The role of vitamin D through SphK1/S1P in the regulation of MS progression.

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-ß, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-ß, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

The Effect of Resveratrol in Sirt1/CST Pathway to Inhibit TNF-α Induced Inflammatory Response in Rat Primary Fibroblast-Like Synoviocytes.

Rheumatoid arthritis has a significant impact on the life quality, but current pharmacological therapies have limitations. As a result, there is growing interest in exploring the potential of natural plant components to intervene in the development of rheumatoid arthritis. Resveratrol, a natural polyphenol and one of the main active components of the Chinese herbal medicine Polygonum cuspidatum, has emerged as a promising candidate for this purpose. In the present study, we investigated the role and mechanism of resveratrol in inhibiting inflammatory response in rat primary fibroblast-like synoviocytes. Tumor necrosis factor (TNF)-α was used to establish a model of inflammation, the Sirtuin1 selective inhibitor Selisistat (EX527) was used to inhibit Sirtuin1 activity, and small interfering RNA was used to silence cortistatin expression. The results showed that pre-treatment with resveratrol could time- and dose-dependently inhibit TNF-α induced cellular interleukin (IL)-1ß and IL-6 secretion, and upregulate Sirtuin1 and cortistatin mRNA and protein expression in the range of 48 h, 100 µM. Selisistat (EX527) could attenuate resveratrol inhibited inflammatory response and upregulated cortistatin expression. Silencing cortistatin expression attenuated the effect of resveratrol on inhibiting inflammatory response, but did not affect its effect on upregulating Sirtuin1 expression. In conclusion, resveratrol effectively inhibited the TNF-α induced inflammatory response in fibroblast-like synoviocytes by a mechanism involving the Sirtuin1/cortistatin pathway.

PVAT targets VSMCs to regulate vascular remodelling: angel or demon.

Vascular remodelling refers to abnormal changes in the structure and function of blood vessel walls caused by injury, and is the main pathological basis of cardiovascular diseases such as atherosclerosis, hypertension, and pulmonary hypertension. Among them, the neointimal hyperplasia caused by abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a key role in the pathogenesis of vascular remodelling. Perivascular adipose tissue (PVAT) can release vasoactive substances to target VSMCs and regulate the pathological process of vascular remodelling. Specifically, PVAT can promote the conversion of VSMCs phenotype from contraction to synthesis by secreting visfatin, leptin, and resistin, and participate in the development of vascular remodelling-related diseases. Conversely, it can also inhibit the growth of VSMCs by secreting adiponectin and omentin to prevent neointimal hyperplasia and alleviate vascular remodelling. Therefore, exploring and developing new drugs or other treatments that facilitate the beneficial effects of PVAT on VSMCs is a potential strategy for prevention or treatment of vascular remodelling-related cardiovascular diseases.

Red nucleus interleukin-1ß evokes tactile allodynia through activation of JAK/STAT3 and JNK signaling pathways.

We previously reported that interleukin-1ß (IL-1ß) in the red nucleus (RN) is involved in pain modulation and exerts a facilitatory effect in the development of neuropathic pain. Here, we explored the actions of signaling pathways, including the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-κB (NF-κB) pathways, on RN IL-1ß-mediated pain modulation. After a single dose of recombinant rat IL-1ß (rrIL-1ß, 10 ng) injected into the RN in normal rats, a tactile allodynia was evoked in the contralateral but not ipsilateral hindpaw, commencing 75 min and peaking 120 min postinjection. Up-regulated protein levels of phospho-STAT3 (p-STAT3) and p-JNK were observed in the RN 120 min after rrIL-1ß injection, the increases of p-STAT3 and p-JNK were blocked by anti-IL-1ß antibody. However, the expression levels of p-ERK, p-p38 MAPK, and NF-κB in the RN were not affected by rrIL-1ß injection. RN neurons and astrocytes contributed to IL-1ß-evoked up-regulation of p-STAT3 and p-JNK. Further studies demonstrated that injection of the JAK2 antagonist AG490 or JNK antagonist SP600125 into the RN 30 min prior to the administration of rrIL-1ß could completely prevent IL-1ß-evoked tactile allodynia, while injection of the ERK antagonist PD98059, p38 MAPK antagonist SB203580, or NF-κB antagonist PDTC did not affect IL-1ß-evoked tactile allodynia. In conclusion, our data provide additional evidence that RN IL-1ß is involved in pain modulation, and that it exerts a facilitatory effect by activating the JAK/STAT3 and JNK signaling pathways.

