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Photohardening therapy, also known as photodesensitization therapy, refers to the phototherapy and photochemotherapy of idiopathic actinic dermatoses, and its goal is to improve the patients′ tolerance to sunlight and prevent disease flares. Its mechanisms of action involve a variety of cellular and inflammatory factors. This therapy is suitable for all idiopathic actinic dermatoses, with definite efficacy and good safety. However, the treatment specificity usually leads to poor compliance. The development of UVA1 rush hardening and home phototherapy is expected to solve this problem.
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Objective:To evaluate the effect of metformin on ultraviolet A (UVA) -induced photoaging of an immortalized human keratinocytes cell line (HaCaT), and to explore its potential mechanisms.Methods:Cell counting kit 8 (CCK8) assay was performed to evaluate the effect of metformin at different concentrations (0 - 100 mmol/L) on the viability of HaCaT cells, and 10 mmol/L metformin was selected for subsequent experiments. Cultured HaCaT cells were divided into a blank control group (conventional culture), a metformin group (treated with culture medium containing 10 mmol/L metformin), a UVA irradiation group (conventional culture for 24 hours followed by 10 J/cm 2 UVA irradiation) and a metformin + UVA group (treated with culture medium containing 10 mmol/L metformin for 24 hours followed by 10 J/cm 2 UVA irradiation) ; UVA irradiation was performed at a dose of 10 J/cm 2 once a day for 3 consecutive days. After 4-day treatment, cells were collected, the β-galactosidase assay was performed to determine the proportion of senescent cells in each group, 2′, 7′-dichlorodihydrofluorescein diacetate assay to detect levels of intracellular reactive oxygen species (ROS), and the comet assay to detect DNA damage levels. Additionally, some HaCaT cells were divided into the blank control group, metformin group, 1.25 μmol/L dorsomorphin (an adenosine monophosphate-activated protein kinase [AMPK] inhibitor) + metformin group, and 2.5 μmol/L dorsomorphin + metformin group, and cells in the latter two groups were treated with 1.25 and 2.5 μmol/L dorsomorphin respectively for 2 hours, followed by the treatment with 10 mmol/L metformin for 24 hours. Western blot analysis was performed to determine the cellular localization and phosphorylation levels of nuclear factor-erythroid 2-related factor 2 (Nrf2). By using the small-interfering RNA (siRNA) -mediated silencing method, siRNA-Nrf2 was transfected into HaCaT cells to knock down Nrf2 expression (siRNA-Nrf2 group) ; 2.5 μmol/L dorsomorphin-treated HaCaT cells or Nrf2-knockdown HaCaT cells were treated with metformin and UVA irradiation (dorsomorphin + metformin + UVA group, siRNA-Nrf2 + metformin + UVA group, respectively), and the proportions of senescent cells were further calculated in each group. Statistical analysis was carried out by using one-way analysis of variance and two-way analysis of variance, and least significant difference (LSD) - t test was used for multiple comparisons. Results:Treatment with different concentrations of metformin for 24 hours could affect the viability of HaCaT cells to varying degrees ( F = 5 206.31, P < 0.001) ; there were no significant differences in the relative survival rates of HaCaT cells between the 10 - 20 mmol/L metformin groups and the control group (0 mmol/L metformin group, all P > 0.05), while the relative cell survival rates were significantly lower in the 25 - 100 mmol/L metformin groups than in the control group (all P < 0.05). After UVA irradiation, HaCaT cells shrank significantly and became narrow and elongated, and the intercellular spaces increased; the relative cell survival rate was significantly lower in the UVA irradiation group (76.13% ± 1.03%) than in the blank control group (100.00% ± 1.24%, LSD- t = 14.86, P < 0.001), but significantly higher in the metformin + UVA group (106.69% ± 2.45%) than in the UVA irradiation group (LSD- t = 11.55, P < 0.001). Moreover, the UVA irradiation group showed significantly increased proportions of senescent cells (45.14% ± 4.98%), intracellular ROS levels (144.61% ± 4.91%), and percentages of DNA in the tail (75.33% ± 1.77%) compared with the blank control group (23.84% ± 1.89%, 55.49% ± 1.57%, 1.88% ± 0.29%, respectively, all P < 0.001), while the metformin + UVA group showed significantly decreased proportions of senescent cells (24.26% ± 1.34%), intracellular ROS levels (58.62% ± 2.17%), percentages of DNA in the tail (15.83% ± 1.23%) compared with the UVA irradiation group (all P < 0.001). Western blot analysis showed that the Nrf2 expression in the cytoplasm was lower in the 10 mmol/L metformin group than in the blank control group, while the phosphorylated Nrf2 expression in the nuclei was higher in the 10 mmol/L metformin group than in the blank control group, suggesting that metformin could effectively induce the phosphorylation of Nrf2 and its nuclear translocation; both the pretreatment with 1.25 and 2.5 μmol/L dorsomorphin could significantly reduce the phosphorylation levels of AMPKα and Nrf2 induced by 10 mmol/L metformin. The proportions of senescent cells in the dorsomorphin + metformin + UVA group and the siRNA-Nrf2 + metformin + UVA group were 67.84% ± 2.74% and 65.94% ± 1.33%, respectively, which were significantly higher than those in the metformin + UVA group (37.76% ± 1.64%, t = 14.45, 13.34, respectively, both P < 0.001) . Conclusion:Metformin may inhibit UVA-induced photoaging of HaCaT cells by activating the AMPK/Nrf2 signaling pathway, scavenging ROS and reducing DNA damage.
