The role of efferocytosis-fueled macrophage metabolism in the resolution of inflammation.

The phagocytosis of dying cells by macrophages, termed efferocytosis, is a tightly regulated process that involves the sensing, binding, engulfment, and digestion of apoptotic cells. Efferocytosis not only prevents tissue necrosis and inflammation caused by secondary necrosis of dying cells, but it also promotes pro-resolving signaling in macrophages, which is essential for tissue resolution and repair following injury or inflammation. An important factor that contributes to this pro-resolving reprogramming is the cargo that is released from apoptotic cells after their engulfment and phagolysosomal digestion by macrophages. The apoptotic cell cargo contains amino acids, nucleotides, fatty acids, and cholesterol that function as metabolites and signaling molecules to bring about this re-programming. Here, we review efferocytosis-induced changes in macrophage metabolism that mediate the pro-resolving functions of macrophages. We also discuss various strategies, challenges, and future perspectives related to drugging efferocytosis-fueled macrophage metabolism as strategy to dampen inflammation and promote resolution in chronic inflammatory diseases.

Low MBOAT7 expression, a genetic risk for MASH, promotes a pro-fibrotic pathway involving hepatocyte TAZ upregulation.

BACKGROUND AND AIMS: The common genetic variant rs641738 C>T is a risk factor for metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH), including liver fibrosis, and is associated with decreased expression of the phospholipid-remodeling enzyme MBOAT7 (LPIAT1). However, whether restoring MBOAT7 expression in established MASLD dampens the progression to liver fibrosis and, importantly, the mechanism through which decreased MBOAT7 expression exacerbates MASH fibrosis remain unclear. APPROACH AND RESULTS: We first showed that hepatocyte MBOAT7 restoration in mice with diet-induced steatohepatitis slows the progression to liver fibrosis. Conversely, when hepatocyte-MBOAT7 was silenced in mice with established hepatosteatosis, liver fibrosis but not hepatosteatosis was exacerbated. Mechanistic studies revealed that hepatocyte-MBOAT7 restoration in MASH mice lowered hepatocyte-TAZ (WWTR1), which is known to promote MASH fibrosis. Conversely, hepatocyte-MBOAT7 silencing enhanced TAZ upregulation in MASH. Finally, we discovered that changes in hepatocyte phospholipids due to MBOAT7 loss-of-function promote a cholesterol trafficking pathway that upregulates TAZ and the TAZ-induced profibrotic factor Indian hedgehog (IHH). As evidence for relevance in humans, we found that the livers of individuals with MASH carrying the rs641738-T allele had higher hepatocyte nuclear TAZ, indicating higher TAZ activity, and increased IHH mRNA. CONCLUSIONS: This study provides evidence for a novel mechanism linking MBOAT7-LoF to MASH fibrosis; adds new insight into an established genetic locus for MASH; and, given the druggability of hepatocyte TAZ for MASH fibrosis, suggests a personalized medicine approach for subjects at increased risk for MASH fibrosis due to inheritance of variants that lower MBOAT7.

Hepatocyte-Specific Fads1 Overexpression Attenuates Western Diet-Induced Metabolic Phenotypes in a Rat Model.

Fatty acid desaturase 1 (FADS1) is a rate-limiting enzyme in long-chain polyunsaturated fatty acid (LCPUFA) synthesis. Reduced activity of FADS1 was observed in metabolic dysfunction-associated steatotic liver disease (MASLD). The aim of this study was to determine whether adeno-associated virus serotype 8 (AAV8) mediated hepatocyte-specific overexpression of Fads1 (AAV8-Fads1) attenuates western diet-induced metabolic phenotypes in a rat model. Male weanling Sprague-Dawley rats were fed with a chow diet, or low-fat high-fructose (LFHFr) or high-fat high-fructose diet (HFHFr) ad libitum for 8 weeks. Metabolic phenotypes were evaluated at the endpoint. AAV8-Fads1 injection restored hepatic FADS1 protein levels in both LFHFr and HFHFr-fed rats. While AAV8-Fads1 injection led to improved glucose tolerance and insulin signaling in LFHFr-fed rats, it significantly reduced plasma triglyceride (by ~50%) and hepatic cholesterol levels (by ~25%) in HFHFr-fed rats. Hepatic lipidomics analysis showed that FADS1 activity was rescued by AAV8-FADS1 in HFHFr-fed rats, as shown by the restored arachidonic acid (AA)/dihomo-γ-linolenic acid (DGLA) ratio, and that was associated with reduced monounsaturated fatty acid (MUFA). Our data suggest that the beneficial role of AAV8-Fads1 is likely mediated by the inhibition of fatty acid re-esterification. FADS1 is a promising therapeutic target for MASLD in a diet-dependent manner.

