LCN2 is involved in various cellular functions, including transport of small hydrophobic molecules, protection of MMP9 from proteolytic degradation, and regulating innate immunity. LCN2 is elevated in multiple human cancers, frequently being associated with tumor size, stage, and invasiveness. Our previous studies have shown that LCN2 expression could be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in esophageal squamous cell carcinoma (ESCC) by the binding of five nucleoproteins (MISP, KLF10, KLF15, PPP1R18, and RXRβ) at a novel TPA-responsive element (TRE), at -152~-60 bp of the 5' flanking region of the LCN2 promoter. However, much is unknown about whether these proteins can respond to TPA stimulation to regulate LCN2 transactivation and which cell signaling pathways mediate this process. In this study, expression plasmids encoding these five nucleoproteins were stably transfected into EC109 cells. Then, stable transfectant was characterized by a Dual-Luciferase Reporter Assay System. RT-PCR, real-time PCR, western blotting, specific kinase inhibitor treatment, and bioinformatics analyses were applied in this study. We found that MISP, KLF10, KLF15, PPP1R18, and RXRβ proteins could strongly respond to TPA stimulation and activate LCN2 transcriptional expression. MEK, ERK, JNK, and P38 kinases were involved in the LCN2 transactivation. Furthermore, the MEK-ERK signal pathway plays a major role in this biological process but does not involve PKCα signaling.

Dendritic spines are divided into four subtypes, namely, Mushroom, Stubby, Thin, and Branched. The mushroom-shaped spines are related to learning and memory. Previous studies have shown that the dendritic cell factor 1 (Dcf1, a transmembrane protein) affects the memory process and regulates the development of dendritic spines by inhibiting the expression of lipocalin 2 (Lcn2, a member of the family containing over 20 small secreted proteins). However, the exact subtype of dendritic spines that are specifically affected by Dcf1 remains unknown. Here, we identified that deletion of Dcf1 leads to developmental defects in mushroom-shaped spines. We provide evidence for memory defects caused by Dcf1-knockout in mice. We discovered and report for the first time that Dcf1 affects the development of mushroom-shaped spines by inhibiting the expression of Lcn2. Further, we demonstrated that environmental enrichment can effectively stimulate Dcf1-knockout mice and rescue development defects in mushroom-shaped spines caused by Dcf1 deletion. Our results provide a novel direction for further studies on dendritic spine development and mechanisms associated with learning and memory.

Acute respiratory distress syndrome (ARDS) is characterized as a neutrophil-dominant disorder without effective pharmacological interventions. Knowledge of neutrophils in ARDS patients at the transcriptome level is still limited. We aimed to identify the hub genes and key pathways in neutrophils of patients with ARDS. The transcriptional profiles of neutrophils from ARDS patients and healthy volunteers were obtained from the GSE76293 dataset. The differentially expressed genes (DEGs) between ARDS and healthy samples were screened using the limma R package. Subsequently, functional and pathway enrichment analyses were performed based on the database for annotation, visualization, and integrated discovery (DAVID). The construction of a protein-protein interaction network was carried out using the search tool for the retrieval of interacting genes (STRING) database and the network was visualized by Cytoscape software. The Cytoscape plugins cytoHubba and MCODE were used to identify hub genes and significant modules. Finally, 136 upregulated genes and 95 downregulated genes were identified. Gene ontology analyses revealed MHC class II plays a major role in functional annotations. SLC11A1, ARG1, CHI3L1, HP, LCN2, and MMP8 were identified as hub genes, and they were all involved in the neutrophil degranulation pathway. The MAPK and neutrophil degranulation pathways in neutrophils were considered as key pathways in the pathogenesis of ARDS. This study improves our understanding of the biological characteristics of neutrophils and the mechanisms underlying ARDS, and key pathways and hub genes identified in this work can serve as targets for novel ARDS treatment strategies.

