Myostatin Knockout Affects Mitochondrial Function by Inhibiting the AMPK/SIRT1/PGC1α Pathway in Skeletal Muscle

Simple SummaryMyostatin (Mstn) is a negative regulator of skeletal muscle mass, and its deletion leads to reduced mitochondrial function. However, the exact regulatory mechanism remains unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. The skeletal muscle of Mstn-KO mice significantly increased, and the basal metabolic rate, muscle ATP synthesis, mitochondrial respiratory chain complex activity, tricarboxylic acid cycle (TCA), and thermogenesis decreased. In the muscle tissue of Mstn-KO mice, the expression of SIRT1 and pAMPK decreased, and the acetylation modification of PGC-1α increased. Furthermore, the treatment of isolated muscle cells from Mstn-KO and wild-type mice with AMPK activator (AICAR) and AMPK inhibitor (Compound C) found that Compound C down-regulated the expression of pAMPK and SIRT1 and the activity of citrate synthase (CS), isocitrate dehydrogenase (ICDHm) and α-ketoglutarate acid dehydrogenase (α-KGDH) similar to that of Mstn-KO. However, AICAR partially reversed the inhibitory effect of Mstn-KO on the expression of pAMPK and SIRT1 and activity of three enzymes. Thus, Mstn-KO affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway.AbstractMyostatin (Mstn) is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes. The deletion of the Mstn gene in mice leads to reduced mitochondrial functions. However, the underlying regulatory mechanisms remain unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. Mstn-KO mice exhibited significantly larger skeletal muscles. Meanwhile, Mstn knockout regulated the organ weights of mice. Moreover, we found that Mstn knockout reduced the basal metabolic rate, muscle adenosine triphosphate (ATP) synthesis, activities of mitochondrial respiration chain complexes, tricarboxylic acid cycle (TCA) cycle, and thermogenesis. Mechanistically, expressions of silent information regulator 1 (SIRT1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were down-regulated, while peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) acetylation modification increased in the Mstn-KO mice. Skeletal muscle cells from Mstn-KO and WT were treated with AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR), and the AMPK inhibitor Compound C, respectively. Compared with the wild-type (WT) group, Compound C treatment further down-regulated the expression or activity of pAMPK, SIRT1, citrate synthase (CS), isocitrate dehydrogenase (ICDHm), and α-ketoglutarate acid dehydrogenase (α-KGDH) in Mstn-KO mice, while Mstn knockout inhibited the AICAR activation effect. Therefore, Mstn knockout affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway. The present study reveals a new mechanism for Mstn knockout in regulating energy homeostasis.     

Short Chain Fatty Acid Acetate Increases TNFα-Induced MCP-1 Production in Monocytic Cells via ACSL1/MAPK/NF-κB Axis

Short-chain fatty acid (SCFA) acetate, a byproduct of dietary fiber metabolism by gut bacteria, has multiple immunomodulatory functions. The anti-inflammatory role of acetate is well documented; however, its effect on monocyte chemoattractant protein-1 (MCP-1) production is unknown. Similarly, the comparative effect of SCFA on MCP-1 expression in monocytes and macrophages remains unclear. We investigated whether acetate modulates TNFα-mediated MCP-1/CCL2 production in monocytes/macrophages and, if so, by which mechanism(s). Monocytic cells were exposed to acetate with/without TNFα for 24 h, and MCP-1 expression was measured. Monocytes treated with acetate in combination with TNFα resulted in significantly greater MCP-1 production compared to TNFα treatment alone, indicating a synergistic effect. On the contrary, treatment with acetate in combination with TNFα suppressed MCP-1 production in macrophages. The synergistic upregulation of MCP-1 was mediated through the activation of long-chain fatty acyl-CoA synthetase 1 (ACSL1). However, the inhibition of other bioactive lipid enzymes [carnitine palmitoyltransferase I (CPT I) or serine palmitoyltransferase (SPT)] did not affect this synergy. Moreover, MCP-1 expression was significantly reduced by the inhibition of p38 MAPK, ERK1/2, and NF-κB signaling. The inhibition of ACSL1 attenuated the acetate/TNFα-mediated phosphorylation of p38 MAPK, ERK1/2, and NF-κB. Increased NF-κB/AP-1 activity, resulting from acetate/TNFα co-stimulation, was decreased by ACSL1 inhibition. In conclusion, this study demonstrates the proinflammatory effects of acetate on TNF-α-mediated MCP-1 production via the ACSL1/MAPK/NF-κB axis in monocytic cells, while a paradoxical effect was observed in THP-1-derived macrophages.     