TAZ-induced Cybb contributes to liver tumor formation in non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is a leading cause of hepatocellular carcinoma (HCC), but mechanisms linking NASH to eventual tumor formation remain poorly understood. Herein, we investigate the role of TAZ/WWTR1, which is induced in hepatocytes in NASH, in the progression of NASH to HCC. METHODS: The roles of hepatocyte TAZ and its downstream targets were investigated in diet-induced and genetic models of NASH-HCC using gene-targeting, adeno-associated virus 8 (AAV8)-H1-mediated gene silencing, or AAV8-TBG-mediated gene expression. The biochemical signature of the newly elucidated pathway was probed in liver specimens from humans with NASH-HCC. RESULTS: When hepatocyte-TAZ was silenced in mice with pre-tumor NASH using AAV8-H1-shTaz (short-hairpin Taz), subsequent HCC tumor development was suppressed. In this setting, the tumor-suppressing effect of shTaz was not dependent of TAZ silencing in the tumors themselves and could be dissociated from the NASH-suppressing effects of shTaz. The mechanism linking pre-tumor hepatocyte-TAZ to eventual tumor formation involved TAZ-mediated induction of the NOX2-encoding gene Cybb, which led to NADPH-mediated oxidative DNA damage. As evidence, DNA damage and tumor formation could be suppressed by treatment of pre-tumor NASH mice with AAV8-H1-shCybb; AAV8-TBG-OGG1, encoding the oxidative DNA-repair enzyme 8-oxoguanine glycosylase; or AAV8-TBG-NHEJ1, encoding the dsDNA repair enzyme non-homologous end-joining factor 1. In surrounding non-tumor tissue from human NASH-HCC livers, there were strong correlations between TAZ, NOX2, and oxidative DNA damage. CONCLUSIONS: TAZ in pre-tumor NASH-hepatocytes, via induction of Cybb and NOX2-mediated DNA damage, contributes to subsequent HCC tumor development. These findings illustrate how NASH provides a unique window into the early molecular events that can lead to tumor formation and suggest that NASH therapies targeting TAZ might also prevent NASH-HCC. LAY SUMMARY: Non-alcoholic steatohepatitis (NASH) is emerging as the leading cause of a type of liver cancer called hepatocellular carcinoma (HCC), but molecular events in pre-tumor NASH hepatocytes leading to HCC remain largely unknown. Our study shows that a protein called TAZ in pre-tumor NASH-hepatocytes promotes damage to the DNA of hepatocytes and thereby contributes to eventual HCC. This study reveals a very early event in HCC that is induced in pre-tumor NASH, and the findings suggest that NASH therapies targeting TAZ might also prevent NASH-HCC.

Activating Adenosine Monophosphate-Activated Protein Kinase Mediates Fibroblast Growth Factor 1 Protection From Nonalcoholic Fatty Liver Disease in Mice.