Infectious endometritis is a major cause of reduced pregnancy rates in horses. The objectives of this study were to establish a timeline of the innate immune response in the uterus of healthy horses and to investigate the oestrous cycle effect on this. Endometrial biopsies were collected from five horses before and at 3, 12, 24, 48 and 72 h after inoculation of Escherichia coli, once in oestrus and once in dioestrus. They were analysed by quantitative real-time PCR, microbiology and histology. Neutrophil numbers increased from very low levels in the absence of inflammation to severe neutrophilia 3 h after inoculation. The concentrations of mRNAs for Toll-like receptor (TLR)2, TLR4, NOD-like receptor NLRC5, tissue inhibitor of metallopeptidases 1 (TIMP1) and chemokines CCL2, CXCL9, CXCL10 and CXCL11 were all increased 3 h after inoculation of E. coli compared to levels detected prior to inoculation. Chemokine mRNA levels remained elevated for 48 h. Concentrations of mRNAs for the antimicrobial peptides equine β-defensin 1 (EBD1), lysozyme, secretory leukoprotease inhibitor (SLPI), lipocalin 2 (LCN2), lactoferrin and uteroferrin were increased between 3 and 12 h post inoculation. The gene for secreted phospholipase A2 (sPLA2) was expressed constitutively. P19 uterocalin mRNA levels were higher in dioestrus than in oestrus over the first 24 h of inflammation. Neutrophils and many innate immune genes responded rapidly to the introduction of E. coli into the uterus, while the oestrous cycle stage had only a relatively minor effect on the response to E. coli. This study has delineated a useful model of innate immunity in infectious endometritis of healthy animals.

Early diagnosis and treatment of osteonecrosis of the femoral head (ONFH) is challenging. Bone trabecula play a vital role in the severity and progression of ONFH. In the present study, the investigators used gene expression profiling of bone trabecula to investigate gene alterations in ONFH patients. Osteonecrotic bone trabecula (ONBT) such as necrosis, fibrosis, and lacuna were confirmed by histological examination in the patients. The adjacent ''normal'' bone trabecula (ANBT) did not show any pathological changes. Gene sequencing data revealed that although ANBT showed no significant histological changes, alteration of mRNA profiling in ANBT was observed, similarly to that in ONBT. Our results indicated that the alteration of mRNA profiling in ANBT may cause normal bone tissue to develop into necrotic bone. RNA-seq data indicated that 2297 differentially abundant mRNAs were found in the ONBT group (1032 upregulated and 1265 downregulated) and 1523 differentially abundant mRNAs in the ANBT group (744 upregulated and 799 downregulated) compared with the healthy control group. GO enrichment analysis suggested that fatty acid metabolism and degradation were the main zones enriched with differentially expressed genes (DEG). KEGG pathway enrichment analysis indicated that PPARγ pathway was the most significantly regulated pathway. Lipocalin-2 (LCN2), an osteoblast-enriched secreted protein, was significantly decreased in ONBT suggesting that downregulation of LCN2 might affect lipid metabolism and lead to hyperlipidemia, and thus promote pathogenesis of ONFH.

OBJECTIVE

Mechanical strain of the lung tissue is a physiological process that affects the behavior of lung cells. Since recent evidence also suggests alterations in the expression of certain genes as a consequence of mechanotransduction, our study aimed at the analysis of the gene expression profile in lung epithelial cells subjected to chronic cyclic strain.

METHODS

Various human lung epithelial cell lines (A549 as principal adherent cell line and four others) were subjected to cyclic strain (16 % surface distension, 12 min(-1)) in a Strain Cell Culture Device for 24 h. In comparison to static controls, expression analyses were performed by gene microarray and qPCR.

RESULTS

Microarray analysis revealed many differences in the gene expression but at moderate levels. Altogether 25 genes were moderately down-regulated (0.86-fold ± 0.06) and 26 genes were up-regulated (1.18-fold ± 0.10) in A549 and the others. Strain-regulated genes often code for transcription factors, such as E2F4 and SRF. qPCR analyses confirmed the up-regulation of both transcription factors and further genes, such as PLAU (urokinase-type plasminogen activator) and S100A4 (S100 protein A4). Moreover, we showed the down-regulations of AGR2 (anterior gradient 2) and LCN2 (lipocalin 2).

