Objective: The aim of this study was to explore the ototoxicity of toluene in the early development of zebrafish embryos/larvae. Methods: Zebrafish were utilized to explore the ototoxicity of toluene. Locomotion analysis, immunofluorescence, and qPCR were used to understand the phenotypes and molecular mechanisms of toluene ototoxicity. Results: The results demonstrated that at 2 mmol/L, toluene induced zebrafish larvae death at 120 hours post fertilization (hpf) at a rate of 25.79% and inhibited the rate of hatching at 72 hpf. Furthermore, toluene exposure inhibited the distance travelled and average swimming velocity of zebrafish larvae while increasing the frequency of movements. As shown by fluorescence staining of hair cells, toluene inhibited the formation of lateral line neuromasts and middle line 1 (Ml Conclusion This study indicated that toluene may affect the development of both the inner ear and lateral line systems in zebrafish, while the lateral line system may be more sensitive to toluene than the inner ear.
Animals ; Ear, Inner/growth & development* ; Embryo, Nonmammalian/drug effects* ; Gene Expression Regulation, Developmental/drug effects* ; Hair Cells, Auditory/metabolism* ; Lateral Line System/growth & development* ; Locomotion/drug effects* ; Ototoxicity/physiopathology* ; Toluene/toxicity* ; Zebrafish

OBJECTIVE: To investigate the effects of stachydrine (STA) on apoptosis of Aβ-induced PC12 cells mimicking Alzheimer's disease and explore the mechanisms. METHODS: The differential genes of STA were analyzed based on GSE85871 data, and the target genes of STA were identified using STITCH database. PC12 cells were treated with Aβ to establish a cell model of Alzheimer's disease, and the changes in cell viability and cell cycle in response to STA treatment were assessed using MTT assay and flow cytometry, respectively. RT-PCR and Western blotting were used to detect the relevant gene or protein expressions in the treated cells. RESULTS: GSE85871 data showed 37 up-regulated genes and 48 down-regulated genes in cells following treatment with STA. Analysis of the data from the STITCH database indicated that RPS8 and EED were the target genes of STA. Treatment of PC12 cells with Aβ significantly lowered the cell viability ( < 0.05) and the expressions of RPS8 and EED at both the mRNA and protein levels ( < 0.05), and obviously inhibited the expression of apoptosis-related proteins Bcl-2 and p53 ( < 0.05). STA treatment of the cells significantly reversed the effect of Aβ and induced cell cycle arrest in G2/M phase, causing also significantly increases in the expression levels of RPS8, EED, Bcl-2 and p53 ( < 0.05). CONCLUSIONS STA plays an important role in inhibiting the apoptosis of PC12 cells induced by Aβ possibly by regulating RPS8 and EED expression to promote the expressions of Bcl-2 and p53.
Alzheimer Disease ; Animals ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; Gene Expression Regulation ; drug effects ; Models, Biological ; PC12 Cells ; Proline ; analogs & derivatives ; pharmacology ; Rats

OBJECTIVE: To investigate the effects of blocking the activation of ERK pathway on the expression of matrix metalloproteinase-9 (MMP-9) and the formation of cerebral edema in SD rats after brain injury. METHODS: Ninety SD rats were randomly divided into 3 equal groups, including a sham-operated group, modified Feeney's traumatic brain injury model group, and ERK inhibition group where the ERK inhibitor SCH772984 (500 μg/kg) was injected via the femoral vein 15 min before brain trauma. At 2 h and 2 days after brain trauma, the permeability of blood-brain barrier was assessed by Evans blue method, the water content of the brain tissue was determined, and the phosphorylation level of ERK and the expression level of MMP-9 mRNA and protein were measured by RT-PCR and Western blotting. RESULTS: Compared with the sham-operated group, the rats with brain trauma exhibited significantly increased level of ERK phosphorylation at 2 h and significantly increased expression of MMP-9 mRNA and protein 2 days after the injury ( < 0.01). Treatment with the ERK inhibitor significantly decreased the phosphorylation level of ERK after the injury ( < 0.01), suppressed over-expression of MMP-9 mRNA and protein 2 days after the injury ( < 0.01). The permeability of blood-brain barrier increased significantly 2 h after brain trauma ( < 0.05) and increased further at 2 days ( < 0.01); the water content of the brain did not change significantly at 2 h ( > 0.05) but increased significantly 2 d after the injury ( < 0.01). Treatment with the ERK inhibitor significantly lowered the permeability of blood-brain barrier and brain water content after brain trauma ( < 0.01). CONCLUSIONS Blocking the activation of ERK pathway significantly reduced the over-expression of MMP-9 and alleviates the damage of blood-brain barrier and traumatic brain edema, suggesting that ERK signaling pathway plays an important role in traumatic brain edema by regulating the expression of MMP-9.
