TLR4-IN-C34 Inhibits Lipopolysaccharide-Stimulated Inflammatory Responses via Downregulating TLR4/MyD88/NF-κB/NLRP3 Signaling Pathway and Reducing ROS Generation in BV2 Cells.

TLR4 signal activated by lipopolysaccharide (LPS) is involved in the pathological process of the central nervous system (CNS) diseases and the suppression of TLR4 signal may become an effective treatment. TLR4-IN-C34, a TLR4 inhibitor, is expected to become a candidate compound with anti-neuroinflammatory response. In the present study, the anti-neuroinflammatory effects and possible mechanism of TLR4-IN-C34 were investigated in BV2 microglia cells stimulated by LPS. The results showed that TLR4-IN-C34 decreased the levels of pro-inflammatory factors and chemokines including NO, TNF-α, IL-1ß, IL-6, and MCP-1 in the supernatant of LPS-stimulated BV2 cells. Further research indicated that TLR4-IN-C34 suppressed the expression or phosphorylation levels of inflammatory proteins regarding TLR4/MyD88/NF-κB/NLRP3 signaling pathway. In addition, TLR4-IN-C34 reduced ROS production in BV2 cells after LPS treatment. In conclusion, our findings suggest that anti-neuroinflammatory activity of TLR4-IN-C34 may be interrelated to the inhibition of TLR4/MyD88/NF-κB/NLRP3 signaling pathway and reduction of ROS generation.

RKC-B1 Blocks Activation of NF-κB and NLRP3 Signaling Pathways to Suppress Neuroinflammation in LPS-Stimulated Mice.

RKC-B1 is a novel fermentation product obtained from the marine micromonospora FIM02-523A. Thus far, there have been few reports about the pharmacological activity of RKC-B1. In our present study, we investigated the anti-neuroinflammatory effects and the possible mechanism of RKC-B1 in LPS-stimulated mice. After treatment with RKC-B1, RNA-seq transcriptome of the cerebral cortex tissue was conducted to find the differentially expressed genes (DEGs). Inflammatory cytokines and proteins were evaluated by ELISA and WB. In RNA-seq analysis, there were 193 genes screened as core genes of RKC-B1 for treatment with neuroinflammation. The significant KEGG enrichment signaling pathways of these core genes were mainly included TNF signaling pathway, IL-17 signaling pathway, NOD-like receptor signaling pathway, NF-κB signaling pathway and others. The corresponding top five KEGG enrichment pathways of three main clusters in PPI network of core genes were closely related to human immune system and immune disease. The results showed that RKC-B1 reduced the levels of pro-inflammatory factors (IL-6, IL-1ß, MCP-1, and ICAM-1) and the expression of COX2 in cerebral cortex tissue. Additionally, we found that the anti-neuroinflammation activity of RKC-B1 might be related to suppress activating of NF-κB and NLRP3/cleaved caspase-1 signaling pathways. The current findings suggested that RKC-B1 might be a promising anti-neuroinflammatory agent.

Circ-RNF111 aggravates the malignancy of gastric cancer through miR-876-3p-dependent regulation of KLF12.

BACKGROUND: The aberrant expression of circular RNAs (circRNAs) plays vital roles in the advancement of human cancers, including gastric cancer (GC). In this study, the functions of circRNA ring finger protein 111 (circ-RNF111) in GC were investigated. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed for the levels of circ-RNF111, microRNA-876-3p (miR-876-3p) and krueppel-like factor 12 (KLF12) mRNA. RNase R assay was conducted for the feature of circ-RNF111. Cell Counting Kit-8 (CCK-8) assay, colony formation assay, wound-healing assay, and transwell assay were applied for cell viability, colony formation, migration, and invasion, respectively. Flow cytometry analysis was used to analyze cell apoptosis and cell cycle process. The glycolysis level was examined using specific commercial kits. Western blot assay was carried out to measure the protein levels of hexokinase 2 (HK-2) and KLF12. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to verify the combination between miR-876-3p and circ-RNF111 or KLF12. Murine xenograft model was constructed for the role of circ-RNF111 in vivo. Immunohistochemistry (IHC) was used for KLF12 level. RESULTS: Circ-RNF111 was higher expressed in GC tissues and cells than normal tissues and cells. Silencing of circ-RNF111 restrained cell viability, colony formation, migration, invasion, cell cycle process and glycolysis and induced apoptosis in GC cells in vitro. Circ-RNF111 positively regulated KLF12 expression via absorbing miR-876-3p. MiR-876-3p downregulation reversed the impacts of circ-RNF111 silencing on GC cell malignant phenotypes. MiR-876-3p overexpression repressed GC cell growth, metastasis and glycolysis, inhibited apoptosis and arrested cell cycle, while KLF12 elevation weakened the effects. Besides, circ-RNF111 knockdown inhibited tumor growth in vivo. CONCLUSION: Circ-RNF111 knockdown relieved the development of GC by regulating miR-876-3p/KLF12 axis.

