Lipopolysaccharide inhibits lipophagy in HepG2 cells via activating mTOR pathway / 生理学报

This study aimed to investigate the effect of lipopolysaccharide (LPS) on lipophagy in hepatocytes and the underlying mechanism. Human hepatoma cell line HepG2 was cultured in vitro, treated with 0.1 mmol/L palmitic acid (PA), and then divided into control group (0 μg/mL LPS), LPS group (10 μg/mL LPS), LPS+DMSO group and LPS+RAPA (rapamycin, 10 μmol/L) group. Lipid accumulation in hepatocytes was observed by oil red O staining. The autophagic flux of the cells was assessed using confocal laser scanning microscope after being transfected with autophagy double-labeled adenovirus (mRFP-GFP-LC3). The level of intracellular lipophagy was visualized by the colocalization of lipid droplets (BODIPY 493/503 staining) and lysosomes (lysosome marker, lysosomal associated membrane protein 1, LAMP1). The expression levels of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), ribosome protein subunit 6 kinase 1 (S6K1), p-S6K1, LC3II/I and P62 protein were examined by Western blot. The results showed that the number of red lipid droplets stained with oil red O was significantly increased in LPS group compared with that in control group (P < 0.001). Moreover, in LPS group, the number of autophagosomes was increased, while the number of autophagolysosomes and the colocalization rate of LAMP1 and BODIPY were significantly decreased (P < 0.05). Meanwhile, the ratios of p-mTOR/mTOR and p-S6K1/S6K1, the ratio of LC3II/LC3I and the protein expression of P62 were significantly increased (P < 0.05) in LPS group. Furthermore, compared with LPS+DMSO group, RAPA treatment obviously reduced the number of lipid droplets and autophagosomes, and raised the number of autophagolysosomes and the colocalization rate of LAMP1 and BODIPY (P < 0.05). In conclusion, the results demonstrate that LPS inhibits lipophagy in HepG2 cells via activating mTOR signaling pathway, thereby aggravating intracellular lipid accumulation.

Deletion of CD36 gene ameliorates glucose metabolism abnormality induced by high-fat diet and promotes liver lipid accumulation / 生理学报

This study aimed to investigate the effects and the underlying mechanism of CD36 gene on glucose and lipid metabolism disorder induced by high-fat diet in mice. Wild type (WT) mice and systemic CD36 knockout (CD36

Pure Laparoscopic Liver Resection for Malignant Liver Tumor: Anatomic Resection Versus Nonanatomic Resection / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Laparoscopic liver resection (LLR) has been considered to be safe and feasible. However, few studies focused on the comparison between the anatomic and nonanatomic LLR. Therefore, the purpose of this study was to compare the perioperative factors and outcomes of the anatomic and nonanatomic LLR, especially the area of liver parenchymal transection and blood loss per unit area.</p><p><b>METHODS</b>In this study, surgical and oncological data of patients underwent pure LLR procedures for malignant liver tumor were prospectively collected. Blood loss per unit area of liver parenchymal transection was measured and considered as an important parameter. All procedures were conducted by a single surgeon.</p><p><b>RESULTS</b>During nearly 5 years, 84 patients with malignant liver tumor received a pure LLR procedure were included. Among them, 34 patients received anatomic LLR and 50 received nonanatomic LLR, respectively. Patients of the two groups were similar in terms of demographic features and tumor characteristics, despite the tumor size was significantly larger in the anatomic LLR group than that in the nonanatomic LLR group (4.77 ± 2.57 vs. 2.87 ± 2.10 cm, P = 0.001). Patients who underwent anatomic resection had longer operation time (364.09 ± 131.22 vs. 252.00 ± 135.21 min, P < 0.001) but less blood loss per unit area (7.85 ± 7.17 vs. 14.17 ± 10.43 ml/cm 2 , P = 0.018). Nonanatomic LLR was associated with more blood loss when the area of parenchymal transection was equal to the anatomic LLR. No mortality occurred during the hospital stay and 30 days after the operation. Moreover, there was no difference in the incidence of postoperative complications. The disease-free and overall survival rates showed no significant differences between the anatomic LLR and nonanatomic LLR groups.</p><p><b>CONCLUSIONS</b>Both anatomic and nonanatomic pure LLR are safe and feasible. Measuring the area of parenchymal transection is a simple and effective method to estimate the outcomes of the liver resection surgery. Blood loss per unit area is an important parameter which is comparable between the anatomic LLR and nonanatomic LLR groups.</p>

