Objective To evaluate the therapeutic effect of hydrogen-rich saline on allergic contact dermatitis (ACD) in mice and to explore its underlying mechanisms.Methods Forty mice were equally divided into 4 groups:control group,control treatment group,ACD group and ACD treatment group.ACD was induced by repetitive topical application of dinitrofluorobene (DNFB) to the left ear of mice on day 1,2 and 5.Hydrogen-rich saline was intraperitoneally given to the mice in the ACD treatment group at a dose of 5 ml/kg per day from day 1 to 5.On day 6,the mice were sacrificed,ear tissue was removed from them and subjected to measurement of thickness and weight,detection of tumor necrosis factor (TNF)-α,interleukin (IL)-6,IL-17 and interferon (IFN)-γ expression by enzyme linked immunosorbent assay,as well as numeration of inflammatory cells in lesions after hematoxylin-eosin (HE) staining.Data were processed by SPSS 18.0 software,and statistical analysis was carried out by one-way analysis of variance and least significant difference (LSD) procedure.Results Compared with the control group,the ACD group showed a significant increase in lesion score (7.33 ± 1.53 vs.0,P ＜ 0.05),differences in the thickness ((0.73 ± 0.15) mm vs.(0.13 ± 0.05) mm,P ＜ 0.05) and swelling degree (expressed as tissue weight:(18.67 ± 3.05) mg vs.(3.33 ± 1.52) mg,P ＜ 0.05) between the left and right ear,expressions of TNF-α ((1475.52 ± 233.81) pg/mg vs.(239.01 ± 52.39) pg/mg,P＜ 0.05),IL-6 ((184.65 ± 78.39) pg/mg vs.(42.28 ± 17.64) pg/mg,P＜ 0.05),IL-17 ((628.56 ± 201.44) pg/mg vs.(127.58 ± 50.28) pg/mg,P＜ 0.05) and IFN-γ ((197.72 ± 37.81) pg/mg vs.(24.57 ± 8.31) pg/mg,P ＜ 0.05),and the number of inflammatory cells per square millimetre in the left ear tissue (752.00 ± 166.06 vs.127.33 ± 77.18,P ＜ 0.05).However,hydrogen-rich saline treatment induced a statistical decrease in all of these parameters in the ACD treatment group compared with the ACD group,including lesion score (3.33 ± 0.58,P ＜ 0.05),difference in thickness ((0.46 ± 0.11) mm,P ＜ 0.05) and swelling degree ((11.00 ± 2.64) mg,P ＜ 0.05),expressions of TNF-α ((817.72 ± 101.13) pg/mg,P＜ 0.05),IL-6 ((95.86 ± 36.65) pg/mg,P＜ 0.05),IL-17 ((373.38 ± 126.74) pg/mg,P＜ 0.05),IFN-γ ((63.31± 17.38) pg/mg,P ＜ 0.05) and the number of inflammatory cells per square millimetre (384.00 ± 97.35,P ＜0.05).Conclusion Hydrogen may inhibit the release of inflammatory factors in ACD.