CONCLUSIONS

We identified many genes of which the expression was moderately altered in lung epithelial cells subjected to chronic cyclic strain. Although many moderate changes in the gene expression profile might affect cellular behavior, it also suggests an effective adaptation of cells to mechanical forces in long-term conditions.

Acne is the eighth most common disease worldwide. Disease burden of acne such as anxiety, reduced self-esteem, and facial scarring lowers the life quality of acne patients. Isotretinoin is the most potent treatment for moderate-severe acne. However, the adverse events of isotretinoin especially teratogenicity limit its use. This study aims at investigating the therapeutical mechanisms of isotretinoin using bioinformatics analysis. Differentially expressed genes (DEGs) were filtered from microarray datasets GSE10432, GSE10433, and GSE11792. Functional and pathway enrichment analyses of DEGs were performed. Protein-protein interaction (PPI) network and module analyses were also conducted based on DEGs. Using isotretinoin for 1 week, LCN2, PTGES, and GDF15 were upregulated and might mediate sebocytes apoptosis and thus decreased sebum production; CCL2 originated from activated TNF signaling pathway and S100A7 could be related with "acne-flare". While treating with isotretinoin for 8 weeks, key genes were downregulated, including HMGCS1, HMGCR, FDFT1, MVD, IDI1, and FDPS, which may be associated with decreased sebum synthesis; HMGCS1, HMGCR, and FDFT1 also probably associated with apoptosis of sebocytes. There were only two common genes including ACSBG1 and BCAT2 which worked in both 1 week and 8 weeks and could associate with decreased sebum synthesis and apoptosis of sebocytes, respectively. These results indicate potential therapeutics and side effect mechanisms of isotretinoin in the acne treatment and provide a research direction to further investigate the therapeutic mechanism of isotretinoin and thus develop retinoid-like compounds with similar curative effect and without teratogenicity.

UNASSIGNED

Recurrent urinary tract infections are associated with uropathogenic Escherichia coli (UPEC) ascending and infecting the urinary tract. Antibiotics provide only symptomatic relief, not prevent recurrence. Clinical evidence suggests that intravesical glycosaminoglycan therapy, such as hyaluronic acid (HA), helps reduce UTI recurrence. This has been investigated here using in vitro systems modelling the urogenital tract tissues.

UNASSIGNED

RT4 bladder cells were preconditioned with high molecular weight HA >> 1500 kDa) at 2 mg mL-1 and challenged with UPEC to analyse barrier protection and bacterial adherence. Untreated and HA-preconditioned VK2 E6/E7 vaginal cells were challenged with E. coli flagellin (50 ng mL-1) to mimic bacterial challenge, and media analysed for lipocalin-2, human β-defensin 2 and interleukin-8 by ELISA. Experiments were repeated after siRNA knockdown of Toll-like receptors 2, 4 and 5, and CD44 to investigate signalling.

UNASSIGNED

Microscopic analyses showed reduced bacterial adherence and urothelial disruption with HA, suggesting that HA functions as a barrier protecting the epithelium from bacterial infection. Cells treated with HA and flagellin simultaneously produced more of the host antimicrobial peptide LCN2 and pro-inflammatory IL-8 (P < 0.05) compared to the no HA/flagellin challenges. Increased gene expression of DEFB4 (P < 0.05), but not the hBD2 peptide, was observed in the HA/flagellin-challenged cells.

UNASSIGNED

These data suggest that exogenous HA has potential to protect the urogenital epithelia from UPEC infection via a two-pronged approach that involves the physical enhancement of the epithelial barrier and augmentation of its innate immune response.

Lipocalin-2 (LCN2) is an immunomodulatory protein holding major metabolic and immune functions. It is involved in several inflammatory processes and induced by cytokines of the interleukin-1 family known as contributors to the morbidity in graft-versus-host disease (GVHD) following hematopoietic stem cell transplantation (HSCT). The possible role of LCN2 in predicting outcome and course of illness has never been elucidated in patients undergoing HSCT for hematologic malignancies. We conducted a prospective cohort study including 40 patients following autologous or allogeneic HSCT by collecting plasma samples at seven time points with respect to GVHD, relapse, and outcome. LCN2 levels were significantly increased in acute patients with GVHD compared with autologous and healthy controls (125.7 ng/mL vs. 65.9 and 71.4 ng/mL) and correlated with its severity. Similarly, LCN2 levels were significantly elevated in chronic GHVD compared with autologous and healthy controls (295.0 ng/mL vs. 54.9 and 76.5 ng/mL). Moreover, LCN2 correlated with mortality. The suspected role of LCN2 as a predictive parameter for outcome and prognosis needs to be further investigated.

