Balance of Macrophage Activation by a Complex Coacervate-Based Adhesive Drug Carrier Facilitates Diabetic Wound Healing.

Uncontrolled and sustained inflammation disrupts the wound-healing process and produces excessive reactive oxygen species, resulting in chronic or impaired wound closure. Natural antioxidants such as plant-based extracts and natural polysaccharides have a long history in wound care. However, they are hard to apply to wound beds due to high levels of exudate or anatomical sites to which securing a dressing is difficult. Therefore, we developed a complex coacervate-based drug carrier with underwater adhesive properties that circumvents these challenges by enabling wet adhesion and controlling inflammatory responses. This resulted in significantly accelerated wound healing through balancing the pro- and anti-inflammatory responses in macrophages. In brief, we designed a complex coacervate-based drug carrier (ADC) comprising oligochitosan and inositol hexaphosphate to entrap and release antioxidant proanthocyanins (PA) in a sustained way. The results from in vitro experiments demonstrated that ADC is able to reduce LPS-stimulated pro-inflammatory responses in macrophages. The ability of ADC to reduce LPS-stimulated pro-inflammatory responses in macrophages is even more promising when ADC is encapsulated with PA (ADC-PA). Our results indicate that ADC-PA is able to polarize macrophages into an M2 tissue-healing phenotype via up-regulation of anti-inflammatory and resolution of inflammatory responses. Treatment with ADC-PA around the wound beds fine-tunes the balance between the numbers of inducible nitric oxide synthase-positive (iNOS+) and mannose receptor-negative (CD206-) M1 and iNOS-CD206+ M2 macrophages in the wound microenvironment compared to controls. Achieving such a balance between the numbers of iNOS+CD206- M1 and iNOS-CD206+ M2 macrophages in the wound microenvironment has led to significantly improved wound closure in mouse models of diabetes, which exhibit severe impairments in wound healing. Together, our results demonstrate for the first time the use of a complex coacervate-based drug delivery system to promote timely resolution of the inflammatory responses for diabetic wound healing by fine-tuning the functions of macrophages.

Proanthocyanidins-loaded complex coacervates-based drug delivery attenuates oral squamous cell carcinoma cells metastatic potential through down-regulating the Akt signaling pathway.

Oral cancer, constituted up to 90% by squamous cell carcinomas, is a significant health burden globally. Grape seed proanthocyanidins (PA) have been suggested as a potential chemopreventive agent for oral cancer. However, their efficacy can be restricted due to the low bioavailability and bioaccessibility. Inspired by sandcastle worm adhesive, we adapted the concept of complex coacervation to generate a new type of drug delivery platform. Complex coacervates are a dense liquid phase formed by the associative separation of a mixture of oppositely charged polyelectrolytes, can serve as a drug delivery platform to protect labile cargo. In this study, we developed a complex coacervates-based delivery of PA. The release kinetics was measured, and anticancer effects were determined in two human tongue squamous cell carcinoma cell lines. The results showed that complex coacervate successfully formed and able to encapsulate PA. Additionally, PA were steadily released from the system in a pH-dependent manner. The drug delivery system could significantly inhibit the cell proliferation, migration, and invasion of cancer cells. Moreover, it could markedly reduce the expression of certain matrix metalloproteinases (MMP-2, 9, and 13) crucial to metastatic processes. We also found that suppression of protein kinase B (Akt) pathway might be the underlying mechanism for these anticancer activities. Taken together, complex coacervates-based delivery of PA can act as an effective anticancer approach for oral cancer therapy.

Transcranial photobiomodulation (808 nm) attenuates pentylenetetrazole-induced seizures by suppressing hippocampal neuroinflammation, astrogliosis, and microgliosis in peripubertal rats.

