Dimethyl Sulfoxide Control Research Articles

Effect of extracellular signal-regulated kinase inhibitor on calpain in rat cerebral cortex after cardiopulmonary resuscitation

To explore the effect of extracellular signal-regulated kinase (ERK) inhibitor PD98059 on calpain-related proteins in the brain, and to understand the pathophysiological changes of calpain in cerebral ischemia/reperfusion injury (CIRI). Forty-two rats were divided into sham operation (Sham) group (n = 6), model group (n = 12), dimethyl sulfoxide (DMSO) control group (n = 12), and PD98059 group (n = 12) by random number table. The rat model of CIRI induced by cardiac arrest-cardiopulmonary resuscitation (CA-CPR) was reproduced by transesophageal electrical stimulation to induce ventricular fibrillation. In the Sham group, only the basic operations such as anesthesia, tracheal intubation, and arteriovenous catheterization were performed without CA-CPR. The rats in the DMSO control group and PD98059 group were injected with DMSO or PD98059 0.30 mg/kg via femoral vein, respectively, 30 minutes after the restoration of spontaneous circulation (ROSC), and rats in the Sham group and model group were given the same amount of normal saline. The duration of CPR, 24-hour survival rate and neurological deficit score (NDS) after ROSC were recorded. Hematoxylin-eosin (HE) staining and Nissl staining were used to observe the pathological changes of the cerebral cortex. The expressions of phosphorylated ERK (p-ERK), ERK, calpastatin, calpain-1, and calpain-2 were detected by Western blotting. The co-expression of p-ERK and calpain-2 was detected by double immunofluorescence. There were no significant differences in the duration of CPR and 24-hour survival rate among all groups. In the model group, the nuclei of the cerebral cortex were obviously deformed and pyknotic, cells vacuoles and tissues were arranged disorderly, Nissl corpuscles were significantly reduced, NDS scores were also significantly reduced, level of ERK phosphorylation was increased, and calpain-2 protein was significantly up-regulated compared with the Sham group. There was no significant difference in the above parameters between the DMSO control group and the model group. After intervention with PD98059, the pathological injury of brain tissue was significantly improved, Nissl corpuscles were significantly increased, the NDS score was significantly higher than that in the model group [75.0 (72.0, 78.0) vs. 70.0 (65.0, 72.0), P < 0.05], the level of ERK phosphorylation and calpain-2 protein expression were significantly lower than those in the model group [p-ERK (p-ERK/ERK): 0.65±0.12 vs. 0.92±0.05, calpain-2 protein (calpain-2/GAPDH): 0.73±0.10 vs. 1.07±0.14, both P < 0.05], while there was no significant difference in the expressions of calpastatin and calpain-1 in the cerebral cortex among all the groups. Double immunofluorescence staining showed that p-ERK and calpain-2 were co-expressed in cytosol and nucleus, and the co-expression rate of p-ERK and calpain-2 in the model group was significantly higher than that in the Sham group [(38.6±4.3)% vs. (9.2±3.5)%, P < 0.05], while it was significantly lowered in the PD98059 group compared with the model group [(18.2±7.0)% vs. (38.6±4.3)%, P < 0.05]. ERK together with calpain-2 participated in CIRI induced by CA-CPR. PD98059 inhibited the expression of calpain-2 and ERK phosphorylation. Therefore, ERK/calpain-2 may be a novel therapeutic target for CIRI.

Postponement of the opacification of lentoid bodies derived from human induced pluripotent stem cells after lanosterol treatment-the first use of the lens aging model in vitro in cataract drug screening.

Purpose: Our previous study observed that human induced pluripotent stem cell (HiPSC)-derived lentoid bodies (LBs) became cloudy with extended culture time, partially mimicking the progress of human age-related cataracts (ARCs) in a dish. In the present study, lanosterol, a potential anticataract drug, was used to further verify the value of this model in drug screening for cataract treatment. Methods: Mature LBs on day 25, which were differentiated from HiPSCs using the “fried egg” method, were continually cultured and treated with either dimethyl sulfoxide (control) or lanosterol. The LBs’ shape and opacity alterations were examined using light microscopy and mean gray value evaluation. The soluble and insoluble proteins were examined through SDS-PAGE gel electrophoresis combined with Coomassie blue staining. The protein aggregations were examined with immunofluorescence. Results: The mature LBs became cloudy with an extended culture time, and the opacification of the LBs was partially prevented by lanosterol treatment. There was less increase in insoluble proteins in the lanosterol-treated LBs than in the control group. There were also fewer cells containing aggregated protein (αA‐crystallin and αB‐crystallin) puncta in the lanosterol-treated LBs than in the control LBs. Conclusion: It was found that the opacification of LBs could be delayed by lanosterol treatment, which could be achieved by reducing protein aggregation, suggesting a promising HiPSC-derived drug-screening model for Age-related cataract.

Optimization of Benzoxazinorifamycins to Minimize hPXR Activation for the Treatment of Tuberculosis and HIV Coinfection.

