Custodiol® Supplemented with Synthetic Human Relaxin Decreases Ischemia-Reperfusion Injury after Porcine Kidney Transplantation.

Objective. Ischemia-reperfusion injury (IRI) is inevitable after kidney transplantation (KT), impairing outcomes. Relaxin-2 (RLX) is a promising insulin-related peptide hormone that protects against renal IRI in rodents, although large animal models are needed before RLX can be tested in a human setting. Methods. In this blinded, randomized, and placebo-controlled experimental study kidneys from 19 donor pigs were retrieved after perfusion with Custodiol® ± RLX (5 or 20 nmol/L) and underwent static cold storage (SCS) for 24 and 48 h, respectively. Subsequently, KT was performed after unilateral right nephrectomy. Study outcomes included markers for kidney function, oxidative stress, lipid peroxidation, and endothelial cell damage. PCR analysis for oxidative stress and apoptosis-related gene panels as well as immunohistochemistry were performed. Results. RLX upregulated SOD2 and NFKB expression to 135% (p = 0.042) and 125% (p = 0.019), respectively, while RIPK1 expression was downregulated to 82% (p = 0.016) of corresponding controls. Further RLX significantly downregulated RIPK1 and MLKL expression and decreased the number of Caspase 3- and MPO-positive cells in grafts after SCS. Conclusions. RLX supplemented Custodiol® significantly decreased IRI via both antioxidant and anti-apoptotic mechanisms. Clinical trials are warranted to implement synthetic human RLX as a novel additive to preservation solutions against IRI.

Curcuma longa L. Effects on Akt/mTOR Pathway and NF-κB Expression During Skin Wound Healing: An Immunohistochemical Study.

Skin ulcers, wounds, or burns represent a burden for health care worldwide. Our aim was to explore the effects of mucoadhesive formulation with Curcuma longa L. extract mucoadhesive formulation containing curcumin (MFC) on skin healing in Wistar rats. Fifty-four rats were randomly allocated into 3 groups: control, vehicle, and MFC. A full-thickness circular wound was induced on the back of each animal. Two daily applications of the products were performed according to the experimental group. On days 3, 10, and 21, 6 animals in each group were euthanized. Clinical analysis was based on wound area. Histologic analysis was performed in hematoxylin and eosin-stained sections, with re-epithelization and inflammation being assessed by means of semiquantitative scores. To analyze the Akt/mTOR pathway, immunohistochemistry for phospho Akt (pAkt) and phospho ribosomal protein S6 were investigated. In addition, nuclear factor kappa-light-chain-enhancer of activated B cells immunolabeling was performed. Clinical analysis revealed wounds with a smaller area on days 3 and 10 in curcumin-treated animals. Histologically, MFC had a significant impact on inflammatory events on days 3 and 10 and promoted faster re-epithelization, which was evidenced on day 10. MFC-treated wounds exhibited pAkt upregulation on day 10 and both pAkt and phospho ribosomal protein S6 downregulation on day 21. Nuclear factor kappa-light-chain-enhancer of activated B cells expression varied through the evaluation periods; however, no significant difference was observed between groups. Collectively, our results indicate that MFC is efficient in accelerating cutaneous wound repair through modulation of the inflammatory process and stimulus of re-epithelization by an Akt/mTOR-dependent mechanism.

Neuroprotective Effects of Telmisartan and Nifedipine Against Cuprizone-Induced Demyelination and Behavioral Dysfunction in Mice: Roles of NF-κB and Nrf2.

