Transient receptor potential ankyrin 1 channels in the bladder mediate low temperature elicited bladder overactivity in rats.

AIMS: This study aimed to investigate whether pathways involving transient receptor potential ankyrin 1 (TRPA1) channels in the urinary bladder mediate the bladder overactivity elicited by exposure to a low temperature in rats. METHODS: At postnatal week 10, female Sprague-Dawley (SD) rats were intraperitoneally injected with the TRPA1 channel antagonist, HC030031, at room temperature (RT) and subsequently exposed to low temperature (LT). Bladder specimens treated with HC030031 were evaluated for contractions through cumulative addition of the TRPA1 channel agonist trans-cinnamaldehyde. Two days before cystometric investigation, small interfering RNA (siRNA) targeting TRPA1 was transfected into urinary bladders. Then, cystometric investigations were performed on rats subjected to TRPA1 siRNA transfection at both RT and LT. Expression of TRPA1 channels in the urinary bladder was assessed through immunohistochemistry and real-time reverse transcription-polymerase chain reaction. RESULTS: At RT, micturition patterns were unaffected by HC030031 treatment. However, upon exposure to LT, rats treated with HC030031 exhibited a reduction of LT-elicited bladder overactivity, as evidenced by inhibited decreases in voiding interval, micturition volume, and bladder capacity. Additionally, HC030031 inhibited trans-cinnamaldehyde-induced contractions. Immunohistochemical analysis showed the presence of TRPA1 channels in the urinary bladder. Notably, rats with TRPA1 siRNA-transfected bladders could partially inhibit bladder overactivity during LT exposure. CONCLUSIONS: These findings indicate that pathways involving TRPA1 channels expressed in the urinary bladder could mediate the LT-elicited bladder overactivity.

Combination of TRP channel dietary agonists induces energy expending and glucose utilizing phenotype in HFD-fed mice.

BACKGROUND: Bioactive dietary constituents activating Transient receptor potential (TRP) channels have emerged as promising candidates for the prevention of metabolic disorders. OBJECTIVE: The present study is an attempt to evaluate anti-obesity potential of a dietary TRP-based tri-agonist, combination of sub-effective doses of capsaicin (TRPV1 agonist), menthol (TRPM8 agonist), and cinnamaldehyde (TRPA1 agonist) in high-fat diet (HFD)-fed mice. DESIGN: Male C57BL/6 J mice divided into three groups (n = 8), were fed on normal pellet diet (NPD), or high-fat diet (HFD) (60% energy by fat) and HFD + CB (combination of capsaicin 0.4 mg/Kg, menthol 20 mg/Kg, and cinnamaldehyde 2 mg/Kg; p.o) for 12 weeks. Effects on HFD-induced weight gain, biochemical, histological and genomic changes in the WAT, BAT, liver and hypothalamus tissues were studied. RESULTS: Administration of tri-agonist prevented HFD-induced increase in weight gain, improved altered morphometric parameters, glucose homeostasis, and adipose tissue hypertrophy. Tri-agonist supplementation was found to induce browning of white adipose tissue and promote brown adipose tissue activation. Enhanced glucose utilization and prevention of lipid accumulation and insulin resistance in the liver was observed in mice supplemented with a tri-agonist. CONCLUSION: The present work provides evidence that the new approach based on combination of sub-effective doses of TRP channel agonists (TRI-AGONIST) can be employed to develop concept-based functional food for therapeutic and preventive strategies against HFD-associated pathological complications.

Dietary cinnamaldehyde nanoemulsion boosts growth and transcriptomes of antioxidant and immune related genes to fight Streptococcus agalactiae infection in Nile tilapia (Oreochromis niloticus).

