Assessment of the mode of action of perchloroethylene-induced mouse liver tumors.

Perchloroethylene (PCE) is used as a solvent and chemical intermediate. Following chronic inhalation exposure, PCE selectively induced liver tumors in mice. Understanding the mode of action (MOA) for PCE carcinogenesis in mice is important in defining its possible human cancer risk. The proposed MOA is based on the extensive examination of the peer-reviewed studies that have assessed the mouse liver effects of PCE and its major oxidative metabolite trichloroacetic acid (TCA). Similar to PCE, TCA has also been demonstrated to liver tumors selectively in mice following chronic exposure. The Key Events (KE) of the proposed PCE MOA involve oxidative metabolism of PCE to TCA [KE 1]; activation of the peroxisome proliferator-activated receptor alpha (PPARα) [KE 2]; alteration in hepatic gene expression including cell growth pathways [KE 3]; increase in cell proliferation [KE 4]; selective clonal expansion of hepatic preneoplastic foci [KE 5]; and formation of hepatic neoplasms [KE 6]. The scientific evidence supporting the PPARα MOA for PCE is strong and satisfies the requirements for a MOA analysis. The PPARα liver tumor MOA in rodents has been demonstrated not to occur in humans; thus, human liver cancer risk to PCE is not likely.

Sodium butyrate alleviates free fatty acid-induced steatosis in primary chicken hepatocytes via the AMPK/PPARα pathway.

Fatty liver hemorrhagic syndrome (FLHS) is a prevalent metabolic disorder observed in egg-laying hens, characterized by fatty deposits and cellular steatosis in the liver. Our preliminary investigations have revealed a marked decrease in the concentration of butyric acid in the FLHS strain of laying hens. It has been established that sodium butyrate (NaB) protects against metabolic disorders. However, the underlying mechanism by which butyrate modulates hepato-lipid metabolism to a great extent remains unexplored. In this study, we constructed an isolated in vitro model of chicken primary hepatocytes to induce hepatic steatosis by free fatty acids (FFA). Our results demonstrate that treatment with NaB effectively mitigated FFA-induced hepatic steatosis in chicken hepatocytes by inhibiting lipid accumulation, downregulating the mRNA expression of lipo-synthesis-related genes (sterol regulatory element binding transcription factor 1 (SREBF1), acetyl-CoA carboxylase 1(ACC1), fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), liver X receptor α (LXRα), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR)) (P < 0.05), and upregulating the mRNA and protein expression of AMP-activated protein kinase α1 (AMPKα1), peroxisome proliferator-activated receptor α (PPARα), and carnitine palmitoyl-transferase 1A (CPT1A) (P < 0.05). Moreover, AMPK and PPARα inhibitors (Compound C (Comp C) and GW6471, respectively) reversed the protective effects of NaB against FFA-induced hepatic steatosis by blocking the AMPK/PPARα pathway, leading to lipid droplet accumulation and triglyceride (TG) contents in chicken primary hepatocytes. With these findings, NaB can alleviate hepatocyte lipoatrophy injury by activating the AMPK/PPARα pathway, promoting fatty acid oxidation, and reducing lipid synthesis in chicken hepatocytes, potentially being able to provide new ideas for the treatment of FLHS.

Osteocalcin activates lipophagy via the ADPN-AMPK/PPARα-mTOR signaling pathway in chicken embryonic hepatocyte.

Fatty liver hemorrhage syndrome (FLHS) is the leading cause of noninfectious mortality in caged layers worldwide. Osteocalcin (OCN) is a protein secreted by osteoblasts, and its undercarboxylated form (ucOCN) acts as a multifunctional hormone that protects laying hens from FLHS. Lipophagy is a form of selective autophagy that breaks down lipid droplets (LDs) through lysosomes, and defective lipophagy is associated with FLHS. The aim of this study was to investigate the effects of ucOCN on the lipophagy of chicken embryonic hepatocytes and associated the function of the adiponectin (ADPN) signaling pathway. In this study, chicken embryonic hepatocytes were divided into 5 groups: control (CONT), fat emulsion (FE, 10% FE, v/v), FE with ucOCN at 1 ng/mL (FE-LOCN), 3 ng/mL (FE-MOCN), and 9 ng/mL (FE-HOCN). In addition, 4 µM AdipoRon, an adiponectin receptor agonist, was used to investigate the function of ADPN. The results showed that compared with CONT group, FE promoted the levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (P < 0.05) and decreased the mRNA expression of ADNP receptors (AdipoR1 and AdipoR2). Compared with FE group, 3 and 9 ng/mL ucOCN inhibited the levels of autophagy adaptor p62 and p-mTOR (P < 0.05), increased the ratios of LC3-II/LC3-I (P < 0.05) and phosphorylated adenosine 5'-monophosphate-activated protein kinase (p-AMPK)/AMPK (P < 0.05), as well as the levels of peroxisome proliferator-activated receptor α (PPAR-α) and ADPN (P < 0.05). In addition, ucOCN at the tested concentrations increased the colocalization of LC3 and LDs in fatty hepatocytes. Administrated 4 µM AdipoRon activated AdipoR1 and AidpoR2 mRNA expression (P < 0.05), decreased the concentrations of triglyceride (P < 0.05), without effects on cell viability (P > 0.05). AdipoRon also increased the LC3-II/LC3-I ratio (P < 0.05) and the levels of p-AMPK/AMPK and PPAR-α (P < 0.05). In conclusion, the results reveal that ucOCN regulates lipid metabolism by activating lipophagy via the ADPN-AMPK/PPARα-mTOR signaling pathway in chicken embryonic hepatocytes. The results may provide new insights for controlling FLHS in laying hens.