Significance: Transcranial photobiomodulation (tPBM) at 808 nm attenuates pentylenetetrazole (PTZ)-induced seizures and convulsive status epilepticus (CSE) in peripubertal rats by protecting neurons from injury and parvalbumin-positive interneurons from apoptosis, and preserving the integrity of perisomatic inhibitory networks. However, the effects of tPBM on neuroinflammation, astrogliosis, and microgliosis in epileptic rat brains are unknown. Thus, further study to unveil these aspects is needed for understanding the phenomena of tPBM on pediatric CSE prevention. Aim: To evaluate the effects of tPBM on neuroinflammation, astrogliosis, and microgliosis in peripubertal rat hippocampus with PTZ-induced seizures and SE. Approach: An 808-nm diode laser was applied transcranially to peripubertal rats prior to PTZ injection. Immunofluorescence staining of neuron-specific enolase (NSE) was used as a marker of neuroinflammation, glial fibrillary acid protein (GFAP) for astrogliosis, ionized calcium-binding adapter molecule 1 (Iba-1) for microgliosis, and mitochondrial cytochrome c oxidase subunit 1 (MT-CO1) for confirming the involvement of cytochrome c oxidase (CCO). Results: tPBM significantly reduced NSE immunoreactivity in CA3 in PTZ-treated rats, GFAP immunoreactivity in CA1, and Iba-1 immunoreactivity in CA3. Enhancement of hippocampal MT-CO1 reflected that tPBM acted in CCO-dependent manner. Conclusions: tPBM (808) attenuated PTZ-induced seizures and SE by suppressing neuroinflammation, astrogliosis, and microgliosis in peripubertal rats.

Transcranial photobiomodulation add-on therapy to valproic acid for pentylenetetrazole-induced seizures in peripubertal rats.

BACKGROUND: Convulsive status epilepticus (CSE) prevention is critical for pediatric patients with epilepsy. Immediate intervention before CSE reduce severity. Despite its wide usage as an anticonvulsant, valproic acid (VPA) results in harmful side effects such as dose-dependent hepatotoxicity. Hence, reducing VPA dosage to minimize side effects while maintaining its efficacy is necessary, and transcranial photobiomodulation (tPBM) add-on therapy could facilitate this. We recently demonstrated for the first time that tPBM at a wavelength of 808 nm attenuated CSE in peripubertal rats. However, the effects of VPA with the add-on therapy of tPBM prior to seizures have not yet been explored. This study investigated whether adding tPBM to VPA exerts synergistic effect for CSE prevention in peripubertal rats. METHODS: A gallium-aluminum-arsenide laser (wavelength of 808 nm with an exposure duration of 100 s and irradiance of 1.333 W/cm2 at the target) was applied transcranially 30 min after VPA injection in Sprague Dawley rats. All the rats received 90 mg/kg of pentylenetetrazole (PTZ). Except for the saline (n = 3), tPBM + saline (n = 3), and PTZ group (n = 6), all the rats received a PTZ injection 30 min after VPA injection. The rats received add-on tPBM with PTZ immediately after tPBM. In the VPA + PTZ group, the rats received low-dose (100 mg/kg, n = 6), medium-dose (200 mg/kg, n = 6), and high-dose (400 mg/kg, n = 7) VPA. In the VPA + tPBM + PTZ group, the rats received low (100 mg/kg, n = 5), medium (200 mg/kg, n = 6), and high (400 mg/kg, n = 3) doses of VPA. Seizures were evaluated according to the revised Racine's scale in a non-blinded manner. RESULTS: Adding tPBM to low-dose VPA reduced the incidence of severe status epilepticus and significantly delayed the latency to stage 2 seizures. However, adding tPBM to high-dose VPA increased the maximum seizure stage, prolonged the duration of stage 4-7 seizures, and shortened the latency to stage 6 seizures. CONCLUSIONS: Adding tPBM to low-dose VPA exerted a synergistic prevention effect on PTZ-induced seizures, whereas adding tPBM to high-dose VPA offset the attenuation effect.

Exerting the Appropriate Application of Methylprednisolone in Acute Spinal Cord Injury Based on Time Course Transcriptomics Analysis.

Methylprednisolone (MP) is an anti-inflammatory drug approved for the treatment of acute spinal cord injuries (SCIs). However, MP administration for SCIs has become a controversial issue while the molecular effects of MP remain unexplored to date. Therefore, delineating the benefits and side effects of MP and determining what MP cannot cure in SCIs at the molecular level are urgent issues. Here, genomic profiles of the spinal cord in rats with and without injury insults, and those with and without MP treatment, were generated at 0, 2, 4, 6, 8, 12, 24, and 48 h post-injury. A comprehensive analysis was applied to obtain three distinct classes: side effect of MP (SEMP), competence of MP (CPMP), and incapability of MP (ICMP). Functional analysis using these genes suggested that MP exerts its greatest effect at 8~12 h, and the CPMP was reflected in the immune response, while SEMP suggested aspects of metabolism, such as glycolysis, and ICMP was on neurological system processes in acute SCIs. For the first time, we are able to precisely reveal responsive functions of MP in SCIs at the molecular level and provide useful solutions to avoid complications of MP in SCIs before better therapeutic drugs are available.

