Integrating Constituents Absorbed into Blood, Network Pharmacology, and Quantitative Analysis to Reveal the Active Components in Rubus chingii var. suavissimus that Regulate Lipid Metabolism Disorder.

Rubus chingii var. suavissimus (S. K. Lee) L. T. Lu (RS)-a sweet plant also known as Tiancha distributed in the south of China where it is used as a beverage-recently gained extensive attention as adjuvant therapy of diabetes and hypertension. Although pharmacological studies indicate that RS has beneficial effects in regulating lipid metabolism disorder characteristics, the active chemicals responsible for this effect remains unclear. The present study aims to predict the effective substances of RS on regulating lipid metabolism disorder through the analysis of the chemical profile of RS, the absorbed prototype components in rat plasma, and network pharmacology. Also, a UPLC method able to quantify the screened potential effective chemicals of RS products was established. First, a total of 69 components-including diterpene, triterpenoids, flavonoids, polyphenols, and lignans-were systematically characterized in RS. Of those, 50 compounds were detected in the plasma of rats administered with RS extract. Through network pharmacology, 9 potential effective components, 71 target genes, and 20 pathways were predicted to be involved in RS-mediated regulation of lipid metabolism disorder. The quantitative analysis suggested that the contents of potential effective components varied among samples from different marketplaces. In conclusion, the presented results provide a chemical basis for further research of Rubus chingii var. suavissimus.

Rubus chingii var. suavissimus alleviates high-fat diet-induced lipid metabolism disorder via modulation of the PPARs/SREBP pathway in Syrian golden hamsters.

While the underlying mechanism remains unknown, Rubus chingii var. suavissimus (S. K. Lee) L. T. Lu or Rubus suavissimus S. Lee (RS), a sweet plant distributed in southwest of China, has been used as beverage and folk medicine. Pharmacological studies indicated the potential of RS improving the obesity phenotype and hyperlipidemia. The mechanism is still not yet to be put forward. To verify the substantial effects of RS on lipid metabolism, a Syrian golden hamster model was adopted. The physiological and pathological evaluation of experimental animals demonstrated that RS can relieve the lipid metabolism disorder induced by high-fat diet and alleviated liver injury. RS upregulation the expressions of peroxisome proliferator-activated receptor α (PPARα), PPARγ and CCAAT/enhancer binding protein α (C/EBPα), as well as adipocyte-specific genes, glucose transporter 4 (Glut4), lipoprotein lipase (LPL) and fatty acid binding protein 4 (aP2). On the other side, RS suppressed the sterol regulatory element binding protein 1 (SREBP1) and downstream acetyl-CoA carboxylase 1 (ACC1), stearoyl-CoA desaturase-1 (SCD1) and fatty acid synthase (FAS). In conclusion, RS alleviated lipid metabolism disorder symptoms caused by high-fat diet accompanied with 8 weeks of treatment, involving enhanced ß-oxidation, increased adipogenesis and decreased the metabolism of fatty acids, via modulation of the PPARs/SREBP pathway in Syrian golden hamsters.

MRCKß links Dasm1 to actin rearrangements to promote dendrite development.

Proper dendrite morphogenesis and synapse formation are essential for neuronal development and function. Dasm1, a member of the immunoglobulin superfamily, is known to promote dendrite outgrowth and excitatory synapse maturation in vitro. However, the in vivo function of Dasm1 in neuronal development and the underlying mechanisms are not well understood. To learn more, Dasm1 knockout mice were constructed and employed to confirm that Dasm1 regulates dendrite arborization and spine formation in vivo. We performed a yeast two-hybrid screen using Dasm1, revealing MRCKß as a putative partner; additional lines of evidence confirmed this interaction and identified cytoplasmic proline-rich region (823-947 aa) of Dasm1 and MRCKß self-activated kinase domain (CC1, 410-744 aa) as necessary and sufficient for binding. Using co-immunoprecipitation assay, autophosphorylation assay, and BS3 cross-linking assay, we show that Dasm1 binding triggers a change in MRCKß's conformation and subsequent dimerization, resulting in autophosphorylation and activation. Activated MRCKß in turn phosphorylates a class 2 regulatory myosin light chain, which leads to enhanced actin rearrangement, causing the dendrite outgrowth and spine formation observed before. Removal of Dasm1 in mice leads to behavioral abnormalities. Together, these results reveal a crucial molecular pathway mediating cell surface and intracellular signaling communication to regulate actin dynamics and neuronal development in the mammalian brain.