Chrysomycin A Attenuates Neuroinflammation by Down-Regulating NLRP3/Cleaved Caspase-1 Signaling Pathway in LPS-Stimulated Mice and BV2 Cells.

Chrysomycin A (Chr-A), an antibiotic chrysomycin, was discovered in 1955 and is used to treat cancer and tuberculosis. In the present study, the anti-neuroinflammatory effects and possible mechanism of Chr-A in BALB/c mice and in BV2 microglia cells stimulated by lipopolysaccharide (LPS) were investigated. Firstly, the cortex tissues of mice were analyzed by RNA-seq transcriptome to identify differentially expressed genes (DEGs) regulated by Chr-A in LPS-stimulated mice. Inflammatory cytokines and inflammatory proteins were detected by enzyme-linked immunosorbent assay and Western blot. In RNAseq detection, 639 differential up-regulated genes between the control group and LPS model group and 113 differential down-regulated genes between the LPS model group and Chr-A treatment group were found, and 70 overlapping genes were identified as key genes for Chr-A against neuroinflammation. Subsequent GO biological process enrichment analysis showed that the anti-neuroinflammatory effect of Chr-A might be related to the response to cytokine, cellular response to cytokine stimulus, and regulation of immune system process. The significant signaling pathways of KEGG enrichment analysis were mainly involved in TNF signaling pathway, cytokine-cytokine receptor interaction, NF-κB signaling pathway, IL-17 signaling pathway and NOD-like receptor signaling pathway. Our results of in vivo or in vitro experiments showed that the levels of pro-inflammatory factors including NO, IL-6, IL-1ß, IL-17, TNF-α, MCP-1, CXCL12, GM-CSF and COX2 in the LPS-stimulated group were higher than those in the control group, while Chr-A reversed those conditions. Furthermore, the Western blot analysis showed that its anti-neuroinflammation appeared to be related to the down-regulation of NLRP3/cleaved caspase-1 signaling pathway. The current findings provide new insights into the activity and molecular mechanisms of Chr-A for the treatment of neuroinflammation.

Pinocembrin Ameliorates Cognitive Impairment Induced by Vascular Dementia: Contribution of Reelin-dab1 Signaling Pathway.

BACKGROUND: As a substrate of apoER2, Reelin has been verified to exert neuroprotection by preventing memory impairment. Pinocembrin is the most abundant natural flavonoid found in propolis, and it has been used to exert neuroprotection, blood-brain barrier protection, anti-oxidation, and inflammation diminishing, both in vitro and in vivo. However, the roles and molecular mechanisms of pinocembrin in neurobehavioral outcomes and neuronal repair after vascular dementia are still under investigation. PURPOSE: To explore the role of pinocembrin in the involvement of the Reelin-dab1 signaling pathway in improving memory impairment, both in cell culture and animals experiments. MATERIAL AND METHODS: Behavioral tests were conducted on day 48 to confirm the protection of pinocembrin against cognitive impairment. Cell and molecular biology experiments demonstrated that the Reelin-dab1 pathway mediates the underlying mechanism of cognitive improvement by pinocembrin. RESULTS: It was showed that pinocembrin alleviated learning and memory deficits induced by vascular dementia, by inducing the expression of Reelin, apoER2, and p-dab1 in the hippocampus. The expression of Reelin and p-dab1 was both inhibited following Reelin RNA interference in SH-SY5Y prior to oxygen glucose deprivation (OGD) injury, suggesting that Reelin played a core role in pinocembrin's effect on OGD in vitro. CONCLUSION: Pinocembrin improves the cognition via the Reelin-dab1 signaling pathway.

