Effects of keratinocyte-derived and fibroblast-derived exosomes on human epidermal melanocytes

Background: Exosomes have been demonstrated to carry proteins, membrane lipids, mRNAs and microRNAs which can be transferred to surrounding cells and regulate the functions of those recipient cells. Objectives: The objective of the study was to investigate the effects of exosomes released by keratinocytes and fibroblasts on the proliferation, tyrosinase activity and melanogenesis of melanocytes. Methods: Melanocytes, keratinocytes and fibroblasts obtained from human foreskin were cultured and exosomes secreted by keratinocytes and fibroblasts were harvested from the culture supernatants by ultracentrifugation. Each exosome fraction was divided into two parts; one part was subjected to high-throughput sequencing using an Illumina HiSeq sequencer to characterize the microRNA expression profiles, while the other part was labeled with the fluorescent dye PKH67 and was then co-cultivated with epidermal melanocytes. Results: High-throughput sequencing analysis showed 168 differentially expressed microRNA within exosomes derived from keratinocytes and from fibroblasts, 97 of those being up-regulated with the other 71 down-regulated. Gene ontology analysis showed that the target genes responsible for these differentially expressed microRNAs were mainly enriched in the protein-binding region of molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that target genes regulated by differentially expressed microRNA were mainly involved in mitogen-activated protein kinase (MAPK) signaling pathway, Ras signaling pathway, cAMP signaling pathway and Wnt signaling pathway. Keratinocyte-derived exosomes were taken up by melanocytes co-cultured with them and promoted the proliferation, tyrosinase activity and melanin synthesis of those melanocytes. However, fibroblast-derived exosomes had no similar effects on melanocytes. Conclusion: Keratinocyte-derived exosomes but not fibroblast-derived exosomes were taken up by melanocytes in co-culture and significantly stimulated their proliferation, tyrosinase activity and melanin synthesis. Those different effects may be mainly due to the differential expression of microRNAs in exosomes derived from the different types of cells. Limitations: Electron microscopy of the obtained exosomes and in-depth study of apparently differentially expressed microRNAs were not performed

Melanocyte spheroids are formed by repetitive long-term trypsinization

Background: Autologous melanocyte transplantation plays an important role in the treatment of vitiligo. Objective: Previous studies have indicated that, compared with melanocytes growing in monolayers, melanocyte spheroids have a better survival in growth factor- and serum-deprived conditions. Methods: Melanocyte spheroids were obtained from human epidermis by repetitive long-term trypsinization and maintained an aggregated morphology for a short period in certain conditions. Results: Melanocyte spheroids were capable of growing into normal dendritic melanocytes in monolayer when they were harvested and reinoculated in 24-well plates. Immunohistochemical analysis of the melanocyte spheroids revealed that they were positive for HMB45, a melanosome-specific marker. No melanomas occurred when melanocyte spheroids were transplanted into mice. Conclusion: Our study provides a promising approach for melanocyte transplantation to treat vitiligo.

Effects of neuroactive steroid allopregnanolone on the damage of cortical neurons / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the protective mechanism of neuroactive steroid allopregnanolone on N-methyl-D-aspartate (NMDA) induced toxicity in primary mouse cortical neurons.</p><p><b>METHODS</b>Primary cultured mouse cortical neurons were subjected to allopregnanolone, the expression of beta-aminobutyric acid receptor beta2 subunit (beta2-GABA-R) mRNAs was detected by RT-PCR and Akt phosphorylation was assayed by Western blot using Akt-phosphoserine 473-specific antibody. After the cultured mouse cortical neurons were pretreated with or without allopregnanolone prior to treatment with NMDA , DNA isolated was analyzed by agarose gel electrophoresis and proteins collected were analyzed by Western blot with anti-cleaved-PARP, anti-cleaved caspase-3, and anti-cleaved caspase-9 antibodies.</p><p><b>RESULTS</b>When cultured mouse cortical neurons were exposed to allopregnanolone both the expression of beta2-GABA-R mRNAs and Akt phosphorylation increased. Allopregnanolone inhibited the NMDA-induced apoptosis and decreased the level of active-PARP, active-caspase-3 and active-caspase-9 notably at a final concentration of 5 x 10(6) mol/L.</p><p><b>CONCLUSION</b>Pretreatment with allopregnanolone may be neuroprotective on NMDA-induced neuronal cells apoptosis by increasing beta2-GABA-R expression and Akt phosphorylation.</p>

