Methyl Thiazolyl Tetrazolium Method Research Articles

Breviscapine reverses doxorubicin resistance in breast cancer and its related mechanisms.

Based on the effect of breviscapine (BRE) on reversing drug resistance of human breast cancer cell line MCF-7/doxorubicin (Dox), the mechanism was preliminarily explored. The methyl thiazolyl tetrazolium (MTT) method was introduced to detect inhibitory effect of Dox alone or in combination with BRE on MCF-7 (M) and MCF-7/Dox (MD) cells, and the inhibitory concentration (IC50 ) was obtained. Cell apoptosis and Dox concentration was assessed by flow cytometry. The drug resistance multiple and reversal fold were calculated. Western blot was performed to evaluate the expression of Bcl-2, Bax, EGFR, p-EGFR, P-gp, caspase-3, and cleaved-caspase-3 protein in cells. The efflux of Rho-123 was measured by flow cytometry and fluorescence microscopy. The IC50 of Dox on MD and M cells was 16.67 and 0.71 μg/mL, respectively, with a drug resistance ratio of 23.48 times. The IC50 of Dox combined with BRE on MD cells was 5.62 μg/mL, with a reversal ratio of 2.97 times. BRE greatly enhanced Dox-induced apoptosis of MD cells. Bax and cleaved-caspase-3 (proapoptotic protein) expression were obviously increased, while Bcl-2 (antiapoptotic protein) expression was significantly decreased after BRE treatment. BRE inhibited EGFR activation and P-gp expression. BRE increased the intracellular accumulation of Dox in MD cells by P-gp. BRE could increase the MD sensitivity to Dox via increasing Bax and cleaved-caspase-3 expression and inhibiting Bcl-2 expression, thereby promoting cell apoptosis. BRE reversed Dox resistance of MD cells by increasing Dox intracellular accumulation through inhibiting P-gp expression via EGFR.

Therapeutic action of Kushen recipe extractive and its inhibitory effect on eotaxin in mouse models with contact dermatitis.

Treatment of skin allergic diseases remains a challenging research topic. To investigate the effect of Kushen recipe extractive (KS) gel on contact dermatitis (CD) of mouse. Allergic contact dermatitis (ACD) model of mouse was established. Immunohistochemical method (ICH) and flow cytometry method (FCM) were used to detect CD4+ and CD8+ T lymphocytes and explore the regulation effect of KS on the immune status of the organism. The expression status of eotaxin tissue was evaluated by real-time polymerase chain reaction (RT-PCR), ICH, and western blotting method. The survival rates of HaCaT cell and Fibroblasts affected by KS were detected by methyl thiazolyl tetrazolium (MTT) method. The inhibitory effect of KS on eotaxin produced by HaCaT cell and FBs induced by TNF-α and interleukin (IL)-4 were evaluated using RT-PCR and enzyme-linked immunosorbent assay methods. The inhibitory effect of KS on nuclear factor-κB (NF-κB) and Signal transducers and activators of transcription 6 (STAT6) activation induced by TNF-α and IL-4 was detected by electrophoretic mobility shift assay and western blotting methods. We confirmed that KS shows favorable therapeutic effect on CD, which can obviously inhibit eotaxin expression and Eosinophils recruitment in allergic skin of mouse, as well as regulate the immune status of the organism. Furthermore, KS and its main effective components can inhibit TNF-α and IL-4 induced upregulation of eotaxin via the two signal transduction pathways, NF-κB and STAT6. The great importance of traditional Chinese recipe KS is evidenced by its therapeutic effect and mechanism in ACD of mouse.

Study on the anti-sepsis mechanism of ursolic acid by targeting myeloid differentiation protein-2