Multiple sclerosis is a chronic inflammatory neurodegenerative disease of the central nervous system which injures the myelin sheath. Telmisartan and nifedipine are antihypertensive drugs that recently showed neuroprotective properties against neurodegenerative diseases. This study evaluated the neuroprotective effect of telmisartan or nifedipine in cuprizone-induced demyelination in mice by examining the underlying mechanisms. C57BL/6 mice received a diet containing 0.7% (w/w) cuprizone for 7 days followed by 3 weeks on a 0.2% cuprizone diet. Telmisartan (5 mg/kg/day, p.o.) or nifedipine (5 mg/kg/day, p.o.) was administered for 3 weeks starting from the second week. Telmisartan or nifedipine improved locomotor activity and enhanced motor coordination as demonstrated by open field, rotarod, and grip strength tests. Furthermore, telmisartan or nifedipine restored myelin basic protein mRNA and protein expression and increased luxol fast blue-staining intensity. Telmisartan or nifedipine attenuated cuprizone-induced oxidative stress and apoptosis by decreasing brain malondialdehyde and caspase-3 along with restoring reduced glutathione and brain-derived neurotrophic factor levels. Telmisartan or nifedipine exerted an anti-inflammatory effect by reducing the expression of nuclear factor kappa B (NF-κB p65) as well as pro-inflammatory cytokines and elevating the expression of IκB-α. In parallel, telmisartan or nifedipine upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and the levels of heme oxygenase-1 and NADPH quinone oxidoreductase 1 enzymes. In conclusion, the current study provides evidence for the protective effect of telmisartan and nifedipine in cuprizone-induced demyelination and behavioral dysfunction in mice possibly by modulating NF-κB and Nrf2 signaling pathways.

Protective Effect of Vitamin D3 Against Pb-Induced Neurotoxicity by Regulating the Nrf2 and NF-κB Pathways.

Lead (Pb) is a known toxic heavy metal which accumulates in different tissues and causes oxidative stress (OS) and inflammation. The brain tissue is considered as one of the most vulnerable organs to the Pb-induced toxicity. The aim of this study was to investigate the therapeutic effects of vitamin D3 (VD) supplementation against the damages caused by chronic Pb toxicity in the cerebral cortex. Forty Wistar rats were divided into four equal groups and were treated as follows: control group received no treatment, VD group received 1000 IU/kg of VD by intramuscular injection every other day, Pb group received 1000 mg/L of Pb in drinking water, and Pb + VD group received VD and Pb simultaneously. The experiment lasted for 4 weeks and the analyses were conducted 24 h after the last administrations. The obtained results demonstrated that Pb significantly increased cortical lipid peroxidation and reactive oxygen species (ROS) levels. At the same time, there was a significant reduction in glutathione (GSH) content, catalase (CAT), and superoxide dismutase (SOD) activities, as well as a significant increase in the tissue level of inflammatory cytokines. Furthermore, Pb increased the messenger RNA (mRNA) expression level of nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor-kappa B (NF-κB). Anyhow, VD administration during the period of Pb exposure suppressed the OS and inflammation by increasing the antioxidant molecules and decreasing the inflammatory cytokines and consequently repaired Pb-induced cortical tissue damages. Remarkably, these responses were concomitant with the alterations in Nrf2 and NF-κB gene expressions. In conclusion, the present study discloses the potential protective effects for VD against Pb-induced neurotoxicity via anti-inflammatory and antioxidative mechanisms.

Mid-dose losartan mitigates diabetes-induced hepatic damage by regulating iNOS, eNOS, VEGF, and NF-κB expressions

Background/aim: Losartan, an antihypertensive drug, is highly preferred in patients with diabetes mellitus (DM) and hypertension because of its retarding effect on diabetic nephropathy. In this study, we investigated the potential therapeutic effect of different doses of losartan on hepatic damage in a streptozotocin (STZ, 50 mg/kg)-induced DM model in rats. Materials and methods: In this study, five different groups were formed: control, DM, low-dose losartan (5 mg/kg), mid-dose losartan (20 mg/kg), and high-dose losartan (80 mg/kg). Liver tissues of experimental groups were evaluated immunohistochemically for TUNEL, iNOS, eNOS, VEGF, and NF-κB pathways. In addition to immunohistochemical analysis, analyses of SOD and MDA, which are oxidative stress markers, were also performed and the results were evaluated together. Results: When biochemical and immunohistochemical findings were evaluated together, it was found that the results obtained from the mid-dose losartan group were closer to those of the control than the other groups. Conclusion: This study indicated that mid-dose losartan administration may have a therapeutic effect by inhibiting apoptosis and regulating iNOS, eNOS, VEGF, and NF-κB protein expressions in DM-induced hepatic damage.

