Phytochemical constituents and anticancer activities of Tarchonanthus camphoratus essential oils grown in Saudi Arabia.

Tarchonanthus Camphoratus L. is traditionally known for its various medicinal purposes. In this study, the T. camphoratus essential oil (TCEO) was isolated via steam distillation, and its chemical constituents were determined using GC-MS. The in vitro antiproliferative effects of TCEO on A549, HepG2, MCF-7 cancer cells, and HUVEC non-tumor cells was investigated using an MTT assay. Flow cytometry analysis was conducted to evaluate cell cycle distribution using propidium iodide staining, and cell death mode using Annexin V-FITC/PI assays. The expression of some apoptosis related genes was investigated using qRT-PCR. Major constituents of TCEO included fenchol, borneol, 3-cyclohexene-1-methanol and 3-ethyl-3-methyl. Cell viability test showed that TCEO is highly effective against MCF-7 cells with IC50 12.5 µg/mL. Cell cycle arrest at the G1/S phase, and apoptosis mediation were evident in the presence of TCEO. Gene expression analysis of several pro-apoptotic and anti-apoptotic genes revealed the initiation of apoptosis in TCEO-MCF-7 cells. In conclusion, our study confirms the antiproliferative activity of the T. camphoratus essential oil.

Fat composition in infant formula contributes to the severity of necrotising enterocolitis.

Necrotising enterocolitis (NEC) is a devastating disease that typically affects formula-fed premature infants, suggesting that dietary components may influence disease pathogenesis. TAG are the major fat components of infant formula, and their digestion requires pancreatic lipases, which may be naturally deficient in premature neonates. We hypothesise that NEC develops partly from the accumulation of incompletely digested long-chain TAG-containing unsaturated fatty acids within the intestinal epithelial cells, leading to oxidative stress and enterocyte damage. We further hypothesise that the administration of a formula that contains reduced TAG ('pre-digested fat') that do not require lipase action may reduce NEC severity. To test these hypotheses, we induced NEC in neonatal mice using three different fat formulations, namely 'standard fat', 'pre-digested fat' or 'very low fat', and determined that mice fed 'standard fat' developed severe NEC, which was significantly reduced in mice fed 'pre-digested fat' or 'very low fat'. The expression level of the critical fat-digesting enzyme carboxyl ester lipase was significantly lower in the newborn compared with older pups, leading to impaired fat digestion. The accumulation of mal-digested fat resulted in the significant accumulation of fat droplets within the intestinal epithelium of the distal ileum, resulting in the generation of reactive oxygen species and intestinal inflammation. Strikingly, these changes were prevented in pups fed 'pre-digested fat' or 'very low fat' formulas. These findings suggest that nutritional formula containing a pre-digested fat system may overcome the natural lipase deficiency of the premature gut, and serve as a novel approach to prevent NEC.

Lactobacillus acidophilus modulates inflammatory activity by regulating the TLR4 and NF-κB expression in porcine peripheral blood mononuclear cells after lipopolysaccharide challenge.

A total of forty weaned pigs ((Landrace × Yorkshire) × Duroc) were used to evaluate the effects of Lactobacillus acidophilus on inflammatory activity after lipopolysaccharide (LPS) challenge. Experimental treatments were as follows: (T1) control diet+saline challenge; (T2) control diet with 0·1% L. acidophilus+saline challenge; (T3) control diet+LPS challenge; and (T4) control diet with 0·1% L. acidophilus+LPS challenge. On d-14, piglets were challenged with saline (T1 and T2) or LPS (T3 and T4). Blood samples were obtained at 0, 2, 4, 6 and 12 h after being challenged and analysed for immune cell cytokine production and gene expression pattern. The L. acidophilus treatment increased the average daily weight gain (ADWG) and average daily feed intake (ADFI) compared with the control diet. With the control diet, the LPS challenge (T3) increased the number of immune cells and expression of TNF-α and IL-6 compared with the saline challenge (T1). Whereas with the saline challenge L. acidophilus treatment (T2) increased the number of leucocytes and CD4 compared with the control diet (T1), with the LPS challenge L. acidophilus treatment (T4) decreased the number of leucocytes, lymphocytes, CD4+ and CD8+ and expression of TNF-α and IL-6 compared with the control diet (T3). L. acidophilus treatment decreased the expression of TRL4 and NF-κB in peripheral blood mononuclear cells (PBMC) after LPS challenge, which leads to inhibition of TNF-α, IFN-γ, IL-6, IL-8 and IL1B1 and to induction of IL-4 and IL-10. We suggested that L. acidophilus improved ADWG and ADFI and protected against LPS-induced inflammatory responses by regulating TLR4 and NF-κB expression in porcine PBMC.

