NK1R/5-HT1AR interaction is related to the regulation of melanogenesis.

Substance P (SP) is a candidate mediator along the brain-skin axis and can mimic the effects of stress to regulate melanogenesis. Previously, we and others have found that the regulation of SP for pigmentary function was mediated by neurokinin 1 receptor (NK1R). Emerging evidence has accumulated that psychologic stress can induce dysfunction in the cutaneous serotonin 5-hydroxytryptamine (5-HT)-5-HT1A/1B receptor system, thereby resulting in skin hypopigmentation. Moreover, NK1R and 5-HTR (except 5-HT3) belong to GPCR. The present study aimed at assessing the possible existence of NK1R-5-HTR interactions and related melanogenic functions. Western blot and PCR detection revealed that SP reduced expression of 5-HT1A receptor via the NK1 receptor. Biochemical analyses showed that NK1R and 5-HT1AR could colocalize and interact in a cell and in the skin. When the N terminus of the NK1R protein was removed NK1R surface targeting was prevented, the interaction between NK1R-5-HT1AR decreased, and the depigmentation caused by SP and WAY100635 could be rescued. Importantly, pharmaceutical coadministration of NK1R agonist (SP) and 5-HT1A antagonist (WAY100635) enhanced the NK1-5-HT1A receptor coimmunoprecipitation along with the depigmentary response. SP and WAY100635 cooperation elicited activation of a signaling cascade (the extracellular, regulated protein kinase p-JNK signaling pathway) and inhibition of p70S6K1 phosphorylation and greatly reduced melanin production in vitro and in vivo in mice and zebrafish. Moreover, the SP-induced depigmentation response did not be occur in 5-htr1aa+/- zebrafish embryos. Taken together, the results of our systemic study increases our knowledge of the roles of NK1R and 5-HT1AR in melanogenesis and provides possible, novel therapeutic strategies for treatment of skin hypo/hyperpigmentation.-Wu, H., Zhao, Y., Huang, Q., Cai, M., Pan, Q., Fu, M., An, X., Xia, Z., Liu, M., Jin, Y., He, L., Shang, J. NK1R/5-HT1AR interaction is related to the regulation of melanogenesis.

IFN-γ inhibits 5-HT-induced melanin biosynthesis via downregulation of 5-HT receptors in vivo/in vitro.

Hypopigmentation disorders, such as vitiligo, are difficult for treatment due to complicated pathogenesis, resulting from multiple factors including neural and immune elements. 5-HT and IFN-γ both play crucial roles in these skin diseases. However, the interactions between 5-HT and IFN-γ in regulation of melanogenesis is still unknown. Our study aimed at exploring whether IFN-γ affects 5-HT-induced melanogenesis and searching the mechanism. In our study, IFN-γ attenuated 5-HT-induced pigmentation and green fluorescent protein (GFP) expression in zebrafishes. In addition, we found that IFN-γ decreased serum serotonin levels as well as the cutaneous expression of tryptophan hydroxylase 1 (TPH1), 5-HT1A receptor (5-HT1AR) and 5-HT1B receptor (5-HT1BR) in C57BL/6 mice. Moreover, IFN-γ attenuated 5-HT-induced melanin biosynthesis as well as the expression of 5-HT1AR, 5-HT1BR and 5-HT2A receptor (5-HT2AR) in B16F10 cells, which blocked by interferon-γ receptor 1 and interferon-γ receptor 2 (IFNGR1/IFNGR2) antibodies. In summary, IFN-γ not only affects melanogenesis alone, but also inhibits 5-HT response on melanin biosynthesis. Mediated by IFNGR1/IFNGR2, IFN-γ downregulated 5-HT receptors expression, which directly affect 5-HT-induced melanin biosynthesis. Our work may give insights into the drug development of hypopigmentation disorders with neuro-immune derangement.

Effect of the Tibetan Medicine Zuotai on Degranulation and Inflammatory Mediator Release in RBL-2H3 Cells.

Zuotai is a drug containing mercury considered to be the king of Tibetan medicine. The biosafety of Zuotai led people's attention and so far little is known about the toxicity of Zuotai to mast cells. RBL-2H3 cells which used as an alternative model of mast cells were treated with Zuotai, ß-HgS and positive drug Compound 48/80 respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the toxicity of drugs to RBL-2H3 cells. The degranulation of RBL-2H3 cells was studied from ß-hexosaminidase, histamine, interleukin (IL)-4 and tumor necrosis factor-α (TNF-α). The result showed that Zuotai can affect the cytotoxicity and degranulation of RBL-2H3 cells and the results can provide reference for the toxicity evaluations of Tibetan medicine Zuotai.

Influence of Residue Soil on the Properties of Fly Ash-Slag-Based Geopolymer Materials for 3D Printing.

