Neuroprotective Effects of Sinomenine on Experimental Autoimmune Encephalomyelitis via Anti-Inflammatory and Nrf2-Dependent Anti-Oxidative Stress Activity.

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). Sinomenine (SIN), a bioactive alkaloid extracted from the Chinese medicinal plant Sinomenium acutum, has powerful anti-inflammatory and immunosuppressive therapeutic benefits. In our previous research, we found that SIN increased resistance to oxidative stress via the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in PC12 neuronal cells. However, whether SIN can improve the symptoms and pathological features of experimental autoimmune encephalomyelitis (EAE), a murine model of MS, via the Nrf2 signaling pathway remains unclear. EAE was immunized followed by SIN treatment. Then we evaluated the effects of SIN in EAE. Subsequently, primary microglia were cultured to explore the effect of SIN on microglia activation. Further, the levels of Nrf2 and its downstream molecules were detected to assess the molecular mechanisms of SIN. We demonstrated that SIN effectively ameliorated the severity of EAE, accompanied by a reduction in the demyelination, axonal damage and inhibition of inflammatory cell infiltration. Mechanistically, SIN decreased the inflammatory cytokines expression, and suppressed microglia and astrocytes activation in EAE mice. Furthermore, SIN suppressed lipopolysaccharide (LPS)-induced microglial activation and the production of pro-inflammatory factors in vitro. Moreover, SIN inhibited oxidative stress via the activation of the Nrf2 signaling pathway. Our work proves that SIN exerts its neuroprotective effects by the Nrf2-dependent anti-oxidative stress and diminishing neuroinflammation, suggesting that the "antioxiflammation" effect of SIN is expected to be an ideal treatment strategy for MS/EAE.

Branched-chain amino acids promotes the repair of exercise-induced muscle damage via enhancing macrophage polarization.

The repair of exercise-induced muscle damage (EIMD) is closely related with inflammation. Branched-chain amino acids (BCAAs), as a nutritional supplement, promote EIMD repair; however, the underlying mechanism remains unclear. In vivo, Sprague-Dawley rats were subjected to Armstrong's eccentric exercise (a 120-min downhill run with a slope of -16° and a speed of 16 m min-1) to induce EIMD and BCAA supplement was administered by oral gavage. Protein expression of macrophages (CD68 and CD163) and myogenic regulatory factors (MYOD and MYOG) in gastrocnemius was analyzed. Inflammatory cytokines and creatine kinase (CK) levels in serum was also measured. In vitro, peritoneal macrophages from mice were incubated with lipopolysaccharide (LPS) or IL-4 with or without BCAAs in culture medium. For co-culture experiment, C2C12 cells were cultured with the conditioned medium from macrophages prestimulated with LPS or IL-4 in the presence or absence of BCAAs. The current study indicated BCAA supplementation enhanced the M1/M2 polarization of macrophages in skeletal muscle during EIMD repair, and BCAAs promoted M1 polarization through enhancing mTORC1-HIF1α-glycolysis pathway, and promoted M2 polarization independently of mTORC1. In addition, BCAA-promoted M1 macrophages further stimulated the proliferation of muscle satellite cells, whereas BCAA-promoted M2 macrophages stimulated their differentiation. Together, these results show macrophages mediate the BCAAs' beneficial impacts on EIMD repair via stimulating the proliferation and differentiation of muscle satellite cells, shedding light on the critical role of inflammation in EIMD repair and the potential nutritional strategies to ameliorate muscle damage.

Activation of Sirtuin1 by lyceum barbarum polysaccharides in protection against diabetic cataract.