Red nucleus interleukin-6 participates in the maintenance of neuropathic pain through JAK/STAT3 and ERK signaling pathways.

We previously reported that interleukin-6 (IL-6) in the red nucleus (RN) is up-regulated at 3weeks after spared nerve injury (SNI), and plays facilitated role in the later maintenance of neuropathic pain. The current study aimed to reveal the roles of different signaling pathways, including Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase/protein kinase B (PI3K/AKT), in RN IL-6-mediated pain modulation. In accord with the increase of IL-6 in the RN following SNI, the protein levels of phospho-STAT3 (p-STAT3), p-ERK and p-JNK were also up-regulated in the RN contralateral to the nerve injury side at 3weeks after SNI. The increases of p-STAT3 and p-ERK (but not p-JNK) were associated with IL-6 and could be blocked by anti-IL-6 antibody. Microinjection of JAK2 inhibitor AG490, ERK inhibitor PD98059 and also JNK inhibitor SP600125 into the RN significantly increased the paw withdrawal threshold (PWT) and alleviated SNI-induced mechanical allodynia. Further studies showed that microinjection of recombinant rat IL-6 (rrIL-6, 20ng) into the RN of normal rats significantly decreased the PWT of rats and increased the local protein levels of p-STAT3 and p-ERK, but not p-JNK. Pre-treatment with AG490 and PD98059 could prevent IL-6-induced mechanical allodynia. Whereas, p-p38 MAPK and p-AKT did not show any expression changes in the RN of rats with SNI or rats treated with rrIL-6. These results suggest that RN IL-6 participates in the later maintenance of SNI-induced neuropathic pain and plays facilitated role through activating JAK/STAT3 and ERK signaling pathways.

Human cathelicidin LL-37 enhance the antibiofilm effect of EGCG on Streptococcus mutans.

BACKGROUND: Streptococcus mutans forms biofilms as a resistance mechanism against antimicrobial agents in the human oral cavity. We recently showed that human cathelicidin LL-37 exhibits inhibitory effects on biofilm formation of S. mutans through interaction with lipoteichoic acid (LTA), but without antibacterial or biofilm dispersal abilities. (-)-Epigallocatechin gallate (EGCG) is the most abundant constituent of tea catechins that has the greatest anti-infective potential to inhibit the growth of various microorganisms and biofilm formation. Therefore, in this study, we evaluated whether LL-37 interacts with EGCG to enhance the antibiofilm effect of EGCG on S. mutans biofilm formation. METHODS: Clinical S. mutans strains (n = 10) isolated from children's saliva were tested in a biofilm formation assay. The antibiofilm effect of EGCG with and without LL-37 was analyzed by the minimum biofilm eradication concentration assay and confirmed using field emission-scanning electron microscopy. In addition, the interaction among EGCG, LL-37, and LTA of S. mutans was determined using quartz crystal microbalance analysis. RESULTS: EGCG killed 100 % of planktonic S. mutans within 5 h, inhibited biofilm formation within 24 h, and reduced bacteria cells in preformed biofilms within 3 h at a concentration of 0.2 mg/mL. However, EGCG did not appear to interact with LTA. LL-37 effectively enhanced the bactericidal activity of EGCG against biofilm formation and preformed biofilms as determined by quantitative crystal violet staining and field emission-scanning electron microscopy. In addition, quartz crystal microbalance analysis revealed that LL-37 interacted with EGCG and promoted binding between EGCG and LTA of S. mutans. CONCLUSIONS: We show that LL-37 enhances the antibiofilm effect of EGCG on S. mutans. This finding provides new knowledge for dental treatment by using LL-37 as a potential antibiofilm compound.

The Red Nucleus Interleukin-6 Participates in the Maintenance of Neuropathic Pain Induced by Spared Nerve Injury.