Objective To investigate whether hydrogen can regulate the expressions of inflammatory factors by ultraviolet B (UVB)-induced human HaCaT keratinocytes through the autophagy pathway.Methods Cultured HaCaT keratinocytes were divided into several groups:blank control group receiving no treatment,hydrogen group cultured in hydrogen-rich medium,three UVB groups irradiated with UVB at 1,10,50 mJ/cm2 respectively,three UVB + hydrogen groups irradiated with UVB at 1,10,50 mJ/cm2 respectively followed by culture in hydrogen-rich medium,UVB + 3MA group pretreated with the autophagy inhibitor 3MA for 1 hour followed by UVB radiation at 50 mJ/cm2,UVB + rapamycin group pretreated with the autophagy activator rapamycin for 1 hour followed by UVB radiation at 50 mJ/cm2,UVB + 3MA +hydrogen group pretreated with 3MA for 1 hour followed by UVB radiation at 50 mJ/cm2 and culture in hydrogen-rich medium,UVB + rapamycin + hydrogen group pretreated with rapamycin for 1 hour followed by UVB radiation at 50 mJ/cm2 and culture in hydrogen-rich medium.After additional culture with or without hydrogen for 12 hours,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cellular proliferative activity,Western-blot analysis to measure the expressions of autophagy-associated protein 1 light chain 3 (LC3) and Beclin 1,and enzyme-linked immunosorbent assay (ELISA) to measure the supernatant levels of inflammatory factors including tumor necrosis factor (TNF)-α,interleukin (IL)-1β,IL-6 and high mobility group protein B1 (HMGB1),and a test kit was used to determine the level of lactate dehydrogenase (LDH).Results Compared with the blank control group,the 10-and 50-mJ/cm2 UVB groups showed significantly increased release of LDH,expressions of LC3 and Beclin1 and supernatant levels of TNF-α,IL-1 β,IL-6 and HMGB 1,but decreased cellular proliferative activity (all P ＜ 0.05).Hydrogen significantly attenuated the release of LDH,down-regulated the supernatant levels of TNF-α,IL-1β,IL-6 and HMGB1,but up-regulated cellular proliferative activity as well as LC3 and Beclin1 expressions in the 10-and 50-mJ/cm2 UVB + hydrogen groups compared with the 10-and 50-mJ/cm2 UVB groups respectively (all P ＜ 0.05).In addition,the levels of TNF-α,IL-1β,II-6 and HMGB1 were significantly higher in the 50-mJ/cm2 UVB + 3MA group than in the 50-mJ/cm2 UVB group,and higher in the 50-mJ/cm2 UVB + 3MA + hydrogen group than in the 50-mJ/cm2 UVB + hydrogen group,but lower in the 50-mJ/cm2 UVB + rapamycin group than in the 50-mJ/cm2 UVB group (all P＜ 0.05).Conclusion UVB radiation can increase the expressions ofautophagy-associated proteins,and hydrogen-rich medium can down-regulate the expressions of inflammatory factors by UVB-induced HaCaT cells through the autophagy pathway.

BACKGROUND: In clinical practice, the selection of dental implants with different specifications is based on the patient' s mandibular plane angle, and the long-term success rate is different in dental implants with different specifications. Therefore, it is reasonable to assume that the mandibular plane angle could affect the mandibular stress distribution of dental implants, thus affecting the success rate of dental implantation.OBJECTIVE: To perform an orthogonal experimental study on the mechanical effects of different implant specifications, mandibular plane angles and mandibular bone densities on stress distribution of the first mandibular molar implants.METHODS: A L9(33) orthogonal experiment was designed with 3 three-level factors, including mandibular plane angle (14°, 22°, 34°), implant diameter (6.6, 8.2, 9.6 mm), and mandibular bone density (types II, III, IV). Different dental implants with different combinations were implanted into an alveolar bone model, and placed onto a pressure testing machine under 500 N load. Then, strain (stress) data were collected in real-time and analyzed.RESULTS AND CONCLUSION: The optimal combination was as follows: low angle; 4.8 mm in diameter; mandibular bone with type II bone density. The relations of all the three factors were the diameter of dental implant > the mandibular plane angle > mandibular bone density. To conclude, the mandibular plane angle has some certain influences on the stability of dental implants. If the biting force is the same, dental implant bears the largest stress under the high angle,subsequently followed by the average angle and the low angle.

Objective To determine the distribution profile of apocrine sweat glands in axillary region of patients with axillary osmidrosis,and to compare their distribution at different sites.Methods Fifteen patients with axillary osmidrosis were enrolled in this study from September to December 2010.All the patients underwent surgical removal of apocrine sweat glands under direct vision.Full-thickness skin tissue measuring 2 mm in width was excised down to the axillary superficial fascia at the incisional surgical sites from five patients.Five points,which were at the center of axillary region (point 1),1 cm away from the center of axillary region (point 2),1 cm inside the edge of axillary hair-bearing area (point 3),the edge of axillary hair-bearing area (point 4),and 1 cm outside the edge of axillary hair-bearing area (point 5),were marked,and dark red,rough granular subcutaneous tissue was obtained at these points in 10 patients with axillary osmidrosis.Results The secretory portion of apocrine sweat glands was mainly distributed in the reticular dennis and superficial subcutaneous adipose tissue,but no apocrine sweat glands were obs erred in the epidermis,dermal papilla or axillary superficial fascia.The distribution profile of apocrine sweat glands was consistent with that of axillary hairs.There were numerous apocrine sweat glands in the center of axillary region,but only a small number at the edge of axillary hair-bearing area,and no apocrine sweat gland was observed at 1 cm outside the edge.The percentage of apocrine sweat gland area at point 1-5 was 74.1％,46.6％,25.3％,2.1％,and 0 respectively,with significant differences between point 1 and 2 (t--29.78,P＜ 0.01),point 2 and 3 (t--9.76,P＜ 0.01),point 3 and 4 (t =20.83,P＜ 0.01),but not between point 4 and 5 (t =1.96,P ＞ 0.05).Conclusions During the surgical treatment of axillary osmidrosis,the removal of apocrine sweat glands should be extended to the reticular dennis and superficial subcutaneous adipose tissue in depth and the edge of axillary hair-bearing area in width,and there is no need to blindly increase the extent of removal.