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ObjectiveTo detect the mutations of GJB3 and GJB4 genes in two sporadic cases of erythrokeratodermia variabilis(EKV).MethodsGenomic DNA was extracted from two sporadic patients with EKV,their family members,and 100 normal human controls.All the exons and adjacent splice sites of GJB3 and GJB4 genes were amplified by PCR.Mutation scanning was carried out via direct bidirectional DNA sequencing.ResultsA G134C mutation was found at the GJB3 gene in patient 1,which caused a substitution of glycine by alanine at codon 45 (G45A).No mutation was found in the GJB4 gene in case 1 or GJB3 and GJB4 genes in case 2.ConclusionA missence mutation G45A in GJB3 gene is found in a patient with EKV.
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Objective To investigate the effect of acitretin on the histopathology and ultrastructure of lesions from patients with bullous ichthyosiform erythrodermia (BIE), and to explore mechanisms underlying the modulation of keratinization process by acitretin. Methods Lesional tissue was obtained from the back of 4 patients with BIE before and after the treatment with acitretin. Light microscopy and transmission electron microscopy were performed to observe histopathological and ultrastructural changes in these lesions. Results After treatment, the improvement in clinical manifestations was more than 75% in all the 4 patients, and reached 90% in 1 of the 4 patients. As histopathology and ultrastructural study showed, there was an obvious improvement in hyperkeratosis and continuity of extra cellular lamellar membrane, and a decrease in keratin deposition in prickle and granular layer, but no remarkable changes were observed for the proliferation of prickle cells or acantholysis. Conclusions Acitretin shows a favorable efficacy in clinical treatment of BIE,with histopathological and ultrastructural improvement mainly located in the stratum corneum. The modulation of keratinization process in keratinocytes by acitretin appears more apparent in granular and corneum layers.
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OBJECTIVE: To observe the release mode and the amount of retention in skin of podophyllotoxin liposomes in topical application. METHODS: Two kinds of podophyllotoxin liposome suspension (DPPC liposome and bean lecithin liposome) and podophyllotoxin tincture in same concentration were topically applied on the skin of porkets and the amounts of retention of drug in skin of different preparations were detected with confocal laser scanning microscope.RESULTS: The quantity of retention of podophyllotoxin DPPC liposome in epidermis and superficial layer of dermis was larger than those of other two preparations and the drug concentration in applying DPPC liposome kept higher for 48 hours.CONCLUSION:Podophyllotoxin liposome coated by DPPC is much more targetable to skin and is a fairly good topical preparation for applying on skin.
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Objective To search the clinical manifestations,data of laboratory examination, features of CT and MRI, therapy, prognosis in order to evaluate the progress in therapy of patients with crypotococcus meningitis(CM).Methods The findings of clinical features were analyzed and summarized for therapy of 43 CM cases identified with India-ink capsule staining.Results Primary positive rates of India-ink capsule staining were 58 1%(25/43), secondary positive rates were 30 2%(13/43),7 0%(3/43),patients diagnosed with cultivate positive. Clinical cure was achieved in 22 of 32 patients (68 7%) treated with amphotericin B, 4 patients of without efficacy using amphotericin B were cured by liposomal amphotericin B treatment.6 patients was died from cerebral hernia.Conclusions It is indicated that amphotericin B and fluconazole are effective and well tolerated in treatment of crypotococcus meningitis. If amphotericin B should inefficiency therapy of CM,liposomal amphotericin B would be consider.
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Objective To explore the best formula of the 0 5% liposome podophyllotoxin chitosan film. Methods Chitosan,acetic acid and gelatin were selected as three factors to prepare film, each factor including three levels, and 0 5% liposome podophyllotoxin served as blank factor. The films of different formula were prepared according to orthogonal design. The evaluation was made on the basis of the characteristics of conglutination and dissolution, then the best formula was determined finally. Results The results showed that the conglutination and dissolution of the film which was made of 2% chitosan, 1%acetic acid, 2% gelatin and 0 5% liposome podophyllotoxin chitosan was the most perfect. Conclusion The film designed according to orthogonal experiment was coincidenced with the requirements in aspect of conglutination and dissolution, and its appearance was perfect.
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Objective To study the distribution pattern of liposome podophyllotoxin(LP)in rat skin.Methods The rats were divided into two groups:0.5%liposome podophyllotoxin suspension was ap-plied to LP group,0.5%podophyllotoxin tincture was applied to control group.The skin specimens were ob-tained1?2?4?6?12and24h after drug application,the amount of fluorescent stain was observed under con-focal laser scanning microscope and converted to the values of area under the curve(AUC).Results The epidermal AUC of fluorescent amount in LP group was1.5-fold than that in control group,dermal AUC was2.3-fold higher.The unit area fluorescent amount in both epidermis and dermis was highest2hours after topical medication in control group(1585.52/?m 2 and2005.66?m 2 ),and quickly reduced after4hours.But the epidermal and dermal unit area fluorescent amount in LP group was rather low in4hours after topi-cal medication,and gradually increased after6hours,and peaked after12hours(750.28/?m 2 and1073.08/?m 2 ).Conclusion Liposome preparation of podophyllotoxin can be slowly released and lasts longer in the skin.