Assessment of the Oral Delivery of a Myelin Basic Protein Gene Promoter with Antiapoptotic bcl-xL (pMBP-bcl-xL) DNA by Cyclic Peptide Nanotubes with Two Aspect Ratios and Its Biodistribution in the Brain and Spinal Cord.

Cyclo-(D-Trp-Tyr) peptide nanotubes (PNTs) were reported to be potential carriers for oral gene delivery in our previous study; however, the effect of the aspect ratio (AR) of these PNTs on gene delivery in vivo could affect penetration or interception in biological environments. The aim of this study was to assess the feasibility of cyclo-(D-Trp-Tyr) PNTs with two ARs as carriers for oral pMBP-bcl-xL-hRluc delivery to the spinal cord to treat spinal cord injury (SCI). We evaluated the biodistribution of oligodendrocyte (OLG)-specific myelin basic protein gene promoter-driven antiapoptotic DNA (pMBP-bcl-xL) to the brain and spinal cord delivered with cyclo-(D-Trp-Tyr) PNTs with large (L) and small (S) PNTs with two ARs. After complex formation, the length, width, and AR of the L-PNTs/DNA were 77.86 ± 3.30, 6.51 ± 0.28, and 13.75 ± 7.29 µm, respectively, and the length and width of the S-PNTs/DNA were 1.17 ± 0.52 and 0.17 ± 0.05 µm, respectively, giving an AR of 7.12 ± 3.17 as detected by scanning electron microscopy. Each of these three parameters exhibited significant differences (p < 0.05) between L-PNTs/DNA and S-PNTs/DNA. However, there were no significant differences (p > 0.05) between the L-PNTs and S-PNTs for either their DNA encapsulation efficiency (29.72 ± 14.19 and 34.31 ± 16.78%, respectively) or loading efficiency (5.15 ± 2.58 and 5.95 ± 2.91%). The results of the in vitro analysis showed that the S-PNT/DNA complexes had a significantly higher DNA release rate and DNA permeation in the duodenum than the L-PNT/DNA complexes. Using Cy5 and TM-rhodamine to individually and chemically conjugate the PNTs with plasmid DNA, we observed, using laser confocal microscopy, that the PNTs and DNA colocalized in complexes. We further confirmed the complexation between DNA and the PNTs using fluorescence resonance energy transfer (FRET). Data from an in vivo imaging system (IVIS) showed that there was no significant difference (p > 0.05) in PNT distribution between L-PNTs/DNA and S-PNTs/DNA within 4 h. However, the S-PNT/DNA group had a significantly higher DNA distribution (p < 0.05) in several organs, including the ilium, heart, lungs, spleen, kidneys, testes, brain, and spinal cord. Finally, we determined the bcl-xL protein expression levels in the brain and spinal cord regions for the L-PNT/DNA and S-PNT/DNA complex formulations. These results suggested that either L-PNTs or S-PNTs may be used as potential carriers for oral gene delivery to treat SCI.

Synergistic effects of electronegative-LDL- and palmitic-acid-triggered IL-1ß production in macrophages via LOX-1- and voltage-gated-potassium-channel-dependent pathways.

Electronegative LDL (LDL(-)) and free fatty acids (FFAs) are circulating risk factors for cardiovascular diseases (CVDs) and have been associated with inflammation. Interleukin-1 beta (IL-1ß) represents a key cytokine in the development of CVD; however, the initial trigger of IL-1ß in CVD remains to be explored. In this study, we investigated the combined effects of LDL(-) from the plasma of ST-segment elevation myocardial infarction (STEMI) patients or diet-induced hypercholesterolemic rabbits and bovine serum albumin bound palmitic acid (PA-BSA) on IL-1ß production in macrophages. Macrophages derived from THP-1 cells or human peripheral blood mononuclear cells were independently treated with LDL(-), PA-BSA or cotreated with LDL(-) and PA-BSA. The results showed that nLDL and/or PA-BSA had no effect on IL-1ß, and LDL(-) slightly increased IL-1ß; however, cotreatment with LDL(-) and PA-BSA resulted in abundant secretion of IL-1ß in macrophages. Rabbit LDL(-) induced the elevation of cellular pro-IL-1ß and p-Iκ-Bα, but PA-BSA had no effect on pro-IL-1ß or p-Iκ-Bα. In potassium-free buffer, LDL(-)-induced IL-1ß reached a level similar to that induced by cotreatment with LDL(-) and PA-BSA. Moreover, LDL(-) and PA-BSA-induced IL-1ß was inhibited in lectin-type oxidized LDL receptor-1 (LOX-1) knockdown cells and by blockers of voltage-gated potassium (Kv) channels. LDL(-) from diet-induced hypercholesterolemic rabbit had a similar effect as STEMI LDL(-) on IL-1ß in macrophages. These results show that PA-BSA cooperates with LDL(-) to trigger IL-1ß production in macrophages via a mechanism involving the LOX-1 and Kv channel pathways, which may play crucial roles in the regulation of inflammation in CVD.

