Phospho-nuclear factor-κB Research Articles

An Isofibrous Diet with Fiber Konjac Glucomannan Ameliorates Salmonella typhimurium-Induced Colonic Injury by Regulating TLR2-NF-κB Signaling and Intestinal Microbiota in Mice.

This study aimed to investigate the hypothesis that dietary konjac glucomannan (KGM) could alleviate Salmonella typhimurium-induced colitis by modulating intestinal microbiota. Mice were fed an isocaloric and isofibrous diet supplemented with either 7% KGM or cellulose and were treated with 5 × 108 CFU of S. typhimurium. The results showed that KGM had an average molecular weight of 936 kDa and predominantly consisted of mannose and glucose at a molar ratio of 1:1.22. In vivo studies demonstrated that dietary KGM effectively mitigated colonic lesions, oxidative stress, disruption of tight junction protein 2 and occludin, and the inflammatory response induced by S. typhimurium. Moreover, KGM administration alleviated the dramatic upregulation of toll-like receptor 2 (TLR2) and phosphonuclear factor κB (NF-κB) protein abundance, induced by Salmonella treatment. Notably, dietary KGM restored the reduced Muribaculaceae and Lactobacillus abundance and increased the abundance of Blautia and Salmonella in S. typhimurium-infected mice. Spearman correlation analysis revealed that the gut microbiota improved by KGM contribute to inhibit inflammation and oxidative stress. These results demonstrated the protective effects of dietary KGM against colitis by modulating the gut microbiota and the TLR2-NF-κB signaling pathway in response to Salmonella infection.

Astragalus Polysaccharide Promotes Neuronal Injury Repair via the Notch1/NFκB Signaling Axis in the Ventroposterior Thalamic Nucleus in Rats with Focal Cerebral Ischemia.

Ischemic stroke is the most common form of stroke and the second most common cause of death and incapacity worldwide. Its pathogenesis and treatment have been the focus of considerable research. In traditional Chinese medicine, the root of Mongolian astragalus has been important in the treatment of stroke since ancient times. Astragalus polysaccharide (APS) is a key active ingredient of astragalus and offers therapeutic potential for conditions affecting the neurological system, the heart, cancer, and other disorders. However, it is not yet known how APS works to protect against ischemic stroke. Rats were subjected to middle cerebral artery occlusion (MCAO) to imitate localized cerebral ischemia. Each of four experimental groups (normal, sham, MCAO, and MCAO+APS) contained 12 adult male Sprague-Dawley (SD) rats selected randomly from a total of 48 rats. Following successful establishment of the model, rats in the MCAO+APS group received intraperitoneal injection of APS (50 mg/kg) once daily for 14 days, whereas all other groups received no APS. The Bederson nerve function score and the forelimb placement test were used to detect motor and sensory function defects, while Nissl staining was used to investigate pathological defects in the ventroposterior thalamic nucleus (VPN). Immunohistochemical staining and Western blot were used to evaluate the expression of Neurogenic locus notch homolog protein 1 (Notch1), hairy and enhancer of split 1 (Hes1), phospho-nuclear factor-κB p65 (p-NFκB p65), and nuclear factor-κB p65 (NFκB p65) proteins in the VPN on the ischemic side of MCAO rats. APS promoted the recovery of sensory and motor function, enhanced neuronal morphology, increased the number of neurons, and inhibited the expression of Notch1/NFκB signaling pathway proteins in the VPN of rats with cerebral ischemia. After cerebral ischemia, APS can alleviate symptoms of secondary damage to the VPN, which may be attributed to the suppression of the Notch1/NFκB pathway.

Amelioration of Atherosclerosis by lycopene is linked to the modulation of gut microbiota dysbiosis and related gut-heart axis activation in high-fat diet-fed ApoE−/− mice

BackgroundInterplay between gut microbiota and heart, termed “gut-heart” axis, has a crucial role in the pathogenesis of atherosclerosis. Our previous study showed that lycopene possesses anti-inflammatory and anti-atherosclerotic effects, but its link to the gut microbiota is poorly understood. Herein, we surmised that lycopene could regulate the gut microbiota, exert anti-atherosclerotic effect by regulating the “gut-heart” axis.MethodsMale ApoE−/− mice were fed a high-fat diet (HFD) with or without lycopene (0.1% w/w) for 19 weeks. Gut microbiota was analyzed by 16 S rRNA sequencing, the protein levels of zonula occludens-1 (ZO-1), occludin, toll-like receptor 4 (TLR4) and phospho-nuclear factor-κB (NF-κB) p65 were measured by Western blotting, the levels of serum inflammatory factors including monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 were assayed using ELISA kits. Also, the concentrations of serum lipopolysaccharide (LPS), D-lactic acid (D-LA) and diamine peroxidase (DAO) were measured through ELISA method.ResultsThe aortic sinus sections revealed that lycopene supplementation significantly reduced the extent of atherosclerotic lesions and inhibited atherosclerosis development caused by HFD. The analysis of gut microbiota showed that lycopene reduced the ratio of Firmicutes/Bacteroides and increased the relative abundance of Verrucomicrobia, Akkermansia and Alloprevotella, which were related to elevated intestinal barrier function and reduced inflammation. Moreover, lycopene up-regulated the expression of intestinal ZO-1 and occludin and decreased serum LPS, D-LA and DAO levels. In addition, lycopene inhibited the expression of TLR4 and phospho-NF-κB p65 in aortic sinus plaque, serum MCP-1, TNF-α, IL-1β, and IL-6 levels were also lowered by lycopene treatment.ConclusionsOur results indicated the protective effect of lycopene against atherosclerosis induced by HFD and further revealed that its mechanism might be its prebiotic effect on maintaining gut microbiota homeostasis and improving intestinal barrier function, consequently reducing serum LPS-triggered inflammatory response in the heart.