Tuberculosis (TB) is one of the most significant world health problems, responsible for 1.5 M deaths in 2020, and yet, current treatments rely largely on 40 year old paradigms. Although the rifamycins (RIFs), best exemplified by the drug rifampin (RMP), represent a well-studied and therapeutically effective chemotype that targets the bacterial RNA polymerase (RNAP), these agents still suffer from serious drawbacks including the following: 3-9 month treatment times; cytochrome P450 (Cyp450) induction [particularly problematic for human immunodeficiency virus-Mycobacterium tuberculosis (MTB) co-infection]; and the existence of RIF-resistant (RIFR) MTB strains. We have utilized a structure-based drug design approach to synthesize and test 15 benzoxazinorifamycins (bxRIFs), congeners of the clinical candidate rifalazil, to minimize human pregnane X receptor (hPXR) activation while improving potency against MTB. We have determined the compounds' activation of the hPXR [responsible for inducing Cyp450 3A4 (CYP3A4)]. Compound IC50s have been determined against the wild-type and the most prevalent RIFR (β-S450L) mutant MTB RNAPs. We have also determined their bactericidal activity against "normal" replicating MTB and a model for non-replicating, persister MTB. We have identified a minimal substitution and have probed larger substitutions that exhibit negligible hPXR activation (1.2-fold over the dimethyl sulfoxide control), many of which are 5- to 10-fold more potent against RNAPs and MTB than RMP. Importantly, we have analogues that are essentially equipotent against replicating MTB and non-replicating persister MTB, a property that is correlated with faster kill rates and may lead to shorter treatment durations. This work provides a proof of principle that the ansamycin core remains an attractive and effective scaffold for novel and dramatically improved RIFs.

Formulation and Development of Adapalene Topical Nanohydrogel Using Different Surfactants and Cosurfactants for Antiacne Activity: In Vitro and Ex Vivo Evaluation

A formulation of an adapalene nanohydrogel (ADP‐NH) using different surfactants and cosurfactants for topical application was developed and characterized. The best formulation was obtained with nanohydrogel (NH) containing Tween‐80‐ (NH‐Tween‐80‐) incorporated carbopol‐940 and ethanol, 0.67% and 3.00% w/w, respectively. The optimized formulations of NH‐Tween‐80, nanohydrogel containing sodium lauryl sulphate (NH‐SLS), and nanohydrogel containing glycerol (NH‐glycerol) were separately evaluated to examine their in vitro and ex vivo permeability characteristics and compared with 0.3% dimethyl sulfoxide (control) solution. The polydispersity index of NH‐Tween‐80, NH‐SLS, and NH‐glycerol were found to be 0.264 ± 0.312, 0.382 ± 0.0045, and 0.310 ± 0.412, respectively. All NH formulations showed pH within human skin pH ranges throughout the stability period. The NH‐Tween‐80 revealed 191.22 μg/mL of ADP permeation through Strat‐M® membrane which was statistically significant (p &lt; 0.05) compared to NH‐SLS, NH‐glycerol, and control solution. At 24 h, NH‐Tween‐80 demonstrated 305.11 μg/mL of ADP permeation in Wistar rat abdominal skin which was 1.99‐, 1.56‐, and 4.89‐fold higher in comparison with NH‐SLS, NH‐glycerol, and control solution, respectively. Moreover, the ex vivo permeability of NH‐Tween‐80 was also compared with conventional gel (market sample) which was 3.38‐fold greater at 24 h. During the 6th month of accelerated stability analysis, the NH‐Tween‐80, NH‐SLS, and NH‐glycerol demonstrated 99.25% ± 0.15, 91.23% ± 0.41, and 96.08% ± 0.20 drug content, respectively. There were no noticeable physical changes observed up to 6 months for NH‐Tween‐80, while color change was observed in the 1st month and 3rd month of accelerated stability samples of NH‐SLS and NH‐glycerol, respectively. In this study, only NH‐Tween‐80 was considered both physically and chemically stable formulation. Therefore, it was concluded that the topical application of ADP‐NH containing Tween‐80 could be a very promising alternative for the treatment of acne vulgaris.

Cytopathological characteristics for provoked lesions of squamous epithelia of the digestive system in animal models

Relevance: Noticeable spreading of malignant neoplasms of the upper section of the digestive system and the worsening of these patients’ quality of life in case of late diagnostics stimulate the development of protocols for early detection of precancerous changes.&#x0D; The purpose of the study was to investigate the morphological characteristics of changes in the squamous epithelium of the upper part of the digestive tube in model organisms under provoked carcinogenesis when exposed to substances with proven carcinogenic activity.&#x0D; Methods: 40 female nonlinear white rats from the Orekhovo-Zuevsky urban settlement veterinary station were used as model organisms. The rats were divided into four equal groups. The first control group was not exposed to any specific influences. In groups 2-4, the rats underwent sedation and mechanical scarification microtraumatization of the oral mucosa. Then, they were applied twice a week by 0.9% NaCl solution (control group 2), 1% aqueous solution of dimethyl sulfoxide (DMSO, Akrikhin, Russia) (control group 3), or 1% aqueous solution of DMSO containing 0.1 mg/ml 4-nitroquinoline-N-oxide (4-NQO, Acros Organics, USA) (Experimental Group 4).&#x0D; Results: The cytological picture associated with chronic trauma to the mucous membrane of the upper digestive tract was characterized by nonspecific reactions: inflammation, hyper- and parakeratosis, and atrophic changes. There were no statistically significant differences in the frequency of such changes between the experimental and control groups. Specific changes in squamous epithelium towards a precancerous state in the Experimental Group with chronic exposure to a substance with carcinogenic activity occurred significantly more often than in the control groups (p &lt;0.05), as proven by the results of comparative histological verification. Conclusion: Cytological study could be used in clinical practice as a screening tool for precancerous oropharynx and upper esophagus conditions.

