Optimization of method for achieving a single-cell suspension from mouse corneas.

The single-cell RNA-sequencing (scRNA-seq) technique is used to explore the biological characteristics of tissues under pathological and physiological conditions that include certain chronic eye diseases. Harvesting of single-cell suspensions is one challenge inherent to scRNA-seq procedures. This study aimed to use an optimized method to digest a whole mouse cornea to harvest single-cell suspensions. We utilized five different mouse cornea digestion methods to obtain single-cell suspensions: (1) 5 dissected mouse corneas were cut into pieces (∼0.5 mm) and digested in trypsin for 10 min, and this digestion was repeated for 10 cycles; (2) 5 dissected mouse corneas were cut into pieces and incubated with 5 mg/ml collagenase A at 37 °C for 1h and then further digested in trypsin at 37 °C for 10 min; (3) used the same approach as that used in method 2, but the second digestion step was performed in TrypLE for 20 min; (4) used the same approach as that used in method 2, but the concentration of collagenase A was 2 mg/ml and the incubation time was 2h; (5) used the same approach as that used in method 3, but the corneas were incubated in 2 mg/ml collagenase A at 37 °C for 2h. Trypan blue staining was used to calculate the cell viability and agglomeration rate. The cell types and percentages were determined using immunofluorescence staining. RNA integrity number (RIN) was measured by Agilent 2100. Method 1 showed the lowest cell yield (0.375 × 105), epithelial cell percentage, and less than 70% cell viability, thus not a proper protocol. Method 2 showed the highest cell viability (over 90%), percentage of single-cell (89.53%), and high cell quantity (1.05 × 105). Method 3 had a significantly lower cell viability (55.30%). Cell agglomeration rates of method 4 and 5 reached up to 20% and 13%, and with lower cell viability (72.51%, 59.87%, respectively) and decreased epithelial cell rate compared to method 2 and 3. The results suggest that method 2 (5 mg/ml collagenase A and trypsin) is a preferred protocol for digesting mouse cornea to obtain single-cell suspension which achieves the criterion of single-cell RNA sequencing.

[Prediction of anti-liver fibrosis effect of Piperis Longi Fructus based on network pharmacology].

Network pharmacology and liver fibrosis(LF) model in vitro were used to analyze the underly mechanism of anti-liver fibrosis effect that induced by Piperis Longi Fructus and its major active compounds. TCMSP and TCMIP were used to search for the chemical constituents of Piperis Longi Fructus, as well as the oral bioavailability(OB), drug-likeness(DL), intercellular permeability of intestinal epithelial cells(Caco-2) and Drug-likeness grading were set as limiting conditions. The related target genes of Piperis Longi Fructus were queried by TCMSP database, while related targets of LF were screened by GeneCards databases. Interaction network was constructed using Cytoscape 3.7.1. These above data were imported into STRING database for PPI network analysis. Enrichment of gene ontology(GO) and pathway analysis(KEGG) within Bioconductor database were utilized to note functions of related targets of Piperis Longi Fructus. Finally, the core targets and pathways were preliminarily verified by in vitro experiments. The effects of piperlongumine(PL), the major active component of Piperis Longi Fructus, on proliferation of rat liver stellate cells(HSC-T6) and expression of α smooth muscle actin(α-SMA) and collagen Ⅰ were investigated. The major factors TNF-α of tumor necrosis factor(TNF) pathway and NF-κB p65, IL-6 protein expressions of LF process were examined. A total of 12 active compounds such as PL were obtained by analyzing the bioavailability and drug-like properties, which inferred to 48 targets. The functional enrichment analysis of GO obtained 1 240 GO items, mainly involving in process of biology and molecular function. A total of 99 signaling pathways were enriched in the KEGG pathway enrichment analysis, including TNF signaling pathway, cGMP-PKG signaling pathway, calcium signaling pathways. CCK-8 assay showed that PL inhibited proliferation of HSC-T6 induced by transforming growth factor-ß1(TGF-ß1). Western blot analysis found that treated with PL suppressed the protein expressions of α-SMA, collagen Ⅰ, TNF-α and p65 in HSC-T6. Enzyme linked immunosorbent assay(ELISA) showed that PL inhibited the expressions of TNF-α and IL-6 in the cluture supertant of HSC-T6 cells. In conclusion, PL could play an anti-liver fibrosis role by regulating TNF/NF-κB signaling pathway. This study provided the mechanism basis of anti-LF effects induced by Piperis Longi Fructus and its major active compounds, which might help for the further study of the mechanism and key targets of Piperis Longi Fructus.

