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Objective:To investigate the protective effect and mechanism of Acronychia pedunculata water extracts on UV-induced light damage of human keratinocytes.Methods:The experiment was conducted from December 2018 to April 2020 in the Guangxi Medical University Laboratory of Genetics. The photoaged keratinocyte model was used, the cells were co-cultured with different concentrations of Acronychia pedunculata water extracts. The cell proliferation rate was detected by CCK-8 method. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and total antioxidant capacity (T-AOC) of cells were detected by a test kit. The levels of IL-1β, IL-6 and tumor necrosis factor-alpha (TNF-α) were determined by ELISA.Results:The proliferation of HaCaT cells was promoted by 0.5 mg/L-2.0 mg/L of the extracts. Compared with control group, the proliferation rate of HaCaT cells in the experimental group was significantly increased ( P<0.05). Compared with control group, the contents of ROS was decreased ( F=214.67, P<0.05), MDA was decreased ( F=811.88, P<0.05), SOD was increased ( F=28.95, P<0.05), CAT was increased ( F=213.31, P<0.05), GPX was increased ( F=65.10, P<0.05), T-AOC was increased ( F=305.58, P<0.05), IL-1β was decreased ( F=15.46, P<0.05), IL-6 was decreased ( F=59.2, P<0.05), and TNF-α was decreased ( F=33.13, P<0.05). Conclusions:The extracts of 0.5-2.0 mg/L of Acronychia pedunculata have protective effects on the photoaging cell model, which may be related to the increase of SOD, CAT, GPX and other antioxidant enzymes and the level of T-AOC in photoaging HaCaT cells, and the decrease of ROS, MDA content and the expression of inflammatory cytokines.
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Objective To preliminarily evaluate the effect of levocetirizine hydrochloride at different concentrations on the growth of in vitro cultured human dermal papilla cells,and to explore its mechanism.Methods Human dermal papilla cells were divided into several groups to be cultured with Dulbecco's modified eagle medium (DMEM) containing 0 (control group),1,10,100,1 000,10 000 μg/L levocetirizine hydrochloride respectively for 48 hours.Immunofluorescence staining was performed to observe the growth of the dermal papilla cells,and methyl thiazolyl tetrazolium (MTT) assay to evaluate the proliferative activity of the dermal papilla cells.Real-time fluorescence-based quantitative PCR was conducted to measure the mRNA expression of cyclooxygenase 2 (COX-2),prostaglandin D2 synthase (PTGDS),prostaglandin E2 (PGE2),prostaglandin F2alpha (PGF2α),G protein-coupled receptor 44 (GPR44),protein kinase B (AKT) and glycogen synthase kinase 3β (GSK3β),and Western blot analysis to determine the protein expression of PTGDS.After 24-hour culture with DMEM containing levocetirizine hydrochloride at different concentrations,enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of prostaglandin D2 (PGD2) and PGD2R receptor in the culture supernatant of the human dermal papilla cells.Statistical analysis was carried out with SPSS 17.0 software using one-way analysis of variance for the comparison of the above indices among the groups,and least significant difference (LSD)-t test for multiple comparisons.Results Immunofluorescence staining showed that human dermal papilla cells grew well and reached over 90％ confluence in the 100 μg/L levocetirizine hydrochloride group.MTT assay revealed that there were significant differences in the proliferation rate among all the groups (F =42.22,P ＜ 0.05),and the proliferation rate was significantly higher in the 100 μg/L levocetirizine hydrochloride group (115.80％ ± 5.10％) than in the control group (100％,t =28.26,P ＜ 0.05).The mRNA expression(2-△△Ct) of COX-2,PGF2a,PTGDS,GPR44 and AKT all significantly differed among these groups (F =1.97,3.66,2.17,2.66 and 7.32 respectively,all P ＜ 0.05),while no significant difference in the mRNA expression of PGE2 and GSK3β was observed among these groups (F =0.87 and 1.19 respectively,both P ＞ 0.05).The 100 μg/L levocetirizine hydrochloride group showed significantly decreased mRNA expression of COX-2,PTGDS and GPR44 (0.84± 0.08,0.81±0.10 and 0.85 ± 0.09 respectively) compared with the control group (t =1.97,2.17 and 2.66 respectively,all P ＜ 0.05),but significantly increased mRNA expression of PGF2α and AKT (1.96 ± 0.25 and 1.74 ± 0.32 respectively) compared with the control group (t =3.66,7.32 respectively,both P ＜ 0.05).Moreover,the protein expression of PTGDS,PGD2 and PGD2R significantly differed among these groups (all P ＜ 0.05),and was significantly lower in the 100 μg/L levocetirizine hydrochloride group than in the control group (P ＜ 0.05).Conclusion Levocetirizine hydrochloride can promote the in vitro growth of human dermal papilla cells,likely by inhibiting the PGD2-GPR44 pathway and activating the AKT signal pathway.