Effect of Taspine hydrochloride on the repair of rat skin wounds by regulating keratinocyte growth factor signal

ABSTRACT To explore the regulation of keratinocyte growth factor (KGF) in the process of repairing rat skin wounds by taspine hydrochloride (TA/HCl), 45 male Sprague-Dawley (SD) rats were purchased and divided into an experimental group, a dimethyl sulfoxide (DMSO) control group, and a basic fibroblast growth factor (bFGF) control group, each with 15 only. A back trauma model was innovatively adopted to prevent rats from biting and contaminating. The wound healing time and healing rate of the rat, and the Hydroxyproline (Hyp) and KGF expressions were observed. Morphological changes of wound tissue and the number of capillaries were observed after hematoxylin-eosin (HE) staining. The results showed that wound healing rate of experimental group and bFGF group was significantly higher than that of DMSO group (P < 0.05) after 2–15 days, and wound healing time of experimental group was 18 days, which was significantly lower than that of the DMSO group (P < 0.05). Expression levels of Hyp and KGF in the granulation tissue of rats in the experimental group were much higher than those in the DMSO control group after trauma (P < 0.05). In early stage of wound tissue repair, the number of new capillaries formed in experimental group was significantly higher than that in DMSO control group (P < 0.05). In summary, this study innovatively focused on KGF. The mechanism of TA/HCL promoting rat skin wound healing was closely related to KGF.

The Sumoylation Inhibitor TAK-981 in Combination with the CD19-Targeting Antibody Tafasitamab Shows Enhanced Anti-Tumor Activity in Preclinical B-Cell Lymphoma Models

IntroductionTAK-981 is a first-in-class small molecule inhibitor of the SUMO activating enzyme currently in Phase I/II clinical trials. TAK-981 has been shown to increase NK cell activation and M1 macrophage polarization via upregulation of Type I interferon (IFN) signaling, leading to enhanced antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in combination with rituximab (Nakamura 2019, AACR).Tafasitamab (MOR208) is a CD19-targeting antibody with enhanced Fc effector function mediating ADCC, ADCP and direct cytotoxic activities against B-lymphoma cells. Based on the Phase II clinical study L-MIND (Salles et al., 2020 and Duell et al., 2021), tafasitamab in combination with lenalidomide received accelerated approval by the Food and Drug Administration for the treatment of transplant-ineligible adult patients with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL).Due to the potential for TAK-981 to enhance the activity of tafasitamab via activation of innate effector cells, we aimed to investigate the effects of this drug combination on ADCC, ADCP and tumor cell viability in vitro. Additionally, combinatorial activity of TAK-981 plus tafasitamab was evaluated in lymphoma xenograft models.MethodsA panel of 9 aggressive lymphoma cell lines was analyzed (7 DLBCL and 2 Burkitt lymphoma). For ADCC, PBMC effector cells from healthy human donors were pre-treated with 0.1 or 1 µM TAK-981 or dimethyl sulfoxide (DMSO) control for 24 hours. Tumor cells were incubated with/without 1 nM tafasitamab in the presence of TAK-981 pretreated PBMCs at effector-to-target (E:T) ratios of 5:1 to 10:1 for 2 hours. Degranulation of NK cells was determined via CD107a surface expression after co-incubation of TAK-981 pre-treated PBMCs with tumor cells and 0.1 or 10 nM tafasitamab for 3 hours. Cytokine levels in the supernatant were investigated upon incubation of PBMCs with lymphoma cells, 1 µM TAK-981 and/or 10 nM tafasitamab for 24 hours. For the ADCP assays, in vitro differentiated macrophages were treated with 1 µM TAK-981 for 24 hours. Next, macrophages were incubated with lymphoma cells and 1 or 10 nM tafasitamab at an E:T ratio of 2:1 for 3 hours. For cell viability assays, tumor cells were treated with 1-1000 nM TAK-981 and/or 5 nM tafasitamab for 24 hours in the absence of effector cells. Cytotoxicity, phagocytosis, degranulation and cytokine release were analyzed by flow cytometry. Cell viability was assessed by determination of ATP levels. For in vivo analysis, effects of TAK-981 (7.5 mg/kg IV twice weekly) in combination with tafasitamab (3, 10 or 20 mg/kg IP twice weekly) on tumor growth were evaluated in Daudi and WSU-DLCL2 xenograft models of Burkitt lymphoma and DLBCL grown in SCID mice.ResultsIn ADCC experiments, increased cytotoxicity was observed upon combination treatment with TAK-981 and tafasitamab compared to the respective mono treatments in 5/8 tested lymphoma cell lines (Daudi, SU-DHL-2, SU-DHL-6, TMD8, OCI-LY10). Moreover, TAK-981 plus tafasitamab enhanced degranulation of NK cells and cytokine release compared to mono treatments. In ADCP assays, combination of TAK-981 and tafasitamab resulted in increased phagocytosis rates in comparison to mono treatments in 2/2 tested cell lines (Daudi, Ramos). Cell viability analysis revealed a combination benefit by increased direct cytotoxic effects against SU-DHL-6 cells. Finally, TAK-981 and tafasitamab were investigated in Daudi and WSU-DLCL2 xenograft models with 3 weeks of dosing. In the Daudi model, the combination treatments of TAK-981 with 10 or 20 mg/kg tafasitamab performed better than either treatment alone, and in the WSU-DLCL2 model, the combination treatments of TAK-981 with 3, 10 or 20 mg/kg tafasitamab performed better than the single agent treatments.ConclusionsThe combination of TAK-981 with tafasitamab significantly enhanced anti-tumor effects compared to the respective monotherapies in vitro and in vivo. These preclinical data support a clinical evaluation of this drug combination in patients with lymphoma including aggressive subtypes such as Burkitt lymphoma and DLBCL.The study was funded by MorphoSys AG and Takeda Development Center Americas, Inc. DisclosuresPatra-Kneuer: MorphoSys AG: Current Employment. Nakamura: Takeda Development Center Americas, Inc.: Current Employment. Song: Takeda Pharmaceuticals International Co.: Current Employment. Grossman: Takeda Development Center, Cambridge MA: Current Employment. Polzer: MorphoSys: Current Employment. Ginzel: MorphoSys: Current Employment. Steidl: MorphoSys AG: Current Employment. Berger: Takeda Development Center Americas, Inc.: Current Employment. Proscurshim: Takeda Pharmaceuticals: Current Employment, Current holder of individual stocks in a privately-held company. Heitmüller: MorphoSys AG: Current Employment.