The present study was conducted to investigate the effects of dietary cinnamaldehyde nanoemulsion (CNE) on growth, digestive activities, antioxidant and immune responses and resistance against Streptococcus agalactiae (S. agalactiae) in Nile tilapia. Four experimental diets were formulated containing CNE at levels of 0, 100, 200 and 300 mg/kg diet for 12 weeks. At the end of the experiment, all fish were challenged by S. agalactiae. The results showed that the final body weight was increased in fish groups fed 200 and 300 mg CNE/kg diet by 18.4 and 17.2% with respect to the control group. Moreover, feed conversion ratio and digestive enzymes' activities were improved in groups fed 200 and 300 then 100 mg of dietary CNE/kg diet. Groups fed CNE exhibited a significant increase in serum immune-related parameters when compared with control group. Additionally, the hypocholesterolemic effects was achieved after CNE feeding unlike the control group in a dose dependent manner. With increasing dietary CNE levels, genes expression of cytokines and antioxidant enzymes were upregulated. Less severe adverse clinical symptoms and respectable cumulative mortalities associated with S. agalactiae infection were observed in fish fed CNE. To our knowledge, this study was the first offering a protective effect of CNE against S. agalactiae infection in Nile tilapia with a maximum down-regulation of cylE and hylB virulence genes expression noticed in group fed 300 mg of CNE/kg diet (up to 0.10 and 0.19- fold, respectively). Therefore, the present study recommended that an incorporation of CNE at level of 300 mg/kg diet for Nile tilapia could promote their growth, enhance their immunity and antioxidant status and provide protection against virulent S. agalactiae.

Anti-oomycetes and immunostimulatory activity of natural plant extract compounds against Saprolegnia spp.: Molecular docking and in-vitro studies.

This study aimed to investigate the effectiveness of five natural plant extract compounds Curcumin (CUR); Eugenol (EUG), Cinnamaldehyde (CIN), Stigmasterol (ST) and Morin (MOR), on two species of Saprolegnia; Saprolegnia parasitica and S. australis. Selective compounds were screened for the minimum inhibitory concentration, first for anti-oomycetes activity and then mycelium growth inhibition, spore germination inhibition and colonisation test. Nitric oxide production and myeloperoxidase activity of the compounds were tested in head kidney leukocytes of rainbow trout, Oncorhynchus mykiss to assess the immunostimulatory potential. Molecular docking of effective compounds was carried out with effector proteins of S. parasitica to investigate the target binding sites. Among all, CUR could completely inhibit zoospore production and significantly (p ≤ .05) inhibit hyphal growth at 16 mg l-1 against S. parasitica and S. australis. CIN at the concentration of 50 mg l-1 completely inhibited hyphal growth of both Saprolegnia spp., although the zoospore production of S. parasitica and S. australis was reduced at 25 mg l-1 and 10 mg l-1. In the case of EUG, significant inhibition of the hyphal growth and germination of S. parasitica zoospores was observed at 50 mg l-1. ST and MOR did not show antioomycetes activity. The molecular docking results were consistent with in vitro studies, possibly due to the binding with the vital proteins (Plasma membrane ATPase, V-type proton ATPase, TKL protein kinase, Host targeting protein 1) of S. parasitica and ultimately inhibiting their activity. CUR and CIN showed increased nitric oxide production at the highest concentration of 250 and 256 mg l-1 but the value was not significant (p ≤ .05) with control. CUR showed significantly higher peroxidase activity (p ≤ .05) at a concentration of 256 mg l-1 though values were significantly similar with concentration from 16 to 128 mg l-1. The nitric oxide and total peroxidase activity of rainbow trout leukocytes in the case of CIN showed a significant difference only at 250 mg l-1 against the control. The results conclude that CUR, CIN showed the better anti-Saprolegnia activity and could be used as phyto-additives in aquaculture. Among all, the inclusion of CUR as phyto-additives will provide additional immunostimulatory activity.

Impact of cinnamaldehyde on innate immunity and immune gene expression in Channa striatus against Aphanomyces invadans.

The effect of cinnamaldehyde (CM) enriched diet on immunity and cytokine gene expression in Channa striatus against Aphanomyces invadans is reported. C. striatus was uniformly divided into eight groups (n = 25 fish each) and fed with formulated diets with 0, 5, 10, and 15 mg kg-1 CM enriched diet. In healthy and infected groups fed with 5 mg kg-1 diet the leukocytes count increased significantly after 4th week; with 10 mg kg-1 CM diet the increase manifested after 6th week, but with 15 mg kg-1 not even after 8th week. In both groups, 5 mg kg-1 CM diet resulted in a significant increase in the serum total protein, albumin, and globulin levels after 4th week, whereas with other diets this effect was observed only after 6th week. Similarly, with any enriched diet the lysozyme activity increased significantly, but with 15 mg kg-1 CM diet only after 6th week. In both groups the complement activity and lymphocyte production increased significantly when fed with 5 mg kg-1 CM diet after 4th week while with other enriched diets only after 6th week. The phagocytic activity increased significantly in both groups fed with 5 mg kg-1 CM diet after 6th week, whereas the SOD activity increased after 4th week. The IgM production increased significantly in both groups fed with 5 mg kg-1 CM diet after 2nd week, while with 5 and 10 mg kg-1 CM diet after 4th week. In both groups, the expression of CXCR3α was significant on 4th week when fed with 10 mg kg-1 CM diet, while in the healthy group fed with 15 mg kg-1 CM diet the expression manifested earlier than 4th week. However, when fed with 10 and 15 mg kg-1 CM diets the increase was observed on 6th week; whereas, the expression of MHC-I reached the maximum on 6th week with any enriched diet. The results indicate that in C. striatus the innate immunity and expression of cytokine and immune related genes were significantly modulated when fed with 5 mg kg-1 CM diet on 4th week against A. invadans.