Identification of Celastramycin as a Novel Therapeutic Agent for Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) accompanying increased production of inflammatory factors and adaptation of the mitochondrial metabolism to a hyperproliferative state. However, all the drugs in clinical use target pulmonary vascular dilatation, which may not be effective for patients with advanced PAH. OBJECTIVE: We aimed to discover a novel drug for PAH that inhibits PASMC proliferation. METHODS AND RESULTS: We screened 5562 compounds from original library using high-throughput screening system to discover compounds which inhibit proliferation of PASMCs from patients with PAH (PAH-PASMCs). We found that celastramycin, a benzoyl pyrrole-type compound originally found in a bacteria extract, inhibited the proliferation of PAH-PASMCs in a dose-dependent manner with relatively small effects on PASMCs from healthy donors. Then, we made 25 analogs of celastramycin and selected the lead compound, which significantly inhibited cell proliferation of PAH-PASMCs and reduced cytosolic reactive oxygen species levels. Mechanistic analysis demonstrated that celastramycin reduced the protein levels of HIF-1α (hypoxia-inducible factor 1α), which impairs aerobic metabolism, and κB (nuclear factor-κB), which induces proinflammatory signals, in PAH-PASMCs, leading to reduced secretion of inflammatory cytokine. Importantly, celastramycin treatment reduced reactive oxygen species levels in PAH-PASMCs with increased protein levels of Nrf2 (nuclear factor erythroid 2-related factor 2), a master regulator of cellular response against oxidative stress. Furthermore, celastramycin treatment improved mitochondrial energy metabolism with recovered mitochondrial network formation in PAH-PASMCs. Moreover, these celastramycin-mediated effects were regulated by ZFC3H1 (zinc finger C3H1 domain-containing protein), a binding partner of celastramycin. Finally, celastramycin treatment ameliorated pulmonary hypertension in 3 experimental animal models, accompanied by reduced inflammatory changes in the lungs. CONCLUSIONS: These results indicate that celastramycin ameliorates pulmonary hypertension, reducing excessive proliferation of PAH-PASMCs with less inflammation and reactive oxygen species levels, and recovered mitochondrial energy metabolism. Thus, celastramycin is a novel drug for PAH that targets antiproliferative effects on PAH-PASMCs.

Evodiamine prevents dextran sulfate sodium-induced murine experimental colitis via the regulation of NF-κB and NLRP3 inflammasome.

Evodiamine (EVO), an extraction from the traditional Chinese medicine Evodia rutaecarpa, has been reported to possess anti-inflammatory, anti-tumor and other pharmacological activities. However, the effectiveness of EVO to relieve dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) has not been evaluated. In this study, the protective effects and mechanisms of EVO on DSS-induced UC mice were investigated. The results indicated that treatment with EVO ameliorated DSS-induced UC mice body weight loss, disease activity index (DAI), colon length shortening, colonic pathological damage, and myeloperoxidase (MPO) activity. The production of TNF-α, IL-1ß and IL-6 was also significantly inhibited by EVO. Further mechanistic results showed that EVO restrained the inflammation by regulating NF-κB signal and NLRP3 inflammasome. Furthermore, results also showed that EVO contributed to the tight junction (TJ) architecture integrity by modulating the expression of zonula occludens-1 (ZO-1) and occludin during colitis. Surprisingly, treatment with EVO reduced the concentration of plasmatic lipopolysaccharide (LPS) and re-balanced the levels of Escherichia coli and Lactobacillus. These findings suggested that EVO may have a potential protective effect on DSS-induced colitis and may be useful for the prevention and treatment of UC.

Anti-inflammatory and antioxidant effect of melatonin on recovery from muscular trauma induced in rats.

In recent decades, the number of people who practice sports has grown exponentially, increasing the number of muscular injuries. Trauma injury occurs when the muscle is exposed to a sudden compression force. Melatonin (MLT) has often been cited in the literature as a potent antioxidant and anti-inflammatory agent. This study was designed to evaluate MLT action on muscle tissue in Wistar rats in an experimental model of muscle trauma. Twenty-eight Wistar rats were used, divided into four groups: CO (Control), CO + MLT (Control + Melatonin), T (Trauma) and T + MLT (Trauma + Melatonin). MLT (20 mg/kg) was administered (ip) daily at dusk until day 7. The trauma occurred on day 1, 2 h before the first MLT application. On day 8, muscle tissue was collected for histological analysis (HE), immunohistochemistry (TNF-α and NFκB), evaluation of oxidative stress through analysis of lipoperoxidation by TBARS and activity of SOD and GPx enzymes, and analysis of nitrites and nitrates. In the evaluation of TBARS and SOD, we observed a significant increase in the T group and a significant decrease in the T + MLT group. In the evaluation of GPx, there was a significant increase in the T group and a significant decrease in the T + MLT group. The histological analysis of muscle tissue revealed structural changes of muscle fibers and inflammatory infiltrate in the T group but a decrease in this damage in the T + MLT group. In the immunohistochemical evaluation, increased expression of TNFα and NFκB proteins in the T group was observed and a significant decrease of this expression in the T + MLT group. MLT was shown to attenuate oxidative damage and to diminish the expression of inflammatory proteins and tissue damage in this experimental model.