Previous studies have demonstrated that the red nucleus (RN) is involved in the regulation of neuropathic pain and plays both facilitated and inhibitory roles through different cytokines. Here, we aim to investigate the expression changes and roles of interleukin-6 (IL-6), a pleiotropic cytokine, as well as its receptor (IL-6R) in the RN of rats with neuropathic pain induced by spared nerve injury (SNI). Immunohistochemistry indicated that IL-6 and IL-6R were weakly expressed in the RN of normal rats, and were mainly co-localized with neurons and oligodendrocytes. Following SNI, the expression levels of IL-6 and IL-6R in the RN did not show obvious changes at 1 week and 2 weeks postinjury. However, both of them were significantly increased in the RN contralateral (but not ipsilateral) to the nerve ligation side at 3 weeks postinjury, and co-localized not only with neurons and oligodendrocytes, but also with numerous astrocytes. Injection of different doses of anti-IL-6 antibody (100, 250, 500 ng) into the RN contralateral to the nerve ligation side at 3 weeks postinjury dose-dependently increased the paw withdrawal threshold (PWT) of rats and alleviated SNI-induced mechanical allodynia. Conversely, injection of different doses of recombinant rat IL-6 (5.0, 10, 20 ng) into the unilateral RN of normal rats dose-dependently decreased the PWT of contralateral (but not ipsilateral) hind paw and evoked significant mechanical allodynia, which was similar to SNI-induced neuropathic allodynia. These results further support the conclusion that the RN is involved in the modulation of neuropathic pain, and suggest that IL-6 and IL-6R in the RN play a facilitated role in the later maintenance of SNI-induced neuropathic pain.

Influence of ALDH2 Glu504Lys polymorphism on nitroglycerin response in chronic heart failure and involvement of Calcitonin Gene Related Peptide (CGRP).

OBJECTIVE: The aim of this study was to investigate whether the neuropeptide calcitonin gene related peptide (CGRP) contributes to nitroglycerin (GTN) response in patients with chronic heart failure (CHF) and the association with the mitochondrial aldehyde dehydrogenase-2 (ALDH2) Glu504Lys (ALDH2*2) polymorphism. METHODS: This is a 2-period, placebo-controlled clinical study. An intravenous infusion of saline followed by GTN (20 µg/min), each for 2 hours, respectively, was given to 49 stable CHF patients. Blood pressure (BP), heart rate (HR) and respiratory rate (RR) were measured at baseline, at 10 min, 30 min, 1.0 h, 1.5 h, and 2.0 h after initiation of saline infusion and initiation of GTN therapy. Blood samples were drawn for the determination of plasma CGRP for 49 patients at baseline, and at 2.0 h after initiation of saline and GTN infusion, respectively. Global clinical status of the patients was evaluated. Left ventricular ejection (LVEF), left ventricular end-diastolic volume (LVEDV), stroke volume (SV) and cardiac output (CO) were measured with 2D echocardiography with Simpson's biplane method (Pillip HP sonos 5500) by the same investigator at baseline and at 2.0 h after initiation of saline and GTN infusion. RESULTS: Systolic blood pressure (SBP), diastolic blood pressure (DBP), and left ventricular end-diastolic volume (LVEDV) were decreased, while left ventricular ejection fraction (LVEF) was increased at the end of GTN infusion (p < 0.001, respectively). Saline infusion showed no hemodynamic effects. At the end of GTN infusion, ALDH2*1/*1 homozygous patients showed higher degrees of both the absolute decrease in SBP (DSBP) (p < 0.001) and increase in LVEF (p < 0.001) than carriers of the ALDH2*2 allele. Mean plasma concentration of CGRP was increased after GTN infusion (p < 0.001), but not changed after saline infusion (p > 0.05). Changes in plasma concentration of CGRP correlated positively with the improvement in LVEF (r = 0.400, p = 0.004), while correlated negatively with changes in SBP (r = -0.300, p = 0.036) and LVEDV (r = -0.290, p = 0.043). CONCLUSIONS: ALDH2*2 polymorphism is associated with contributions of CGRP to GTN response in CHF patients.

The ALDH2 Glu504Lys polymorphism is associated with coronary artery disease in Han Chinese: Relation with endothelial ADMA levels.