Trichinella spiralis cathepsin L induces macrophage M1 polarization via the NF-κB pathway and enhances the ADCC killing of newborn larvae

BackgroundDuring the early stages of Trichinella spiralis infection, macrophages predominantly undergo polarization to the M1-like phenotype, causing the host’s inflammatory response and resistance against T. spiralis infection. As the disease progresses, the number of M2-type macrophages gradually increases, contributing to tissue repair processes within the host. While cysteine protease overexpression is typically associated with inflammation, the specific role of T. spiralis cathepsin L (TsCatL) in mediating macrophage polarization remains unknown. The aim of this study was to assess the killing effect of macrophage polarization mediated by recombinant T. spiralis cathepsin L domains (rTsCatL2) on newborn larvae (NBL).MethodsrTsCatL2 was expressed in Escherichia coli strain BL21. Polarization of the rTsCatL2-induced RAW264.7 cells was analyzed by enzyme-linked immunosorbent assay (ELISA), quantitative PCR (qPCR), western blot, immunofluorescence and flow cytometry. The effect of JSH-23, an inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), on rTsCatL2-induced M1 polarization investigated. Cytotoxic effects of polarized macrophages on NBL were observed using in vitro killing assays.ResultsFollowing the co-incubation of rTsCatL2 with RAW264.7 murine macrophage cells, qPCR and ELISA revealed increased transcription and secretion levels of inducible nitric oxide synthase (iNOS), interleukin (IL)-6, IL-1β and tumor necrosis factor alpha (TNF-α) in macrophages. Western blot analysis showed a significant increase in iNOS protein expression, while the expression level of arginase-1 protein remained unchanged. Flow cytometry revealed a substantial increase in the number of CD86-labeled macrophages. The western blot results also indicated that rTsCatL2 increased the expression levels of phospho-NF-κB and phospho-nuclear factor-κB inhibitor alpha (IκB-α) proteins in a dose-dependent manner, while immunofluorescence revealed that rTsCatL2 induced nuclear translocation of the p65 subunit of NF-κB (NF-κB p65) protein in macrophages. The inhibitory effect of JSH-23 suppressed and abrogated the effect of rTsCatL2 in promoting M1 macrophage polarization. rTsCatL2 mediated polarization of macrophages to the M1-like phenotype and enhanced macrophage adhesion and antibody-dependent cell-mediated cytotoxicity (ADCC) killing of NBL.ConclusionsThe results indicated that rTsCatL2 induces macrophage M1 polarization via the NF-κB pathway and enhances the ADCC killing of NBL. This study provides a further understanding of the interaction mechanism between T. spiralis and the host.Graphical

Calycosin attenuates Angiostrongylus cantonensis-induced parasitic meningitis through modulation of HO-1 and NF-κB activation.

Angiostrongylus cantonensis causes a form of parasitic meningitis in humans. Albendazole (ABZ) kills nematode larvae in the brain. However, dead larvae can trigger a severe inflammatory response, resulting in brain damage. Accumulating evidence suggests that calycosin represents a potential anti-inflammatory therapeutic candidate. In this study, we investigated the combined effects of ABZ and calycosin in angiostrongyliasis caused by A. cantonensis in BALB/c mice. Inflammatory mediators (such as phospho-nuclear factor-κB, cyclooxygenase-2, matrix metalloproteinase-9, tumour necrosis factor-α and interleukin-1β) are associated with the development of meningitis and immune inflammatory reactions. We found that A. cantonensis significantly induces inflammatory mediator production and increases the blood–brain barrier (BBB) permeability. However, co-administration of both ABZ and calycosin markedly suppressed meningitis and inflammatory mediator production and decreased the BBB permeability compared to treatment with a single drug. Furthermore, calycosin and ABZ plus calycosin treatment facilitated production of the antioxidant haem oxygenase-1 (HO-1). Moreover, co-therapy with ABZ and calycosin failed to mitigate angiostrongyliasis in the presence of tin-protoporphyrin IX, an HO-1-specific inhibitor. This finding suggests that the beneficial effects of ABZ plus calycosin treatment on the regulation of inflammation are mediated by the modulation of HO-1 activation. The present results provide new insights into the treatment of human angiostrongyliasis using co-therapy with ABZ and calycosin.