Moringa oleifera extract attenuates the CoCl2 induced hypoxia of rat's brain: Expression pattern of HIF-1α, NF-kB, MAO and EPO.

Hypoxia-induced oxidative stress and apoptosis are the major hallmark explanations underlying brain dysfunction. Hypoxia in the current study was induced by Cobalt chloride (CoCl2) treatment in rats. The aim of this experiment was to explore the potential ameliorative potency of Moringa oleifera ethanolic extract (MO) against experimentally induced hypoxia on the structure and function of the rat's brain. Fifty male rats were allocated to five groups (10 rats each): a control group, a MO-treated group (400 mg/kg bw, orally), a CoCl2-treated group (40 mg/kg bw/day, orally), a prophylaxis group, and a therapeutic co-treated group. Oxidative stress biomarkers and monoamine neurotransmitter were evaluated in brain tissue. In addition, qRT-PCR for expression pattern of HIF-1α, EPO, CYTO, NF-kB, and MAO-A. Glial fibrillary acidic protein (GFAP), apoptotic markers (BCL-2 and caspase 3) were detected immunohistochemically in brain cells. The results revealed a significantly lower concentration of GABA, monoamine neurotransmitter in hypoxic rat's brain. Moreover, an evident up-regulation of the mRNA expression of HIF-1α, EPO, CYTO, NF-kB, and MAO-A. There was marked encephalopathy manifested by pyknotic neurons with eosinophilic cytoplasm, vacuolations and cerebral congestions in the hypoxic rat brains. Additionally, the score of neuronal expression occupied by GFAP- positive astroglia, Caspase-3 and microglial CD68 were elevated but Bcl-2 expression was found decreased in the hypoxic group than control. The endpoints of this study clearly stated that MO ethanolic extract suggestively counteracted neurotoxic impacts caused by hypoxia, particularly when it administered prior to and concurrently with CoCl2 administration.

Effect of Omega-3 Fatty Acid on STAMP2 Expression in the Heart and Kidney of 5/6 Nephrectomy Rat Model.

Six transmembrane protein of prostate 2 (STAMP2) is a critical modulator of inflammation and metabolism in adipose tissue. There are no data on the expression of STAMP2 in chronic kidney disease, which is an inflammatory disease related to metabolic disorders. This study aimed to investigate STAMP2 expression in the kidney and heart in 5/6 nephrectomy (Nx) rats, and the effect of omega-3 fatty acid (FA) on STAMP2 expression. Male Sprague Dawley rats were divided into three groups: sham control (0.9% saline), 5/6 Nx (0.9% saline), and 5/6 Nx treated with omega-3 FA (300 mg per kg per day by gastric gavage). The expression of STAMP2 in the kidney and heart were examined by western blotting. Serum creatinine levels were higher in 5/6 Nx rats than in controls. Compared with sham controls, the expression of IκB, NF-κB, NOX4, SREBP-1, and LXR were upregulated and STAMP2 and phosphorylated-AMPK expression were downregulated in the kidney and heart of 5/6 Nx rats. Omega-3 FA supplementation prevented these changes in biomarkers related to inflammation and metabolic lipid disorders. Omega 3-FA supplementation induced the upregulation of STAMP2 protein in 5/6 Nx rats, which was associated with an attenuation of inflammation- and metabolic disease-related markers.

Paeonia lactiflora Pall. regulates the NF-κB-NLRP3 inflammasome pathway to alleviate cholestasis in rats.