Design, synthesis, and biological evaluation of a novel indoleamine 2,3-dioxigenase 1 (IDO1) and thioredoxin reductase (TrxR) dual inhibitor.

Targeting the Trp-Kyn pathway is an attractive approach for cancer immunotherapy. Thioredoxin reductase (TrxR) enzymes are reactive oxygen species (ROS) modulators that are involved in the tumor cell growth and survival processes. The 4-phenylimidazole scaffold is well-established as useful for indoleamine 2,3-dioxygenase 1 (IDO1) inhibition, while piperlongumine (PL) and its derivatives have been reported to be inhibitors of TrxR. To take advantage of both immunotherapy and TrxR inhibition, we designed a first-generation dual IDO1 and TrxR inhibitor (ZC0101) using the structural combination of 4-phenylimidazole and PL scaffolds. ZC0101 exhibited better dual inhibition against IDO1 and TrxR in vitro and in cell enzyme assays than the uncombined forms of 4-phenylimidazole and PL. It also showed antiproliferative activity in various cancer cell lines, and a selective killing effect between normal and cancer cells. Furthermore, ZC0101 effectively induced apoptosis and ROS accumulation in cancer cells. Knockdown of TrxR1 and IDO1 expression induced cellular enzyme inhibition and ROS accumulation effects during ZC0101 treatment, but only reduced TrxR1 expression was able to improve ZC0101's antiproliferation effect. This proof-of-concept study provides a novel strategy for cancer treatment. ZC0101 represents a promising lead compound for the development of novel antitumor agents that can also be used as a valuable probe to clarify the relationships and mechanisms of cancer immunotherapy and ROS modulators.

Shape-based virtual screen for the discovery of novel CDK8 inhibitor chemotypes.

With the aim of discovering novel cyclin-dependent kinase 8 (CDK8) inhibitors, a combined similarity search and molecular docking approach was employed, which led to 32 hits. Biological tests led to the discovery of several novel submicromolar inhibitors. In particular, compound C768-0769 (ZC0201) showed good CDK8 inhibitory activity, and compound ZC0201 effectively suppressed HCT-116 colorectal cancer cell proliferation by inducing G1/S transition arrest. Furthermore, modulation of phosphorylated signal transducer and activator of transcription 1 (Ser 727) (STAT1SER727), a pharmacodynamic biomarker of CDK8 activity, demonstrated that ZC0201 may cause G1/S transition arrest through CDK8 activity inhibition. Due to its good cellular activity, ZC0201 may be an ideal lead compound for further modification as a potential cancer therapeutic agent.

Microbial diversity of mangrove sediment in Shenzhen Bay and gene cloning, characterization of an isolated phytase-producing strain of SPC09 B. cereus.

