Chaihu-Longgu-Muli decoction improves sleep disorders by restoring orexin-A function in CKD mice.

Introduction: Chaihu-Longgu-Muli decoction (CLMD) is a well-used ancient formula originally recorded in the "Treatise on Febrile Diseases" written by the founding theorist of Traditional Chinese Medicine, Doctor Zhang Zhongjing. While it has been used extensively as a therapeutic treatment for neuropsychiatric disorders, such as insomnia, anxiety and dementia, its mechanisms remain unclear. Methods: In order to analyze the therapeutic mechanism of CLMD in chronic renal failure and insomnia, An adenine diet-induced chronic kidney disease (CKD) model was established in mice, Furthermore, we analyzed the impact of CLMD on sleep behavior and cognitive function in CKD mice, as well as the production of insomnia related regulatory proteins and inflammatory factors. Results: CLMD significantly improved circadian rhythm and sleep disturbance in CKD mice. The insomnia related regulatory proteins, Orexin, Orexin R1, and Orexin R2 in the hypothalamus of CKD mice decreased significantly, while Orexin and its receptors increased remarkably after CLMD intervention. Following administration of CLMD, reduced neuron loss and improved learning as well as memory ability were observed in CKD mice. And CLMD intervention effectively improved the chronic inflflammatory state of CKD mice. Discussion: Our results showed that CLMD could improve sleep and cognitive levels in CKD mice. The mechanism may be related to the up-regulation of Orexin-A and increased phosphorylation level of CaMKK2/AMPK, which further inhibits NF-κB downstream signaling pathways, thereby improving the disordered inflammatory state in the central and peripheral system. However, More research is required to confirm the clinical significance of the study.

c-Ski inhibits the proliferation of vascular smooth muscle cells via suppressing Smad3 signaling but stimulating p38 pathway.

Proliferation of vascular smooth muscle cells (VSMCs) plays key roles in the progression of intimal hyperplasia, but the molecular mechanisms that trigger VSMC proliferation after vascular injury remain unclear. c-Ski, a co-repressor of transforming growth factor ß (TGF-ß)/Smad signaling, was detected to express in VSMC of rat artery. During the course of arterial VSMC proliferation induced by balloon injury in rat, the endogenous protein expressions of c-Ski decreased markedly in a time-dependent manner. In vivo c-Ski gene delivery was found to significantly suppress balloon injury-induced VSMC proliferation and neointima formation. Further investigation in A10 rat aortic smooth muscle cells demonstrated that overexpression of c-Ski gene inhibited TGF-ß1 (1 ng/ml)-induced A10 cell proliferation while knockdown of c-Ski by RNAi enhanced the stimulatory effect of TGF-ß1 on A10 cell growth. Western blot for signaling detection showed that suppression of Smad3 phosphorylation while stimulating p38 signaling associated with upregulation of cyclin-dependent kinase inhibitors p21 and p27 was responsible for the inhibitory effect of c-Ski on TGF-ß1-induced VSMC proliferation. These data suggest that the decrease of endogenous c-Ski expression is implicated in the progression of VSMC proliferation after arterial injury and c-Ski administration represents a promising role for treating intimal hyperplasia via inhibiting the proliferation of VSMC.

Upregulation of ski in fibroblast is implicated in the peroxisome proliferator--activated receptor δ-mediated wound healing.

BACKGROUND/AIM: Both peroxisome proliferator-activated receptor (PPAR) δ and Ski are investigate the interaction of PPARδ and Ski and this interaction-associated effect in wound healing. METHODS: Effect of PPARδ activation on Ski expression was detected in rat skin fibroblasts by real-time PCR and western blot. Luciferase assay, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay were performed to identify the binding site of PPARδ in the promoter region of rat Ski gene. And the functional activity of PPARδ regulation to Ski was detected in fibroblast proliferation and rat skin wound healing model. RESULTS: PPARδ agonist GW501516 upregulated Ski expression in a dose-dependent manner. Direct repeat-1 (DR1) response element locating at -865∼-853 in Ski promoter region was identified to mediate PPARδ binding to Ski and associated induction of Ski. Furthermore, PPARδ upregulated Ski to promote fibroblasts proliferation and rat skin wound repair, which could be largely blocked by pre-treated with Ski RNA interference. CONCLUSION: This study demonstrates that Ski is a novel target gene for PPARδ and upregulation of Ski to promote fibroblast proliferation is implicated in the PPARδ-mediated wound healing.

