Inflammatory response in the mid colon of ICR mice treated with polystyrene microplastics for two weeks

Background The oral administration of polystyrene-microplastics (PS-MPs) causes chronic constipation of ICR mice, but there are no reports on their effects on the inflammatory response in the colon. To determine if the oral administration of MPs causes inflammation in the colon, the changes in the apoptosis-associated speck like protein containing a caspase recruitment domain (ASC)-inflammasome pathway, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway, and inflammatory cytokine expression were evaluated in the mid colon of ICR mice treated with 0.5 μm size PS-MPs for two weeks. Results The thicknesses of the mucosa, muscle, flat luminal surface, and crypt layer were decreased significantly (p < 0.01) in the mid colon of the MPs treated group compared to the Vehicle treated group. On the other hand, a remarkable increase in the expression levels of NOD-like receptor pyrin domain-containing protein (NLRP) 3, ASC, and Cleaved Caspase (Cas)-1 protein was observed in the MPs treated group. In addition, similar increasing pattern in the levels of p-NF-κB and phospho-inhibitory subunit of NF-κB (p-IkB) α protein was detected. Four inflammatory cytokines, including NF-κB, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β, showed an increased expression level after the MPs treatment. Conclusions Therefore, the present study suggests that PS-MPs can be a novel cause of an inflammatory response in the mid colon of ICR mice.

Anti-Obesity Effect of α-Cubebenol Isolated from Schisandra chinensis in 3T3-L1 Adipocytes

The efficacy of α-cubebenol isolated from Schisandra chinensis has been studied in several diseases, including cecal ligation, puncture challenge-induced sepsis, and degranulation of neutrophils. To identify the novel functions of α-cubebenol on lipid metabolism, alterations on the regulation of lipogenesis, lipolysis, and inflammatory response were observed in 3T3-L1 adipocytes treated with α-cubebenol. Most lipogenic targets, including lipid accumulation, level of lipogenic transcription factors, and expression of lipogenic regulators, were suppressed in MDI (3-isobutyl-1-methylxanthine, dexamethasone, and insulin)-stimulated 3T3-L1 adipocytes treated with α-cubebenol without significant cytotoxicity. In addition, similar inhibition effects were observed in the iNOS-induced COX-2 mediated pathway and NLRP3 inflammasome pathway of MDI-stimulated 3T3-L1 cells treated with α-cubebenol. Lipolytic targets, such as cAMP concentration, expression of adenylyl cyclase and PDE4, and their downstream signaling pathway, in MDI-stimulated 3T3-L1 cells were stimulated by the α-cubebenol treatment. The levels of transcription factors and related proteins for β-oxidation were significantly higher in the MDI + α-cubebenol treated group than in the MDI + Vehicle treated group. These results show that α-cubebenol has a novel role as a lipogenesis inhibitor, lipolysis and β-oxidation stimulator, and inflammasome suppressor in MDI-stimulated 3T3-L1 adipocytes.

YAP/TAZ Promote Hematopoietic Regeneration Via Accelerating the Recovery of Bone Marrow Niche