BACKGROUND AND AIMS: Fibroblast growth factor (FGF) 1 demonstrated protection against nonalcoholic fatty liver disease (NAFLD) in type 2 diabetic and obese mice by an uncertain mechanism. This study investigated the therapeutic activity and mechanism of a nonmitogenic FGF1 variant carrying 3 substitutions of heparin-binding sites (FGF1△HBS ) against NAFLD. APPROACH AND RESULTS: FGF1△HBS administration was effective in 9-month-old diabetic mice carrying a homozygous mutation in the leptin receptor gene (db/db) with NAFLD; liver weight, lipid deposition, and inflammation declined and liver injury decreased. FGF1△HBS reduced oxidative stress by stimulating nuclear translocation of nuclear erythroid 2 p45-related factor 2 (Nrf2) and elevation of antioxidant protein expression. FGF1△HBS also inhibited activity and/or expression of lipogenic genes, coincident with phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and its substrates. Mechanistic studies on palmitate exposed hepatic cells demonstrated that NAFLD-like oxidative damage and lipid accumulation could be reversed by FGF1△HBS . In palmitate-treated hepatic cells, small interfering RNA (siRNA) knockdown of Nrf2 abolished only FGF1△HBS antioxidative actions but not improvement of lipid metabolism. In contrast, AMPK inhibition by pharmacological agent or siRNA abolished FGF1△HBS benefits on both oxidative stress and lipid metabolism that were FGF receptor (FGFR) 4 dependent. Further support of these in vitro findings is that liver-specific AMPK knockout abolished therapeutic effects of FGF1△HBS against high-fat/high-sucrose diet-induced hepatic steatosis. Moreover, FGF1△HBS improved high-fat/high-cholesterol diet-induced steatohepatitis and fibrosis in apolipoprotein E knockout mice. CONCLUSIONS: These findings indicate that FGF1△HBS is effective for preventing and reversing liver steatosis and steatohepatitis and acts by activation of AMPK through hepatocyte FGFR4.

Dioxin-like and non-dioxin-like PCBs differentially regulate the hepatic proteome and modify diet-induced nonalcoholic fatty liver disease severity.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants associated with metabolic disruption and non-alcoholic fatty liver disease (NAFLD). Based on their ability to activate the aryl hydrocarbon receptor (AhR), PCBs are subdivided into two classes: dioxin-like (DL) and non-dioxin-like (NDL) PCBs. Previously, we demonstrated that NDL PCBs compromised the liver to promote more severe diet-induced NAFLD. Here, the hepatic effects and potential mechanisms (by untargeted liver proteomics) of DL PCBs, NDL PCBs or co-exposure to both in diet-induced NAFLD are investigated. Male C57Bl/6 mice were fed a 42% fat diet and exposed to vehicle control; Aroclor1260 (20 mg/kg, NDL PCB mixture); PCB126 (20 µg/kg, DL PCB congener); or a mixture of Aroclor1260 (20 mg/kg)+PCB126 (20 µg/kg) for 12 weeks. Each exposure was associated with a distinct hepatic proteome. Phenotypic and proteomic analyses revealed increased hepatic inflammation and phosphoprotein signaling disruption by Aroclor1260. PCB126 decreased hepatic inflammation and fibrosis at the molecular level; while altering cytoskeletal remodeling, metal homeostasis, and intermediary/xenobiotic metabolism. PCB126 attenuated Aroclor1260-induced hepatic inflammation but increased hepatic free fatty acids in the co-exposure group. Aroclor1260+PCB126 exposure was strongly associated with multiple epigenetic processes, and these could potentially explain the observed non-additive effects of the exposures on the hepatic proteome. Taken together, the results demonstrated that PCB exposures differentially regulated the hepatic proteome and the histologic severity of diet-induced NAFLD. Future research is warranted to determine the AhR-dependence of the observed effects including metal homeostasis and the epigenetic regulation of gene expression.

Proteomic Analysis Reveals Novel Mechanisms by Which Polychlorinated Biphenyls Compromise the Liver Promoting Diet-Induced Steatohepatitis.