ETHNOPHARMACOLOGICAL RELEVANCE: Lycium barbarum polysaccharide (LBP) extracted from the Lycium barbarum L. has been widely used to improve diabetes and its relative complications. However, the mechanisms have not fully understood. A recent study has demonstrated that LBP upregulates suituin 1 (SIRT1). OBJECTIVE: This study was to define the role of Sirt1 and its downstream signaling pathways in diabetic cataract using in vitro and in vivo models. MATERIALS AND METHODS: Human lens epithelial cell line SRA01/04 cells were cultured under high glucose (HG) medium with treatment of LBP or vehicle. Cell viability, apoptosis, protein and/or mRNA levels of Sirt1, BAX, Bcl-2, active-caspase-3, FOXO1, p27 and acetylated p53 were measured. SIRT1 upregulated- and knocked-down cells were generated and tested in high glucose culture. Diabetes mellitus was induced in rats by streptozotocin injection. Body weight, blood glucose levels, lens transparency and retinal function were assessed and SIRT1, as well as the aforementioned biomarkers were measured using Western blotting and qPCR in the animal lens samples. RESULTS: The results showed that HG decreased cell viability and LBP prevented the decrease. The reduced viability in HG cultured SRA01/04 cells was associated with increased levels of BAX, active caspase 3, FOXO1, p27, and p53 and decreased levels of SIRT1 and Bcl-2. Further experiments using sirt1 gene modulated cells showed that upregulation of Sirt1 improved viability, increase cell division as reflected by an increased proportion of S phase in the cell cycle, reduced the number of apoptotic cell death and suppressed p53 acetylation and caspase 3 activation. Opposite results were observed in SIRT1 knock-down cells. Treating diabetic animals with LBP reduced body weight loss and blood glucose content in diabetic animals. Similarly, LBP hindered the development of cataract in lenses and improved retinal function. The beneficial effect of LBP on diabetic cataract was associated with the supression of p53, caspase 3, FOXO1, BAX, p27 and elevation of SIRT1 and Bcl-2, which were consistent with the in vitro findings. CONCLUSION: Our findings showed that diabetes caused cataract is associated with suppression of SIRT1 and Bcl-2 and activation of other cell death related genes. LBP prevented diabetic cataract in animals by upregulating Sirt1 and Bcl-2 and suppressing cell death related genes.

[Research progress of relationship between continuous cropping obstacles and microRNAs in Rehmannia glutinosa].

The efficacy of Rehmannia glutinosa which as a large quantity of traditional Chinese medicine is significant. However, the land must be given up after one season of R. glutinosa cultivation or replanted after a period of 8-10 years because of the severe continuous cropping obstacles. MicroRNAs is a class of endogenous non-coding small RNAs, which participate in regulation of physiological activities by target mRNA cleavage or translational repression in plants. In recent years,studies on the role of miRNAs in plants have made significant progresses,especially in medicinal plants．MiRNAs from some different medicinal plant species have been identified with regulatory effects．When plants are exposed to environmental stress, miRNAs act on stress-related genes and initiate stress-resistance mechanisms in the body against adverse factors. R. glutinosa is also a kind of environmental stress. It is conducive to deciphering the molecular mechanism of continuous cropping obstacles for us by researching miRNAs. This article reviews the production of miRNAs, mechanism, research approaches and characteristics of resisting the environmental stresses in plants, the development trends and future prospect of R. glutinosa miRNAs research.

[Advances on molecular mechanisms of Rehmannia glutinosa consecutive monoculture problem formation in multi-omics era].

Although consecutive monoculture problems have been studied for many years, no effective treatments are currently available. The complexity of systems triggered the formation of consecutive monoculture problems was one major cause. This paper elaborated the physiological and ecological mechanisms of consecutive monoculture problem formation based on the interaction relationship among multiple factors presented in the rhizosphere soil of consecutive monoculture plants. At same time, in this paper the multiple interactions among cultivated medicinal plants, autotoxic allelochemicals and rhizosphere microbial were proposed to be most important causes that derived the formation of consecutive monoculture problem. The paper also highlighted the advantage of 'omics' technologies integrating plant functional genomics and metabolomics as well as microbial macro-omics in understanding the multiple factor interaction under a particular ecological environment. Additionally, taking R. glutinosa as an example, the paper reviewed the molecular mechanism for the formation of R. glutinosa consecutive monoculture problem from the perspective of the accumulation of allelopathic autotoxins, the rhizosphere microecology catastrophe and theresponding of consecutive monoculture plants. Simultaneously, the roles of mutilple 'omics' technologies in comprehending these formation mechanism were described in detail. This paper provides finally a new insight to solve systematically the mechanism of consecutive monoculture problem formation on molecular level.

Differential proteomic analysis of replanted Rehmannia glutinosa roots by iTRAQ reveals molecular mechanisms for formation of replant disease.