Phytases hydrolyze phytate to release inorganic phosphate, which decreases the requirement for phosphorus in fertilizers for crops and thus reduces environmental pollutants. This study analyzed microbial communities in rhizosphere sediment, collected in September 2012 from Shenzhen Bay, Guangdong, China, using high-throughput pyrosequencing; the results showed that the dominant taxonomic phyla were Chloroflexi, Firmicutes, and Proteobacteria, and the proportion of the beneficial bacteria, Bacillus, was 4.95 %. Twenty-nine culturable, phytase-producing bacteria were isolated, their phosphorus solubilization capacity was analyzed, and they were taxonomically characterized. Their phylogenetic placement was determined using 16S ribosomal RNA (rRNA) gene sequence analysis. The result shows that most of the isolates are members of the order Bacillales, although seven strains of Enterobacteriales, two strains of Pseudomonadales, and one strain of Oceanospirillales were also identified. The phytase gene was cloned from SPC09, Bacillus cereus, which showed the highest phosphorus solubilizing ability among the isolated strains. The gene encoded a primary translation product of 335 amino acids. A construct including the 1005-nt ORF fragment, Bc-phy, was transformed into Escherichia coli. The recombinant phytase was produced and purified, which revealed the temperature optima at 60 °C and pH optima at 6.5. The assessment by quantitative PCR (qPCR) showed an abundance of bacteria containing the Bc-phy gene; the level was generally higher in the mangrove forest than in the tidal flats and in surface soil compared to bottom soil, and the highest value was obtained in June. Herein, we report on the cloning, characterization, and activity of a novel phytase isolated from a mangrove system.

Water solubility differentiates the impact of tea polyphenols and rutin on the postprandial glycemic response to cooked maize starch.

Postprandial hyperglycemia is an independent risk factor for cardiovascular diseases, and the impact of tea polyphenols (TP) and rutin, representative phenolic compounds with different water solubilities, on the postprandial glycemic response to cooked normal corn starch (CCS) was investigated. Comparatively, TP (DPPH50 = 0.12 mmol L-1) are more potent than rutin (DPPH50 = 0.50 mmol L-1) in scavenging the free radicals of DPPH, but both TP and rutin inhibited the activity of porcine pancreatic α-amylase (PPA), the major enzyme in starch digestion, with an IC50 of 4.09 mmol L-1 and 2.71 mmol L-1, respectively. However, an in vivo study showed that a significant reduction in postprandial blood glucose was only observed in the presence of rutin, and TP had no effect on the glycemic response to CCS. To find out the underlying mechanism, fluorescence spectroscopy and molecular docking were carried out and they showed that, compared to TP, rutin bound to the active site of PPA with higher affinity and a lower free energy (ΔG) driven by hydrogen bonds and π-stacking, and rutin also greatly increased the viscosity of starch. Collectively, water-soluble TP have a higher antioxidant property and a lower potency to inhibit PPA compared to water-insoluble rutin, and the weaker interaction between TP and PPA, and starch as well might synergistically contribute to TP's ineffectiveness in lowering the postprandial glycemic response, and water solubility linking the molecular structures and functions of phenolic compounds might be the fundamental basis for the observed difference in their biological functions, and water solubility can also be used to enrich specific phenolic compounds for desired functions.

Effects of different dietary lipid level on the growth, survival and immune-relating genes expression in Pacific white shrimp, Litopenaeus vannamei.

Five feeding trials based on the isonitrogenous and isoenergetic experimental diets containing 34% protein, 6%, 8%, 10%, 12% or 14% lipid respectively in the circulating water culture system for both 30 and 60 days were conducted to investigate the effect of the dietary lipid level on the growth and immunity in white shirmp, Litopenaeus vannamei adults. The body weight and specific growth rate of white shrimp in different treatments indicated that shrimps fed the diet of 12% lipid level for 30d and 10% lipid level for 60d had the best developmental status. The ability of respiratory burst in hemocytes was improved as the increase of dietary lipid level. The transcripts of LGBP and pPO were sensitive to the dietary lipid in hemocyte and hepatopancreas respectively. The activities of CAT, GPx and AKP were increased to a certain extend according to dietary lipid level. Qualification of MDA showed the lowest level in the sample subjected to 12% lipid level diet, indicating an optimal utilization of the dietary lipid and an efficient clearance of MDA in vivo. These results suggested that dietary lipid level of 10-12% significantly tunes the growth and enhance the immune abilities mainly via ROS pathway of L. vannamei.

Trascriptome analysis of the Pacific white shrimp Litopenaeus vannamei exposed to nitrite by RNA-seq.