To explore the mechanism of ursolic acid in treating sepsis using myeloid differentiation protein-2 (MD-2) as the research carrier. The affinity of ursolic acid and MD-2 was determined by biofilm interferometry technique, and the bonding mode between ursolic acid and MD-2 was tested with the aid of molecular docking technique. Raw 264.7 cells were cultured in RPMI 1640 medium and subcultured was conducted when the cell density reached 80%-90%. The second-generation cells were used for in the experiment. The effects of 8, 40 and 100 mg/L ursolic acid on cell viability were assessed by methyl thiazolyl tetrazolium (MTT) method. Cells were divided into blank group, lipopolysaccharide (LPS) group (LPS 100 μg/L) and ursolic acid group (100 μg/L LPS treatment after addition of 8, 40 or 100 mg/L ursolic acid). The effect of ursolic acid on the release of cytokines nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1β) were evaluated by enzyme-linked immunosorbent assay (ELISA). The influence of ursolic acid on the mRNA expressions of TNF-α, IL-6, IL-1β, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR). The implication of ursolic acid on the protein expressions of LPS-Toll-like receptor 4 (TLR4)/MD-2-nuclear factor-κB (NF-κB) pathway were tested by Western blotting. Ursolic acid could bind to the hydrophobic cavity of MD-2 through hydrophobic bond with the amino acid residues of the protein. Therefore, ursolic acid showed high affinity with MD-2 [dissociation constant (KD) = 1.43×10-4]. The cell viability were decreased slightly, with the concentration of ursolic acid increasing, and the cell viability of 8, 40 and 100 mg/L ursolic acid were 96.01%, 94.32% and 92.12%, respectively, and there was no significant difference compared with the blank group (100%). Compared with the blank group, the cytokine level of the LPS group was significantly increased. The level of cytokines were significantly reduced by the treatment of 8, 40 and 100 mg/L ursolic acid, and the higher the concentration, the more obvious effect [compared between 100 mg/L ursolic acid group and LPS group: IL-1β (μmol/L): 38.018±0.675 vs. 111.324±1.262, IL-6 (μmol/L): 35.052±1.664 vs. 115.255±5.392, TNF-α (μmol/L): 39.078±2.741 vs. 119.035±4.269, NO (μmol/L): 40.885±2.372 vs. 123.405±1.291, all P < 0.01]. Compared with the blank group, the mRNA expressions of TNF-α, IL-6, IL-1β, iNOS and COX-2 in the LPS group were significantly increased, and the protein expressions of MD-2, myeloid differentiation factor 88 (MyD88), phosphorylation NF-κB p65 (p-NF-κB p65) and iNOS in the LPS-TLR4/MD-2-NF-κB pathway were significantly up-regulated. Compared with the LPS group, the mRNA expressions of TNF-α, IL-6, IL-1β, iNOS and COX-2 were significantly reduced by the treatment of 100 mg/L ursolic acid bound with MD-2 protein [TNF-α (2-ΔΔCt): 4.659±0.821 vs. 8.652±0.787, IL-6 (2-ΔΔCt): 4.296±0.802 vs. 11.132±1.615, IL-1β (2-ΔΔCt): 4.482±1.224 vs. 11.758±1.324, iNOS (2-ΔΔCt): 1.785±0.529 vs. 4.249±0.811, COX-2 (2-ΔΔCt): 5.591±1.586 vs. 16.953±1.651, all P < 0.01], and the proteins expressions of MD-2, MyD88, p-NF-κB p65 and iNOS in the LPS-TLR4/MD-2-NF-κB pathway were significantly down-regulated (MD-2/β-actin: 0.191±0.038 vs. 0.704±0.049, MyD88/β-actin: 0.470±0.042 vs. 0.875±0.058, p-NF-κB p65/β-actin: 0.178±0.012 vs. 0.571±0.012, iNOS/β-actin: 0.247±0.035 vs. 0.549±0.033, all P < 0.01). However, there was no difference in protein expression of NF-κB p65 among the three groups. Ursolic acid inhibits the release and expression of cytokines and mediators and regulates LPS-TLR4/MD-2-NF-κB signaling pathway by blocking MD-2 protein, and thus plays an anti-sepsis role.

Protective effect and mechanism research of Phyllanthus emblica Linn. fruit extract on UV-induced photodamage in keratinocytes.

Ultraviolet (UV) irradiation causes acute and chronic cutaneous effects that may result in photodamage and photoaging. Epidermis keratinocytes, as the closest surface of skin, are susceptible to damage from UV rays. Phyllanthus emblica Linn. fruit (PE) extract, as a medicine and food dual-use plant, contains high levels of polyphenols and possesses multiple pharmacological properties. The present study investigated common and different molecular mechanisms and signaling pathway activations of UVA and UVB stimulated cell damage and photoprotective effect of PE extract against UVA and UVB by Methyl Thiazolyl Tetrazolium (MTT) method, Elisa assay, flow cytometry, differentially expressed genes analysis and western blot analysis. The results showed that UVA exposure (10J/cm2) reduced HaCaT cell viability significantly, increased the apoptosis rate, elevated intracellular reactive oxygen species level and reduced antioxidant enzyme activities. And UVA irradiation could inhibit the ERK/TGF-β/Smad signaling pathway to downregulate collagen I, collagen III and elastin expressions, resulting in the photoaging of skin cells. We also found UVB exposure (30mJ/cm2) caused HaCaT cell damage, promoted apoptosis, increased ROS production and induced the release of proinflammatory cytokines (IL-1α, IL-6 and PGE2). Further, in HaCaT cells, UVB ray was able to induce the activation of apoptosis markers (cleaved PARP1 and cleaved caspase3) through the MAPK/AP-1 signaling pathway using western blot analysis. Pre-treatment of PE extract prevented the UVA and UVB induced photoaging and injury in HaCaT cells through activation of ERK/TGF-β/Smad pathway and inhibition of MAPK/AP-1 pathway, respectively. Therefore, PE extract has the potential to be used as an oral and topical preparation against skin aging and injury induced by UVA and UVB.

Alpha-tocopherol enhances spermatogonial stem cell proliferation and restores mouse spermatogenesis by up-regulating BMI1.

Spermatogonial stem cells (SSCs) are essential for maintaining reproductive function in males. B-lymphoma Mo-MLV insertion region 1 (BMI1) is a vital transcription repressor that regulates cell proliferation and differentiation. However, little is known about the role of BMI1 in mediating the fate of mammalian SSCs and in male reproduction. This study investigated whether BMI1 is essential for male reproduction and the role of alpha-tocopherol (α-tocopherol), a protective agent for male fertility, as a modulator of BMI1 both in vitro and in vivo. Methyl thiazolyl tetrazolium (MTT) and 5-ethynyl-2'-deoxyuridine (EDU) assays were used to assess the effect of BMI1 on the proliferative ability of the mouse SSC line C18-4. Real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence were applied to investigate changes in the mRNA and protein expression levels of BMI1. Male mice were used to investigate the effect of α-tocopherol and a BMI1 inhibitor on reproduction-associated functionality in vivo. Analysis revealed that BMI1 was expressed at high levels in testicular tissues and spermatogonia in mice. The silencing of BMI1 inhibited the proliferation of SSCs and DNA synthesis and enhanced the levels of γ-H2AX. α-tocopherol enhanced the proliferation and DNA synthesis of C18-4 cells, and increased the levels of BMI1. Notably, α-tocopherol rescued the inhibition of cell proliferation and DNA damage in C18-4 cells caused by the silencing of BMI1. Furthermore, α-tocopherol restored sperm count (Ctrl vs. PTC-209, p = 0.0034; Ctrl vs. PTC-209 + α-tocopherol, p = 0.7293) and normalized sperm malformation such as broken heads, irregular heads, lost and curled tails in vivo, as demonstrated by its antagonism with the BMI1 inhibitor PTC-209. Analysis demonstrated that α-tocopherol is a potent in vitro and in vivo modulator of BMI1, a transcription factor that plays an important role in in SSC proliferation and spermatogenesis. Our findings identify a new target and strategy for treating male infertility that deserves further pre-clinical investigation.