Baicalein attenuates caspase-independent cells death via inhibiting PARP-1 activation and AIF nuclear translocation in cerebral ischemia/reperfusion rats.

It is reported that baicalein can activate PI3K/AKT pathway, inhibit caspase activation and reduce cerebral infarct volume in middle cerebral artery occlusion (MCAO) rats. However, a caspase-independent mechanism initiated by poly (ADP-ribose) polymerase-1 (PARP-1) activation has been reported to make more contribution to cells death after ischemic stroke. In the present study, we established a cerebral ischemia/reperfusion (I/R) rat model through middle cerebral artery occlusion following reperfusion to investigate the mechanisms of ischemic tissue recovery following baicalein treatment. The data showed that baicalein treatment at dose of 100 mg/kg for 7 days significantly inhibited the release of cytokines, activation of PARP-1, nuclear translocation of apoptosis-inducing factor (AIF) and macrophage migration inhibitory factor (MIF) in cerebral I/R rats, therefore decreased cerebral infarct volume and neurological scores. Then, we further investigated the signal transduction mechanisms of ischemic tissue protection by baicalein in vitro. Following oxygen and glucose deprivation (OGD) in SH-SY5Y cells, the mitochondrial AIF was translocated into nucleus after 12 h. The co-immunoprecipitation analysis showed that the interaction between AIF and MIF was activated by OGD and subsequently resulted in MIF nuclear translocation. Also, the baicalein inhibited apoptosis, reduced oxidative stress, protected mitochondrial function and restored mitochondrial membrane potential in OGD cells. The results obtained from both in vivo and in vitro study demonstrated the PARP-1/AIF pathway involved in mechanisms of baicalein to protect the cerebral tissues from ischemic injury.

Dynamic Alterations of Brain Injury, Functional Recovery, and Metabolites Profile after Cerebral Ischemia/Reperfusion in Rats Contributes to Potential Biomarkers.

Cerebral ischemia-reperfusion (I/R) is characterized by initial transient cerebral ischemia followed by reperfusion. Various pathophysiological processes are involved in brain injury and functional recovery during cerebral I/R. There are few studies on dynamic metabolic process after cerebral I/R. The present study was to observe dynamic alteration of brain injury, functional recovery, and metabolites after cerebral I/R in rats and discover potential metabolic markers. The cerebral I/R model was established by middle cerebral artery occlusion (MCAO) for 90 min, following reperfusion in rats. The results of cerebral infarction area, cerebral edema, and behavior test showed that there were dynamic changes in brain injury and functional recovery at different periods after cerebral I/R. Further analysis showed that the brain injury was severe on the first day of cerebral I/R, and there was a significant functional recovery from the 7th day of cerebral I/R, followed by an aggravation trend of brain injury from the days 7 to 28. Furthermore, Matrix-assisted laser desorption ionization mass spectrometry imaging analysis showed that the expression of ATP, glucose, and citric acid on 7th day was the highest during cerebral I/R, which indicated that energy metabolism and oxidative phosphorylation played important roles during cerebral I/R. In addition, the untargeted metabolomic results showed that the level of isocitric acid, the ratio of oxyglutaric acid/glutamic acid, and the level of pyruvic acid associated with the TCA cycle were also the highest on the 7th day during cerebral I/R, which indicated that the transient spontaneous recovery of ischemic brain on the 7th day after ischemia-reperfusion might be related to oxidative phosphorylation and energy metabolism in the brain in this period. In conclusion, the results suggest that some small molecule metabolites participate in the brain injury and functional recovery during cerebral I/R, which is of great significance to the development of therapeutic drugs and diagnostic markers.

Cerebral ischemia-reperfusion aggravated cerebral infarction injury and possible differential genes identified by RNA-Seq in rats.

