Five Constituents Contributed to the Psoraleae Fructus-Induced Hepatotoxicity via Mitochondrial Dysfunction and Apoptosis

Backgrounds: Psoraleae Fructus (PF)-induced hepatotoxicity has been reported in clinical and animal experiments. However, the hepatotoxic constituents and mechanisms underlying PF-induced toxicity have remained unclear. Therefore, this study explored the potentially toxic PF components and revealed their relative mechanisms.Methods: The hepatotoxicity of PF water (PFW) and ethanol (PFE) extracts was compared using Kunming mice. The different compositions between PFW and PFE, which were considered toxic compositions, were identified using the UHPLC-Q-Exactive MS method. Then, L02 and HepG2 cell lines were used to evaluate the toxicity of these compositions. Cell viability and apoptosis were determined through the Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. An automatic biochemical analyzer detected the aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). Lastly, we used high-content screening (HCS) to determine the levels of reactive oxygen species (ROS), lipid, and mitochondrial membrane potential (MMP).Results: The ethanol extraction process aggravated the hepatotoxicity of PF, causing more severe injuries. The content of psoralen, isopsoralen, bavachin, psoralidin, bavachinin, neobavaisoflavone, and bakuchiol was higher in the PFE than PFW. Bavachin, psoralidin, bavachinin, neobavaisoflavone, and bakuchiol induced cell apoptosis and the AST, ALT, and ALP leakages. Furthermore, these five constituents increased intracellular lipid accumulation and ROS levels but decreased the MMP level.Conclusion: The ethanol extraction process could induce severe PF hepatotoxicity. Bavachin, psoralidin, bavachinin, neobavaisoflavone, and bakuchiol are the main hepatotoxic ingredients. This mechanism could be associated with oxidative stress and mitochondrial damage-mediated apoptosis. Taken together, this study provides a basis for the clinical application of PF that formulates and improves its herbal standards.

HO-3867 Induces ROS-Dependent Stress Response and Apoptotic Cell Death in Leishmania donovani

Lack of vaccine and increasing chemotherapeutic toxicities currently necessitate the development of effective and safe drugs against various forms of leishmaniases. We characterized the cellular stress induced by a novel curcumin analogue, HO-3867, encapsulated within the phosphatidylcholine-stearylamine (PC-SA) liposome for the first time against Leishmania. The liposomal formulation of HO-3867 (i.e., PC-SA/HO-3867) initiated oxidative stress-induced apoptosis in L. donovani, revealed by altered cell morphology, phosphatidylserine externalization, mitochondrial depolarization, intracellular lipid accumulation, and cell cycle arrest in promastigotes. Liposomal HO-3867 was observed to be a strong apoptosis inducer in L. donovani and L. major in a dose-dependent manner, yet completely safe for normal murine macrophages. Moreover, PC-SA/HO-3867 treatment induced L. donovani metacaspase and PARP1 activation along with downregulation of the Sir2 gene. PC-SA/HO-3867 arrested intracellular L. donovani amastigote burden in vitro, with reactive oxygen species (ROS) and nitric oxide (NO)-mediated parasite killing. These data suggest that liposomal HO-3867 represents a highly promising and non-toxic nanoparticle-based therapeutic platform against leishmaniasis inspiring further preclinical developments.

Characterization of cellular development and fatty acid accumulation in thraustochytrid Aurantiochytrium strains of Taiwan

Long-chain saturated and polyunsaturated fatty acids of two new thraustochytrid isolates cultured from Taiwan mangroves, Aurantiochytrium sp. IMB169 and Aurantiochytrium sp. IMB171, were characterized through their cell growth and development in relation to their intracellular lipid accumulation using transmission electron microscopy. Flow cytometry in combination with the lipophilic fluorescent dye BODIPY 505/515 was used to stain and characterize intracellular lipid bodies in the two isolates. The transmission electron microscopy and flow cytometry analyses revealed a progressive accumulation of lipid products in IMB169 and IMB171. Further, selective BODIPY stained cells were successfully separated and enriched using flow cytometry at single cell level. Among the two isolates, IMB169 was found to produce a high level of docosahexaenoic acid. The qualitative and analytical results obtained using electron microscopy and flow cytometry studies were validated by gas chromatography (GC). In addition, a quantitative baseline was established using cell growth, flow cytometry and GC analyses for developing an efficient bioprocessing methodology to selectively enrich thraustochytrids phenotypes with desirable characteristics.