Levistolide A and periplogenin inhibit the growth of gastric cancer cells in vitro and in vivo

Aim: In the present study, the natural products levistolide A (LA) and periplogenin (PPG) were studied for their growth inhibitory effects on the development of gastric cancer cells in vitro and, more critically, in vivo, alone or in combination with the therapeutic medication 5-fluorouracil (5-FU). Methods: Methyl thiazolyl tetrazolium (MTT), also known as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays were used for the cell viability study. Apoptosis was detected by western blot to detect the cleavage of caspase substrate poly (ADP-ribose) polymerase (PARP) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assays. The nude mice bearing gastric cancer cells were used for the anti-cancer activity detection of LA and its combinational treatment effect with 5-FU. Results: The results in the present study shown that the two compounds were able to inhibit the viability of the cancer cells in a dose- and time-dependent manner by MTT method. They could trigger apoptosis when used alone, and more potently, in combination with 5-FU detected by TUNEL positivity and the cleavage of caspase substrate PARP. In nude mice bearing gastric cancer cells, injection (i.p.) of LA or PPG alone inhibited the growth of the cancer cells. The treatment using one of the compounds in combination with 5-FU inhibited the cancer cell growth at a higher level than the treatment by a compound alone. Conclusions: LA and PPG could inhibit the growth of the cancer cells, alone or in combination with 5-FU, in vitro and in vivo, suggesting that they are promising investigational drugs for therapeutic development.

MiR-223-3p increases resistance of colorectal cancer cells to 5-fluorouracil via targeting SORBS1.

5-Fluorouracil (5-FU) is the first-line drug for treating colorectal cancer (CRC), and the resistance of tumor cells to 5-FU is the main cause of chemotherapeutic failure. However, the resistant mechanism is still unclear. This study aims to explore the tumor suppressor genes involved in 5-FU resistance in CRC, and to find the microRNA (miRNA) that regulates these genes. CRC data sets GSE28702 and GSE69657 were downloaded from Gene Expression Omnibus (GEO) database, and gene expression profiles of patients in the FOLFOX chemotherapeutic response group and the non-response group were analyzed, and differential expression genes were identified between the 2 groups. Target gene was then selected. Online bioinformatics databases TargetScan, miRwalk, and miRDB were used to predict miRNA targeting the interested gene sorbin and SH3 domain containing 1 (SORBS1). siSORBS1, HA-SORBS1, miR-223-3p mimic, anti-miR-223-3p, and their corresponding negative controls (siNC, HA, miR-NC, and anti-miR-NC) were transfected into CRC cell lines of HCT116 and SW620 by transient transfection technique, respectively. Co-transfection was done with miRNA and plasmid (miR-NC+HA, miR-223-3p mimic+HA, or miR-223-3p mimic+HA-SORBS1) or anti-miRNA and siRNA (anti-miR-NC+siNC, anti-miR-223-3p+siNC, or anti-miR-223-3p+siSORBS1) in HCT116 cells. Real-time reverse transcription PCR (real-time RT-PCR) and/or Western blotting were used to detect the expression levels of SORBS1 and miR-223-3p in cells. After transfection, the cells were treated with different concentrations of 5-FU, and the cell viability was detected by methyl thiazolyl tetrazolium (MTT) method. The targeting relationship between miR-223-3p and SORBS1 was comfirmed by dual luciferase reporter gene assay. There were 409 and 528 highly expressed genes in the FOLFOX chemotherapeutic response group of GSE69657 and GSE28702, respectively. There were 22 overlapping genes in the response group, among which exist 3 tumor suppressor genes might be involved in chemosensitivity in CRC, and SORBS1 was selected as the target gene for further study. Three online bioinformatics databases predicted miRNAs targeting SORBS1 and obtained an intersection molecule miR-223-3p. After treatment with 5-FU (25 µmol/L) for 12-36 h, the levels of miR-223-3p in HCT116 and SW620 cells were significantly down-regulated (all P<0.05). After transfection with siSORBS1 or miR-223-3p mimic, the expression levels of SORBS1 in HCT116 and SW620 cells were down-regulated, and the cell viability was increased (all P<0.05). After transfection with HA-SORBS1 or anti-miR-223-3p, the expression levels of SORBS1 in HCT116 and SW620 cells were up-regulated, and the cell viability was decreased (all P<0.05). The result of dual luciferase reporter gene assay showed that the luciferase activity of cells co-transfected with SORBS1 3'-UTR wild plasmid and miR-223-3p mimic was significantly lower than that of the 3'-UTR wild plasmid and miR-NC cells (P<0.05). Compared with co-transfection with miR-223-3p mimic and HA, the cell viability of cells co-transfected with miR-223-3p mimic and HA-SORBS1 was decreased significantly (P<0.01). Compared with the co-transfected anti-miR-223-3p and siNC, the cell viability of the co-transfected anti-miR-223-3p and siSORBS1 was significantly increased (P<0.05). MiR-223-3p increases 5-FU resistance in CRC cells by targeting SORBS1,and miR-223-3p is expected to become a new target for clinical treatment of CRC.