GenX Disturbs the Indicators of Hepatic Lipid Metabolism Even at Environmental Concentration in Drinking Water via PPARα Signaling Pathways.

Hexafluoropropylene oxide dimer acid (HFPO-DA; trade name GenX), as a substitute for perfluorooctanoic acid (PFOA), has been attracting increasing attention. However, its impact and corresponding mechanism on hepatic lipid metabolism are less understood. To investigate the possible mechanisms of GenX for hepatotoxicity, a series of in vivo and in vitro experiments were conducted. In in vivo experiment, male mice were exposed to GenX in drinking water at environmental concentrations (0.1 and 10 µg/L) and high concentrations (1 and 100 mg/L) for 14 weeks. In in vitro experiments, human hepatocellular carcinoma cells (HepG2) were exposed to GenX at 10, 160, and 640 µM for 24 and 48 h. GenX exposure via drinking water resulted in liver damage and disruption of lipid metabolism even at environmental concentrations. The results of triglycerides (TG) and total cholesterol (TC) in this study converged with the results of the population study, for which TG increased in the liver but unchanged in the serum, whereas TC increased in both liver and serum concentrations. KEGG and GO analyses revealed that the hepatotoxicity of GenX was associated with fatty acid transport, synthesis, and oxidation pathways and that Peroxisome Proliferator-Activated Receptor (PPARα) contributed significantly to this process. PPARα inhibitors significantly reduced the expression of CD36, CPT1ß, PPARα, SLC27A1, ACOX1, lipid droplets, and TC, suggesting that GenX exerts its toxic effects through PPARα signaling pathway. In general, GenX at environmental concentrations in drinking water causes abnormal lipid metabolism via PPARα signaling pathway.

The impact of vaccine-linked chemotherapy on liver health in a mouse model of chronic Trypanosoma cruzi infection.

BACKGROUND: Chagas disease, chronic infection with Trypanosoma cruzi, mainly manifests as cardiac disease. However, the liver is important for both controlling parasite burdens and metabolizing drugs. Notably, high doses of anti-parasitic drug benznidazole (BNZ) causes liver damage. We previously showed that combining low dose BNZ with a prototype therapeutic vaccine is a dose sparing strategy that effectively reduced T. cruzi induced cardiac damage. However, the impact of this treatment on liver health is unknown. Therefore, we evaluated several markers of liver health after treatment with low dose BNZ plus the vaccine therapy in comparison to a curative dose of BNZ. METHODOLOGY: Female BALB/c mice were infected with a bioluminescent T. cruzi H1 clone for approximately 70 days, then randomly divided into groups of 15 mice each. Mice were treated with a 25mg/kg BNZ, 25µg Tc24-C4 protein/ 5µg E6020-SE (Vaccine), 25mg/kg BNZ followed by vaccine, or 100mg/kg BNZ (curative dose). At study endpoints we evaluated hepatomegaly, parasite burden by quantitative PCR, cellular infiltration by histology, and expression of B-cell translocation gene 2(BTG2) and Peroxisome proliferator-activated receptor alpha (PPARα) by RT-PCR. Levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were quantified from serum. RESULTS: Curative BNZ treatment significantly reduced hepatomegaly, liver parasite burdens, and the quantity of cellular infiltrate, but significantly elevated serum levels of ALT, AST, and LDH. Low BNZ plus vaccine did not significantly affect hepatomegaly, parasite burdens or the quantity of cellular infiltrate, but only elevated ALT and AST. Low dose BNZ significantly decreased expression of both BTG2 and PPARα, and curative BNZ reduced expression of BTG2 while low BNZ plus vaccine had no impact. CONCLUSIONS: These data confirm toxicity associated with curative doses of BNZ and suggest that while dose sparing low BNZ plus vaccine treatment does not reduce parasite burdens, it better preserves liver health.

Si-Ni-San Reduces Hepatic Lipid Deposition in Rats with Metabolic Associated Fatty Liver Disease by AMPK/SIRT1 Pathway.