Effect of inflammatory stress on hepatic cholesterol accumulation and hepatic fibrosis in C57BL/6J mice / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether inflammatory stress exacerbates hepatic cholesterol accumulation and liver fibrosis using a C57BL/6J mouse model of chronic inflammation.</p><p><b>METHODS</b>Twelve male C57BL/6J mice were given a high-fat diet (15.0% fat, 1.25% cholesterol, 0.5% cholic acid) and randomly assigned to the normal control group (n=6; subcutaneously injected with 0.5 mL of isotonic saline, every other day for 14 weeks) or the chronic inflammation model group (n=6; subcutaneously injected with of 0.5 mL of 10% casein, every other day for 14 weeks). At the end of week 14, the animals were sacrificed and blood was collected from the left ventricle for serological analysis of inflammatory markers and lipid profile, including serum amyloid A (SAA), interleukin-6 (IL-6), total cholesterol (TC) and free cholesterol (FC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL)). Extracted liver tissues were divided for use in histological analysis (lipid accumulation and fibrosis evaluated by Oil Red O, Sirius red and Masson's trichrome staining) and quantitative fluorescence real-time PCR (to measure b-actin normalized expression of TNF-a MCP1, SREBP-2, LDLr, HMGCoA-r, ATF-6, GRP78, BMP-7, TGF-b, and collagens type I and type IV). Comparisons between groups were made by the two-samples t-test or Satterthwaite t-approximation test, collagen type I and type IV.</p><p><b>RESULTS</b>Compared to the normal control group, the inflammation model group showed elevated serum IL-6 (12.55+/-4.75 vs. 32.41+/-7.42 pg/mL, P less than 0.01), reduced serum TC (14.82+/-1.56 vs. 10.62+/-0.48 mmol/L, P less than 0.01), up-regulated hepatic TNF-a mRNA expression (1.05+/-0.35 vs. 2.12+/-0.72, P less than 0.01), and elevated hepatic TC (12.10+/-2.57 vs. 23.21+/-8.75 mmol/L, P less than 0.05). In addition, the inflammation group showed abnormal lipid deposition, and increased and thickened reticular fibers. The livers of the inflammation group also showed up-regulated mRNA expression of SREBP-2 (normal control: 1.01+/-0.19 vs. 2.63+/-0.13, P less than 0.05), GRP78 (1.07+/-0.47 vs. 2.21+/-0.99, P less than 0.05), TGF-b (1.01+/-0.14 vs. 1.38+/-0.28, P less than 0.05), and collagen type I (1.02+/-0.27 vs. 1.71+/-0.51, P less than 0.05) and down-regulation of BMP-7 (1.01+/-0.15 vs. 0.55+/-0.25, P less than 0.01).</p><p><b>CONCLUSION</b>Activation of the inflammatory system exacerbates hepatic cholesterol accumulation and hepatic fibrosis in C57BL/6J mice.</p>

Effect of RNAi-mediated silencing of SREBP2 gene on inflammatory cytokine-induced cholesterol accumulation in HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of RNA interference (RNAi)-mediated silencing of the SREBP2 on inflammatory cytokine-induced cholesterol accumulation in HepG2 cells.</p><p><b>METHODS</b>Short-hairpin (sh)RNA targeting SREBP2 or negative control (NC) shRNA were transfected into HepG2 cells by a liposomal method. G418-selective culturing was used to obtain the SREBP2 shRNA HepG2 and NC shRNA HepG2 cell lines. The two cell lines were cultured in serum-free medium and left untreated (control) or treated with TNF-a (20 ng/ml), low-density lipoprotein (LDL) loading (100 mug/ml), or a combination LDL plus TNF-a treatment. Lipid accumulation was evaluated by oil red O (ORO) staining. Intracellular cholesterol level was measured by enzymatic assay. The mRNA and protein levels of SREBP2 and its downstream target genes, LDL receptor (LDLr), and HMGCoA reductase, were measured by real-time PCR and Western blotting, respectively.</p><p><b>RESULTS</b>SREBP2 shRNA HepG2 and NC shRNA HepG2 stable cell lines were successfully established. ORO staining and cholesterol quantitative analysis showed that LDL loading significantly increased intracellular cholesterol and that expression of SREBP2 further exacerbated the inflammatory cytokine-induced lipid accumulation, as seen in NC shRNA HepG2 cells. LDL loading of NC shRNA HepG2 decreased the gene and protein expressions of SREBP2, LDLr, and HMGCoA reductase, but the suppressive effect was overridden by inflammatory cytokine. SREBP2 shRNA HepG2 cells showed lower levels of cholesterol accumulation under LDL loading and inflammatory stress conditions. Moreover, the mRNA and protein levels of SREBP2, LDLr, and HMGCoA reductase were much lower than in NC shRNA HepG2 cells under the same conditions.</p><p><b>CONCLUSION</b>Inflammatory cytokine exacerbated cholesterol accumulation in HepG2 via disrupting SREBP2. RNAi-mediated inhibition of SREBP2 expression significantly ameliorated the cholesterol accumulation induced by inflammatory cytokine.</p>