CRISPR/Cas9-Mediated Knockout of GmFATB1 Significantly Reduced the Amount of Saturated Fatty Acids in Soybean Seeds

Soybean (Glycine max) oil is one of the most widely used vegetable oils across the world. Breeding of soybean to reduce the saturated fatty acid (FA) content, which is linked to cardiovascular disease, would be of great significance for nutritional improvement. Acyl-acyl carrier protein thioesterases (FATs) can release free FAs and acyl-ACP, which ultimately affects the FA profile. In this study, we identified a pair of soybean FATB coding genes, GmFATB1a and GmFATB1b. Mutants that knock out either or both of the GmFATB1 genes were obtained via CRISPR/Cas9. Single mutants, fatb1a and fatb1b, showed a decrease in leaf palmitic and stearic acid contents, ranging from 11% to 21%. The double mutant, fatb1a:1b, had a 42% and 35% decrease in palmitic and stearic acid content, displayed growth defects, and were male sterility. Analysis of the seed oil profile revealed that fatb1a and fatb1b had significant lower palmitic and stearic acid contents, 39–53% and 17–37%, respectively, while that of the unsaturated FAs were the same. The relative content of the beneficial FA, linoleic acid, was increased by 1.3–3.6%. The oil profile changes in these mutants were confirmed for four generations. Overall, our data illustrate that GmFATB1 knockout mutants have great potential in improving the soybean oil quality for human health.     

New Therapeutic Targets for Hepatic Fibrosis in the Integrin Family, α8β1 and α11β1, Induced Specifically on Activated Stellate Cells

A huge effort has been devoted to developing drugs targeting integrins over 30 years, because of the primary roles of integrins in the cell-matrix milieu. Five αv-containing integrins, in the 24 family members, have been a central target of fibrosis. Currently, a small molecule against αvβ1 is undergoing a clinical trial for NASH-associated fibrosis as a rare agent aiming at fibrogenesis. Latent TGFβ activation, a distinct talent of αv-integrins, has been intriguing as a therapeutic target. None of the αv-integrin inhibitors, however, has been in the clinical market. αv-integrins commonly recognize an Arg-Gly-Asp (RGD) sequence, and thus the pharmacophore of inhibitors for the 5-integrins is based on the same RGD structure. The RGD preference of the integrins, at the same time, dilutes ligand specificity, as the 5-integrins share ligands containing RGD sequence such as fibronectin. With the inherent little specificity in both drugs and targets, “disease specificity” has become less important for the inhibitors than blocking as many αv-integrins. In fact, an almighty inhibitor for αv-integrins, pan-αv, was in a clinical trial. On the contrary, approved integrin inhibitors are all specific to target integrins, which are expressed in a cell-type specific manner: αIIbβ3 on platelets, α4β1, α4β7 and αLβ2 on leukocytes. Herein, “disease specific” integrins would serve as attractive targets. α8β1 and α11β1 are selectively expressed in hepatic stellate cells (HSCs) and distinctively induced upon culture activation. The exceptional specificity to activated HSCs reflects a rather “pathology specific” nature of these new integrins. The monoclonal antibodies against α8β1 and α11β1 in preclinical examinations may illuminate the road to the first medical agents.     

Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid β-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal β-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid β-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid β-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.     

Mutation of GmIPK1 Gene Using CRISPR/Cas9 Reduced Phytic Acid Content in Soybean Seeds

Phytic acid (PA) acts as an antinutrient substance in cereal grains, disturbing the bioavailability of micronutrients, such as iron and zinc, in humans, causing malnutrition. GmIPK1 encodes the inositol 1,3,4,5,6-pentakisphosphate 2-kinase enzyme, which converts myo-inopsitol-1,3,4,5,6-pentakisphosphate (IP₅) to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP₆) in soybean (Glycine max L.). In this study, for developing soybean with low PA levels, we attempted to edit the GmIPK1 gene using the CRISPR/Cas9 system to introduce mutations into the GmIPK1 gene with guide RNAs in soybean (cv. Kwangankong). The GmIPK1 gene was disrupted using the CRISPR/Cas9 system, with sgRNA-1 and sgRNA-4 targeting the second and third exon, respectively. Several soybean Gmipk1 gene-edited lines were obtained in the T₀ generation at editing frequencies of 0.1–84.3%. Sequencing analysis revealed various indel patterns with the deletion of 1–9 nucleotides and insertions of 1 nucleotide in several soybean lines (T₀). Finally, we confirmed two sgRNA-4 Gmipk1 gene-edited homozygote soybean T₁ plants (line #21-2: 5 bp deletion; line #21-3: 1 bp insertion) by PPT leaf coating assay and PCR analysis. Analysis of soybean Gmipk1 gene-edited lines indicated a reduction in PA content in soybean T₂ seeds but did not show any defects in plant growth and seed development.     