Environmental pollution contributes to fatty liver disease pathogenesis. Polychlorinated biphenyl (PCB) exposures have been associated with liver enzyme elevation and suspected steatohepatitis in cohort studies. Male mice treated with the commercial PCB mixture, Aroclor 1260 (20 mg/kg), and fed high fat diet (HFD) for 12 weeks developed steatohepatitis. Receptor-based modes of action including inhibition of the epidermal growth factor (EGF) receptor were previously proposed, but other mechanisms likely exist. Objectives were to identify and validate the pathways, transcription factors, and mechanisms responsible for the steatohepatitis associated with PCB and HFD coexposures. Comparative proteomics analysis was performed in archived mouse liver samples from the aforementioned chronic exposure study. Pathway and transcription factor analysis (TFA) was performed, and selected results were validated. Liver proteomics detected 1103 unique proteins. Aroclor 1260 upregulated 154 and downregulated 93 of these. Aroclor 1260 + HFD coexposures affected 55 pathways including glutathione metabolism, intermediary metabolism, and cytoskeletal remodeling. TFA of Aroclor 1260 treatment demonstrated alterations in the function of 42 transcription factors including downregulation of NRF2 and key nuclear receptors previously demonstrated to protect against steatohepatitis (e.g., HNF4α, FXR, PPARα/δ/γ, etc.). Validation studies demonstrated that Aroclor 1260 significantly reduced HNF4α protein levels, while Aroclor 1260 + HFD reduced expression of the HNF4α target gene, albumin, in vivo. Aroclor 1260 attenuated EGF-dependent HNF4α phosphorylation and target gene activation in vitro. Aroclor 1260 reduced levels of NRF2, its target genes, and glutathione in vivo. Aroclor 1260 attenuated EGF-dependent NRF2 upregulation, in vitro. Aroclor 1260 indirectly activated hepatic stellate cells in vitro via induction of hepatocyte-derived TGFß. PCB exposures adversely impacted transcription factors regulating liver protection, function, and fibrosis. PCBs, thus, compromised the liver by reducing its protective responses against nutritional stress to promote diet-induced steatohepatitis. The identified mechanisms by which environmental pollutants influence fatty liver disease pathogenesis require confirmation in humans.

Melatonin ameliorates intervertebral disc degeneration via the potential mechanisms of mitophagy induction and apoptosis inhibition.

Intervertebral disc degeneration (IDD) is a complicated disease in patients. The pathogenesis of IDD encompasses cellular oxidative stress, mitochondrion dysfunction and apoptosis. Melatonin eliminates oxygen free radicals, regulates mitochondrial homoeostasis and function, stimulates mitophagy and protects against cellular apoptosis. Therefore, we hypothesize that melatonin has beneficial effect on IDD by mitophagy stimulation and inhibition of apoptosis. The effects of melatonin on IDD were investigated in vitro and in vivo. For the former, melatonin diminished cellular apoptosis caused by tert-butyl hydroperoxide in nucleus pulposus (NP) cells. Mitophagy, as well as its upstream regulator Parkin, was activated by melatonin in both a dose and time-dependent manner. Mitophagy inhibition by cyclosporine A (CsA) partially eliminated the protective effects of melatonin against NP cell apoptosis, suggesting that mitophagy is involved in the protective effect of melatonin on IDD. In addition, melatonin was demonstrated to preserve the extracellular matrix (ECM) content of Collagen II, Aggrecan and Sox-9, while inhibiting the expression of matrix degeneration enzymes, including MMP-13 and ADAMTS-5. In vivo, our results demonstrated that melatonin treatment ameliorated IDD in a puncture-induced rat model. To conclude, our results suggested that melatonin protected NP cells against apoptosis via mitophagy induction and ameliorated disc degeneration, providing the potential therapy for IDD.

Polychlorinated biphenyl exposures differentially regulate hepatic metabolism and pancreatic function: Implications for nonalcoholic steatohepatitis and diabetes.

The endocrine disrupting chemicals, polychlorinated biphenyls (PCBs), have been associated with nonalcoholic steatohepatitis (NASH) and diabetes. However, an integrative analysis of the effects of PCBs on the liver and pancreas has never been performed for the two major PCB subtypes, dioxin-like (DL) and nondioxin-like (NDL), and a mixture of NDL/DL PCBs. Therefore, male C57BL/6 J mice fed a control synthetic diet were treated with either a NDL PCB mixture, Aroclor 1260 (20 mg/kg); a single DL PCB congener, PCB 126 (20 µg/kg); a NDL/DL mixture, Aroclor 1260 plus PCB 126; or vehicle control for 2 weeks. PCB126 had the greatest impact on hepatic lipid metabolism. It caused steatosis due to increased hepatic lipid import with associated hypolipidemia. However, all PCB exposures impacted expression of hepatic lipid metabolism genes in different manners. The 'NASH gene', Pnpla3, was elevated by Aroclor 1260, but decreased by all other exposures. The expression of hepatokines implicated in metabolic syndrome (Fgf21, Igf1, and betatrophin) were differentially regulated. The NDL/DL PCB mixture had the greatest effects on pancreatic histology, including acinar cell atrophy, mild steatosis, and fibrosis without ductal changes or immune cell infiltration. It decreased expression of insulin and altered the expression of genes regulating islet identity. None of these exposures was associated with altered HOMA-IR or HOMA-B. In summary, PCB exposures differentially regulated liver and pancreas structure and function. Novel mechanisms for PCB-induced endocrine/metabolic disruption included altered hepatokines and Pnpla3 as well as 'PCB pancreatopathy' that was associated with altered expression of pancreatic islet identity factors. More research is required to understand fully these findings in the context of human NASH and diabetes.