Background & aims: Interleukin-20 (IL-20) and IL-22 belong to the IL-10 family. IL-10 is a well-documented anti-inflammatory cytokine while IL-22 is well-known for its epithelial protection and anti-bacterial function, showing great therapeutic potential for organ damage; but the function of IL-20 remains largely unknown.

Methods: Il20 knockout (Il20^-/-) mice and wild-type littermates were generated and injected with Concanavalin A (ConA) and Klebsiella pneumoniae (K.P.) to induce acute hepatitis and bacterial infection, respectively.

Results: Il20^-/- mice were resistant to acute hepatitis with selective elevation of the hepatoprotective cytokine IL-6 levels without affecting most other cytokines. Such selective inhibition of IL-6 by IL-20 was due to IL-20 targeting-hepatocytes that produce high levels of IL-6 but a limited number of other cytokines. Mechanistically, IL-20 upregulated NAD(P)H: quinone oxidoreductase 1 (NQO1) expression and subsequently promoted the protein degradation of transcription factor IκBζ, resulting in selective downregulation of the IκBζ-dependent gene Il6 as well several other IκBζ-dependent genes including lipocalin-2 (Lcn2). Given an important role of IL-6 and LCN2 in limiting bacterial infection, we examined the effect of IL-20 on bacterial infection and found Il20^-/- mice were resistant to K.P. infection accompanied with an elevation of hepatic IκBζ-dependent antibacterial genes. Moreover, IL-20 upregulated hepatic NQO1 by activating ERK/p38MAPK/NRF2 signaling pathways via the binding of IL-22R1/IL-20R2. Finally, hepatic IL1B, IL20, and IκBζ target genes are elevated and correlated each other in patients with acute alcoholic hepatitis.

Conclusions: IL-20 selectively inhibits hepatic IL-6 production rather than exerts an IL-10 like broad anti-inflammatory properties and has opposing functions compared to IL-22 by aggravating acute hepatitis and bacterial infection. Thus, anti-IL-20 therapy may have benefits to control acute hepatitis and bacterial infection.

Keywords: IL-10; IL-22; Klebsiella pneumoniae; NQO1; liver.

BACKGROUND

Early diagnosis of complications after severe trauma by specific biomarkers remains difficult.

OBJECTIVE

Identify potential new biomarkers for early diagnosis of post-traumatic complications.

METHODS

Mice underwent pressure-controlled hemorrhage or sham procedure. Four hours later, genome-wide expression of isolated Kupffer cells was compared with controls using Affymetrix-Genechip-Expression-Analysis and real-time-PCR.

RESULTS

Expression analysis and real-time-PCR revealed a significant increase of gene expression of Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5.

CONCLUSIONS

Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5 might represent new biomarkers for early diagnosis of post-traumatic complications, if they are linked to the development of post-traumatic complications.

Subarachnoid hemorrhage (SAH) is devastating disease with high mortality, high disability rate, and poor clinical prognosis. It has drawn great attentions in both basic and clinical medicine. Therefore, it is necessary to explore the therapeutic drugs and effective targets for early prediction of SAH. Firstly, we demonstrate that LCN2 can effectively intervene or treat SAH from the perspective of cell signaling pathway. Next, three potential genes that we explored have been validated by manually reviewed experimental evidences. Finally, we turn out that the SAH early ensemble learning predictive model performs better than the classical LR, SVM, and Naïve-Bayes models.