In the present study, transcriptome of nitrite-exposed Litopenaeus vannamei was performed using a newly developed high-throughput sequencing technology (Illumina RNA-seq). As many as 42,336 unigenes were generated with 561 bp of average length and 736 bp of unigene N50 after filtering and assembly. These unigenes from the de novo assembly were further annotated using BLAST and BLAST2GO softwares. A total of 23,532 unigenes were unambiguous alignments to the reference when BLAST against non-redundant protein sequence (Nr), non-redundant nucleotide (Nt), Swiss-Prot, Gene Ontology database (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases available at NCBI. Numerous candidate genes associated with immune response, detoxification, apoptosis pathway were identified. Ten candidate genes related to immune responses and apoptosis were selected for validating the results of assembly and annotation by real-time quantitative PCR. Results revealed that the expressions of all these ten genes were up-regulated after nitrite exposure. Combining to our previous study, we speculate that all these selected genes may be involved in the response to nitrite stress. The study shows a systematic overview of the transcriptome analysis in L. vannamei, and provides valuable gene information for studying molecular mechanisms under nitrite exposure.

Measurement of intracellular nitric oxide (NO) production in shrimp haemocytes by flow cytometry.

A flow cytometric method to measure the production of intracellular nitric oxide (NO) was adapted for use with shrimp haemocytes. We applied fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) for NO detection in haemocytes from the tiger shrimp Penaeus monodon, and used flow cytometry to quantify fluorescence intensity in individual haemocyte. The optimized protocol for intracellular NO analysis consists to incubate haemocytes with DAF-FM DA at 10 µM for 60 min to determine the mean fluorescence intensity. Result showed that NO was also produced in the untreated shrimp haemocytes. NO level in granular cells and semigranular cells were much higher than that in hyaline cells. Defined by different characteristic of NO content, three subsets of haemocytes were observed. Zymosan A at dose of 10 or 100 particles per haemocyte triggered higher DAF-FM fluorescence intensity in granular and semigranular cells, than PMA that had no significant impact on all three cell types. These results indicate that granular and semigranular cells are the primary cells for NO generation. Cytochalasin B significantly inhibited the NO level induced by zymosan A. NG-Monomethyl-L-arginine (L-NMMA) and diphenylene iodonium chloride (DPI) significantly suppressed the DAF-FM fluorescence in haemocytes, but apocynin could not modulate it, indicating that the DAF-FM fluorescence was closely related to the activity of NO-synthase pathway. The NO donor sodium nitroprusside (SNP) improved the DAF-FM fluorescence in haemocytes, while the NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) significantly decreased the fluorescence, demonstrating that the fluorescence intensity of DAF-FM is mainly dependent on the intracellular NO level.

Exploration of active sites of ethyl alcohol electro-oxidation on porous gold nanoparticles with enhanced Raman spectroscopy.

Novel porous gold nanospheres are prepared by calcination of the gold-urea complexes. The enhanced Raman spectra of ethanol catalyzed by different doses of porous gold nanospheres are measured with a 532 nm laser as the excitation source, and an enhanced charge coupled device served in spectral detection and microscopic imaging. The electrochemical experiments show that the catalytic oxidation products of ethanol with porous gold nanoparticles are acetaldehyde, acetic acid, and water, which further proved that the porous gold nanoparticles can activate the -CH2 of ethanol.

Non-alcoholic fatty liver disease combined with rheumatoid arthritis exacerbates liver fibrosis by stimulating co-localization of PTRF and TLR4 in rats.