BACKGROUND: The normal growth of Rehmannia glutinosa, a widely used medicinal plant in China, is severely disturbed by replant disease. The formation of replant disease commonly involves interactions among plants, allelochemicals and microbes; however, these relationships remain largely unclear. As a result, no effective measures are currently available to treat replant disease. RESULTS: In this study, an integrated R. glutinosa transcriptome was constructed, from which an R. glutinosa protein library was obtained. iTRAQ technology was then used to investigate changes in the proteins in replanted R. glutinosa roots, and the proteins that were expressed in response to replant disease were identified. An integrated R. glutinosa transcriptome from different developmental stages of replanted and normal-growth R. glutinosa produced 65,659 transcripts, which were accurately translated into 47,818 proteins. Using this resource, a set of 189 proteins was found to be significantly differentially expressed between normal-growth and replanted R. glutinosa. Of the proteins that were significantly upregulated in replanted R. glutinosa, most were related to metabolism, immune responses, ROS generation, programmed cell death, ER stress, and lignin synthesis. CONCLUSIONS: By integrating these key events and the results of previous studies on replant disease formation, a new picture of the damaging mechanisms that cause replant disease stress emerged. Replant disease altered the metabolic balance of R. glutinosa, activated immune defence systems, increased levels of ROS and antioxidant enzymes, and initiated the processes of cell death and senescence in replanted R. glutinosa. Additionally, lignin deposition in R. glutinosa roots that was caused by replanting significantly inhibited tuberous root formation. These key processes provide important insights into the underlying mechanisms leading to the formation of replant disease and also for the subsequent development of new control measures to improve production and quality of replanted plants.

Conservative treatment and percutaneous catheter drainage improve outcome of necrotizing pancreatitis.

BACKGROUND/AIMS: To investigate the clinical effects of the maximum conservative treatment algorithm with percutaneous catheter drainage (PCD) as the first choice for necrotizing pancreatitis (NP). METHODOLOGY: Retrospectively analyzed NP patients who had fine needle aspiration (FNA) for proven infection of necrosis which was considered an indication for surgery (n=22, group 1) compared to patients subjected to maximum conservative treatment with PCD in NP patients (n=30, group 2). RESULTS: On admission, most baseline data did not show any statistical difference between the two groups, In group 2, all patients were implemented maximum conservative treatment, 25 of 30 patients were cured by PCD (83.3%), open necrosectomy were needed for 3 patients (10.0%) and 2 dead during hospitalization (6.7%). Whereas, in group 1, surgical operation rate was 45.6% and hospital mortality 31.8%, both of the ratios differed significantly compared with group 2 (45.6% vs. 10%, P=0.004; 31.8% vs. 6.7%, P=0.046 respectively). Furthemore, Hospital stay were significantly higher in group 1 compared with group 2 (90±18.5 vs. 39±13.4; P=0.033). CONCLUSIONS: A conservative approach with PCD as the first choice to treatment NP might decrease the rate of surgical operation and mortality, and improve the outcome of NP.

[The clinical and imaging study of thalamic venous infarction].

OBJECTIVE: To explore the clinical and radiological features of bilateral thalamus venous infarction. METHODS: The cases definitely diagnosed as thalamus venous infarction were collected and the corresponding clinical and radiological data were retrospectively analyzed. RESULTS: Four cases confirmed as thalamus venous infarction by digital substraction angiography (DSA) were collected. Bilateral thalamus lesions were detected in all cases by brain MRI scans which mainly presented as thalamus edema with high T1 and T2 signals with partial enhancement. Mild hemorrage was also shown in one case. Acute or subacute onset with clinical manifestations of headache, hypomnesia and hypersomnia were reported in all cases. The neurological examination showed conscious disturbance, memory impairment and positive Babinski sign. The venous thrombi were formed mainly in the transverse and the straight sinuses in 3 cases with the deep cerebral venous involved in 2 cases. All patients were improved after the anticoagulation therapy. Dural arteriovenous fistula was found in the other case drained by the Rosenthal's vein, and the symptoms were ameliorated after the embolotherapy. CONCLUSIONS: As the thalamus is drained by the thalamostriate vein and the lateral thalamic vein towards the internal cerebral vein with the caudate portion drained particularly by the Rosenthal's vein, venous thrombosis or fistula drainage into these veins would probably disturb the normal drainage of the thalamus and result in further edema and infarction. Thus, the venous infarction should be taken into consideration whenever bilateral thalamus lesions are encountered in clinical practice and DSA is necessary to confirm the diagnosis.