Irisin Attenuates Pathological Neovascularization in Oxygen-Induced Retinopathy Mice.

PurposeAbnormal angiogenesis is a defining feature in a couple of ocular neovascular diseases. The application of anti-VEGFA therapy has achieved certain benefits in the clinic, accompanying side effects and poor responsiveness in many patients. The present study investigated the role of irisin in retinal neovascularization.MethodsWestern blot and quantitative PCR were used to determine irisin expression in the oxygen-induced retinopathy mice model. The pathological angiogenesis and inflammation index were examined after irisin administration. Primary retinal astrocytes were cultured and analyzed for VEGFA expression in vitro. Astrocyte-conditioned medium was collected for transwell assay and tube formation assay in human microvascular endothelial cells-1.ResultsIrisin was downregulated in the oxygen-induced retinopathy mice retinae. Additional irisin attenuated pathological angiogenesis, inflammation, and apoptosis in vivo. In vitro, irisin decreased astrocyte VEGFA production, and the conditioned medium suppressed human microvascular endothelial cells-1 migration. Last, irisin inhibited hypoxia-inducible factor-2α, nuclear factor-κB, and pNF-κB (Phospho-Nuclear Factor-κB) expression.ConclusionsIrisin mitigates retinal pathological angiogenesis. Chinese

Anticancer effects of oleanolic acid on human melanoma cells

Owing to the ineffectiveness of the currently used therapies against melanoma, there has been a shift in focus toward alternative therapies involving the use of natural compounds. This study assessed the anticancer effects of oleanolic acid (OA) and its ability to induce apoptosis in A375SM and A375P melanoma cells in vivo. Compared to the control group, viability of A375P and A375SM cells decreased following OA treatment. In OA-treated A375SM and A375P cells, 4′,6-diamidino-2-phenylindole staining showed an increase in the apoptotic body, and flow cytometry revealed increased number of apoptotic cells compared to that in the control group. OA-treated A375SM cells exhibited an increased expression of the apoptotic proteins, cleaved poly (ADP-ribose) polymerase (PARP) and B-cell lymphoma (Bcl)-2-associated X protein (Bax) as well as decreased expression of the antiapoptotic protein Bcl-2 compared to that in the control group. In OA-treated A375P cells, expression patterns of cleaved PARP and Bcl-2 were similar to those in OA-treated A375SM cells; however, no difference was reported in the expression of Bax compared to that in the control group. Additionally, OA-treated melanoma cells showed decreased expression of phospho-nuclear factor-κB (p–NF–κB), phospho-inhibitor of nuclear factor-κBα (p-IκBα), and phospho-IκB kinase αβ than that in the control group. Moreover, immunohistochemistry showed a comparatively decreased level of p–NF–κB in the OA-treated group than that in the control group. Xenograft analysis confirmed the in vivo anticancer effects of OA against A375SM cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay revealed an increased number of TUNEL-positive cells in the OA-treated group compared to that in the control group. In conclusion, the study results suggest that OA induces apoptosis of A375SM and A375P cells in vitro and apoptosis of A375SM cells in vivo. Furthermore, the in vitro and in vivo anticancer effects were mediated by the NF-κB pathway.

Epigallocatechin-3-gallate ameliorates LPS-induced inflammation by inhibiting the phosphorylation of Akt and ERK signaling molecules in rat H9c2 cells.

The inflammatory response has been implicated in various cardiac and systemic diseases. Epigallocatechin-3-gallate (EGCG), the major polyphenol extracted from green tea, has various biological and pharmacological properties, such as anti-inflammation, anti-oxidative and anti-tumorigenesis. To some extent, the mechanism of EGCG in the inflammatory response that characterizes myocardial dysfunction is not fully understood. The present study aimed to investigate the inhibiting effect of EGCG on lipopolysaccharide (LPS)-induced inflammation in vitro. Treatment with LPS affected rat H9c2 cardiomyocytes and induced an inflammatory response. However, the LPS-induced effects were attenuated after treatment with EGCG. The present results demonstrated that EGCG treatment repressed several inflammatory mediators, such as vascular endothelial growth factor, chemokine ligand 5, chemokine ligand 2, intercellular adhesion molecule-1, matrix metalloproteinase-2, tumor necrosis factor-α and nitric oxide (induced by LPS), and the repressing effect of EGCG on inflammatory response was dose-dependent in the range of 6.25-100 µM. EGCG inhibited these marked inflammatory key signaling molecules by reducing the expression of phospho-nuclear factor-κB p65, -Akt, -ERK and -MAPK p38 while the total protein level of these signal proteins were not affected. In conclusion, the present findings suggested that EGCG possesses cardiomyocyte-protective action in reducing the LPS-induced inflammatory response due to the inhibition of the phosphorylation of Akt and ERK signaling molecules.