Background: Metabolic associated fatty liver disease (MAFLD) is a chronic disease characterized by excessive lipid deposition in the liver without alcohol or other clear liver-damaging factors. AMP-activated protein kinase (AMPK)/silencing information regulator (SIRT)1 signaling pathway plays an important role in MAFLD development. Si-Ni-San (SNS), a traditional Chinese medicine, has shown reducing hepatic lipid deposition in MAFLD rats, however, the underlying mechanisms of SNS are barely understood. Purpose: The aim of this research was to investigate the mechanisms of SNS in reducing hepatic lipid deposition in MAFLD rats by regulating AMPK/SIRT1 signaling pathways. Methods: The components of SNS were determined by high performance liquid chromatography with mass spectrometry (HPLC-MS) analysis. MAFLD rats were induced by high-fat and high-cholesterol diet (HFHCD), and treated by SNS. SNS-containing serum and Compound C (AMPK inhibitor) were used to treat palmitic acid (PA)-induced HepG2 cells. To elucidate the potential mechanism, lipid synthesis-related proteins (SREBP-1c and FAS), fatty acid oxidation (PPARα and CPT-1), and AMPK/SIRT1 signaling pathway (p-AMPK and SIRT1) were assessed by Western blot. Results: SNS improved serum lipid levels, liver function and reduced hepatic lipid deposition in MAFLD rats. SNS-containing serum reduced lipid deposition in PA-induced HepG2 cells. SNS could up-regulate protein expressions of PPARα, CPT-1, p-AMPK and SIRT1, and down-regulate protein expressions of SREBP-1c and FAS. Similar effects of SNS-containing serum were observed in PA-induced HepG2 cells. Meanwhile, Compound C weakened the therapeutic effects of SNS-containing serum on lipid deposition. Conclusion: SNS could reduce hepatic lipid deposition by inhibiting lipid synthesis and promoting fatty acid oxidation, which might be related with activating the AMPK/SIRT1 signaling pathway. This study could provide a theoretical basis for the clinical use of SNS to treat MAFLD.

FMO1 Promotes Nonalcoholic Fatty Liver Disease Progression by Regulating PPARα Activation and Inducing Ferroptosis.

BACKGROUND: The function of flavin containing dimethylaniline monooxygenase 1 (FMO1), which is known to play a part in lipid metabolism, remains unclear in the development of nonalcoholic fatty liver disease (NAFLD). This research has the objective of examining the contributions of FMO1 in the progression of NAFLD and the associated mechanisms, particularly the peroxisome proliferator activated receptor alpha (PPARα) and ferroptosis pathways. METHODS: An in vitro NAFLD model was established by treating L02 cells with free fatty acids (FFAs). The FMO1 and ferroptosis levels were examined in the cellular NAFLD model. FMO1 was knocked down using short-interfering RNA transfection. The effects of FMO1 knockdown on lipid accumulation, PPARα expression, and ferroptosis were examined in the cellular NAFLD model. Additionally, the effects of FMO1 and/or PPARα overexpression on lipid metabolism and ferroptosis were analyzed. Furthermore, L02 cells were pre-treated with GW7647 (PPARα agonist) or RSL3 (ferroptosis activator) and stimulated with FFAs. RESULTS: The levels of FMO1 and ferroptosis were upregulated in the in vitro NAFLD model. FMO1 knockdown suppressed the FFA-induced accumulation of lipids in hepatocytes, downregulation of PPARα expression, and upregulation of ferroptosis. In contrast, FMO1 overexpression dysregulated lipid metabolism and downregulated PPARα levels. Meanwhile, PPARα overexpression mitigated the FMO1 overexpression-induced upregulation of ferroptosis and lipid accumulation. Treatment with RSL3 suppressed the effects of PPARα overexpression on lipid accumulation and FMO1 expression. CONCLUSIONS: FMO1 upregulates ferroptosis by suppressing PPARα in NAFLD, which leads to the dysregulation of lipid metabolism.

Mulberry fruit repairs alcoholic liver injury by modulating lipid metabolism and the expression of miR-155 and PPARα in rats.

As alcohol consumption increases, alcoholic liver disease (ALD) has become more popular and is threating our human life. In this study, we found mulberry fruit extract (MFE) repaired alcohol-caused liver diseases by regulating hepatic lipid biosynthesis pathway and oxidative singling in alcoholically liver injured (ALI) rats. MFE administration inhibited hepatic lipid accumulation and improved liver steatosis in ALI rats. MFE also enhanced the antioxidant capacity and alleviated the inflammatory response by increasing the activities of antioxidant enzymes and decreasing the contents of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α. Additionally, MFE regulated the expression of miRNA-155 and lipid metabolism-related PPARα protein in rats. Both miR-155 and PPARα play important roles in liver function. The results indicate that MFE has hepatoprotective effects against ALI in rats.

Lactational retrorsine exposure changes maternal milk components and disturbs metabolism homeostasis of offspring rats.