OBJECTIVES: Cholestasis is a critical risk factor for severe hepatic disease or cirrhosis. The anti-inflammatory effect of Paeonia lactiflora Pall. (PLP), named Chishao in traditional Chinese medicine (TCM), on alpha-naphthylisothiocyanate (ANIT)-induced cholestasis model was tried to be elucidated in this research. METHODS: Therapeutic effect indices on hepatic function, including ALT, AST, TBIL, DBIL, ALP, TBA and γ-GT, were measured. To further investigate the protective mechanism of PLP, the mRNA and protein expression levels of NF-κB-NLRP3 inflammasome pathway were detected. RESULTS: Our results showed that compared with the model group, PLP could significantly reduce the increased serum indices such as ALT, AST, TBIL, DBIL, ALP, TBA and γ-GT induced by ANIT in a dose-dependent way. Moreover, we found that PLP downregulated the mRNA expression levels including IKK, p65, NLRP3, caspase-1 and IL-1ß, especially at the large dose. Furthermore, PLP also significantly inhibited NF-κB-NLRP3 inflammasome pathway by decreasing the protein levels of p65, p-p65, p-IKK, NLRP3, caspase-1 and IL-1ß. CONCLUSIONS: The results indicated that PLP could ameliorate ANIT-induced cholestasis in rats and the anti-inflammatory effect of PLP might be related to regulating NF-κB-NLRP3 inflammasome pathway. This study will provide scientific evidence for PLP as a potential drug candidate for cholestasis.

Treating murine Kala-azar with a Mayan plant induces immunochemical changes.

Pentalinon andrieuxii Muell Arg is a Mexican-Central American plant anciently used by local people to treat cutaneous leishmaniasis. We evaluated a hexane extract of the root we called PAE for its chemical content and for its immunochemical and in vitro activity against Leishmania donovani and healing of experimental Kala-azar. Chemical analysis using gas chromatography coupled to mass spectrometry (GC-MS) identified hexadecanoic acid, hexadecanoic acid ethyl ester, 9, 12-octadecadienoic acid ethyl ester, octadecanoic acid ethyl ester, 9-octadecenoic acid ethyl ester and diethyl phthalate as the main compounds present in PAE. We also demonstrated PAE kills promastigotes and amastigotes in vitro and significantly reduces parasite loads in liver and spleen of infected Balb/c mice. PAE induces expression of NFkB/AP-1 transcription factors and production of IL-2 and IFN-γ by spleen cells of PAE treated but not in the untreated control mice. Furthermore, there were not IL-6, IL-10 nor TNF production in macrophages treated in vitro with PAE. We developed an affordable extract of P. andrieuxii effective to treat experimental Kala-azar in Balb/c mice.

Identification and application of anti-inflammatory compounds screening system based on RAW264.7 cells stably expressing NF-κB-dependent SEAP reporter gene.

BACKGROUND: NF-κB is one of the key transcription factors in the inflammatory response, transactivates a series of pro-inflammatory genes and is therefore regarded as an important target for anti-inflammatory drug screening. METHOD: We recombined the reporter gene vector with inserting the "neo" transcript into the vector pNF-κB-SEAP, made the reporter gene vector stable in a eukaryotic cell line. The recombinant reporter gene vector we named pNF-κB-SEAP-Neo was transfected into RAW264.7. We selected the transfected RAW264.7 cell line with G418 for 15 days and then get RAW264.7 cells stably expressing NF-κB-dependent SEAP named as RAW264.7-pNF-κB-SEAP cells. We treated the RAW264.7-pNF-κB-SEAP cells with NF-κB agonists as LPS, PolyI:C and TNF-α, NF-κB inhibitor as PDTC and BAY117085, in different concentrations and time points and tested the expression of the SEAP, constructed the drug screening system on the base of the RAW264.7-pNF-κB-SEAP cell line. 130 chemicals were screened with the drug screening system we constructed and one of these chemicals numbered w10 was found could inhibit the NF-κB significantly. At last, we verified the inhibition of w10 to expression of genes promoted with NF-κB in HepG2 and Hela, and to migration of Hela. RESULT: In this study, we established a drug screening system based on RAW264.7 cells that stably expressed the NF-κB-dependent, SEAP reporter gene. To develop a standard method for drug screening using this reporter-gene cell line, the test approach of SEAP was optimized and basic conditions for drug screening were chosen. This included the initial cell number inoculated in a 96-well plate, the optimum agonist, inhibitor of NF-κB pathway and their concentrations during screening. Subsequently, 130 newly synthesized compounds were screened using the stable reporter-gene cell line. The anti-inflammatory effects of the candidate compounds obtained were further verified in 2 cancer cell lines. The results indicated that compound W10 (methyl 4-(4-(prop-2-yn-1-ylcarbamoyl) phenylcarbamoyl) benzoate) significantly inhibited SEAP production under the screening conditions. Further results confirmed that the precursor compound significantly inhibited the transcription of NF-κB target genes. CONCLUSION: In conclusion, RAW264.7 cells, stably expressing the NF-κB-dependent SEAP-reporter gene, may provide a new, feasible, and efficient cellular drug-screening system.