Numerous studies have shown that local excessive inflammatory response in brain tissue was an important pathogenesis of secondary injury following cerebral ischemia-reperfusion (I/R). However, the inflammatory-related targets and pathways after cerebral I/R injury are still unclear. This study was to investigate possible targets and mechanisms after cerebral I/R injury. Rats were subjected to transient or permanent middle cerebral artery occlusion (MCAO). Neurological deficit scores test was used to evaluate neurological function. Cerebral infarction was evaluated by MRI, TTC staining and Nissl staining. Microglia activation was detected by immunofluorescence using Iba-1 antibody. Inflammatory factors were detected by ELISA assay. RNA-sequencing transcriptome analysis was processed and the differential genes were verified by real-time quantitative PCR (qPCR) and western blotting. The results showed that neurological function of rats in I/R group was more severe than that in I group on the 7th after cerebral I/R. Therefore, the differences between cerebral ischemia and cerebral I/R for 7 days were studied in further study. The results showed that the levels of pro-inflammatory factors in I/R group were higher and the levels of anti-inflammatory factors were lower than those in I group. KEGG pathway and gene network enrichment analysis revealed that some common differential up- and down-regulated genes were involved in most of significant pathways. These common differential up-regulated genes belonged to TLR4/MYD88 inflammatory signaling pathway and common differential down-regulated genes belonged to HRAS/RAF1 neurotrophic signaling pathway. Interestingly, according to the genetic interaction analysis of string database, these up-regulated differential genes might promote the development of inflammation, while the down-regulated differential genes might inhibit the development of inflammation. Furthermore, qPCR and WB results verified that these pro-inflammatory genes in the I/R group were higher than those in the I group, while possible anti-inflammatory genes in the I/R group were lower than those in the I group. It is concluded that TLR4/MYD88 inflammatory signaling pathway and HRAS/RAF1 neurotrophic signaling pathway may play different roles after cerebral I or I/R and may be therapeutic targets for stroke recovery.

Myricitrin blocks activation of NF-κB and MAPK signaling pathways to protect nigrostriatum neuron in LPS-stimulated mice.

Myricitrin, a bioactive and natural flavonoids, is well known for its anti-inflammatory and antioxidant properties. However, the anti-neuroinflammation and possible mechanism has not been fully elucidated. Therefore, the present study was to investigate the possible mechanism of its neuroprotection and anti-neuroinflammation in the nigrostriatum of LPS-stimulated mice. The results showed that myricitrin improved neuron injury and raised the expressions of PSD-95 protein and TH protein in the nigrostriatum of LPS-stimulated mice. In addition, myricitrin decreased the production of pro-inflammatory factors including IL-1ß, IL-6 and TNFα, decreased the level of chemokine MCP-1, and suppressed the expressions of COX-2 and iNOS. Meanwhile, myricitrin suppressed HMGB1, TLR4, and MyD88 expression in the nigrostriatum of LPS-stimulated mice. Furthermore, myricitrin inhibited NF-κB and MAPK signaling pathways activated by LPS. In conclusion, our studies suggest that myricitrin blocks activation of protects NF-κB and MAPK signaling pathways to nigrostiatum neuron from injury in LPS-stimulated mice and is beneficial to treatment nigrostriatum inflammation of PD.

Kaempferol attenuates neuroinflammation and blood brain barrier dysfunction to improve neurological deficits in cerebral ischemia/reperfusion rats.

Kaempferol has been reported to act as an anti-inflammatory agent in LPS-induced neuroinflammation in vitro and in vivo, but its role in the inflammation after cerebral ischemia/reperfusion (I/R) is unclear. The present study was to investigate the effect of kaempferol on inflammation in ischemic brain tissue and explore its mechanisms in cerebral I/R rats. Cerebral I/R rat model was established by middle cerebral artery occlusion for 60 min and following reperfusion. Kaempferol at doses of 25, 50 and 100 mg/kg was administered for 7 days after cerebral I/R. Kaempferol treatment significantly reduced cerebral infarct volume, attenuated inflammation and blood-brain barrier (BBB) disruption after cerebral I/R, thus improved neurological outcomes at the day 7 after cerebral I/R. Furthermore, the results also showed kaempferol treatment decreased the phosphorylation and nuclear transposition of transcription factor NF-κB p65, thus inhibited expression of various pro-inflammatory proteins. In conclusion, kaempferol attenuates neuroinflammation and blood brain barrier dysfunction to improve neurological deficits in cerebral I/R rats, its mechanism is related to NF-κB pathway.

Evaluation and Identification of the Neuroprotective Compounds of Xiaoxuming Decoction by Machine Learning: A Novel Mode to Explore the Combination Rules in Traditional Chinese Medicine Prescription.