Pyrrolizidine alkaloids (PAs) are a type of plant-derived environmental toxins, which pose a health hazard to human and livestock via contaminating soil, water, plants and food. In this study, we aimed to investigate the effect of lactational retrorsine (RTS, a typical toxic PA) exposure on breastmilk components and glucose-lipid metabolism of offspring rats. Dams were intragastrically administered with 5 mg/(kg·d) RTS during lactation. After metabolomic analyses, 114 differential constituents were identified in breastmilk between control and RTS groups, featured by reduction of lipids and lipid-like molecules, while presence of abundant RTS and its derivative in RTS-exposed milk. RTS exposure induced liver injury in pups, but the leakage of transaminases in serum recovered in their adulthood. Serum glucose levels were lower in pups but higher in male adult offspring from RTS group. RTS exposure also induced hypertriglyceridemia, hepatic steatosis and decreased glycogen content in both pups and adult offspring. Additionally, suppression of PPARα-FGF21 axis persisted in offspring liver after RTS exposure. These data indicated that inhibition of PPARα-FGF21 axis induced by milk deficient in lipid contents, together with hepatotoxic injury caused by RTS in breastmilk, may disrupt glucose and lipid metabolism of pups, and the persistent suppression of PPARα-FGF21 axis may program metabolic disorder of glucose and lipid in adult offspring.

Melatonin alleviates cadmium-induced nonalcoholic fatty liver disease in ducks by alleviating autophagic flow arrest via PPAR-α and reducing oxidative stress.

Cadmium (Cd) is an important environmental pollutant that causes liver damage and induces nonalcoholic fatty liver disease (NAFLD). NAFLD is a fat accumulation disease and has significant effects on the body. Melatonin (Mel) is an endogenous protective molecule with antioxidant, anti-inflammatory, antiobesity, and antiaging effects. However, whether Mel can alleviate Cd-induced NAFLD and its mechanism remains unclear. First, in vivo, we found that Mel maintained mitochondrial structure and function, inhibited oxidative stress, and reduced Cd-induced liver injury. In addition, Mel alleviated lipid accumulation in the liver induced by Cd. In this process, Mel inhibits fatty acid production and promotes fatty acid oxidation. Interestingly, Mel regulated PPAR-α expression and alleviated Cd-induced autophagy blockade. In vitro model, the oil Red O staining, and WB results showed that Mel alleviated Cd-induced lipid accumulation. In addition, RAPA was used to activate autophagy to alleviate Cd-induced lipid accumulation, and TG was used to block autophagy flux to aggravate Cd-induced autophagy accumulation. After knocking down PPAR-α, the autophagosome fusion with lysosomes, and autophagic flux was inhibited and increased Cd-induced lipid accumulation. Mel alleviates mitochondrial damage and oxidative stress, and attenuates Cd-induced NAFLD by restoring the expression of PPAR-α and restoring autophagy flux.

[Ketogenic diet improves low temperature tolerance in mice by up-regulating PPARα in the liver and brown adipose tissue].

The aim of the present study was to investigate the effects of short-term ketogenic diet on the low temperature tolerance of mice and the involvement of peroxisome proliferator-activated receptor α (PPARα). C57BL/6J mice were divided into two groups: normal diet (WT+ND) group and ketogenic diet (WT+KD) group. After being fed with normal or ketogenic diet at room temperature for 2 d, the mice were exposed to 4 °C low temperature for 12 h. The changes in core temperature, blood glucose, blood pressure of mice under low temperature condition were detected, and the protein expression levels of PPARα and mitochondrial uncoupling protein 1 (UCP1) were detected by Western blot. PPARα knockout mice were divided into normal diet (PPARα-/-+ND) group and ketogenic diet (PPARα-/-+KD) group. After being fed with the normal or ketogenic diet at room temperature for 2 d, the mice were exposed to 4 °C low temperature for 12 h. The above indicators were also detected. The results showed that, at room temperature, the protein expression levels of PPARα and UCP1 in liver and brown adipose tissue of WT+KD group were significantly up-regulated, compared with those of WT+ND group. Under low temperature condition, compared with WT+ND, the core temperature and blood glucose of WT+KD group were increased, while mean arterial pressure was decreased; The ketogenic diet up-regulated PPARα protein expression in brown adipose tissue, as well as UCP1 protein expression in liver and brown adipose tissue of WT+KD group. Under low temperature condition, compared to WT+ND group, PPARα-/-+ND group exhibited decreased core temperature and down-regulated PPARα and UCP1 protein expression levels in liver, skeletal muscle, white and brown adipose tissue. Compared to the PPARα-/-+ND group, the PPARα-/-+KD group exhibited decreased core temperature and did not show any difference in the protein expression of UCP1 in liver, skeletal muscle, white and brown adipose tissue. These results suggest that the ketogenic diet promotes UCP1 expression by up-regulating PPARα, thus improving low temperature tolerance of mice. Therefore, short-term ketogenic diet can be used as a potential intervention to improve the low temperature tolerance.

