Isolation and antitumor activities of acidic polysaccharide from Gynostemma pentaphyllum Makino.

Two acidic polysaccharides (GP-B1 and GP-C1) were obtained from Gynostemma pentaphyllum. The molecular weights (Mw) of the two fractions were 79 kDa for GP-B1 and 126 kDa for GP-C1. GP-B1 was composed of Gal, Ara, Man, Rha, Xyl, Glc, GalA and GlcA in a molar ration of 3.5:3.2:0.6:0.9:0.3:0.5:0.6:0.4. GP-C1 consisted of Gal, Ara, Man, Rha, Glc, and GlcA in the proportions of 2.1:1.0:0.3:0.5:0.4:0.9. Among them, GP-B1 treatment had a significant inhibitory effect on the growth of melanoma B16 in vivo and in vitro. Meanwhile GP-B1 could increase the relative spleen weight and stimulate the splenocyte proliferation alone or combined with ConA. Moreover, GP-B1 treatment induced an evident increase in the level of serum TNF-α, IFN-γ, and IL-12 and a reduction for IL-10 production. These results indicate that the antitumor effects of GP-B1 are associated with immunostimulation.

Purification of a polysaccharide from Gynostemma pentaphyllum Makino and its therapeutic advantages for psoriasis.

In current study, a water-soluble polysaccharide (GP-I), with a molecular mass of 33 kDa, was purified from Gynostemma pentaphyllum. Gas chromatography (GC) analysis suggested that it was composed of Glc, Gal, Man, Rha and Ara with a ratio of 5.3: 4.2: 3.0: 0.7: 0.8. The GP-I (25, 50, 100, 200 and 400 µg/ml) was found to have significant anti-proliferative effects on HaCat cells in a dose-dependent manner, as measured by MTT assay. On the contrary, Trypan blue exclusion experiment indicated that GP-I had no cytotoxicity to HaCat cells. Moreover, the decrease of mitochondrial membrane potential (MMP) in GP-I treated cells was also observed, indicating apoptosis in HaCat cells. Besides, tumor necrosis factor-α (TNF-α), a vital pro-inflammatory cytokine in psoriasis, in the supernatant of HaCat cells was dramatically reduced by GP-I. Collectively, these findings suggested that GP-I was a promising agent to be developed for psoriasis treatment in clinical therapy.

[Effect of curcumin on IL-17-induced nitric oxide production and expression of iNOS in human keratinocytes].

AIM: To investigate the effect of curcumin on IL-17-induced NO production, mRNA and protein expression of iNOS in human keratinocyte cell lines(HaCaT cells). METHODS: HaCaT cells were stimulated with IL-17 and incubated with three doses of curcumin for 24h in vitro. After collections of supernatant, total RNA and protein, NO levels in supernatant were detected and fluorescence quantitative PCR and Western blot were performed to determine the effect of curcumin on NO levels and iNOS. RESULTS: IL-17 increased NO levels, and expression of iNOS in HaCaT cells(P<0.01). Curcumin decreased IL-17 induced NO production and the iNOS expression at mRNA (P<0.01) and protein (P<0.01) levels significantly. CONCLUSION: Curcumin down-regulates IL-17-induced NO secretions and iNOS expression in HaCaT cells, thus provides a theoretical basis for the treatment of inflammatory diseases of skin related to keratinocytes.

[Expression of CD1a and CD207 in condyloma acuminatum epidermis].

AIM: To investigate the expression of CD1a and CD207 in condyloma acuminatum (CA) epidermis and to understand its significance. METHODS: The mRNA expression of CD1a and CD207 in six CA epidermal lesions and in six normal controls were detected using oligonucleotide microarrys and confirmed by semi-quantitative RT-PCR, and the protein level of CD1a and CD207 in six CA epidermal lesions and in six normal controls were measured by Western blot. RESULTS: With microarrys, the mRNA expression of CD1a and CD207 were detected markedly down-regulated in six CA epidermal lesions as compared with that in six normal controls. Moreover, the down-regulation of CD1a and CD207 was verified by semi-quantitative RT-PCR. Western blot analysis showed that the protein expression of CD1a and CD207 in six CA epidermal lesions were significantly lower than that in normal controls. CONCLUSION: The expression of CD1a and CD207 is markedly down-regulated in CA epidermis compared with that in normal epidermis, and the results may suggest that the number of LC in CA epidermis is decreased and the function is impaired.