Effect of ginkgolide B on plasma levels of interleukin-12 and PAF in severe acute pancreatitis in rats / 中国应用生理学杂志

<p><b>AIM</b>To observe the levels of interleukin-12 (IL-12) and platelet activating factor (PAF) in severe acute pancreatitis (SAP) in rats and the efficacy of Ginkgolide B (BN52021) in treating SAP.</p><p><b>METHODS</b>Wistar rats were randomly divided into 3 groups: model group (SAP), treatment group (BN) and negative control group (NC). SAP was induced by retrograde infusion of 5% sodium taurocholate into the pancreatic duct in Wistar rats. NC rats only receive abdominal incision. In groups of SAP and NC rats received the femoral vein injection of isotonic Na chloride 15 minutes after induction of SAP; in BN group,rats received BN52021 instead. After operation rats were sacrificed at 1, 6 and 12 hour for plasma IL-12 and PAF determined with ELISA.</p><p><b>RESULTS</b>An increase of IL-12 in group BN was observed VS group SAP or group NC at 1 h stage (p = 0.011, P < 0.01). At 6 h or 12 h stage,an increase of IL-12 in group SAP was observed VS group NC (P < 0.01, P < 0.05). The plasma level of PAF in group SAP or group BN was increased significantly at 1 h time stage VS group NC (P < 0.001). At 6 h or 12 h stage, a decrease of PAF in group BN or group NC was observed VS group SAP (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>It confirmed that the plasma level of cytokine IL-12 in SAP group was decreased significantly in early stage and it witnessed a remarkable increase of cytokine PAF. The plasma level of IL-12 was increased in early stage but PAF was decreased in rats treatment by BN52021 which inhibited the development of SAP.</p>

Pharmacological action and mechanisms of ginkgolide B / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To review the recent research progress in pharmacological actions and mechanisms of ginkgolide B. Data sources Information included in this article was identified by searching of PUBMED (1987 - 2006) online resources using the key terms "ginkgolide B", "platelet activating factor", and "pharmacological". Study selection Mainly original milestone articles and critical reviews written by major pioneer investigators of the field were selected.</p><p><b>RESULTS</b>The key issues related to the pharmacological actions and mechanisms of ginkgolide B were summarized. The ginkgolide B possesses a number of beneficial effects such as anti-inflammatory, anti-allergic, antioxidant, and neuroprotective effects. Meantime, their mechaniams were discussed.</p><p><b>CONCLUSIONS</b>The Ginkgolide B is the most potent antagonist of platelet activating factor (PAF) and exhibits therapeutic action in a variety of diseases mainly by the PAF receptor.</p>

Dynamic changes of pro- and anti-inflammatory cytokines in acute pancreatitis / 第三军医大学学报

Objective　To explore the significance of the dynamic changes of pro-and anti-inflammatory cytokines in the onset and development of acute panreatitis (AP). Methods　Pro-inflammatory cytokines TNFα, IL-1β, IL-6, and IL-8 and anti-inflammatory cytokines IL-10 and IL-1ra in the plasma of 48 patients with AP and 20 healthy individuals were determined with ELISA. Results　The levels of all pro-and anti-inflammatory cytokines in plasma was significantly higher in AP patients than in control group (P<0.05) in early stage of the disease, and then all levels were decreased gradually, consistent with the alterations of clinical symptoms of the AP patients. Conclusion　The dynamic changes of pro-and anti-inflammatory cytokines might play important role in the onset and development of AP.

Dynamic changes of pro- and anti-inflammatory cytokines in acute pancreatitis / 第三军医大学学报

Objective　To explore the significance of the dynamic changes of pro-and anti-inflammatory cytokines in the onset and development of acute panreatitis (AP). Methods　Pro-inflammatory cytokines TNFα, IL-1β, IL-6, and IL-8 and anti-inflammatory cytokines IL-10 and IL-1ra in the plasma of 48 patients with AP and 20 healthy individuals were determined with ELISA. Results　The levels of all pro-and anti-inflammatory cytokines in plasma was significantly higher in AP patients than in control group (P<0.05) in early stage of the disease, and then all levels were decreased gradually, consistent with the alterations of clinical symptoms of the AP patients. Conclusion　The dynamic changes of pro-and anti-inflammatory cytokines might play important role in the onset and development of AP.