Objective To observe the effect of hydrogen on ultraviolet B (UVB)-induced oxidative damage to skin fibroblasts.Methods Primary human skin fibroblasts from foreskin tissues were divided into five groups:normal control group receiving no treatment,hydrogen control group treated with hydrogen-rich saline,UVB group receiving irradiation only,post-treatment group irradiated with UVB followed by hydrogen-rich saline treatment,and pre-treatment group treated with hydrogen-rich saline followed by UVB irradiation.The dose of UVB was 30,60 and 90 mJ/cm2 in the cell proliferation assay and 90 mJ/cm2 in the other experiments.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of fibroblasts,a chemiluminescence method to estimate the activity of superoxide dismutase (SOD) and catalase as well as to determine the level of malondialdehyde in the culture supernatant of fibroblasts,enzyme linked immunosorbent assay (ELISA) to determine the supernatant level of 8-isoprostane-prostaglandin F2α (8-iso-PGF2α),Western blot to detect the expression of heme oxygenase-1 (HO-1) in fibroblasts.One-factor analysis of variance was conducted to assess differences in these parameters among these groups.Results UVB irradiation decreased the proliferative activity (absorbence value at 490 nm) of fibroblasts in a dose-dependent manner.Both the pre-treatment group and post-treatment group showed a statistical increase in proliferative activity of cells compared with the corresponding UVB control groups (all P ＜ 0.05).The activity of SOD and catalase as well as the protein expression of HO-1 were significantly higher (all P ＜ 0.05),whereas the supernatant levels of malondialdehyde and 8-iso-PGF2α were statistically lower (both P ＜ 0.05) in the pre-treatment group and post-treatment group than in the UVB control group.Conclusion Hydrogen may mitigate UVB-induced oxidative damage to skin fibroblasts.

Objective:To explore intrinsic mechanisms underlying the inhibitory effect of resveratrol on neovascularization in cutaneous squamous cell carcinoma from the perspective of ubiquitin/ubiquitin-like protein modification balance.Methods:The human cutaneous squamous cell carcinoma cell line A431 was used as the research object. Cultured A431 cells at exponential growth phase were divided into 3 groups (control group, 50 μmol/L resveratrol group, and 100 μmol/L resveratrol group) to be cultured with mediums containing 0, 50, and 100 μmol/L resveratrol, respectively. Cell proliferation activity was assessed by the 3- (4,5) -dimethylthiazol (-z-y1) -2,5-di-phenytetrazoliumromide (MTT) assay after 48-hour culture; the vasculogenic mimicry formation assay was performed to evaluate the vasculogenic mimicry formation ability of A431 cells after 12-hour treatment with resveratrol; Western blot analysis was conducted to detect the relative protein expression levels of ubiquitin, small ubiquitin-related modifier-1 (SUMO1), hypoxia-inducible factor 1α (HIF-1α), and vascular endothelial growth factor receptor (VEGFR) in different groups after 48-hour treatment with resveratrol. Then, 24 8-week-old BALB/c male thymectomized mice were randomly and equally divided into 3 groups to be subcutaneously inoculated with A431 cells in the inguinal region, followed by intraperitoneal injections of 1 mg/kg or 2 mg/kg resveratrol (1 mg/kg or 2 mg/kg resveratrol group), or the same volume of physiological sodium chloride solutions (control group) ; the intraperitoneal injections were done once every 3 days in all groups; all the above mice were sacrificed on the 21st day, and the tumors were resected and weighed. Immunohistochemistry assay was performed to determine the CD31 expression in tumor tissues. One-way analysis of variance was used for comparisons among multiple groups, and least significant difference (LSD) - t test was used for multiple comparisons. Results:The proliferation rate of A431 cells significantly differed among the control group, 50 μmol/L resveratrol group, and 100 μmol/L resveratrol group ( F = 17.75, P = 0.017), and was significantly lower in the 50 μmol/L resveratrol group (66.53% ± 5.09%) and the 100 μmol/L resveratrol group (35.88% ± 4.28%) than in the control group (100%, LSD- t = 21.17, 29.04, P = 0.011, 0.004, respectively) ; the total length of vessel wall-like structures formed by A431 cells significantly differed among the 3 groups ( F = 21.37, P = 0.004), and was significantly lower in the 50 μmol/L resveratrol group (102.73 ± 11.36 μm) and the 100 μmol/L resveratrol group (37.83 ± 4.19 μm) than in the control group (185.26 ± 8.02 μm, both P < 0.05) ; the relative protein expression levels of ubiquitin, SUMO1, HIF-1α, and VEGFR also significantly differed among the 3 groups, the ubiquitin protein expression was significantly higher in the 50 μmol/L resveratrol group (2.09 ± 0.13) and the 100 μmol/L resveratrol group (3.53 ± 0.16) than in the control group (0.68 ± 0.11, both P < 0.05), while the protein expression of SUMO1, HIF-1α, and VEGFR was significantly lower in the 50 μmol/L resveratrol group (1.87 ± 0.13, 0.81 ± 0.06, 0.73 ± 0.09, respectively) and the 100 μmol/L resveratrol group (1.02 ± 0.11, 0.45 ± 0.06, 0.39 ± 0.05, respectively) than in the control group (3.10 ± 0.11, 0.97 ± 0.08, 0.98 ± 0.07, respectively, all P < 0.05). In the mice experiment, the weight of subcutaneous tumors and the proportion of CD31-positive cells in tumor tissues significantly differed among the control group, 1 mg/kg resveratrol group, and 2 mg/kg resveratrol group (weight: 3.29 ± 0.57 g, 2.91 ± 0.49 g, 2.55 ± 0.52 g; proportion: 76.24% ± 5.51%, 39.45% ± 5.48%, 12.07% ± 3.54%; F = 14.33, 15.34, P = 0.019, 0.021, respectively), and were significantly lower in the 1 mg/kg resveratrol group and 2 mg/kg resveratrol group than in the control group (all P < 0.05) . Conclusion:Resveratrol could inhibit tumor growth and neovascularization in tumor tissues, which were possibly associated with the inhibitory effect of resveratrol on neovascularization in cutaneous squamous cell carcinoma by suppressing the SUMOylation of HIF-1α protein via ubiquitin/ubiquitin-like protein modification pathways.