[Alteration of tazarotene induced gene-2 expression in psoriasis vulgaris].

OBJECTIVE: To investigate the role of tazarotene induced gene-2 (TIG2) in psoriasis vulgaris. METHODS: TIG2 protein and mRNA expressions in normal tissues, psoriatic lesions and uninvolved skin tissues were detected by immunohistochemistry and in situ hybridization, respectively. RESULTS: TIG2 protein and mRNA were expressed in all the layers of normal and uninvolved epidermis. TIG2 expression was detected in the upper layers of the stratum spinosum of the marginal region of the psoriatic lesions, but not in the central area of the lesions. TIG2 expression was significantly lower in the basal layers of the central area of the paoriasis than that in the normal skin and uninvolved tissues (P < 0.01), and also lower in the marginal regions of the lesions (P < 0.01).The suprabasal layers of the marginal region in the lesion showed significantly lower TIG2 expression than the central area of the lesion (P < 0.01). CONCLUSION: TIG2 may maintain the normal differentiation of epidermal keratinocytes and implicate in the pathogenesis and development of psoriasis vulgaris.

[Alteration of retinoid X receptor alpha mRNA level after acitretin and/or narrow-band UVB treatment in normal human keratinocytes].

OBJECTIVE: To investigate the alteration of retinoid X receptor alpha (RXRalpha) mRNA level in normal human keratinocytes after acitretin and/or NB-UVB irradiation treatment. METHODS: After a 12-hour incubation with 10(-7)-10(-6) mol/L acitretin and/or following 50-100 mJ/cm2 NB-UVB irradiation in normal human keratinocytes, RXRalpha mRNA expression was examined by reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR. RESULTS: The expression of RXRalpha mRNA was obviously decreased by NB-UVB irradiation, but not by acitretion single treatment. When combining acitretin treatment with NB-UVB irradiation, greater decreased RXRalpha mRNA expression was observed than that of single treatment. CONCLUSION: Narrow-band UVB irradiation treatment can decrease RXRalpha mRNA expression, but not acitretin single treatment. Combining treatment with both can produce synergistic inhibition effects.

[Expression of E-cadherin, beta-catenin and cyclin D1 in epidermal skin lesions of patients with active psoriasis vulgaris].

OBJECTIVE: To examine the expressions of E-cadherin, beta-catenin and cyclin D1 in the skin lesions of patients with psoriasis vulgaris, and understand their possible roles in keratinocyte hyperproliferation in these patients. METHODS: Immunohistochemistry was performed to detect the expressions of E-cadherin, beta-catenin and cyclin D1 in the normal skin tissues and psoriatic lesions. RESULTS: In normal skin tissues, positive staining for E-cadherin and beta-catenin was detected in all layers of the normal epidermis at the sites of cell-cell junctions, and downregulation of E-cadherin and beta-catenin expression was found in the granular layer and basal layer of the psoriatic lesions. Cyclin D1 overexpression was observed mainly in the basal layer of the lesions, which was correlated to abnormal expression of beta-catenin. CONCLUSION: Downregulation of E-cadherin and beta-catenin expression and cyclin D1 overexpression in psoriatic skin are probably involved in keratinocyte hyperproliferation in psoriasis vulgaris.

[A novel retinoid CD437 induces apoptosis of human epidermoid carcinoma A431 cells].