Transformation of 15-ene steviol by Aspergillus niger, Cunninghamella bainieri, and Mortierella isabellina.

Transformation of 15-ene steviol (ent-13-hydroxy-kaur-15-en-19-oic acid) by growth cultures of Aspergillus niger BCRC 32720, Cunninghamella bainieri ATCC 9244, and Mortierella isabellina ATCC 38063 was conducted to generate various derivatives for the development of bioactive compounds. Four previously undescribed compounds along with six known compounds were obtained. The newly identified isolates were characterized using 1D and 2D NMR, IR, and HRESIMS, and three compounds were further confirmed by X-ray crystallographic analyses. Subsequently, the effects of 15-ene steviol and its derivatives on lipopolysaccharide (LPS)-induced cytokine production by THP-1 cells were examined, with dexamethasone used as a positive control. Results indicated that most of the tested compounds showed lower inhibitory effects than those detected in the dexamethasone-treated group, except that 15-ene steviol showed better effects than dexamethasone on the reduction of LPS-induced monocyte chemoattractant protein (MCP)-1, -2, and -3 release. Three specialized products similarly showed better effects than dexamethasone on the inhibition of LPS-induced secretion of regulated on activation, normal T cell expressed and secreted (RANTES). Moreover, none of the tested compounds showed any cytotoxicity or triggered cell apoptosis, and none affected the protein integrity of toll-like receptor 4 (TLR4) or MyD88, suggesting that these compounds may exert the anti-inflammatory activity downstream of membrane-associated TLR4 and MyD88 molecules.

Electronegative LDL Induces M1 Polarization of Human Macrophages Through a LOX-1-Dependent Pathway.

In response to environmental stimuli, monocytes undergo polarization into classically activated (M1) or alternatively activated (M2) states. M1 and M2 macrophages exert opposing pro- and anti-inflammatory properties, respectively. Electronegative low-density lipoprotein (LDL) (LDL(-)) is a naturally occurring mildly oxidized LDL found in the plasma of patients with hypercholesterolemia, diabetes, and acute myocardial infarction, and has been shown to involve in the pathogenesis of atherosclerosis. In this study, we examined the effects of LDL(-) on macrophage polarization and the involvement of lectin-like oxidized LDL receptor-1 (LOX-1) in this process. THP-1 macrophages were treated with native LDL (nLDL) or LDL(-), and then the expression of M1/M2-related surface markers and cytokines were evaluated. The results show that treatment with LDL(-) resulted in profound increase in proinflammatory cytokines, IL-1ß, IL-6, and TNF-α, and M1-surface marker CD86; however, M2-related cytokines, IL-10 and TGF-ß, and M2-surface marker CD206 were not changed by LDL(-). Untreated or nLDL-treated cells were used as control. LDL(-)-induced M1 polarization and secretion of proinflammatory cytokines were diminished in LOX-1 knockdown cells. Taken together, the results show that LDL(-) promotes differentiation of human monocytes to M1 macrophages through a LOX-1-dependent pathway, and explore the contribution of LDL(-) and LOX-1 to the development of chronic inflammation in atherosclerosis.

Transcranial photobiomodulation attenuates pentylenetetrazole-induced status epilepticus in peripubertal rats.

Convulsive status epilepticus is the most common neurological emergency in children. Transcranial photobiomodulation (tPBM) reverses elevated rodent neurotransmitters after status epilepticus (SE) yet whether tPBM can attenuate seizure behaviors remains unknown. Here, we applied near-infrared laser at wavelength 808 nm transcranially to peripubertal Sprague-Dawley rats prior to pentylenetetrazole (PTZ) injection. Hematoxylin-eosin, immunofluorescence (IF) staining with anti-parvalbumin (PV) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay after IF staining was performed. Behaviorally, tPBM attenuated the mean seizure score and reduced the incidence of SE and mortality. Histochemically, tPBM reduced dark neurons in the cortex, hippocampus, thalamus and hypothalamus, lessened the apoptotic ratio of parvalbumin-positive interneurons (PV-INs) and alleviated the aberrant extent of PV-positive unstained somata of PCs in the hippocampus. Conclusively, tPBM attenuated PTZ-induced seizures, SE and mortality in peripubertal rats and reduced PTZ-induced neuronal injury, apoptosis of PV-INs and preserved PV positive perisomatic inhibitory network in the hippocampus.