Low-Dose Acrolein, an Endogenous and Exogenous Toxic Molecule, Inhibits Glucose Transport via an Inhibition of Akt-Regulated GLUT4 Signaling in Skeletal Muscle Cells.

Urinary acrolein adduct levels have been reported to be increased in both habitual smokers and type-2 diabetic patients. The impairment of glucose transport in skeletal muscles is a major factor responsible for glucose uptake reduction in type-2 diabetic patients. The effect of acrolein on glucose metabolism in skeletal muscle remains unclear. Here, we investigated whether acrolein affects muscular glucose metabolism in vitro and glucose tolerance in vivo. Exposure of mice to acrolein (2.5 and 5 mg/kg/day) for 4 weeks substantially increased fasting blood glucose and impaired glucose tolerance. The glucose transporter-4 (GLUT4) protein expression was significantly decreased in soleus muscles of acrolein-treated mice. The glucose uptake was significantly decreased in differentiated C2C12 myotubes treated with a non-cytotoxic dose of acrolein (1 µM) for 24 and 72 h. Acrolein (0.5-2 µM) also significantly decreased the GLUT4 expression in myotubes. Acrolein suppressed the phosphorylation of glucose metabolic signals IRS1, Akt, mTOR, p70S6K, and GSK3α/ß. Over-expression of constitutive activation of Akt reversed the inhibitory effects of acrolein on GLUT4 protein expression and glucose uptake in myotubes. These results suggest that acrolein at doses relevant to human exposure dysregulates glucose metabolism in skeletal muscle cells and impairs glucose tolerance in mice.

The Onset of Systemic Oxidative Stress Associated with the Accumulation of Lipid Peroxidation Product Acrolein in the Skin of Patients with Small-Vessel Vasculitis.

Small-vessel vasculitis (SVV) is the inflammation of the vessel wall that can result in hemorrhage and/or ischemia. Among the histological findings in SVV are increased infiltrating neutrophils, which, due to their oxidative burst and myeloperoxidase activity, release excessive reactive oxygen species, triggering a chain reaction of lipid peroxidation and yielding reactive aldehydes such as acrolein. The implication of oxidative stress in the pathogenesis of SVV was studied, focusing on acrolein immunohistochemistry in the affected skin vessels and systemic stress response. Samples from SVV patients and healthy subjects were collected and analyzed for total serum peroxides, total antioxidant capacity, inflammatory and immunological parameters, as well as for the presence of acrolein-protein adducts in the skin tissue specimens. The obtained data showed that systemic redox homeostasis and iron metabolism are altered in SVV patients. Possible biomarkers in the evaluation of oxidative status, disease activity and prevalence were indicated. Furthermore, a strong correlation between the accumulation of acrolein-protein adducts in the skin and the progression of the disease was revealed. Thus, the results of this study demonstrate that SVV is not only associated with systemic oxidative stress but also with tissue-specific oxidative stress that promotes acrolein formation and protein modification correlating with the severity of cutaneous vasculitis.

Combination of herbal components (curcumin, carvacrol, thymol, cinnamaldehyde) in broiler chicken feed: Impacts on response parameters, performance, fatty acid profiles, meat quality and control of coccidia and bacteria.