Identification and validation of a novel lead compound targeting 4-diphosphocytidyl-2-C-methylerythritol synthetase (IspD) of mycobacteria.

Tuberculosis is a serious threat to world-wide public health usually caused in humans by Mycobacterium tuberculosis (M. tuberculosis). It exclusively utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), the precursors of all isoprenoid compounds. The 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD; EC 2.7.7.60) is the key enzyme of the MEP pathway. It is also of interest as a new chemotherapeutic target, as the enzyme is absent in mammals and ispD is an essential gene for growth. A high-throughput screening method was therefore developed to identify compounds that inhibit IspD. This process was applied to identify a lead compound, domiphen bromide (DMB), that may effectively inhibit IspD. The inhibitory action of DMB was confirmed by over-expressing or down-regulating IspD in Mycobacterium smegmatis (M. smegmatis), demonstrating that DMB inhibit M. smegmatis growth additionally through an IspD-independent pathway. This also led to higher levels of growth inhibition when combined with IspD knockdown. This novel IspD inhibitor was also reported to exhibit antimycobacterial activity in vitro, an effect that likely occurs as a result of perturbation of cell wall biosynthesis.

[Spatiotemporal expression and analysis of responding consecutive monoculture genes in Rehmannia glutinosa].

OBJECTIVE: Based on previous study, authors used the suppression subtractive hybridization (SSH) technique to construct the forward and reverse subtractive cDNA libraries of consecutive monoulture Rehmannia glutinosa. Five genes related with consecutive monoculture problem of R. glutinosa were chosen from the each of two subtractive libraries. And their spatiotemporal expression was measured in order to explore the functions in consecutive monoculture problem of R. glutinosa. METHOD: Using the real-time quantitative PCR, we tested the relative expression values of the genes in different development stages and tissues of normal growth (one-year culturing) and consecutive monoculture (two-year culturing) R. glutinosa. RESULT: The five genes (calcium-dependent protein kinase, s-adenosyl-methionine synthetase, Aminocyclopropane-1-carboxylate oxidase, methyltransferase, calpain), which were chosen from the forward library had high expression in consecutive monoculture R. glutinosa, especially in root, and were hardly expression in normal growth R. glutinosa. On the contrary, the other five genes (RNA-dependent RNA polymerase, RNA replicase, DNA-directed RNA polymerase IIa, cyclin D, RNA binding protein) chosen from the reverse library had high expression in one-year R. glutinosa, but were down regulated or shut down in consecutive monoculture R. glutinosa. CONCLUSION: The key genes, which regulate inessential metabolism parthway (such as cyclin D, DNA-directed RNA polymerase IIa), were restrained or shut down in consecutive monoculture R. glutinosa. Calcium and ethylene signaling might played key roles in the formation of consecutive monoculture problem, resulting in disturbing normal metabolic process and syndrome of disease in R. glutinosa appeared in turn.

Changes in thalamus connectivity in mild cognitive impairment: evidence from resting state fMRI.

PURPOSE: The subcortical region such as thalamus was believed to have close relationship with many cerebral cortexes which made it especially interesting in the study of functional connectivity. Here, we used resting state functional MRI (fMRI) to examine changes in thalamus connectivity in mild cognitive impairment (MCI), which presented a neuro-disconnection syndrome. MATERIALS AND METHODS: Data from 14 patients and 14 healthy age-matched controls were analyzed. Thalamus connectivity was investigated by examination of the correlation between low frequency fMRI signal fluctuations in the thalamus and those in all other brain regions. RESULTS: We found that functional connectivity between the left thalamus and a set of regions was decreased in MCI; these regions are: bilateral cuneus, middle occipital gyrus (MOG), superior frontal gyrus (SFG), medial prefrontal cortex (MPFC), precuneus, inferior frontal gyrus (IFG) and precentral gyrus (PreCG). There are also some regions showed reduced connectivity to right thalamus; these regions are bilateral cuneus, MOG, fusiform gyrus (FG), MPFC, paracentral lobe (PCL), precuneus, superior parietal lobe (SPL) and IFG. We also found increased functional connectivity between the left thalamus and the right thalamus in MCI. CONCLUSION: The decreased connectivity between the thalamus and the other brain regions might indicate reduced integrity of thalamus-related cortical networks in MCI. Furthermore, the increased connectivity between the left and right thalamus suggest compensation for the loss of cognitive function. Briefly, impairment and compensation of thalamus connectivity coexist in the MCI patients.