Animals ; Brain Edema ; drug therapy ; etiology ; Brain Injuries, Traumatic ; complications ; drug therapy ; Gene Expression Regulation, Enzymologic ; drug effects ; Indazoles ; pharmacology ; therapeutic use ; MAP Kinase Signaling System ; drug effects ; Matrix Metalloproteinase 9 ; genetics ; Piperazines ; pharmacology ; therapeutic use ; Protein Kinase Inhibitors ; pharmacology ; therapeutic use ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To explore the effects of taurolithocholic acid (tLCA) and chenodeoxycholic acid (CDCA) on the expression of aorexigenic neuropeptide in mouse hypothalamus GT1-7 cells. METHODS: Mouse hypothalamic GT1-7 cells were treated with culture medium containing 10% FBS (control group, =3) or with 10 nmol/L, 100 nmol/L, 1 μmol/L and 10 μmol/L tLCA (tLCA group, =3) or CDCA (CDCA group, =3) for 12, 24 or 48 h. Real-time PCR was performed to determine the expression levels of proopiomelanocortin (POMC) mRNA in the cells, and the production levels of α-melanocyte-stimulating hormone (α-MSH) were assessed using an ELISA kit. Signal transduction and activator of transcription 3 phosphorylation (p-STAT3), threonine kinase phosphorylation (p-AKT), suppressor of cytokine signaling 3 (SOCS3), G protein-coupled bile acid receptor-1 (TGR5) and farnesoid X receptor (FXR) protein were detected by Western blotting. RESULTS: Western blotting results showed that mouse hypothalamic GT1-7 cells expressed two bile acid receptors, TGR5 and FXR, whose expressions were regulated by bile acids. Real-time PCR showed that the expression of POMC mRNA was significantly increased in the cells after treatment with 10 μmol/L tLCA or CDCA for 24 h. POMC-derived anorexigenic peptide α-MSH increased significantly in GT1-7 cells after treatment with 10 μmol/L tLCA or CDCA for 24 h. Treatment of the cells with tLCA or CDCA significantly increased the expressions of intracellular signaling proteins including p-STAT3, p-AKT and SOCS3. CONCLUSIONS Mouse hypothalamic GT1-7 cells express bile acid receptors TGR5 and FXR. Bile acids tLCA or CDCA can promote the expression of POMC mRNA and increase the production of the anorexigenic peptide α-MSH. The intracellular signaling proteins p-AKT, p-STAT3 and SOCS3 are likely involved in bile acid-induced anorexigenic peptide production.