The objective of this study was to determine whether curcumin and a commercial microencapsulated phytogenic supplement containing thymol, cinnamaldehyde and carvacrol in broiler chicken feed would improve health and meat quality (fatty acid profile), as well as to determine the coccidiostatic and bactericidal potential of the additives. The broiler chickens were divided into five groups: NC - negative control feed; PC - positive control; CU - with 50 mg/kg of curcumin, PHY - 100 mg/kg phytogenic; and PHY + CU, a combination of both additives at 50 mg/kg (curcumin) and 100 mg/kg (phytogenic). We observed significantly higher levels of total proteins associated with increased circulating globulins, as well as lower levels of uric acid, cholesterol and triglycerides in the PHY + CU group than in the NC. There were significantly fewer oocysts in birds supplemented with additives in the NC group on day 21; on day 35, the NC, PHY and PHY + CU groups had significantly lower counts than the PC and CU groups; however, at 44 days, the lowest counts were in PC group. The bacterial counts were significantly lower on day 21 in all groups that received additives than those of the control group; however, at 44 days, the bacterial and Escherichia coli counts in these groups were significantly higher than those of the control. Curcumin with or without phytogenic agent improved meat quality, with increased antioxidant levels and reduction of lipid peroxidation. There were significantly lower total saturated fatty acid levels and significantly greater monounsaturated/polyunsaturated fatty acid levels in broilers that consumed additives individually and in combination. The combination of additives significantly increased the crypt/villus ratio, a marker of improved intestinal health and performance. Additives potentiated their individual effects, suggesting they can replace conventional growth promoters without compromising health, intestinal mucosa or meat quality.

Laser speckle contrast imaging, the future DBF imaging technique for TRP target engagement biomarker assays.

A comparison was made between the established laser Doppler imaging (LDI) technique and the more recently developed laser speckle contrast imaging (LSCI) method to measure changes in capsaicin- and cinnamaldehyde-induced dermal blood flow (DBF) as an indicator of TRPV1 and TRPA1 activation, respectively. METHODS: Capsaicin (1000 µg/20 µl) and cinnamaldehyde (10%) solutions were applied on the forearm of 16 healthy male volunteers, alongside their corresponding vehicle solutions. Pre challenge and 10, 20, 30, 40 and 60 min post challenge application, changes in DBF were assessed with the LSCI technique, followed by LDI. The area under the curve from 0 to 60 min (AUC0-60) post capsaicin and cinnamaldehyde application was calculated as a summary measure of the response. Correlation between the LDI and LSCI instrument was assessed using a simple linear regression analysis. Sample size calculations (SSC) were performed for future studies using either the LDI or LSCI technique. RESULTS: Higher arbitrary perfusion values were obtained with LDI compared to LSCI, yet a complete discrimination between the challenge and vehicle responses was achieved with both techniques. A strong degree of correlation was observed between LDI and LSCI measurements of the capsaicin- (R = 0.84 at Tmax and R = 0.92 for AUC0-60) and cinnamaldehyde-induced (R = 0.78 at Tmax and R = 0.81 for AUC0-60) increase in DBF. SSC revealed that LSCI requires considerably less subjects to obtain a power of 80% (about 15 versus 27 subjects in case of capsaicin and 7 versus 13 for cinnamaladehyde). CONCLUSIONS: The LSCI technique was identified as the preferred method to capture capsaicin- and cinnamaldehyde-induced changes in DBF. Besides its reduced variability, the shorter scan time provides a major advantage, allowing real-time DBF measurements.

Stephalagine, an aporphine alkaloid from Annona crassiflora fruit peel, induces antinociceptive effects by TRPA1 and TRPV1 channels modulation in mice.

Pain relief represents a critical unresolved medical need. Consequently, the search for new analgesic agents is intensively studied. Annona crassiflora, a native species of the Brazilian Savanna, represents a potential source for painful treatment. This study aimed to investigate the antinociceptive potential of A. crassiflora fruit peel, focusing on its major alkaloid, stephalagine, in animal models of pain evoked by the activation of transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) channels. Male C57BL/6/J mice were submitted to formalin-, cinnamaldehyde-, and capsaicin-induced nociception tests to assess nociceptive behavior, and to the open-field and rotarod tests for motor performance analyses. Moreover, the stephalagine's effect was tested on capsaicin- and cinnamaldehyde-induced Ca2+ influx in spinal cord synaptosomes. In silico assessments of the absorption, distribution, metabolism and central nervous system permeability of stephalagine were carried out. The ethanol extract and alkaloidal fraction reduced the nociception induced by formalin. When administered by oral route (1 mg/kg), stephalagine reduced the spontaneous nociception and paw edema induced by TRPV1 agonist, capsaicin, and by TRPA1 agonists, cinnamaldehyde- and formalin, without altering the animals' locomotor activity. The prediction of in silico pharmacokinetic properties of stephalagine suggests its capacity to cross the blood-brain barrier. Furthermore, this alkaloid reduces the capsaicin- and cinnamaldehyde-mediated Ca2+ influx, indicating a possible modulation of TRPV1 and TRPA1 channels, respectively. Together, our results support the antinociceptive and anti-edematogenic effects of the A. crassiflora fruit peel and suggest that these effects are triggered, at least in part, by TRPV1 and TRPA1 modulation by stephalagine.