Luteolin Mitigates Diabetic Dyslipidemia in Rats by Modulating ACAT-2, PPARα, SREBP-2 Proteins, and Oxidative Stress.

Diabetic dyslipidemia is a crucial link between type-2 diabetes mellitus (T2DM) and atherosclerotic cardiovascular diseases (ASCVD). Natural biologically active substances have been advocated as complementary remedies for ASCVD and T2DM. Luteolin, a flavonoid, exhibits antioxidant, hypolipidemic, and antiatherogenic effects. Hence, we aimed to determine influence of luteolin on lipid homeostasis and hepatic damage in rats with T2DM induced by high-fat-diet (HFD) and streptozotocin (STZ). After being fed HFD for 10 days, male Wistar rats received 40 mg/kg STZ intraperitoneal injection on 11th day. Seventy-two hours later, hyperglycemic rats (fasting glucose > 200 mg/dL) were randomized into groups, and oral hydroxy-propyl-cellulose, atorvastatin (5 mg/kg), or luteolin (50 mg/kg or 100 mg/kg) administered daily, while continuing HFD for 28 days. Luteolin significantly ameliorated dyslipidemia levels and concomitantly improved atherogenic index of plasma in a dose-dependent manner. Increased levels of malondialdehyde and diminished levels of superoxide dismutase, catalase, and glutathione in HFD-STZ-diabetic rats were significantly regulated by luteolin. Luteolin significantly intensified PPARα expression while decreasing expression of acyl-coenzyme A:cholesterol acyltransferase-2 (ACAT-2) and sterol regulatory element binding protein-2 (SREBP-2) proteins. Moreover, luteolin effectively alleviated hepatic impairment in HFD-STZ-diabetic rats to near-normal control levels. The findings of the present study expound mechanisms by which luteolin mitigated diabetic dyslipidemia and alleviated hepatic impairment in HFD-STZ-diabetic rats by amelioration of oxidative stress, modulation of PPARα expression, and downregulation of ACAT-2 and SREBP-2. In conclusion, our results imply that luteolin may be efficacious in management of dyslipidemia in T2DM, and future research may be essential to substantiate our findings.

Regulation of NOX/p38 MAPK/PPARα pathways and miR-155 expression by boswellic acids reduces hepatic injury in experimentally-induced alcoholic liver disease mouse model: novel mechanistic insight.

Alcoholic liver disease (ALD) refers to hepatic ailments induced by excessive alcohol intake. The pathogenesis of ALD comprises a complex interplay between various mechanistic pathways, among which inflammation and oxidative stress are key players. Boswellic acids (BAs), found in Boswellia serrata, have shown hepatoprotective effects owing to their antioxidant and anti-inflammatory activities, nevertheless, their therapeutic potential against ALD has not been previously investigated. Hence, this study was performed to depict the possible protective effect of BAs and detect their underlying mechanism of action in an experimentally-induced ALD mouse model. Male BALB/c mice were equally categorized into six groups: control, BAs-treated, ALD, and ALD that received BAs at three-dose levels (125, 250, and 500 mg/kg) by oral gavage for 14 days. Results showed that the high dose of BAs had the most protective impact against ALD according to histopathology examination, blood alcohol concentration (BAC), and liver function enzymes. Mechanistic investigations revealed that BAs (500 mg/kg) caused a significant decrease in cytochrome P450 2E1(CYP2E1), nicotine adenine dinucleotide phosphate oxidase (NOX) 1/2/4, p38 mitogen-activated protein kinase (MAPK), and sterol regulatory element-binding protein-1c (SREBP-1c) levels, and the expression of miR-155, yet increased peroxisome proliferator-activated receptor alpha (PPARα) levels. This led to an improvement in lipid profile and reduced hepatic inflammation, oxidative stress, and apoptosis indices. In summary, our study concludes that BAs can protect against ethanol-induced hepatic injury, via modulating NOX/p38 MAPK/PPARα pathways and miR-155 expression.

Oxidized corn oil changes the liver lipid metabolism of broilers by upregulating peroxisome proliferators activate receptor-α.