miR-214 promotes osteoclastogenesis by targeting Pten/PI3k/Akt pathway.

microRNA is necessary for osteoclast differentiation, function and survival. It has been reported that miR-199/214 cluster plays important roles in vertebrate skeletal development and miR-214 inhibits osteoblast function by targeting ATF4. Here, we show that miR-214 is up-regulated during osteoclastogenesis from bone marrow monocytes (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) induction, which indicates that miR-214 plays a critical role in osteoclast differentiation. Overexpression of miR-214 in BMMs promotes osteoclastogenesis, whereas inhibition of miR-214 attenuates it. We further find that miR-214 functions through PI3K/Akt pathway by targeting phosphatase and tensin homolog (Pten). In vivo, osteoclast specific miR-214 transgenic mice (OC-TG214) exhibit down-regulated Pten levels, increased osteoclast activity, and reduced bone mineral density. These results reveal a crucial role of miR-214 in the differentiation of osteoclasts, which will provide a potential therapeutic target for osteoporosis.

Delivery of siRNAs against MERS-CoV in Vero and HEK-293 cells: A comparative evaluation of transfection reagents.

Background: A new coronavirus was identified in Jeddah, Saudi Arabia in 2012 and designated as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). To date, this virus has been reported in 27 countries. The virus transmission to humans has already been reported from camels. Currently, there is no vaccine or antiviral therapy available against this virus. Methods: The siRNAs were in silico predicted, designed, and chemically synthesized by using the MERS-CoV-orf1ab region as a target. The antiviral activity was experimentally evaluated by delivering the siRNAs with Lipofectamine™ 2000 and JetPRIMER as transfection reagents in both Vero cell and HEK-293-T cell lines at two different concentrations (10.0 nM and 5.0 nM). The Ct value of quantitative Real-Time PCR (qRT-PCR) was used to calculate and determine the reduction of viral RNA level in both cell supernatant and cell lysate isolated from both cell lines. Results: The sequence alignment resulted in the selection of highly conserved regions. The orf1ab region was used to predict and design the siRNAs and a total of twenty-one siRNAs were finally selected from four hundred and twenty-six siRNAs generated by online software. Inhibition of viral replication and significant reduction of viral RNA was observed against selected siRNAs in both cell lines at both concentrations. Based on the Ct value, the siRNAs # 11, 12, 18, and 20 were observed to be the best performing in both cell lines at both concentrations. Conclusion: Based on the results and data analysis, it is concluded that the use of two different transfection reagents was significantly effective. But the Lipofectamine™ 2000 was found to be a better transfection reagent than the JetPRIMER for the delivery of siRNAs in both cell lines.

Exosomes from Human Omental Adipose-Derived Mesenchymal Stem Cells Secreted into Ascites Promote Peritoneal Metastasis of Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) patients frequently develop peritoneal metastasis, especially in the human omentum. However, the mechanism underlying this propensity remains unknown. A previous study found that human omental adipose-derived mesenchymal stem cells are potentially involved in ovarian cancer growth and metastasis, but the results were inconsistent and even contradictory. In addition, the underlying mechanisms of visceral adipose metastasis remain poorly understood. Here, our goal is to clarify the role and mechanism of human omental adipose-derived mesenchymal stem cells (HO-ADSCs) in EOC cancer growth and metastasis. We first found that human omental tissue conditioned medium (HO-CM) enhances EOC cell function. Subsequent coculture studies indicated that HO-ADSCs increase the growth, migratory and invasive capabilities of ovarian cancer cells. Then, we demonstrated that exosomes secreted by HO-ADSCs (HO-ADSC exosomes) enhanced ovarian cancer cell function, and further mechanistic studies showed that the FOXM1, Cyclin F, KIF20A, and MAPK signaling pathways were involved in this process. In addition, subcutaneous tumorigenesis and peritoneal metastatic xenograft experiments provided evidence that HO-ADSC exosomes promote ovarian cancer growth and metastasis in vivo. Finally, our clinical studies provided evidence that ascites from ovarian cancer patients enhance EOC cell line proliferation, migration, and invasion in vitro. The present study indicated that HO-ADSC exosomes are secreted into ascites and exert a tumor-promoting effect on EOC growth and metastasis, providing a new perspective and method to develop future novel therapeutic strategies for the treatment of ovarian cancer.

