LPS-induced Tumor Necrosis Factor-alpha Research Articles

Increased Gene Expression of LITAF, TNF-α and BCL6 in Endometrial Tissues of Women with Endometriosis: A Case-Control Study.

Endometriosis, as a common inflammatory chronic disease is characterized by endometrial tissue growth outside the uterine cavity. It was reported that lipopolysaccharides (LPS) activate a transcription factor called LPSinduced tumor necrosis factor-alpha (LITAF) in macrophages, which induced transcription of cytokine genes such as tumor necrosis factor alpha (TNF-α). B-cell lymphoma 6 protein (BCL6) is a transcription factor which expression was increased in endometrial tissues of infertile women with endometriosis. In addition, it was shown that mRNA and protein of LITAF and BCL6 were inversely related in mature B lymphocytes and B-Cell lymphomas. Accordingly, we investigated gene expression of LITAF, BCL6 and ,TNF-α in eutopic and ectopic endometrial tissues of women with endometriosis compared to the controls. In this case-control study, 10 women with endometriosis (endometriosis group) and 10 women without endometriosis (control group) enrolled after diagnostic laparoscopy. Real-time polymerase chain reaction (PCR) technique was used to quantitatively analyze gene expression. One-Way ANOVA was used for data analysis. This study showed that LITAF gene expression was significantly higher in ectopic endometrial tissues compared to the control samples. Expression level of BCL6 gene was significantly increased in the ectopic tissues of women with endometriosis compared to the control and eutopic samples. Although TNF-ɑ gene expression was increased in the ectopic lesions compared to the eutopic and control endometrial samples, these differences were not significant. The results suggested that over-expression of these inflammatory genes in ectopic endometrial lesions can be considered as a molecular scenario in pathophysiology of endometriosis by induction of inflammatory cascades and cellular proliferation.

The Role of the TLR4-MyD88 Signaling Pathway in the Immune Response of the Selected Scallop Strain “Hongmo No. 1” to Heat Stress

The innate immunity of marine bivalves is challenged upon exposure to heat stress, especially with increases in the frequency and intensity of heat waves. TLR4 serves a classical pattern recognition receptor in recognizing pathogenic microorganisms and activating immune responses. In this study, three genes, HMTLR4, HMMyD88 and HMTRAF6, were characterized as homologs of genes in the TLR4-MyD88 signaling pathway in the selected scallop strain “Hongmo No. 1”. According to RT-PCR, acute heat stress (32 °C) inhibited genes in the TLR4-MyD88 signaling pathway, and LPS stimulation-induced activation of TLR4-MyD88 signal transduction was also negatively affected at 32 °C. ELISA showed LPS-induced tumor necrosis factor alpha (TNF-α) or lysozyme (LZM) activity, but this was independent of temperature. RNA interference (RNAi) confirmed that HMTLR4 silencing suppressed the expression of its downstream gene, whether at 24 °C or at 32 °C. The level of TNF-α and the activity of LZM also decreased after injection with dsRNA, indicating a negative effect on the innate immunity of scallops. Additionally, acute heat stress affected the suppression of downstream gene expression when compared with that at 24 °C, which led us to the hypothesis that heat stress directly influences the downstream targets of HMTLR4. These results enrich the knowledge of scallop immunity under heat stress and can be beneficial for the genetic improvement of new scallop strains with higher thermotolerance.

Protective effects of Stephania pierrei tuber-derived oxocrebanine against LPS-induced acute lung injury in mice.

Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) have high mortality rates. Though corticosteroids are commonly used for the treatment of these conditions, their efficacy has not been conclusively demonstrated and their use can induce various adverse reactions. Hence, the application of corticosteroids as therapeutic modalities for ALI/ARDS is limited. Meanwhile, the aporphine alkaloid oxocrebanine isolated from Stephania pierrei tubers has demonstrated anti-inflammatory efficacy in murine/human macrophage cell lines stimulated by lipopolysaccharide (LPS). Accordingly, the primary objectives of the present study are to investigate the anti-inflammatory effects of oxocrebanine on LPS-induced murine alveolar epithelial (MLE-12) cells and its efficacy against LPS-induced murine ALI. Results show that oxocrebanine downregulates the abundance of interleukin (IL)-1beta, IL-6, and inducible nitric oxide synthase, as well as the phosphorylation of nuclear factor-kappaB (NF-κB), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), p38, protein kinase B (Akt), and glycogen synthase kinase-3beta signalling proteins in LPS-inducedMLE-12 cells. Moreover, in a murine ALI model, oxocrebanine lowers lung injury scores and lung wet/dry weight ratios while reducing inflammatory cell infiltration. It also suppresses LPS-induced tumour necrosis factor-alpha and IL-6 in the bronchoalveolar lavage fluid and plasma. Moreover, oxocrebanine downregulates NF-κB, SAPK/JNK, p38, and Akt phosphorylation in the lung tissues of LPS-treated mice. Taken together, the foregoing results show that oxocrebanine provides significant protection against LPS-induced ALI in mice primarily by suppressing various inflammatory signalling pathways in alveolar epithelial cells and lung tissues. Hence, oxocrebanine might prove effective as an anti-inflammatory agent for the treatment of lung inflammation.

Ivacaftor therapy post myocardial infarction augments systemic inflammation and evokes contrasting effects with respect to tissue inflammation in brain and lung

Acquired cystic fibrosis transmembrane regulator (CFTR) dysfunctions have been associated with several conditions, including myocardial infarction (MI). Here, CFTR is downregulated in brain, heart, and lung tissue and associates with inflammation and degenerative processes. Therapeutically increasing CFTR expression attenuates these effects. Whether potentiating CFTR function yields similar beneficial effects post-MI is unknown. The CFTR potentiator ivacaftor is currently in clinical trials for treatment of acquired CFTR dysfunction associated with chronic obstructive pulmonary disease and chronic bronchitis. Thus, we tested ivacaftor as therapeutic strategy for MI-associated target tissue inflammation that is characterized by CFTR alterations.MI was induced in male C57Bl/6 mice by ligation of the left anterior descending coronary artery. Mice were treated with ivacaftor starting ten weeks post-MI for two consecutive weeks.Systemic ivacaftor treatment ameliorates hippocampal neuron dendritic atrophy and spine loss and attenuates hippocampus-dependent memory deficits occurring post-MI. Similarly, ivacaftor therapy mitigates MI-associated neuroinflammation (i.e., reduces higher proportions of activated microglia). Systemically, ivacaftor leads to higher frequencies of circulating Ly6C+ and Ly6Chi cells compared to vehicle-treated MI mice. Likewise, an ivacaftor-mediated augmentation of MI-associated pro-inflammatory macrophage phenotype characterized by higher CD80-positivity is observed in the MI lung. In vitro, ivacaftor does not alter LPS-induced CD80 and tumor necrosis factor alpha mRNA increases in BV2 microglial cells, while augmenting mRNA levels of these markers in mouse macrophages and differentiated human THP-1-derived macrophages.Our results suggest that ivacaftor promotes contrasting effects depending on target tissue post-MI, which may be largely dependent on its effects on different myeloid cell types.

Triterpenoid CDDO-EA inhibits lipopolysaccharide-induced inflammatory responses in skeletal muscle cells through suppression of NF-κB.