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Objective To explore the miRNA regulating the potential cancer-promoting gene CCL18 in cutaneous malignant melanoma.Methods Bioinformatics analysis was conducted by using online software miRanda and TargetScan,so as to predict the miRNA targeting CCL18 gene.Three kinds of C CL18 3'UTR dual-luciferase reporter vectors,including mutant 3'UTR vector (mutant 3'UTR group),wildtype 3'UTR vector (wild-type 3'UTR group) and empty vector (blank control group),as well as miRNA vectors carring selected miRNAs were constructed according to human gene sequence analysis,and then were used to co-transfect 293T cells.After 48-hour treatment,the cells were lysed for detection of luciferase activity.Real-time fluorescence-based quantitative PCR was performed to measure the expression of CCL 18 and selected miRNA in 14 fresh malignant melanoma tissue specimens and 14 paracancerous normal skin tissue specimens (control tissues),and their correlations were analyzed.Results Online software analysis showed that some miRNAs were identified to target the 3'UTR of CCL18 gene,including miR-183,miR-128 and miR-33a.Luciferase reporter vectors and miRNA vectors were constructed successfully.As luciferase activity assay showed,when miR-183 and miR-128 were bound to the CCL18 3'UTR,the luciferase activities were significantly higher in their mutant 3'UTR groups (11.63 ± 0.42;8.80 ± 0.49) than in their wild-type 3'UTR groups (4.86 ± 0.39;5.01 ± 0.54;both P ＜ 0.05) and blank control groups (2.41 ± 0.13;2.39 ± 0.05;both P ＜ 0.01),while there were no significant differences between miR-33a-hinding mutant 3'UTR group (6.41 ± 0.47) and miR-33a-binding wild-type 3'UTR group (6.16 ± 0.22,P ＞ 0.05).Real-time fluorescence-based quantitative PCR revealed higher mRNA expression of the CCL18 gene (3.52 ± 1.68),but lower expression of miR-183 (0.49 ± 0.32),miR-128 (0.30 ± 0.20) and miR-33a (0.46 ± 0.40) in the malignant melanoma tissues compared with the control tissues.The mRNA expression of the CCL18 gene was negatively correlated with the expression of miR-128 (rs =-0548,P ＜ 0.05),but showed no significant correlations with the expression of miR-183 and miR-33a (both P ＞ 0.05).Conclusion miR-128 may play a role in regulating the potential malignant melanoma-promoting gene CCL18.
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Objective To screen microRNAs(miRNAs)related to early mycosis fungoides(MF). Methods A high?throughput miRNA PCR array was used to determine miRNA expression profiles in skin lesions of 6 patients with early MF (early MF group) and 6 patients with lichen planus (control group), followed by screening of differentially expressed miRNAs between the two groups. Then, real?time fluorescence?based quantitative PCR(RT?qPCR)was performed to verify the differentially expressed miRNAs in lesional specimens from 13 patients with early MF and 13 patients with eczema or lichen planus, as well as in Myla cells and normal human T?lymphocytes. Results The high?throughput miRNA PCR array showed that the expressions of hsa?miR?378a?5p, hsa?miR?107 and hsa?miR?302c?3p were significantly higher in the early MF group than in the control group(all P<0.05). For skin lesions, the results from RT?qPCR were similar to those from the miRNA array assay. Compared with normal human peripheral blood T?lymphocytes, Myla cells showed significantly increased expressions of hsa?miR?378a?5p and hsa?miR?107, which was consistent with the results from the miRNA array assay. However, no significant difference was observed in the expression of hsa?miR?302c?3p between the two kinds of cells. Conclusion MiRNA expression profiles in early MF are different from those in inflammatory skin diseases.