ROCK2 inhibition attenuates profibrogenic immune cell function to reverse thioacetamide-induced liver fibrosis.

BACKGROUND & AIMS: Fibrosis, the primary cause of morbidity in chronic liver disease, is induced by pro-inflammatory cytokines, immune cell infiltrates, and tissue resident cells that drive excessive myofibroblast activation, collagen production, and tissue scarring. Rho-associated kinase 2 (ROCK2) regulates key pro-fibrotic pathways involved in both inflammatory reactions and altered extracellular matrix remodelling, implicating this pathway as a potential therapeutic target. METHODS: We used the thioacetamide-induced liver fibrosis model to examine the efficacy of administration of the selective ROCK2 inhibitor KD025 to prevent or treat liver fibrosis and its impact on immune composition and function. RESULTS: Prophylactic and therapeutic administration of KD025 effectively attenuated thioacetamide-induced liver fibrosis and promoted fibrotic regression. KD025 treatment inhibited liver macrophage tumour necrosis factor production and disrupted the macrophage niche within fibrotic septae. ROCK2 targeting in vitro directly regulated macrophage function through disruption of signal transducer and activator of transcription 3 (STAT3)/cofilin signalling pathways leading to the inhibition of pro-inflammatory cytokine production and macrophage migration. In vivo, KDO25 administration significantly reduced STAT3 phosphorylation and cofilin levels in the liver. Additionally, livers exhibited robust downregulation of immune cell infiltrates and diminished levels of retinoic acid receptor-related orphan receptor gamma (RORγt) and B-cell lymphoma 6 (Bcl6) transcription factors that correlated with a significant reduction in liver IL-17, splenic germinal centre numbers and serum IgG. CONCLUSIONS: As IL-17 and IgG-Fc binding promote pathogenic macrophage differentiation, together our data demonstrate that ROCK2 inhibition prevents and reverses liver fibrosis through direct and indirect effects on macrophage function and highlight the therapeutic potential of ROCK2 inhibition in liver fibrosis. LAY SUMMARY: By using a clinic-ready small-molecule inhibitor, we demonstrate that selective ROCK2 inhibition prevents and reverses hepatic fibrosis through its pleiotropic effects on pro-inflammatory immune cell function. We show that ROCK2 mediates increased IL-17 production, antibody production, and macrophage dysregulation, which together drive fibrogenesis in a model of chemical-induced liver fibrosis. Therefore, in this study, we not only highlight the therapeutic potential of ROCK2 targeting in chronic liver disease but also provide previously undocumented insights into our understanding of cellular and molecular pathways driving the liver fibrosis pathology.

Immunomodulatory receptor VSIG4 is released during spontaneous bacterial peritonitis and predicts short-term mortality.

BACKGROUND & AIMS: V-set Ig-domain-containing 4 (VSIG4) is an immunomodulatory macrophage complement receptor modulating innate and adaptive immunity and affecting the resolution of bacterial infections. Given its expression on peritoneal macrophages (PMs), we hypothesised a prognostic role of peritoneal VSIG4 concentrations in patients with spontaneous bacterial peritonitis (SBP). METHODS: We isolated PMs from patients with cirrhosis and analysed VSIG4 expression and release by flow cytometry, quantitative real-time PCR, ELISA, and confocal microscopy. We measured soluble VSIG4 concentrations in ascites from 120 patients with SBP and 40 patients without SBP and investigated the association of soluble VSIG4 in ascites with 90-day survival after SBP using Kaplan-Meier statistics, Cox regression, and competing-risks regression analysis. RESULTS: VSIG4 expression was high on resting, large PMs, which co-expressed CD206, CD163, and tyrosine-protein kinase Mer (MERTK). VSIG4 gene expression in PMs decreased in patients with SBP and normalised after resolution. During SBP, VSIG4hi PMs were depleted (25% vs. 57%; p <0.001) and soluble VSIG4 in ascites were higher in patients with SBP than in patients without (0.73 vs. 0.35 µg/ml; p <0.0001). PM activation by Toll-like receptor (TLR) agonists or infection with live bacteria in vitro resulted in a loss of surface VSIG4 and the release of soluble VSIG4. Mechanistically, shedding of VSIG4 from PMs was protease-dependent and susceptible to microtubule transport inhibition. Soluble VSIG4 in ascites exceeded serum concentrations and correlated with serum creatinine, model for end-stage liver disease score and C-reactive protein during SBP. Concentrations of 1.0206 µg/ml or higher indicated increased 90-day mortality (hazard ratio 1.70; 95% CI 1.01-2.86; p = 0.046). CONCLUSIONS: VSIG4 is released from activated PMs into ascites during SBP. Higher peritoneal VSIG4 levels indicate patients with organ failure and poor prognosis. LAY SUMMARY: Patients with liver cirrhosis who develop ascites have an increased risk of infection and mortality. Our study shows that in patients with infected ascites, the complement receptor VSIG4 is released by resident macrophages into the abdominal fluid where it can be measured. Patients with elevated levels of this protein in ascites are at high risk of dying within 90 days.