Chronic inflammation is a major contributor to the development of obesity-induced insulin resistance, which then can lead to the development of type 2 diabetes (T2D). Skeletal muscle plays a pivotal role in insulin-stimulated whole-body glucose disposal. Therefore, dysregulation of glucose metabolism by inflammation in skeletal muscle can adversely affect skeletal muscle insulin sensitivity and contribute to the pathogenesis of T2D. The mechanism underlying insulin resistance is not well known; however, macrophages are important initiators in the development of the chronic inflammatory state leading to insulin resistance. Skeletal muscle consists of resident macrophages which can be activated by lipopolysaccharide (LPS). These activated macrophages affect myocytes via a paracrine action of pro-inflammatory mediators resulting in secretion of myokines that contribute to inflammation and ultimately skeletal muscle insulin resistance. Therefore, knowing that synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acids (CDDOs) can attenuate macrophage pro-inflammatory responses in chronic disorders, such as cancer and obesity, and that macrophage pro-inflammatory responses can modulate skeletal muscle inflammation, we first examined whether CDDO-ethyl amide (CDDO-EA) inhibited chemokine and cytokine production in macrophages since this had not been reported for CDDO-EA. CDDO-EA blocked LPS-induced tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukine-1beta (IL-1β), and interleukine-6 (IL-6) production in RAW 264.7 mouse and THP-1 human macrophages. Although many studies show that CDDOs have anti-inflammatory properties in several tissues and cells, little is known about the anti-inflammatory effects of CDDOs on skeletal muscle. We hypothesized that CDDO-EA protects skeletal muscle from LPS-induced inflammation by blocking nuclear factor kappa B (NF-κB) signaling. Our studies demonstrate that CDDO-EA prevented LPS-induced TNF-α and MCP-1 gene expression by inhibiting the NF-κB signaling pathway in L6-GLUT4myc rat myotubes. Our findings suggest that CDDO-EA suppresses LPS-induced inflammation in macrophages and myocytes and that CDDO-EA is a promising compound as a therapeutic agent for protecting skeletal muscle from inflammation.

Anti-Inflammatory Effects of Mitrephora sirikitiae Leaf Extract and Isolated Lignans in RAW 264.7 Cells.

Mitrephora sirikitiae Weeras., Chalermglin & R.M.K. Saunders has been reported as a rich source of lignans that contribute to biological activities and health benefits. However, cellular anti-inflammatory effects of M. sirikitiae leaves and their lignan compounds have not been fully elucidated. Therefore, this study aimed to investigate the anti-inflammatory activities of methanol extract of M. sirikitiae leaves and their lignan constituents on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 mouse macrophage cells. Treatment of RAW 264.7 cells with the methanol extract of M. sirikitiae leaves and its isolated lignans, including (−)-phylligenin (2) and 3′,4-O-dimethylcedrusin (6) significantly decreased LPS-induced prostaglandin E2 (PGE2) and nitric oxide (NO) productions. These inhibitory effects of the extract and isolated lignans on LPS-induced upregulation of PGE2 and NO productions were derived from the suppression of cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) production, respectively. In addition, treatment with 2-(3,4-dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (3) and mitrephoran (5) was able to suppress LPS-induced tumor necrosis factor alpha (TNF-α) secretion and synthesis in RAW 264.7 cells. These results demonstrated that M. sirikitiae leaves and some isolated lignans exhibited potent anti-inflammatory activity through the inhibition of secretion and synthesis of PGE2, NO, and TNF-α.

Lupalbigenin Inhibiting NF-κB Translocation Associated with Anti-Inflammatory Responses in Lipopolysaccharide Stimulated RAW 264.7 Macrophages

Background: The dried stem of Derris scandens Benth. is well known as an Asian medicinal plant and is used for a variety of ailments. It is claimed to used for the relief of muscle aches and pain and revealed the presence of compounds that appear to modify inflammatory processes. Chemical analysis of D. scandens have revealed the presence of numerous isoflavone derivatives. Lupalbigenin, prenylated isoflavone is a key component of D. scandens stem ethanolic extract. Its anti-inflammatory activity in cell culture had never previously been reported which is why this study was performed. Objective: To investigate the inflammatory activity through molecular signaling pathways of lupalbigenin from D. scandens aqueous ethanol extract using LPS-induced cell changed in Raw 264.7 macrophages cells. Materials and Methods: Lupalbigenin was purified from aqueous extract of dried stem of D. scandens. Cell viability of lupalbigenin in Raw 264.7 cell was determined by MTT assay. Griess reagent has been used for nitric oxide determination. The protein expression of iNOS, cyclooxygenase-2 (COX-2), TNF-α, NF-κB and MAPK were performed by Western blotting. In addition, we also used immunofluorescent assay to examine the NF-κB translocation from nucleus to cytoplasm. Results: Lupalbigenin at 1.25 and 2.5 mM effectively inhibited the LPS-induced tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) as well as nuclear factor kappa B (NF-κB). In addition, lupalbigenin prevented LPSinduced inflammation by decreasing p38 and JNK expression in mitogen-activated protein kinase (MAPK) pathway. Conclusion: Lupalbigenin at low concentrations showed down-regulation of inflammatory gene and protein expressions as well as inhibiting NF-κB translocation. Lupalbigenin could be used as an anti-inflammatory agent. Keywords: Lupalbigenin; Anti-inflammatory activity; Nitric oxide (NO); NF-κB translocation