Concentration dependence of human and mouse aryl hydrocarbon receptor responsiveness to polychlorinated biphenyl exposures: Implications for aroclor mixtures.

1. Aryl hydrocarbon receptor (AhR) ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs), are endocrine disrupting chemicals associated with nonalcoholic fatty liver disease. This study documents the species-specific differences between mouse (high affinity mAhR) and human AhR (hAhR) activation by PCB congeners and Aroclor mixtures. 2. AhR activation by TCDD or PCBs 77, 81, 114, 114, 126, and 169 was measured using luciferase reporter constructs transfected into either Hepa1c1c7 mouse or HepG2 human liver cell lines. The EC50 values were lower in Hepa1c1c7 cells than HepG2 cells for all compounds tested except PCB 81. The results for TCDD and PCB 126 were validated in primary human and mouse hepatocytes by measuring CYP1A1 gene transcript levels. 3. Because humans are exposed to PCB mixtures, several mixtures (Aroclors 1254; 1260; and 1260 + 0.1% PCB126 each at 10 µg/ml) were then tested. Neither Aroclor 1254 nor Aroclor 1260 increased luciferase activity by the transfected AhR reporter construct. The Aroclor 1260 + 0.1% PCB 126 mixture induced mAhR-mediated transactivation, but not hAhR activation in cell lines. 4. In summary, significant concentration-dependent differences exist between human and mouse AhR activation by PCBs. Relative effect potencies differed, in some cases, from published toxic equivalency factors.

Dietary copper-fructose interactions alter gut microbial activity in male rats.

Dietary copper-fructose interactions contribute to the development of nonalcoholic fatty liver disease (NAFLD). Gut microbiota play critical roles in the pathogenesis of NAFLD. The aim of this study was to determine the effect of different dietary doses of copper and their interactions with high fructose on gut microbiome. Male weanling Sprague-Dawley rats were fed diets with adequate copper (6 ppm CuA), marginal copper (1.5 ppm CuM) (low copper), or supplemented copper (20 ppm CuS) (high copper) for 4 wk. Deionized water or deionized water containing 30% fructose (wt/vol) was given ad libitum. Copper status, liver enzymes, gut barrier function, and gut microbiome were evaluated. Both low- and high-copper diets led to liver injury in high-fructose-fed rats, and this was associated with gut barrier dysfunction, as shown by the markedly decreased tight junction proteins and increased gut permeability. 16S rDNA sequencing analysis revealed distinct alterations of the gut microbiome associated with dietary low- and high-copper/high-fructose feeding. The common features of the alterations of the gut microbiome were the increased abundance of Firmicutes and the depletion of Akkermansia. However, they differed mainly within the phylum Firmicutes. Our data demonstrated that a complex interplay among host, microbes, and dietary copper-fructose interaction regulates gut microbial metabolic activity, which may contribute to the development of liver injury and hepatic steatosis. The distinct alterations of gut microbial activity, which were associated with the different dietary doses of copper and fructose, imply that separate mechanism(s) may be involved. NEW & NOTEWORTHY First, dietary low- and high-copper/high-fructose-induced liver injury are associated with distinct alterations of gut microbiome. Second, dietary copper level plays a critical role in maintaining the gut barrier integrity, likely by acting on the intestinal tight junction proteins and the protective commensal bacteria Akkermansia. Third, the alterations of gut microbiome induced by dietary low and high copper with or without fructose differ mainly within the phylum Firmicutes.

Neural Stem Cells Expressing bFGF Reduce Brain Damage and Restore Sensorimotor Function after Neonatal Hypoxia-Ischemia.