Animals ; Cell Line ; Chenodeoxycholic Acid ; pharmacology ; Gene Expression Regulation ; drug effects ; Hypothalamus ; cytology ; Mice ; Neuropeptides ; genetics ; metabolism ; Pro-Opiomelanocortin ; genetics ; RNA, Messenger ; genetics ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; Suppressor of Cytokine Signaling 3 Protein ; metabolism ; Taurolithocholic Acid ; pharmacology ; alpha-MSH ; genetics

As a member of the AFF (AF4/FMR2) family, AFF4 is a transcription elongation factor that is a component of the super elongation complex. AFF4 serves as a scaffolding protein that connects transcription factors and promotes gene transcription through elongation and chromatin remodelling. Here, we investigated the effect of AFF4 on human dental follicle cells (DFCs) in osteogenic differentiation. In this study, we found that small interfering RNA-mediated depletion of AFF4 resulted in decreased alkaline phosphatase (ALP) activity and impaired mineralization. In addition, the expression of osteogenic-related genes (DLX5, SP7, RUNX2 and BGLAP) was significantly downregulated. In contrast, lentivirus-mediated overexpression of AFF4 significantly enhanced the osteogenic potential of human DFCs. Mechanistically, we found that both the mRNA and protein levels of ALKBH1, a critical regulator of epigenetics, changed in accordance with AFF4 expression levels. Overexpression of ALKBH1 in AFF4-depleted DFCs partially rescued the impairment of osteogenic differentiation. Our data indicated that AFF4 promoted the osteogenic differentiation of DFCs by upregulating the transcription of ALKBH1.
Biomarkers ; metabolism ; Cell Differentiation ; Cells, Cultured ; Dental Sac ; drug effects ; metabolism ; Gene Expression Regulation ; Humans ; Osteogenesis ; genetics ; Repressor Proteins ; Transcription Factors ; genetics ; metabolism ; Transcriptional Elongation Factors ; metabolism

With the expanded application of heavy metal cadmium, soil cadmium pollution is more and more serious. In this study, using Salix matsudana as a phytoremediation candidate, we observed changes of gene expression and metabolic pathway after 1, 7 and 30 days under 2.5 mg/L and 50 mg/L cadmium stress. The result of transcriptome sequencing showed that we obtained 102 595 Unigenes; 26 623 and 32 154 differentially expressed genes (DEG) in the same concentration and different stress time; 8 550, 3 444 and 11 428 DEG with different concentrations at the same time; 25 genes closely related to cadmium stress response were screened. The changes of genes expression (such as metallothionein, ABC transporter, zinc and manganese transporter) depended on both concentration of cadmium and exposure time. The expression of several genes was obviously up-regulated after cadmium stress, for example 3,6-deoxyinosinone ketolase (ROT3) in brassinolide synthesis pathway and flavonoid synthase (FLS), flavanone-3-hydroxylase (F3H) in the synthesis pathway of brassinolide. In addition, GO analysis shows that GO entries were mainly enriched in metabolic processes including cellular processes, membranes, membrane fractions, cells, cellular fractions, catalytic activation and binding proteins in response to cadmium stress, whose number would increase along with cadmium concentration and exposure time. The reliability of transcriptome information was verified by qPCR and physiological experimental data. Response mechanisms of S. matsudana after cadmium stress were analyzed by transcriptome sequencing, which provided theoretical guidance for remediation of cadmium pollution in soil by S. matsudana.
Biodegradation, Environmental ; Cadmium ; toxicity ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; drug effects ; Plant Proteins ; genetics ; Reproducibility of Results ; Salix ; drug effects ; genetics ; Stress, Physiological ; genetics ; Transcriptome ; drug effects

Proteasome inhibitors have shown remarkable success in the treatment of hematologic neoplasm. There has been a lot of attention to applying these drugs for solid tumor treatment. Recent preclinical study has signified the effectiveness on cell proliferation inhibition in lung adenocarcinoma treated by carfilzomib (CFZ), a second generation proteasome inhibitor. However, no insight has been gained regarding the mechanism. In this study, we have systematically investigated the CFZ functions in cell proliferation and growth, cell cycle arrest, and apoptosis in lung adenocarcinoma cells. Flow cytometry experiments showed that CFZ significantly induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma. MTS and colony formation assays revealed that CFZ substantially inhibited survival of lung adenocarcinoma cells. All results were consistently correlated to the upregulation expression of Gadd45a, which is an important gene in modulating cell cycle arrest and apoptosis in response to physiologic and environmental stresses. Here, upregulation of Gadd45a expression was observed after CFZ treatment. Knocking down Gadd45a expression suppressed G2/M arrest and apoptosis in CFZ-treated cells, and reduced cytotoxicity of this drug. The protein expression analysis has further identified that the AKT/FOXO3a pathway is involved in Gadd45a upregulation after CFZ treatment. These findings unveil a novel mechanism of proteasome inhibitor in anti-solid tumor activity, and shed light on novel preferable therapeutic strategy for lung adenocarcinoma. We believe that Gadd45a expression can be a highly promising candidate predictor in evaluating the efficacy of proteasome inhibitors in solid tumor therapy.