Antidepressant-like effect of ginsenoside Rb1 on potentiating synaptic plasticity via the miR-134-mediated BDNF signaling pathway in a mouse model of chronic stress-induced depression.

Background: Brain-derived neurotrophic factor (BDNF)-tropomyosin-related kinase B (TrkB) plays a critical role in the pathogenesis of depression by modulating synaptic structural remodeling and functional transmission. Previously, we have demonstrated that the ginsenoside Rb1 (Rb1) presents a novel antidepressant-like effect via BDNF-TrkB signaling in the hippocampus of chronic unpredictable mild stress (CUMS)-exposed mice. However, the underlying mechanism through which Rb1 counteracts stress-induced aberrant hippocampal synaptic plasticity via BDNF-TrkB signaling remains elusive. Methods: We focused on hippocampal microRNAs (miRNAs) that could directly bind to BDNF and are regulated by Rb1 to explore the possible synaptic plasticity-dependent mechanism of Rb1, which affords protection against CUMS-induced depression-like effects. Results: Herein, we observed that brain-specific miRNA-134 (miR-134) could directly bind to BDNF 3'UTR and was markedly downregulated by Rb1 in the hippocampus of CUMS-exposed mice. Furthermore, the hippocampus-targeted miR-134 overexpression substantially blocked the antidepressant-like effects of Rb1 during behavioral tests, attenuating the effects on neuronal nuclei-immunoreactive neurons, the density of dendritic spines, synaptic ultrastructure, long-term potentiation, and expression of synapse-associated proteins and BDNF-TrkB signaling proteins in the hippocampus of CUMS-exposed mice. Conclusion: These data provide strong evidence that Rb1 rescued CUMS-induced depression-like effects by modulating hippocampal synaptic plasticity via the miR-134-mediated BDNF signaling pathway.

Organophosphate esters cause thyroid dysfunction via multiple signaling pathways in zebrafish brain.

Organophosphate esters (OPEs) are widespread in various environmental media, and can disrupt thyroid endocrine signaling pathways. Mechanisms by which OPEs disrupt thyroid hormone (TH) signal transduction are not fully understood. Here, we present in vivo-in vitro-in silico evidence establishing OPEs as environmental THs competitively entering the brain to inhibit growth of zebrafish via multiple signaling pathways. OPEs can bind to transthyretin (TTR) and thyroxine-binding globulin, thereby affecting the transport of TH in the blood, and to the brain by TTR through the blood-brain barrier. When GH3 cells were exposed to OPEs, cell proliferation was significantly inhibited given that OPEs are competitive inhibitors of TH. Cresyl diphenyl phosphate was shown to be an effective antagonist of TH. Chronic exposure to OPEs significantly inhibited the growth of zebrafish by interfering with thyroperoxidase and thyroglobulin to inhibit TH synthesis. Based on comparisons of modulations of gene expression with the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases, signaling pathways related to thyroid endocrine functions, such as receptor-ligand binding and regulation of hormone levels, were identified as being affected by exposure to OPEs. Effects were also associated with the biosynthesis and metabolism of lipids, and neuroactive ligand-receptor interactions. These findings provide a comprehensive understanding of the mechanisms by which OPEs disrupt thyroid pathways in zebrafish.