The polysaccharide from Camellia oleifera fruit shell enhances immune responses via activating MAPKs and NF-κB signaling pathways in RAW264.7 macrophages.

Macrophage plays an important role in innate immune responses by secreting immune molecules and phagocytosis. Camellia oleifera fruit shell, accounting for approximately 60% weight of the single C. oleifera fruit, is rich in polysaccharides and has several biological activities such as anti-oxidation, lipid regulation and anticancer. However, the immunomodulatory activity of the polysaccharide from C. oleifera fruit shells (CPS) has not been reported. This study aimed to investigate the immunomodulatory activities and mechanisms of CPS in RAW264.7 macrophages. The Methyl Thiazolyl Tetrazolium (MTT) method was used to evaluate the effect of CPS on the cell viability of RAW264.7 macrophages, and cell morphology was pictured using microscope. The production of immune-related molecules, including nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor α (TNFα), interleukin (IL)-1β and IL-6, was detected by Griess assay and enzyme-linked immunosorbent assay (ELISA). The protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) and the phosphorylation level of mitogen-activated protein kinases (MAPKs) were analyzed through western blotting. The mRNA levels of related genes were tested using reverse transcription-polymerase chain reaction (RT-PCR). The nuclear translocation of nuclear factor-kappa B (NF-κB) was detected using immunofluorescence technology. The results indicated that CPS treatment stimulated the production of NO and PGE2 and significantly enhanced the protein expression of iNOS and COX2 with little effect on the cell morphology and viability. Also, the secretion and mRNA levels of TNFα were increased by the treatment of CPS. In addition, CPS treatment markedly upregulated the phosphorylation level of MAPKs including Extracellular Signal Regulated Kinase (ERK), P38, and c-Jun N-terminal Kinase (JNK) at different time points and caused the activation and nuclear translocation of NF-κB. Our data implied that CPS exerts immunomodulatory activities by activating MAPKs and NF-κB signaling pathways in RAW264.7 macrophages.

Regulatory effects and signaling mechanism of sodium ferulate on the proliferation and apoptosis of human skin hypertrophic scar fibroblasts

Objective: To investigate the regulatory effects and signaling mechanism of sodium ferulate on the proliferation and apoptosis of human skin hypertrophic scar fibroblasts (HSFbs). Methods: The experimental research methods were used. The 4th-6th passage of HSFbs from human skin were used for the following experiments. HSFbs were co-cultured with sodium ferulate at final mass concentrations of 1, 1×10-1, 1×10-2, 1×10-3, 1×10-4, 1×10-5, and 1×10-6 mg/mL for 48 hours, and methyl thiazolyl tetrazolium method was used to determine the cell absorbance values and linear regression was used to analyze the half lethal concentration (LC50) of sodium ferulate (n=6). HSFbs were co-cultured with sodium ferulate at final mass concentrations of 0.1, 0.2, 0.3, and 0.4 mg/mL for 24, 48, 72, and 96 hours, and methyl thiazolyl tetrazolium method was used to determine the cell absorbance values and the cell proliferation inhibition rate was calculated (n=3). According to the random number table, the cells were divided into 0.300 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, 0.003 mg/mL sodium ferulate group treated with sodium ferulate at corresponding final mass concentrations, and negative control group without any treatment. After 72 hours of culture, the cell absorbance values were determined by methyl thiazolyl tetrazolium method (n=5), the microscopic morphology of cells was observed by transmission electron microscope (n=3), the cell apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay and the apoptosis index was calculated (n=4), the protein expressions of B lymphocystoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and cysteine aspartic acid specific protease-3 (caspase-3) were determined by immunohistochemistry (n=4), and the protein expressions of transformed growth factor β1 (TGF-β1), phosphorylated Smad2/3, phosphorylated Smad4, and phosphorylated Smad7 were detected by Western blotting (n=4). Data were statistically analyzed with one-way analysis of variance and Dunnett test. Results: The LC50 of sodium ferulate was 0.307 5 mg/mL. After being cultured for 24-96 hours, the cell proliferation inhibition rates of cells treated with sodium ferulate at four different mass concentrations tended to increase at first but decrease later, which reached the highest after 72 hours of culture, so 72 hours was chosen as the processing time for the subsequent experiments. After 72 hours of culture, the cell absorbance values in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group were 0.57±0.06, 0.53±0.04, 0.45±0.05, respectively, which were significantly lower than 0.69±0.06 in negative control group (P<0.01). After 72 hours of culture, compared with those in negative control group, the cells in the three groups treated with sodium ferulate showed varying degrees of nuclear pyknosis, fracture, or lysis, and chromatin loss. In the cytoplasm, mitochondria were swollen, the rough endoplasmic reticulum was expanded, and local vacuolation gradually appeared. After 72 hours of culture, compared with that in negative control group, the apoptosis indexes of cells were increased significantly in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group (P<0.05 or P<0.01). After 72 hours of culture, compared with those in negative control group, the protein expressions of Bcl-2 of cells in 0.300 mg/mL sodium ferulate group was significantly decreased (P<0.01), the protein expressions of Bax of cells in 0.030 mg/mL sodium ferulate group and 0.300 mg/mL sodium ferulate group were significantly increased (P<0.05), and the protein expression of caspase-3 of cells in 0.300 mg/mL sodium ferulate group was significantly increased (P<0.01). After 72 hours of culture, compared with those in negative control group, the protein expression levels of TGF-β1, phosphorylated Smad2/3, and phosphorylated Smad4 of cells in 0.030 mg/mL sodium ferulate group and 0.300 mg/mL sodium ferulate group were significantly decreased (P<0.05 or P<0.01), and the protein expression levels of phosphorylated Smad7 of cells in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group were significantly increased (P<0.01). Conclusions: Sodium ferulate can inhibit the proliferation of HSFbs of human skin and promote the apoptosis of HSFbs of human skin by blocking the expression of key proteins on the TGF-β/Smad signaling pathway and synergistically activating the mitochon- drial apoptosis pathway.