The Hypoglycemic and Renal Protection Properties of Crocin via Oxidative Stress-Regulated NF-κB Signaling in db/db Mice.

BackgroundAs the main ingredient of Crocus sativus L. (Iridaceae) extract, crocin- I (CR) has been reported to show various pharmacological activities. The aim of this study was to investigate the hypoglycemic and renal protection properties of CR in db/db mice.MethodsEight-week-old db/db mice were treated with metformin (Met) (100 mg/kg) and CR (50 mg/kg) for eight weeks.ResultsCR treatment showed hypoglycemic functions indicated by reduced bodyweight, food and water intake, plasma glucose, and serum levels of glycated hemoglobin A1c. Additionally, the CR group showed increased serum levels of insulin and pyruvate kinase, hypolipidemic functions indicated by the suppressed levels of total cholesterol and triglyceride, and enhanced levels of high-density lipoprotein cholesterol, which are also indicators of hypoglycemic functions. The renal protection function of CR was demonstrated by its protection of renal structures and its regulation of potential indicators of nephropathy. The anti-oxidation and anti-inflammation effects of CR were verified by enzyme-linked immunosorbent assay. In the kidneys of db/db mice, CR decreased the expression of phospho-IκBα and phospho-nuclear factor-κB (NF-κB), whereas it enhanced the expression of nuclear respiratory factor 2, manganese superoxide dismutase 1, heme oxygenase-1, and catalase.ConclusionsThe anti-diabetic and anti-diabetic nephritic effects of CR were related to its modulation of oxidative stress-mediated NF-κB signaling.

Latifolin protects against myocardial infarction by alleviating myocardial inflammatory via the HIF-1α/NF-κB/IL-6 pathway

Context The Traditional Chinese herb medicine Dalbergia odorifera T. Chen (Fabaceae), exerted a protective effect on myocardial ischaemia. Latifolin is a neoflavonoid extracted from Dalbergia odorifera. It has been reported to have the effects of anti-inflammation and cardiomyocyte protection. Objective To investigate whether latifolin can improve myocardial infarction (MI) through attenuating myocardial inflammatory and to explore its possible mechanisms. Materials and methods Left coronary artery was ligated to induce a rat model of MI, and the rats were treated with sodium carboxymethyl cellulose (CMC-Na) or different doses of latifolin (25, 50, 100 mg/kg/d) by oral gavage for 28 days. Serum contents of myocardial enzyme were measured at seven and fourteen days after treatment. Cardiac function, infarct size, histopathological changes and inflammatory cells infiltration was assessed at 28 days after treatment. Western blotting was used to investigate the underlying mechanisms. Results Latifolin treatment markedly decreased the contents of myocardial enzymes, and increased left ventricular ejection fraction (85.27% vs. 59.11%) and left ventricular fractional shortening (62.71% vs. 45.53%). Latifolin was found to significantly reduced infarction size (27.78% vs. 39.07%), myocardial fibrosis and the numbers of macrophage infiltration (436 cells/mm2 vs. 690 cells/mm2). In addition, latifolin down-regulated the expression levels of hypoxia-inducible factor-1α (0.95-fold), phospho-nuclear factor-κB (0.2-fold) and interleukin-6 (1.11-fold). Discussion and conclusions Latifolin can protect against myocardial infarction by improving myocardial inflammation through the HIF-1α/NF-κB/IL-6 signalling pathway. Accordingly, latifolin may be a promising drug for pharmacological treatment of ischaemic cardiovascular disease.

The α7 nicotinic acetylcholine receptor positive allosteric modulator prevents lipopolysaccharide-induced allodynia, hyperalgesia and TNF-α in the hippocampus in mice.

Previous studies have shown that α7 nicotinic acetylcholine receptor (nAChR) has a critical role in the regulation of pain sensitivity and neuroinflammation. However, pharmacological effects of α7 nAChR activation in the hippocampus on neuroinflammatory mechanisms associated with allodynia and hyperalgesia remain unknown. We have determined the effects of 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS), an α7 nAChR positive allosteric modulator, on lipopolysaccharide (LPS)-induced allodynia and hyperalgesia in mice. We also evaluated the effects of TQS on immunoreactivity of microglial marker ionized-calcium binding adaptor molecule 1 (Iba-1), phospho-nuclear factor-κB (p-NF-κB p65), tumor necrosis factor-alpha (TNF-α), and norepinephrine (NE) level. Mice were treated with (0.25, 1 or 4 mg/kg, ip) followed by LPS (1 mg/kg, ip) administration. Allodynia and hyperalgesia were determined using von Frey filaments and hot plate respectively. Immunoreactivity of Iba-1, p-NF-κB p65, and TNF-α, were measured in the hippocampus using immunofluorescence assay. Hippocampal NE level was evaluated using high performance liquid chromatography. LPS administration resulted in allodynia and hyperalgesia in mice after six h. Systemic administration of TQS prevented LPS-induced allodynia and hyperalgesia. TQS pretreatment significantly decreased the immunoreactivity of Iba-1, p-NF-κB, and TNF-α in CA1 and DG regions of the hippocampus. In addition, TQS reversed LPS-induced NE reduction in the hippocampus. Taken together, our results suggest that TQS prevented LPS-induced allodynia and hyperalgesia, upregulation of TNF-α expression and NE level reduction involving microglial α7 nAChR in part in the hippocampus. Therefore, these findings highlight the important effects of α7 nAChR allosteric modulator against symptoms of inflammatory pain.