Lipocalin 2 (Lcn2, also called as neutrophil gelatinase-associated lipocalin) is a member of the lipocalin family and a known target for breast cancer. Therefore, it is of interest to use Docetaxel as a scaffold to design molecules with improved efficiency from naturally derived phytochemicals. We document 10 analogues (4Deacetyltaxol, 7Acetyltaxol, Cabazitaxel, Cephalomannine, Docetaxal, Deacetyltaxol, Docetaxeltrihydrate, Ortataxel, Paclitaxel, Taxoline) having optimal binding with Lipocalin 2 in comparison with Docetaxel. This data is highly useful for consideration in the design and development of drugs for breast cancer.

Keywords: Lipocalin 2; analogues; docetaxel; molecular docking.

Melamine is an important and widely used organic industrial chemical. Recently, clinical findings of renal failure and kidney stones in infants have been associated with ingestion of melamine-contaminated infant formula. To understand the toxicity and clinical outcome of melamine exposure, repeated oral dose studies in rats and monkeys were performed to characterize the subchronic toxicity of melamine. Assessment of toxicity was based on mortality, clinical signs, body weights, ophthalmic findings, clinical pathology, gross pathology, organ weights, and microscopic observations. The first rat study was intended to be a 14-day oral study followed by an 8-day recovery period. The dose levels were 140, 700, and 1,400 mg/kg/day (lowered to 1,000 mg/kg/day subsequently due to mortality). Oral administration of melamine at 700 mg/kg/day for 14 consecutive days in rats produced compound-related clinical signs (red urine), decreased body weights, and changes in clinical pathology (increased serum urea nitrogen and creatinine) and anatomical pathology (renal tubular cell debris, crystal deposition, and hyperactive regeneration of renal tubular epithelium). The kidney was identified as the target organ. Oral administration at 1,400 mg/kg/day (subsequently lowered to 1,000 mg/kg/day) resulted in animal death and moribundity. There were no treatment-related findings in the 140 mg/kg/day group. There were no compound-related findings in the high-dose recovery animals. The second rat study was a 5-day oral toxicity study with genomic biomarkers assayed in the kidney tissues. At the top dose of 1,050 mg/kg/day, similar clinical and anatomical pathology findings as described above were observed. The genes measured, Kim-1, Clu, Spp1, A2m, Lcn2, Tcfrsf12a, Gpnmb, and CD44, were significantly up-regulated (fivefold to 550-fold), while Tff3 was significantly down-regulated (fivefold). These results indicated that genomic markers could sensitively diagnose melamine-induced kidney injury. A 3-month oral study with 4-week recovery in monkeys was also conducted. In this monkey study, the animals were treated with melamine at doses of 60, 200, or 700 mg/kg/day. The administration of 700 mg/kg/day melamine by nasal-gastric gavage to monkeys resulted in test article-related clinical signs including turbid and whitish urine, urine crystals, red blood cell changes, increased serum alanine aminotransferase and kidney and/or liver weights, and microscopic findings including nephrotoxicity, pericarditis, and increased hematopoiesis. Nephrotoxicity was also noted at 200 mg/kg/day. It was concluded that the kidney is the primary target organ and the NOAEL was estimated to be 140 mg/kg/day in rats following a 14-day oral administration and 60 mg/kg/day in the monkey study.

A seminal study recently demonstrated that bromide (Br-) has a critical function in the assembly of type IV collagen in basement membrane (BM), and suggested that Br- supplementation has therapeutic potential for BM diseases. Because salts of bromide (KBr and NaBr) have been used as antiepileptic drugs for several decades, repositioning of Br- for BM diseases is probable. However, the effects of Br- on glomerular basement membrane (GBM) disease such as Alport syndrome (AS) and its impact on the kidney are still unknown. In this study, we administered daily for 16 weeks 75 mg/kg or 250 mg/kg (within clinical dosage) NaBr or NaCl (control) via drinking water to 6-week-old AS mice (mouse model of X-linked AS). Treatment with 75 mg/kg NaBr had no effect on AS progression. Surprisingly, compared with 250 mg/kg NaCl, 250 mg/kg NaBr exacerbated the progressive proteinuria and increased the serum creatinine and blood urea nitrogen in AS mice. Histological analysis revealed that glomerular injury, renal inflammation and fibrosis were exacerbated in mice treated with 250 mg/kg NaBr compared with NaCl. The expressions of renal injury markers (Lcn2, Lysozyme), matrix metalloproteinase (Mmp-12), pro-inflammatory cytokines (Il-6, Il-8, Tnf-α, Il-1β) and pro-fibrotic genes (Tgf-β, Col1a1, α-Sma) were also exacerbated by 250 mg/kg NaBr treatment. Notably, the exacerbating effects of Br- were not observed in wild-type mice. These findings suggest that Br- supplementation needs to be carefully evaluated for real positive health benefits and for the absence of adverse side effects especially in GBM diseases such as AS.