The Mycobacterial Adjuvant Analogue TDB Attenuates Neuroinflammation via Mincle-Independent PLC-γ1/PKC/ERK Signaling and Microglial Polarization.

Microglial activation has long been recognized as a hallmark of neuroinflammation. Recently, the bacillus Calmette-Guerin (BCG) vaccine has been reported to exert neuroprotective effects against several neurodegenerative disorders. Trehalose-6,6'-dibehenate (TDB) is a synthetic analogue of trehalose-6,6'-dimycolate (TDM, also known as the mycobacterial cord factor) and is a new adjuvant of tuberculosis subunit vaccine currently in clinical trials. Both TDM and TDB can activate macrophages and dendritic cells through binding to C-type lectin receptor Mincle; however, its action mechanism in microglia and their relationship with neuroinflammation are still unknown. In this article, we found that TDB inhibited LPS-induced M1 microglial polarization in primary microglia and BV-2 cells. However, TDB itself had no effects on IKK, p38, and JNK activities or cytokine expression. In contrast, TDB activated ERK1/2 through PLC-γ1/PKC signaling and in turn decreased LPS-induced NF-κB nuclear translocation. Furthermore, TDB-induced AMPK activation via PLC-γ1/calcium/CaMKKß-dependent pathway and thereby enhanced M2 gene expressions. Interestingly, knocking out Mincle did not alter the anti-inflammatory and M2 polarization effects of TDB in microglia. Conditional media from LPS-stimulated microglial cells can induce in vitro neurotoxicity, and this action was attenuated by TDB. Using a mouse neuroinflammation model, we found that TDB suppressed LPS-induced M1 microglial activation and sickness behavior, but promoted M2 microglial polarization in both WT and Mincle-/- mice. Taken together, our results suggest that TDB can act independently of Mincle to inhibit LPS-induced inflammatory response through PLC-γ1/PKC/ERK signaling and promote microglial polarization towards M2 phenotype via PLC-γ1/calcium/CaMKKß/AMPK pathway. Thus, TDB may be a promising therapeutic agent for the treatment of neuroinflammatory diseases.

AMPK-dependent and independent actions of P2X7 in regulation of mitochondrial and lysosomal functions in microglia.

BACKGROUND: P2X7 is ubiquitously expressed in myeloid cells and regulates the pathophysiology of inflammatory diseases. Since mitochondrial function in microglia is highly associated with microglial functions in controlling neuronal plasticity and brain homeostasis, we interested to explore the roles of P2X7 in mitochondrial and lysosomal functions as well as mitophagy in microglia. METHODS: P2X7-/- bone marrow-derived macrophages (BMDM), primary microglia and BV-2 immortalized microglial cells were used to detect the particular protein expression by immunoblotting. Mitochondrial reactive oxygen species (mitoROS), intracellular calcium, mitochondrial mass and lysosomal integrity were examined by flow cytometry. Mitochondrial oxygen consumption rate (OCR) was recorded using Seahorse XF flux analyzer. Confocal microscopic images were performed to indicate the mitochondrial dynamics and mitophagy after P2X7 activation. RESULTS: In primary microglia, BV-2 microglial cells and BMDM, P2X7 agonist BzATP triggered AMPK activation and LC3II accumulation through reactive oxygen species (ROS) and CaMKKII pathways, and these effects were abolished by P2X7 antagonist A438079 and P2X7 deficiency. Moreover, we detected the dramatic decreases of mitochondrial OCR and mass following P2X7 activation. AMPK inhibition by compound C or AMPK silencing reversed the P2X7 actions in reduction of mitochondrial mass, induction of mitochondrial fission and mitophagy, but not in uncoupling of mitochondrial respiration. Interestingly, we found that P2X7 activation induced nuclear translocation of TFEB via an AMPK-dependent pathway and led to lysosomal biogenesis. Mimicking the actions of BzATP, nigericin also induced ROS-dependent AMPK activation, mitophagy, mitochondrial fission and respiratory inhibition. Longer exposure of BzATP induced cell death, and this effect was accompanied by the lysosomal instability and was inhibited by autophagy and cathepsin B inhibitors. CONCLUSION: Altogether ROS- and CaMKK-dependent AMPK activation is involved in P2X7-mediated mitophagy, mitochondrial dynamics and lysosomal biogenesis in microglial cells, which is followed by cytotoxicity partially resulting from mitophagy and cathepsin B activation.