Pterostilbene Ameliorates Nephropathy Injury in Streptozotocin-Induced Diabetic Rats

Background: Our study investigated the therapeutic role and potential mechanisms of pterostilbene (PS) in diabetic nephropathy (DN) rats. Methods: DN models were established by high-fat diet after streptozotocin injection. A total of 50 Sprague-Dawley rats were randomly divided into control, DN, PS-treated groups (PS-H, PS-M, PS-L). PS was administered to rats by gavage for 8 weeks at 3 different doses (25, 10, and 5 mg/kg/day). The levels of oxidative stress activity (superoxide dismutase [SOD], malondialdehyde [MDA], glutathione peroxidase [GSH-PX]) and inflammatory factors (tumor necrosis factor [TNF]-α, interleukin (IL)-6, IL-1β, monocyte chemoattractant factor [MCP]-1) were detected by ­ELISA. TGF-β, Smad1, and fibronectin (FN) were measured through immunohistochemistry. The relative expressions of phospho-IκBα/IκBα, phospho-IκB kinases (IKK)β/IKKβ, phospho-nuclear factor-κB (NF-κB) p65/NF-κB p65 were detected by western blot. Results: Compared with DN group, the levels of TNF-α, IL-6, IL-1β, and MCP-1 were decreased in the PS-H group (p < 0.05). Meanwhile, the levels of SOD, MDA, GSH-PX improved in kidney and serum in PS-H groups (p< 0.05). PS also significantly decreased the level of phospho-NF-κB p65 and increased the levels of phospho- IKKβ and phospho-Iκ-Bα (p < 0.05). The results showed that PS treatment decreased TGF-β, Smad1, and FN expressions. Conclusion: PS had potential therapeutic effects on DN, which may be related to the regulation of NF-κB pathway.

Alginate oligosaccharide enhances intestinal integrity of weaned pigs through altering intestinal inflammatory responses and antioxidant status.

Alginate oligosaccharide (AOS), prepared from depolymerised alginate, a natural polysaccharide occurring in the cell walls of brown algae, provides beneficial effects for intestinal health. However, the underlying mechanisms by which AOS supplementation maintains the intestinal integrity of weaned pigs remain obscure. Here, we aimed to determine how AOS modulates the intestinal integrity of weaned pigs. Twenty-four weaned pigs were assigned to two treatments: a control group (basal diet) and an AOS group (the basal diet supplemented with 100 mg kg−1 AOS). On day 15, eight pigs per treatment were randomly selected and sacrificed for serum and intestinal samples. We observed that AOS supplementation enhanced the intestinal integrity, as evidenced by the increased (P < 0.05) intestinal occludin protein abundance. Compared to the control group, AOS ingestion both elevated (P < 0.05) the jejunal and ileal catalase activity and decreased (P < 0.05) the duodenal and jejunal tumour necrosis factor-α concentration and mast cell tryptase expression. Furthermore, AOS down-regulated (P < 0.05) the duodenal toll-like receptor 4 (TLR4) and its down-stream signals, myeloid differentiation factor 88 (MyD88), interleukin-1 receptor-associated kinase 1 (IRAK1) and tumour necrosis factor receptor-associated factor 6 (TRAF6) mRNA levels, as well as jejunal nucleotide-binding oligomerisation domain protein 1 (NOD1) and its adaptor molecule, receptor-interacting serine/threonine-protein kinase 2 (RIPK2), mRNA levels. Additionally, phospho-nuclear factor-κB (p-NF-κB) p65 protein abundance in the duodenum and jejunum was down-regulated (P < 0.05) following AOS supplementation. According to the above results, the enhanced intestinal integrity in AOS-supplemented pigs appears to be associated with the elevated antioxidant capacity and the reduced mast cell degranulation, as well as the inhibited pro-inflammatory cytokines production via inhibiting the TLR4/NF-κB and NOD1/NF-κB signalling pathways.