Design, Synthesis, Molecular Modelling, and Biological Evaluation of Oleanolic Acid-Arylidene Derivatives as Potential Anti-Inflammatory Agents.

IntroductionOleanolic acid, a pentacyclic triterpenic acid, is widely distributed in medicinal plants and is the most commonly studied triterpene for various biological activities, including anti-allergic, anti-cancer, and anti-inflammatory.MethodsThe present study was carried out to synthesize arylidene derivatives of oleanolic acid at the C-2 position by Claisen Schmidt condensation to develop more effective anti-inflammatory agents. The derivatives were screened for anti-inflammatory activity by scrutinizing NO production inhibition in RAW 264.7 cells induced by LPS and their cytotoxicity. The potential candidates were further screened for inhibition of LPS-induced interleukin (IL-6) and tumour necrosis factor-alpha (TNF-α) production in RAW 264.7 cells.ResultsThe results of in vitro studies revealed that derivatives 3d, 3e, 3L, and 3o are comparable to that of the oleanolic acid on the inhibition of TNF-α and IL-6 release. However, derivative 3L was identified as the most potent inhibitor of IL-6 (77.2%) and TNF-α (75.4%) when compared to parent compound, and compounds 3a (77.18%), 3d (71.5%), and 3e (68.8%) showed potent inhibition of NO than oleanolic acid (65.22%) at 10µM. Besides, from docking score and Cyscore analysis analogs (3e, 3L, 3n) showed greater affinity towards TNF-α and IL-1β than dexamethasone.ConclusionHerein, we report a series of 15 new arylidene derivatives of oleanolic acid by Claisen Schmidt condensation reaction. All the compounds synthesized were screened for their anti-inflammatory activity against NO, TNF-α and IL-6. From the data, it was evident that most of the compounds exhibited better anti-inflammatory activity.

Rutin attenuates inflammatory responses induced by lipopolysaccharide in an in vitro mouse muscle cell (C2C12) model

White striping (WS) and woody breast (WB) are 2 of the major myopathies in the modern poultry industry. Even though the exact etiology for WS and WB is still unknown, differentially expressed genes in broiler breast muscle affected by WS and WB indicate that oxidative stress and inflammation could be involved in their occurrences. Therefore, it is very important to identify natural compounds with anti-oxidative stress and anti-inflammation properties that can reduce the occurrences of WS and WB in broiler chickens. Rutin is a polyphenol antioxidant that has been reported to be present in several plant extracts. In the current study, we established an in vitro inflammation model by using mouse muscle cells (C2C12) and evaluated the effects of rutin on lipopolysaccharide (LPS)-induced inflammatory responses in the muscle cells. Interleukin 6 (IL-6) secretion was measured by enzyme-linked immunosorbent assay. The mRNA abundance of cytokines and inducible nitric oxide synthase (iNOS) was measured by real-time PCR. Nuclear factor kappa B (NF-κB) activation was detected by electrophoretic mobility shift assay. The results showed that LPS (25ng/ml) stimulation quickly activated NF-κB and induced significant IL-6 expression on both mRNA and protein levels (P < 0.05) in cells when compared with control cells without the LPS treatment. The rutin treatment decreased IL-6 mRNA abundance induced by LPS in a dose-dependent manner (P < 0.05). LPS-induced tumor necrosis factor-alpha and iNOS gene expression was significantly attenuated by 100μM of rutin (P < 0.05). Moreover, LPS induced reactive oxygen species (ROS) production, and NF-κB activation was significantly blocked by 100μM of rutin. These results suggest that rutin can attenuate LPS-induced inflammation in muscle cells and supplementation of rutin or rutin-containing plant extracts may present a promising approach to control WS and WB in broiler chickens.