Korean Red Ginseng Improves Astrocytic Mitochondrial Function by Upregulating HO-1-Mediated AMPKα–PGC-1α–ERRα Circuit after Traumatic Brain Injury

Heme oxygenase-1 (HO-1) exerts beneficial effects, including angiogenesis and energy metabolism via the peroxisome proliferator-activating receptor-γ coactivator-1α (PGC-1α)–estrogen-related receptor α (ERRα) pathway in astrocytes. However, the role of Korean red ginseng extract (KRGE) in HO-1-mediated mitochondrial function in traumatic brain injury (TBI) is not well-elucidated. We found that HO-1 was upregulated in astrocytes located in peri-injured brain regions after a TBI, following exposure to KRGE. Experiments with pharmacological inhibitors and target-specific siRNAs revealed that HO-1 levels highly correlated with increased AMP-activated protein kinase α (AMPKα) activation, which led to the PGC-1α-ERRα axis-induced increases in mitochondrial functions (detected based on expression of cytochrome c oxidase subunit 2 (MTCO2) and cytochrome c as well as O₂ consumption and ATP production). Knockdown of ERRα significantly reduced the p-AMPKα/AMPKα ratio and PGC-1α expression, leading to AMPKα–PGC-1α–ERRα circuit formation. Inactivation of HO by injecting the HO inhibitor Sn(IV) protoporphyrin IX dichloride diminished the expression of p-AMPKα, PGC-1α, ERRα, MTCO2, and cytochrome c in the KRGE-administered peri-injured region of a brain subjected to TBI. These data suggest that KRGE enhanced astrocytic mitochondrial function via a HO-1-mediated AMPKα–PGC-1α–ERRα circuit and consequent oxidative phosphorylation, O₂ consumption, and ATP production. This circuit may play an important role in repairing neurovascular function after TBI in the peri-injured region by stimulating astrocytic mitochondrial biogenesis.     

Effect of Overexpression of γ-Tocopherol Methyltransferase on α-Tocopherol and Fatty Acid Accumulation and Tolerance to Salt Stress during Seed Germination in Brassica napus L.

Rapeseed (Brassica napus L.) is an important oil crop and a major source of tocopherols, also known as vitamin E, in human nutrition. Enhancing the quality and composition of fatty acids (FAs) and tocopherols in seeds has long been a target for rapeseed breeding. The gene γ-Tocopherol methyltransferase (γ-TMT) encodes an enzyme catalysing the conversion of γ-tocopherol to α-tocopherol, which has the highest biological activity. However, the genetic basis of γ-TMT in B. napus seeds remains unclear. In the present study, BnaC02.TMT.a, one paralogue of Brassica napus γ-TMT, was isolated from the B. napus cultivar “Zhongshuang11” by nested PCR, and two homozygous transgenic overexpression lines were further characterised. Our results demonstrated that the overexpression of BnaC02.TMT.a mediated an increase in the α- and total tocopherol content in transgenic B. napus seeds. Interestingly, the FA composition was also altered in the transgenic plants; a reduction in the levels of oleic acid and an increase in the levels of linoleic acid and linolenic acid were observed. Consistently, BnaC02.TMT.a promoted the expression of BnFAD2 and BnFAD3, which are involved in the biosynthesis of polyunsaturated fatty acids during seed development. In addition, BnaC02.TMT.a enhanced the tolerance to salt stress by scavenging reactive oxygen species (ROS) during seed germination in B. napus. Our results suggest that BnaC02.TMT.a could affect the tocopherol content and FA composition and play a positive role in regulating the rapeseed response to salt stress by modulating the ROS scavenging system. This study broadens our understanding of the function of the Bnγ-TMT gene and provides a novel strategy for genetic engineering in rapeseed breeding.     