Adult hematopoietic stem cells (HSCs) reside and are protected in a unique bone marrow (BM) microenvironment, termed the HSC niche, which consists mainly of vascular endothelial cells (EC) and EC-associated mesenchymal stromal cells (MSC). Myeloablative stresses, such as ionizing radiation (IR) and chemotherapy, induce not only depletion of hematopoietic cells but also disruption of HSC niche components, as exemplified by dilation and leakiness of BM vasculature and depletion and dysfunction of BM MSCs. These structural and functional changes in the HSC niche restrain efficient hematopoietic recovery, which often compromises the efficacy of HSC transplantation (HSCT) and chemotherapy. YAP/TAZ are the two transcriptional coactivators normally repressed by LATS kinases downstream of the Hippo pathway. Although cumulative evidence has established a critical role of YAP/TAZ activation in tissue regeneration of various solid organs, their role in BM regeneration remains poorly understood. Our quantitive RT-PCR revealed that YAP/TAZ are abundantly expressed in steady-state mouse ECs (CD45 -Ter119 -CD31 +Sca1 +CD105 hi) and MSCs (CD45 -Ter119 -CD31 -PDGFRα +CD51 +LepR +) but scarcely in hematopoietic cells including HSCs (Lin -cKit +Sca1 +Flk2 -CD150 +CD48 -CD34 lo), which was confirmed by reanalysis of the published single cell RNA-seq datasets (GSE128423). Immunofluorescent imaging of BM sections revealed that YAP/TAZ are distributed mainly in the cytoplasm of ECs but evenly in the cytoplasm and nuclei of MSCs, indicating their differential basal activity in these two HSC niche components. Kinetic transcriptome analysis revealed that YAP/TAZ activity is transiently activated in ECs at 24 hours and returns to a basal repressive state by day 3 after sublethal IR. This transient activation of endothelial YAP/TAZ was critical for vascular integrity, as conditional deletion of YAP/TAZ in ECs (Cdh5-Cre ERT2Yap1 f/fTaz f/f) caused 100% lethality of mice within 10 days following sublethal IR. In sharp contrast, the kinetic expression analysis of a YAP/TAZ target gene CTGF indicated their transient inhibition in MSCs after sublethal IR, and the conditional YAP/TAZ deletion in BM MSCs (Ebf3-Cre ERT2Yap1 f/fTaz f/f) led to their reduced colony forming ability when assessed by colony forming unit fibroblast (CFU-F) assay. Recently, we discovered a novel and potent LATS inhibitor GA-003 that selectively induces mouse and human YAP/TAZ activation in vitro (IC 50 against LATS1 = 1.06 ± 0.08 nM). To analyze the effect of pharmacological YAP/TAZ activation on BM regeneration in vivo, we treated mice with intraperitoneal injection of GA-003 (50 mg/kg per day, for 8 days) following sublethal IR. Remarkably, we observed an accelerated recovery of hematopoiesis, with the absolute numbers of BM cellularity, GMP (Lin -cKit +Sca1 -FcγR +CD34 +) and HSC EPCR (Lin -cKit +Sca1 +CD150 +EPCR +) on day 14 increased by 3.50-fold (p=0.0002), 6.49-fold (p=0.0022) and 11.41-fold (p=0.022), respectively in the GA-003-treated group compared to vehicle-treated group. In addition, GA-003 also promoted hematopoietic recovery after 5-FU injection (150 mg/kg) and HSCT. Nonetheless, consistent with the scarce expression of YAP/TAZ in hematopoietic stem and progenitor cells (HSPC), in vitro GA-003 treatment did not enhance HSPC growth, suggesting niche-mediated effects by GA-003. Indeed, in vitro tube formation assay indicated accelerated angiogenesis by GA-003-treated human umbilical vein ECs, and CFU-F assays revealed significant enhancement of colony formation by mouse BM-derived MSCs upon GA-003 treatment. To reveal the effect of GA-003 on the HSC niche components in vivo, we performed whole BM immunofluorescent imaging at various time points following sublethal IR and GA-003 treatment. We observed alleviated vascular dilation and leakiness and earlier restoration of vascular damage in GA-003-treated group compared to vehicle-treated group, which was associated with increased VE-Cadherin expression in ECs. These results suggest that reinforcing YAP/TAZ activity upon myelosuppression promotes HSC niche integrity and recovery and accelerates hematopoietic regeneration. Taken together, our results establish YAP/TAZ as novel regulators of HSC niche and highlight YAP/TAZ as promising therapeutic targets to boost hematopoietic recovery after myeloablative interventions such as chemotherapy and HSCT. Disclosures Aihara: Nissan Chemical Corporation: Current Employment. Iwawaki: Nissan Chemical Corporation: Current Employment. Nishino: Nissan Chemical Corporation: Current Employment. Iwama: Nissan Chemical Corporation: Research Funding.