Xiaoxuming decoction (XXMD), a classic traditional Chinese medicine (TCM) prescription, has been used as a therapeutic in the treatment of stroke in clinical practice for over 1200 years. However, the pharmacological mechanisms of XXMD have not yet been elucidated. The purpose of this study was to develop neuroprotective models for identifying neuroprotective compounds in XXMD against hypoxia-induced and H2O2-induced brain cell damage. In this study, a phenotype-based classification method was designed by machine learning to identify neuroprotective compounds and to clarify the compatibility of XXMD components. Four different single classifiers (AB, kNN, CT, and RF) and molecular fingerprint descriptors were used to construct stacked naïve Bayesian models. Among them, the RF algorithm had a better performance with an average MCC value of 0.725±0.014 and 0.774±0.042 from 5-fold cross-validation and test set, respectively. The probability values calculated by four models were then integrated into a stacked Bayesian model. In total, two optimal models, s-NB-1-LPFP6 and s-NB-2-LPFP6, were obtained. The two validated optimal models revealed Matthews correlation coefficients (MCC) of 0.968 and 0.993 for 5-fold cross-validation and of 0.874 and 0.959 for the test set, respectively. Furthermore, the two models were used for virtual screening experiments to identify neuroprotective compounds in XXMD. Ten representative compounds with potential therapeutic effects against the two phenotypes were selected for further cell-based assays. Among the selected compounds, two compounds significantly inhibited H2O2-induced and Na2S2O4-induced neurotoxicity simultaneously. Together, our findings suggested that machine learning algorithms such as combination Bayesian models were feasible to predict neuroprotective compounds and to preliminarily demonstrate the pharmacological mechanisms of TCM.

Baicalein administered in the subacute phase ameliorates ischemia-reperfusion-induced brain injury by reducing neuroinflammation and neuronal damage.

Ischemic stroke is a cerebrovascular disease with high morbidity, high mortality, and high disability, representing a serious threat to human life and health. Clinically, the extensive injury caused by ischemic stroke results from ischemia-reperfusion (I/R) injury thrombolytic treatment. However, there are few reports on the use of medications in the subacute stage of cerebral I/R. Baicalein (5,6,7-trihydroxyflavone) is a biologically active ingredient extracted from the root of Scutellaria baicalensis Georgi. In the present study, we investigated the therapeutic effect of baicalein administered in the subacute phase of cerebral I/R injury in a rat model of ischemia induced by occlusion of the middle cerebral artery (MCA). Rats were treated daily with baicalein (200 mg/kg, i.g.) in the subacute phase (24 h after reperfusion) for 7 days. The results showed that baicalein significantly reduced neurobehavioral deficits and decreased brain infarct volume from 18.99% to 7.41%. Immunofluorescence analysis of the ischemic penumbra showed that baicalein significantly reduced expression of the M1 marker, cluster of differentiation (CD) 16 and CD86, and increased expression of the M2 marker, CD 163 and CD206, indicating that baicalein inhibited M1 transformation and promoted M2 transformation of microglia/macrophage to inhibit neuroinflammation. Moreover, baicalein suppressed NF-κB signaling by reducing IκBα phosphorylation and nuclear translocation of NF-κB/p65, which decreased the release of the pro-inflammatory factors IL-6, IL-18, and TNF-α. In addition, baicalein reduced phosphorylation of JNK, ERK and p38, which are involved modulation of microglia/macrophage M1/M2 polarization. Western blot analysis of apoptosis- and autophagy-related proteins showed that baicalein increased the Bcl-2/Bax ratio and reduced caspase-3 expression to decrease neuronal apoptosis and ameliorate neuronal loss. Baicalein also decreased the LC3-II/LC3-I ratio and promoted phosphorylation of the PI3K/Akt/mTOR signaling pathway which implied inhibition of autophagy. These observations suggest that baicalein exerts neuroprotective effects by reducing neuroinflammation, apoptosis and autophagy, and protects against cerebral I/R injury in the subacute phase in vivo.

Kaempferol Attenuates LPS-Induced Striatum Injury in Mice Involving Anti-Neuroinflammation, Maintaining BBB Integrity, and Down-Regulating the HMGB1/TLR4 Pathway.

