondrial transcription factor a (Tfam), uncoupling protein 3 (Ucp 3), and nuclear respiratory factor 1 (Nrf 1) were analyzed. The HF diet caused a weight gain of +1820 % comparing the control group (C) with the HF group (from 0.32±0.31 g to 5.5±0.39 g, P<0.001). The HF group showed fat droplets in the hepatocyte cytoplasm (steatosis) and less dense and large mitochondria in transmission electron microscopy. The mitochondria size (cross-section) did not show a significant difference between the groups C and HF. However, the mitochondria numerical density per area was 30 % less, the mitochondrial surface density (outer membrane) was 20 % less, and the mitochondrial volume density was 22 % less in the HF group than the C group. The gene expressions of molecular markers of mitochondrial biogenesis Cpt 1α, Tfam, Ucp 3, and Nrf 1 decreased in the HF group compared to the C group. The quantitative results match perfectly with the molecular ones of mitochondrial biogenesis markers. In the future, it will be crucial to verify if and how these data recover with the reduction of obesity, which would be of significant interest given the current obesity epidemic that affects the world population.

RESUMEN: La población mundial atraviesa una epidemia de obesidad que tiene graves consecuencias para el sistema de salud. Este estudio se centró en el análisis de las mitocondrias hepáticas en animales obesos inducidos por una dieta alta en grasas (HF) y utilizó la estereología basada en modelos en micrografías electrónicas para el estudio cuantitativo. Además, se analizaron las expresiones génicas de los marcadores moleculares de la biogénesis mitocondrial carnitina palmitoiltransferasa 1a (Cpt 1α), factor de transcripción mitocondrial a (Tfam), proteína desacoplante 3 (Ucp 3) y factor respiratorio nuclear 1 (Nrf 1). La dieta HF provocó un aumento de peso de +1820 % comparando el grupo de control (C) con el grupo HF (de 0,32 ± 0,31 g a 5,5 ± 0,39 g, P <0,001). El grupo HF mostró gotas de grasa en el citoplasma de los hepatocitos (esteatosis) y mitocondrias menos densas y grandes en la microscopía electrónica de transmisión. El tamaño de las mitocondrias (sección transversal) no mostró una diferencia significativa entre los grupos C y HF. Sin embargo, la densidad numérica de mitocondrias por área fue 30% menor, la densidad de superficie mitocondrial (membrana externa) fue 20 % menor y la densidad de volumen mitocondrial fue 22 % menor en el grupo HF que en el grupo C. Las expresiones génicas de los marcadores moleculares de la biogénesis mitocondrial Cpt 1α, Tfam, Ucp 3 y Nrf 1 disminuyeron en el grupo HF en comparación con el grupo C. Los resultados cuantitativos coinciden perfectamente con los moleculares de los marcadores de biogénesis mitocondrial. En el futuro, será crucial verificar si estos datos se recuperan y cómo se recuperan con la reducción de la obesidad, lo que sería de gran interés dada la actual epidemia de obesidad que afecta a la población mundial.

The role of pioglitazone in antioxidant, anti-inflammatory, and insulin sensitivity in a high fat-carbohydrate diet-induced rat model of insulin resistance

We explored the cascade effects of a high fat-carbohydrate diet (HFCD) and pioglitazone (an anti-diabetic therapy used to treat type 2 diabetes mellitus (T2DM)) on lipid profiles, oxidative stress/antioxidant, insulin, and inflammatory biomarkers in a rat model of insulin resistance. Sixty albino rats (80-90 g) were randomly divided into three dietary groups; 1) standard diet; 2) HFCD diet for 12 weeks to induce an in vivo model of insulin resistance; and 3) HFCD diet plus pioglitazone. Blood and tissue samples were taken to assess hepatic function, lipid profiles, oxidative biomarkers, malondialdehyde (MDA) levels, antioxidant defense biomarkers, including reduced glutathione (GSH), superoxide dismutase (SOD), and the inflammatory markers interleukin-6 (IL-6) and tumor necrotic factor (TNF-α). HFCD-fed rats had significantly (P≤0.05) increased serum triacylglycerol (TG), total cholesterol (TC), low-density lipoprotein (LDL), alanine transaminase (ALT), and bilirubin levels, but decreased high-density lipoprotein (HDL) levels compared with the normal group. Moreover, serum leptin, resistin, TNF-α, and IL-6 levels were increased significantly in HFCD animals compared with controls. Similarly, HFCD-induced insulin resistance caused antioxidant and cytokine disturbances, which are important therapy targets for pioglitazone. Importantly, administration of this drug ameliorated these changes, normalized leptin and resistin and inflammatory markers by reducing TNF-α levels. Metabolic cascades of elevated lipid profiles, oxidative stress, insulin, and inflammatory biomarkers are implicated in insulin resistance progression. HFCD induced metabolic cascades comprising hypertriglyceridemia, hyperglycemia, insulin resistance, obesity-associated hormones, and inflammatory biomarkers may be alleviated using pioglitazone.