Co-hybridized composite nanovesicles for enhanced transdermal eugenol and cinnamaldehyde delivery and their potential efficacy in ulcerative colitis.

Percutaneous absorption of drugs can be enhanced by ethosomes, which are nanocarriers with excellent deformability and drug-loading properties. However, the ethanol within ethosomes increases phospholipid membrane fluidity and permeability, leading to drug leakage during storage. Here, we developed and characterized a new phospholipid nanovesicles that is co-hybridized with hyaluronic acid (HA), ethanol and the encapsulated volatile oil medicines (eugenol and cinnamaldehyde [EUG/CAH]) for transdermal administration. In comparison with EUG/CAH-loaded ethosomes (ES), the formulation stability and percutaneous drug absorption of EUG/CAH-loaded HA-immobilized ethosomes (HA-ES) were significantly improved. After transdermal administration of HA-ES, the interstitial cells of Cajal in the colon of rats with trinitrobenzene sulfonate-induced ulcerative colitis (UC) were significantly increased, and the stem cell factor/c-kit signaling pathway was partly repaired. Overall, HA-ES possesses excellent deformability and showed improved efficacy against UC compared with ES, which is demonstrated as a promising transdermal delivery vehicle for volatile oil medicines.

Does Hyaluronidase Enhance Drug Penetration to Mechanoreceptors?

BACKGROUND: The pharmacological study of mechanoreceptors embedded within tissue is hampered by tissue barriers to applied research drugs. METHODS: Hyaluronidase increases the permeability of tissues and is used clinically to facilitate the distribution of injected drugs. An in vitro rat sinus hair preparation was used to determine whether hyaluronidase (1,500 or 3,000 IU/10 mL) had an effect on drug access to receptor sites on slowly adapting St I and St II mechanoreceptors. Electrical recordings were made from single mechanoreceptor units that were activated by trapezoid ramp stimuli. Cinnamaldehyde (500-1,500 µM) and capsazepine (100 µM) were used as test drugs. Changes in onset time and degree of depression of firing due to test drugs were compared to control experiments not employing hyaluronidase. RESULTS: There were no statistical effects on any of the observed measures. Often the effects were opposite to those predicted. Using a likelihood approach, it was calculated that there was strong evidence (log-likelihood ratios from -0.5 to -6.5) to support a null effect over a facilitatory effect. There was no evidence of loss of integrity of mechanoreceptor mechanotransduction mechanisms following hyaluronidase applications. Comparison with Existing Method: The use of hyaluronidase does not facilitate drug access to receptors. CONCLUSIONS: In the in vitro sinus hair preparation, the addition of hyaluronidase does not allow easier access to slowly adapting mechanoreceptors within the follicle.

Cinnamaldehyde improves the growth performance and digestion and absorption capacity in grass carp (Ctenopharyngodon idella).

The present study evaluated the effect of cinnamaldehyde (CIN) on the growth performance and digestion and absorption capacity of grass carp (Ctenopharyngodon idella). Fish were fed five diets including graded levels of CIN for 60 days. The results indicated that (1) appropriate CIN supplementation increased the growth performance and promoted the intestine growth of grass carp; (2) dietary appropriate CIN supplementation increased the digestion and absorption capacity by increasing the activities of intestinal and hepatopancreas digestive enzymes (lipase, chymotrypsin, trypsin, and amylase) and intestinal brush border enzymes (creatine kinase (CK), Na+/K+-ATPase, γ-glutamyl transpeptidase (γ-GT), and alkaline phosphatase (AKP)); (3) dietary CIN increased the absorption capacity which may be associated with the upregulated messenger RNA (mRNA) abundances of their amino acid transporters (AATs) in the intestine, which might be associated with activating the target of rapamycin (TOR) signaling pathway. The best CIN supplementation in the diets of grass carp was estimated to be 76.40 mg kg-1 diet based on the best percent weight gain (PWG). In general, CIN increased the digestion and absorption capacity of grass carp and raised the mRNA abundances of AATs which may be partly related to activation of the TOR signaling pathway.