Clinical features and prognosis of epilepsy in the elderly in western China.

PURPOSE: To investigate the characteristics and prognosis of epilepsy amongst older people hospitalized in southwestern China with newly diagnosed epilepsy. METHODS: We prospectively enrolled people older than 65 years who were admitted to a tertiary epilepsy center in West China between January 2008 and January 2013. Participants were divided into early-onset group (those who had a first seizure before age of 65) and late-onset group (those in whom the first seizure occurred after age of 65). Clinical data were collected and all participants were followed for two years. RESULTS: Of 340 people enrolled, focal seizure (84%) was the most frequent seizure type. Status epilepticus (64.4% vs. 46.7%, p=0.022) and structural epilepsy (59.3% vs. 40.0%, p=0.015) were more prevalent in late-onset group than early-onset group. Ischemic stroke was the leading putative cause (22.6%) in elderly epilepsies. Around 80% were given anti-epileptic drugs (AEDs) for treatment. Forty-two people did not complete the study, of whom 26 were lost to follow-up and 16 died for causes other than epilepsy. Of the 298 who completed the follow-up, 240 (80.5%) achieved significant seizure reduction. Logistic regression analysis indicated that late-onset epilepsies and AEDs treatment were associated with more favorable seizure outcome at two-year follow-up (OR=4.029 and 92.007, respectively). The number of AEDs intake exerted no significant impact on seizure outcome. CONCLUSIONS: In older people, late-onset epilepsies differed in several aspects from early-onset epilepsies. The overall effectiveness of AEDs treatment in older people was satisfactory.

Effectiveness of levetiracetam use following resective surgery in patients with refractory epilepsy: a prospective observational study.

PURPOSE: This study aimed to evaluate the effectiveness of levetiracetam (LEV) use for seizure control in patients who had undergone resective surgery for intractable epilepsy in routine clinical practice. METHODS: This was a prospective, observational study. Refractory epilepsy patients who underwent epilepsy surgery from January 2008 to December 2011 in the Department of Neurosurgery, West China Hospital were prospectively analyzed. Patients were divided into two groups according to antiepileptic drug (AED) treatment used immediately after epilepsy surgery (group A: therapy with LEV; group B: therapy without LEV). AED regimens were compared with regard to seizure-outcome for a period of more than 2 years. The International League Against Epilepsy (ILAE) classification was used to categorize seizure outcome. RESULTS: A total of 319 patients (184 male and 135 female patients; mean age 28.2±13.4 years) were studied. The mean postoperative follow-up period was 3.9±1.2 years. The two groups showed was no significant difference in preoperative baseline data. At the 6-month follow-up, the proportion of patients with seizure freedom was significantly higher in group A than in group B (78.8% vs. 67.5%, p=0.03). Seizure outcomes after 2 years were assessed using the ILAE classification. The proportion of patients under ILAE seizure-outcome classification I (seizure freedom) was significantly higher in group A than in group B (74.3% vs. 60.7%, p=0.01). Seizure recurrence rates at the final assessment, after planned reduction or withdrawal, were 26.3% for group A and 40.6% for group B (p=0.04). CONCLUSIONS: AED strategy after resective surgery may be a potentially modifiable prognostic indicator influencing seizure outcome in patients with intractable epilepsy. Compared to other AEDs, LEV appears to be more effective in controlling postoperative seizures in our long-term follow-up, and the advantage can be seen in early stage after surgery.

[Chemical constituents from Embelia laeta].

OBJECTIVE: To study the chemical constituents from Embelia laeta. METHODS: The constituents from the EtOAc fraction of ethanol extract of Embelia laeta were separated and purified by column chromatography with silica gel and polyamide. The compounds were identified by their physiochemical proerties and spectral data. RESULTS: Eleven compounds were isolated and identified as p-sitosterol (1), beta-daucosterel (2), gallic acid (3), vanillic acid (4), rutin (5), hyperin (6), quercetin (7), kaemperol ( 8), chrysoeriol (9), physcion(10) and apigenin-7-O-glucoside(ll). CONCLUSION: Compounds 5-11 are isolated from this genus for the first time.