Rheumatoid arthritis (RA) has a high prevalence in patients with non-alcoholic fatty liver disease (NAFLD); however, the underlying mechanism is unclear. To address this, our study established a rat model with both NAFLD and RA by feeding a high-fat diet (HFD) and administering intradermal injection of Freund's complete adjuvant (FCA) with bovine type II collagen. Collagen-induced RA (CIA) was confirmed by hind paw swelling and histological examination. The histomorphological characteristics of NAFLD were evaluated by Masson's trichrome and hematoxylin-eosin staining. The development of NAFLD was further evaluated by measuring serum concentrations of triglyceride (TG), total cholesterol (T-CHO), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lipopolysaccharide (LPS). The results showed that HFD feeding exacerbated secondary inflammation in CIA rats, whereas FCA/bovine type II collagen injection increased serum levels of ALT, AST, TG, T-CHO, and LPS and exacerbated hepatic fibrosis in both normal and NAFLD rats. Interestingly, NAFLD + CIA significantly promoted the expression of PTRF, a caveolae structure protein involved in hepatic lipid metabolism and affecting downstream signaling of Toll-like receptor 4 (TLR4) and PI3K/Akt activation. High resolution confocal microscopy revealed increased PTRF and TLR4 co-localization in hepatic small vessels of NAFLD + CIA rats. AAV9-mediated PTRF knockdown inhibited TLR4 signaling and alleviated hepatic fibrosis in NAFLD + CIA rats. Together, these findings indicate that NAFLD combined with CIA causes synovial injury and enhances non-alcoholic fatty liver fibrosis in rats. PTRF could attenuate the symptoms of NAFLD + CIA likely by affecting TLR4/PTRF co-expression and downstream signaling.

Enhancing the affinity of novel GLS1 allosteric inhibitors by targeting key residue Lys320.

Aim: A series of novel GLS1 irreversible allosteric inhibitors targeting Lys320 might have robust enzyme inhibitory activity and potent antitumor activity. Materials & methods: Novel GLS1 allosteric inhibitors targeting Lys320 were synthesized and their anticancer activity was assessed. Moreover, GLS1 protein was used as a model system to analyze the reactivity of these electrophilic groups in GLS1 irreversible allosteric inhibitors with other amino acids, including tyrosine, histidine, serine and threonine, using biochemical and biophysical assays. Results: AC16 exhibited robust GLS1 inhibitory activity, antiproliferative effect in vitro, good plasma stability and potential covalent addition with GLS1 K320. Conclusion: This study opens a novel avenue for the design of robust irreversible GLS1 inhibitors targeting the allosteric site K320.

Discovery of novel hydroxyamidine based indoleamine 2,3-dioxygenase 1 (IDO1) and thioredoxin reductase 1 (TrxR1) dual inhibitors.

In order to take advantage of both immunotherapeutic and metabolic antitumor agents, novel dual indoleamine 2,3- dioxygenase 1 (IDO1) and thioredoxin reductase 1 (TrxR1) inhibitors were designed. Thioredoxin reductase 1 (TrxR1) is a main ROS modulator within CRC cells. Indoleamine 2,3-dioxygenase (IDO1) is crucial controller for tryptophan (Trp) metabolism that is also important for CRC immunotherapy. Herein, ten compounds 12a-j containing hydroxyamidine scaffold were designed, synthesized and evaluated for inhibitory activities against IDO1/TrxR1 enzyme and CRC cells. Among these compounds, the most active compound 12d (ZC0109) showed excellent and balanced activity against both IDO1 (IC50 = 0.05 µM) and TrxR1 (IC50 = 3.00 ± 0.25 µM) were selected for further evaluation. Compound ZC0109 exhibited good dual inhibition against IDO1 and TrxR1 both in vitro and in vivo. Further mechanistic studies reveal that, through IDO1 and TrxR1 inhibition by ZC0109 treatment, accumulated ROS effectively induced apoptosis and G1/S cell cycle arrest in cancer cells. In vivo evaluation demonstrated excellent anti-tumor effect of ZC0109 with the notable ability of promoting ROS-induced apoptosis, reducing kynurenine level in plasma and restoring anti-tumor immune response. Thus, ZC0109 represents a potential CRC therapy agent for further development.

Effect of nitrite on immunity of the white shrimp Litopenaeus vannamei at low temperture and low salinity.