Claudins, VEGF, Nrf2, Keap1, and Nonspecific Airway Hyper-Reactivity Are Increased in Mice Co-Exposed to Allergen and Acrolein.

Acrolein, an α/ß-unsaturated aldehyde, is volatile at room temperature. It is a respiratory irritant found in environmental tobacco smoke, which can be generated during cooking or endogenously at sites of injury. An acute high concentration of uncontrolled irritant exposure can lead to an asthma-like syndrome known as reactive airways dysfunction syndrome (RADS). However, whether acrolein can induce RADS remains poorly understood. The aim of study is to develop a RADS model of acrolein inhalation in mice and to clarify the mechanism of RADS. Mice were treated with ovalbumin (OVA) and exposed to acrolein (5 ppm/10 min). Airway hyper-responsiveness (AHR) was measured on days 24 and 56, and samples were collected on days 25 and 57. Tight junction protein, antioxidant-associated protein, and vascular endothelial growth factor (VEGF) levels were estimated by Western blotting and immunohistochemical staining. Reactive oxygen species (ROS) was calculated using enzyme linked immunosorbent assays. Acrolein or OVA groups exhibited an increase in airway inflammatory cells and AHR compared to a sham group. These effects were further increased in mice in the OVA + acrolein exposure group than in the OVA exposure group and persisted in the acrolein exposure group for 8 weeks. CLDNs, carbonyls, VEGF, Nrf2, and Keap1 were observed in the lungs. Our data demonstrate that acrolein induces RADS and that ROS, angiogenesis, and tight junction proteins are involved in RADS in a mouse model.

Dual drug-loaded cubic liquid crystal gels for transdermal delivery: inner structure and percutaneous mechanism evaluations.

The goal of this paper was to develop and evaluate dual component-loaded with the hydrophilic sinomenine hydrochloride (SH) and lipophilic cinnamaldehyde (CA) cubic liquid crystal gels for transdermal delivery. The gels was prepared with a vortex method using phytantriol/water (70:30, w/w) and characterized by polarized light microscopy, small-angle X-ray scattering and rheology. The inner structure of the gels were Pn3m cubic phase and exhibited a pseudoplastic fluid behavior. Furthermore, the in vitro release profile showed that the release behavior of the two drugs from cubic liquid crystal gels conformed to Higuchi equation and were dominated by Fick's diffusion (n < 0.45). The ex vivo penetration experiment indicated that dual components-loaded liquid crystal gels can enhance and extend the skin permeation of these two drugs, especially the ratio of SH to CA is 1: 0.5. Finally, transdermal mechanisms were evaluated using laser scanning confocal microscopy and attenuated total reflectance-fourier transform infrared, hinting that hydrophilic and lipophilic drugs weaken each other's transdermal velocity at the initial stage of penetration. In short, the dual drug-loaded liquid crystal gels was a promising strategy for transdermal applications in treatment of chronic disease.

Effects of phytogenic feed additive based on thymol, carvacrol and cinnamic aldehyde on body weight, blood parameters and environmental bacteria in broilers chickens.