Evaluation of different antiepileptic drug strategies in medically refractory epilepsy patients following epilepsy surgery.

PURPOSE: This study aimed to explore the most appropriate antiepileptic drug strategies after successful epilepsy surgery. METHODS: A total of 131 refractory epilepsy patients who underwent epilepsy surgery from January 2005 to December 2008 in the Department of Neurosurgery, West China Hospital, were retrospectively reviewed. Patients were divided into three groups (monotherapy, duotherapy, and polytherapy) according to drug combinations used immediately after epilepsy surgery. Seizure outcomes were followed up for more than 2 years. Engel classification was used to evaluate seizure outcomes. RESULTS: The mean postoperative follow-up period was 3.7±1.0 years. Preoperative baseline data among the three groups were comparable. Seizure recurrence rate in monotherapy was obviously higher than in other groups (34.1% vs. 15.1%, 7.1%) at 6-month follow-up, which showed a statistically significant difference (p=0.02). Seizure outcomes for 2 years were assessed using Engel classification. In the duotherapy group, the rate of Engel class I was definitely higher than in the other two groups (69.9% vs. 47.7%, 57.1%, p=0.02). Seizure relapse rates at the 2-year follow-up, after planned reduction or withdrawal, were 46.4% for monotherapy, 16.9% for duotherapy, and 25.0% for polytherapy (p=0.01). CONCLUSIONS: Monotherapy may be not sufficient enough to control seizures completely. It appears to have a higher risk for seizure relapse when considering drug reduction. It suggests that duotherapy may be more effective and safer than monotherapy. Even after successful epilepsy surgery, duotherapy seems preferable to monotherapy or polytherapy for control of residual seizures.

Alpha-synuclein functions as a negative regulator for expression of tyrosine hydroxylase.

Previous studies have shown that over-expression of alpha-Synuclein (alpha-Syn), a protein whose abnormality is implicated in the pathogenesis of Parkinson's disease (PD), reduces tyrosine hydroxylase (TH) expression and dopamine synthesis. To explore the possible mechanism for the regulation of TH expression by alpha-Syn, luciferase reporter gene carrying a -493/+27bp fragment of human TH gene (pGL3-TH520) and pcDNA carrying halpha-Syn gene (pcDNA-halpha-Syn) were co-transfected into T293 cells. The results showed that alpha-Syn was only detected in pcDNA-halpha-Syn-transfected cells but not in pcDNA vector control cells. In alpha-Syn-transfected cells, the luciferase activity was dramatically reduced compared with the vector control cells. These results suggest that alpha-Syn may function as a negative regulator for TH expression by affecting the activity of TH promoter.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry combined with magnetic beads for detecting serum protein biomarkers in parkinson's disease.

BACKGROUND: Biomarkers for neurodegenerative diseases are essential to facilitate disease diagnosis. Application of proteomics has greatly hastened the search for novel biomarkers. In this study, new potential biomarkers were discovered, and a diagnostic pattern was established for idiopathic Parkinson's disease (PD) by using proteomic technology. METHODS: Serum proteins from PD patients and controls were captured by magnetic bead-based weak cation exchange. The molecular weight of the proteins in bead-binding fraction was detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Biomarker Wizard 3.1 and Biomarker Patterns Software were used for data analysis and constructing a model of biomarkers. A blinded testing set was used to validate the model. RESULTS: A total of 17 discriminating m/z peaks related to PD were identified. The model based on the 5 biomarkers generated an excellent separation between PD and healthy controls with 98.36% for the sensitivity and 83.05% for the specificity. Blind test data demonstrated the model could recognize patients with PD with a sensitivity of 85.0% and a specificity of 70.0%. CONCLUSIONS: The preliminary data suggested a potential application of MALDI-TOF-MS combined with magnetic beads. The model comprising 5 promising biomarkers can differentiate individuals with PD and the healthy subjects precisely.

[Remote passive sensing of aeroengine exhausts using FTIR system].