Effect of Peptide Receptor Radionuclide Therapy in Combination with Temozolomide against Tumor Angiogenesis in a Glioblastoma Model

Cell adhesion receptor integrin avb3 is a promising biomarker for developing tumor-angiogenesis targeted theranostics. In this study, we aimed to examine the therapeutic potential of peptide receptor radionuclide therapy (PRRT) with 188Re-IDA-D-[c(RGDfK)]2 (11.1 MBq). The results showed that the tumor volume was significantly decreased by 81% compared with the vehicle-treated group in U87-MG xenografts. The quantitative in vivo anti-angiogenic responses of PRRT were obtained using 99mTc-IDA-D-[c(RGDfK)]2 SPECT and corresponded to the measured tumor volume. PRRT combined with temozolomide (TMZ) resulted in a 93% reduction in tumor volume, which was markedly greater than that of each agent used individually. In addition, histopathological characterization showed that PRRT combined with TMZ was superior to PRRT or TMZ alone, even when TMZ was used at half dose. Overall, our results indicated that integrin-targeted PRRT and TMZ combined therapy might be a new medical tool for the effective treatment of glioblastoma.

Therapeutic role of Prostaglandin E2 receptor 4 stimulation in inflammatory cardiomyopathy

Background Prostaglandin E2 receptor 4 (EP4) plays a crucial role in inflammatory diseases. Inflammatory cardiomyopathy often leads to refractory heart failure. Purpose We aimed to evaluate the role of EP4 in the development of inflammatory cardiomyopathy. Methods Experimental autoimmune myocarditis (EAM) was induced by immunization with cardiac myosin in balb/c mice. EP4 selective antagonist (CJ-42794, Cayman Chemical: 30 mg/kg/day), EP4 selective agonist (20 mg/kg/day), both, or vehicle alone was daily administrated after the immunization. Cardiac function and dimensions were assessed by echocardiography. Blood pressure and heart rate were assessed with tail-cuff method. Cardiac inflammation and fibrosis were immunohistologically examined. Molecular examination was performed by RT-PCR and immunoblotting. Results Cardiac dysfunction and dilatation were worsened on day 21 in EP4 antagonist-treated group compared with in the vehicle-treated group, accompanied by an increase in cellular infiltration area (21.7±1.9 vs. 11.0±2.7%, P=0.0367, respectively). Conversely, cardiac dysfunction and dilatation were improved in EP4 agonist-treated group compared with in the vehicle-treated group (Left ventricular fractional shortening: 69±3% vs. 40±4%, P&lt;0.0001; Left ventricular systolic dimension: 0.7±0.1mm vs. 1.9±0.3mm, P=0.0007; respectively). These parameters did not show significant differences between both-treated group and the vehicle-treated group. The protective effect of EP4 stimulation in EAM was also kept on day 56. Moreover, cardiac fibrosis area as well as mRNA expressions of Type III collagen and brain natriuretic peptide in the bulk hearts was significantly reduced on day 56 in EP4 agonist-treated group compared with in the vehicle-treated group (12.3±2.4% vs. 24.7±3.0%, P=0.0278, respectively). Cardiac expression of phosphorylated smad 2/3 protein as well as TGF-β1 mRNA did not show significant differences between the 2 groups, while cardiac expression of RORgammat protein, the master regulator of Th17 immunity was increased in the EP4 antagonist-treated group. Conclusions EP4 activation negatively regulated the induction of cardiac autoimmunity, which alleviated cardiac dysfunction, dilatation, and fibrosis. EP4 may be a therapeutic target for preventing the development of inflammatory cardiomyopathy. FUNDunding Acknowledgement Type of funding sources: None.

Hydrogen Sulfide Prevents Mesenteric Adipose Tissue Damage, Endothelial Dysfunction, and Redox Imbalance From High Fructose Diet-Induced Injury in Aged Rats