Ixeris dentata (Thunb. Ex Thunb.) Nakai Extract Inhibits Proliferation and Induces Apoptosis in Breast Cancer Cells through Akt/NF-κB Pathways.

Ixeris dentata (Thunb. Ex Thunb.) Nakai (ID) exhibits various physiological activities, and its related plant derived-products are expected to represent promising cancer therapeutic agents. However, the anticancer effects of ID extract on breast cancer cells classified as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are still unknown. In this study, we investigated the anti-cancer effects and analyzed the molecular mechanism of ID extract in T47D, MCF-7 (ER-, PR-positive, HER2-negative), SK-BR-3(ER-, PR-negative, HER2-positive), and MDA-MB-231 (Triple-negative) through in vitro studies. Additionally, we examined its anti-tumor effects through in vivo studies. Our findings indicated that ID extract-induced apoptosis was mediated via various survival pathways on four breast cancer cells by identifying the factors including Bcl-2 family, phospho-Akt and phospho-nuclear factor-κB (NF-κB). Based on in vitro findings that induced apoptosis via Akt-NF-κB signaling, we investigated the effects of ID extract on mice bearing MDA-MB-231 cells. The results showed that ID extract significantly decreased MDA-MB-231 tumor volume and weight via inducing apoptosis by suppressing phospho-Akt. Overall, these results indicate that ID extract induces apoptosis through the Akt-NF-κB signaling pathway in MDA-MB-231 breast cancer cells and tumors, and it may serve as a therapeutic agent for triple-negative human breast cancer.

Wound-healing Activity of Zanthoxylum bungeanum Maxim Seed Oil on Experimentally Burned Rats.

Background:The seed oil of Zanthoxylum bungeanum Maxim (ZBSO) is considered to be rich source of fatty acids, mainly oleic and linoleic acids, and has been used for the treatment of burns in Chinese medicine.Objective:We evaluated the healing efficacy of ZBSO and explored its possible mechanism on scalded rats.Materials and Methods:Sprague-Dawley rat models with deep second-degree burns were set up, and ZBSO (500 and 1000 μl/wound) was topically applied twice daily for 7 days and then once daily until wound healing. The therapeutic effects of ZBSO were evaluated by observing wound closure time, decrustation time, wound-healing ratio, and pathological changes. Collagen type-III, matrix metalloproteinase-2 (MMP-2), MMP-9, phospho-nuclear factor-κB (p-NF-κB) p65, inhibitor of NF-κB subunit α p-IκBα, and inhibitor of NF-κB subunit α (IκBα) expression were determined using Western blotting.Results:The ZBSO-treated group showed a higher wound-healing ratio and shorter decrustation and wound closure times than the untreated group. The topical application of ZBSO increased collagen synthesis as evidenced by an increase in hydroxyproline level and upregulated expression of collagen type-III on days 7, 14, and 21 posttreatment. A reduction in MMP-2 and MMP-9 expressions also confirmed the collagen formation efficacy of ZBSO. Furthermore, there was a significant increase in superoxide dismutase levels and a decrease in malondialdehyde levels in ZBSO-treated wounds. ZBSO also decreased tumor necrosis factor alpha, interleukin-1 (IL-1) β, and IL-6 levels in serum, upregulated IκBα, and downregulated p-NF-κB p65 and p-IκBα expression in vivo, indicating the anti-inflammatory action of ZBSO.Conclusion:ZBSO has significant potential to treat burn wounds by accelerating collagen synthesis and the anti-inflammatory cascade of the healing process.SUMMARY The seed oil of Zanthoxylum bungeanum Maxim (ZBSO) is rich of fatty acidsThe healing efficacy of ZBSO on experimentally scalded rats was evaluatedZBSO has significant potential to treat deep second-degree burn woundsZBSO could accelerate collagen synthesis and inhibit the inflammatory signaling. Abbreviations used: ECL: Enhanced chemiluminescence; ECM: Extracellular matrix; ELISA: Enzyme-linked immunosorbent assay; GC-MS: Gas chromatography-mass spectrometry; HRP: Horseradish peroxidase; HYP: Hydroxyproline; IκBα: Inhibitor of NF-κB subunit α; IL: Interleukin; MDA: Malondialdehyde; MMP: Matrix metalloproteinase-2; NF-κB: Nuclear factor-κB; SFE: Supercritical fluid extraction; SOD: Superoxide dismutase; SSD: Silver sulfadiazine; TCM: Traditional Chinese medicine; TNF: Tumor necrosis factor.