Neuroinflammation has been demonstrated to be linked with Parkinson's disease (PD), Alzheimer's disease, and cerebral ischemia. Our previous investigation had identified that kaempferol (KAE) exerted protective effects on cortex neuron injured by LPS. In this study, the effects and possible mechanism of KAE on striatal dopaminergic neurons induced by LPS in mice were further investigated. The results showed that KAE improved striatal neuron injury, and increased the levels of tyrosine hydroxylase (TH) and postsynaptic density protein 95 (PSD95) in the striatum of mice. In addition, KAE inhibited the production of pro-inflammatory cytokines, including interleukin 1ß (IL-1ß), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), reduced the level of monocyte chemotactic protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1), and cyclooxygenase-2 (COX-2) in the striatum tissues. Furthermore, KAE protected blood-brain barrier (BBB) integrity and suppressed the activation of the HMGB1/TLR4 inflammatory pathway induced by LPS in striatum tissues of mice. In conclusion, these results suggest that KAE may have neuroprotective effects against striatum injury that is induced by LPS and the possible mechanisms are involved in anti-neuroinflammation, maintaining BBB integrity, and down-regulating the HMGB1/TLR4 pathway.

Xiao-Xu-Ming decoction extract regulates differentially expressed proteins in the hippocampus after chronic cerebral hypoperfusion.

Xiao-Xu-Ming decoction has been widely used to treat stroke and sequelae of stroke. We have previously shown that the active fractions of Xiao-Xu-Ming decoction attenuate cerebral ischemic injury. However, the global protein profile and signaling conduction pathways regulated by Xiao-Xu-Ming decoction are still unclear. This study established a two-vessel occlusion rat model by bilateral common carotid artery occlusion. Rats were intragastrically administered 50 or 150 mg/kg Xiao-Xu-Ming decoction for 4 consecutive weeks. Learning and memory abilities were measured with Morris water maze. Motor ability was detected with prehensile test. Coordination ability was examined using the inclined screen test. Neuronal plasticity was observed by immunofluorescent staining. Differentially expressed proteins of rat hippocampus were analyzed by label-free quantitative proteomics. Real time-polymerase chain reaction and western blot assay were used to identify the changes in proteins. Results showed that Xiao-Xu-Ming decoction dramatically alleviated learning and memory deficits, and motor and coordination dysfunction, and increased the expression of microtubule-associated protein 2. Xiao-Xu-Ming decoction extract remarkably decreased 13 upregulated proteins and increased 39 downregulated proteins. The regulated proteins were mainly involved in oxidation reduction process, intracellular signaling cascade process, and protein catabolic process. The signaling pathways were mainly involved in ubiquitin mediated proteolysis and the phosphatidylinositol signaling system. Furthermore, there was an interaction among Rab2a, Ptpn1, Ppm1e, Cdk18, Gorasp2, Eps15, Capza2, Syngap1 and Mt-nd1. Protein analyses confirmed the changes in expression of MT-ND1. The current findings provide new insights into the molecular mechanisms of Xiao-Xu-Ming decoction extract's effects on chronic cerebral hypoperfusion.

Kaempferol alleviates LPS-induced neuroinflammation and BBB dysfunction in mice via inhibiting HMGB1 release and down-regulating TLR4/MyD88 pathway.

Kaempferol is a natural flavonoid with many biological activities including anti-oxidation and anti-inflammation. Nevertheless, its anti-neuroinflammation role and the relevant mechanism remain unclear. The present study was to investigate effects of kaempferol against LPS-induced neuroinflammation and blood-brain barrier dysfunction as well as the mechanism in mice. BALB/c mice were treated with LPS 5mg/kg to induce inflammation after pre-treatment with kaempferol 25, 50, or 100mg/kg for 7days. The results showed that kaempferol reduced the production of various pro-inflammatory factors and inflammatory proteins including IL-1ß, IL-6, TNF-α, MCP-1, COX-2 and iNOS in brain tissues. In addition, kaempferol also protected BBB integrity and increased BBB related proteins including occludin-1, claudin-1 and CX43 in brain of LPS-induced mice. Furthermore, kaempferol significantly reduced HMGB1 level and suppressed TLR4/MyD88 inflammatory pathway in both transcription level and translation level. These results collectively suggested that kaempferol might be a promising neuroprotective agent for alleviating inflammatory responses and BBB dysfunction by inhibiting HMGB1 release and down-regulating TLR4/MyD88 inflammatory pathway.