Objective To evaluate the effect of luteolin on the growth,migration and vasculogenic mimicry formation of a melanoma cell line B16.Methods In vitro cultured B16 melanoma cells were divided into 4 groups:low-,middle-and high-dose luteolin groups treated with 2.5,5,10 μmol/L luteolin respectively,and control group treated with 0.1％ dimethyl sulfoxide (DMSO).Scratch assay,Transwell invasion assay and vascular channel formation assay were performed to assess the migration,invasion of and vascular channel formation by melanoma cells.A model of subcutaneous transplanted B 16 melanoma was established in 12 C57 mice,which were randomly and equally divided into 4 groups:control group gavaged with ultrapure water,low-,middle-and high-dose luteolin groups gavaged with 10,20,40 mg/kg luteolin respectively every day.The above treatment for the tumor-bearing mice lasted till day 28,and then these mice were sacrificed.Meanwhile,the lung and tumor tissues of the mice were excised,and the growth,metastasis and vasculogenic mimicry of transplanted melanoma were observed.Immunofluorescence and immunohistochemical studies were performed to evaluate the effects of luteolin on the expression of vascular endothelial cadherin (VE-cadherin),vascular endothelial growth factor receptor 1 (VEGFR1),VEGFR2,matrix metalloproteinase-2 (MMP-2) and MMP-9 in the transplanted melanoma.Means were compared among several groups by using one-way analysis of variation or rank sum test.Results In vitro study showed that the relative scratch width at 48 hours significantly differed among the control group,low-,middle-and high-dose luteolin groups (0.47 ± 0.04,0.64 ± 0.04,0.73 ± 0.03,0.84 ± 0.04 respectively;F =34.51,P ＜ 0.001),and the migration ability of B16 cells was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (all P ＜ 0.05).At 24 hours,there were significant differences in the number of cells crossing the Transwell membrane among the control group,low-,middle-and high-dose luteolin groups (281.00 ± 8.79,169.00 ± 15.35,92.00 ± 14.79 and 57.00 ± 13.72 respectively;F =275.30,P ＜ 0.001),and the invasive ability was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (P ＜ 0.01).Meanwhile,the number of formed vascular channels also differed among the above 4 groups (20.00 ± 2.77,11.00 ± 1.28,7.00 ± 1.86 and 2.00 ± 1.32 respectively;F =48.61,P ＜ 0.001),and the number of vascular channels was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (all P ＜ 0.01).In vivo study showed that the tumor size significantly differed among the control group,low-,middle-and high-dose luteolin groups (5.10 ± 1.72,4.02 ± 2.13,2.98 ± 0.92,1.49 ± 1.13 cm3 respectively;F =28.76,P ＜ 0.001),and was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (t =3.86,7.11 and 13.06 respectively,all P ＜ 0.01).CD31-PAS double staining showed that the number of vasculogenic mimicry was significantly higher in the control group than in the low-,middle-and high-dose luteolin groups (all P ＜ 0.01).In vivo and in vitro studies both showed that the expression of vasculogenic mimicry-related markers in the cells or mouse tumor tissues was significantly lower in the high-dose luteolin group than in the control group (P ＜ 0.05).Conclusion Luteolin can effectively inhibit the growth,metastasis and vasculogenic mimicry formation of melanoma.