Effect of HBV on the expression of SREBP in the hepatocyte of chronic hepatitis B patients combined with hepatic fatty change / 中华肝脏病杂志

To investigate the effect of HBV on the expression of Sterol regulatory element binding proteins( SREBP ) in the hepatocyte of patients with chronic hepatitis B (CHB) combined with hepatic fatty change. 55 cases diagnosed as CHB combined with hepatic fatty change in our department were selected and liver biopsies were carried out. The patients were dividied into 3 groups, group A: HBV DNA is less than or equal to 1000 copies/ml(15 cases), group B: 1000 copies/ml less than HBV DNA less than 100000 copies/ml (18 cases) and group C: HBV DNA is more than or equal to 100000 copies/ml (22 cases). 10 patients with HBV DNA in less than or equal to 1000 copies/ml after antiviral therapy with Nucleoside analogues were seen as group C1 (before treatment) and group C2 (after treatment) respectively; 12 patients with HBV DNA is more than or equal to 100000 copies/ml after antiviral therapy were classified as group C3 (before treatment) and group C4 (after treatment). Lipid droplets in the hepatic tissue were observed with oil red staining. Real time PCR were performed to detect the expressions of SREBP-1c and SREBP-2 mRNA in the liver. The protein expressions of SREBP-1c and SREBP-2 were detected with immunohistochemistry staining. Statistic data were analysed with SPSS11.5 software. (1) Red integrated optical densities (IOD) reflected by lipid drops in group A, B and C are 1004.27+/-218.63, 1937.01+/-401.47 and 4133.79+/-389.28 respectively, the degree of oil red O in each group was different (F = 385.69, P is less than to 0.01), which is increased as HBV DNA load increasing; Red IOD in group C1, C2 and C3, C4 are 4020.84+/-326.64, 1012.02+/-244.89, 4189.18+/-329.21 and 4121.76+/-304.09 respectively. Compared with group C1, the degree of oil red O in group C2 is decreased and the difference is statistically significant (t = 22.55, P is less than to 0.01); However, the difference of the degree of oil red O between group C4 and C3 is not statistically significant. (2) Compared with group A, the expressions of SREBP-1c mRNA in group B and C are raised by 1.218+/-0.130 and 1.798+/-0.118 times respectively, among group A, B, C, the expressions of SREBP-1c mRNA are statistically significant different ( F = 297.47, P is less than to 0.01). The expressions of SREBP-2 mRNA in group B and C are decreased by 0.956+/-0.118 and 0.972+/-0.153 times as compared to group A. However, the difference of SREBP-2 mRNA expression among the 3 groups is not statistically significant ( F = 0.568, P is more than to 0.05). Compared with group C1, SREBP-1c mRNA in group C2 is decreased by 0.714+/-0.081 folds (t=11.224, P is less than to 0.01), while SREBP-2 mRNA in group C2 is raised by1.034+/-0.155 times(t=0.692, P is more than to 0.05). SREBP-1c mRNA and SREBP-2 mRNA in group C4 are raised by 1.012+/-0.206 times and decreased by 0.998+/-0.183 times as compared to group C3 without difference found (t=0.196 or 0.031, P is more than to 0.05). (3) the expressions of SREBP-1c protein in group A, B and C are 36257.21+/-5709.79, 50413.47+/-4989.28 and 71025.83+/-6047.13 respectively, and the difference is statistically significant among the 3 groups (F = 178.26, P is less than to 0.01); the expressions of SREBP-2 protein in group A, B and C are 32913.52+/-3951.21, 32625.91+/-4025.06 and 34173.44+/-5316.25 respectively, but the difference is not statistically significant among the 3 groups ( F = 0.562, P is more than to 0.05), SREBP-1c protein levels in group C1, C2, C3, C4 are 69832.16+/-4941.36, 48735.47+/-5471.41, 70871.69+/-5083.14 and 68913.32+/-5343.22 respectively, the difference of SREBP-1c protein levels between group C1 and C2 is statistically significant (t=10.260, P is less than to 0.01); while the difference between group C3 and group C4 is not statistically significant(t=1.558, P is more than to 0.05). The expressions of SREBP-2 protein in group C1, C2, C3 and C4 are 33 980.21+/-4081.80, 34011.50+/-3859.27, 33610.12+/-4761.10 and 32915.66+/-5023.61 respectively, the difference of SREBP-2 protein levels in group C1 and group C2 is not statistically significant (t=0.038, P is more than to 0.05) and same result exists between group C3 and group C4 (t=0.459, P is more than to 0.05). HBV DNA may participate in the hepatic steatosis formation through interfering with the SREBP-1c expression.