Honokiol attenuates lipotoxicity in hepatocytes via activating SIRT3-AMPK mediated lipophagy

Background Non-alcoholic fatty liver disease (NAFLD) is characterized by ectopic accumulation of triglycerides in the liver. Emerging evidence has demonstrated that lipophagy regulates lipid mobilization and energy homeostasis in the liver. Sirtuin 3 (SIRT3), a mitochondrial NAD+-dependent deacetylase, modulates the activities of several substrates involving in autophagy and energy metabolism. Honokiol (HK) is a natural lignan from the plants of Magnolia genus that exhibits potent liver protective property. Methods AML12 was challenged with 500 μM palmitic acid and 250 μM oleic acid mixture solution to induce lipotoxicity. C57BL/6J mice were fed with a choline-deficient high fat diet (CDHFD) to generate liver steatosis. The expression of autophagy-related and AMP-activated protein kinase (AMPK) pathway proteins was evaluated by Western blotting and immunofluorescence staining. Intracellular lipid accumulation was validated by Nile red staining. Molecular docking analysis was performed on AutoDock 4.2. Results HK (5 and 10 μM) was found to attenuate lipid accumulation through promoting SIRT3-AMPK-mediated autophagy, mainly on lipid droplets. HK had hydrophobic interaction with amino acid residues (PHE294, GLU323 and VAL324) and NAD+. Moreover, HK improved mitochondrial function to enhance lipolysis, through decreasing the acetylated long-chain acyl-CoA dehydrogenase level. In CDHFD-fed mice, HK (2.5 and 10 mg/Kg) treatment obviously prevented lipid accumulation in the liver. And co-treatment of the AMPK inhibitor, Compound C, almost abolished the above changes. Conclusions These results suggest that HK could ameliorate lipotoxicity in hepatocytes by activating SIRT3-AMPK-lipophagy axis, which might be a potential therapeutic agent against NAFLD.

Curcumin Conjugated Gold Nanoclusters as Perspective Therapeutics for Diabetic Cardiomyopathy

Accumulation of lipids in the myocardium contributes to the development of cardiac dysfunctions and various chronic diseases, such as diabetic cardiomyopathy (DCM). Curcumin (Cur) can relieve lipid accumulation problems, but its efficiency is limited by poor water solubility and biocompatibility. Herein, gold nanoclusters (AuNCs) were used to improve the efficiency of Cur, and the conjugates Curcumin-AuNCs (AuCur) were developed. In the treatment of high-fat-induced myocardial cell damage, we found that AuCur could effectively reduce intracellular lipid accumulation, the increase of reactive oxygen species (ROS), the increase of mitochondrial division, and the increase of apoptosis compared with Cur. AuCur decreased the expression of the peroxisome proliferator-activated receptors-α subtype (PPARα), and the therapeutic effect of AuCur was canceled when the expression of PPARα was enhanced. For the above reasons, AuCur treated the toxic effect of high lipid on cardiomyocytes by regulating PPARα, providing a new idea and method for the treatment of DCM.

Overexpression of lipA or glpD_RuBisCO in the Synechocystis sp. PCC 6803 Mutant Lacking the Aas Gene Enhances Free Fatty-Acid Secretion and Intracellular Lipid Accumulation

Although engineered cyanobacteria for the production of lipids and fatty acids (FAs) are intelligently used as sustainable biofuel resources, intracellularly overproduced FAs disturb cellular homeostasis and eventually generate lethal toxicity. In order to improve their production by enhancing FFAs secretion into a medium, we constructed three engineered Synechocystis 6803 strains including KA (a mutant lacking the aas gene), KAOL (KA overexpressing lipA, encoding lipase A in membrane lipid hydrolysis), and KAOGR (KA overexpressing quadruple glpD/rbcLXS, related to the CBB cycle). Certain contents of intracellular lipids and secreted FFAs of all engineered strains were higher than those of the wild type. Remarkably, the KAOL strain attained the highest level of secreted FFAs by about 21.9%w/DCW at day 5 of normal BG11 cultivation, with a higher growth rate and shorter doubling time. TEM images provided crucial evidence on the morphological changes of the KAOL strain, which accumulated abundant droplets on regions of thylakoid membranes throughout the cell when compared with wild type. On the other hand, BG11-N condition significantly induced contents of both intracellular lipids and secreted FFAs of the KAOL strain up to 37.2 and 24.5%w/DCW, respectively, within 5 days. Then, for the first time, we shone a spotlight onto the overexpression of lipA in the aas mutant of Synechocystis as another potential strategy to achieve higher FFAs secretion with sustainable growth.

Intracellular Lipid Accumulation and Mitochondrial Dysfunction Accompanies Endoplasmic Reticulum Stress Caused by Loss of the Co-chaperone DNAJC3

Recessive mutations in DNAJC3, an endoplasmic reticulum (ER)-resident BiP co-chaperone, have been identified in patients with multisystemic neurodegeneration and diabetes mellitus. To further unravel these pathomechanisms, we employed a non-biased proteomic approach and identified dysregulation of several key cellular pathways, suggesting a pathophysiological interplay of perturbed lipid metabolism, mitochondrial bioenergetics, ER-Golgi function, and amyloid-beta processing. Further functional investigations in fibroblasts of patients with DNAJC3 mutations detected cellular accumulation of lipids and an increased sensitivity to cholesterol stress, which led to activation of the unfolded protein response (UPR), alterations of the ER-Golgi machinery, and a defect of amyloid precursor protein. In line with the results of previous studies, we describe here alterations in mitochondrial morphology and function, as a major contributor to the DNAJC3 pathophysiology. Hence, we propose that the loss of DNAJC3 affects lipid/cholesterol homeostasis, leading to UPR activation, β-amyloid accumulation, and impairment of mitochondrial oxidative phosphorylation.