Validation of Promoters and Codon Optimization on CRISPR/Cas9-Engineered Jurkat Cells Stably Expressing αRep4E3 for Interfering with HIV-1 Replication

Persistent and efficient therapeutic protein expression in the specific target cell is a significant concern in gene therapy. The controllable integration site, suitable promoter, and proper codon usage influence the effectiveness of the therapeutic outcome. Previously, we developed a non-immunoglobulin scaffold, alpha repeat protein (αRep4E3), as an HIV-1 RNA packaging interference system in SupT1 cells using the lentiviral gene transfer. Although the success of anti-HIV-1 activity was evidenced, the integration site is uncontrollable and may not be practical for clinical translation. In this study, we use the CRISPR/Cas9 gene editing technology to precisely knock-in αRep4E3 genes into the adeno-associated virus integration site 1 (AAVS1) safe harbor locus of the target cells. We compare the αRep4E3 expression under the regulation of three different promoters, including cytomegalovirus (CMV), human elongation factor-1 alpha (EF1α), and ubiquitin C (UbC) promoters with and without codon optimization in HEK293T cells. The results demonstrated that the EF1α promoter with codon-optimized αRep4E3mCherry showed higher protein expression than other promoters with non-optimized codons. We then performed a proof-of-concept study by knocking in the αRep4E3mCherry gene at the AAVS1 locus of the Jurkat cells. The results showed that the αRep4E3mCherry-expressing Jurkat cells exhibited anti-HIV-1 activities against HIV-1NL4-3 strain as evidenced by decreased capsid (p24) protein levels and viral genome copies as compared to the untransfected Jurkat control cells. Altogether, our study demonstrates that the αRep4E3 could interfere with the viral RNA packaging and suggests that the αRep4E3 scaffold protein could be a promising anti-viral molecule that offers a functional cure for people living with HIV-1.     

Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopamine neurons and the deposition of misfolded proteins known as Lewy bodies (LBs), which contain α-synuclein (α-syn). The causes and molecular mechanisms of PD are not clearly understood to date. However, misfolded proteins, oxidative stress, and impaired autophagy are believed to play important roles in the pathogenesis of PD. Importantly, α-syn is considered a key player in the development of PD. The present study aimed to assess the role of Ellagic acid (EA), a polyphenol found in many fruits, on α-syn aggregation and toxicity. Using thioflavin and seeding polymerization assays, in addition to electron microscopy, we found that EA could dramatically reduce α-syn aggregation. Moreover, EA significantly mitigated the aggregated α-syn-induced toxicity in SH-SY5Y cells and thus enhanced their viability. Mechanistically, these cytoprotective effects of EA are mediated by the suppression of apoptotic proteins BAX and p53 and a concomitant increase in the anti-apoptotic protein, BCL-2. Interestingly, EA was able to activate autophagy in SH-SY5Y cells, as evidenced by normalized/enhanced expression of LC3-II, p62, and pAKT. Together, our findings suggest that EA may attenuate α-syn toxicity by preventing aggregation and improving viability by restoring autophagy and suppressing apoptosis.     

Development of Atropa belladonna L. Plants with High-Yield Hyoscyamine and without Its Derivatives Using the CRISPR/Cas9 System

Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6β-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost.     

Cellular Integrin α5β1 and Exosomal ADAM17 Mediate the Binding and Uptake of Exosomes Produced by Colorectal Carcinoma Cells

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size−exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5β1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes.     

Profound Modification of Fatty Acid Profile and Endocannabinoid-Related Mediators in PPARα Agonist Fenofibrate-Treated Mice

Fenofibrate (FBR), an oral medication used to treat dyslipidemia, is a ligand of the peroxisome proliferator-activated receptor α (PPARα), a nuclear receptor that regulates the expression of metabolic genes able to control lipid metabolism and food intake. PPARα natural ligands include fatty acids (FA) and FA derivatives such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), known to have anti-inflammatory and anorexigenic activities, respectively. We investigated changes in the FA profile and FA derivatives by HPLC and LC-MS in male C57BL/6J mice fed a standard diet with or without 0.2% fenofibrate (0.2% FBR) for 21 days. Induction of PPARα by 0.2% FBR reduced weight gain, food intake, feed efficiency, and liver lipids and induced a profound change in FA metabolism mediated by parallel enhanced mitochondrial and peroxisomal β-oxidation. The former effects led to a steep reduction of essential FA, particularly 18:3n3, with a consequent decrease of the n3-highly unsaturated fatty acids (HUFA) score; the latter effect led to an increase of 16:1n7 and 18:1n9, suggesting enhanced hepatic de novo lipogenesis with increased levels of hepatic PEA and OEA, which may activate a positive feedback and further sustain reductions of body weight, hepatic lipids and feed efficiency.     

Collagen I Modifies Connexin-43 Hemichannel Activity via Integrin α2β1 Binding in TGFβ1-Evoked Renal Tubular Epithelial Cells

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2β1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFβ1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2β1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFβ1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2β1 signaling, ahead of establishing Peptide 5 as a potential intervention.     