The aim of this study was to evaluate whether a phytogenic feed additive (PFA) based on essential oils such as carvacrol, thymol and cinnamic aldehyde, could be considered a replacement for antimicrobials used as growth promoters in broiler chickens, as well as to investigate its effect on total bacterial count, biochemical profiles, meat quality and meat fatty acid profile. A total of 240 broiler chicks were randomly distributed into 4 groups with 4 replicates of 15 animals each, as follow: T1 (basal diet only; the control group), T2 (basal diet supplemented with zinc bacitracin), T3 (basal diet with 0.5% of the PFA), T4 (basal diet with 1.0% of the PFA). The addition of 0.5% of the PFA improved live body weight of supplemented birds compared to the control group at 35 and 42 days of age, while the total bacterial count in the environment was reduced when 1.0% of the PFA was used. In addition, intestinal villi height and crypt depth suffered variations during the entire experiment in birds treated with both concentrations of the PFA and zinc bacitracin. Total erythrocyte counts were higher on days 14, 28 and 42 in both treated groups (PFA) compared to the control group, as well as hemoglobin content on days 28 and 42. On the other hand, leukocyte counts were lower on days 14, 28 and 42 due to reduced lymphocyte counts in both PFA treated groups compared to the control group. Serum levels of alanine aminotransferase (ALT) were lower in broilers fed with either concentration of PFA on day 14 of life, and the same was observed regarding aspartate aminotransferase (AST) in broiler treated with 0.5% of the PFA. Also, total protein and globulin levels were lower on days 14 and 28 in groups fed with phytogenic compared to the control group. Regarding meat quality, breast meat showed higher red intensity and shear force in groups fed with both concentrations of phytogenic compared to the control group, while weight loss by cooking was lower. Finally, 1.0% of phytogenic showed lower docosadienoic acid (C22:2) content in breast meat. In conclusion, results showed that the use of PFAs based on carvacrol and thymol may be considered an interesting alternative to increase broilers performance, replacing the use of antimicrobials as growth promoters, as well as an interesting alternative to reduce the total bacterial count in the environment of broiler chickens. Moreover, the diet containing phytogenic also showed hepaprotective effects but deserves attention regarding possible alterations on the immune response.

Administration of cinnamaldehyde promotes osteogenesis in ovariectomized rats and differentiation of osteoblast in vitro.

To explore the effect of cinnamaldehyde on the distal femur in ovariectomized rats and its influence on osteoblast in vitro. Female Sprague-Dawley rats which underwent either bilateral ovariectomy or sham operation were divided into five groups randomly: group OVX (OVX, N = 10) and group sham (SHAM, N = 10) received normal saline (NS) by gavage at a dose of 50 ml/kg·d; group low dose, group middle dose and group high dose received cinnamaldehyde by gavage at a dose of 25 mg/kg·d (OLD, N = 10), 50 mg/kg·d (OMD, N = 10), and 75 mg/kg·d (OHD, N = 10) respectively. Distal femurs were harvested for hematoxylin and eosin (HE) staining, micro-ct scanning and immunohistochemical analysis. Murine mesenchymal stem cells were cultured and dealt with the presence of either cinnamaldehyde at a dose of 15ug/ml (OLD), 30ug/ml (OMD), 60ug/ml (OHD) or vehicle. ALP staining and western blot were performed to observe the influence of cinnamaldehyde on the differentiation of osteoblast. HE and micro-ct results indicated that osteogenesis were promoted with the treatment of cinnamaldehyde. Immunohistochemical results showed that cinnamaldehyde increased the number of osteoblast and decreased the number of osteoclast. In vitro studies indicated that cinnamaldehyde promoted expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and collagen type Iɑ1 (COL1ɑ1). The treatment effect behaved as dose-dependently. Thus, cinnamaldehyde inhibits osteoclastogenesis and promotes osteoblastogenesis, and may plays an important role in the treatment of osteoporosis clinically.

Short communication: Investigation of antibiotic alternatives to improve health and growth of veal calves.

The inherent disease susceptibility of veal calves results in frequent antimicrobial use. Improvements in antimicrobial stewardship necessitate alternative therapies to improve calf health and growth, while reducing the need for antimicrobials important to human health. This study investigated the effect of 2 alternative therapies, lactoferrin (an iron-binding protein found in colostrum) and cinnamaldehyde (an essential oil of the cinnamon plant) on growth, disease incidence, and mortality risk in special-fed veal calves. On the day of arrival to the growing facility (3 to 7 d of age), calves (n = 80 per treatment) were randomized to 1 of 3 treatments: (1) control (no supplement), (2) lactoferrin (1 g/d in milk replacer for 7 d), or (3) cinnamaldehyde (1 g/d in milk replacer for 21 d). Body weight was measured on the day of arrival (d 0), 21, and 42 d postarrival. Health assessments were performed twice weekly through 21 d, and mortality records were obtained through 6 wk postarrival. A repeated-measures ANOVA was used to compare growth between treatment groups, and a Poisson regression model (PROC GENMOD, SAS v. 9.4, SAS Institute Inc., Cary, NC) was used to test differences between groups in the incidence of diarrhea (fecal score ≥2 with and without depression and temperature) and disease through 3 wk postarrival. Body weight and average daily gain were similar between treatments. Neither lactoferrin nor cinnamaldehyde had an effect on diarrhea incidence. However, the risk of navel inflammation was significantly lower for calves that received cinnamaldehyde compared with calves in the control group. Mortality through 6 wk postarrival was low, with 4, 1, and 0 deaths from the control, lactoferrin, and cinnamaldehyde treatment groups, respectively. Additional research is needed to investigate various doses of these alternative therapies on calf health and growth, in addition to different routes of administration.

