[Effect of methotrexate on regulation for the number of regulatory T cells and expression of Foxp3 in psoriasis].

OBJECTIVE: To explore the role of methotrexate (MTX) in regulating the number of regulatory T cells (Treg) and the mRNA expression of transcription factor Foxp3.
 Methods: 1) We analyzed the number of Treg and the mRNA expression of Foxp3 by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR) respectively in patients with psoriasis vulgaris, patients with psoriasis vulgaris after the 8-week treatment of MTX, and healthy people. 2) BALB/c female mice were smeared with imiquimod (IMQ) cream for 6 days. We recorded the change of the lesion in mice every day. The morphological changes of lesion in mice were evaluated by the psoriasis area and severity index (PASI) and HE staining. 3) The mouse model was randomly divided into a control group and an MTX group. The MTX group was treated with different doses of MTX (38.5 and 77.0 nmol/L) on the third day of this experiment. The morphological changes of lesion in mice were evaluated by PASI and HE staining. We tested the number of Treg and the expression level of Foxp3 mRNA in splenic lymphocytes.
 Results: 1) The number of Treg and the expression level of Foxp3 mRNA were lower in psoriasis vulgaris patients than those in the healthy control group (P<0.05). After 8-week treatment of MTX, the number of Treg was increased (P<0.05) and Foxp3 mRNA level was up-regulated (P<0.01). 2) Typical psoriasis-like skin lesions, such as red scaly skin plaque were found after topical application of IMQ. Both the number of Treg in the splenic lymphocytes of mice and the Foxp3 mRNA level of Treg were reduced by IMQ (P<0.01 and P<0.05). 3) Different doses of MTX for mice showed the ability to improve skin lesion, increase the number of Treg in the spleen of mice and Foxp3 mRNA level in psoriatic dermatitis of mice (P<0.05).
 Conclusion: MTX is able to regulate the number of Treg and Foxp3 mRNA expression in psoriasis.

Topical Sunitinib ointment alleviates Psoriasis-like inflammation by inhibiting the proliferation and apoptosis of keratinocytes.

Psoriasis is a chronic auto-immune inflammation disease with skin lesions and abnormal keratinocyte proliferation. Sunitinib, a multi-targeted tyrosine kinase inhibitor, is known to selectively inhibit several growth factor receptors, including vascular endothelial growth factor receptor, platelet-derived growth factor receptor and stem cell factor. It was reported that a patient with renal cell carcinoma (RCC) whose psoriatic lesion was resolved dramatically during treatment with Sunitinib, however, the mechanism is still unclear. We applied Sunitinib ointment to treat imiquimod-induced mouse model of psoriasis and found that Sunitinib ointment could alleviate imiquimod-induced psoriasis-like inflammation and reduce the Ki67 expression, while Sunitinib ointment couldn't reduce imiquimod-induced splenomegaly of the mouse model, then we concentrated on studying the effect of Sunitinib on the proliferation and apoptosis of keratinocytes, we cultivated HaCaT cells with epidermal growth factor (HaCaT/E cells) to represent as a state of highly proliferative psoriatic keratinocytes. We found that Sunitinib could inhibit the proliferation of Hacat/E cell in a time and concentration dependent manner by influencing the expression level of cell cycle protein D1, cycle protein E1, in addition, Sunitinib could induce the apoptosis of Hacat/E cell and up-regulate the expression of poly ADP-ribose polymerase (PARP). Sunitinib down-regulated the expression of phosphorylated signal transduction and activator of transcription 3 (p-Stat3) of Hacat/E cells significantly. We conclude that Sunitinib alleviates imiquimod-induced psoriasis-like inflammation by regulating the proliferation and apoptosis of HaCaT cells through inhibiting the expression of p-Stat3.

TRAF6 regulates EGF-induced cell transformation and cSCC malignant phenotype through CD147/EGFR.

TRAF6, a well-known adapter molecule, plays pivotal role in TLR/IL-1R associated signaling pathway. Although TRAF6 has been shown to have oncogenic activity in various malignant tumors, the details remain unclear. In this study, we demonstrated that TRAF6 facilitates Ras (G12V) and EGF-induced cellular transformation through EGFR. Silencing of TRAF6 expression significantly downregulated AP-1 activity, as well as MMP-2,9 expression after EGF stimulation. Furthermore, we found that TRAF6 plays an essential role in cutaneous squamous cell carcinoma (cSCC) malignant phenotypes, affecting cell growth and migration. CD147/Basigin, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, is over-expressed in tumors and induces tumorigenesis. Our results showed that CD147 formed complex with EGFR and TRAF6. Knockdown of TRAF6 disrupted the CD147-EGFR complex, thereby inducing EGFR endocytosis. Therefore, TRAF6 might be a novel molecular target for cSCC prevention or therapy.

