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To observe the occurrence time of neuralgia and the expression of purinergic ligand-gated ion channel 7 receptor (P2X7R) in the dorsal horn of the spinal cord after intraperitoneal injection of streptozotocin (STZ) in diabetic rats, and to explore the effect of electroacupuncture (EA) and pretreatment of EA on the heat pain threshold and expression of P2X7R in the spinal dorsal horn in rats with diabetic neuropathic pain (DNP), and to explore the possible mechanism of EA for DNP.PartⅠ: Thirty male SD rats were randomly selected from 64 male SD rats as the control group; the remaining rats were given intraperitoneal injection of STZ (10 mg/mL) at a dose of 65 mg/kg to establish the diabetes model, and 30 rats were successfully modeled as the model group. The control group and the model group were divided into three subgroups respectively at 7, 14 and 21 days, with 10 rats in each subgroup. Body mass, fasting blood glucose (FBG) and thermal pain threshold were recorded at 7, 14 and 21 days after injection; the expression of P2X7R in spinal dorsal horn was detected by Western blot. PartⅡ: Eight SD rats were randomly selected from 35 male SD rats as the blank group, and the remaining 27 rats were given intraperitoneal injection of STZ (10 mg/mL) at a dose of 65 mg/kg to establish the diabetes model. The 24 rats with successful diabetes model were randomly divided into a DNP group, an EA group and a pre-EA group, 8 rats in each group. Fifteen to 21 days after STZ injection, the EA group received EA at "Zusanli" (ST 36) and "Kunlun" (BL 60), continuous wave, frequency of 2 Hz, 30 min each time, once a day; the intervention method in the pre-EA group was the same as that in the EA group. The intervention time was 8 to 14 days after STZ injection. The body mass, FBG and thermal pain threshold were recorded before STZ injection and 7, 14 and 21 days after STZ injection; the expression of P2X7R in spinal dorsal horn was detected by Western blot 21 days after injection.PartⅠ: Compared with the control group, in the model group, the body mass was decreased and FBG was increased 7, 14 and 21 days after STZ injection (The diabetic neuropathic pain is observed 14 days after STZ injection. EA could not only treat but also prevent the occurrence of DNP, and its mechanism may be related to down-regulation of P2X7R expression in the dorsal horn of the spinal cord.

Sympathetic nervous system(SNS) is involved in the mechanism of immune suppression after stroke. Furthermore, as the pro-inflammatory effect of nuclear factor kappa B(NF-kB) is inhibited after stroke, which is regulated by cyclic adenosine monophosphate(cAMP) and proteinkinase A(PKA). The cAMP-PKA-NF-kB pathway might play an important role in noradrenergic-mediated immune dysfunction.The purpose of our research is to analyze how SNS interfere with the immune system after acute stroke and the underlying mechanism of cAMP-PKA-NF-kB pathway in regulating the inflammation.32 healthy male Sprague-Dawley rats were divided into 4 groups equally and randomly (1) Sham operation group; (2) middle cerebral artery occlusion; (MCAO) control group; (3) propranolol MCAO group; (4) isopropylarterenol sham group. 72h later after MCAO or sham operation, tumor necrosis factor-α(TNF-α)and interleukine-10(IL-10) in serum as well as cAMP, PKA and NF-kB in spleen cells were tested.TNF-α decreased while IL-10 increased in serum after acute ischemia stroke (p0.05). Meanwhile, the levels of cAMP and PKA in spleen both increased in MCAO model while the expression of NF-kB was inhibited (p0.05). When propranolol was used to inhibit SNS, all of the results reversed (p0.05). But the reversed results were still significantly different from the sham operation group (p0.05). Isopropylarterenol administrated rats appeared the same trend as MCAO group when compared to the sham operation group (p0.05). However, the differences still existed (p0.05).On account of the SNS activation after stroke, epinephrine activates the expression of cAMP, which further increases the level of PKA. Therefore, the level of nuclear factor NF-kB is down-regulated. Since the pro-inflammatory effect of NF-kB slacked, the immune system may be inhibited after stroke.

The troponin complex, composed of troponin T (TnT), troponin I (TnI), and troponin C (TnC), is the major calcium-dependent regulator of muscle contraction, which is present widely in both vertebrates and invertebrates. Little is known about evolutionary aspects of troponin in the animal kingdom. Using a combination of data mining and functional analysis of TnI, we report evidence that an N-terminal extension of TnI is present in most of bilaterian animals as a functionally important domain. Troponin components have been reported in species in most of representative bilaterian phyla. Comparison of TnI sequences shows that the core domains are conserved in all examined TnIs, and that N- and C-terminal extensions are variable among isoforms and species. In particular, N-terminal extensions are present in all protostome TnIs and chordate cardiac TnIs but lost in a subset of chordate TnIs including vertebrate skeletal-muscle isoforms. Transgenic rescue experiments in Caenorhabditis elegans striated muscle show that the N-terminal extension of TnI (UNC-27) is required for coordinated worm locomotion but not in sarcomere assembly and single muscle-contractility kinetics. These results suggest that N-terminal extensions of TnIs are retained from a TnI ancestor as a functional domain.