Role of TRPA1 in acute cardiopulmonary toxicity of inhaled acrolein.

Acrolein is a highly toxic, volatile, unsaturated aldehyde generated during incomplete combustion as in tobacco smoke and indoor fires. Because the transient receptor potential ankyrin 1 (TRPA1) channel mediates tobacco smoke-induced lung injury, we assessed its role in high-level acrolein-induced toxicity in mice. Acrolein (100-275ppm, 10-30min) caused upper airway epithelial sloughing, bradypnea and oral gasping, hypothermia, cardiac depression and mortality. Male wild-type mice (WT, C57BL/6; 5-52weeks) were significantly more sensitive to high-level acrolein than age-matched, female WT mice. Both male and female TRPA1-null mice were more sensitive to acrolein-induced mortality than age- and sex-matched WT mice. Acrolein exposure increased lung weight:body weight ratios and lung albumin and decreased plasma albumin to a greater extent in TRPA1-null than in WT mice. Lung and plasma protein-acrolein adducts were not increased in acrolein-exposed TRPA1-null mice compared with WT mice. To assess TRPA1-dependent protective mechanisms, respiratory parameters were monitored by telemetry. TRPA1-null mice had a slower onset of breathing rate suppression ('respiratory braking') than WT mice suggesting TRPA1 mediates this protective response. Surprisingly, WT male mice treated either with a TRPA1 antagonist (HC030031; 200mg/kg) alone or with combined TRPA1 (100mg/kg) and TRPV1 (capsazepine, 10mg/kg) antagonists at 30min post-acrolein exposure (i.e., "real world" delay in treatment) were significantly protected from acrolein-induced mortality. These data show TRPA1 protects against high-level acrolein-induced toxicity in a sex-dependent manner. Post-exposure TRPA1 antagonism also protected against acrolein-induced mortality attesting to a complex role of TRPA1 in cardiopulmonary injury.

TRPA1 mediates changes in heart rate variability and cardiac mechanical function in mice exposed to acrolein.

Short-term exposure to ambient air pollution is linked with adverse cardiovascular effects. While previous research focused primarily on particulate matter-induced responses, gaseous air pollutants also contribute to cause short-term cardiovascular effects. Mechanisms underlying such effects have not been adequately described, however the immediate nature of the response suggests involvement of irritant neural activation and downstream autonomic dysfunction. Thus, this study examines the role of TRPA1, an irritant sensory receptor found in the airways, in the cardiac response of mice to acrolein and ozone. Conscious unrestrained wild-type C57BL/6 (WT) and TRPA1 knockout (KO) mice implanted with radiotelemeters were exposed once to 3ppm acrolein, 0.3ppm ozone, or filtered air. Heart rate (HR) and electrocardiogram (ECG) were recorded continuously before, during and after exposure. Analysis of ECG morphology, incidence of arrhythmia and heart rate variability (HRV) were performed. Cardiac mechanical function was assessed using a Langendorff perfusion preparation 24h post-exposure. Acrolein exposure increased HRV independent of HR, as well as incidence of arrhythmia. Acrolein also increased left ventricular developed pressure in WT mice at 24h post-exposure. Ozone did not produce any changes in cardiac function. Neither gas produced ECG effects, changes in HRV, arrhythmogenesis, or mechanical function in KO mice. These data demonstrate that a single exposure to acrolein causes cardiac dysfunction through TRPA1 activation and autonomic imbalance characterized by a shift toward parasympathetic modulation. Furthermore, it is clear from the lack of ozone effects that although gaseous irritants are capable of eliciting immediate cardiac changes, gas concentration and properties play important roles.