Ramulus Cinnamomi extract attenuates neuroinflammatory responses via downregulating TLR4/MyD88 signaling pathway in BV2 cells.

Ramulus Cinnamomi (RC), a traditional Chinese herb, has been used to attenuate inflammatory responses. The purpose of this study was to investigate the effect of RC extract on lipopolysaccharide (LPS)-induced neuroinflammation in BV2 microglial cells and the underlying mechanisms involved. BV2 cells were incubated with normal medium (control group), LPS, LPS plus 30 µg/mL RC extract, or LPS plus 100 µg/mL RC extract. The BV2 cell morphology was observed under an optical microscope and cell viability was detected by MTT assay. Nitric oxide level in BV2 cells was detected using Griess regents, and the levels of interleukin-6, interleukin-1ß, and tumor necrosis factor α in BV2 cells were determined by ELISA. The expression levels of cyclooxygenase-2, Toll-like receptor 4 and myeloid differentiation factor 88 proteins were detected by western blot assay. Compared with the LPS group, both 30 and 100 µg/mL RC extract had no significant effect on the viability of BV2 cells. The levels of nitric oxide, interleukin-6, interleukin-1ß and tumor necrosis factor α in BV2 cells were all significantly increased after LPS induction, and the levels were significantly reversed after treatment with 30 and 100 µg/mL RC extract. Furthermore, RC extract significantly inhibited the protein expression levels of cyclooxygenase-2, Toll-like receptor 4 and myeloid differentiation factor 88 in LPS-induced BV2 cells. Our findings suggest that RC extract alleviates neuroinflammation by downregulating the TLR4/MyD88 signaling pathway.

Association between TNFAIP3 Gene Polymorphisms and Risk of Allergic Rhinitis in a Chinese Han Population.

Tumor necrosis factor alpha-inducible protein 3 (TNFAIP3) gene polymorphisms have been reported to be associated with the susceptibility to several immune-related diseases. Here we investigated the effect of TNFAIP3 gene polymorphisms on the risk of allergic rhinitis (AR) in a Chinese Han population. The case-control study included 540 AR patients and 524 healthy controls. Genotyping for TNFAIP3 polymorphisms (rs5029928, rs9494885, rs10499194, rs610604, and rs7753873) were performed using restriction fragment length polymorphism analysis and DNA sequencing. Allele and genotype frequencies were compared between patients and controls. The rs9494885 TC genotype (corrected p (p=0.0032); odds ratio (OR)=2.06, 95% confidence intervals (CI): 1.40-3.04) and C allele (p=0.0056; OR=1.94, 95% CI: 1.35-2.76) were more frequent in AR patients compared with controls. The frequencies of the rs9494885 TT genotype (p=0.0029; OR=0.49, 95% CI: 0.33-0.72) and T allele (p= 0.0056; OR=0.52, 95% CI: 0.36-0.74) were lower in AR patients than that in controls. A higher frequency of the rs7753873 AC genotype (p=0.0023; OR=1.96, 95 %CI: 1.38-2.77) and C allele (p=0.0012; OR=1.74, 95% CI: 1.26-2.40) and a lower frequency of the rs7753873 AA genotype (p=0.0040; OR=0.53, 95% CI: 0.38-0.75) and A allele (p=0.0012; OR=0.58, 95% CI: 0.42-0.80) were observed in AR patients. TNFAIP3 gene polymorphisms (rs9494885 and rs7753873) are associated with the susceptibility to AR in the Chinese Han population.

[Relationships of the mRNA and protein expression of gastrin with Fas/FasL and caspases in colorectal carcinoma].