White shrimp Litopenaeus vannamei Boone (6-7 cm) were individually exposed during 8 h to 0 (control), 0.5, 1.0, and 10.0 mg/l NO(2)(-)-N at 22 °C and 10 ‰ salinity. Nitrite concentration was measured, and hemolymph and hepatopancreas were sampled at 0, 4 and 8 h. Superoxide dismutase (SOD) activity, catalase (CAT) activity, and malondialdehyde (MDA) in haemolymph and hepatopancreas of L. vannamei Boone, and hemocyanin were examined. The results indicated significant relationships among hemocyanin concentration (Hc), nitrite concentration (nt), and exposure time (t) was as following: Hc = 1.250 + 0.0360 nt + 0.0636t, (R(2) = 0.702, P < 0.01), and Gompertz models with 3-parameters was used to describe well the increase trend of Hc with increase of nitrite concentration at 4, 8 h, respectively (R(2) > 0.99, P ≤ 0.05). MDA level and CAT activity in hemolymph decreased significantly at 8 h, and MDA level and CAT activity in hepatopancreas increased markedly. The SOD activity in hepatopancreas remained almost stably in the range of 0.22-0.24 U/g Hb within 8 h. The results showed there existed a immune difference between in hemolymph and hepatopancreas after white shrimp exposed to ambient nitrite within 8 h, and further experiments should be designed to answer the reason.

An improved explants culture method: Sustainable isolation of keloid fibroblasts with primary characteristics.

BACKGROUND: Keloid (KD) is a complex fibroproliferative disease, but the exact mechanisms underlying keloid pathogenesis remain to be elucidated. The primary keloid fibroblasts (KFs) culture in vitro has always been a fundamental measure to study the pathogenesis of keloid. However, the traditional primary culture methods have some limitations, such as a long culture cycle, low specimen utilization rate and so on. AIMS: Improve the keloid explants culture method sts. MATERIALS & METHODS: We proposed an improved new "explants multiple culture method"-reusing keloid explants for primary culture and harvesting the primary KFs in specific culture times. Meanwhile, the purity, proliferation, apoptosis, migration, invasion, extracellular matrix synthesis, and some fibrosis and inflammation-related proteins of KFs obtained from the first, fifth, and tenth explants cultures were detected. RESULTS: The results showed that the culture cycle of this new method (Cell Isolation: 2.67 ± 0.86 days, Explants removal: 8.83 ± 0.79 days, Cell Passage: 15.17 ± 1.39 days) was significantly shorter than that of the traditional method (Cell Isolation: 8.67 ± 1.84 days, Explants removal: 17.67 ± 2.17 days, Cell Passage: 22.67 ± 1.84 days). No significant difference was observed between the phenotypes of the fibroblasts obtained from the first explants culture and cultures less than 10 times (p > 0.05). DISSCUSSION: Taken together, this study provides an effective method for the primary culture of KFs with a higher specimen utilization rate and shorter culture cycle. CONCLUSION: This method breaks through the limitation of traditional explants culture requiring a large number of keloid specimens and provides a rich source of KFs for the study of keloid.

Paeoniflorin Ameliorates Colonic Fibrosis in Rats with Postinfectious Irritable Bowel Syndrome by Inhibiting the Leptin/LepRb Pathway.