BACKGROUND/AIMS: Neonatal hypoxia-ischemia (HI) causes severe brain damage and significantly increases neonatal morbidity and mortality. Increasing evidences have verified that stem cell-based therapy has the potential to rescue the ischemic tissue and restore function via secreting growth factors after HI. Here, we had investigated whether intranasal neural stem cells (NSCs) treatment improves the recovery of neonatal HI, and NSCs overexpressing basic fibroblast growth factor (bFGF) has a better therapeutic effect for recovery than NSCs treatment only. METHODS: We performed permanent occlusion of the right common carotid artery in 9-day old ICR mice as animal model of neonatal hypoxia-ischemia. At 3 days post-HI, NSC, NSC-GFP, NSC-bFGF and vehicle were delivered intranasally. To determine the effect of intranasal NSC, NSC-GFP and NSC-bFGF treatment on recovery after HI, we analyzed brain damage, sensor-motor function and cell differentiation. RESULTS: It was observed that intranasal NSC, NSC-GFP and NSC-bFGF treatment decreased gray and white matter loss area in comparison with vehicle-treated mouse. NSC, NSC-GFP and NSC-bFGF treatment also significantly improved sensor motor function in cylinder rearing test and adhesive removal test, however, NSC-bFGF-treatment was more effective than NSC-treatment in the improvement of somatosensory function. Furthermore, compared with NSC and NSC-GFP, NSC-bFGF treatment group appeared to differentiate into more neurons. CONCLUSION: Taken together, intranasal administration of NSCs is a promising therapy for treatment of neonatal HI, but NSCs overexpressing bFGF promotes the survival and differentiation of NSCs, and consequently achieves a better therapeutic effect in improving recovery after neonatal HI.

Nuclear receptors and nonalcoholic fatty liver disease.

Nuclear receptors are transcription factors which sense changing environmental or hormonal signals and effect transcriptional changes to regulate core life functions including growth, development, and reproduction. To support this function, following ligand-activation by xenobiotics, members of subfamily 1 nuclear receptors (NR1s) may heterodimerize with the retinoid X receptor (RXR) to regulate transcription of genes involved in energy and xenobiotic metabolism and inflammation. Several of these receptors including the peroxisome proliferator-activated receptors (PPARs), the pregnane and xenobiotic receptor (PXR), the constitutive androstane receptor (CAR), the liver X receptor (LXR) and the farnesoid X receptor (FXR) are key regulators of the gut:liver:adipose axis and serve to coordinate metabolic responses across organ systems between the fed and fasting states. Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and may progress to cirrhosis and even hepatocellular carcinoma. NAFLD is associated with inappropriate nuclear receptor function and perturbations along the gut:liver:adipose axis including obesity, increased intestinal permeability with systemic inflammation, abnormal hepatic lipid metabolism, and insulin resistance. Environmental chemicals may compound the problem by directly interacting with nuclear receptors leading to metabolic confusion and the inability to differentiate fed from fasting conditions. This review focuses on the impact of nuclear receptors in the pathogenesis and treatment of NAFLD. Clinical trials including PIVENS and FLINT demonstrate that nuclear receptor targeted therapies may lead to the paradoxical dissociation of steatosis, inflammation, fibrosis, insulin resistance, dyslipidemia and obesity. Novel strategies currently under development (including tissue-specific ligands and dual receptor agonists) may be required to separate the beneficial effects of nuclear receptor activation from unwanted metabolic side effects. The impact of nuclear receptor crosstalk in NAFLD is likely to be profound, but requires further elucidation. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

Valproate Attenuates Endoplasmic Reticulum Stress-Induced Apoptosis in SH-SY5Y Cells via the AKT/GSK3ß Signaling Pathway.