Effects of ligustilide on the extracellular recombinant human heat shock protein 60 induced inflammatory reactions in the THP-1 cells and the related mechanisms

Objective: To investigate the effects of ligustilide(LIG) on extracellular recombinant human heat shock protein 60 (HSP60) induced inflammatory reactions in the THP-1 cells and the related mechanisms. Methods: THP-1 cells were differentiated to macrophages by incubation with phorbol-12-myristate-13-acetate (PMA). The immunofluorescence method was used to screen the optimum transfection concentration of MyD88 siRNA.The macrophages were divided into six groups(n=3), including blank control(siRNA transfection reagent), model(siRNA transfection reagent+ HSP60 10 mg/L), negative control(MyD88 negative control+ HSP60 10 mg/L), LIG group(siRNA transfection reagent+ HSP60 10 mg/L+ LIG 20 mg/L), RNA interfering(RNAi) group(MyD88 siRNA+ HSP60 10 mg/L) and RNAi+ LIG group(MyD88 siRNA+ HSP60 10 mg/L+ LIG 20 mg/L). The protein expression level of MyD88 and phospho-nuclear factor-κB(p-NF-κB) in macrophages and the level of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in the culture supernatant were assessed by Western blot analyses or ELISA, respectively. Results: (1)The protein expression levels of MyD88 (1.196±0.125 vs. 0.341±0.063, P<0.01) and p-NF-κB(0.817±0.034 vs. 0.312±0.046, P<0.01) were significantly higher in the model group than those in the blank control group.The protein expression levels of MyD88(0.554±0.043) and p-NF-κB(0.538±0.063) in the RNAi group were significantly lower than those in the model group (all P<0.01) but significantly higher than those in the blank control group (all P<0.05). The protein expression levels of MyD88(0.694±0.087, P<0.05) and p-NF-κB(0.669±0.043, P<0.01)in the LIG group were markedly lower than those in the model group, but higher than those in the RNAi group (P<0.05) and the blank control group (P<0.01). The protein expression levels of MyD88(0.409±0.069) and p-NF-κB(0.395±0.046) in the RNAi+ LIG group were significantly lower than in the model group (all P<0.01) and in the LIG group(P<0.05 or 0.01), and were similar to the blank control group(P>0.05). The expression level of p-NF-κB in the RNAi+ LIG group was significantly lower than in the RNAi group (P<0.05). (2) The contents of TNF-α((312.24±28.69) ng/L vs. (5.99±1.03) ng/L, P<0.01) and IL-6((233.45±57.77) ng/L vs. (2.25±0.67) ng/L, P<0.01) were significantly higher in the model group than in the blank control group. The contents of TNF-α((235.66±25.12) ng/L) and IL-6((131.59±13.99) ng/L) were significantly lower in the RNAi group than in the model group (P<0.01). The contents of TNF-α((258.13±44.80) ng/L) and IL-6((175.92±28.27) ng/L) were also significantly lower in the LIG group than in the model group(P<0.05) while the content of IL-6 was significantly higher in the LIG group than in the RNAi group(P<0.01). The contents of TNF-α((88.57±16.10) ng/L) and IL-6((59.99±10.31) ng/L) were significantly lower in the RNAi+ LIG group than those in the model group, the RNAi group and the LIG group(P<0.05 or 0.01). Conclusions: The MyD88/NF-κB signaling pathway is one of the key signaling pathways of human HSP60 induced inflammation in THP-1 cells. Ligustilide could exhibit the anti-inflammatory effect probably by inhibiting the MyD88/NF-κB signaling pathway.

Attenuation of exercise effect on inflammatory responses via novel role of TLR4/PI3K/Akt signaling in rat splenocytes

Moderate exercise diminishes proinflammation cytokine production in various types of immune cells, but the intracellular signaling pathways involved are not completely understood. Phosphoinositide 3-kinase (PI3K)/Akt, a crucial downstream protein of toll-like receptor 4 (TLR4), may modulate inflammation. The present study aimed to investigate the effects of exercises on lipopolysaccharide (LPS)-stimulated inflammatory response in splenocytes and to explore potential mechanisms of the PI3K/Akt pathway. Male rats were divided into sedentary and exercise groups. Animals in the exercise group underwent endurance training 30 min/day, 7 days/wk, at the speed of 20 m/min on a treadmill for 1 wk. Here, we showed that exercise 1) attenuated TLR4, 2) increased PI3K/phospho-Akt (p-Akt), and 3) diminished phospho-nuclear factor-κB (p-NF-κB) expression. In addition, administration of splenocytes isolated from trained rats with LPS in vitro showed 1) reduced tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and nitric oxide secretion and 2) decreased splenocyte proliferation. The plasma corticosterone (CCS) level in the exercise group was higher than that in the sedentary group. We confirmed that CCS down-regulated TNF-α and IL-6 secretion in response to LPS in rat splenocytes. Dexamethasone also significantly attenuated LPS-evoked release of TNF-α and IL-6 in a dose-dependent manner. These findings suggested that exercise dampened the secretion of inflammation mediators probably through partial inhibition of TLR4 and p-NF-κB and activation of PI3K/p-Akt expression in the spleen.

Angiotensin II receptor blocker irbesartan attenuates cardiac dysfunction induced by myocardial infarction in the presence of renal failure.