Lipidomics profiling reveals the role of glycerophospholipid metabolism in psoriasis.

Psoriasis is a common and chronic inflammatory skin disease that is complicated by gene-environment interactions. Although genomic, transcriptomic, and proteomic analyses have been performed to investigate the pathogenesis of psoriasis, the role of metabolites in psoriasis, particularly of lipids, remains unclear. Lipids not only comprise the bulk of the cellular membrane bilayers but also regulate a variety of biological processes such as cell proliferation, apoptosis, immunity, angiogenesis, and inflammation. In this study, an untargeted lipidomics approach was used to study the lipid profiles in psoriasis and to identify lipid metabolite signatures for psoriasis through ultra-performance liquid chromatography-tandem quadrupole mass spectrometry. Plasma samples from 90 participants (45 healthy and 45 psoriasis patients) were collected and analyzed. Statistical analysis was applied to find different metabolites between the disease and healthy groups. In addition, enzyme-linked immunosorbent assay was performed to validate differentially expressed lipids in psoriatic patient plasma. Finally, we identified differential expression of several lipids including lysophosphatidic acid (LPA), lysophosphatidylcholine (LysoPC), phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidic acid (PA); among these metabolites, LPA, LysoPC, and PA were significantly increased, while PC and PI were down-regulated in psoriasis patients. We found that elements of glycerophospholipid metabolism such as LPA, LysoPC, PA, PI, and PC were significantly altered in the plasma of psoriatic patients; this study characterizes the circulating lipids in psoriatic patients and provides novel insight into the role of lipids in psoriasis.

Epidermal CD147 expression plays a key role in IL-22-induced psoriatic dermatitis.

Psoriasis is a chronic inflammatory skin disease characterized by abnormal keratinocyte proliferation and terminal differentiation. Interleukin-22 (IL-22) and the transcription factor Stat3 play pivotal roles in the pathogenesis of psoriasis. CD147 is a transmembrane glycosylation protein that belongs to the immunoglobulin superfamily. Our previous studies have shown that CD147 is a marker of high keratinocyte proliferation and poor keratinocyte differentiation as well as a psoriasis susceptibility gene. The current study demonstrates that CD147 is highly expressed in psoriatic skin lesions. Specific CD147 over-expression in the epidermis of K5-promoter transgenic mice promotes imiquimod (IMQ)-induced psoriasis-like inflammation characterized by acanthosis, granular layer loss and inflammatory cell infiltration. We also found that IL-22 increases CD147 transcription in vitro and in vivo and that Stat3 binds directly to the CD147 promoter between positions -854 and -440, suggesting that CD147 expression is up-regulated in patients with psoriasis through Stat3 activation. In addition, CD147 knockdown dramatically blocks IL-22-mediated Stat3 activation as well as IL-22-induced cytokine, chemokine and antimicrobial factor expression. Together, these findings show that CD147 is a novel and key mediator of IL-22-induced psoriatic alterations in the epidermis and might be a therapeutic target in patients with psoriasis.

TRAF6 regulates melanoma invasion and metastasis through ubiquitination of Basigin.

TRAF6 plays a crucial role in the regulation of the innate and adaptive immune responses. Although studies have shown that TRAF6 has oncogenic activity, the role of TRAF6 in melanoma is unclear. Here, we report that TRAF6 is overexpressed in primary as well as metastatic melanoma tumors and melanoma cell lines. Knockdown of TRAF6 with shRNA significantly suppressed malignant phenotypes including cell proliferation, anchorage-independent cell growth and metastasis in vitro and in vivo. Notably, we demonstrated that Basigin (BSG)/CD147, a critical molecule for cancer cell invasion and metastasis, is a novel interacting partner of TRAF6. Furthermore, depletion of TRAF6 by shRNA reduced the recruitment of BSG to the plasma membrane and K63-linked ubiquitination, in turn, which impaired BSG-dependent MMP9 induction. Taken together, our findings indicate that TRAF6 is involved in regulating melanoma invasion and metastasis, suggesting that TRAF6 may be a potential target for therapy or chemo-prevention in melanoma.

Au@Ag/Au nanoparticles assembled with activatable aptamer probes as smart "nano-doctors" for image-guided cancer thermotherapy.

Although nanomaterial-based theranostics have increased positive expectations from cancer treatment, it remains challenging to develop in vivo "nano-doctors" that provide high-contrast image-guided site-specific therapy. Here we designed an activatable theranostic nanoprobe (ATNP) via self-assembly of activatable aptamer probes (AAPs) on Au@Ag/Au nanoparticles (NPs). As both quenchers and heaters, novel Au@Ag/Au NPs were prepared, showing excellent fluorescence quenching and more effective near-infrared photothermal therapy than Au nanorods. The AAP comprised a thiolated aptamer and a fluorophore-labeled complementary DNA; thus, the ATNP with quenched fluorescence in the free state could realize signal activation through target binding-induced conformational change of the AAP, and then achieve on-demand treatment under image-guided irradiation. By using S6 aptamer as the model, in vitro and in vivo studies of A549 lung cancer verified that the ATNP greatly improved imaging contrast and specific destruction, suggesting a robust and versatile theranostic strategy for personalized medicine in future.