A high fructose diet (HFD) and advanced age are key factors for the gradual loss of physiological integrity of adipose tissue. Endogenous hydrogen sulfide (H2S) has beneficial effects on cytoprotection and redox balance. But its interactive effects on age-related damage of mesenteric vessels and connective and adipose tissues (MA) during HFD which could be the base of the development of effective physiological-based therapeutic strategy are unknown. The aim of study was to investigate age- and HFD-induced mesenteric cellular changes and activities of enzymes in H2S synthesis and to test the effects of sodium hydrosulfide (NaHS) which is considered an H2S donor on them. Adult and aged male rats on a standard diet (SD) or 4-week HFD were exposed to acute water-immersion restraint stress (WIRS) for evaluation of mesenteric subcellular and cellular adaptive responses by electron microscopy. The effects of exogenous NaHS (5.6 mg/kg/day for 9 days) versus vehicle on mesentery changes were investigated. Serum glucose level, thiobarbituric acid reactive substances (TBARS), and activities of cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), thiosulfate-dithiol sulfurtransferase (TST), and sulfite oxidase (SO) were examined by spectrophotometry. In both adult and aged SD groups, treatment with NaHS protected mesenteric cells after WIRS. In both groups, the treatment with NaHS also protected MA mitochondria, microvascular endothelial and sub-endothelial structures, and fibroblasts versus the vehicle-treated group that had signs of damage. HFD increased MA injury and mitochondrial changes in both aged and adult rats. HFD-associated malfunction is characterized by low activities of CSE, CBS, TST, SO, and increased TBARS. Finally, we demonstrated that pretreatment with NaHS inhibited MA and mitochondria alterations in aged rats exposed to HFD and WIRS, lowered TBARS, and enhanced H2S enzyme activities in contrast to the vehicle-treated group. Mitochondrial integrity alterations, endothelial damage, and redox imbalance are key factors for rat mesenteric adipose tissue damage during advanced age. These alterations and MA hypertrophic changes retain the central for HFD-induced damage. Moreover, H2S signaling contributes to MA and mitochondria redox balance that is crucial for advanced age and HFD injury. The future study of H2S donors’ effects on mesenteric cells is fundamental to define novel therapeutic strategies against metabolic changes.

Abstract P422: Chronic Nicotine Exposure Increases Hematoma Growth Following Intracerebral Hemorrhage in Young Rats of Both Sexes

Spontaneous intracerebral hemorrhage (sICH) is the deadliest stroke subtype. There is strong evidence that tobacco use / smoking increases the risk of sICH, and some epidemiological studies have observed sex differences in sICH outcomes. However, systematic controlled studies on the effect of tobacco / smoking on post-sICH outcomes in both sexes are lacking. Therefore, we determined the effect of nicotine exposure on outcomes following collagenase-induced sICH both sexes. Young animals of both sexes were randomly divided into nicotine (4.5 mg / kg / day b.w.) and vehicle (saline) treatment groups (using osmotic pumps for two to three weeks). sICH in females was induced during the diestrous stage of estrous cycle. sICH was induced by collagenase injection into the right striatum and ~24 hours later, neurological scores were evaluated, rats were euthanized, and brains were sectioned to measure hematoma volume. Hematoma volumes for male rats was 42% higher (p<0.01) in the nicotine-treated group (139 ± 9 mm 3 , n=10) versus vehicle-treated group (98 ± 9 mm 3 , n=10). Hematoma volumes for female rats was 48% higher (p<0.01) in the nicotine-treated group (134 ± 11 mm 3 , n=10) versus vehicle-treated group (90 ± 7 mm 3 , n=10). Hematoma volumes for the vehicle and nicotine-treated male groups were not different from their respective female groups. The neurological score for the nicotine-treated male group (9.3 ± 0.6) was significantly higher (p<0.05) when compared to vehicle group (7.4 ± 0.6). The neurological score for the nicotine-treated female group (10.7 ± 0.2) was significantly higher (p<0.001) than the vehicle group (7.7 ± 0.7). The neurological score for the vehicle-treated male group was not different from its respective female group. However, the neurological score for the nicotine-treated male group was significantly lower than the female group. Our results show that chronic nicotine exposure increases hematoma volume post-sICH in animals of both sexes. Future studies into the mechanism of nicotine-induced increase in hematoma growth following sICH are required. Support: James and Esther King Biomedical Research Grant 9JK08

Cissus Quadrangularis enhances UCP1 mRNA, indicative of white adipocyte browning and decreases central obesity in humans in a randomized trial