This study investigates the impact of residue soil (RS) powder on the 3D printability of geopolymer composites based on fly ash and ground granulated blast furnace slag. RS is incorporated into the geopolymer mixture, with its inclusion ranging from 0% to 110% of the combined mass of fly ash and finely ground blast furnace slag. Seven groups of geopolymers were designed and tested for their flowability, setting time, rheology, open time, extrudability, shape retention, buildability, and mechanical properties. The results showed that with the increase in RS content, the fluidity of geopolymer mortar decreases, and the setting time increases first and then decreases. The static yield stress, dynamic yield stress, and apparent viscosity of geopolymer mortar increase with the increase in RS content. For an RS content between 10% and 90%, the corresponding fluidity is above 145 mm, and the yield stress is controlled within the range of 2800 Pa, which meets the requirements of extrusion molding. Except for RS-110, geopolymer mortars with other RS contents showed good extrudability and shape retention. The compressive strength of 3D printing samples of geopolymer mortar containing RS has obvious anisotropy.

[The chemical and structural analysis of Tibetan medicine Zhuxi].

Zhuxi is a mineral medicine widely used in traditional Tibetan medicine throughout history. However, the bioactive component in Zhuxi still remains unclear. In order to enunciate the material basis of its pharmacological activity, the present research has determined the chemical component and structure of Zhuxi. X-ray fluorescence spectroscopy (XRF), inductively coupled plasma optical emission spectrometer (ICP-OES) and X-ray diffraction (XRD) were utilized to assay two samples of Zhuxi. XRF and ICP-OES analysis indicated that the main elements in Zhuxi are Fe, S and O, also containing some minor elements, such as Si, Na, Mg, Al, K, Ni, Ca, Ti and so on. XRD analysis suggested that the main crystal compound in Zhuxi is FeS2 (Cubic, Pa-3), also existing a few of Fe(+3)O(OH) (orthorhombic, Pbnm) and other some unknown compounds. These studies has highlighted the potential the element components and compound structures of Zhuxi, so it may be a good starting point for exploring the material basis of its pharmacological activity.

[Chemical and structural analysis of Nengchi Bajin ashes in refining of Tibetan medicine gTSo thal].

OBJECTIVE: To investigate the chemical components and microstructure of Nengchi Bajin ashes which are adjuvant material in the refining of Tibetan medicine gTSo thal, in order to explore the material basis of the refining of gTSo thal. METHOD: Scanning electron microscope-energy dispersive spectrometer (SEM-EDX) and X-ray diffraction (XRD) were used to measure the Nengchi Bajin ashes. RESULT: SEM-EDX analysis show that except of themselves elements of Nengchi Bajin ashes, Nengchi Bajin ashes contain the major elements, such as S, O, C and so on, also contain small amount other elements. XRD analysis show that the structures are AuPb2, PbO (tetragonal and orthorhombic) and Pb in gold ash, Ag2S and PbO in silver ash, Cu1.98 (Zn0.73 Fe0.29)Sn0.99 S4, CuS, SiO2, NaCu2S2 and Ca (Fe(+2), Mg) (CO3)2 in bronze ash, Cu7S4 (orthorhombic and monoclinic) and CuO in red copper ash, Cu7 S4, PbS, ZnS, CaCO3and NaCu2S2 in brass ash, FeS, Fe+2 Fe(2+3)O4 and SiO2 in iron ash, SnS and SiO2 tin ash, PbS, PbSO4 and SnS2 in lead ash. CONCLUSION: We have acquired the datum of elements and microstructure of Nengchi Bajin ashes by SEM-EDX and XRD techniques, and that is benefit to explore the material basis of refining gTSo thal.

[Quality control of traditional Tibetan Medicine Zsuotai].

OBJECTIVE: To establish the method of quality control for traditional Tibetan Medicine Zsuotai. METHODS: Collecting the samples of Tsuotai from Qinghai, Tibet, Sichuan, and Gansu province, to detect Hg2+ by Zsuotai reacted with HCl-HNO3 (3:1), and to determine the quantity of HgS in Zsuotai by sulfocyanate volumetric method. RESULTS: The method for the determination of HgS in Zsuotai was in good reproducibility (RSD = 0.68%). The calibration curve was linear (r = 0.9999) within -0.0002 - 0.2123 g of mercuric sulfide. The recovery was 100.94% (RSD = 0.66%). CONCLUSIONS: This method is convenient and accurate, so it can be used to establish quality control of the medicinal material.

A Novel Zebrafish Larvae Hypoxia/Reoxygenation Model for Assessing Myocardial Ischemia/Reperfusion Injury.