Postinfectious irritable bowel syndrome (PI-IBS) is a highly prevalent gastrointestinal disorder associated with immune dysregulation and depression- and anxiety-like behaviors. Through traditional medicine, the active ingredient of Paeoniae Radix called paeoniflorin (PF) was previously found to prevent the symptoms of PI-IBS. However, there is limited information on the effects of PF on intestinal function and depression- and anxiety-like symptoms in PI-IBS animal models. Here, we aimed to determine the effects of PF treatment on the symptoms of PI-IBS in a rat model. The PI-IBS rat model was established via early postnatal sibling deprivation (EPSD), trinitrobenzenesulfonic acid (TNBS), and chronic unpredictable mild stress (CUMS) stimulation and then treated with different dosages of PF (10, 20, and 40 mg/kg) and leptin (1 and 10 mg/kg). The fecal water content and body weight were measured to evaluate the intestinal function, while the two-bottle test for sucrose intake, open field test (OFT), and elevated plus maze test (EMT) were performed to assess behavioral changes. The serum leptin levels were also measured using an enzyme-linked immunosorbent assay. Furthermore, the expressions of leptin and its receptor, LepRb, were detected in colonic mucosal tissues through an immunohistochemical assay. The activation of the PI3K/AKT signaling pathway and the expression of brain-derived neurotrophic factor (BDNF) were also detected via western blotting. After the experimental period, the PI-IBS rats presented decreased body weight and increased fecal water content, which coincided with elevated leptin levels and heightened depression- and anxiety-like behaviors (e.g., low sucrose intake, less frequency in the center areas during OFT, and fewer activities in the open arms during EMT). However, the PF treatment ameliorated these observed symptoms. Furthermore, PF not only inhibited leptin/LepRb expression but also reduced the PI3K/AKT phosphorylation and BDNF expression in PI-IBS rats. Notably, cotreatment with leptin (10 mg/kg) reduced the effects of PF (20 mg/kg) on colonic fibrosis, leptin/LepRb expression, and PI3K/AKT activation. Therefore, our findings suggest that leptin is targeted by PF via the leptin/LepRb pathway, consequently ameliorating the symptoms of PI-IBS. Our study also contributes novel insights for elucidating the pharmacological action of PF on gastrointestinal disorders and may be used for the clinical treatment of PI-IBS in the future.

The inhibition of HeLa cells proliferation through SPARCL1 mediated by SPP1.

Secreted protein acidic and rich in cysteines-like 1 (SPARCL1) is implicated in tumor progression and considered as a tumor suppressor. Aim of the study is to investigate the role of SPARCL1 in the regulation of tumor biology. SPARCL1 expression in human cervical cells was determined through western blot and RT-PCR. The effects of SPARCL1 overexpression on cell proliferation, migration and invasion were evaluated through CCK8 assay, colony formation assay, Wound healing assay and Transwell assay, respectively. The gain function of Secreted phosphor protein 1 (SPP1) was also evaluated in these cell functions. We observed that SPARCL1 expression at protein levels and transcription levels was lower in HeLa cells than that in Ect1/E6E7 cells. When SPARCL1 was overexpressed in HeLa cells, cell proliferation, migration and invasion were greatly repressed. Additionally, SPARCL1 overexpression markedly downregulated SPP1 expression at transcription levels. Mechanistical study revealed that SPP1 overexpression could greatly counteract the effects of SPARCL1 overexpression on the aforementioned cell processes and inhibit the phosphorylation of focal adhesion kinase (FAK) and extracellular regulated protein kinases (ERK). Our findings indicated that HeLa cells overexpressing SPARCL1 showed weaker abilities of proliferation, migration and invasion, and its effects could be neutralized by SPP1 overexpression possibly via FAK/ERK pathway. The relationship of SPARCL1 and SPP1 could help us to further understand the pathogenesis of cervical cancer and SPARCL1/SPP1 could be beneficial therapeutic targets in cervical cancer.

Suppression of PTRF Alleviates Post-Infectious Irritable Bowel Syndrome via Downregulation of the TLR4 Pathway in Rats.