The traditional method of measuring the aeroengine exhausts is intrusive gas sampling analysis techniques. The disadvantages of the techniques include complex system, difficult operation, high costs and potential danger because of back-pressure effects. The non-intrusive methods have the potential to overcome these problems. So the remote FTIR passive sensing is applied to monitor aeroengine exhausts and determine the concentration of the exhausts gases of aeroengines. The principle of FTIR remote passive sensing is discussed. The model algorithm for the calibration of FTIR system, the radiance power distribution and gas concentration are introduced. TENSOR27 FTIR-system was used to measure the spectra of infrared radiation emitted by the hot gases of exhausts in a test rig. The emission spectra of exhausts were obtained under different thrusts. By analyzing the spectra, the concentrations of CO2, CO and NO concentration were calculated under 4 thrusts. Researches on the determination of concentration of the exhausts gases of aeroengines by using the remote FTIR sensing are still in early stage in the domestic aeronautics field. The results of the spectra and concentration in the aeroengine test are published for the first time. It is shown that the remote FTIR passive sensing techniques have a great future in monitoring the hot gas of the aeroengines exhausts.

Upregulation of Ryk expression in rat dorsal root ganglia after peripheral nerve injury.

To study changes of Ryk expression in dorsal root ganglia (DRG) after peripheral nerve injury, we set up an animal model of unilateral sciatic nerve lesioned rats. Changes of Ryk protein expression in DRG neurons after unilateral sciatic nerve injury were investigated by immunostaining. Changes of Ryk mRNA were also tested by semi-quantitative PCR concurrently. We found, both at the level of protein and mRNA, that Ryk could be induced in cells of ipsilateral DRG after unilateral sciatic nerve lesion. Further investigation by co-immunostaining confirmed that the Ryk-immunoreactive (Ryk-IR) cells were NeuN-immunoreactive (NeuN-IR) neurons of DRG. We also showed the pattern of Ryk induction in DRG neurons after sciatic nerve injury: the number of Ryk IR neurons peaked at 2 weeks post-lesion and decreased gradually by 3 weeks post-lesion. The proportions of different sized Ryk IR neurons were also observed and counted at various stages after nerve lesion. Analysis of Ryk mRNA by RT-PCR showed the same induction pattern as by immunostaining. Ryk mRNA was not expressed in normal or contralateral DRG, but was expressed 1, 2 and 3 weeks post-lesion in the ipsilateral DRG. Ryk mRNA levels increased slightly from 1 to 2 weeks, decreased then by 3 weeks post-lesion. These results indicate that Ryk might be involved in peripheral nerve plasticity after injury. This is a novel function apart from its well-known fundamental activity as a receptor mediating axon guidance and outgrowth.

[Aberrant immunophenotypes in 126 patients with acute leukemia].

The existence of leukemia aberrant immunophenotypes (LAIP) has been suggested to be a valuable tool for the detection of minimal residual disease (MRD), as they could distinguish leukemic cells from normal hematopoietic progenitors. This study was purposed to analyze the characteristics of LAIP in acute leukemia and further explore the proportion of different types of LAIP in acute leukemia patients. Flow cytometry (FCM) with four color and CD45/SSC gating were used to detect the antigen expression in samples of bone marrow from 126 patients with acute leukemia. The results showed that definite LAIP could be detected in about 76% patients. The LAIP could be divided into four groups as cross-lineage antigen expression, asynchronous antigen expression, antigen overexpression and antigen lack expression. The percentages of these LAIPs were 39%, 46%, 21% and 29% respectively. About 11% out of analyzed cases showed the existence of only one aberrant phenotype while two or more of aberrant phenotypes could be detected in majority cases. It is concluded that the LAIP with four subgroups can be detected in the majority of patients with acute leukemia and immunophenotyping based on LAIP is applicable for the detection of MRD.

Effect of alpha-synuclein on the promoter activity of tyrosine hydroxylase gene.