Adenocarcinoma of Lung/pathology* ; Apoptosis/drug effects* ; Cell Cycle Checkpoints/drug effects* ; Cell Cycle Proteins/genetics* ; Cell Line, Tumor ; Forkhead Box Protein O3/physiology* ; Gene Expression Regulation, Neoplastic/drug effects* ; Humans ; Lung Neoplasms/pathology* ; Oligopeptides/pharmacology* ; Proto-Oncogene Proteins c-akt/physiology* ; Up-Regulation

Seed vigor is a key factor affecting seed quality. The mechanical drying process exerts a significant influence on rice seed vigor. The initial moisture content (IMC) and drying temperature are considered the main factors affecting rice seed vigor through mechanical drying. This study aimed to determine the optimum drying temperature for rice seeds according to the IMC, and elucidate the mechanisms mediating the effects of drying temperature and IMC on seed vigor. Rice seeds with three different IMCs (20%, 25%, and 30%) were dried to the target moisture content (14%) at four different drying temperatures. The results showed that the drying temperature and IMC had significant effects on the drying performance and vigor of the rice seeds. The upper limits of drying temperature for rice seeds with 20%, 25%, and 30% IMCs were 45, 42, and 38 °C, respectively. The drying rate and seed temperature increased significantly with increasing drying temperature. The drying temperature, drying rate, and seed temperature showed extremely significant negative correlations with germination energy (GE), germination rate, germination index (GI), and vigor index (VI). A high IMC and drying temperature probably induced a massive accumulation of hydrogen peroxide (H₂O₂) and superoxide anions in the seeds, enhanced superoxide dismutase (SOD) and catalase (CAT) activity, and increased the abscisic acid (ABA) content. In the early stage of seed germination, the IMC and drying temperature regulated seed germination through the metabolism of H₂O₂, gibberellin acid (GA), ABA, and α-amylase. These results indicate that the metabolism of reactive oxygen species (ROS), antioxidant enzymes, GA, ABA, and α-amylase might be involved in the mediation of the effects of drying temperature on seed vigor. The results of this study provide a theoretical basis and technical guidance for the mechanical drying of rice seeds.