In vitro and in vivo assessment of delivery of hydrophobic molecules and plasmid DNAs with PEO-PPO-PEO polymeric micelles on cornea.

The stability and bio-distribution of genes or drug complexes with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO, Pluronic F-68) polymeric micelles (PM) are essential for an effective nanosized PM delivery system. We used Förster resonance energy transfer (FRET) pairs with PM and measured the FRET ratio to assess the stability of PM in vitro and in vivo on the cornea. The FRET ratio reached a plateau at 0.8 with 3% PM. Differential scanning calorimetry measurement confirmed the complex formation of FRET pairs with PM. Confocal imaging with the fluorophores fluorescein isothiocyanate isomer I (FITC) and rhodamine B base (RhB) also showed the occurrence of FRET pairs in vitro. The fluorophores were mixed with 3% PM solution or the FITC-labeled PEO-PPO-PEO polymers (FITC-P) were mixed with RhB-labeled plasmids (RhB-DNA). In addition, the in vitro corneal permeation of FRET pair complexes with PM reached a 0.8 FRET ratio. One hour after eye drop administration, FRET pairs colocalized in the cytoplasm, and surrounded and entered the nuclei of cells in the cornea, and the polymers were located in the corneal epithelial layers, as detected through anti-PEG immunohistochemistry. Furthermore, fluorescence colocalization in the cytoplasm and cell nucleus of the corneal epithelium was confirmed in tissues where RhB or RhB-DNA complexed with FITC-P was found to accumulate. We demonstrate that at a concentration of 3%, PM can encapsulate FRET pairs or RhB-DNA and retain their integrity within the cornea 1 h after administration, suggesting the feasibility and stability of PEO-PPO-PEO polymers as a vehicle for drug delivery.

Coordinate effects of P2X7 and extracellular acidification in microglial cells.

Extracellular adenosine 5'-triphosphate (ATP) is a damage-associated molecular pattern and contributes to inflammation associated diseases including cancer. Extracellular acidosis is a novel danger signal in the inflammatory sites, where it can modulate inflammation, immunity and tumor growth. Extracellular acidification was shown to inhibit P2X7-mediated channel currents, while it remains unknown how acidification and P2X7 together affect cellular responses. Here, we treated BV-2 microglial cells with ATP in a short period (<15 min) or a sustained acidified condition. For short acidification we compared the actions of neutralized ATP and acidic ATP in a condition with pH buffering. For sustained acidification, we treated cells with neutralized ATP in acidic medium or acidic ATP in medium without pH buffering. In the short acidified condition, neutralized ATP induced higher responses than acidic ATP to increase intracellular calcium and reactive oxygen species, decrease intracellular potassium and induce cell death. In contrast, these cellular responses and mitochondrial fission caused by neutralized ATP were enhanced by pH 6.0 and pH 4.5 media. P2X7 activation can also rapidly block mitochondrial ATP turnover and respiration capacity, both of which were mimicked by nigericin and enhanced by acidity. Taken together P2X7-mediated ionic fluxes and reactive oxygen species production are attenuated under short acidification, while sustained acidification itself can induce mitochondrial toxicity which deteriorates the mitochondrial function under P2X7 activation.

Electronegative LDL is linked to high-fat, high-cholesterol diet-induced nonalcoholic steatohepatitis in hamsters.

The pathogenesis of nonalcoholic steatohepatitis (NASH), like that of atherosclerosis, involves lipid accumulation, inflammation and fibrosis. Recent studies suggest that oxidized LDL (oxLDL) may be a risk factor for NASH, but oxLDL levels were not directly measured in these studies. The aim of this study was to examine whether there was an association between electronegative LDL [LDL(-)], a mildly oxLDL found in the blood, and the development of NASH using two animal models. Golden Syrian hamsters and C57BL/6 mice were fed a high-fat, high-cholesterol (HFC) diet for 6 or 12weeks, then liver lipid and histopathology, plasma lipoprotein profile and LDL(-) levels were examined. The HFC-diet-fed hamsters and mice had similar levels of hepatic lipid but different histopathological changes, with microvesicular steatosis, hepatocellular hypertrophy, inflammation and bridging fibrosis in the hamsters, but only in mild steatohepatitis with low inflammatory cell infiltration in the mice. It also resulted in a significant increase in plasma levels of LDL cholesterol and LDL(-) in hamsters, but only a slight increase in mice. Moreover, enlarged Kupffer cells, LDL(-) and accumulation of unesterified cholesterol were detected in the portal area of HFC-diet-fed hamsters, but not HFC-diet-fed mice. An in vitro study showed that LDL(-) from HFC-diet-fed hamsters induced TNF-α secretion in rat Kupffer cell through a LOX-1-dependent pathway. Our results strongly suggest that LDL(-) is one of the underlying causes of hepatic inflammation and plays a critical role in the development of NASH.