The objective of the following study was to investigate the effects of naturally oxidized corn oil on the antioxidant capacity and lipid metabolism of broilers. A total of 450, 1-day-old Arbor Acres male broilers were randomly divided into 5 treatments with 6 replicate cages and 15 birds/cage. The dietary treatment array consisted of ratios of naturally oxidized corn oil to non-oxidized corn oil from 0:100, 25:75, 50:50, 75:25, and 100:0, respectively. Serum, liver, and abdominal fat samples were taken at 42 d. The results showed that the liver organ index, liver catalase (CAT) activity, malondialdehyde (MDA) content, and the serum aspartate aminotransferase (AST) content had significant quadratic relationships with the ratio of naturally oxidized corn oil (P < 0.05). Inflammatory infiltrating cells appeared in the liver of the 50% and 75% oxidized corn oil group. The percentage of abdominal fat, and serum free fatty acids (FFA) content increased linearly with the increased proportion of oxidized corn oil (P < 0.05). The mRNA expression of NADH quinone oxidoreductase 1 (NQO-1), nuclear factor kappa B (NF-κB), toll-like receptor-4 (TLR-4), peroxisome proliferators activate receptor-α (PPARα), carnitine acyltransferase (CPT1), and acyl-coenzyme oxidase (ACO) of the liver increased linearly while oxidized corn oil increased in the diet (P < 0.05). Diets containing 100% oxidized corn oil significantly changed the mRNA expression of liver Caveolin compared with other treatment groups (P < 0.05). Taken together, this study demonstrated that naturally oxidized corn oil could change liver lipid metabolism and accelerate lipid deposition of broilers by upregulating PPARα.

FNDC5/PPARa Pathway Alleviates THP-1-derived Macrophage Pyroptosis and Its Mechanism.

Context: Atherosclerosis (AS) is a chronic inflammatory disease. Pyroptosis is a newly discovered, pro-inflammatory cell death that can trigger and amplify the occurrence and progression of AS. Researchers are still uncertain about the anti-atherosclerotic mechanism of "fibronectin type III domain-containing protein 5" (FNDC5). Objective: The study aimed to investigate the ability of FNDC5-mediated, "peroxisome proliferator activated receptor alpha" (PPARa) to inhibit oxidized low-density lipoprotein (ox-LDL)-induced, THP-1-derived macrophage pyroptosis and to determine a potential molecular mechanism at the cellular level. Design: The research team performed a laboratory study. Setting: The study took place in the Department of Cardiovascular Medicine at the Affiliated Hospital of Guzhou Medical University at the Medical Research Institute at Guizhou Medical University in Guiyang, Guizhou, China. Outcome Measures: The research team: (1) constructed and stably transfected FNDC5 gene-overexpressing and FNDC5 gene-silencing lentiviral vectors into THP-1 cells; (2) observed the cell morphology under an inverted fluorescence microscope and screened the stably transfected THP-1 cells with puromycin; (3) verified the transfection efficiency using quantitative real-time polymerase chain reaction (qRT-PCR) and Western Blot; (4) used phorbol to induce THP-1 cells into macrophages; (5) cultured the THP-1-derived macrophages with different concentrations of ox-LDL-25, 50, 75, and 100 µg/ml-for 24 h; (6) performed Hoechst 33342/ propidium iodide (PI) double staining and examined lactate dehydrogenase (LDH) and interleukin-1 beta (IL-1ß) activity to determine the effects of ox-LDL on THP-1-derived macrophage pyroptosis; (7) selected the optimal ox-LDL concentration; (8) divided the THP-1-derived macrophages into seven groups: NC group (no ox-LDL intervention), ox-LDL group, PBS group, Mock1 group, Ad-FNDC5 group, Mock2 group, and Sh-FNDC5 group; (9) examined the expressions of functional proteins and the pyroptosis of THP-1-derived macrophages, including FNDC5, PPARa, and "nuclear factor kappa-light chain enhancer of activated B cells P65" (NF-κB P65), and those related to the pyroptosis pathway, using Western Blot and Hoechst 33342/PI double staining, respectively; (10) treated the THP-1-derived macrophages with FNDC5 expression with GW6471, a specific PPARα antagonist; (11) determined the expressions of functional proteins and the pyroptosis of THP-1-derived macrophages, including FNDC5, PPARa, and NF-κB P65, and those related to the pyroptosis pathway, using Western Blot and Hoechst 33342/PI double staining and detection of the LDH and IL-1ß activity, respectively. Results: With the stably transfected THP-1 cells with FNDC5 overexpression or silencing the ox-LDL-induced, THP-1-derived, macrophage pyroptosis occurred in a concentration-dependent manner. Compared with the ox-LDL, phosphate buffered saline (PBS), Mock1, and Mock2 groups, the Ad-FNDC5 group had a significant increase in expression of FNDC5 and of peroxisome proliferator activated receptor alpha (PPARa) proteins (P < .05). The "nuclear factor kappa-light chain enhancer of activated B cells P65: (NF-κB P65), NOD-like receptor thermal protein domain associated protein 3, (NLRP3), Caspase-1, gasdermin D (GSDMD, IL-1ß and IL-18 protein expressions, percentage of PI-positive cells, LDH activity, and IL-1ß activity decreased significantly (P < .05); the results in the Sh-FNDC5 group were opposite to those in the Ad-FNDC5 group. 3. Intervention with GW6471 (PPARa antagonist) in the stably transfected THP-1-derived macrophages with FNDC5 overexpression abolished the protective effect of FNDC5 against ox-LDL-induced THP-1-derived macrophage pyroptosis. Conclusions: Irisin/PPARa inhibited THP-1-derived macrophage pyroptosis and inflammation and delayed AS by inhibiting the NF-κB/NLRP3 pathway.