[Construction and analysis of suppression subtractive cDNA libraries of continuous monoculture Rehmannia glutinosa].

OBJECTIVE: To explore the molecular mechanism of continuous monoculture problem by constructing the cDNA libraries of continuous monoculture Rehmannia glutinosa. METHOD: To use the suppression subtractive hybridization (SSH) technique to construct the forward and reverse subtractive cDNA libraries of continuous monoculture R. glutinosa to adopt blue-white colony screening and PCR to detect the positive clones which would be sequenced and analyzed by bioinformatics. RESULT: The subtracted cDNA libraries of continuous monoculture R. glutinosa. were successfully constructed, and the result showed that the forward and reverse subtracted libraries obtained 300 positive clones, respectively. The forward and reverse libraries got different ESTs, and produced 232 (forward library) and 214 (reverse library) unique ESTs by sequencing. Based on homology search of BLASTX and BLASTN in NCBI, 200 and 195 of unique ESTs were homologous to known genes in the forward and reverse libraries, respectively. Categories of orthologous group (COG) showed that the forward and reverse libraries got 60 and 61 ESTs with the corresponding gene annotation, involving 21 metabolic pathways. CONCLUSION: The information of differential expression genes in continuous monoculture R. glutinosa, and their functional annotation of differentially expressed genes indicate that continuous monoculture has a profound effect on expression of the genes in R. glutinosa. Furthermore, the research analyzed several key genes in response to replant problem, which provided a foundation for revealing the molecular mechanism of continuous monoculture R. glutinosa.

Specific and nonspecific thalamocortical functional connectivity in normal and vegetative states.

Recent theoretical advances describing consciousness from information and integration have highlighted the unique role of the thalamocortical system in leading to integrated information and thus, consciousness. Here, we examined the differential distributions of specific and nonspecific thalamocortical functional connections using resting-state fMRI in a group of healthy subjects and vegetative-state patients. We found that both thalamic systems were widely distributed, but they exhibited different patterns. Nonspecific connections were preferentially associated with brain regions involved in higher-order cognitive processing, self-awareness and introspective mentalizing (e.g., the dorsal prefrontal and anterior cingulate cortices). In contrast, specific connections were prevalent in the ventral and posterior part of the prefrontal and precuneus, known involved in representing externally-directed attentions. Significant reductions of functional connectivity in both systems, especially the nonspecific system, were observed in VS. These data suggest that brain networks sustaining information and integration may be differentiated by the nature of their thalamic functional connectivity.

Altered effective connectivity and anomalous anatomy in the basal ganglia-thalamocortical circuit of stuttering speakers.

Combining structural equation modeling (SEM) and voxel-based morphometry (VBM), this study investigated the interactions among neural structures in the basal ganglia-thalamocortical circuit (BGTC) in the left hemisphere of stuttering and non-stuttering speakers. Stuttering speakers (n=12) and non-stuttering controls (n=12) were scanned while performing a picture-naming task and a passive-viewing (baseline) task. Results showed significant differences between stuttering and non-stuttering speakers in both effective connectivity and anatomical structures in the BGTC in the left brain. Specifically, compared to non-stuttering speakers, stuttering speakers showed weaker negative connectivity from the left posterior middle temporal gyrus (PMTG) to the putamen, but stronger positive connectivity from the putamen to the thalamus, from the thalamus to the PMTG and anterior supplementary motor area (preSMA), and from the anterior superior temporal gyrus (ASTG) to the preSMA. Accompanying such altered connectivity were anatomical differences: compared to non-stuttering controls, stuttering speakers showed more grey matter (GM) volume concentration in the left putamen, less GM volume concentration in the left medial frontal gyrus and ASTG, and less white matter volume concentration underlying the left posterior superior temporal gyrus inside the BGTC. These results shed significant light on the neural mechanisms (in terms of both functional connectivity and neural anatomy) of stuttering.