Identification of ferroptosis as a novel mechanism for antitumor activity of natural product derivative a2 in gastric cancer.

Ferroptosis is a type of cell death accompanied by iron-dependent lipid peroxidation, thus stimulating ferroptosis may be a potential strategy for treating gastric cancer, therapeutic agents against which are urgently required. Jiyuan oridonin A (JDA) is a natural compound isolated from Jiyuan Rabdosia rubescens with anti-tumor activity, unclear anti-tumor mechanisms and limited water solubility hamper its clinical application. Here, we showed a2, a new JDA derivative, inhibited the growth of gastric cancer cells. Subsequently, we discovered for the first time that a2 induced ferroptosis. Importantly, compound a2 decreased GPX4 expression and overexpressing GPX4 antagonized the anti-proliferative activity of a2. Furthermore, we demonstrated that a2 caused ferrous iron accumulation through the autophagy pathway, prevention of which rescued a2 induced ferrous iron elevation and cell growth inhibition. Moreover, a2 exhibited more potent anti-cancer activity than 5-fluorouracil in gastric cancer cell line-derived xenograft mice models. Patient-derived tumor xenograft models from different patients displayed varied sensitivity to a2, and GPX4 downregulation indicated the sensitivity of tumors to a2. Finally, a2 exhibited well pharmacokinetic characteristics. Overall, our data suggest that inducing ferroptosis is the major mechanism mediating anti-tumor activity of a2, and a2 will hopefully serve as a promising compound for gastric cancer treatment.

Study on RNAi-based herbicide for Mikania micrantha.

The invasive plant Mikania micrantha Kunth (M. micrantha) from South America poses a significant threat to the stability and biodiversity of ecosystems. However, an effective and economical method to control M. micrantha is still lacking. RNA interference (RNAi) has been widely studied and applied in agriculture for trait improvement. Spray-induced gene silencing (SIGS) can produce RNAi silencing effects without introducing heritable modifications to the plant genome and is becoming a novel nontransformation strategy for plant protection. In this study, the genes encoding chlorophyll a/b-binding proteins were selected as targets of RNAi, based on high-throughput sequencing of M. micrantha transcriptome and bioinformatic analyses of sequence specificity. Three types of RNAi molecules, double-stranded RNA, RNAi nanomicrosphere, and short hairpin RNA (shRNA), with their corresponding short interfering RNA sequences were designed and synthesized for SIGS vector construction, from which each RNAi molecule was transcribed and extracted to be sprayed on M. micrantha leaves. Whereas water-treated control leaves remained green, leaves treated with RNAi molecules turned yellow and eventually wilted. Quantitative real-time PCR showed that the expression levels of target genes were significantly reduced in the RNAi-treated groups compared with those of the control, suggesting that all three types of RNAi herbicides effectively silenced the endogenous target genes, which are essential for the growth of M. micrantha. We also found that shRNA showed better silencing efficiency than the other two molecules. Taken together, our study successfully designed three types of RNAi-based herbicides that specifically silenced endogenous target genes and controlled the growth of M. micrantha. Moreover, we identified a gene family encoding chlorophyll a/b-binding proteins that is important for the growth and development of M. micrantha and could serve as potential targets for controlling the spread of M. micrantha.

Anti-tumor activities of Panax quinquefolius saponins and potential biomarkers in prostate cancer.

BACKGROUND: Prostate carcinoma is the second most common cancer among men worldwide. Developing new therapeutic approaches and diagnostic biomarkers for prostate cancer (PC) is a significant need. The Chinese herbal medicine Panax quinquefolius saponins (PQS) have been reported to show anti-tumor effects. We hypothesized that PQS exhibits anti-cancer activity in human PC cells and we aimed to search for novel biomarkers allowing early diagnosis of PC. METHODS: We used the human PC cell line DU145 and the prostate epithelial cell line PNT2 to perform cell viability assays, flow cytometric analysis of the cell cycle, and FACS-based apoptosis assays. Microarray-based gene expression analysis was used to display specific gene expression patterns and to search for novel biomarkers. Western blot and quantitative real-time PCR were performed to demonstrate the expression levels of multiple cancer-related genes. RESULTS: Our data showed that PQS inhibited the viability of DU145 cells and induced cell cycle arrest at the G1 phase. A significant decrease in DU145 cell invasion and migration were observed after 24 h treatment by PQS. PQS up-regulated the expression levels of p21, p53, TMEM79, ACOXL, ETV5, and SPINT1 while it down-regulated the expression levels of bcl2, STAT3, FANCD2, DRD2, and TMPRSS2. CONCLUSION: PQS promoted cells apoptosis and inhibited the proliferation of DU145 cells, which suggests that PQS may be effective for treating PC. TMEM79 and ACOXL were expressed significantly higher in PNT2 than in DU145 cells and could be novel biomarker candidates for PC diagnosis.