SB203580 increases G-CSF production via a stem-loop destabilizing element in the 3' untranslated region in macrophages independently of its effect on p38 MAPK activity.

BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) is a major regulator of the production and survival of neutrophils. Regulation of G-CSF expression is complex and occurs at both transcription and post-transcription levels. Two distinct types of cis-acting elements in the 3' untranslated region (3'UTR) of G-CSF mRNA have been identified as destabilizing elements; these consist of adenylate uridylate-rich elements (AUREs) and a stem-loop destabilizing element (SLDE). Regulation of the stability of mRNA by p38 mitogen-activated protein kinase (MAPK) has been indicated to be linked to AUREs in the 3'UTR. However, whether p38 MAPK is involved in the regulation of the stability of G-CSF mRNA has not been elucidated. This study investigated the effect of SB203580, an inhibitor of p38 MAPK, on the lipopolysaccharide-induced G-CSF expression in macrophages at the post-transcription level. RESULTS: Our study showed surprising results that SB203580 augmented the lipopolysaccharide-induced increase in the G-CSF mRNA levels in RAW264.7 mouse macrophages, mouse bone marrow-derived macrophages and in THP-1 human macrophages. This effect was also seen in p38α MAPK knockdown RAW264.7 cells, showing that it was not due to inhibition of p38 MAPK activity. In the presence of actinomycin D, the decay of G-CSF mRNA was slower in SB203580-treated cells than in control cells, showing that SB203580 increased the stability of G-CSF mRNA. Reporter genes containing luciferase with or without the 3'UTR of G-CSF were constructed and transfected into RAW264.7 cells and the results showed that the presence of the 3'UTR reduced the luciferase mRNA levels and luciferase activity. Furthermore, SB203580 increased the luciferase mRNA levels and activity in RAW264.7 cells transfected with the luciferase reporter containing the 3'UTR, but not in cells transfected with the luciferase reporter without the 3'UTR. Mutations of the highly conserved SLDE in the 3'UTR abolished these effects, showing that the SLDE was essential for the SB203580-induced increase in the stability of mRNA. CONCLUSIONS: SB203580 increases G-CSF expression in macrophages by increasing the stability of G-CSF mRNA via its 3'UTR, and the effect was not due to its inhibition of p38 MAPK activity. The results of this study also highlight a potential target for boosting endogenous production of G-CSF during neutropenia.

YC-1 induces lipid droplet formation in RAW 264.7 macrophages.

BACKGROUND: 3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) is a potential anticancer drug that may activate soluble guanylyl cyclase (sGC) and increase the level of cyclic guanosine monophosphate (cGMP). The aim of this study was to explore the effects of YC-1 on lipid droplet accumulation and foam cell formation in macrophages. RESULTS: Human-oxidized low density lipoprotein (ox-LDL) was used to induce accumulation of lipid droplets in a murine macrophage cell line, RAW 264.7. Oil red O staining showed that treatment with 20 µM YC-1 for 24 h increased the area of intracellular lipid droplets in macrophages. The results of high content screening (HCS) with the AdipoRed™ assay further revealed that YC-1 enhanced ox-LDL-induced foam cell formation. This was evidenced by an increase in the total area of lipid droplets and the mean fluorescence intensity per cell. Inhibition of cGMP-dependent protein kinase (PKG) using KT5823 significantly reduced YC-1-enhanced lipid droplet formation in ox-LDL-induced macrophage foam cells. CONCLUSION: YC-1 induces lipid droplet formation in macrophages, possibly through the sGC/cGMP/PKG signaling pathway. This chemical should be tested with caution in future clinical trials.

Gene methylation of human ovarian carcinoma stromal progenitor cells promotes tumorigenesis.