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Objective To investigate clinical and pathological features of primary cutaneous CD30 + anaplastic large cell lymphoma(PC-ALCL). Methods Clinical and pathological data were collected from 7 patients with PC-ALCL and analyzed retrospectively. Results Of the 7 patients, 6 were male and 1 was female, with an average age of 52 years. PC-ALCL was characterized by solitary (n = 3)or multiple (n = 4)erythematous nodules, lumps and/or plaques with (n = 6)or without (n = 1)ulceration. Systemic involvement was observed in none of the 7 patients. Histopathological examination showed diffuse distribution of tumor cells in the dermis, which were large with rich cytoplasm and atypical nuclei. Mitotic figures were seen. An immunohistochemical study of tumor cells showed positive staining for CD30 and cytotoxic protein, but negative staining for CD20, CD56,anaplastic lymphoma kinase(ALK). Epstein-Barr virus-encoded RNA in situ hybridization was negative. Conclusions PC-ALCL is a rare primary cutaneous low-grade malignant T-cell lymphoma, which can be confirmed by clinical manifestations as well as histopathological and immunohistochemical examinations. It usually has good prognosis with rare systemic involvement and metastasis.
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Objective To measure histidine triad nucleotide?binding protein 1(HINT1)protein expression and gene promoter methylation, and to analyze the relationship between HINT1 gene promoter methylation and clinical pathological features of melanoma. Methods Fifty?six patients with melanoma and 51 patients with nevus were enrolled as subjects and controls, respectively. Methylation?specific PCR (MSP) was performed to measure the methylation of HINT1 gene promoter in lesional and paratumoral tissue specimens from the patients with melanoma, as well as in lesional specimens from the patients with nevus. Immunohistochemistry was carried out to quantify the expression of HINT1 protein in these tissue specimens. Results MSP showed that the methylation rate of HINT1 gene promoter was significantly higher in melanoma tissues than in paratumoral and nevus tissues(76.8%[43/56]vs. 33.9%[19/56]and 35.3%[18/51], χ2 = 20.810 and 18.749, respectively, both P 0.05). Immunohistochemistry revealed that the expression rate of HINT1 was 21.4%(12/56)in melanoma tissues, compared to 82.4%(42/51)in nevus tissues(χ2 = 39.633, P <0.01). There was a significant difference in the methylation rate of HINT1 promoter between HINT1?positive and ?negative melanoma tissues(6/12 vs. 37/44[84.1%], P<0.05), and between Clark levelⅠ-ⅡandⅢ-Ⅴmelanoma tissues(59.1%[13/22]vs. 88.2%[30/34],χ2=6.365,P=0.012). Conclusions HINT1 protein is lowly expressed in melanoma, which may be associated with high methylation of its gene promoter. Moreover, the high methylation ofHINT1 gene promoter may be involved in the initiation and progression of melanoma.
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Objective To construct a eukaryotic expression plasmid carrying the PKCI-1/HINT1 gene,to investigate its expression in A375 melanoma cells after transfection,and to evaluate its effects on apoptosis and autophagy of A375 cells.Methods The PKCI-1/HINT1 gene sequence was amplified by reverse transcription PCR (RT-PCR) with total RNA extracted from A375 cells as the template,then inserted into the eukaryotic expression plasmid PCDNA3.1 (+) to construct a recombinant plasmid,PCDNA3.1 (+)-PKCI-1/HINT1.Some A375 cells were classified into two groups to be transiently transfected with the recombinant plasmid (PCDNA3.1 (+)-PKCI-1/HINT1 group) or the empty plasmid PCDNA3.1 (+) (control group).After additional 48-hour culture,RT-PCR and Western blot analysis were performed to quantify the mRNA and protein expressions of PKCI-1/HINT1 respectively,Hoechst 33342 staining was conducted to detect apoptosis of A375 cells,Western blot analysis to detect the expressions of intracellular caspase-3 and autophagy-associated protein beclin1,and cell autophagy was observed by using the green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) labelling method combined with confocal laser scanning microscopy.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of A375 cells at 24,48,72 and 96 hours after transfection.Results Enzyme digestion and sequencing analysis confirmed that the eukaryotic expression plasmid PCDNA3.1 (+)-PKCI-1/HINT1 was successfully constructed and effectively expressed in the transfected A375 cells.MTT assay showed that PKCI-1/HINT1 could obviously inhibit the proliferation of A375 cells,and the number of live cells was decreased by 17.0％,25.6％ and 29.4％ in the PCDNA3.1 (+)-PKCI-1/HINT1 group at 48,72 and 96 hours,respectively,compared with the control group (all P ＜ 0.05).Hoechest 33258 staining revealed that PKCI-1/HINT1 could promote the formation of apoptotic bodies in A375 cells.Confocal laser scanning microscopy demonstrated that the overexpression of PKCI-1/HINT1 increased GFP-LC3 puncta formation in A375 cells.In addition,Western blot analysis indicated that PKCI-1/HINT1 up-regulated the protein expressions of caspase-3 and beelin1 in A375 cells.Conclusions The eukaryotic expression plasmid PCDNA3.1 (+)-PKCI-1/HINT1 was successfully constructed,and PKCI-1/HINT1 could be effectively expressed in A375 cells.High-level expression of PKCI-1/HINT1 could suppress cellular proliferation,promote apoptosis,and induce autophagy,of A375 cells.