Acetylshikonin exerts anti-tumor effects on non-small cell lung cancer through dual inhibition of STAT3 and EGFR

BackgroundLung cancer is one of the most common types of malignant tumor. It has one of the highest morbidity and mortality rates worldwide, and approximately 85% of cases are non-small cell lung cancer (NSCLC). Clinically, several EGFR inhibitors have been used to treat NSCLC, but resistance can develop. Studies have shown that cross talk between signal transducer and activator of transcription 3 (STAT3) and epidermal growth factor receptor (EGFR) can mediate drug resistance. Acetylshikonin has obvious antitumor effects, but the mechanism of action is still unclear. PurposeTo analyze the antitumor activity of acetylshikonin in lung cancer and clarify its molecular mechanism. MethodsMethyl thiazolyl tetrazolium (MTT), colony formation and 5-ethynyl-2’-deoxyuridine (EDU) assays were performed to examine the effects of acetylshikonin in inhibiting the proliferation of NSCLC cells (PC-9, H1975 and A549). Scratch wound and transwell assays were used to evaluate the migration and invasion of NSCLC cells. Flow cytometry was employed to determine whether acetylshikonin could induce apoptosis. Proteome sequencing was used to identify the targets of acetylshikonin. Immunofluorescence staining and western blotting were utilized to verify the inhibition of STAT3 and EGFR phosphorylation. A xenotransplantation model was established to evaluate the efficacy of acetylshikonin in nude mice. ResultsOur data demonstrated that acetylshikonin significantly decreased the survival rate of human NSCLC cells, increased the apoptotic rate and inhibited cell migration dose-dependently. Immunofluorescence staining and western blotting analyses revealed that acetylshikonin inhibited EGFR and STAT3 pathways. Acetylshikonin also inhibited tumor growth in a xenograft model better than inhibitors of EGFR and STAT3. ConclusionAcetylshikonin has anti-cancer effects on NSCLC cells by inhibiting EGFR and STAT3, indicating that acetylshikonin may be a new antitumor drug to treat NSCLC.

Comparison of the Effects of 3D Printing Bioactive Porous Titanium Alloy Scaffolds and Nano-biology for Direct Treatment of Bone Defects.

This study was to compare the effects of three-dimensional (3D) printed bioactive porous titanium alloy scaffolds (3DP-BPTAS) and rhBMP-2/PLA-loaded sustained-release nanospheres (SRNs) in the treatment of bone defects. In this study, the bioactive porous titanium alloy scaffolds (BPTAS) with different pore sizes were prepared by selective laser melting (SLM) technology. The rhBMP-2/PLA SRNs were prepared by the double emulsion solvent volatilization method. The morphology of the two nanomaterials was observed under a scanning electron microscope (SEM). The encapsulation rate (ER), drug loading (DL), and in vitro release rate of the SRNs were detected by enzyme-linked immunosorbent assay (ELISA); and the effects of different particle sizes of BPTAS and SRNs on the proliferation of BMSCs were measured using the Methyl Thiazolyl Tetrazolium (MTT) method. 42 healthy male rabbits were selected and rolled into a control group (no treatment), a model group (the femoral condyle defect model), an A800 group (model + 800 μm of BPTAS), and an A1000 group (model + 1000 μm of BPTAS), an A1200 group (model + 1200 μm of BPTAS), an A1500 group (model + 1500 μm of BPTAS), and an SNR group (model + rhBMP-2/PLA SRNs). There were 6 rabbits in each group, and they were sacrificed 4, 8, and 12 weeks after the surgery. They were performed with general observation, X-ray photography, and histological and biomechanical examinations. According to the Lane-Sandhu bone defect repair tissue X-ray and histological scoring standard, the effect of bone defect repair was evaluated. It was found that the actual pore structure of the scaffold prepared by the SLM process was consistent with the theoretical design. The observation under TEM showed that rhBMP-2/PLA SRNs were approximately round, with an average particle size of 835 nm, and its encapsulation efficiency and drug loading rate were 89.02 ± 5.14% and 0.033 ± 0.004%, respectively. The rhBMP-2/PLA SRNs and BPTAS had no statistically obvious increase in the number of cells after cell treatment compared with the control group (P> 0.05). At 12 weeks postoperatively, the stent bone tissue growing distance (SBTGD) in the SRN group was longer than that in the A1000 group (P< 0.01), and that in the A1000 group was better in contrast to the A800, A1200, and A1500 groups (P< 0.01). The Lane-Sandhu X-ray score of the SRN group was better than other groups (P< 0.05). It suggested that 3DP-BPTAS and rhBMP-2/PLA SRNs could repair the bone defects, and rhBMP-2/PLA SRNs were more conducive to the formation of new bone tissue.