Forsythiaside attenuates Escherichia coli lipopolysaccharide-induced liver acute inflammatory response in chicken

This study investigated the effects of forsythiaside on the acute inflammatory response induced by Escherichia coli lipopolysaccharide (LPS) in liver of broiler chickens. Fifteen-day-old chickens were randomly assigned to three groups (n = 20 for each group, orally treated with 0, 30, or 60 mg/kg BW of forsythiaside) for 7 days. At 21 days of age, the chickens were intravenously injected with either LPS (200 μg/kg BW) or sterile saline (200 μg/kg BW, control group). All the chickens were humanely euthanized by cervical dislocation 2 h after the LPS injection. The results showed that the injection of LPS induced some indexes, including total proteins, nitric oxide (NO), interleukin-1beta (IL-1β), interleukin-6 (IL-6), and interleukin-17 (IL-17) production ( P &lt; 0.05) and increased the mRNA expression of LPS-induced tumor necrosis factor-alpha (LITAF), IL-1β, IL-17, IL-6, and inducible nitric oxide synthase (iNOS) ( P &lt; 0.05). Forsythiaside supplementation alleviated the LPS-induced inflammatory response by inhibiting the production of total proteins, NO, LITAF, IL-1β, IL-17, and IL-6 and down-regulating the mRNA expression of pro-inflammatory cytokines and iNOS. In conclusion, forsythiaside is a potential treatment for LPS-induced liver acute inflammation in chicken.

Chelerythrine Attenuates the Inflammation of Lipopolysaccharide-Induced Acute Lung Inflammation Through NF-κB Signaling Pathway Mediated by Nrf2.

Chelerythrine (CH), is a kind of benzo[c] phenanthridine alkaloid isolated from plants such as Chelidonium, with pharmacological activities as antitumor, antibiosis and anti-inflammation. However, few studies have demonstrated whether CH could protect against lipopolysaccharide (LPS)-induced acute lung injury (ALI), and the underlying mechanism is also uncertain. The purpose of the present study was to investigate the anti-inflammatory effects of CH on LPS-induced ALI in mice and in RAW264.7 cells. In this study, we demonstrated that treatment with CH significantly ameliorated LPS-induced pathological changes in the lung. CH also attenuated LPS-induced W/D ratio, inflammatory cell infiltration. Meanwhile, LPS-induced Tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1β (IL-1β) production and oxidative stress were markedly suppressed by CH. Furthermore, western blot showed that CH suppressed LPS-stimulated inflammation of RAW264.7 cells through activation of nuclear factor kappa-B (NF-κB) pathway. Knocking down of nuclear factor erythroid 2-related factor 2 (Nrf2) led to the reduction of nuclear translocation of the NF-κB p65, which triggered inflammation. These experimental results provided evidence that CH could be a potential therapeutic candidate for the intervention of ALI caused by LPS.