Posidonia oceanica (L.) Delile Extract Reduces Lipid Accumulation through Autophagy Activation in HepG2 Cells

Posidonia oceanica (L.) Delile is a marine plant traditionally used as an herbal medicine for various health disorders. P. oceanica leaf extract (POE) has been shown to be a phytocomplex with cell-safe bioactivities, including the ability to trigger autophagy. Autophagy is a key pathway to counteract non-alcoholic fatty liver disease (NAFLD) by controlling the breakdown of lipid droplets in the liver. The aim of this study was to explore the ability of POE to trigger autophagy and reduce lipid accumulation in human hepatoma (HepG2) cells and then verify the possible link between the effect of POE on lipid reduction and autophagy activation. Expression levels of autophagy markers were monitored by the Western blot technique in POE-treated HepG2 cells, whereas the extent of lipid accumulation in HepG2 cells was assessed by Oil red O staining. Chloroquine (CQ), an autophagy inhibitor, was used to study the relationship between POE-induced autophagy and intracellular lipid accumulation. POE was found to stimulate an autophagy flux over time in HepG2 cells by lowering the phosphorylation state of ribosomal protein S6, increasing Beclin-1 and LC3-II levels, and decreasing p62 levels. By blocking autophagy with CQ, the effect of POE on intracellular lipid accumulation was clearly reversed, suggesting that the POE phytocomplex may reduce lipid accumulation in HepG2 cells by activating the autophagic process. This work indicates that P. oceanica may be considered as a promising molecule supplier to discover new natural approaches for the management of NAFLD.

Influencing Factors of HLA-DR Expression in Orbital Preadipocytes of Patients With Thyroid Associated Ophthalmopathy

Purpose: To investigate the effects of IFN-γ, IL-6, PPAR-γ agonist pioglitazone and PPAR-γ antagonist GW9662 on preadipocyte proliferation and HLA-DR expression.Methods: Preadipocytes were cultured from thyroid associated ophthalmopathy patients and health ocular trauma patients and induced to differentiate in vitro. IFN-γ, IL-6, PPAR-γ agonist pioglitazone and PPAR-γ antagonist GW9662 were added to preadipocytes to observe their effects on proliferation and HLA-DR expression.Results: IFN-γ significantly increased the levels of HLA-DR mRNA and protein in preadipocytes which are cultured from thyroid associated ophthalmopathy patients. IFN-γ also reduced the accumulation of lipid in the cytoplasm of preadipocytes. On the contrary, IL-6 had no effect on the expression of HLA-DR. Pioglitazone increased the expression of HLA-DR in orbital preadipocytes of TAO patients, and promoted the proliferation and intracellular lipid accumulation of preadipocytes. On the contrary, GW9662 decreased the expression of HLA-DR in orbital preadipocytes of TAO patients, and reduced the proliferation and intracellular lipid accumulation of preadipocytes.Conclusion: IFN-γ increase the expression of HLA-DR in TAO orbital preadipocytes after differentiation. PPAR - γ antagonists may be beneficial to the treatment of TAO.

Honokiol Attenuates Lipotoxicity in Hepatocytes via Activating SIRT3-AMPK Mediated Lipophagy

Background: Non-alcoholic fatty liver disease (NAFLD) is characterized by ectopic accumulation of triglycerides in the liver. Emerging evidence has demonstrated that lipophagy regulates lipid mobilization and energy homeostasis in liver. Sirtuin 3 (SIRT3), a mitochondrial NAD+-dependent deacetylase, modulates the activities of several substrates involving in autophagy and energy metabolism. Honokiol (HK) is a natural lignan from the plants of Magnolia genus that exhibits potent liver protective property. Methods: AML12 was challenged with 500 μM palmitic acid and 250 μM oleic acid mixture solution to induce lipotoxicity. The expression of autophagy-related and AMP-activated protein kinase (AMPK) pathway proteins was evaluated by Western blotting and immunofluorescence staining. Intracellular lipid accumulation was validated by Nile red staining. Molecular docking analysis was performed on AutoDock 4.2.Results: HK (5 and 10 μM) was found to attenuate lipid accumulation through promoting SIRT3-AMPK-mediated autophagy, mainly on lipid droplets. HK had hydrophobic interaction with amino acid residues (PHE294, GLU323 and VAL324) and NAD+. Moreover, HK improved mitochondrial function to enhance lipolysis, through decreasing the acetylated long-chain acyl-CoA dehydrogenase level. Conclusions: These results suggest that HK could ameliorate lipotoxicity in hepatocytes by activating SIRT3-AMPK-lipophagy axis, which might be a potential therapeutic agent against NAFLD.