Endoplasmic reticulum (ER) stress-induced apoptosis plays an important role in a range of neurological disorders, such as neurodegenerative diseases, spinal cord injury, and diabetic neuropathy. Valproate (VPA), a typical antiepileptic drug, is commonly used in the treatment of bipolar disorder and epilepsy. Recently, VPA has been reported to exert neurotrophic effects and promote neurite outgrowth, but its molecular mechanism is still unclear. In the present study, we investigated whether VPA inhibited ER stress and promoted neuroprotection and neuronal restoration in SH-SY5Y cells and in primary rat cortical neurons, respectively, upon exposure to thapsigargin (TG). In SH-SY5Y cells, cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and the expression of ER stress-related apoptotic proteins such as glucose­regulated protein (GRP78), C/EBP homologous protein (CHOP), and cleaved caspase-12/-3 were analyzed with Western blot analyses and immunofluorescence assays. To explore the pathway involved in VPA-induced cell proliferation, we also examined p-AKT, GSK3ß, p-JNK and MMP-9. Moreover, to detect the effect of VPA in primary cortical neurons, immunofluorescence staining of ß-III tubulin and Anti-NeuN was analyzed in primary cultured neurons exposed to TG. Our results demonstrated that VPA administration improved cell viability in cells exposed to TG. In addition, VPA increased the levels of GRP78 and p-AKT and decreased the levels of ATF6, XBP-1, GSK3ß, p-JNK and MMP-9. Furthermore, the levels of the ER stress-induced apoptosis response proteins CHOP, cleaved caspase-12 and cleaved caspase-3 were inhibited by VPA treatment. Meanwhile, VPA administration also increased the ratio of Bcl-2/Bax. Moreover, VPA can maintain neurite outgrowth of primary cortical neurons. Collectively, the neurotrophic effect of VPA is related to the inhibition of ER stress-induced apoptosis in SH-SY5Y cells and the maintenance of neuronal growth. Collectively, our results suggested a new approach for the therapeutic function of VPA in neurological disorders and neuroprotection.

Epidermal growth factor attenuates blood-spinal cord barrier disruption via PI3K/Akt/Rac1 pathway after acute spinal cord injury.

After spinal cord injury (SCI), disruption of blood-spinal cord barrier (BSCB) elicits blood cell infiltration such as neutrophils and macrophages, contributing to permanent neurological disability. Previous studies show that epidermal growth factor (EGF) produces potent neuroprotective effects in SCI models. However, little is known that whether EGF contributes to the integrity of BSCB. The present study is performed to explore the mechanism of BSCB permeability changes which are induced by EGF treatment after SCI in rats. In this study, we demonstrate that EGF administration inhibits the disruption of BSCB permeability and improves the locomotor activity in SCI model rats. Inhibition of the PI3K/Akt pathways by a specific inhibitor, LY294002, suppresses EGF-induced Rac1 activation as well as tight junction (TJ) and adherens junction (AJ) expression. Furthermore, the protective effect of EGF on BSCB is related to the activation of Rac1 both in vivo and in vitro. Blockade of Rac1 activation with Rac1 siRNA downregulates EGF-induced TJ and AJ proteins expression in endothelial cells. Taken together, our results indicate that EGF treatment preserves BSCB integrity and improves functional recovery after SCI via PI3K-Akt-Rac1 signalling pathway.

Basic fibroblast growth factor promotes melanocyte migration via activating PI3K/Akt-Rac1-FAK-JNK and ERK signaling pathways.

Vitiligo is a depigmentation disorder characterized by loss of functional melanocytes of the skin epidermis. The pathogenesis of vitiligo remains elusive. The purpose of this study is to investigate the effects of basic fibroblast growth factor (bFGF) on melanocyte migration, including its biochemical mechanism using transwell assay in vitro. We found that melanocyte treated with bFGF showed a significant increase in migration and cytoskeletal rearrangement. These changes were associated with increased activation of PI3K/Akt, Rac1, FAK, JNK, and ERK. Likewise, reduction of PI3K/Akt, Rac1, FAK, JNK, and ERK activity using selective inhibitors or siRNA was associated with impediment of bFGF-induced melanocyte migration. In addition, activity of Rac1, FAK, and JNK was reduced in cells in which PI3K/Akt was inhibited, activity of FAK and JNK was reduced in cells in which the Rac1 was inhibited, and activity of JNK was reduced in cells in which the FAK was inhibited. Collectively, these data demonstrate that bFGF facilitated melanocyte migration via PI3K/Akt-Rac1-FAK-JNK and ERK signaling pathways. © 2016 IUBMB Life, 68(9):735-747, 2016.

Heparin-Based Coacervate of FGF2 Improves Dermal Regeneration by Asserting a Synergistic Role with Cell Proliferation and Endogenous Facilitated VEGF for Cutaneous Wound Healing.