IN VITRO ANTI-INFLAMMATORY ACTIVITY OF THE COMPONENTS OF AMOMUM TSAO-KO IN MURINE MACROPHAGE RAW 264.7 CELLS

Background: Plants still remain the prime source of drugs for the treatment of inflammation and can provide leads for the development of novel anti-inflammatory agents. &#x0D; Material and methods: An in vitro bioassay guide revealed that the 80% ethanol (EtOH) extract of the whole plant, Amomum tsao-ko (Zingiberaceae), displayed anti-inflammatory activity after assessing its effects on murine macrophage RAW 264.7 cells. &#x0D; Result: Phytochemical study of the 80% EtOH extract of Amomum tsao-ko led to the isolation of eight compounds: 4-hydroxy-3-methoxy-benzoic acid (1), meso-hannokinol (2), (+)-hannokinol (3), coumaric acid (4), 4-hydroxy-benzoic acid (5), (+)-epicatechin (6), (-)-catechin (7), and myrciaphenone A (8). The results indicated that two of the isolated components, (+)-epicatechin (6) and (-)-catechin (7), inhibited the production of nitric oxide (NO) significantly in lipopolysaccharide treated RAW 264.7 cells. &#x0D; Conclusion: LPS-induced interleukin tumor necrosis factor-alpha (TNF-), IL-1β and IL-10 production was also decreased in a dose-dependent manner. In addition, western blot analysis revealed that (+)-epicatechin (6) and (-)-catechin (7) reduced the expression of inducible nitric oxide synthase and inhibited nuclear localization of nuclear factor kappa-B (NF-κB).

Encapsulated Hsp70 decreases endotoxin-induced production of ROS and TNFα in human phagocytes.

Human heat shock protein Hsp70 was experimentally inserted into polyelectrolyte microcapsules. Encapsulated recombinant Hsp70 was studied in terms of its effects on neutrophil apoptosis, the production of reactive oxygen species, and the secretion of tumor necrosis factor alpha by promonocytic THP-1 cells. It was found that encapsulated Hsp70 effectively inhibits neutrophil apoptosis, unlike free exogenous protein used in solution. In THP-1 cells, encapsulated and free Hsp70 reduced LPS-induced tumor necrosis factor alpha production with a similar efficiency. Encapsulated Hsp70 reduces LPS-induced reactive oxygen species production by neutrophils in the course of its release from the microcapsules but not as much as free Hsp70. Thus, the polyelectrolyte microcapsules can be used as containers for the effective delivery of Hsp70 to neutrophils and monocytes to significantly improve the functioning of the innate immune system.

Novel factors of Anopheles gambiae haemocyte immune response to Plasmodium berghei infection.

BackgroundInsect haemocytes mediate cellular immune responses (e.g., phagocytosis) and contribute to the synthesis of humoral immune factors. In previous work, a genome-wide molecular characterization of Anopheles gambiae circulating haemocytes was followed by functional gene characterization using cell-based RNAi screens. Assays were carried out to investigate the role of selected haemocyte-specific or enriched genes in phagocytosis of bacterial bio-particles, expression of the antimicrobial peptide cecropin1, and basal and induced expression of the mosquito complement factor LRIM1 (leucine-rich repeat immune gene I).FindingsHere we studied the impact of a subset of genes (37 candidates) from the haemocyte-specific dsRNA collection on the development of Plasmodium in the mosquito by in vivo gene silencing. Our screening identifies 10 novel factors with a role in the mosquito response to Plasmodium. Analysis of in vivo screening phenotypes reveals a significant anti-correlation between the prevalence of oocysts and melanised ookinetes.ConclusionsAmong novel immune genes are putative pattern recognition proteins (leucine-rich repeat, fibrinogen-domain and R-type lectins), immune modulation and signalling proteins (LPS-induced tumor necrosis factor alpha factor, LITAF and CLIP proteases), and components of extracellular matrix such as laminin and collagen. Additional identified proteins such as the storage protein hexamerin and vesicular-type ATPase (V-ATPase) are associated for the first time with the mosquito response against Plasmodium.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1359-y) contains supplementary material, which is available to authorized users.