A Laboratory Method to Measure Skin Surface Staining by Cigarette Smoke, Tobacco Heating Products and E-Cigarettes

Summary Exhaled or side-stream cigarette smoke (CS) may visually stain a consumer's skin over time. Tobacco heating products (THPs) and e-cigarettes (ECs) have reduced staining potential because they do not produce side-stream aerosols and their exhaled aerosols have significantly reduced levels of toxicants, particles and odour. Here we assess discolouration of porcine skin in vitro after exposure to particulate matter (PM) or aerosols from CS (3R4F), two THPs (glo and glo sens) and an EC (iSwitch Maxx). PM was prepared by capturing aerosols on Cambridge filter pads and eluting with dimethyl sulfoxide (DMSO). Abattoir-obtained porcine skin samples were incubated with PM or DMSO control at 37 °C between 0 and 6.0 h. For aerosol assessment, porcine skin samples were exposed to between 50 and 400 puffs of the products, or air control, using a smoking machine. Colour profiles and staining levels of each skin sample were measured at different timepoints and puff thresholds using a spectrophotometer. Staining increased with time and dose, the greatest changes being observed following exposure to aerosols and PM from CS. THP, EC and control values were significantly different from CS after 0.5 h exposure to PM or 50 puffs of aerosols. The minimal staining induced by THPs and EC was comparable to controls. These data suggest that THPs and ECs could offer hygiene benefits to consumers who switch from smoking cigarettes. Further studies are required to assess the longer-term effects of THPs and ECs on skin discoloration. [Contrib. Tob. Nicotine Res. 30 (2021) 158–166]

Animal Experimental Study of the Role of Menthol and Cineole, Two Tobacco Additives, on Nicotine Dependence

To establish a nicotine intravenous self-administration rat model, and to examine, with this model, the effects of two flavoring additives, menthol and cineole, on nicotine dependence. Thirty male Sprague-Dawley (SD) rats were included in the study. After jugular venous catheterization was performed, fixed concentration of nicotine was administered in order to train the rats and establish the rat model of intravenous self-administration groups, receiving intraperitoneal injection of menthol, cineole, and dimethyl sulfoxide (DMSO), the vehicle that was used for the control group. The rats were tested with different fixed-ratio (FR) schedules, including FR1 schedule, in which the rat received one nicotine infusion for every active nose poke, FR2 schedule, in which the rat received one nicotine infusion for every two active nose pokes, and FR5 schedule, in which the rat received one nicotine infusion for every five active nose pokes. The number of active and inactive poke responses and the number of nicotine infusion were documented accordingly. After 10 days of training in nicotine self-administration, the 30 rats demonstrated significant increase in the number of active poke responses and the number of nicotine infusion, which were maintained at a stable and relatively high level. The number of active poke responses was significantly higher that of inactive poke responses ( P< 0.001). The rat model of intravenous nicotine self-administration was successfully established. In the testing phase, under the FR2 schedule, the menthol group showed a reduced number of active poke responses ( P=0.020). Under the FR5 schedule, the groups showed obvious interaction between time and the number of active poke responses ( P<0.011), with the menthol group showing reduced number of active poke responses on day three ( P=0.011) and the cineole group showing rising number of active poke responses on day three ( P=0.003). The DMSO control group did not show any significant change. Menthol and cineole are shown to have an effect on nicotine dependence. When there is relative difficulty involved in obtaining nicotine, menthol suppresses nicotine dependence, whereas cineole enhances nicotine dependence.

Prediction and Verification of the Major Ingredients and Molecular Targets of Tripterygii Radix Against Rheumatoid Arthritis.