Polyacrylic acid modified upconversion nanoparticles for simultaneous pH-triggered drug delivery and release imaging.

A poly(acrylicacid)-modified NaYF4:Yb, Er upconversion nanoparticles (PAA-UCNPs) with dual functions of drug delivery and release imaging have been successfully developed. The PAA polymer coated on the surface of UCNPs serve as a pH-sensitive nanovalve for loading drug molecules via electrostatic interaction. The drug-loading efficiency of the PAA-UCNPs was investigated by using doxorubicin hydrochloride (DOX) as a model anticancer drug to evaluate their potential as a delivery system. Results showed loading and releasing of DOX from PAA-UCNPs were controlled by varying pH, with high encapsulation rate at weak alkaline conditions and an increased drug dissociation rate in acidic environment, which is favorable for construct a pH-responsive controlled drug delivery system. The in vitro cytotoxicity test using HeLa cell line indicated that the DOX loaded PAA-UCNPs (DOX@PAA-UCNPs) were distinctly cytotoxic to HeLa cells, while the PAA-UCNPs were highly biocompatible and suitable to use as drug carriers. Furthermore, the upconversion fluorescence resonance energy transfer (UFRET) imaging through the two-photon laser scanning microscopy (TLSM) revealed the time course of intracellular delivery of DOX from DOX@PAA-UCNPs. Thus, PAA-UCNPs are effective for constructing pH-responsive controlled drug delivery systems for multi-functional cancer therapy and imaging.

A highly selective sandwich-type FRET assay for ATP detection based on silica coated photon upconverting nanoparticles and split aptamer.

In this paper, we report a highly selective sandwich-type fluorescence resonance energy transfer (FRET) assay for ATP detection by combining the unique optical properties of silica coated photon upconverting NaYF4:Yb(3+), Er(3+) nanoparticles (Si@UCNPs) with the high specific recognition ability of ATP aptamer. In the protocol, a single aptamer of ATP was split into two fragments. One of which was covalently attached to the Si@UCNPs at the 5' end, and the other was labeled with Black Hole Quencher-1 (BHQ1) at the 3' end. In the presence of ATP, the two fragments bound ATP with high affinity to form the sandwich complexes on the surface of Si@UCNPs. ATP induced association of the two fragments, thus bringing the Si@UCNPs and BHQ1 into close proximity. Under the illumination of 980 nm laser, energy transfer took place between the Si@UCNPs as the donor and BHQ1 as the acceptor, creating an optical "sandwich-type" assay for ATP detection. By monitoring the fluorescence change of the Si@UCNPs at 550 nm, the presence of the ATP could be quantitatively detected with a detection limit of 1.70 µM. The linear response range was 2 µM-16 µM. The background of this assay was ignorable because the fluorescence intensity of Si@UCNPs at 550 nm was not changed in the absence of ATP. This assay was also able to discriminate ATP from its analogs.

Highly sensitive label-free fluorescent detection of Hg2+ ions by DNA molecular machine-based Ag nanoclusters.

We present here a highly selective and sensitive label-free method to detect Hg(2+) ions in aqueous solution by using DNA molecular machine-based fluorescent Ag nanoclusters (AgNCs). This mechanism is based on the Hg(2+) ions triggering machine-like operations of DNA and the "product" of the machine being used to stabilize fluorescent AgNCs. In this method, a tailored DNA, containing a sequence for Hg(2+) ions recognition, a sequence-specific nicking site for Nb BbvC I and a sequence complementary to the DNA as a template for the synthesis of fluorescent AgNCs, was firstly designed. In the presence of Hg(2+) ions, the machine's function operations were triggered. A series of machine-like operations, including replication, scission, and displacement then occurred with the addition of polymerase/dNTPs/Nb BbvC I, which manufactured lots of "product" DNA. The "product" DNA could act as a template for the preparation of fluorescent AgNCs. Thus the fluorescence of the AgNCs could be used as a signal transduction of this DNA machine, which was related to the concentration of the Hg(2+) ions. The repeated synthesis of the "product" and its template effect for AgNCs synthesis led to signal amplification in the assay of Hg(2+) ions. A linear response to the concentration of Hg(2+) ions was observed in the range from 0.08 nM to 20 nM and a detection limit of 0.08 nM was obtained. By contrast, the operation of the machine could not be executed in an Hg(2+) ion-free system. Moreover, the detection was not only label-free but also specific for Hg(2+) ions without being affected by other metal ions.