Sesamin ameliorates lipopolysaccharide/d-galactosamine-induced fulminant hepatic failure by suppression of Toll-like receptor 4 signaling in mice

Sesamin has been described to exert anti-oxidant and anti-inflammatory properties. In present study, we investigated the potential effects and mechanisms of sesamin on lipopolysaccharide (LPS)-induced fulminant hepatic failure (FHF) in d-galactosamine (D-GalN)-sensitized mice. Our results showed that pretreatment with sesamin dose-dependently improved LPS/D-GalN-induced mortality and liver injury as indicated by reduced serum levels of aminotransferases and alleviated pathological damage as well as hepatocyte apoptosis in mice. Additionally, sesamin markedly attenuated LPS/D-GalN-induced adhesion molecules expression, and decreased neutrophils recruitment. Furthermore, sesamin inhibited LPS-induced tumor necrosis factor-alpha (TNF-α) production, p38 mitogen-activated protein kinases (MAPK) and NF-κB activation, and Toll like receptor (TLR) 4 expression in mice and in RAW264.7 macrophage cells. In summary, these results demonstrate that sesamin protects mice from LPS-induced FHF and the molecular mechanisms may down-regulate the expression of TLR4, block MAPK and NF-κB activation, decrease the production of TNF-α.

Hepatotoxicants induce cytokine imbalance in response to innate immune system

In recent years, attention has been paid to innate immune systems as mechanisms to initiate or promote drug-induced liver injury (DILI). Kupffer cells are hepatic resident macrophages and might be involved in the pathogenesis of DILI by release of pro- and anti-inflammatory mediators such as cytokines, chemokines, reactive oxygen species, and/or nitric oxides. The purpose of this study was to investigate alterations in mediator levels induced by hepatotoxic compounds in isolated Kupffer cells and discuss the relation between balance of each cytokine or chemokine and potential of innate immune-mediated DILI. Primary cultured rat Kupffer cells were treated with hepatotoxic (acetaminophen, troglitazone, trovafloxacin) or non-hepatotoxic (pioglitazone, levofloxacin) compounds with or without lipopolysaccharide (LPS). After 24 hr treatment, cell supernatants were collected and various levels of mediators released by Kupffer cells were examined. Although hepatotoxicants had no effect on the LPS-induced tumor necrosis factor-alpha (TNF-α) secretion, they enhanced the release of pro-inflammatory cytokine interleukin-1 beta (IL-1β) and suppressed the anti-inflammatory cytokines interleukin-6 (IL-6) and interleukin-10 (IL-10) induced by LPS. These cytokine shifts were not associated with switching the phenotypes of M1 and M2 macrophages in Kupffer cells. In conclusion, the present study suggested that the levels of some specific cytokines are affected by DILI-related drugs with LPS stimulation, and imbalance between pro- and anti-inflammatory cytokines, induced by the up-regulation of IL-1β and the down-regulation of IL-6 or IL-10, plays a key role in innate immune-mediated DILI.

Oleanolic acid analogs as NO, TNF-α and IL-1β inhibitors: Synthesis, biological evaluation and docking studies

A series of oleanolic acid analogs, characterized by structural modifications at position C-3 and C-28 of oleanane skeleton were synthesized and assessed for antiinflammatory potential towards lipopolysaccharide (LPS) induced nitric oxide (NO) production in macrophages. Results revealed that all the synthesized analogs of oleanolic acid inhibit NO production with an IC50 of 2.66–41.7μM as compared to the specific nitric oxide synthase (NOS) inhibitor, L-NAME (IC50=69.21 and 73.18μM on RAW 264.7 and J774A.1 cells, respectively) without affecting the cell viability when tested at their half maximal concentration. The most potent NO inhibitors (2, 8, 9 and 10) at a concentration of 20μg/mL also demonstrated mild inhibition (27.9–51.9%) of LPS-induced tumor necrosis factor alpha (TNF-α) and weak inhibition (11.1–37.5%) towards interleukin 1-beta (IL-1β) production in both the cells. The present study paves a direction that analogs of oleanolic acid can be employed as a lead in the development of potent NO inhibitors. Molecular docking studies also showed that 10 (with top Goldscore docking pose 19.05) showed similar interaction as that of co-crystallized inhibitor and, thereby, helps to design the potent inhibitors of TNF-α.