Tripterygii Radix exhibits good clinical efficacy and safety in rheumatoid arthritis (RA) patients, but its effective components and mechanism of action are still unclear. The purpose of this study was to explore and verify the major ingredients and molecular targets of Tripterygii Radix in RA using drug-compounds-biotargets-diseases network and protein-protein interaction (PPI) network analyses. The processes and pathways were derived from Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The most important compounds and biotargets were determined based on the degree values. RA fibroblast-like synoviocytes (RA-FLS) were separated from RA patients and identified by hematoxylin and eosin (HE) staining and immunohistochemistry. The purity of RA-FLS was acquired by flow cytometry marked with CD90 or VCAM-1. RA-FLS were subjected to control, dimethyl sulfoxide (control), kaempferol, or lenalidomide treatment. Cell migration was evaluated by the transwell assay. The relative expression of biotarget proteins and cytokines was analyzed by western blotting and flow cytometry. In total, 144 chemical components were identified from Tripterygii Radix; kaempferol was the most active ingredient among 33 other components. Fourteen proteins were found to be affected in RA from 285 common biotargets. The tumor necrosis factor (TNF) signaling pathway was predicted to be one of the most latent treatment pathways. Migration of RA-FLS was inhibited and the expression of protein kinase B (AKT1), JUN, caspase 3 (CASP3), TNF receptor 1 and 2 (TNFR1 and TNFR2), interleukin-6 (IL-6), and TNF-α was significantly affected by kaempferol. Thus, this study confirmed kaempferol as the effective component of Tripterygii Radix against RA-FLS and TNF signaling pathway and its involvement in the regulation of AKT1, JUN, CASP3, TNFR1, TNFR2, IL-6, and TNF-α expression.

Inhibition of Caspase-8 Activity Improves Freezing Efficiency of Male Germline Stem Cells in Mice.

Cryopreservation of male germline stem cells (GSCs) is an essential technique for their long-term preservation and utilization in various fields. However, the specific apoptosis pathways involved in cryoinjury during freezing remain unclear. Therefore, our study sought to identify the pathways involved in cryoinjury-induced apoptosis and thereby to improve freezing efficiency during GSC cryopreservation through the creation of a specific molecular-based cryoprotectant. The activities of caspase-8, caspase-9, caspase-3, and caspase-7 were assessed by Western blot analyses to determine the role of specific apoptosis pathways in GSC cryoinjury. Specifically, the role of a specific caspase was identified by recovery rate, relative proliferation rate, Annexin V/propidium iodide co-staining, and caspase activity using its inhibitor and activator. Moreover, the safety of the cryoprotectant was assessed by immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, the efficacy of the molecular-based cryoprotectant was assessed using frozen cells in the presence of dimethyl sulfoxide (DMSO) (control), trehalose, a caspase-8 inhibitor Z-IETD-FMK [ZIF], or a mixture of the aforementioned compounds, after which the changes in Src signaling were measured. Our results demonstrated that caspase-8 plays a major role in cryoinjury-induced apoptosis and therefore its inhibition improves freezing efficiency. Specifically, a significantly higher relative proliferation rate was observed in the Z-IETD-FMK 0.01 μM-treated cells than in the DMSO control (100% ± 6.2% vs. 189.8% ± 9.5%), with decreases in both early apoptosis (16.6% ± 2.2% vs. 7.5% ± 1.0%) and caspase-8 activity (1.0-fold vs. 0.4-fold). The relative proliferation rate was significantly higher in the cryoprotectant mixture (246.0% ± 12.2%) than other individual treatment groups (trehalose 200 mM, 189.8% ± 9.5%; Z-IETD-FMK 0.01 μM, 189.7% ± 2.2%) with no significant differences in Src signaling. Therefore, our findings provide novel insights into the development of freezing protocols to enhance GSC freezing efficiency, thereby facilitating the wider adoption of GSCs in the livestock industry and/or clinical trials.

A comparative study of Caesalpinia bonduc (L.) Roxb. root extracts on sexual behaviour in male Wistar rats.

Caesalpinia bonduc is among the traditionally used plant in Benin, for its enhancement of male sexual activity. This study was undertaken to investigate the potential effect of C.bonduc root extracts on sexual behaviour of male Wistar rats. For that, thirty-six rats were allocated into six groups and orally treated with dimethyl sulfoxide (control), Sildenafil citrate (standard) and C.bonduc root extracts (hexane, ethyl acetate, ethanol and methanol) orally for twenty-eight days. Sexual behaviour parameters such as intromission frequency, intromission latency, mount latency and mount frequency were evaluated on day 0, 14 and 28. After the study completion, the body and reproductive organ weights as well as testicular histology and testosterone level were recorded. C.bonduc root extracts treatments had no significant effect on the body weight of rats. Enhancement in sexual behaviour was observed in ethanolic extract treated rats. An significant increase in mount frequency and intromission frequency as well as significant reduction in mount latency and intromission latency were noticed for ethanolic extract. The same extract caused an improvement in testosterone levels, relative testes weight and histological architecture. The findings demonstrate the aphrodisiac potential of C.bonduc root and lend support to the folkloric use as aphrodisiac.

Co-expression network of long non-coding RNA and mRNA reveals molecular phenotype changes in kidney development of prenatal chlorpyrifos exposure in a mouse model

BackgroundChlorpyrifos (CPF) is one of the most widely used organophosphorus pesticides globally and can accumulate in the kidney. Researchers have confirmed the regulatory functions of long non-coding ribonucleic acid (lncRNA) in the kidney. However, very few studies have examined the effects of prenatal CPF exposure or lncRNA on kidney development.MethodsHigh-throughput ribonucleic acid (RNA) sequencing was performed on embryonic kidneys obtained at E12.5, E14.5, E16.5, and E18.5 of prenatal CPF-exposed mice and the dimethyl sulfoxide (DMSO) control mice. A weighted gene co-expression network analysis (WGCNA) and a functional enrichment analysis were applied to construct a lncRNA-messenger ribonucleic acid (mRNA) network and screen targeted genes. These strategies were used to select the modules and genes correlated with prenatal CPF exposure in mouse kidney development.ResultsA gene ontology (GO) analysis revealed that the hub mRNAs linked to prenatal CPF exposure were mainly involved in the extracellular matrix and collagen degradation. Prss1, Prss2, and Prss3 were the most significantly upregulated mRNAs, and all had strong connections to lncRNAs Gm28760, Gm28139, and Gm26717. Additionally, we analyzed the lncRNA-mRNA network at different developmental kidney stages after prenatal CPF exposure. The results showed that kidney development was blocked at E12.5, which led to ectopic proximal tubule formation at E18.5.ConclusionsIn summary, the RNA-sequencing and weighted gene co-expression network analyses showed that molecular phenotype changes occur in kidney development in a prenatal CPF exposure mouse model.