Background: Accumulating evidence suggests that the polymerase I and transcript release factor (PTRF), a key component of the caveolae structure on the plasma membrane, plays a pivotal role in suppressing the progression of colorectal cancers. However, the role of PTRF in the development of functional gastrointestinal (GI) disorders remains unclear. Post-infectious irritable bowel syndrome (PI-IBS) is a common functional GI disorder that occurs after an acute GI infection. Here, we focused on the role of PTRF in the occurrence of PI-IBS and investigated the underlying mechanisms. Methods: Lipopolysaccharide (LPS) (5 µg/ml) was used to induce inflammatory injury in human primary colonic epithelial cells (HCoEpiCs). Furthermore, a rat model of PI-IBS was used to study the role of PTRF. Intestinal sensitivity was assessed based on the fecal water content. A two-bottle sucrose intake test was used to evaluate behavioral changes. Furthermore, shRNA-mediated knockdown of PTRF was performed both in vitro and in vivo. We detected the expression of PTRF in colonic mucosal tissues through immunohistochemistry (IHC), western blotting (WB), and immunofluorescence (IF) analysis. Luciferase activity was quantified using a luciferase assay. Co-localization of PTRF and Toll-like receptor 4 (TLR4) was detected using IF analysis. The activation of the signaling pathways downstream of TLR4, including the iNOs, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) pathways, was detected via WB. The levels of NO, IL-1ß, IL-6, and TNF-α were measured using enzyme-linked immunosorbent assays. Results: LPS significantly induced PTRF expression and signaling downstream of TLR4, including p38, ERK, and JNK pathways, in HCoEpiCs. Moreover, shRNA-mediated knockdown of PTRF in HCoEpiCs significantly decreased the phosphorylation of JNK, ERK, and p38 and iNOS expression. In PI-IBS rats, the lack of PTRF not only reduced fecal water content and suppressed depressive behavior but also increased the body weight. Furthermore, we found a strong co-localization pattern for PTRF and TLR4. Consistently, the lack of PTRF impaired TLR4 signaling, as shown by the decreased levels of p-JNK, p-ERK, and p-p38, which are upstream factors involved in iNOS expression. Conclusion: PTRF promoted PI-IBS and stimulated TLR4 signaling both in vitro and in vivo. The results of this study not only enlighten the pathogenesis of PI-IBS but also help us understand the biological activity of PTRF and provide an important basis for the clinical treatment of PI-IBS by targeting PTRF.

Discovery of hydroxytyrosol as thioredoxin reductase 1 inhibitor to induce apoptosis and G1/S cell cycle arrest in human colorectal cancer cells via ROS generation.

Colorectal cancer (CRC) is one of the most common cancer types and a leading cause of cancer-associated mortality in China. Increased thioredoxin reductase 1 (TrxR1) levels have been previously identified as possible target for CRC. The present study revealed that the natural product hydroxytyrosol (HT), which exhibits a polyphenol scaffold, is a potent inhibitor of TrxR1. Inhibition of TrxR1 was indicated to result in accumulation of reactive oxygen species, inhibit proliferation and induce apoptosis and G1/S cell cycle arrest of CRC cells. Using a C-terminal mutant TrxR1 enzyme activity assay, TrxR1 RNA interference assay and HT binding model assay, the present study demonstrated the core character of the selenocysteine residue in the interaction between HT and TrxR1. HT can serve as polyphenol scaffold to develop novel TrxR1 inhibitors for CRC treatment in the future.

LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1.

Abundant evidence has illustrated that long non-coding RNA (lncRNA) plays a vital role in the regulation of tumor development and progression. Most lncRNAs have been proven to have biological and clinical significance in acute myeloid leukemia (AML), but further investigation remains necessary. In this study, we investigated lncRNA NR-104098 in AML and its specific mechanism. The microarray analysis was performed on NB4 cells. Based on the related analysis results, we identified that lncRNA NR-104098 is a suppressor gene that is significantly upregulated in AML cells. LncRNA NR-104098 could inhibit proliferation and induce differentiation in AML cells in vitro and also play main role in the mouse xenografts. Mechanically, it was confirmed that lncRNA NR-104098 may effectively inhibit EZH2 transcription by directly binding to E2F1 and recruiting E2F1 to the EZH2 promoter. In addition, ATPR can significantly increase the expression of lncRNA NR-104098, whereas knocking down NR104098 can inhibit the inhibitory effect of ATPR on the proliferation and induction differentiation of AML cells. Taken together, these results lead to deeper insight into the mechanism of ATPR-induced AML differentiation and prevent proliferation by inhibiting EZH2 on the transcriptional level.