Protective effect of Qingluotongbi formula against Tripterygium wilfordii induced liver injury in mice by improving fatty acid ß-oxidation and mitochondrial biosynthesis.

CONTEXT: Qingluotongbi formula (QLT) is a Chinese medicine compound consisting of Tripterygium wilfordii Hook. f. (Celastraceae, TW), Panax notoginseng (Burkill) F.H.Chen (Araliaceae, PN), Rehmannia glutinosa (Gaertn.) DC. (Orobanchaceae, RG), Sinomenium acutum (Thunb.) Rehder & E.H. Wilson (Menispermaceae, SA), and Bombyx mori L. (Bombycidae, BM). OBJECTIVE: This study investigated the protective effect and possible mechanism of QLT against TW-induced liver injury in mice. MATERIALS AND METHODS: To establish the model of TW-induced liver injury in mice, C57BL/6J mice were randomly divided into 4 groups: control group, low-dose TW group, middle-dose TW group, and high-dose TW group. To observe the effects of QLT and its individual ingredients against TW-induced liver injury, C57BL/6J mice were randomly divided into 7 groups: control group, TW group, QLT group, PN group, RG group, SA group, BM group.After administration for 7 days, C57BL/6J mice were tested for biochemical indicators and liver pathological changes. Then, we evaluated the mitochondrial function and analysed the gene and protein expression related to the peroxisome proliferator-activated receptor alpha (PPARα)/peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) pathway by quantitative real-time PCR (qRT-PCR) and Western blotting. RESULTS: Compared with the control group (0.30 ± 0.35), TW significantly increased mice liver histological score (L, 0.95 ± 1.14; M, 1.25 ± 1.16; H, 4.00 ± 1.13). QLT and its ingredients significantly improved the pathology scores (CON, 0.63 ± 0.74; TW, 4.19 ± 1.53; QLT, 1.56 ± 0.62; PN, 1.94 ± 0.68; RG, 2.75 ± 1.39; SA, 4.13 ± 0.99; BM, 4.13 ± 0.99). Western blot and qRT-PCR analysis revealed that QLT and its ingredients reversed TW-induced suppression of PPARα/PGC1-α pathway.Discussion and conclusions: These findings provide valuable information for compound compatibility studies and TW clinical applications.

Ketogenic diet improves low temperature tolerance in mice by up-regulating PPARα in the liver and brown adipose tissue / 生理学报

The aim of the present study was to investigate the effects of short-term ketogenic diet on the low temperature tolerance of mice and the involvement of peroxisome proliferator-activated receptor α (PPARα). C57BL/6J mice were divided into two groups: normal diet (WT+ND) group and ketogenic diet (WT+KD) group. After being fed with normal or ketogenic diet at room temperature for 2 d, the mice were exposed to 4 °C low temperature for 12 h. The changes in core temperature, blood glucose, blood pressure of mice under low temperature condition were detected, and the protein expression levels of PPARα and mitochondrial uncoupling protein 1 (UCP1) were detected by Western blot. PPARα knockout mice were divided into normal diet (PPARα-/-+ND) group and ketogenic diet (PPARα-/-+KD) group. After being fed with the normal or ketogenic diet at room temperature for 2 d, the mice were exposed to 4 °C low temperature for 12 h. The above indicators were also detected. The results showed that, at room temperature, the protein expression levels of PPARα and UCP1 in liver and brown adipose tissue of WT+KD group were significantly up-regulated, compared with those of WT+ND group. Under low temperature condition, compared with WT+ND, the core temperature and blood glucose of WT+KD group were increased, while mean arterial pressure was decreased; The ketogenic diet up-regulated PPARα protein expression in brown adipose tissue, as well as UCP1 protein expression in liver and brown adipose tissue of WT+KD group. Under low temperature condition, compared to WT+ND group, PPARα-/-+ND group exhibited decreased core temperature and down-regulated PPARα and UCP1 protein expression levels in liver, skeletal muscle, white and brown adipose tissue. Compared to the PPARα-/-+ND group, the PPARα-/-+KD group exhibited decreased core temperature and did not show any difference in the protein expression of UCP1 in liver, skeletal muscle, white and brown adipose tissue. These results suggest that the ketogenic diet promotes UCP1 expression by up-regulating PPARα, thus improving low temperature tolerance of mice. Therefore, short-term ketogenic diet can be used as a potential intervention to improve the low temperature tolerance.

Capparis spinosa improves non-alcoholic steatohepatitis through down-regulating SREBP-1c and a PPARα-independent pathway in high-fat diet-fed rats.