OBJECTIVE: To investigate the effect of a novel retinoid CD437 and all-trans retinoic acid (ATRA) in inducing cell apoptosis and inhibiting the proliferation of human epidermoid carcinoma A431 cells and normal human epidermal keratinocytes. METHODS: MTT assay was used to determine the inhibitory effects of CD437 and ATRA on the growth of A431 cells and normal human epidermal keratinocytes, and the cell morphological changes were observed microscopically. Flow cytometry was used to investigate the effect of CD437 and ATRA on the cell cycle and apoptosis. RESULTS: CD437 was more effective than ATRA in inhibiting the proliferation of A431 cells and normal human epidermal keratinocytes. CD437 increased the percentage of sub-G1 populations in A431 cells and induced G1 arrest in normal human epidermal keratinocytes. ATRA appeared to be relatively ineffective for inducing apoptosis in A431 cells as compared to CD437. CD437 did not duce obvious apoptosis in normal human epidermal keratinocytes. CONCLUSION: CD437 is more effective than ATRA in inhibiting the proliferation and inducing apoptosis in A431 cells and shows selective apoptosis-inducing effect against malignant keratinocytes, suggesting its potential in the prevention or treatment of cutaneous carcinoma.

[Mutation detection of ATP2C1 gene in Chinese patients with Hailey-Hailey disease].

OBJECTIVE: To investigate the mutations of ATP2C1 gene in Chinese patients with Hailey-Hailey disease (HHD). METHODS: Genomic DNA was extracted from peripheral blood leukocytes. PCR and direct DNA sequencing were used to detect the mutations in all 27 exons of ATP2C1 gene in patients of two Chinese families and a sporadic patient with HHD. RESULTS: Three mutations in ATP2C1 gene were found, including 1 nonsense mutation, 1 deletion/frameshift mutation and 1 missense mutation. All of them were novel mutations. CONCLUSION: All the three mutations could affect the transcription and translation, and further the function of protein encoded by ATP2C1 gene.

[Inhibition of COL1A1 and COL3A1 expression by RNA interference in human skin fibroblasts].

OBJECTIVE: To suppress COL1A1 and COL3A1 gene expressions in human skin fibroblasts (HSFs) by means of RNA interference (RNAi). METHODS: Three small interfering RNA (siRNA) expression cassette (SEC) sequences were designed for each of the COL1A1 and COL3A1 gene sequences available in GenBank. The synthesized SECs capable of effective gene suppression were transfected into cultured HSFs, either after cloning into the expression vector or mediated by Lipofectamine 2000, and the suppression of the target genes at both mRNA and protein levels was determined by quantitative fluorescence RT-PCR and Western blotting, respectively. RESULTS: Transfection of the SECs into HSFs resulted in specific depression of COL1A1 and COL3A1 expressions (down to 5.00% and 6.48%, respectively). The expression vector-mediated RNAi established a HSF cell line with persistent gene knockdown for over 30 days (to 25.21% and 22.12%, respectively). CONCLUSION: COL1A1 and COL3A1 gene expressions can be specifically and efficiently inhibited in HSFs by either liposome- or vector-mediated SEC transfection.

Immunohistochemical and ultrastructural features of Langerhans cells in condyloma acuminatum.

BACKGROUND: There are few studies on the abnormal morphology of Langerhans cells (LCs) in condyloma acuminatum (CA) lesions and the essence of the abnormal morphology of LCs in CA lesions is still not well elucidated. The aim of this study was to further investigate the morphological features of LCs in CA lesions. METHODS: CD1a(+) LCs in 13 CA lesions and in 13 normal controls were labeled using immunohistochemistry and examined by light microscopy. Ultrastructural investigation on LCs in six CA lesions and in six normal controls was performed by electron microscopy. RESULTS: Compared with those in normal controls, most CD1a(+) LCs in CA lesions exhibited dysplastic dendrities and abnormal distribution. The number of CD1a(+) LCs in CA lesions (26.31 +/- 18.84) was statistically lower (p < 0.001) than that in normal controls (72.00 +/- 27.40). Electron microscopy showed that the number of Birbeck granules within lesional LCs (4.00 +/- 2.94) was significantly decreased (p < 0.001) than that within normal LCs (10.80 +/- 4.78). The ultrastructures of most lesional LCs displayed degenerative changes. CONCLUSIONS: The morphology of most LCs in CA lesions shows degenerative changes, which suggest that these LCs have been functionally impaired.