Quercetin ameliorates Aspergillus fumigatus keratitis by inhibiting fungal growth, toll-like receptors and inflammatory cytokines

PurposeTo investigate the antifungal and anti-inflammatory effects of quercetin on Aspergillus fumigatus (A. fumigatus) keratitis. MethodsHuman corneal epithelial cells (HCECs) and C57BL/6 mice were stimulated by A. fumigatus and treated with quercetin or dimethyl sulfoxide (DMSO) after infection. In HCECs, minimum inhibitory concentration (MIC) and cytotoxicity tests (CCK-8) were used to detect the antifungal effect and cytotoxicity of quercetin. In mice with A. fumigatuskeratitis, clinical score, plate counting and hematoxylin-eosin (HE) staining were performed to evaluate the effects of quercetin in vivo. Myeloperoxidase (MPO) assay and immunofluorescence staining were applied to assess neutrophil recruitment and infiltration. Real time PCR (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and western blot were used to detect the mRNA and protein expressions of inflammatory mediators. ResultsCompared with DMSO control, quercetin (16–64 μM) significantly inhibited the growth of A. fumigatus in a concentration-dependent manner without affecting cell viability in HCECs. In corneas of mice with A. fumigatuskeratitis, quercetin decreased clinical score and fungal load, and reduced neutrophil recruitment and infiltration to the corneal stroma. Moreover, quercetin attenuated the expression of inflammatory mediators including toll-like receptor-4 (TLR-4), TLR-2, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and high mobility group box 1 (HMGB1) in vitro and in vivo. ConclusionsOur study demonstrated that quercetin treatment can ameliorate A. fumigatus keratitis by inhibiting the growth of A. fumigatus, decreasing neutrophil recruitment and infiltration, and downregulating the productions of TLR-4, TLR-2, TNF-α, IL-1β and HMGB1, indicating quercetin is likely to become a potential therapeutic agent in FK treatment.

Compact Fat Grafting: A Novel Method to Improve Graft Retention Through Modulation of Adipocyte Size.

The viable zone where adipocytes and/or adipose-derived stem cells survive is present at the surface of graft fat tissue; however, there is controversy regarding the zone thickness. Graft retention could be improved if more adipocytes are included in the zone. We hypothesize that a temporary reduction in adipocyte size prior to grafting could increase the number of adipocytes in the viable zone. We reduced the adipocyte size by treatment with MLN4924, which controls lipid accumulation in adipocytes, and investigated the histological and microenvironmental changes in grafted fat. Subcutaneous fat harvested from wild-type C57BL/6J mice was chopped into small pieces; treated with dimethyl sulfoxide (control group), 0.25 μM MLN4924, or 0.5 μM MLN4924 for 4 days; and grafted into recipient C57BL/6J mice at the supraperiosteal plane of the skull. The reduced adipocyte size in response to MLN4924 treatment was restored within 8 weeks after fat grafting. The MLN4924-treated groups exhibited substantially greater graft volume, lower tissue hypoxia, and higher production of M2 macrophages compared with the control group. Grafting with compact fat that had smaller adipocytes improved the microenvironment by modulating tissue hypoxia and macrophage polarization, leading to improved graft retention. Therefore, compact fat grafting may offer a new clinical strategy without the need for stem cell manipulation.

Antifibrotic effects of sulforaphane treatment on gingival elasticity reduces orthodontic relapse after rotational tooth movement in beagle dogs.

ObjectiveIncreased gingival elasticity has been implicated as the cause of relapse following orthodontic rotational tooth movement and approaches to reduce relapse are limited. This study aimed to investigate the effects of sulforaphane (SFN), an inhibitor of osteoclastogenesis, on gene expression in gingival fibroblasts and relapse after rotational tooth movement in beagle dogs.MethodsThe lower lateral incisors of five beagle dogs were rotated. SFN or dimethylsulfoxide (DMSO) were injected into the supra-alveolar gingiva of the experimental and control group, respectively, and the effect of SFN on relapse tendency was evaluated. Changes in mRNA expression of extracellular matrix components associated with gingival elasticity in beagles were investigated by real-time polymerase chain reaction. Morphology and arrangement of collagen fibers were observed on Masson’s trichrome staining of buccal gingival tissues of experimental and control teeth.ResultsSFN reduced the amount and percentage of relapse of orthodontic rotation. It also decreased the gene expression of lysyl oxidase and increased the gene expression of matrix metalloproteinase (MMP) 1 and MMP 12, compared with DMSO control subjects. Histologically, collagen fiber bundles were arranged irregularly and were not well connected in the SFN-treated group, whereas the fibers extended in parallel and perpendicular directions toward the gingiva and alveolar bone in a more regular and well-ordered arrangement in the DMSO-treated group.ConclusionsOur findings demonstrated that SFN treatment may be a promising pharmacologic approach to prevent orthodontic rotational relapse caused by increased gingival elasticity of rotated teeth in beagle dogs.