The activity of the renin-angiotensin system is known to be a key factor in the pathophysiology of heart failure and renal failure. Irbesartan, an angiotensin II receptor blocker, has non-hemodynamic cardiovascular and renal protective effects. However, the effect of irbesartan on heart failure complicated by renal failure has not yet been elucidated. Thus the purpose of this study was to evaluate the effect of irbesartan on the pathophysiology of cardiorenal syndrome in a rat model. Subtotal nephrectomy (NTX) was performed in rats was using a two-step surgical procedure. Twenty-eight days after NTX, myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery. The animals were orally administered vehicle or irbesartan (10 mg kg(-1) day(-1)) after NTX. The hearts were harvested 28 days after MI. MI with NTX model rats showed an impaired post-MI survival rate and enhanced cardiac inflammation in comparison to MI without NTX rats. Although irbesartan treatment did not improve the survival rate, it suppressed cardiac inflammation, left ventricular function decline, cardiac fibrosis, hypertrophy of cardiomyocytes and renal fibrosis in MI with NTX rats. Moreover, increases in protein expression levels related to oxidative stress and inflammation (NADPH oxidase 4, phospho-nuclear factor-κB and phospho-c-Jun) observed in the hearts of non-treated MI with NTX rats were attenuated by irbesartan treatment. These effects of irbesartan treatment were independent of blood pressure. We conclude that irbesartan has a cardioprotective effect after MI when renal dysfunction is present.

Genetic Ablation of Extra Domain A of Fibronectin in Hypercholesterolemic Mice Improves Stroke Outcome by Reducing Thrombo-Inflammation.

The fibronectin-splicing variant containing extra domain A (Fn-EDA) is present in negligible amounts in the plasma of healthy humans but markedly elevated in patients with comorbid conditions, including diabetes mellitus and hypercholesterolemia, which are risk factors for stroke. It remains unknown, however, whether Fn-EDA worsens stroke outcomes in such conditions. We determined the role of Fn-EDA in stroke outcome in a model of hypercholesterolemia, the apolipoprotein E-deficient (Apoe(-/-)) mouse. In a transient cerebral ischemia/reperfusion injury model, Apoe(-/-) mice expressing fibronectin deficient in EDA (Fn-EDA(-/-)Apoe(-/-) mice) exhibited smaller infarcts and improved neurological outcomes at days 1 and 8 (P<0.05 versus Apoe(-/-) mice). Concomitantly, intracerebral thrombosis [assessed by fibrin(ogen) deposition] and postischemic inflammation (phospho-nuclear factor-κB p65, phospho-IκB kinase α/β, interleukin 1β, and tumor necrosis factor-α) within lesions of Fn-EDA(-/-)Apoe(-/-) mice were markedly decreased (P<0.05 versus Apoe(-/-) mice). In an FeCl3 injury-induced carotid artery thrombosis model, thrombus growth rate and the time to occlusion were prolonged in Fn-EDA(-/-)Apoe(-/-) mice (P<0.05 versus Apoe(-/-) mice). Genetic ablation of TLR4 improved stroke outcome in Apoe(-/-) mice (P<0.05) but had no effect on stroke outcome in Fn-EDA(-/-)Apoe(-/-) mice. Bone marrow transplantation experiments revealed that nonhematopoietic cell-derived Fn-EDA exacerbates stroke through Toll-like receptor-4 expressed on hematopoietic cells. Infusion of a specific inhibitor of Fn-EDA into Apoe(-/-) mouse 15 minutes after reperfusion significantly improved stroke outcome. Hypercholesterolemic mice deficient in Fn-EDA exhibit reduced cerebral thrombosis and less inflammatory response after ischemia/reperfusion injury. These findings suggest that targeting Fn-EDA could be an effective therapeutic strategy in stroke associated with hypercholesterolemia.

Ginsenoside Rg1 relieves tert-Butyl hydroperoxide-induced cell impairment in mouse microglial BV2 cells

Microglial activation plays an important role in neurodegenerative diseases associated with oxidative stress. tert-Butyl hydroperoxide (t-BHP), an analog of hydroperoxide, mimics the oxidative damage to microglial cells. It has been reported that ginsenoside Rg1 (G-Rg1), an active ingredient of Panax ginseng, has anti-stress and anti-inflammatory properties. The present study aims to investigate the ability of G-Rg1 to decrease the t-BHP-mediated cell damage of BV2 microglial cells. We performed flow cytometry assays to facilitate the detection of reactive oxygen species as well as Western blotting analyses and immunofluorescence assays using specific antibodies, such as antibodies against phospho-mitogen-activated protein kinases (p-MAPKs), phospho-nuclear factor-κB (p-NF-κB), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), Caspase-3, autophagy marker light chain 3 (LC3), and Becline-1. We found that treatment with 50 μM G-Rg1 protected microglial cells against oxidative damage induced by 10 μM t-BHP.