Abscisic Acid/metabolism* ; Antioxidants/pharmacology* ; Catalase/metabolism* ; Gene Expression Regulation, Plant/drug effects* ; Germination ; Gibberellins/metabolism* ; Hydrogen Peroxide/chemistry* ; Malondialdehyde/chemistry* ; Oryza/metabolism* ; Oxygen/chemistry* ; Plant Proteins/genetics* ; Reactive Oxygen Species ; Seeds/metabolism* ; Superoxide Dismutase/metabolism* ; Superoxides/chemistry* ; Temperature ; Weather ; alpha-Amylases/metabolism*

This study aimed to investigate the effect of butyl alcohol extract of Baitouweng Decoction( BAEB) on Candida albicans biofilms based on pH signal pathway. The morphology of biofilms of the pH mutants was observed by scanning electron microscope. The biofilm thickness of the pH mutants was measured by CLSM. The biofilm activity of the pH mutants was analyzed by microplate reader.The biofilm damage of the pH mutants was detected by flow cytometry. The expression of pH mutant biofilm-related genes was detected by qRT-PCR. The results showed that the deletion of PHR1 gene resulted in the defect of biofilm,but there were more substrates for PHR1 complementation. BAEB had no significant effect on the two strains. RIM101 gene deletion or complementation did not cause significant structural damage,but after BAEB treatment,the biofilms of both strains were significantly inhibited. For the biofilm thickness,PHR1 deletion or complementation caused the thickness to decrease,after BAEB treatment,the thickness of the two strains did not change significantly. However,RIM101 gene deletion or complementation had little effect on the thickness,and the thickness of the two strains became thinner after adding BAEB. For biofilm activity,PHR1 deletion or complementation and RIM101 deletion resulted in decreased activity,RIM101 complementation did not change significantly; BAEB significantly inhibited biofilm activity of PHR1 deletion,PHR1 complemetation,RIM101 deletion and RIM101 complemetation strains. For the biofilm damage,PHR1 gene deletion or complementation,RIM101 gene deletion or complementation all showed different degrees of damage; after adding BAEB,the damage rate of PHR1 deletion or complementation was not significantly different,but the damage rate of RIM101 deletion or complementation was significantly increased. Except to the up-regulation of HSP90 gene expression,ALS3,SUN41,HWP1,UME6 and PGA10 genes of PHR1 deletion,PHR1 complementation,RIM101 deletion,and RIM101 complementation strains showed a downward expression trend. In a word,this study showed that mutations in PHR1 and RIM101 genes in the pH signaling pathway could enhance the sensitivity of the strains to the antifungal drug BAEB,thus inhibiting the biofilm formation and related genes expression in C. albicans.
1-Butanol ; Biofilms ; drug effects ; Candida albicans ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Fungal Proteins ; Gene Expression Regulation, Fungal ; Hydrogen-Ion Concentration ; Plant Extracts ; pharmacology ; Signal Transduction

The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on pyroptosis of macrophages and the underlying molecular mechanisms. RAW264.7 macrophages were treated with AGE-alb (1, 2, 4 and 6 g/L) and control albumin (C-alb, 4 g/L) for 24 h, or preincubated with MCC950 (1 μmol/L) for 1 h and then treated with AGE-alb (4 g/L) for 24 h. Cell viability and caspase-1 activity were measured by MTT and assay kits, respectively. Lactate dehydrogenase (LDH) activity and the levels of interleukin-1β (IL-1β) and IL-18 in media were detected. Cell death degree was evaluated by TUNEL and Hoechst 33342/PI staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), procaspase-1 and cleaved caspase-1 were assessed by Western blot. The results showed that AGE-alb treatment caused obvious decrease in cell viability and increases in LDH leakage and the percentages of TUNEL- or PI-positive cells in a concentration-dependent manner. Additionally, AGE-alb promoted IL-1β and IL-18 secretion, upregulated NLRP3 expression, and increased caspase-1 activity especially at the dose of 4 and 6 g/L. However, MCC950 (an NLRP3 inhibitor) pretreatment inhibited significantly the decrease in cell viability and the increases in LDH leakage and percentages of TUNEL- or PI-positive cells induced by AGE-alb. Furthermore, MCC950 attenuated obviously AGE-alb-induced IL-1β and IL-18 secretion and caspase-1 activation. These results indicate that AGE-alb may induce macrophage pyroptosis, and the mechanism is at least partially by activating NLRP3-caspase-1 pathway.
Caspase 1 ; Gene Expression Regulation ; drug effects ; Interleukin-1beta ; genetics ; Macrophages ; drug effects ; NLR Family, Pyrin Domain-Containing 3 Protein ; genetics ; Pyroptosis ; drug effects ; Serum Albumin ; pharmacology