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Objective To better characterize the clinical and histopathological manifestations, diagnosis and differential diagnosis of epithelioid sarcoma (ES).Methods Clinical data were collected from nine patients with ES diagnosed and treated at the Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College from 2000 to 2014.The clinical and histopathological manifestations, immunophenotype, treatment and prognosis of ES were reviewed retrospectively.Results The median age at onset of ES was 34.5 years in these patients, and the average disease duration was 8.3 years.Lesions began on the extremities in 8 patients, and manifested as nodules,ulceration and lymphedema.Histopathological examination showed that tumor cells mainly consisted of spindle cells and epithelioid cells, some of which were in a palisade arrangement with central necrosis.Cytokeratin, epithelial membrane antigen (EMA) and vimentin were coexpressed by tumor cells.Recurrence was observed at the original site in 6 patients after lesion resection, and pulmonary metastasis occurred in 4 patients.Five patients were followed up and two of them died of pulmonary metastasis.Conclusions Local recurrence is frequent in patients with ES after lesion removal.Lymph node and pulmonary metastases of ES are common, and pulmonary metastasis is usually associated with a poor prognosis.
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Objective To investigate the diagnosis and differential diagnosis of dermatofibrosarcoma protuberans (DFSP). Methods Totally, 50 patients with DFSP visiting the Institute of Dermatology, Chinese Academy of Medical Sciences from 1998 to 2014 were enrolled. The clinical manifestations, histopathological and immunohistochemical features, treatment and prognosis of DFSP were retrospectively reviewed. Results The average age at onset of DFSP was (29.5 ± 15.9)years in the 50 patients, with a mean disease duration of 9.57 years. Skin lesions most frequently occurred on the trunk(n = 33, 66.0%), followed by the extremities, head and neck. DFSP was characterized by atrophic patches or plaques in 13 cases (26.0%), multiple nodules varying in size and arising on atrophic plaques or patches in 30 cases (60.0%), single or multiple nodules arising on normal skin in 7 cases (14.0%). Histologically, the tumor consisted of uniform infiltrative spindle cells arranged in a storiform or cartwheel pattern. In addition, the tumor cells expressed CD34 and vimentin. Twenty patients experienced recurrence at the primary site after resection of skin lesions with a recurrence rate of 43.5%. No distant metastasis or death occurred in these patients. Conclusions DFSP usually has various skin manifestations, is easily misdiagnosed, and can be confirmed based on histopathological and immunohistochemical findings. Local recurrence of DFSP is common, and may occur for many times after surgical excision, but lymphatic and distant metastases are rare.
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Objective:To observe the correlation between the B7 blocker,CD28 and cytotoxic T lymphocyte-associated antigen 4 immunoglobulin (CTLA-4Ig) impacting on Th17 and Treg cells expressed in the spleen of lupus mice and its intervention role in lupus-like symptoms of B6.MRL-Faslpr/J lupus mice.Methods:Sixteen 4-month-old female B6.MRL-Faslpr/J mice were selected and randomly divided into treatment group ( groupⅠ) and control group ( groupⅡ);injected the same amount of CTLA-4Ig and PBS intra-venously,checked their 24 hour urine protein ,ANA antibody,ds-DNA antibodies before and after the intervention.Two weeks after the intervention ,detected serum IL-17A,and the percentage of Th17 and Treg cells in their spleen.Results:Two weeks after the last inter-vention,24-hour urine protein,serum ANA and ds-DNA in groupⅠdecreased,and all the differences were statistically significant (P<0.05) compared with groupⅡ.Two weeks after the last intervention,serum IL-17A and the proportion of Th17 cells in the spleen in groupⅠwere lower than those in groupⅡ, but Treg cells in CD4+T lymphocytes was higher than that in groupⅡ,all the differences were statistically significant ( P<0.05).Conclusion:CTLA4-Ig can relieve the lupus-like symptoms in lupus mice;raising the number of Treg cells and decreasing the number of Th17 cells may be one of the important mechanisms for CTLA-4Ig to alleviate lupus-like symptoms in B6.MRL-Faslpr/J mice.