Automatic Segmentation of Magnetic Resonance Images of Severe Patients with Advanced Liver Cancer and the Molecular Mechanism of Emodin-Induced Apoptosis of HepG2 Cells under the Deep Learning.

To improve the accuracy of clinical diagnosis of severe patients with advanced liver cancer and enhance the effect of chemotherapy treatment, the U-Net model was optimized by introducing the batch normalization (BN) layer and the dropout layer, and the segmentation training and verification of the optimized model were realized by the magnetic resonance (MR) image data. Subsequently, HepG2 cells were taken as the research objects and treated with 0, 10, 20, 40, 60, 80, and 100 μmol/L emodin (EMO), respectively. The methyl thiazolyl tetrazolium (MTT) method was used to explore the changes in cell viability, the acridine orange (AO)/ethidium bromide (EB) and 4′,6-diamidino-2-phenylindole (DAPI) were used for staining, the Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) (Annexin V-FITC/PI) was adopted to detect the apoptosis after EMO treatment, and the Western blot (WB) method was used with the purpose of exploring the changes in protein expression levels of PARP, Bcl-2, and p53 in the cells after treatment. It was found that compared with the original U-Net model, the introduction of the BN layer and the dropout layer can improve the robustness of the U-Net model, and the optimized U-Net model had the highest dice similarity coefficient (DSC) (98.45%) and mean average precision (MAP) (0.88) for the liver tumor segmentation.

Regulatory Mechanism of MicroRNA-9 / Long Non-Coding RNA XIST Expression on Mouse Macrophage RAW264.7 Apoptosis Induced by Oxidized Low Density Lipoprotein

ABSTRACT It aims to analyze the influential mechanism of microRNA-9 (miR-9) and long non-coding RNA XIST (lncRNA XIST) expression on the proliferation and apoptosis of macrophages induced by oxidized-low density lipoprotein (ox-LDL). Firstly, lncRNA XIST overexpression vector was constructed, and then RAW264.7 cells were used as the research object. Methylthiazolyl tetrazolium (MTT) method, flow cytometry, and Western blot were used to detect and compare the differences of cell proliferation, apoptosis, and the expression levels of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK), matrix metalloproteinase-9 (MMP-9), and B-cell lymphoma-2 (Bcl-2) after ox-LDL induction and transfection of miR-9 mimic, miR-9 inhibitor and XIST expression vector, respectively. The results showed that lncRNA XIST overexpression vector was successfully constructed and transfected into cells, wh5ich can inhibit the expression level of miR-9. Compared with the normal control group, ox-LDL can inhibit cell proliferation, promote cell apoptosis, and increase the expression level of target protein. Moreover, transfection of XIST expression vector based on ox-LDL induction can significantly enhance the inhibition of cell proliferation, and promote cell apoptosis and the expression of target protein. Transfection of miR-9 mimic can improve the biological changes induced by ox-LDL. After co-transfection of miR-9 mimic and XIST expression vector based on ox-LDL induction, cell proliferation, apoptosis, and target protein expression level were not significantly different from those induced by ox-LDL alone. In summary, the increased expression level of miR-9 can inhibit the apoptosis of macrophages induced by ox-LDL. lncRNA XIST can positively regulate the apoptosis of macrophages induced by ox-LDL.

Resveratrol derivative production by high-pressure treatment: proliferative inhibitory effects on cervical cancer cells

Background: In recent years, functional food components have attracted considerable attention. Resveratrol, a food polyphenol, has been widely studied due to its various physiological activities. Previously, we identified a novel resveratrol derivative, named RK4, in food, which is formed by a chemical reaction involving resveratrol and caffeic acid. Furthermore, it was suggested that high-pressure treatment is an important factor in RK4 production.&#x0D; Objectives: The purpose of this study was to clarify relationships between high-pressure processing and component production and to compare RK4 with the known functional ingredient resveratrol to examine the physiological value of RK4. Through this research, we aimed to develop high-pressure treatment technology that adds new usefulness for food.&#x0D; Methods: Resveratrol and caffeic acid were reacted under high-pressure treatment and in various conditions of concentration and temperature. RK4 levels in the reaction solution were quantitatively analyzed using liquid chromatography-mass spectrometry. In addition, HeLa cervical cancer cells were exposed to RK4 and resveratrol, and survival rates were measured using the methyl thiazolyl tetrazolium (MTT) method after culturing for 24 h. Activation of an apoptosis-inducing marker was detected by western blotting of cells cultured for 48 h after addition of the test compounds.&#x0D; Results: By reacting resveratrol and caffeic acid under high-pressure conditions (~100 MPa), the amount of RK4 produced was significantly increased. It was also found that the reaction temperature and time contributed to this reaction. RK4 exhibited stronger cytotoxicity to HeLa cells than resveratrol. It was also shown that RK4 activated p38, cleaved poly ADP ribose polymerase, and induced apoptosis.&#x0D; Conclusions: RK4 is a valuable component for further research as a novel compound with wider functionality than that of resveratrol. High-pressure treatment may substantially contribute to the production of novel food ingredients. Further elucidation of the relationships between high-pressure treatment and production of new ingredients has promising potential to guide development of new applications in food processing.

Long non-coding RNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 regulates the proliferation and osteogenic differentiation of human periodontal ligament stem cells by targeting miR-24-3p.