Anti-metastatic effect of rhodomyrtone from Rhodomyrtus tomentosa on human skin cancer cells.

This study focused on the inhibitory effect of rhodomyrtone, a bioactive compound isolated from the leaves of Rhodomyrtus tomentosa (Aiton) Hassk., on cancer metastasis in epidermoid carcinoma A431 cells and on the verification of the underlying related molecular mechanisms of this event. We demonstrated that rhodomyrtone at the subcytotoxic concentration (0.5 and 1.5 µg/ml) exhibited pronounced inhibition of cancer metastasis by reducing cell migration, cell adhesive ability and cell invasion of A431 cells in a dose-dependent manner. Data demonstrated that rhodomyrtone could inhibit the focal adhesion kinase (FAK) and phosphorylation of protein kinase B (AKT), c-Raf, extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK involved in the downregulation the enzyme activities and protein expression of matrix metalloproteinase-2 (MMP-2) and MMP-9. Moreover, we found that rhodomyrtone increased the expression of TIMP-1 and TIMP-2, which are inhibitors of MMP-9 and MMP-2, respectively. Rhodomyrtone also inhibited the expression of NF-κB and phosphorylation of NF-κB in a dose-dependent manner. These results suggested that rhodomyrtone inhibited A431 cell metastasis by reducing MMP-2/9 activities and expression through inhibiting ERK1/2, p38 and FAK/Akt signaling pathways via NF-κB activities. This finding suggested that rhodomyrtone may be a novel antimetastasis agent for treatment of skin cancer cells.

The effect of n-3/n-6 polyunsaturated fatty acids on acute reflux esophagitis in rats.

BACKGROUND: Polyunsaturated fatty acids (PUFAs) play various roles in inflammation. However, the effect of PUFAs in the development of reflux esophagitis (RE) is unclear. This study is to investigate the potential effect of n-3/n-6 PUFAs on acute RE in rats along with the underlying protective mechanisms. METHODS: Forty Sprague Dawley rats were randomly divided into four groups (n = 10 in each group). RE model was established by pyloric clip and section ligation. Fish oil- and soybean oil-based fatty emulsion (n-3 and n-6 groups), or normal saline (control and sham operation groups) was injected intraperitoneally 2 h prior to surgery and 24 h postoperatively (2 mL/kg, respectively). The expressions of interleukin (IL)-1ß, IL-8, IL-6 and myeloid differentiation primary response gene 88 (MyD88) in esophageal tissues were evaluated by Western blot and immunohistochemistry after 72 h. The malondialdehyde (MDA) and superoxide dismutase (SOD) expression in the esophageal tissues were determined to assess the oxidative stress. RESULTS: The mildest macroscopic/microscopic esophagitis was found in the n-3 group (P < 0.05). The expression of IL-1ß, IL-8, IL-6 and MyD88 were increased in all RE groups, while the lowest and highest expression were found in n-3 and n-6 group, respectively (P < 0.05). The MDA levels were increased in all groups (P < 0.05), in an ascending trend from n-3, n-6 groups to control group. The lowest and highest SOD levels were found in the control and n-3 group, respectively (P < 0.05). CONCLUSION: n-3 PUFAs may reduce acute RE in rats, which may be due to inhibition of the MyD88-NF-kB pathway and limit oxidative damage.

Baicalein inhibits TNF-α-induced NF-κB activation and expression of NF-κB-regulated target gene products.

The nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, immunity, apoptosis and angiogenesis. In our search for NF-κB inhibitors from natural resources, we identified baicalein from Scutellaria baicalensis as an inhibitor of NF-κB activation. As examined by the NF-κB luciferase reporter assay, we found that baicalein suppressed TNF-α-induced NF-κB activation in a dose-dependent manner. It also inhibited TNF-α-induced nuclear translocation of p65 through inhibition of phosphorylation and degradation of IκBα. Furthermore, baicalein blocked the TNF-α-induced expression of NF-κB target genes involved in anti-apoptosis (cIAP-1, cIAP-2, FLIP and BCL-2), proliferation (COX-2, cyclin D1 and c-Myc), invasion (MMP­9), angiogenesis (VEGF) and major inflammatory cytokines (IL-8 and MCP1). The flow cytometric analysis indicated that baicalein potentiated TNF-α-induced apoptosis and induced G1 phase arrest in HeLa cells. Moreover, baicalein significantly blocked activation of p38, extracellular signal-regulated kinase 1/2 (ERK1/2). Our results imply that baicalein could be a lead compound for the modulation of inflammatory diseases as well as certain cancers in which inhibition of NF-κB activity may be desirable.

Pharmacogenomics of Scopoletin in Tumor Cells.

Drug resistance and the severe side effects of chemotherapy necessitate the development of novel anticancer drugs. Natural products are a valuable source for drug development. Scopoletin is a coumarin compound, which can be found in several Artemisia species and other plant genera. Microarray-based RNA expression profiling of the NCI cell line panel showed that cellular response of scopoletin did not correlate to the expression of ATP-binding cassette (ABC) transporters as classical drug resistance mechanisms (ABCB1, ABCB5, ABCC1, ABCG2). This was also true for the expression of the oncogene EGFR and the mutational status of the tumor suppressor gene, TP53. However, mutations in the RAS oncogenes and the slow proliferative activity in terms of cell doubling times significantly correlated with scopoletin resistance. COMPARE and hierarchical cluster analyses of transcriptome-wide mRNA expression resulted in a set of 40 genes, which all harbored binding motifs in their promoter sequences for the transcription factor, NF-κB, which is known to be associated with drug resistance. RAS mutations, slow proliferative activity, and NF-κB may hamper its effectiveness. By in silico molecular docking studies, we found that scopoletin bound to NF-κB and its regulator IκB. Scopoletin activated NF-κB in a SEAP-driven NF-κB reporter cell line, indicating that NF-κB might be a resistance factor for scopoletin. In conclusion, scopoletin might serve as lead compound for drug development because of its favorable activity against tumor cells with ABC-transporter expression, although NF-κB activation may be considered as resistance factor for this compound. Further investigations are warranted to explore the full therapeutic potential of this natural product.

4-Phenylbutyric Acid Reveals Good Beneficial Effects on Vital Organ Function via Anti-Endoplasmic Reticulum Stress in Septic Rats.

OBJECTIVES: Sepsis and septic shock are the common complications in ICUs. Vital organ function disorder contributes a critical role in high mortality after severe sepsis or septic shock, in which endoplasmic reticulum stress plays an important role. Whether anti-endoplasmic reticulum stress with 4-phenylbutyric acid is beneficial to sepsis and the underlying mechanisms are not known. DESIGN: Laboratory investigation. SETTING: State Key Laboratory of Trauma, Burns and Combined Injury. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: Using cecal ligation and puncture-induced septic shock rats, lipopolysaccharide-treated vascular smooth muscle cells, and cardiomyocytes, effects of 4-phenylbutyric acid on vital organ function and the relationship with endoplasmic reticulum stress and endoplasmic reticulum stress-mediated inflammation, apoptosis, and oxidative stress were observed. MEASUREMENTS AND MAIN RESULTS: Conventional treatment, including fluid resuscitation, vasopressin, and antibiotic, only slightly improved the hemodynamic variable, such as mean arterial blood pressure and cardiac output, and slightly improved the vital organ function and the animal survival of septic shock rats. Supplementation of 4-phenylbutyric acid (5 mg/kg; anti-endoplasmic reticulum stress), especially administered at early stage, significantly improved the hemodynamic variables, vital organ function, such as liver, renal, and intestinal barrier function, and animal survival in septic shock rats. 4-Phenylbutyric acid application inhibited the endoplasmic reticulum stress and endoplasmic reticulum stress-related proteins, such as CCAAT/enhancer-binding protein homologous protein in vital organs, such as heart and superior mesenteric artery after severe sepsis. Further studies showed that 4-phenylbutyric acid inhibited endoplasmic reticulum stress-mediated cytokine release, apoptosis, and oxidative stress via inhibition of nuclear factor-κB, caspase-3 and caspase-9, and increasing glutathione peroxidase and superoxide dismutase expression, respectively. CONCLUSIONS: Anti-endoplasmic reticulum stress with 4-phenylbutyric acid is beneficial to septic shock. This beneficial effect of 4-phenylbutyric acid is closely related to the inhibition of endoplasmic reticulum stress-mediated oxidative stress, apoptosis, and cytokine release. This finding provides a potential therapeutic measure for clinical critical conditions, such as severe sepsis.