NLRP3 inflammasome inhibitor CY-09 reduces hepatic steatosis in experimental NAFLD mice

AimsNon-alcoholic fatty liver disease (NAFLD) has become one of the most common chronic liver diseases. The NOD-like receptor protein 3 (NLRP3) inflammasome was suggested to be involved in the pathogenesis of NAFLD. A small-molecule named CY-09 is a new selective and direct inhibitor of the NLRP3 inflammasome. We aimed to investigate whether CY-09 is effective for the treatment of NAFLD in a high-fat diet (HFD)-induced mouse model. MethodsTwenty mice were fed by HFD for 14 weeks, and then were randomly assigned into two groups: (1) control group receiving dimethylsulfoxide (DMSO) solution; (2) CY-09 group receiving CY-09 injection. In an 8-week follow-up, oral glucose tolerance test (OGTT) and homeostasis model assessment of insulin resistance (HOMA-IR) were used to measure glucose metabolism. Liver steatosis was evaluated by the NAFLD activity score (NAS) and deemed as the primary outcome. ResultsThe body weight in CY-09 group was significantly lower than the DMSO control group on 27 weeks (41.0 ± 3.5 g vs. 49.7 ± 5.2 g, P = 0.014). The area under the curve (AUC) of OGTT was less in CY-09 group than that in DMSO group (35.81 ± 6.79 vs. 22.91 ± 2.58 mmol/L·hr, P = 0.004), as well as HOMA-IR (14.36 ± 3.89 vs. 8.82 ± 2.04 mmol.mIU.L-2, P = 0.023). Microscopically, liver lipid droplets dramatically improved and significantly lower NAS was observed in CY-09 group (8.25 ± 1.26 vs. 3.20 ± 0.45, P < 0.001). ConclusionCY-09 reduces hepatic steatosis in experimental NAFLD mice and CY-09 may be a potential therapeutic drug of NAFLD in clinical practice.

Study on mechanisms of interleukin-17A regulating the expressions of interleukin-1β and interleukin-23 in mouse keratinocytes

Objective: To investigate the mechanisms of interleukin-17A (IL-17A) regulating the expressions of IL-1β and IL-23 in mouse keratinocytes (KCs). Methods: Primary KCs were isolated from the skin of 400 newborn male and female wild type C57BL/6 mice and cultured in 24-well plates with Roswell Park Memorial Institute 1640 medium containing fetal bovine serum in the volume fraction of 10% for the following experiments. (1) The cells were divided into phosphate buffer solution (PBS) control group and IL-17A stimulation group according to the random number table (the same grouping method below), which were cultured with 10 μL PBS or 10 μL IL-17A in the mass concentration of 100 ng/mL for 6 hours, respectively. The expression levels of IL-1β and IL-23 mRNA in cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR), with 3 samples in each group. (2) The cells were divided into dimethyl sulfoxide (DMSO) control group, IL-17A+ DMSO group, IL-17A+ nuclear factor κB (NF-κB) inhibitor group, IL-17A+ signal transduction and activator of transcription 3 (STAT3) inhibitor group, IL-17A+ extracellular signal-regulated kinase 1 (ERK1) inhibitor group, IL-17A+ ERK2 inhibitor group, and IL-17A+ c-Jun N-terminal kinase (JNK) inhibitor group. The reagents were added to cells in corresponding groups respectively and cultured for 6 hours. The volume of each reagent was 10 μL, the mass concentration of IL-17A was 100 ng/mL, and the molarity concentrations of NF-κB, STAT3, ERK1, ERK2, JNK signal pathway inhibitors PDTC, S3I-201, SCH772984, SCH772984, SP600125 were 5 μmol/L, 100 μmol/L, 4 nmol/L, 1 nmol/L, and 10 μmol/L, respectively. The expression levels of IL-1β mRNA and IL-23 mRNA in cells were detected by real-time fluorescence quantitative RT-PCR, with 3 samples in each group. (3) The cells were grouped and treated the same as those in experiment (1). The levels of NF-κB phosphorylation, STAT3 phosphorylation, ERK phosphorylation, and JNK phosphorylation were detected by Western blotting, with 3 samples in each group. Data were statistically analyzed with two-tailed Student t test, one-way analysis of variance, t test, and Bonferroni correction. Results: (1) After culture of 6 hours, compared with those in PBS control group, the expression levels of IL-1β and IL-23 mRNA in cells in IL-17A stimulation group were significantly increased (t=13.46, 6.72, P<0.01). (2) After culture of 6 hours, the expression levels of IL-1β and IL-23 mRNA in cells in DMSO control group, IL-17A+ DMSO group, IL-17A+ NF-κB inhibitor group, IL-17A+ STAT3 inhibitor group, IL-17A+ ERK1 inhibitor group, IL-17A+ ERK2 inhibitor group, and IL-17A+ JNK inhibitor group were 1.00±0.11, 4.01±0.32, 0.32±0.06, 1.76±0.43, 3.62±0.24, 3.80±0.43, 4.26±0.74 and 1.03±0.29, 4.08±0.34, 4.76±0.38, 4.70±0.21, 1.06±0.42, 0.92±0.21, 0.39±0.05, respectively. Compared with those in DMSO control group, the expression levels of IL-1β and IL-23 mRNA in cells in IL-17A+ DMSO group were significantly increased (t=9.24, 12.60, P<0.01). Compared with that in IL-17A+ DMSO group, the expression level of IL-1β mRNA was significantly decreased in cells in IL-17A+ NF-κB inhibitor group and IL-17A+ STAT3 inhibitor group (t=11.34, 6.91, P<0.01). Compared with that in IL-17A+ DMSO group, the expression level of IL-23 mRNA was significantly decreased in cells in IL-17A+ ERK1 inhibitor group, IL-17A+ ERK2 inhibitor group, and IL-17A+ JNK inhibitor group (t=12.44, 13.03, 15.21, P<0.01). (3) After culture of 6 hours, compared with those in PBS control group, the levels of NF-κB phosphorylation, STAT3 phosphorylation, ERK phosphorylation, and JNK phosphorylation in cells in IL-17A stimulation group were significantly increased. Conclusions: IL-17A promotes the transcription of IL-1β in mouse KCs through the phosphorylation of NF-κB and STAT3 pathways and IL-23 through the phosphorylation of ERK and JNK pathways.