Lycium barbarum polysaccharides (LBPs) have been known to have a variety of immunomodulatory functions including activation of T cells, B cells and NK cells. Dendritic cells (DC) are potent antigen-presenting cells that play pivotal roles in the initiation of the primary immune response. However, little is known about the immunomodulatory effects of LBPs on murine bone marrow derived dendritic cells (BMDC). In the present study, the effects of LBPs on the phenotypic and functional maturation of murine BMDC were investigated in vitro. Compared to the BMDC that were only subjected to treatment with RPMI1640, the co-expression of I-A/I-E, CD11c and secretion of IL-12 p40 by BMDC stimulated with LBPs (100 microg/ml) were increased. In addition, the endocytosis of FITC-dextran by LBPs-treated BMDC (100 microg/ml) was impaired, whereas the activation of proliferation of allogenic lymphocytes by BMDC was enhanced. Our results strongly suggest that LBPs are capable of promoting both the phenotypic and functional maturation of murine BMDC in vitro.

Forward genetic screen via mutagenesis is a powerful method for identifying regulatory factors in target pathways in model organisms such as the soil-dwelling free-living nematode Caenorhabditis elegans (C. elegans). Currently manual microscopy is the standard technique for conducting such screens; however, it is labor-intensive and time-consuming because screening requires imaging thousands of animals. Recently microfluidic chips have been developed to increase the throughput of some of such experiments; nonetheless, most of these chips are multilayer devices and complicated to fabricate and therefore prone to failure during fabrication and operation. In addition, most sorting decisions are made manually and the criteria used for sorting are subjective. To overcome these limitations, we developed a simple single-layer microfluidic device and an adaptive algorithm to make sorting decisions. The one-layer device greatly improves the reliability, while the quantitative analysis with adaptive algorithm allows for the identification of mutations that generate subtle changes in expression, which would have been hard to be detected by eye. The screening criterion is set based on the mutagenized population, not separate control populations measured prior to actual screening experiments, to account for stochasticity and day-to-day variations of gene expression in mutagenized worms. Moreover, during each experiment, the threshold is constantly updated to reflect the balance between maximizing sorting rate and minimizing false-positive rate. Using this system, we screened for mutants that have altered expression level of tryptophan hydroxylase, a key enzyme for serotonin synthesis in a CaMKII gain-of-function background. We found several putative mutants in this screen. Furthermore, this microfluidic system and quantitative analysis can be easily adapted to study other pathways in C. elegans.

Polysaccharides extracted from various sources are natural active substances, which may lead to the activation of macrophage via multiple pathways and mechanisms. This article intends to illustrate the signaling pathways of polysaccharides from plants, fungi, algae and other sources, to identify the mechanisms on the molecular level, and to explore the novel target immunomodulatory agents.

Astragalus polysaccharide (APS) was obtained by hot water extraction, alcohol precipitation, gel-permeation chromatography and ultrafiltration. Fluorescence material 2-aminoacridone (2-AMAC) labeled APS bind to macrophage in a time- dependent manner and the binding can be remarkably inhibited by APS. Furthermore, the effect of APS on RAW264.7 macrophage demonstrated APS increase the level of cytokines including TNF-α, GM-CSF and the production of NO. NF-κB protein levels are increased in response to APS. Blocking NF-κB with specific inhibitor resulted in decreased levels of NO and TNF-α. The results suggested that APS possess potent immunomodulatory activity by stimulating macrophage and could be used as an immunotherapeutic adjuvant.

To study the effect of Lycium bararum polysaccharides (LBPs) stimulation on the maturation of murine bone marrow derived dendritic cells (BMDCs).Murine bone marrow cells were cultured in GM-CSF and IL-4 for 5 days, then were purified with a MACS column. Respectively, BMDCs were stimulated with LBPs, LPS and RPMI1640 for 2 days. Cell phenotypes and antigens uptake by BMDCs were analyzed by flow cytometry. Cytokines released by BMDCs were detected. The antigen presenting by BMDCs was evaluated by mixed lymphocyte responses.Compared with to the BMDCs that only subjected to RPMI 1640, the expression of I-A/I-E, CD11c and secretion of IL-12 by BMDCs stimulated with LBPs were increased, the phagocytosis of FITC-dextran by BMDCs stimulated with LBPs was impaired but the activation of proliferation of allogenic lymphocytes by BMDCs was strengthened.LBPs promote not only the maturation of cultured murine BMDCs in vitro, but also the immune response initiation induced by BMDCs.