Objective:To investigate the effect of the transcriptional coactivator Mediator 1 (Med1) on mouse hair regeneration, and to explore potential mechanisms.Methods:Med1 flox/flox C57BL/6J mice were mated with K14-Cre mice, and the mice with epidermis-specific knockout of Med1 gene, namely K14-Cre-expressing Med1 flox/flox mice (knockout group) , were obtained by using the Cre-Loxp system, while Med1 flox/flox mice without K14-Cre expression served as control group. Mice in the two groups (3 mice in each group) were raised together for 8 weeks followed by dorsal hair removal. Hair regeneration was observed for 12 consecutive days after hair removal. After 12 days, all mice in the two groups were sacrificed, their depilated and non-depilated dorsal skin tissues were resected, and total RNA was extracted from the tissues. Real-time quantitative PCR was performed to determine the mRNA expression of hair keratin genes, vitamin D receptor/β-catenin pathway-related genes, and genes associated with maintenance of hair follicle stem cell proliferation and quiescence. Paraffin-embedded sections of depilated and non-depilated mouse skin tissues were prepared, and immunofluorescence staining was conducted to determine the number of stem cells in the hair follicle bulge. Two-independent-sample t test was used for comparisons between two groups. Results:From days 0 to 12 after depilation, hair regeneration was delayed in the depilated skin area in the knockout group compared with the control group. Real-time quantitative PCR showed significantly decreased mRNA relative expression levels of hair keratin genes Ha1 and Krt2-16, vitamin D receptor/β-catenin pathway-related genes S100a3, Dlx3 and Tubb3, and genes associated with maintenance of hair follicle stem cell proliferation and quiescence including Lhx2, Sox9 and Nfatc1 in the depilated skin tissues in the knockout group (22.09 ± 12.32, 2.07 ± 0.20, 0.02 ± 0.01, 12.36 ± 2.12, 1.75 ± 0.46, 0.39 ± 0.02, 4.42 ± 0.76, 0.44 ± 0.07, respectively) compared with the control group (70.53 ± 9.46, 7.76 ± 0.49, 0.05 ± 0.01, 26.16 ± 2.96, 2.60 ± 0.14, 0.71 ± 0.09, 11.93 ± 0.42, 0.75 ± 0.04, respectively; t = 5.40, 18.64, 3.89, 6.57, 3.04, 6.10, 15.03, 6.18, respectively, all P < 0.05) . Immunofluorescence staining showed that the number of CD34 +K15 + hair follicle stem cells in the hair follicle bulge in both depilated and non-depilated skin tissues was significantly lower in the knockout group than in the control group. Conclusion:Med1 gene knockout may down-regulate the expression of downstream genes of the vitamin D receptor/β-catenin pathway and genes associated with maintenance of hair follicle stem cell proliferation and quiescence (Sox9, Nfatc1 and Lhx2) , and reduce the number of hair follicle stem cells, leading to hair follicle differentiation disorder and hair regeneration delay.

OBJECTIVEFinite elemental analysis of the mechanical characteristics of a first mandibular implant molar under different mandibular plane angles determines the load conditions on the implant, thereby providing guidance for clinical application.

METHODSCT data of three mandibular plane angles (low, average, high) were collected. A finite elemental combination model of a dental implant was constructed. The orthogonal experimental research was designed. Results followed data collection and analysis.

RESULTSThe optimal combination was a low angle, 4.8 mm, and type Ⅱ bone. The relations among diameter of the implant, angle of mandibular bone, and bone density were determined.

CONCLUSIONSMandibular plane angle influences the stability of a dental implant. Under constant biting force, dental implants bear the stress proportional to the angle, high angles cause high stress, average angles cause average stress, and low angles cause the least stress.