OBJECTIVE: Non-alcoholic steatohepatitis (NASH) has become a global medical problem. Currently, there is no approved pharmacologic treatment for this condition. Previous studies have suggested that in the pathogenesis of this disease, regulatory pathways associated with de novo lipogenesis and ß-oxidation pathways genes are misregulated. Capparis spinosa (CS) belongs to the family of Capparidaceae and is a traditional plant used to treat various diseases, particularly dyslipidemia. The compounds and extracts of this plant in In vivo and in vitro studies resulted in a reduction in lipid profiles and glucose. However, the mechanism of these effects remains unknown. This study aimed to evaluate the effects of (CS) fruit extract on NASH compared to fenofibrate and explored the related molecular mechanism. RESULTS: In the rats (n = 40) model of NASH, biochemical and histopathological examinations showed that liver steatosis, inflammation, and hepatic fibrosis were markedly attenuated in response to CS and fenofibrate interventions. At the molecular level, CS treatment down-regulated sterol regulatory element-binding protein-1c (SREBP-1c) (p < 0.001), acetyl-CoA carboxylase (ACC) (p < 0.001), and up-regulated Carnitine palmitoyltransferase I (CPT1) expression (p < 0.001). In conclusion, CS has favorable therapeutic effects for NASH, which was associated with ameliorating steatosis and fibrosis via regulation of the DNL and ß-oxidation pathway genes.

Melatonin Ameliorates Cisplatin-Induced Renal Tubular Epithelial Cell Damage through PPARα/FAO Regulation.

Previous studies revealed that melatonin ameliorated acute renal injury induced by cisplatin, but the mechanisms remain unclear. Peroxidase proliferative receptor α (PPARα) is considered the major regulator of fatty acid oxidation (FAO), which is an important source of energy for renal tubular epithelial cells. In this study, the aim was to investigate the role of melatonin in cisplatin-induced NRK-52E (rat renal tubular epithelial cell line) cell damage and the underlying mechanisms. We established a cisplatin-stimulated NRK-52E model in vitro. We assessed the levels of apoptotic proteins, including caspase-3, caspase-9, and B-cell lymphoma 2-associated X protein (Bax), as well as PPARα and FAO-related genes (Acadm, Acat1, Acsm2, Acsm3, PGC-1α, Pecr, Bdh2, and Echs1). Furthermore, we detected the effects of miR-21 and PPARα antagonist on the above indicators. We found that melatonin reduced the protein expression levels of caspase-3, caspase-9, and Bax, and increased the expression levels of the PPARα gene and protein and PPARα activity, as well as FAO-related genes, in NRK-52E cells. However, miR-21 mimics and PPARα antagonists partially antagonized the above effects of melatonin. Our data indicated that melatonin could alleviate cisplatin-induced cell damage through the upregulation of PPARα/FAO.

Protective effects of monoammonium glycyrrhizinate on fatty deposit degeneration induced in primary calf hepatocytes by sodium oleate administration in vitro.

In this study we investigated the effect of MAG on fatty deposit-induced degeneration of primary calf hepatocytes induced by sodium oleate. Primary hepatocytes were isolated from dairy calves and cultured before allocation to the following treatment groups: control (untreated), model (starved for 12 h before treatment with 0.25 mM sodium oleate to induce steatosis-like changes, and the MAG group pretreated with MAG (0.1, 0.25, 0.5, and 1.5 mM) for 12 h before sodium oleate treatment (0.25 mM) for 12 h). To evaluate the effect of MAG on fat-induced degradation of primary hepatocytes, we evaluated lipid deposition, cell viability, apoptosis rate, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity in the culture supernatant, and expression of oxidant and antioxidant enzymes (LDH, MDA, GSH, and CAT), as well as the expression of lipid metabolism-related genes (PPARα, SREBP-1c, ChREBP, CPT1, CPT2, and MTP) and apoptosis-related genes (Cyt-c, Caspase 9, Caspase 8, Caspase 3, Bax, and Bcl-2). MAG significantly reduced lipid accumulation in hepatocytes induced by sodium oleate (P < 0.05), increased cell viability, decreased the apoptosis rate (P < 0.05), and significantly decreased ALT and AST activity in the culture supernatant (P < 0.05). MAG significantly decreased MDA levels in cells and LDH levels in the culture supernatant, while GSH and CAT levels were increased (P < 0.05). MAG significantly increased the expression of the lipid transport- and metabolism-related genes MTP, PPARα, CPT1 and CPT2, and decreased ChREBP expression (P < 0.05). At concentrations higher than 0.25 mM, MAG significantly decreased SREBP-1c expression (P < 0.05). MAG significantly decreased the expression of the apoptosis-related genes Cyt-c, Caspase 9, Caspase 8, Caspase3 and Bax, while Bcl-2 expression was increased (P < 0.05). These findings demonstrate that MAG improves the antioxidant capacity of hepatocytes and effectively reduces lipid deposition by inhibiting the expression of lipid metabolism- and apoptosis-related genes.