Inflammation enhances the accumulation of lipid in ApoE/SRA/CD36 KO mice liver / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate if inflammatory stress enhances liver lipid accumulation via SREBPs mediated dysregulation of low density protein receptor (LDLr) expression in apolipoprotein E, scavenger receptors class A and CD36 triple knockout (ApoE/SRA/CD36 KO) mice.</p><p><b>METHODS</b>16 Male ApoE/SRA/CD36 KO mice were subcutaneously injected with 0.5 ml 10% casein or PBS. The mice were fed a Western diet (Harlan, TD88137) containing 21% fat and 0.15% of cholesterol for 14 weeks. Animals were sacrificed and blood samples were collected. The serum amyloid A (SAA), IL-6, total cholesterol (TC), LDL and high density protein (HDL) were assayed. The lipid accumulation in liver was evaluated by Oil Red O staining. The mRNA and protein expression of SREBP-2, SREBPs cleavage activating protein (SCAP) and LDLr were analyzed by Real-Time Polymerase Chain Reaction (RT-PCR) and immunohistochemistry staining.</p><p><b>RESULTS</b>Blood levels of SAA [(26.60+/-3.24) ng/ml vs (14.35+/-1.73) ng/ml, P < 0.01] and IL-6 [(36.37+/-2.20) pg/ml vs (18.02+/-4.87) pg/ml, P < 0.01] were higher, while TC [(7.72+/-1.70) mmol/L vs (13.23+/-3.61)mmol/L, P less than 0.01], LDL-cholesterol [(2.94+/-0.44) mmol/L vs (9.28+/-3.66) mmol/L, P less than 0.01] and HDL cholesterol [(2.24+/-0.63) mmol/L vs (4.13+/-0.42) mmol/L, P less than 0.01] were lower in inflamed mice compared to controls. ORO staining showed that lipid accumulation in the liver was more extensive in inflamed group despite lower blood lipid levels. Meanwhile, Real Time PCR data showed inflammation induced the expression of LDLr (4.56 fold), SCAP (3.14 fold) and SREBP-2 (14.72 fold) in liver. Immunohistochemical staining also indicated increased proteins expression in the liver, which was consistent with mRNA data.</p><p><b>CONCLUSIONS</b>Inflammation causes lipid accumulation in liver via disrupting SREBP-2 and LDLr expression.</p>

Mechanisms of dysregulation of low-density lipoprotein receptor expression in HepG2 cells induced by inflammatory cytokines / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Low-density lipoprotein (LDL) receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated LDL receptor feedback regulation causing intracellular accumulation of unmodified LDL in peripheral cells. Liver is the central organ for lipid homeostasis. The aim of this study was to investigate the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms in human hepatic cell line (HepG2) cells under physiological and inflammatory conditions.</p><p><b>METHODS</b>Intracellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein (SREBP)-2 and SREBP cleavage-activating protein (SCAP) mRNA levels using real-time quantitative PCR. LDL receptor and SREBP-2 protein expression were examined by Western blotting. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the endoplasmic reticulum (ER) to the Golgi by dual staining with anti-human SCAP and anti-Golgin antibodies.</p><p><b>RESULTS</b>LDL loading increased intracellular cholesterol level, thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions. However, interleukin 1 beta (IL-1 beta) further increased intracellular cholesterol level in the presence of LDL by increasing both LDL receptor mRNA and protein expression in HepG2. LDL also reduced the SREBP and SCAP mRNA level under physiological conditions. Exposure to IL-1 beta caused over-expression of SREBP-2 and also disrupted normal distribution of SCAP-SREBP complex in HepG2 by enhancing translocation of SCAP-SREBP from the ER to the Golgi despite a high concentration of LDL in the culture medium.</p><p><b>CONCLUSIONS</b>IL-1 beta disrupts cholesterol-mediated LDL receptor feedback regulation by enhancing SCAP-SREBP complex translocation from the ER to the Golgi, thereby increasing SREBP-2 mediated LDL receptor expression even in the presence of high concentration of LDL. This results in LDL cholesterol accumulation in hepatic cells via LDL receptor pathway under inflammatory stress.</p>