This study aims to explore the effect and molecular mechanism of long non-coding RNA (lncRNA) potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) on proliferation and osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs). The hPDLSCs of normal periodontal tissues were isolated and cultured. The mineralized solution induced the osteoblast differentiation of hPDLSCs. The down-regulation of lncRNA KCNQ1OT1, the overexpression of anti-miR-24-3p on the proliferation and the levels of osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) of hPDLSCs were investigated. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the levels of lncRNA KCNQ1OT1, miR-24-3p, OCN, OPN, and ALP. Methyl thiazolyl tetrazolium (MTT) method was used to detect cell viability and activity. Cell proliferation was evaluated by MTT. Western blot was used to detect protein expression. The targeted relationship between lncRNA KCNQ1OT1 and miR-24-3p was detected by double-luciferase experiment. The expression level of lncRNA KCNQ1OT1 increased, and that of miR-24-3p decreased during the osteogenesis of hPDLSCs (P<0.05). The down-regulation of lncRNA KCNQ1OT1 inhibited cell proliferation and reduced the mRNA and protein expression levels of OCN, OPN, and ALP (P<0.05). LncRNA KCNQ1OT1 targeted and regulated miR-24-3p. The overexpression of miR-24-3p inhibited cell proliferation and reduced the mRNA and protein expression levels of OCN, OPN, and ALP (P<0.05). Inhibition of miR-24-3p reversed the effect of the down-regulation of lncRNA KCNQ1OT1 on cell proliferation and mRNA and protein expression levels of OCN, OPN, and ALP (P<0.05). Down-regulation of lncRNA KCNQ1OT1 inhibited the proliferation and osteogenic differentiation of hPDLSCs by targeting the up-regulated expression of miR-24-3p.

Effect of acidic culture conditions on the proliferation, apoptosis, and migration ability of human tongue squamous cell carcinoma cells and its related mechanism.

This study aims to explore the effect of acidic culture conditions on the proliferation, apoptosis, and migration ability of human tongue squamous cell carcinoma SCC15 and CAL27 cells and its potential molecular mechanism. After acidic culture for different periods, methyl thiazolyl tetrazolium (MTT) method was adop-ted to detect the cell proliferation of SCC15 and CAL27. Flow cytometry was employed to detect the apoptosis level of SCC15 and CAL27 cells. The migration ability of SCC15 and CAL27 after acidic culture was detected by scratch hea-ling test. Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect the mRNA expression of cyclooxygenase 2 (COX-2) and survivin in SCC15 and CAL27 cells after acidic culture. After culture for 24 h under acidic microenvironment, SCC15 and CAL27 cells grew rapidly and reached the stationary phase after adjustment for 3 days. The apoptosis levels of SCC15 and CAL27 cells decreased after acidic culture, but the most significant reduction occurred after 6 h of acidic culture. The scratch healing rates of SCC15 and CAL27 cells increased after acidic culture. The results of FQ-PCR showed that the mRNA expression levels of COX-2 and survivin in SCC15 and CAL27 cells increased after acidic culture. Extracellular acidic microenvironment can inhibit the apoptosis of tongue squamous carcinoma cells, promote their migration, and induce more adaptable and malignant tongue squamous carcinoma cells. The mechanism may be related to COX-2 and survivin and their signal pathways.

Development of Arteannuin B Sustained-Release Microspheres for Anti-Tumor Therapy by Integrated Experimental and Molecular Modeling Approaches.

Arteannuin B (AB) has been found to demonstrate obvious anti-tumor activity. However, AB is not available for clinical use due to its very low solubility and very short half-life. This study aimed to develop AB long sustained-release microspheres (ABMs) to improve the feasibility of clinical applications. Firstly, AB-polylactic-co-glycolic acid (PLGA) microspheres were prepared by a single emulsification method. In vitro characterization studies showed that ABMs had a low burst release and stable in vitro release for up to one week. The particle size of microspheres was 69.10 μm (D50). The drug loading is 37.8%, and the encapsulation rate is 85%. Moreover, molecular dynamics modeling was firstly used to simulate the preparation process of microspheres, which clearly indicated the molecular image of microspheres and provided in-depth insights for understanding several key preparation parameters. Next, in vivo pharmacokinetics (PK) study was carried out to evaluate its sustained release effect in Sprague-Dawley (SD) rats. Subsequently, the methyl thiazolyl tetrazolium (MTT) method with human lung cancer cells (A549) was used to evaluate the in vitro efficacy of ABMs, which showed the IC50 of ABMs (3.82 μM) to be lower than that of AB (16.03 μM) at day four. Finally, in vivo anti-tumor activity and basic toxicity studies were performed on BALB/c nude mice by subcutaneous injection once a week, four times in total. The relative tumor proliferation rate T/C of AMBs was lower than 40% and lasted for 21 days after administration. The organ index, organ staining, and tumor cell staining indicated the excellent safety of ABMs than Cis-platinum. In summary, the ABMs were successfully developed and evaluated with a low burst release and a stable release within a week. Molecular dynamics modeling was firstly applied to investigate the molecular mechanism of the microsphere preparation. Moreover, the ABMs possess excellent in vitro and in vivo anti-tumor activity and low toxicity, showing great potential for clinical applications.