OBJECTIVES: We studied the association between mitochondrial aldehyde dehydrogenase (ALDH2) Glu504Lys (rs671 or ALDH2*2) polymorphism and coronary artery disease (CAD), and sought to clarify the mechanisms underlying this association. METHODS: The ALDH2 rs671 polymorphism was genotyped in 417 CAD patients and 448 age- and gender-matched controls. All participants were Han Chinese. Human umbilical vein endothelial cells (HUVECs) isolated from 11 human umbilical cords were genotyped, cultured, and exposed to angiotensin II (Ang II, 10(-7)-10(-5)mol/L). Dimethylarginine dimethylaminohydrolase 1 (DDAH1) mRNA expression levels were determined by real-time PCR. Levels of asymmetric dimethylarginine (ADMA) in culture media and cell lysates were determined by high performance liquid chromatography-mass spectrometry (HPLC-MS). RESULTS: The frequency of carriers of the ALDH2 rs671 A allele (GA+AA) was significantly higher in patients with CAD (47.5%) than in controls (35.0%, p=0.0002). After adjustment for potential confounders, the odds ratio (OR) for CAD for carriers of the rs671 A allele was 1.85 (95% confidence interval [CI]: 1.38-2.49, p=0.00005) in the entire study cohort, and 1.95 (95% CI: 1.40-2.70, p=0.00007) in non-drinkers. In non-drinking controls, the homozygous rs671 AA genotype was associated with significantly lower high-density lipoprotein cholesterol (HDL-C) concentrations compared with rs671 GG homozygotes (p=0.015). HUVEC cells homozygous for the G allele of rs671 showed a significantly higher DDAH1 mRNA expression and lower intracellular ADMA levels compared with heterozygous GA cells (p<0.05, respectively). In homozygous GG cells, high concentrations of Ang II (10(-5)mol/L) decreased DDAH1 mRNA expression and increased intracellular ADMA concentrations. CONCLUSIONS: The rs671 polymorphism of ALDH2 is associated with CAD in Han Chinese, possibly by influencing HDL-C levels and endothelial ADMA levels.

Embedded ring injury of the middle finger in an amphetamine abuser.

Embedded finger-ring injury is not usually encountered in clinical practice, and most patients present with psychiatric or physical/mental impairment. We report herein an 18-year-old male amphetamine abuser who presented to our emergency department (ED) for embedded finger-ring injury over his left middle finger. The embedded ring was removed in the ED under heavy sedation, and the wound healed uneventfully over 2 weeks. Thereafter, the young man received psychiatric treatment. Embedded finger-ring injury has never been reported in a drug abuser before.

[Regulation of NF-kappaB/P65 by MDM2 in acute lymphoblastic leukemia in childhood].

OBJECTIVE: MDM2 is considered a proto-oncogene due to its ability to inhibit P53 tumor-suppressor function. But, evidence showed that MDM2 might have a P53-independent role in tumorigenesis. MDM2 is over-expressed in human sarcoma and carcinoma. Recent studies showed that MDM2 might act as a transcriptional factor to modulate expressions of other genes involved in cell cycle regulation and transformation. In the present study, the investigators hypothesized that MDM2 directly affected NF-kappaB expression and function in a P53-independent manner. METHODS: MDM2 was transfected to acute lymphoblastic leukemia (ALL) line EU-4 cells lacking P53 expression and expressing very low levels of MDM2. MDM2 and P65 expression in mRNA level and protein level were detected by Western blot and Northern blot after transfection. Since the expression of E-selectin is P65 dependent, E-selectin promoter-CAT construct and P65 and MDM2 expression plasmids were co-transfected to EU-4 cells. CAT activation was determined with ELISA. The effect of adriamycin (ADM) at the concentrations of 15 micro g/ml, 7.5 micro g/ml, 5 micro g/ml and 1 micro g/ml on MDM2-transfected EU-4 cells and the parent cells was detected by MTT assay. RESULTS: The results showed that MDM2 up-regulated P65 expression at both mRNA and protein levels, and MDM2 increased P65-mediated transactivation of E-selectin promoter. Without P65, MDM2 had no effect on the transactivation of E-selectin. Moreover, MDM2 antisense could not change the transactivation of E-selectin. MTT results showed that the survival rate of MDM2 transfected EU-4 cells was higher than that of parental cells. The results suggested that MDM2 transfection increase drug resistance of EU-4 cells to ADM compared with parent cells. CONCLUSION: (1) MDM2 up-regulated transcriptionally P65 expression. (2) MDM2 increased drug resistance of leukemia cells to ADM. (3) MDM2 elevated NF-kappaB activity in a P53-independent manner in childhood lymphoblastic leukemia cell line.