[Synergistic effects of acitretin and narrow-band ultraviolet-B in inducing retinoic acid receptor gamma mRNA expression in normal human keratinocytes].

OBJECTIVE: To investigate the changes in cell proliferation and retinoic acid receptor gamma (RARgamma) mRNA expression in normal human keratinocytes after acitretin treatment and/or narrow-band ultraviolet-B irradiation. METHODS: Normal human keratinocytes were exposed to irradiation with 100 mJ/cm square NB-UVB and/or subsequent 12-hour incubation with 1x10(-6) mol/L acitretin, and the expression of RARgamma mRNA in the cells was examined using RT-PCR and real-time quantitative RT-PCR. RESULTS: A 0.9- and a 2.3-fold increase in RARgamma mRNA expression was induced in the cells by exposure to 100 mJ/cm square NB-UVB and 10(-6) mol/L acitretin, respectively, and the expression was synergistically enhanced by 2.8-fold after their combined treatment. CONCLUSION: Upregulated expression of RARgamma mRNA can be associated with keratinocyte growth inhibition after treatment with acitretin and NB-UVB irradiation.

[Expression of CCL20 and CXCR4 in epidermal condyloma acuminatum lesions].

OBJECTIVE: To detect CCL20 and CXCR4 expressions in epidermis infected with condyloma acuminatum (CA) and normal epidermis and investigate the effect of their expressions on Langerhans cells in CA epidermis. METHODS: Gene expression of CCL20 and CXCR4 in 3 epidermal CA lesions and in 3 normal epidermis specimens were detected using Affymetrix oligonucleotide microarrays HG-U 133A 2.0, and the protein levels of CCL20 and CXCR4 in these specimens were measured by Western blotting. RESULTS: Microarray analysis revealed markedly down-regulated mRNA expressions of CCL20 and CXCR4 in the 3 epidermal CA lesions as compared with those in the normal specimens. Western blot analysis showed that the protein expressions of CCL20 and CXCR4 in the CA lesions were significantly lower than those in normal epidermis. CONCLUSION: The protein and mRNA expressions of CCL20 and CXCR4 are markedly down-regulated in epidermal CA lesions, which may contribute to decreased number and backflow disturbance of Langerhans cells in these lesions.

[Expression of Langerhans cell-related chemokine genes in condyloma acuminatum epidermis].

AIM: To investigate differential gene expression of Langerhans cell-related chemokines in condyloma acuminatum (CA) epidermis and normal epidermis. METHODS: Gene expression of Langerhans cell-related chemokines in three CA epidermal lesions and in three normal controls was screened using Affymetrix oligonucleotide microarrays HG-U 133A 2.0, and part of the above differential gene expression was confirmed by semi-quantitative RT-PCR. RESULTS: With microarrays, seven down-regulated genes of Langerhans cell-related chemokine were detected in three CA epidermal lesions as compared with three normal controls, and the down-regulation of CXCR4 and CCL20 was verified by semi-quantitative RT-PCR. CONCLUSION: Several Langerhans cell-related chemokine genes are found down-regulated in CA epidermis as compared with normal epidermis, and the down-regulation of these genes may contribute to the decreased number and the homing disturbance of LC in CA epidermis.

[Mechanism of receptor for retinoids inducing apoptosis of human melanoma cell line A375].