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Objective To investigate the mRNA expression of toll like receptor-9 (TLR9) and interferon regulatory factors-5 (IRF5) of AS2O3 on peripheral blood mononuclear cells (PBMCs) in systemic lupus erythematosus (SLE) patients.Methods PBMCs of 15 SLE patients and 15 healthy subjects were treated with different concentrations of AS2O3 and cyclophosphamide (CTX) in vitro.Real-time quantitative polymerase chain reaction was used to amplify TLR9 and IRF5 gene before and after 12 and 24 hours drug intervention and the mRNA expressions were measured.Differences between groups were analyzed by paired t test or variance analysis.Results The mRNA expression levels of TLR9 [12 h(1.38±0.26) and 24 h (1.28±0.35)] on PBMCs in SLE patients were significantly higher than those in healthy controls [12 h(1.05±0.35) and 24 h (0.97±0.19)](t=2.37,P=0.03; t=2.44,P=0.02).The IRF5 mRNA expression levels [12 h (0.95±0.27) and 24 h (0.91 ±0.35)] in SLE patients were obviously higher than those in healthy controls [12 h (0.62 ±0.23) and 24 h (0.60±0.39)] (t =3.07,P=0.01 ; t =3.45,P＜0.01).AS2O3 could suppress the mRNA expression of TLR9 on PBMCs and the effect was gradually increasing with the increasing concentration of AS2O3 and processing time [0.2 mg/L AS2O3 group 12 h (0.430±0.110) and 24 h(0.290±0.050),0.4 mg/L AS2O3 group 12 h (0.170±0.038) and 24 h (0.090±0.017),0.8 mg/L AS2O3 group 12 h (0.023±0.011) and 24 h (0.003±0.001)].Comparing with CTX [12 h (0.814±0.081) and 24 h(0.755±0.139)],AS2O3 had a more significant strong effect on inhibiting the expression of TLR9 mRNA in SLE patients [F=165.32(12 h),P＜0.01; F=99.20 (24 h),P＜0.01].The mRNA expression of IRF5 on PBMCs was not suppressed by AS2O3 and CTX and there was no statistically significant difference between groups (P＞0.05).Conclusion There is abnormal expression of IRF5 and TLR9 mRNA in SLE patients.AS2O3 may suppress the TLR9 mRNA expression in SLE patients,which may be one mechanism of clinical effectiveness.
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Objective To assess the association between Toll-like receptor 9 (TLR9) gene single nucleotide polymorphisms (SNPs) and systemic lupus erythematosus (SLE) development in Guangxi Zhuang and Han populations,as well as the difference in TLR9 SNPs between the two populations.Methods Totally,41 SLE patients of Zhuang nationality and 56 of Han nationality,as well as 82 healthy checkup examinees of Zhuang nationality and 120 of Han nationality were enrolled in this study.Venous blood samples were obtained from all of these subjects and subjected to DNA extraction.The single nucleotide polymorphisms in TLR9 gene were detected by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis followed by direct sequencing.Chi-square test and adjusted Chi-square test were conducted to assess the relationship between the genotype and allele frequencies of TLR9 SNPs and some clinical and laboratory parameters of patients with SLE,as well as the differences in genotype and allele frequencies of TLR9 SNPs between the two populations.Results The frequencies of CC,CT and TT genotypes of TLR9 SNP rs352140 were 42.9％,41.1％ and 16.1％ respectively in the patients of Han nationality,compared to 38.3％,55.8％ and 5.8％ in the healthy controls of Han nationality (all P ＜ 0.05),but no significant difference was observed in the frequency of C or T allele of the SNP rs352140 between the patients and controls of Han nationality (both P ＞ 0.05).There was no significant difference in the genotype or allele frequency of TLR9 SNP rs352140 between the patients and healthy controls of Zhuang nationality,or between the patients of Han nationality and Zhuang nationality (all P ＞ 0.05).The patients with anti-dsDNA antibodies showed a significantly higher frequency of TT genotype (P ＜ 0.05),but similar T allele frequency at TLR9 SNP rs352140 (P ＞ 0.05) compared with those without.The frequencies of both TT genotype and T allele of TLR9 SNP rs352140 were significantly increased in the patients with a SLE disease activity index (SLEDAI) ≥ 9 compared with those with a SLEDAI ＜ 9 (both P ＜ 0.05).There was no statistical difference in either the TT genotype or the T allele frequency at TLR9 SNP rs352140 between antinuclear antibody-positive and-negative patients with SLE (both P ＞ 0.05).Conclusions The TLR9 SNP rs352140 is correlated with several clinical and laboratory parameters of SLE,and might contribute to the susceptibility to SLE in Guangxi Han population.