Synthesis of new heterocyclic lupeol derivatives as nitric oxide and pro-inflammatory cytokine inhibitors

A series of heterocyclic derivatives including indoles, pyrazines along with oximes and esters were synthesized from lupeol and evaluated for anti-inflammatory activity through inhibition of lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW 264.7 and J774A.1 cells. All the synthesized molecules of lupeol were found to be more active in inhibiting NO production with an IC50 of 18.4–48.7μM in both the cell lines when compared to the specific nitric oxide synthase (NOS) inhibitor, L-NAME (IC50=69.21 and 73.18μM on RAW 264.7 and J774A.1 cells, respectively). The halogen substitution at phenyl ring of indole moiety leads to potent inhibition of NO production with half maximal concentration ranging from 18.4 to 41.7μM. Furthermore, alkyl (11, 12) and p-bromo/iodo (15, 16) substituted compounds at a concentration of 20μg/mL exhibited mild inhibition (29–42%) of LPS-induced tumor necrosis factor alpha (TNF-α) and weak inhibition (10–22%) towards interleukin 1-beta (IL-1β) production in both the cell lines. All the derivatives were found to be non-cytotoxic when tested at their IC50 (μM). These findings suggest that the derivatives of lupeol could be a lead to potent inhibitors of NO.

Pharmacokinetic and Anti-inflammatory Effects of Sanguinarine Solid Lipid Nanoparticles

The sanguinarine (SG) was studied for its pharmacokinetic and anti-inflammatory activities with prepared solid lipid nanoparticles (SLNs). The sanguinarine solid lipid nanoparticles (SG-SLNs) were prepared by film-ultrasonic dispersion method and the entrapment efficiency of SG was higher at 75.6 %. The drug release profile of SG was examined in pH 7.4 PBS and 85 % of the SG loaded in SLNs was gradually released during 24 h. We used mice endotoxin shock model which was induced by lipopolysaccharide (1 mg/kg) to examine the anti-inflammatory function of SG-SLNs. Healthy Kunming mice were administered orally with saline, SG (10 mg/kg), and SG-SLNs (10 mg/kg), respectively, at 12 and 1 h before lipopolysaccharide (LPS) injection. Mice were sacrificed at 1 and 6 h, respectively, and blood was collect through the venous sinus to access inflammatory mediators. Pharmacokinetic studies proved that the AUC(0→24) and C(max) of SG-SLNs were significantly increased compared that of SG. SG-SLNs revealed significant anti-inflammatory effects through inhibition of LPS-induced tumor necrosis factor-alpha level, interleukin 6 level, and nitric oxide production in serum. Therefore, it can be concluded that SG-SLNs led to a better oral bioavailability.

AChE and RACK1 Promote the Anti-Inflammatory Properties of Fluoxetine

Selective serotonin reuptake inhibitors (SSRIs) show anti-inflammatory effects, suggesting a possible interaction with both Toll-like-receptor 4 (TLR4) responses and cholinergic signaling through as yet unclear molecular mechanism(s). Our results of structural modeling support the concept that the antidepressant fluoxetine physically interacts with the TLR4-myeloid differentiation factor-2 complex at the same site as bacterial lipopolysaccharide (LPS). We also demonstrate reduced LPS-induced pro-inflammatory interleukin-6 and tumor necrosis factor alpha in human peripheral blood mononuclear cells preincubated with fluoxetine. Furthermore, we show that fluoxetine intercepts the LPS-induced decreases in intracellular acetylcholinesterase (AChE-S) and that AChE-S interacts with the nuclear factor kappa B (NFκB)-activating intracellular receptor for activated C kinase 1 (RACK1). This interaction may prevent NFκB activation by residual RACK1 and its interacting protein kinase PKCβII. Our findings attribute the anti-inflammatory properties of SSRI to surface membrane interference with leukocyte TLR4 activation accompanied by intracellular limitation of pathogen-inducible changes in AChE-S, RACK1, and PKCβII.