NF-κB/p65 expression before and after treatment in rectal cancer patients undergoing neoadjuvant (chemo)radiotherapy and surgery: prognostic marker for disease progression and survival.

Nuclear factor-kappaB (NF-κB), especially p65 subunit, has been associated with origin and progression of cancer as well as with the resistance to radiotherapy and chemotherapy in experimental models. The aim of the present study was to determine expression of NF-κB/p65 in tumor specimens before and after treatment of rectal cancer patients and to evaluate possible relationship between expression of NF-κB/p65 before and after (chemo)radiotherapy, other tumor characteristics and the clinical outcome. Furthermore, NF-κB/p65 was studied in relationship to pathologic response to preoperative (chemo)radiotherapy. Fifty patients with rectal cancer undergoing neoadjuvant (chemo)radiotherapy and surgery were included in the study. Pre-treatment rectal cancer specimens were obtained from diagnostic colonoscopy. Post-treatment rectal cancer specimens were obtained from surgically removed part of the rectum with the tumor. NF-κB/p65 expression was determined by immunohistochemistry and analysis was performed both in biopsies and in post-treatment tumor samples. Cytoplasmic positivity in tumor cells and nuclear positivity in lymphocytes were detected. High NF-κB/p65 positivity in pre-treatment tumor samples was significantly associated with shortened overall survival (OS). Disease-free survival (DFS) tends to be shortened as well. In post-treatment tumor samples, high NF-κB/p65 positivity was neither associated with shortened OS nor with shortened DFS. In post-treatment samples residual tumor cells deeply infiltrating the wall of the rectum with high NF-κB/p65 expression were found. The cells were linked to significantly worse clinical outcome in terms of shortened OS and DFS. NF-κB/p65 positivity did not correlate with pathologic response to preoperative (chemo)radiotherapy. In conclusion, our data suggest that high level of NF-κB/p65 subunit may be associated with more aggressive features of the tumor, higher metastatic potential, and shortened overall survival, but it does not correlate with resistance to (chemo)radiotherapy. Consequently, the level of NF-κB/p65 may help to select those patients who have poor prognosis and are candidates for more intensive anticancer therapy. For these purposes both pre-treatment and post-treatment tumor samples may be used.

Saikosaponin-a Attenuates Oxidized LDL Uptake and Prompts Cholesterol Efflux in THP-1 Cells.

Saikosaponins-a (Ssa) is a major bioactive extract of Radix Bupleuri which is a traditional Chinese medicine. The roles of inflammatory response and lipid transportation in the process of atherosclerosis have drawn increasing attention. We explored the regulation of lipid transportation and immune-inflammatory role of Ssa in early atherosclerosis. The antiatherogenic actions and possible molecular mechanisms of Ssa were texted in THP-1 cells. We examined the effect of Ssa on oxidized low-density lipoprotein (ox-LDL)-induced lipid uptake, cholesterol efflux, immune-inflammatory response. THP-1 macrophages were treated with Ssa followed by ox-LDL for 24 hours. Results from western blot showed that Ssa obviously reduced lipoprotein uptake to block foam cell formation and the expression of Density Lipoprotein Receptor-1 and CD36. Ssa also significantly boosted cholesterol efflux and the expression of ATP binding cassettetransporter A1 and peroxisome proliferator-activated receptor γ. The results also indicated that Ssa inhibited ox-LDL-induced activation of AKT and nuclear factor-κB, assembly of NLRP3 inflammasome and production of proinflammatory cytokines. It is suggested that the ability against immune inflammatory response of Ssa is due to modulation of the PI3K/AKT/NF-κB/NLRP3 pathway. In conclusion, this study provides new insight into Ssa's molecular mechanism and its therapeutic potential in the treatment of atherosclerosis.