OBJECTIVE: To investigate the mechanism of receptors for retinoids inducing apoptosis of human melanoma cell line A375. METHODS: The effects of 3 kinds of retinoids (9-cis-RA, at-RA and 13-cis-RA), of TTNPB (RAR agonist) and of Methoprene acid (Ma, RXR agonist) on apoptosis of A375 cells were studied by detecting the expression of Bcl-2/Bax and by using. Annexin V/PI staining analysis, TUNEL detection and active Caspase-3 analysis. RESULTS: Retinoids and TTNPB could up-regulate the expression of Bax and down-regulate the expression of Bcl-2. The results of TUNEL and Annexin V/PI staining analysis showed that all of retinoids and TTNPB could induce apoptosis of A375 cells, compared with control group (P < 0.05); the effect of TTNPB was significantly greater than that of others (P < 0.05), but Ma was similar to the control (P > 0.05). Active Caspase-3 analysis showed that TTNPB and all of retinoids could up-regulate the expression of Caspase-3, and the effect of TTNPB was significantly greater than that of others (P < 0.05). CONCLUSION: Caspase-3 pathway is involved in the process for retinoids inducing apoptosis of A375 cells. The activation of RAR may have relation with retinoids inducing apoptosis of A375 cells, but may have no longer relation with RXR.

[Localization and differentiation of hair follicle stem cells].

OBJECTIVE: To identify the localization of hair follicles stem cell (HFSC) in different stages of hair and explore the differentiating capacity of HFSC into epidermis in vitro. METHODS: HFSC were detected by K19 immunostaining in normal human skin. Then, the isolated HFSC through enzyme digestion were seeded on dermal equivalent (DE) and cultured between the air-liquid interfaces for 14 days. Skin-equivalents was harvested and used for evaluation. RESULTS: HFSC mainly located in outer root sheet in hair follicle and human anagen hair follicles containing two distinct reservoirs for K19-positive cells located in the bulge and bulb of the follicle. These two reservoirs fused in line of outer root sheets during the catagen-telogen transition phase and individualized again in the newly forming anagen hair follicle. Based on DE, growing HFSC built a multilayered and confined epidermis. CONCLUSION: HFSC located in outer root sheets can promote hair cycle and differentiate into epidermis in vitro.

[Inhibition of promyelocytic leukemia gene by tazarotene in hyperproliferative epidermis of psoriasis].

OBJECTIVE: To investigate the mechanism of tazarotene against active psoriasis vulgaris. METHODS: A randomized, controlled trial was conducted in 43 patients with active psoriasis vulgaris, who were divided into tazarotene and control groups. Promyelocytic leukemia (PML) mRNA in active psoriatic lesions before and 14 days after tazarotene treatment was detected by in situ hybridization. RESULTS: PML mRNA expression was detected not only in the basal layer (86.96%), but also in the suprabasal layers of the epidermis in the manner of focal expression (78.26%). After tazarotene treatment, virtually no PML mRNA expression could be detected in the psoriatic lesions (8.69% in the basal layer and 4.35% in the suprabasal layers). PML mRNA expression in the control group underwent no obvious changes during the observation. CONCLUSIONS: Tazarotene may inhibit abnormal proliferation of keratinocytes through down-regulating PML gene expression in active psoriatic epidermis.

[Expression of E-cadherin and beta-catenin in Bowen's disease and squamous cell carcinoma].

OBJECTIVE: To investigate the involvement of E-cadherin-catenin adhesion system in Bowen's disease (BD) and cutaneous squamous cell carcinoma (SCC). METHODS: Fifteen normal skin, 28 BD and 18 SCC specimens were stained with monoclonal antibodies against E-cadherin and beta-catenin. Evaluation of the staining results was performed with semi-quantification of the pattern and intensity of staining, percentage of positive cells, and cytoplasmic staining. RESULT: Normal skins strongly expressed membranous E-cadherin and beta-catenin, but their expression was remarkably reduced in BD and SCC. Abnormal staining of beta-catenin was observed in the cytoplasm or cell nuclei of BD and SCC. CONCLUSION: Abnormal expression of the E-cadherin/catenin complex is common in SCC and BD.