Effects of salinomycin on proliferation and apoptosis of oral squamous cell carcinoma

To investigate the effects of salinomycin on the proliferation and apoptosis of oral squamous carcinoma cells and to further understand the mechanisms of these effects. The human oral squamous carcinoma cell line CAL-27 was cultured in different concentrations of salinomycin and cisplatin. After co-culture with 0, 1, 2, 4, 8, 16 and 32 μmol/L salinomycin or 0, 1.25, 2.5, 5, 10, 20, 40 and 80 μmol/L cisplatin for 24 hours and 48 hours, the proliferation of oral squamous carcinoma cells were detected by cell counting kit-8(CCK-8) assay. After being exposed to 0, 2, 4, 8 μmol/L salinomycin and 0, 5, 10, 20 μmol/L cisplatin for 48 hours, the cell cycle of oral squamous carcinoma cells was detected by flow cytometry assay, and Western blot analysis was performed to analyze the expressions of cysteine-containing aspartate-specific proteases-3(Caspase-3), cysteine-containing aspartate-specific proteases-9(Caspase-9), poly ADP-ribose polymerase (PARP), protein kinase B (Akt) and phosphorylated protein kinase B (p-Akt) protein in oral squamous carcinoma cells. Both salinomycin and cisplatin significantly inhibited the proliferation of oral squamous cell carcinoma CAL-27 cells in a time- and dose-dependent manner. However, compared with the first-line chemotherapeutic drug cisplatin, salinomycin showed stronger anti-proliferation activity in oral squamous carcinoma cells than cisp-latin (P < 0.001). After being exposed to 8 μmol/L salinomycin, CAL-27 cells exhibited markedly higher proportion in quiescent/ first gap phases (40.40%±1.99% vs. 64.46%±0.90%, P < 0.05), and had a significantly lower proportion in synthesis phases and second gap / mitosis phases (24.32%±2.30% vs. 18.73%±0.61%, P < 0.05; 35.01%±1.24% vs. 16.54%±1.31%, P < 0.05) compared with the dimethyl sulfoxide control group; moreover cisplatin didn't show cell-cycle specific effect on CAL-27. Western blot proved that salinomycin could up-regulate the expressions of Caspase-3 and Caspase-9 protein in oral squamous cell carcinoma CAL-27 cells (P < 0.05). At the same time, the levels of PARP, Akt and p-Akt protein were down-regulated (P < 0.05). Compared with cisplatin, salinomycin has a better inhibitory effect on the proliferation of oral squamous carcinoma cells and blocks the cell cycle process at the quiescent / first gap phase. At the same time, salinomycin could trigger apoptosis of oral squamous carcinoma cells and the mechanism is associated with the Akt/p-Akt signaling pathway.

Development of a spermatogonia cryopreservation protocol for blue catfish, Ictalurus furcatus

Sustainability of channel catfish, Ictalurus punctatus ♀ × blue catfish, Ictalurus furcatus ♂ hybrid aquaculture relies on new innovative technologies to maximize fry output. Transplanting spermatogonial stem cells (SSCs) from blue catfish into channel catfish hosts has the potential to greatly increase gamete availability and improve hybrid catfish fry outputs. Cryopreservation would make these cells readily accessible for xenogenesis, but a freezing protocol for blue catfish testicular tissues has not yet been fully developed. Therefore, the objectives of this experiment were to identify the best permeating [dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, methanol] and non-permeating (lactose or trehalose with egg yolk or BSA) cryoprotectants, their optimal concentrations, and the best freezing rates (−0.5, −1.0, −5.0, −10 °C/min until −80 °C) that yield the highest number of viable type A spermatogonia cells. Results showed that all of these factors had significant impacts on post-thaw cell production and viability. DMSO was the most efficient permeating cryoprotectant at a concentration of 1.0 M. The optimal concentration of each cryoprotectant depended on the specific cryoprotectant due to interactions between the two factors. Of the non-permeating cryoprotectants, 0.2 M lactose with egg yolk consistently improved type A spermatogonia production and viability beyond that of the 1.0 M DMSO control. The overall best freezing rate was consistent at −1 °C/min, but similar results were obtained using −0.5 °C/min. Overall, we recommend cryopreserving blue catfish testicular tissues in 1.0 M DMSO with 0.2 M lactose and egg yolk at a rate of either -0.5 or −1 °C/min to achieve the best cryopreservation outcomes. Continued development of cryopreservation protocols for blue catfish and other species will make spermatogonia available for xenogenic applications and genetic improvement programs.