Discovery of the anti-angiogenesis effect of eltrombopag in breast cancer through targeting of HuR protein.

HuR (human antigen R), an mRNA-binding protein responsible for poor prognosis in nearly all kinds of malignancies, is a potential anti-tumor target for drug development. While screening HuR inhibitors with a fluorescence polarization (FP) based high-throughput screening (HTS) system, the clinically used drug eltrombopag was identified. Activity of eltrombopag on molecular level was verified with FP, electrophoretic mobility shift assay (EMSA), simulation docking and surface plasmon resonance (SPR). Further, we showed that eltrombopag inhibited in vitro cell proliferation of multiple cancer cell lines and macrophages, and the in vivo anti-tumor activity was also demonstrated in a 4T1 tumor-bearing mouse model. The in vivo data showed that eltrombopag was efficient in reducing microvessels in tumor tissues. We then confirmed the HuR-dependent anti-angiogenesis effect of eltrombopag in 4T1 cells and RAW264.7 macrophages with qRT-PCR, HuR-overexpression and HuR-silencing assays, RNA stability assays, RNA immunoprecipitation and luciferase assays. Finally, we analyzed the in vitro anti-angiogenesis effect of eltrombopag on human umbilical vein endothelial cells (HUVECs) mediated by macrophages with cell scratch assay and in vitro Matrigel angiogenesis assay. With these data, we revealed the HuR-dependent anti-angiogenesis effect of eltrombopag in breast tumor, suggesting that the existing drug eltrombopag may be used as an anti-cancer drug.

LncRNA MALAT1 promotes wound healing via regulating miR-141-3p/ZNF217 axis.

BACKGROUND: The process of wound healing is complex. Increasing evidences have shown that lncRNA MALAT1 is abundant in fibroblasts and may be engaged in wound healing process. Therefore, we explored the mechanism of MALAT1 affecting wound healing. METHODS: The expression levels of MALAT1, miR-141-3p as well as ZNF217 in human fibroblast cells (HFF-1) were quantified by qRT-PCR. HFF-1 proliferation was measured by MTT, while migration was detected by wound healing assay. SMAD2 activation and matrix proteins expression were detected by western blotting. The interaction between miR-141-3p and MALAT1 or ZNF217 was further confirmed using the luciferase reporter gene assay. In vivo wound healing was assessed by full-thickness wound healing model on C57BL/6 mice. RESULT: Knockdown of MALAT1 as well as overexpression miR-141-3p remarkably inhibited the proliferation, migration and matrix protein expression in HFF-1 cells. MALAT1 directly targeted and inhibited the expression of miR-141-3p. MiR-141-3p suppressed the activation of TGF-ß2/SMAD2 signaling pathway by targeting ZNF217. Knockdown of MALAT1 inhibited wound healing process in mice. CONCLUSIONS: MALAT1 up-regulates ZNF217 expression by targeting miR-141-3p, thus enhances the activity of TGF-ß2/SMAD2 signaling pathway and promotes wound healing process. This investigation shed new light on the understanding of the role of MALAT1 in wound healing, and may provide potential target for the diagnosis or therapy of chronic wounds.

Protective effect of piperine in ischemia-reperfusion induced acute kidney injury through inhibition of inflammation and oxidative stress.