OBJECTIVE: To approach the associated mechanism by which alpha-synuclein (alpha-Syn) might regulate the metabolism of dopamine. METHODS: A DNA fragment, located at -495 to +25 of the human tyrosine hydroxylase (TH) gene, was amplified by PCR and inserted into the pGL(3)-Basic luciferase reporter vector. The recombinant plasmid pGL(3)-THprom was transfected into a dopaminergic cell line MES23.5 or a alpha-Syn over-expressed MES23.5 (named MES23.5/halpha-Syn(+)). The promoter activity was detected by the Dual Luciferase Assay System. RESULTS: The luciferase activities in the MES23.5 cells transfected with pGL(3)-Basic, pGL(3)-THprom, and pGL(3)-Control vectors were 5.60+/-0.67, 26.80+/-4.11, and 32.90+/-4.75, respectively. On the other hand, the luciferase activity of pGL(3)-THprom in the MES23.5 (26.80+/-4.11) was significantly higher than that in the MES23.5/halpha-Syn(+) (14.40+/-0.61) (P<0.01). CONCLUSION: These results indicate that the - 495 to +25 region in the TH gene possesses promoter activity for controlling the gene expression, and that alpha-Syn may negatively regulate the metabolism of dopamine by affecting the function of TH promoter as a trans-acting factor.

Repetition rates of specific interval patterns in single spike train reflect excitation level of specific receptor types, shown by high-speed favored-pattern detection method.

UNLABELLED: Interval patterns in single spike train, e.g. "favored patterns (FPs, the FP is a sequence of successive intervals of action potentials that occur more often than what is reasonably expected at random.)", may represent neural codes containing information. The present study developed a "high-speed FP-detection method" which could qualitatively and quantitatively analyze FPs. By using this method, single spike trains of nucleus paraventricularis (NPV) and rostral ventrolateral medulla (RVL) having different firing patterns, being involved in regulation of arterial pressure, and controlled by different transmitters, were chosen for analysis. RESULTS: (1) Corticotropin releasing factor, substance P and agonists of alpha-, beta- and M-receptor microinjected into these brain areas, respectively, induced dominant change of specific FP. Repetition rates of specific FPs reflect excitation level of specific receptor types. It shows that chemical codes (different transmitters with their receptor types or subtypes) are transformed into electrical codes (different FPs). (2) When alpha-, beta- and M-receptors of RVL neurons were activated simultaneously by intrinsic excitatory transmitters released due to activation of input pathway, only repetition rate of the specific FP that represented the predominant activity of the receptor type (alpha-adrenergic receptor) markedly increased. The activities of other receptor types (beta- and M-receptors) were masked. (3) Intrinsic inhibitory transmitters (GABA, beta-endorphin) in the RVL all decreased specific FP repetition rate of dominant receptor type. These results may provide a new way to further explore how information in the CNS is conveyed and processed.

cDNA cloning, prokaryotic expression and purification of rat alpha-synuclein.

Objective To clone the cDNA of rat alpha-Syn gene, investigate its prokaryotic expression and produce purified recombinant rat alpha-Syn protein. Methods Rat alpha-Syn cDNA was amplified from the rat brain total RNA by RT-PCR and was cloned into pGEX-4T-1, a prokaryotie expressing vector. The recombinant plasmid containing rat alpha-Syn gene was transformed into E. Coli BL21 to express a fusion protein with rat alpha-Syn protein tagged by glutathione-S-transferase (GST). The fusion protein was then cleaved by thrombin during passing through the GST-agarose 4B column to release the recombinant rat alpha-Syn protein. The recombinant rat alpha-Syn protein was further purified using Superdex S200 gel filtration.Results DNA sequencing confirmed that the cloned cDNA contained 420 base pairs encoding 140 amino acids, which was identical to the reported amino acid sequence of rat alpha-Syn. After transformation, the recombinant plasmid pGEX-raSyn expressed a soluble protein that was inducible by IPTG. The purified recombinant protein was shown to be single band on SDS-PAGE, with a molecular size of around 18000, which was identical to the reported molecular size of rat alpha-Syn.Western blot analysis demonstrated that the recombinant protein was recognized by specific antibody against alpha-Syn. Conclusion The rat alpha-Syn gene was successfully expressed in prokaryotic expression system and highly purified rat alpha-Syn recombinant protein was produced.

[Effect of repeated acute hypoxic treatment on the expression of alpha-synuclein in the mouse brain cortex].