Effective wound healing requires complicated, coordinated interactions and responses at protein, cellular, and tissue levels involving growth factor expression, cell proliferation, wound closure, granulation tissue formation, and vascularization. In this study, we develop a heparin-based coacervate consisting of poly(ethylene argininylaspartate digylceride) (PEAD) as a storage matrix, heparin as a bridge, and fibroblast growth factor-2 (FGF2) as a cargo (namely heparin-FGF2@PEAD) for wound healing. First, in vitro characterization demonstrates the loading efficiency and control release of FGF2 from the heparin-FGF2@PEAD coacervate. The following in vivo studies examine the wound healing efficiency of the heparin-FGF2@PEAD coacervate upon delivering FGF2 to full-thickness excisional skin wounds in vivo, in comparison with the other three control groups with saline, heparin@PEAD as vehicle, and free FGF2. Collective in vivo data show that controlled release of FGF2 to the wounds by the coacervate significantly accelerates the wound healing by promoting cell proliferation, stimulating the secretion of vascular endothelial growth factor (VEGF) for re-epithelization, collagen deposition, and granulation tissue formation, and enhancing the expression of platelet endothelial cell adhesion molecule (CD31) and alpha-smooth muscle actin (α-SMA) for blood vessel maturation. In parallel, no obvious wound healing effect is found for the control, vehicle, and free FGF2 groups, indicating the important role of the coavervate in the wound healing process. This work designs a suitable delivery system that can protect and release FGF2 in a sustained and controlled manner, which provides a promising therapeutic potential for topical treatment of wounds.

Pressure Combined with Ischemia/Reperfusion Injury Induces Deep Tissue Injury via Endoplasmic Reticulum Stress in a Rat Pressure Ulcer Model.

Pressure ulcer is a complex and significant health problem in long-term bedridden patients, and there is currently no effective treatment or efficient prevention method. Furthermore, the molecular mechanisms and pathogenesis contributing to the deep injury of pressure ulcers are unclear. The aim of the study was to explore the role of endoplasmic reticulum (ER) stress and Akt/GSK3ß signaling in pressure ulcers. A model of pressure-induced deep tissue injury in adult Sprague-Dawley rats was established. Rats were treated with 2-h compression and subsequent 0.5-h release for various cycles. After recovery, the tissue in the compressed regions was collected for further analysis. The compressed muscle tissues showed clear cellular degenerative features. First, the expression levels of ER stress proteins GRP78, CHOP, and caspase-12 were generally increased compared to those in the control. Phosphorylated Akt and phosphorylated GSK3ß were upregulated in the beginning of muscle compression, and immediately significantly decreased at the initiation of ischemia-reperfusion injury in compressed muscles tissue. These data show that ER stress may be involved in the underlying mechanisms of cell degeneration after pressure ulcers and that the Akt/GSK3ß signal pathway may play an important role in deep tissue injury induced by pressure and ischemia/reperfusion.

bFGF attenuates endoplasmic reticulum stress and mitochondrial injury on myocardial ischaemia/reperfusion via activation of PI3K/Akt/ERK1/2 pathway.

Extensive research focused on finding effective strategies to prevent or improve recovery from myocardial ischaemia/reperfusion (I/R) injury. Basic fibroblast growth factor (bFGF) has been shown to have therapeutic potential in some heart disorders, including ischaemic injury. In this study, we demonstrate that bFGF administration can inhibit the endoplasmic reticulum (ER) stress and mitochondrial dysfunction induced in the heart in a mouse model of I/R injury. In vitro, bFGF exerts a protective effect by inhibiting the ER stress response and mitochondrial dysfunction proteins that are induced by tert-Butyl hydroperoxide (TBHP) treatment. Both of these in vivo and in vitro effects are related to the activation of two downstream signalling pathways, PI3K/Akt and ERK1/2. Inhibition of these PI3K/Akt and ERK1/2 pathways by specific inhibitors, LY294002 and PD98059, partially reduces the protective effect of bFGF. Taken together, our results indicate that the cardioprotective role of bFGF involves the suppression of ER stress and mitochondrial dysfunction in ischaemic oxidative damage models and oxidative stress-induced H9C2 cell injury; furthermore, these effects underlie the activation of the PI3K/Akt and ERK1/2 signalling pathways.