BACKGROUND: This study aimed to investigate whether the DNA methylation of human ovarian carcinoma stromal progenitor cells (OCSPCs) could promote the tumorigenesis of ovarian carcinoma. METHODS: OCSPCs were first isolated from fresh tumor tissues and ascites of ovarian cancer patients. In vivo and in vitro experiments on the effect of the OCSPCs on tumorigenesis and the effects of DNA demethylation on the OCSPCs were then performed. RESULTS: The OCSPCs possessed self-renewal and multipotent differentiation capacity with elevated expressions of OCT4, NANOG, BMP2, BMP4, Rex-1, AC133 and TGF-ß. The OCSPCs, when combined with tumor cells in vivo could promote tumor growth. The methylation profiles of tumor suppressor genes (TSGs) were significantly higher in the OCSPCs than in ovarian cancer cells (p < 0.001). 5-aza-2-dC could alter the methylation levels of TSGs in OCSPCs and also inhibit the tumor promoting capabilities of the OCSPCs by decreasing the proliferation of tumors cells. The expression levels of TSGs were re-expressed by 5-aza-2-dC to inhibit the self-renewal and growth of OCSPCs. CONCLUSIONS: OCSPCs with decreased TSG expressions in the ovarian tumor microenvironment were able to promote tumorigenesis which could be reversed by DNA demethylation. DNA demethylation reversing the expression of TSGs in OCSPCs may represent a potential therapeutic target for ovarian cancer.

Demethylation of HIN-1 reverses paclitaxel-resistance of ovarian clear cell carcinoma through the AKT-mTOR signaling pathway.

BACKGROUND: Methylation of HIN-1 is associated with poor outcomes in patients with ovarian clear cell carcinoma (OCCC), which is regarded to be an aggressive, chemo-resistant histological subtype. This study aimed to evaluate whether 5-aza-2-deoxycytidine (5-aza-2-dC) can reverse methylation of the HIN-1 gene to restore chemo-sensitivity of OCCC and the possible mechanism. METHODS: In vitro flow cytometric analysis and evaluation of caspase-3/7 activity of paclitaxel-sensitive and resistant OCCC cell lines were performed. Methylation status and expression changes of HIN-1 in the OCCC cell lines treated with 5-aza-2-dC were evaluated, and immunohistochemical staining of HIN-1 in OCCC tissues was performed. In vivo tumor growth with or without 5-aza-2-dC treatment was analyzed, and Western blotting of AKT-mTOR signaling-related molecules was performed. RESULTS: G2-M phase arrest was absent in paclitaxel-resistant OCCC cells after treatment with the cytotoxic drug. The caspase activities of the chemo-resistant OCCC cells were lower than those of the chemo-sensitive OCCC cells when treated with paclitaxel. Methylation of HIN-1 was noted in paclitaxel-resistant OCCC cell lines and cancerous tissues. 5-aza-2-dC reversed the methylation of HIN-1, re-activated the expression of HIN-1, and then suppressed the in vivo tumor growth of paclitaxel-resistant OCCC cells. Immunoblotting revealed that phospho-AKT473 and phospho-mTOR were significantly increased in HIN-1-methylated paclitaxel-resistant OCCC cell lines. However, the expressions of phospho-AKT at Ser473 and Thr308 and phospho-mTOR decreased in the OCCC cells with a high expression of HIN-1. CONCLUSIONS: Demethylating agents can restore the HIN-1 expression in paclitaxel-resistant OCCC cells through the HIN-1-AKT-mTOR signaling pathway to inhibit tumor growth.

Anti-Apoptotic Gene Delivery with cyclo-(d-Trp-Tyr) Peptide Nanotube via Eye Drop Following Corneal Epithelial Debridement.

Corneal keratocyte apoptosis triggered by cornel debridement is one mechanism of corneal disorders. In this study, the feasibility of cyclo-(d-Trp-Tyr) peptide nanotubes (PNTs) as carriers of caspase 3 silence shRNA delivery was assessed. A model of epithelial injury by epithelial debridement was applied to investigate the feasibility of PNTs as gene delivery carriers on corneal injury. First, the PNTs were found within 2 µm in length and 300 nm in width by an atomic force microscope and confocal laser microscope system. Plasmid DNAs were observed to be associated with PNTs by atomic force microscope and confocal laser scanning microscope. The plasmids were associated with tyrosine of PNTs with a binding constant of 2.7 × 108 M-1. The stability of plasmid DNA with PNTs against the DNase was found at 60 min. Using thioflavin T pre-stained PNTs on the corneal eye drop delivery, the distribution of PNTs was in the epithelial and stroma regions. After corneal debridement, the rhodamine-labeled plasmid DNA and thioflavin T pre-stained PNTs were also delivered and could be observed in the stroma of cornea. PNTs complexed with anti-apoptotic plasmid caspase 3 silencing shRNA eye drop delivery decreased 41% of caspase 3 activity after the first dose by caspase 3 activity and Western blot analysis.