An anti-alpha-synuclein (alpha-SYN) monoclonal antibody produced in our laboratory was used to investigate the effect of repeated acute hypoxic treatments on the expression of alpha-SYN in the mouse cerebral cortex. Western blot analysis showed that the expression levels of alpha-SYN in the cortex changed accordingly upon hypoxic exposure times, as that the alpha-synuclein level significantly increased after the first hypoxic exposure and then dropped down to the background level after the fourth hypoxic exposure. Immunohistochemical staining revealed that the alpha-SYN-immunopositive substance was localized not only in the nerve endings, but also within the nuclei of some neurons. The cell density of the neurons with alpha-SYN immunopositive nuclei was increased significantly after the first hypoxic exposure but returned back to control levels after the fourth hypoxic exposure. Our results indicate that both of the alpha-SYN expression level in the brain and the number of the neurons with alpha-SYN positive nuclei are affected by the repeated acute hypoxic treatments and that this modification is hypoxic time-dependent. The mechanism and the physiological significance underlying these changes need to be further investigated.

Inhibitory effect of atriopeptinergic neurons in AV3V region on angiotensinII pressor system in rat brain.

In the central nervous system and the periphery, atrial natriuretic peptide (ANP) and angiotensinII(AngII) play important and opposite roles in regulating blood pressure and fluid electrolyte balance. Their central mechanisms are unclear. In the brain the anteroventral third ventricle region (AV3V) contains the most prominent collection of atriopeptin-like immunoreactive perikarya. Our previous studies show that: (1) AV3V stimulation by glutamate produces a fall in blood pressure; (2) there is an AngII pressor system composed of the lateral hypothalamus/perifornical region (LH/PF), subfornical organ (SFO), nucleus paraventricularis (NPV) and rostral ventrolateral medulla (RVL). The present study was to examine whether ANPergic projections from the AV3V could act on nuclei involved in the above-mentioned AngII pressor system. Here we demonstrate that: (1) Injection of atriopeptinIII into the LH/PF, SFO, NPV, or RVL induces a depressor response; whereas injection of normal saline has no effect. (2) Pre-injection of A 71915 (an atriopeptinIII antagonist) into the LH/PF, SFO, NPV, or RVL reverses the depressor response of the AV3V to glutamate (Glu). The results suggest that excitation of atriopeptinergic neurons in the AV3V by Glu produces an inhibitory effect on each nucleus in the LH/PF-SFO-NPV-RVL AngII pressor system.

[Study on quality analysis of essential oil from twig and leaf of Baeckea frutescens].

OBJECTIVE: To provide scientific methods for quality criterion by studying the chemical components of essential oil from Baeckea frutescens. METHOD: The chemical components of essential oil from B. frutescens were identified by GC-MS-DS, TLC and capillary GC. The relative contents of main components were determined by area normalization. RESULT: More than 50 peaks were separated, and 38 components were identified, which accounted for over 94% of the total GC peaks areas of the essential oil. The methods for quality evaluation of essential oil from B. frutescens by TLC and capillary GC were established. CONCLUSION: The chemical components of essential oil from B. frutescens collected from different habitats and collecting periods have common characteristics as well as differences. Some components, such as linalool, can be used as a standard and chromatography fingerprint to analyze the quality of essential oil from B. frutescens.

Subfornical organ-angiotensin II pressor system takes part in pressor response of emotional circuit.

It has been proved that there are the subfornical organ (SFO)-nucleus paraventricularis (NPV)-rostral ventrolateral medulla (RVL) angiotension II (AngII) pressor system and the central amygdaloid nucleus (AC)-lateral hypothalamus/perifornical region (LH/PF) emotional pressor system in the brain. Because the LH/PF contains abundant AngII ergic neurons projecting to the SFO, the purpose of the present study was to examine whether the (SFO-NPV-RVL) AngII pressor system takes part in the AC-pressor response via AngII ergic neurons in the LH/PF. The results showed that (1) L-glutamate microinjection into the AC or LH/PF induced pressor responses. (2) Both the AC- and LH/PF-pressor responses could be reversed by preinjection of [Sar(1), Thr(8)]-angiotensin II (an antagonist of AngII) into either the SFO, NPV or RVL. Taken together with our previous findings that the projections of the CRF-ergic and SP-ergic neurons in the AC could activate the LH/PF, the above findings prove that: besides several known mechanisms of the brain AngII inducing pressor response, the (SFO-NPV-RVL) AngII pressor system also takes part in the AC-emotional pressor response via AngII ergic projections from the LH/PF to the SFO, which may be the neurophysiological basis of the brain AngII playing an important role in developing hypertension of the SHRs.