Predictive biomarkers which can diagnose the onset of non-communicable diseases and the associated comorbid conditions are lacking for clinical utility. Highly sensitive and specific biomarkers for early disease detection and risk stratification may provide timely intervention to patients and prevent secondary complications. However, till the time patients are diagnosed, cellular events and biomolecules get active effecting multiple organs at the same time. This series of events lead to disruption in normal functioning of the organs and their coordinative crosstalk, hence, increase in mortality rate of patients. The primary functional molecules of inflammatory pathways are active in NCDs. YKL-40, an anti-apoptotic molecule in inflammatory pathways, is overexpressed in patient fluids in different organs under diseased conditions. We performed a preliminary network analysis to study YKL-40 co-expression with diagnostic markers: TNNT2/I3 (Cardiac Troponin T/I) for cardiovascular diseases, LCN2 (NGAL) and CKM (Creatinine kinase M-type) in acute kidney injury and HbA1c in type-2-diabetes. It is observed that YKL-40 is actively co-expressed and linked with standard diagnostic markers and may be influencing the pathways active in organ crosstalk. The pathways may be regulating the signaling events in patients with non-communicable diseases leading to comorbidities. We, hence, postulate that if YKL-40 and disease specific pathways influenced are clinically utilized, this will provide the foundation of establishing tailored and specific approach in diagnosis and monitoring non-communicable diseases and predict the onset of comorbid conditions due to phenomenon influencing organ cross talks.

Keywords: Biomarker; Comorbidity; Non-communicable diseases; Organ crosstalk; YKL-40 (CHI3L1 gene).

Feeding rats with high-fat diet (HFD) with a single streptozotocin (STZ) injection induced obesity, slightly elevated fasting blood glucose and impaired glucose and insulin tolerance, and caused cardiac hypertrophy and mild diastolic dysfunction as published before by Koncsos et al. in 2016. Here we aimed to explore the renal consequences in the same groups of rats. Male Long-Evans rats were fed normal chow (CON; n = 9) or HFD containing 40% lard and were administered STZ at 20 mg/kg (i.p.) at week four (prediabetic rats, PRED, n = 9). At week 21 blood and urine samples were taken and kidney and liver samples were collected for histology, immunohistochemistry and for analysis of gene expression. HFD and STZ increased body weight and visceral adiposity and plasma leptin concentration. Despite hyperleptinemia, plasma C-reactive protein concentration decreased in PRED rats. Immunohistochemistry revealed elevated collagen IV protein expression in the glomeruli, and Lcn2 mRNA expression increased, while Il-1β mRNA expression decreased in both the renal cortex and medulla in PRED vs. CON rats. Kidney histology, urinary protein excretion, plasma creatinine, glomerular Feret diameter, desmin protein expression, and cortical and medullary mRNA expression of TGF-β1, Nrf2, and PPARγ were similar in CON and PRED rats. Reduced AMPKα phosphorylation of the autophagy regulator Akt was the first sign of liver damage, while plasma lipid and liver enzyme concentrations were similar. In conclusion, glomerular collagen deposition and increased lipocalin-2 expression were the early signs of kidney injury, while most biomarkers of inflammation, oxidative stress and fibrosis were negative in the kidneys of obese, prediabetic rats with mild heart and liver injury.

OBJECTIVE

Chlorella vulgaris (CV) has exhibited immune-enhancing and protective activities against cancer and infections. However, there is an increasing concern about the use of Chlorella species in human, regarding its various molecules with antigenic features found in infectious microorganisms. Our goal was to investigate the impact of higher concentrations of CV on tumor growth in spontaneous mouse mammary tumor (SMMT) models.