Strategies to reduce reperfusion injury after ischemia have been considered in clinical practice, but few interventions have successfully passed the proof-of-concept stage. In this study, we developed a novel zebrafish larvae hypoxia/reoxygenation (H/R) model to simulate myocardial ischemia/reperfusion injury (MIRI), with potential utility as a drug screening tool. After H/R treatment, videos of transgenic [Tg(cmlc:EGFP)] larval zebrafish hearts were captured using a digital high-speed camera, and the heart rate, diastolic area, systolic area, and total fraction of area changed were quantified. The mRNA expression of tnnt2, bnp, and hif1α was quantified, and red blood cells (RBCs) were detected by O-dianisidine staining. We found that a decline in cardiac contractility occurred in zebrafish larvae 48 h after hypoxia treatment. Reoxygenation for 2-5 h after 48 h of hypoxia caused heart dysfunction in zebrafish larvae, and were determined to be the optimum conditions for simulating MIRI similar to mammalian models. Our results indicated that heart dysfunction after reoxygenation in zebrafish larvae was accompanied by an upregulated gene expression of a number of myocardial injury biomarkers and increased numbers of RBCs. In conclusion, the novel larval zebrafish H/R model developed in this study could be used for rapid in vivo screening and efficacy assessment of MIRI therapeutics.

A simple, efficient and rapid HPLC-UV method for the detection of 5-HT in RIN-14B cell extract and cell culture medium.

5-Hydroxytryptamine (also known as 5-HT, serotonin) is one of the monoamine neurotransmitters which is distributed widely in plasma and brain of mammals and plays important roles in physiological manipulations. In the present method, we describe the development of a simple, efficient and rapid high performance liquid chromatographic method coupled with ultraviolet (HPLC-UV) detector for the qualitative and quantitative analysis of 5-HT in both cell extract and cell culture medium (RIN-14B). The experiments use repeated freeze-thaw cycles followed by centrifugation and direct injection of the supernatant into the chromatography. An analytical C18 column (Agilent Zorbax Extend, 4.6 × 250 mm, 5 µm.) was taken for chromatographic separation; the mobile phase was 0.05 mol/L potassium dihydrogen phosphate (KH2PO4)/acetonitrile (90:10 v/v). Isocratic elution is established at the flow rate of 1.0 mL/min. The time required for this chromatographic run is 8 min. Over the concentration range of 0.1-10 µg/mL, the calibration curve is linear in this method. Other unique characteristics and advantages include high accuracy (92.02-103.28%) and high precision (intra- and inter-day coefficients of variation ≤ 4.69%). This method is applicable for the investigation of drug/condition-response relationships in the function of synthesis and secretion of 5-HT in cultured RIN-14B cells in various in vitro studies.

Simvastatin therapy in adolescent mice attenuates HFD-induced depression-like behavior by reducing hippocampal neuroinflammation.

BACKGROUND: A high-fat diet (HFD)-induced obesity/hyperlipidemia is accompanied by hormonal and neurochemical changes that can be associated with depression. Emerging studies indicate that simvastatin (SMV, decreasing cholesterol levels) has therapeutic effects on neurological and neuropsychiatric diseases through hippocampal-dependent function. However, the studies on the HFD exposure in adolescent animals, which investigate the neuroprotective effects of SMV on the hippocampal morphology, serotonin (5-HT) system and inflammation, are limited. Hence, the aim of this study was to determine whether SMV attenuates HFD-induced major depressive disorders in adolescent animals and, more specifically, acts as an anti-neuroinflammatory response. METHODS: Twenty-four male C57BL/6 mice were fed a control (n = 8), HFD (n = 8) and HFD + SMV (n = 8) for 14 weeks. In HFD + SMV group, SMV (10 mg/kg) was administrated from the 10th week of HFD feeding. The open field test (OFT) and the tail suspension test (TST) were used to examine the effect of SMV on behavioral performance. HE and Nissl staining were conducted to detect hippocampal morphology and neural survival. Expression of the inflammatory cytokine genes was assayed by quantitative polymerase chain reaction (Q-PCR). RESULTS: Firstly, alterations in lipid parameters were minimized after SMV treatment. HFD-induced depression-like behavior, which was evidenced by an increase in immobility time in TST along with considerable decrease in locomotion activity, was significantly attenuated by SMV therapy for 4 weeks. Additionally, SMV could reduce HFD-induced structural abnormality, neuronal injury, serotonergic system disturbance and pro-inflammatory cytokine over-expression in the hippocampus. Neuroimmunological changes in central hippocampus displayed a similar characteristic (only IL-1ß, IL-6, TNF-α) with that in periphery spleen, whereas they appeared in an entirely opposite trend with that in cerebral cortex. CONCLUSION: Our results suggest that SMV may be a promising treatment for HFD-induced depression-like behavior during adolescent period through brain region-specific neuroninflammatory mechanisms.