[PPARγ up-regulates TGFß/smad signal pathway repressor c-Ski].

TGFß/smad pathway is recognized as an important signal pathway to promote the pathogenesis of atherosclerosis (AS). Peroxisome proliferator-activated receptor γ (PPARγ) activation is considered to be important in modulating AS. Herein, we investigated the regulation of PPARγ on c-Ski, the repressor of TGFß/smad pathway, in rat AS model and cultured vascular smooth muscle cells (VSMCs). c-Ski mRNA and protein expression were detected by real-time PCR and Western blot, respectively, in vivo and in vitro with treatment of PPARγ agonist rosiglitazone and antagonist GW9662. The proliferation and collagen secretion of VSMCs after c-Ski transfection were investigated. The underlying mechanism was further investigated by online program NUBIScan and luciferase reporter gene analysis. Results showed that both mRNA and protein expressions of c-Ski in the AS lesions was down-regulated in vivo, while in cultured VSMCs, c-Ski transfection significantly suppressed the proliferation and collagen secretion of rat VSMCs. Rosiglitazone significantly up-regulated mRNA and protein levels of c-Ski in VSMCs, which could be blocked by GW9662. Online NUBIScan analysis suggested possible PPARγ binding sites in the promoter region of c-Ski. In addition, luciferase activity of c-Ski reporter gene was also increased obviously in the presence of rosiglitazone. These results indicate that c-Ski is one of the newly found target genes of PPARγ and thus involved in the anti-AS effect of PPARγ.

Adherent cells in granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived dendritic cell culture system are qualified dendritic cells.

A widely-used method for generating dendritic cell (DC) is to culture bone marrow cells in granulocyte-macrophage colony-stimulating factor (GM-CSF)-containing medium for 6-10 days. Usually, non-adherent cells are used as qualified dendritic cells while the adherent ones are discarded as "non-dendritic cells" or macrophages. In this study, we show that the adherent cells are nearly identical to the non-adherent cells in both dendritic cell surface markers expression and main dendritic cell-related functions, hence to prove that these "junk cells" are actually qualified dendritic cells.

A single low dose of dendritic cells modified with lentivirus containing a truncated neu gene can effectively suppress neu-overexpressing tumors.

BACKGROUND: Several types of viral-transduced HER2/neu-modified dendritic cells (DC(HER2/neu)) have been used for preventing and/or treating HER2/neu-overexpressing tumors. However, to date, lentivirus has not been used to generate HER2/neu-modified DCs. METHODS: In the present study, we used recombinant lentivirus containing a truncated neu gene (rLVneu) to transduce murine bone marrow-derived dendritic cells and investigated their preventive and therapeutic effects on HER2/neu-overexpressing tumors. RESULTS: The data obtained show that a single low dose of lentiviral-transduced DC(HER2/neu) could significantly elevate serum anti-neu antibody level, stimulate the proliferation of CD4 and CD8 T lymphocytes and interferon-gamma secretion, induce a long lasting preventive effect against HER2/neu-overexpressing tumors and significantly suppress the growth of established HER2/neu-overexpressing tumors. CONCLUSIONS: The present study demonstrates that a single low dose of DCs modified with rLV containing a truncated neu oncogene can achieve a strong and long lasting effect on neu-overexpressing tumors, suggesting the possible clinical application of this strategy.

Gene delivery efficiency in bone marrow-derived dendritic cells: comparison of four methods and optimization for lentivirus transduction.

Many gene delivery methods have been used to transduce or transfect bone marrow-derived dendritic cells (BMDCs) for genetic engineered DC vaccine research. The present study, for the first time, evaluated the efficiencies of four methods (lipofection, DNA electroporation, recombinant adeno-associated virus type 2 (rAAV2) transduction, and recombinant lentivirus (rLV) transduction) using EGFP as a report gene in the same BMDC culture system. Our data demonstrate that rLV transduction is the most effective method; both lipofection and DNA electroporation transfect BMDCs at lower efficiencies; rAAV2 can hardly transduce BMDCs. Furthermore, our results, for the first time, demonstrate that rLV transduction efficiency on BMDCs can be improved significantly by co-centrifugation and repeated transduction.