Evaluation of the Anti-Atopic Dermatitis Effects of α-Boswellic Acid on Tnf-α/Ifn-γ-Stimulated HaCat Cells and DNCB-Induced BALB/c Mice

Boswellic acids, triterpenoids derived from the genus Boswellia (Burseraceae), are known for their anti-inflammatory and anti-tumor efficacy. Atopic dermatitis is a chronic, non-infectious inflammatory skin disease. However, the effects of α-boswellic acid on atopic dermatitis have not been studied. Therefore, in this study we examined the expression level of pro-inflammatory cytokines, histopathological analysis, and physiological data from BALB/c mice with atopic-like dermatitis induced by 2,4-dinitrochlorobenzene and TNF-α/IFN-γ-stimulated HaCaT cells to better understand the agent’s anti-atopic dermatitis efficacy. First, we found that α-boswellic reduced the epidermal thickening, mast cell numbers, and dermal infiltration of 2,4-dinitrochlorobenzene-induced atopic-like dermatitis in BALB/c mice. Furthermore, we also found that α-boswellic acid can restore transepidermal water loss and skin reddening in mice. In human keratinocytes inflamed by TNF-α/IFN-γ, α-boswellic acid inhibited MAP kinase activation and showed a reduction in NF-κB nuclear translocation. Finally, α-boswellic acid can reduce the expression level of cytokines (IL-1β, IL-6, and IL-8) following the stimulation of TNF-α/IFN-γ in HaCaT cells. Taken together, our study suggests that α-boswellic acids are a potential component for the development of anti-atopic dermatitis drugs.     

Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B₄ (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.     

Evaluation of Cytotoxic and Mutagenic Effects of the Synthetic Cathinones Mexedrone, α-PVP and α-PHP

Mexedrone, α-PVP and α-PHP are synthetic cathinones. They can be considered amphetamine-like substances with a stimulating effect. Actually, studies showing their impact on DNA are totally absent. Therefore, in order to fill this gap, aim of the present work was to evaluate their mutagenicity on TK6 cells. On the basis of cytotoxicity and cytostasis results, we selected the concentrations (35–100 µM) to be used in the further analysis. We used the micronucleus (MN) as indicator of genetic damage and analyzed the MNi frequency fold increase by flow cytometry. Mexedrone demonstrated its mutagenic potential contrary to the other two compounds; we then proceeded by repeating the analyzes in the presence of extrinsic metabolic activation in order to check if it was possible to totally exclude the mutagenic capacity for α-PVP and α-PHP. The results demonstrated instead the mutagenicity of their metabolites. We then evaluated reactive oxygen species (ROS) induction as a possible mechanism at the basis of the highlighted effects but the results did not show a statistically significant increase in ROS levels for any of the tested substances. Anyway, our outcomes emphasize the importance of mutagenicity evaluation for a complete assessment of the risk associated with synthetic cathinones exposure.     

P2Y2R Deficiency Ameliorates Hepatic Steatosis by Reducing Lipogenesis and Enhancing Fatty Acid β-Oxidation through AMPK and PGC-1α Induction in High-Fat Diet-Fed Mice

Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic liver disease associated with obesity and insulin resistance. Activation of the purinergic receptor P2Y2R has been reported to promote adipogenesis, inflammation and dyslipidemia in adipose tissues in obese mice. However, the role of P2Y2R and its mechanisms in NAFLD remain unknown. We hypothesized that P2Y2R deficiency may play a protective role in NAFLD by modulating lipid metabolism in the liver. In this study, we fed wild type and P2Y2R knockout mice with a high-fat diet (HFD) for 12 weeks and analyzed metabolic phenotypes. First, P2Y2R deficiency effectively improved insulin resistance with a reduction in body weight and plasma insulin. Second, P2Y2R deficiency attenuated hepatic lipid accumulation and injury with reduced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Third, P2Y2R deficiency decreased the expression of fatty acid synthesis mediators (cluster of differentiation (CD36), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1)); and increased the expression of adipose triglyceride lipase (ATGL), a lipolytic enzyme. Mechanistically, P2Y2R deficiency increased the AMP-activated protein kinase (AMPK) activity to improve mitochondrial fatty acid β-oxidation (FAO) by regulating acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase 1A (CPT1A)-mediated FAO pathway. In addition, P2Y2R deficiency increased peroxisome proliferator-activated gamma co-activator-1α (PGC-1α)-mediated mitochondrial biogenesis. Conclusively, P2Y2R deficiency ameliorated HFD-induced hepatic steatosis by enhancing FAO through AMPK signaling and PGC-1α pathway, suggesting P2Y2R as a promising therapeutic target for NAFLD.