BACKGROUND AND AIM: Renal ischemia-reperfusion is associated with inflammation and oxidative stress. As a major compound in black pepper, piperine has anti-inflammatory and anti-oxidative properties. In present study, the protective effects of oral administration of piperine in renal ischemia-reperfusion (IR) induced acute kidney injuries (AKI) were investigated. EXPERIMENTAL PROCEDURE: Male Wistar rats received piperine (10 or 20 mg/kg.bw) or vehicle for 10 days. The artery and vein of both kidneys were then clamped for 30 min, followed by a 24-h reperfusion period. Concentrations of creatinine and urea-nitrogen in descending aorta blood were measured, and malondialdehyde (MDA) and ferric reducing/antioxidant power (FRAP) levels were measured in kidney tissue to evaluate the oxidative stress. Inflammation was evaluated by measuring the TNF-α and ICAM-1 mRNA expression levels in renal cortical tissue using Real Time PCR method and counting leukocytes infiltration to interstitium. Further measured were tissue damages in H & E stained sections. RESULTS: Renal IR reduced FRAP, while increasing the plasma concentrations of creatinine and urea-nitrogen, tissue MDA level, TNF-α and ICAM-1 mRNA expressions, leukocyte infiltration and histopathologic injuries. Piperine administration significantly reduced the plasma concentrations of creatinine and urea-nitrogen, expression of pro-inflammatory factors, oxidative stress and renal histopathologic injuries. It is to be noted that 20 mg/kg dose was more effective. CONCLUSION: Our results suggest piperine protects the kidney against ischemia-reperfusion induced acute kidney injuries by its anti-inflammatory and anti-oxidative properties.

The mechanism of lipopolysaccharide administration-induced cognitive function impairment caused by glucose metabolism disorder in adult rats.

This essay aims to make investigation on the mechanism of glucose metabolism disorder and Lipopolysaccharide administration-induced cognitive function impairment in adult rats with surgery. METHODS: Divide the objects, 40 male Sprague-Dawley rats at the age of 9 months, into 4 groups. Provide unilateral nephrectomy surgery and/or lipopolysaccharide intraperitoneal injection. Postoperative cognitive function evaluation would be tested by the Morris water maze. Rats with Postoperative Cognitive Dysfunction (POCD) were scanned to analyze the brain glucose metabolism by means of 18F-FDG PET/CT. Phosphatidylinositol 3-Kinase (PI3K), Protein Kinase ß (AKT), Insulin Substrates Receptor-2 (IRS-2) and Glucose Transporter 4 (GLUT4) were detected as well. Data will be captured through gene expression in POCD rats via Quantitative Real-Time PCR (QRT-PCR). On the other side, Western Blot was used to measure the expression levels of IRS-2, p-IRS-2, p-PI3K, PI3K, p-AKT, AKT, GLUT4, and p-GLUT4. RESULTS: During the Morris water maze test, the staging time (latency) of rats in each group was becoming short gradually as the training progressed. The incubation time of Day 5 of each group was shorter than that of Day 1 (P < 0.05). On the Day 3 after the surgery, the average target quadrant residence time of Group S+L (100 µg/Kg) was shorter, compared with Group C, L and S. Of which, the average number of perforation was reduced greater than that of Group C (P < 0.05). The average swimming speed of the groups is of no distinct difference (P > 0.05). After the operation, there was no great difference shown among the subjects (P > 0.05) in the average residence time of the target quadrant, the mean number of passages, and the mean swimming speed. On Day 3, the average latency of Group S+L (100 µg/Kg) was longer than Group C (P < 0.05) in the working memory test after the operation. The average latency of rats in Group L and S was showed longer than that in Group C, with tiny difference (P > 0.05). In the 7-Day working memory test, the average latency of the rats in Group L, S and S+L (100 µg/Kg) was obviously longer than that in Group C. Comparing to preoperative rats, POCD rats of Group S+L (100 µg/Kg) were scanned by 18F-FDG PET/CT three days later after the operation. Its SUVmax of the frontal and temporal lobe areas were decreased significantly (P < 0.05). However, difference degree was not significantly shown in the SUVmax between Group C and the preoperative rats (P > 0.05). In comparison with the gene expression of of Group C, the PI3K, IRS-2, AKT and GLUT4 mRNA genes are the key genes in the insulin signaling pathways of the hippocampus of the POCD rats. The expression level was reduced. The expression level of all protein of PI3K, IRS-2, GLUT4 and AKT in the POCD rats was of no great contrast with that in Group C. But for IRS-2 protein, the phosphorylation level has increased, and meanwhile decreased for AKT, PI3K and GLUT4 proteins (P < 0.05). CONCLUSIONS: Adult SD rats cognitive dysfunction model treated with unilateral nephrectomy combined and 100 µg/kg LPS intraperitoneal injection were led to abnormal both brain glucose metabolism and insulin expression. The proved phenomenal results signal pathway-related proteins PI3K, IRS-2, AKT and GLUT4. It reached the conclusion that surgical trauma, rather than anesthesia, leads to impaired cognitive function. PI3K, IRS-2, AKT, and GLUT4pathway of brain can be partial explanations of the pathogenesis of POCD.