METHODS

Balb/c mice were daily given CV powder at doses of 0, 200, or 300 mg/kg for 42 days (CONTROL, CV200, and CV300 groups, respectively; n = 6/group). On day 14, the SMMT was inoculated. Tumor volume (TV) and body weight (BW) were monitored on 5-day intervals following tumor challenge. On day 43, blood, spleen, lungs, and tumor tissues were collected. Histopathological examinations on lungs and tumor tissues were performed following hematoxylin-eosin staining. Intratumor expression of 27 genes was assessed by real-time PCR. Total IgG, IFNγ, and IL-4 levels in serum and spleen culture supernatant were measured by ELISA.

RESULTS

The TV/BW index showed significant increase in the CV200 group compared to the CONTROL (p = 0.047). The CV200 tumors exhibited more malignant phenotype, higher angiogenesis rate, and lower peritumoral neutrophil and macrophage-to-lymphocyte infiltration ratio compared to the CONTROL. Serum concentrations of IFNγ, IL-4, and IgG were declined, and the spleen IFNγ and IgG production was higher in the CV200 compared to the CONTROL. The IL-1β, IL-10, TGFβ1, FOXP3, HO-1, Gr1, CD11b, PCNA, LCN2, iNOS2, VEGFR2, CD31, and CD105L expressions were markedly increased in the CV200 tumors compared to the CONTROL (p = 0.001, 0.002, 0.006, 0.021, 0.004, 0.030, 0.016, 0.031, 0.025, 0.008, 0.014, 0.022, and 0.037, respectively). The changes in cytokine, IgG and gene expression values considerably correlated with tumor size, as well as with each other.

CONCLUSIONS

Our data provided evidence that C. vulgaris at a specific dose (200 mg/kg) promoted tumor growth in a mammary tumor model. This consequence might reflect an immune derangement in favor of developing a protumor microenvironment. However, this hypothesis needs to be further investigated in future.

Cisplatin is one of the most useful chemotherapeutics which performs its cytotoxic effect via accumulation of platinum resulting in oxidative stress, and destruction of cell DNA. This could probably cause secondary cancers in healthy tissues. Lipocalin2 (Lcn2) is a protein which its expression is increased in oxidative stresses. Therefore, the present study was performed to evaluate the protective effects of Lcn2 up-regulation on cisplatin genotoxicity. In order to up-regulate Lcn2 expression, HEK293 cells were transfected with pcDNA3.1-Lcn2 vector. Afterwards, stable cells consistently expressing Lcn2 were selected via screening with G418 antibiotic. Next, overexpression of Lcn2 was evaluated by RT-PCR and ELISA, comparing to the control non-transfected cells. Then, in order to evaluate the cytoprotective effects of Lcn2 overexpression, transfected and non-transfected cells were subjected to cisplatin treatment followed by MTT and alkaline Comet assays. RT-PCR and ELISA assays confirmed up-regulation of Lcn2 by the stable cells. MTT assay of the Lcn2 over-expressing cells showed higher IC50 values comparing to the non-transfected cells. Furthermore, the Comet assay confirmed Lcn2 protective effects on the cisplatin (1 µg/mL) induced genotoxicity. In the present study, for the first time, we showed the protective effect of Lcn2 on cisplatin induced genotoxicity. Therefore, one of the probable mechanisms of Lcn2 cytoprotctive effects under oxidative stress conditions could be due to the prevention of genotoxicity. However, further evaluations in this regard must be considered.