Chemical compositions and experimental and computational modeling activity of sea cucumber Holothuria parva ethanolic extract against herpes simplex virus type 1

Sea cucumber has antiviral activities against various viruses including herpes simplex virus type 1 (HSV-1). The purpose of the current study was to determine the chemical profile and inhibitory effects of tegument ethanolic extract of Holothuria parva on HSV-1 infection and to elucidate the mechanism of antiviral action of this marine invertebrate. Cytotoxic activity of the extract on Vero cell line was determined using the methyl thiazolyl tetrazolium (MTT) method. The different components in H. parva were determined by GC-MS analysis. To assess the antiviral activity of the extract, MTT and 50% tissue culture infective dose (TCID50) were applied. Finally, computational molecular docking was performed to screen the potential binding ability of extract contents with HSV-1 surface glycoproteins and host cell surface receptors. Using MTT assay, the non-cytotoxic concentration of the extract was measured 46.5 μg/mL. Octadecanoic acid 2-hydroxy-1-(hydroxymethyl) ethyl ester and 2′,6′-acetoxylidide were two major constituents in the H. parva extract. Pre-treatment of HSV-1 with the ethanolic extract of H. parva led to a 2.1 log10 TCID50 reduction in virus titers when compared to the control group (P = 0.002). The log10 TCID50 reductions relative to the control group for co-penetration and post-penetration assays were 1.5 (P = 0.009) and 0.7 (P = 0.09), respectively. The tegument ethanolic extract of H. parva has significant antiviral properties against HSV-1. Docking analysis demonstrated that compounds of the extract [lidocaine and 2-hydroxy-1-(hydroxymethyl) ethyl ester octadecanoic acid] may cover similarly both virus and host cells binding domains leading to interference in virus attachment to cell receptors.

Knockdown of lncRNA TUG1 attenuates cerebral ischemia/reperfusion injury through regulating miR-142-3p.

Cerebral ischemia-reperfusion injury (CI/RI) is one of the most common diseases of the central nervous system. At present, there is no specific treatment for CI/RI. It is necessary to explore the mechanism of CI/RI and find new ways to prevent and treat CI/RI. An oxygen and glucose deprivation/recovery (OGD/R) model was established to evaluate the effects of mouse astrocytes (MA-C) cell viability and apoptosis of stepwise exposure to oxygen and glucose deprivation followed by their replenishment. This assessment included using taurine upregulated gene 1-small interfering RNAs (TUG1-siRNA) transfection to determine the effects of TUG1 knockdown on MA-C survival and apoptosis. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to evaluate TUG1 and miR-142-3p expression levels. The luciferase gene reporter assay was performed to validate that miR-142-3p is a TUG1 target. Accordingly, the effects of miR-142-3p knockdown on TUG1-induced MA-C apoptosis were determined using flow cytometry. Methyl thiazolyl tetrazolium (MTT) method was used to detect cell growth viability. Western blotting analysis was performed to detect the expression levels of apoptosis-related proteins. TUG1 was upregulated, while miR-142-3p was downregulated in the OGD/R model of MA-C cells. Inhibiting the expression of TUG1 could protect MA-C cells and reverse the decrease in growth viability and increasing apoptosis of MA-C cells caused by OGD/R stimulation. On the other hand, the inhibition of miR-142-3p offset the effect of TUG1 knockdown on cell viability and apoptosis. Inhibition of OGD/R-induced increases in TUG1 expression that in turn reduces miR-142-3p upregulation may suppress reperfusion-induced losses in cell viability.

Effects of miR-7 on Hcy-induced rat cerebral arterial vascular smooth muscle cell proliferation, migration and inflammatory factor expression by targeting MMP-14 to regulate TLR4/NF-κB signaling pathway

The current research aimed to investigate the effect of miR-7 targeting matrix metalloproteinase 14 (MMP-14) on homocysteine (Hcy)-induced rat cerebral artery vascular smooth muscle cells (VSMCs) proliferation, migration and inflammatory factor expression and its possible mechanism. The expression of miR-7 and MMP-14 in Hcy-induced VSMCs were detected by real-time fluorescent quantitative PCR (RT-qPCR) and Western blot. Methyl Thiazolyl Tetrazolium (MTT) method, Transwell assays and enzyme-linked immunosorbent assay (ELISA) were performed to detect the effect of miR-7 and MMP-14 expression on the proliferation and migration, as well as interleukin 6 (IL-6) and tumor necrosis factor ɑ (TNF-ɑ) expression of Hcy-induced VSMCs. The interaction between miR-7 and MMP-14 was detected by dual-luciferase reporter gene assay. Western blot was applied to analyse the effects of miR-7 and MMP-14 expression on the Toll-like receptor (TLR4)/nuclear transcription factor-KB (NF-κB) signaling pathway. The results showed that after induced by Hcy, the expression of miR-7 in VSMCs was significantly reduced, the expression of MMP-14 was significantly increased, and the cell viability, the number of migrating cells, IL-6 and TNF-ɑ expression were significantly increased (P&lt;0.05). After overexpression of miR-7, the viability, migration cell numbers, IL-6 and TNF-ɑ expression of Hcy-induced VSMCs were significantly reduced (P&lt;0.05). miR-7 directly binds to MMP-14 and negatively regulates the expression of MMP-14. After overexpression of miR-7, the levels of TLR4 and p-NF-κB p65 in VSMCs were significantly reduced (P&lt;0.05); overexpression of MMP-14 could reduce the effect of miR-7 overexpression on TLR4 and p-NF-κB p65 expression in VSMCs (P&lt;0.05). Overexpression of MMP-14 and/or activation of the TLR4/NF-κB signaling pathway could reverse the effect of miR-7 overexpression on the proliferation, migration and IL-6 and TNF-ɑ expression of Hcy-induced VSMCs (P&lt;0.05). It is concluded that miR-7 can inhibit Hcy-induced rat cerebral artery VSMCs proliferation, migration, and inflammatory factor expression by targeting the regulation of MMP-14 expression and inhibiting the activation of the TLR4/NF-κB signaling pathway.