Traditional Tibetan medicine Anzhijinhua San attenuates ovalbumin-induced diarrhea by regulating the serotonin signaling system in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Tibetan medicine has been practiced for 3800 years. Anzhijinhua San (AZJHS), which is a traditional Tibetan medicine, has been effective in the treatment of indigestion, anorexia and cold diarrhea. However, the effects of AZJHS on allergic diarrhea have not been reported. AIM OF THE STUDY: The aim of the present study was to elucidate the effect of AZJHS on experimental ovalbumin-induced diarrhea and elucidate its possible mechanism. MATERIALS AND METHODS: Female BALB/c mice were sensitized by intraperitoneal injection with 50 µg ovalbumin (OVA) and 1 mg alum in saline twice during a 2-week period. From day 28, mice were orally challenged with OVA (50 mg) every other day for a total of ten times. AZJHS (46.8 and 468.0 mg/kg) was orally administered every other day from day 0-46. Food allergy symptoms were evaluated. OVA- specific IgE, 5-HT and its metabolites in serum were determined. Immunohistochemical and histopathology were performed in gastrointestinal tract tissues. 5-HT-related gene expression was assayed in the colon. RESULTS: Severe symptoms of allergic diarrhea were observed in the model group (diarrhea, anaphylactic response, and rectal temperature). AZJHS (46.8 and 468.0 mg/kg) significantly reduced mouse diarrhea and significantly prevented the increases in OVA-specific IgE levels (P < 0.05), which challenge with OVA. AZJHS (46.8 and 468.0 mg/kg) significantly prevented the increases in 5-HT-positive cells. The nuclei of EC cells in the AZJHS (46.8 and 468.0 mg/kg) group increased in size and the secretory granules were fewer in number compared with those in the model group. AZJHS (46.8 and 468.0 mg/kg) significantly increased the relative fold changes of 5-HTP and 5-HT compared with the model group. The mRNA expression of the serotonin transporter (Sert) and serotonin receptor 3A (Htr3a) was significantly decreased after the 10th challenge with OVA, and AZJHS (46.8 and 468.0 mg/kg) significantly increased these levels. CONCLUSIONS: We demonstrated that the administration of AZJHS attenuated OVA-induced diarrhea by regulating the serotonin pathway. These results indicated that AZJHS may be a potential candidate as an anti-allergic diarrhea agent.

[Metabolic analysis of serotonin system in serum and gastric tissues of ovalbumin-induced allergic mice].

Objective To investigate the changes of 5-HT (serotonin) signaling system in allergic diarrhea mice sensitized with ovalbumin (OVA). Methods The seven-to-eight-week-old BALB/c female mice were randomly divided into model group, sodium chromate group and negative control group. The model group and sodium chromate group were intraperitoneally injected with OVAI (50 µg per mouse) at day 0 and day 14 respectively. And starting from the 28th day, OVAII was orally administered (50 mg per mousee) every other day (8 times in total), and the sodium chromate group was given the sodium chromate (78.0 mg/kg) before the oral administration of OVA every other day (8 times in total). The allergic symptoms, including the systemic score, faeces score and body temperature were recorded following the OVA administration for sensitization. The mice were executed 43 days later. Eyeball blood sample was collected, and then serum was seperated by centrifugation, the gastric tissues was taken out. The serum OVA-specific IgE (OVA-SIgE) was detected by ELISA. The serum content of 5-HT and its related metabolites including kynurenine (KYN), tryptophan (TRP), 5-hydroxytryptophan (5-HTP), and 5-hydroxyindoleacetic acid (5-HIAA) were examined by liquid chromatography-mass spectrometry (LC-MS). The mRNA levels of tryptophan hydroxylase-1 (TPH1), indolamine-2, 3-dioxygenase 1 (IDO1), monoamine oxidase A (MAO-A), 5-hydroxytryptamine 1A receptor (HTR1A), 5-hydroxytryptamine 3 receptor (HTR3), 5-hydroxytryptamine 4 receptor (HTR4) and serotonin reuptake transporter (SERT) were determined by real-time quantitative PCR. Results OVA sensitization caused severe allergic diarrhea in mice. Serum OVA-SIgE increased significantly in mice sensitized by OVA. serum KYN increased remarkably, while 5-HT, 5-HIAA and 5-HTP decreased significantly. The mRNA levels of IDO1, HTR1A and HTR3A increased in gastric tissues, while the levels of TPH1 and MAO-A mRNA decreased. Compared with the model group, the sodium chromate group had lowed systemic score, faeces score, body temperature and OVA-SIgE as well as diarrhea rate. The mRNA levels of 5-HIAA and MAO-A increased in the gastric tissues, and IDO1, 5-HT1A and 5-HT3A mRNAs decreased in the sodium chromate group. Conclusion The serotonin signaling system in ovalbumin-sensitized allergic diarrhea mice has been activated. The administration of sodium chromate can alleviate the allergic symptoms, and change the levels of serum metabolites and the gene expressions of the 5-HT metabolic pathway and its receptors in the stomach.