LCN2 (Lipocalins) was first identified as iron transporter through associating with its siderophores and also involved in many cancer metastases, but its function is still paradoxical. We questioned that whether LCN2 might also associate exogenous iron chelator as does in inherent way and the association may influence their respective function. To address this issue, we investigated the effect of LCN2 on action of DpdtC (2,2'-dipyridine ketone hydrazone dithiocarbamte), an iron chelator in proliferation and metastasis-related gene expression. The results showed that exogenous LCN2 and DpdtC could inhibit growth of HepG2 cells, while the combination treatment enhanced their inhibitory effect both in proliferation and colony formation. This encouraged us to investigate the effect of the interaction on metastasis-related gene expression. The results revealed that both LCN2 and DpdtC impaired the wound healing of HepG2, but the inhibitory effect of DpdtC was significantly enhanced upon association with LCN2. Undergoing epithelium-mesenchymal transition (EMT) is a crucial step for cancer metastasis, LCN2 and DpdtC had opposite effects on EMT markers, the binding of DpdtC to LCN2 significantly weakened the regulation of it (or its iron chelate) on EMT markers. To insight into the interaction between LCN2 and DpdtC-iron, fluorescence titration and molecular docking were performed to obtain the association constant (~ 10⁴ M^-1) and thermodynamic parameters (ΔG = - 26.10 kJ/mol). Importantly this study provided evidence that siderophores-loading state of LCN2 may influence its function, which be helpful for understanding the contradictory role of LCN2 in the metastasis of cancer.

Keywords: DpdtC; EMT; Gene regulation; Interaction; Iron chelator; LCN2.

Polyhexamethylene guanidine phosphate (PHMG-p) was used as a humidifier disinfectant in Korea. PHMG induced severe pulmonary fibrosis in Koreans. The objective of this study was to elucidate mechanism of pulmonary toxicity caused by PHMG-p in rats using multi-omics analysis. Wistar rats were intratracheally instilled with PHMG-p by single (1.5 mg/kg) administration or 4-week (0.1 mg/kg, 2 times/week) repeated administration. Histopathologic examination was performed with hematoxylin and eosin staining. Alveolar macrophage aggregation and granulomatous inflammation were observed in rats treated with single dose of PHMG-p. Pulmonary fibrosis, chronic inflammation, bronchiol-alveolar fibrosis, and metaplasia of squamous cell were observed in repeated dose group. Next generation sequencing (NGS) was performed for transcriptome profiling after mRNA isolation from bronchiol-alveoli. Bronchiol-alveoli proteomic profiling was performed using an Orbitrap Q-exactive mass spectrometer. Serum and urinary metabolites were determined using ¹H-NMR. Among 418 differentially expressed genes (DEGs) and 67 differentially expressed proteins (DEPs), changes of 16 mRNA levels were significantly correlated with changes of their protein levels in both single and repeated dose groups. Remarkable biological processes represented by both DEGs and DEPs were defense response, inflammatory response, response to stress, and immune response. Arginase 1 (Arg1) and lipocalin 2 (Lcn2) were identified to be major regulators for PHMG-p-induced pulmonary toxicity based on merged analysis using DEGs and DEPs. In metabolomics study, 52 metabolites (VIP > 0.5) were determined in serum and urine of single and repeated-dose groups. Glutamate and choline were selected as major metabolites. They were found to be major factors affecting inflammatory response in association with DEGs and DEPs. Arg1 and Lcn2 were suggested to be major gene and protein related to pulmonary damage by PHMG-p while serum or urinary glutamate and choline were endogenous metabolites related to pulmonary damage by PHMG-p.

Boron-containing mesoporous bioactive glass (B-MBG) scaffolds could be capable of promoting osteogenesis by activating Wnt/β-catenin signaling pathway during the process of bone defect repair. Despite this, more involving molecular controls are still largely unclear. In the present study, we identified that the downstream of Wnt/β-catenin signaling pathway named transcription factor 7-like 2 (TCF7L2) served as a key effector to promote boron-induced bone regeneration and osteogenesis through lipocalin 2 (LCN2). TCF7L2 was highly expressed in osteoblasts when treated with B-MBG scaffold extraction than MBG. LCN2, as a secreted bone factor, positively affected osteogenic differentiation of MC3T3-E1 and osteogenesis in vivo, which could be induced by TCF7L2. In addition, interference of TCF7L2 decreased the osteogenic differentiation of osteoblasts. Finally, we identified that rLCN2 could rescue the poor ability of osteogenic differentiation of MC3T3-E1 whose Tcf7l2 gene was knocked down by lentiviral transfection of shRNA. Our findings provide some new insights into the molecular controls of boron-associated bone regeneration and potential therapeutic targets for the treatment of bone defects.