Obesity is associated with the growth and expansion of adipocytes which could be decreased via several mechanisms. Cissus Quadrangularis (CQ) extract has been shown to reduce obesity in humans; however, its effect on human white adipocytes (hWA) has not been elucidated. This study aimed to investigate the effects of CQ on obesity, lipolysis, and browning of hWA. CQ treatment in obese humans significantly decreased waist circumference at week 4 and week 8 when compared with the baseline values (p < 0.05 all) and significantly decreased hip circumference at week 8 when compared with the baseline and week 4 values (p < 0.05 all). Serum leptin levels of the CQ-treated group were significantly higher at week 8 compared to baseline levels (p < 0.05). In hWA, glycerol release was reduced in the CQ-treated group when compared with the vehicle-treated group. In the browning experiment, pioglitazone, the PPAR-γ agonist, increased UCP1 mRNA when compared to vehicle (p < 0.01). Interestingly, 10, 100, and 1000 ng/ml CQ extract treatment on hWA significantly enhanced UCP1 expression in a dose-dependent manner when compared to pioglitazone treatment (p < 0.001 all). In conclusion, CQ decreased waist and hip circumferences in obese humans and enhanced UCP1 mRNA in hWA suggestive of its action via browning of hWA.

Gynura procumbens Root Extract Ameliorates Ischemia-Induced Neuronal Damage in the Hippocampal CA1 Region by Reducing Neuroinflammation

Gynura procumbens has been used in Southeast Asia for the treatment of hypertension, hyperglycemia, and skin problems induced by ultraviolet irradiation. Although considerable studies have reported the biological properties of Gynura procumbens root extract (GPE-R), there are no studies on the effects of GPE-R in brain damages, for example following brain ischemia. In the present study, we screened the neuroprotective effects of GPE-R against ischemic damage and neuroinflammation in the hippocampus based on behavioral, morphological, and biological approaches. Gerbils received oral administration of GPE-R (30 and 300 mg/kg) every day for three weeks and 2 h after the last administration, ischemic surgery was done by occlusion of both common carotid arteries for 5 min. Administration of 300 mg/kg GPE-R significantly reduced ischemia-induced locomotor hyperactivity 1 day after ischemia. Significantly more NeuN-positive neurons were observed in the hippocampal CA1 regions of 300 mg/kg GPE-R-treated animals compared to those in the vehicle-treated group 4 days after ischemia. Administration of GPE-R significantly reduced levels of pro-inflammatory cytokines such as interleukin-1β, -6, and tumor necrosis factor-α 6 h after ischemia/reperfusion. In addition, activated microglia were significantly decreased in the 300 mg/kg GPE-R-treated group four days after ischemia/reperfusion compared to the vehicle-treated group. These results suggest that GPE-R may be one of the possible agents to protect neurons from ischemic damage by reducing inflammatory responses.

EFFECT OF CLOVE OIL ON THE BIOCHEMICAL PROFILE OF OVOTESTIS AND HEPATOPANCREAS OF Achatina fulica

The giant African snail Achatina fulica is widespread across the subtropical and tropical countries and has picked up the global pest status. Its voracious feeding capacity and protandrous reproduction makes it almost invincible once established. Measures to combat its menace have for long focussed on chemical molluscicides. However the rising environmental sensitivity in the past few decades has attracted the use of natural compounds to target this pest. The current study was aimed to evaluate the impact of clove oil on the biochemical profile of its ovotestis and hepatopancreas. The snails were randomly divided into three groups i.e., control, vehicle treated group (1% Tween 80) and clove oil treated group. Snails in the clove oil treated group were subjected to subacute doses of clove oil (20% and 60% of LD50 value/24 hrs) by topical application. The activity levels of vital enzymes namely acetylcholinesterase, acid phosphatase, alkaline phosphatase, protease and lactic dehydrogenase were pertinently altered in the ovotestis and hepatopancreas of the clove oil treated snails as compared to the control. Contents of important biomolecules like DNA, RNA, protein, and phospholipids were reduced, the level of lipid peroxidation was significantly enhanced with a concomitant decrease in glutathione content in clove oil treated groups vis-a-vis control in both the tissues studied, highlighting the toxic effect of clove oil to the snail. Thus it is suggested that clove oil can be used in controlling the population of this harmful pest.