Evaluation of BCL2 and TNFα as mRNA biomarkers for monitoring the immune response in critically ill children.

BACKGROUND: Hospital acquired infection (HAI) and multiple organ dysfunctions (MODS) remain a leading cause of death in pediatric intensive care unit (PICU) despite the great efforts to control it. OBJECTIVE: Our objective was to assess the mRNA of TNFα and BCL2 for prediction of HAI and/or MODS in our community. PATIENTS AND METHODS: Fifty children, admitted to PICU, were included in the study after exclusion of cases of end-stage renal failure, end-stage liver failure and congenital immune deficiency. Serial Blood samples were collected for complete blood count (CBC) and other routine investigations. Gene expression of (TNFα and BCL2) was quantified using quantitative real time PCR (qRT-PCR). Centers of disease control (CDC) criteria were used to detect HAI, and organ failure index (OFI). Pediatric logistic organ dysfunction (PELOD) and pediatric risk of mortality (PRISM) scores were used for follow up. The results were compared between the group who acquired HAI and who didn't. Gene expression was tested with a ROC curve to detect its ability to predict HAI. MAIN RESULTS: The overall complication (HAI and/or MODS) rate was 52%, Complicated cases had a significantly longer duration of stay in PICU (0.002) and in overall hospital stay (p = 0.013) and a higher death rate (p = 0.000). On day1; TNFα, BCL2 and lymphocytic count were lower in patients who developed complications (p = 0.02, p = 0.000 and p = 0.04, respectively), all had the ability to predict the complications with AUC (0.7, 0.8 and 0.67 respectively). On day 4: TNFα and BCL2 returned to normal levels while the lymphocytic count still lower in complicated cases, p = 0.001 and AUC = 0.73. CONCLUSIONS: TNFα and BCL2 on admission can predict HAI and MODS (AUC = 0.7 and AUC = 0.8), but were of no use in the follow-up, however, the lymphocytic count is a rapid, easy and cheap test to assess the immune state with a good predictive and follow up values.

Knockdown of long non-coding RNA CCAT2 suppressed proliferation and migration of glioma cells.

Long non-coding RNA colon cancer-associated transcript 2 (CCAT2) is commonly investigated in a number of cancers. However, little is known of its expression and biological function in glioma biology. In the current study, we used quantitative real-time PCR (qRT-PCR) to determine the expression of CCAT2 in glioma tissues. We found that expression of CCAT2 was up-regulated in glioma tissues and significantly correlated with the advanced tumor stage (III/IV). Functional assays in vitro and in vivo demonstrated that knockdown of CCAT2 could inhibit proliferation, cell cycle progression and migration of glioma cells. Further analysis indicated the effect of CCAT2 knockdown on glioma cell phenotype through inhibiting Wnt/ß-catenin signal pathway activity. Thus, our study provides evidence that CCAT2 may function as a potential biomarker for glioma.

Identification of secretaglobin Scgb2a1 as a target for developmental reprogramming by BPA in the rat prostate.

Secretoglobins are a superfamily of secreted proteins thought to participate in inflammation, tissue repair, and tumorigenesis. Secretoglobin family 2A member 1 (Scgb2a1) is a component of prostatein, a major androgen-binding protein secreted by the rat prostate. Using a rat model for developmental reprogramming of susceptibility to prostate carcinogenesis, we identified, by RNA-seq, that Scgb2a1 is significantly upregulated (>100-fold) in the prostate of adult rats neonatally exposed to bisphenol A (BPA), with increased gene expression confirmed by quantitative RT-PCR and chromatin immunoprecipitation for histone H3 lysine 9 acetylation. Bisulfite analysis of both CpG islands located within 10 kb of the Scgb2a1 promoter identified significant hypomethylation of the CpG island upstream of the transcription start site of this gene in the reprogrammed prostate. These data suggest that expression of Scgb2a1 in the adult prostate could be epigenetically reprogrammed by BPA exposure during prostate development, with potential implications for cancer risk and response to chemotherapeutics associated with prostatein binding.