OBJECTIVE: To examine the correlation between the mRNA and proteins expressions of gastrin(GAS), and the association of protein expression of GAS with apoptosis index(AI) and apoptosis regulation gene Fas/FasL, caspases in colorectal cancer. METHODS: The expressions of GAS mRNA in tumor tissues of 79 cases with colorectal cancer were detected by nested RT-PCR. Cell apoptosis was detected by molecular biology in situ apoptosis detecting technic(TUNEL). Protein expressions of GAS, Fas/FasL, and caspases were detected by immunohistochemical staining (SP method). RESULTS: The positive correlation was found between the mRNA and proteins expressions of GAS(rGAS=0.99, P<0.01). The mRNA and protein expressions of GAS in well and moderately differentiated cancers were significantly lower than those in poorly differentiated cancers (chi(2)(high vs low)=10.47, 10.23, P<0.01, chi(2)(middle vs low)=6.68, 4.95, P<0.05). The mRNA and protein expressions of GAS in papillary and tubular adenocarcinomas were significantly lower than those in mucinous adenocarcinomas, signet-ring cell carcinoma and undifferentiated carcinoma (chi(2)(papillary vs mucinous and signet-ring)=4.80, 6.22, chi(2)(papillary vs undifferentiation)=5.44, 8.43, chi(2)(tubular vs mucinous and signet-ring)=4.40, 4.38, chi(2)(tubular vs undifferentiation)=4.92, 6.43, P<0.05, respectively). The mRNA and protein expressions of GAS in Dukes' stages A, B were significantly lower than those in Dukes stages C, D (chi(2)=4.84, 4.45, P<0.01). The AI in GAS high and moderate expression groups of colorectal cancer were significantly lower than that in low expression group (q(high vs low)=6.71, q(middle vs low)=4.60, P<0.01). The positive expression rate of FasL was significantly different among GAS high, moderate and low expression groups of colorectal cancer (chi(2)=9.35, P<0.01). The positive expression rate of FasL in GAS high and moderate expression groups was higher than that in low expression group (chi(2)high vs low=6.24, chi(2)(middle vs low)=4.74, P<0.05). CONCLUSIONS: GAS plays an important role in the regulation of cell apoptosis in colorectal carcinoma, whose mechanism may be related to the aberrant expression of Fas/FasL. GAS will be one of the indicators of the biological behavior in colorectal carcinoma.

Relationship between expression of gastrin, somatostatin, Fas/FasL and caspases in large intestinal carcinoma.

AIM: To explore the correlation between the mRNAs and protein expression of gastrin (GAS), somatostatin (SS) and apoptosis index (AI), apoptosis regulation gene Fas/FasL and caspases in large intestinal carcinoma (LIC). METHODS: Expression of GAS and SS mRNAs were detected by nested RT-PCR in 79 cases of LIC. Cell apoptosis was detected by molecular biology in situ apoptosis detecting methods (TUNEL). Immunohistochemical staining for GAS, SS, Fas/FasL, caspase-3 and caspase-8 was performed according to the standard streptavidin-biotin-peroxidase (S-P) method. RESULTS: There was a significant positive correlation between mRNA and protein expression of GAS and SS (GASrs = 0.99, P < 0.01; SSrs = 0.98, P < 0.01). There was significant difference in positive expression rates of GAS, SS mRNAs and protein among different histological differentiation, histological types and Dukes' stage of LIC. The AI in GAS high and moderate expression groups was significantly lower than that in low expression groups (3.75 +/- 2.38 vs 7.82 +/- 2.38, P < 0.01; 5.51 +/- 2.66 vs 7.82 +/- 2.38, P < 0.01), and the AI in SS high and moderate expression groups was significantly higher than that in low expression groups (9.03 +/- 1.76 vs 5.35 +/- 3.00, P < 0.01; 7.44 +/- 2.67 vs 5.35 +/- 3.00, P < 0.01). There was a significant negative correlation between the integral ratio of GAS to SS and the AI (r(s) = -0.41, P < 0.01). The positive expression rate of FasL in GAS high and moderate expression groups was higher than that in low expression group (90.9% and 81.0% vs 53.2%, P < 0.05). The positive expression rates of Fas, caspase-8 and caspase-3 in SS high (90.0%, 90.0% and 100%) and moderate (80.0%, 70.0%, 75.0%) expression groups were higher than that in low expression group (53.1%, 42.9%, 49.0%) (90.0% and 80.0% vs 53.1%, P < 0.05; 90.0% and 70.0% vs 42.9%, P < 0.05; 100.0% and 75.0% vs 49.0%, P < 0.05). There was a significant positive correlation between the integral ratio of GAS to SS and the semiquantitative integral of FasL (rs = 0.32, P < 0.01). CONCLUSION: GAS and SS play important roles in the regulation and control of cell apoptosis in LIC, and the mechanism may be directly related to the aberrant expression of Fas/FasL. The GAS and SS will be valuable targets of the biological behavior of LIC.