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Objective To explore the inhibitory effect of tetramethylpyrazine (TMP) on ultraviolet A-induced senescence as well as matrix metalloproteinase-1 (MMP-1) and-3 (MMP-3) mRNA expressions in human dermal fibroblasts (HDFs).Methods HDFs were isolated from the prepuce by enzymatic digestion, and subjected to primary culture.Cultured HDFs were randomly divided into several groups: control group cultured in high-glucose DMEM medium and receiving no treatment, three TMP groups treated with 20, 50 and 100 mg/L TMP respectively, UVA group receiving UVA radiation alone, UVA + TMP groups pretreated with 20, 50 and 100 mg/L TMP respectively for different durations followed by UVA radiation.UVA radiation was given once daily for 5 consecutive days.The 55th passage HDFs served as the P55 group (senescence control group).Subsequently, CCK-8 assay was performed to evaluate the proliferative activity of HDFs in vitro, optical microscopy to observe the morphologic changes of HDFs after UVA radiation, β-galactosidase staining to estimate the senescence in HDFs, and real-time fluorescence-based quantitative PCR to quantify the mRNA expressions of MMP-1 and MMP-3 in HDFs.Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by least significant difference (LSD)-t test or Dunnett's T3 test.Results Compared with the control group, the proliferation of HDFs was significantly but transiently inhibited in vitro after the treatment with 100 mg/L TMP for 48 hours (P ＜ 0.05), but showed no significant changes after the treatment with 20 or 50 mg/L TMP for 24, 48 or 72 hours or after the treatment with 100 mg/L TMP for 24 or 72 hours (all P ＜ 0.05).The pretreatments with TMP of 20, 50 and 100 mg/L for 24, 48 and 72 hours all promoted the proliferation of HDFs to a certain degree in the UVA + TMP groups compared with the UVA group, with significant differences in cellular proliferative activity among the UVA group, UVA + TMP groups and control group at 24, 48 and 72 hours (F =17.451,15.231, 23.535, all P ＜ 0.01).Compared with the UVA group, the proliferative activity of HDFs was significantly increased in UVA + 100-mg/L TMP group at 24, 48, 72 hours, UVA + 50-mg/L TMP group at 24 and 72 hours and UVA + 20-mg/L TMP group at 72 hours.After repetitive UVA radiation, HDFs in the UVA group experienced an increase in cell volume, granule acount, and β-galactosidase expression, which was similar to the changes in the P55 group, while the pretreatments with 20, 50 and 100 mg/L TMP for 24 hours suppressed these UVA-induced changes in HDFs.The percentage of β-galactosidase-positive HDFs was 68.417％ ± 1.181％ in the UVA group, 58.167％ ± 5.620％ in the UVA + 20-mg/L TMP group, 45.167％ ± 5.502％ in the UVA + 50-mg/L TMP group, 43.000％ ± 2.000％ in the UVA + 100-mg/L TMP group, 33.667％ ± 5.865％ in the control group, and 76.000％ ± 6.557％ in the P55 group, with significant differences among these groups (F =45.918, P ＜ 0.01).Furthermore, the UVA group significantly differed from the UVA + TMP groups and control group in the percentage of β-galactosidase-positive HDFs and mRNA expressions of MMP-1 and MMP-3 (all P ＜ 0.05).Conclusion TMP can protect HDFs against senescence induced by repetitive UVA radiation, and down-regulate the mRNA expressions of MMP-1 and MMP-3 during senescence.
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Objective To screen for genes associated with low-dose UVA irradiation-induced adaptation reaction in cultured human melanocytes, and to explore the molecular mechanism of the adaptation.Methods Cultured human melanocytes of fifth to tenth passage were divided into two groups, experimental group was subjected to an irradiation with UVA at 7.2 J/cm2 once daily for 4 times and an additional irradiation at a lethal dose of 86.4 J/cm2 6 hours after the above 4-session irradiation, and control group subjected to a single irradiation with UVA at 86.4 J/cm2. A human genome-wide oligonucleotide chip was used to screen for differentially expressed genes between the two groups of cells followed by a functional classification based on international standard. Moreover, a part of these genes were analyzed and identified by real-time fluorescent quantitative PCR. Results In the adaptation reaction induced by low-dose UVA irradiation in cultured melanocytes, there were 129 differentially expressed genes, including 81 up-regulated genes and 48 down-regulated genes. These genes were found to be mainly involved in metabolism, transport, signal transduction, apoptosis, DNA synthesis and repair, and some of them were oncogenes or anti-oncogenes. Real-time PCR confirmed some of the differentially expressed genes. Conclusions The whole genome-wide oligonucleotide chip could screen with high efficiency for differentially expressed genes in low-dose UVA irradiation-induced adaptation reaction in cultured melanocytes.
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Aim To design and synthesize new phenyloxyisobutyric acid analogues as antidiabetic compounds. Methods Eight new target compounds were synthesized by combination of lipophilic moieties and acidic moiety with nucleophilic replacement or Mitsunobu condensation. The eight compounds were confirmed by 1H NMR, 13C NMR, IR and MS. Results In vitro insulin-sensitizing activity (3T3-L1adipocyte) demonstrated, that the cultured glucose concentration of up-clear solution detected with GODpioglitazone, compounds A and B were added to the insulin-resistant system. Conclusion In vitro insulin-sensitizing activity of target compound A is in between that of rosiglitazone and pioglitazone, and activity of target compound B is slightly less than that of pioglitazone.
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Objective To investigate the influences of photochemotherapy with psoralen and ultraviolet A (PUVA) on skin photoaging and its possible mechanism. Methods HE stain, Verhoeff stain, electron microscopy, enzyme cytochemistry and immunocytochemistry were used to study influences of PUVA on the skin photoaging with characteristic biological markers in the non-lesion back skin of patients with psoriasis vulgaris. Results After treatment with PUVA, the degenerated collagen and elastic fibers were increased with derangement profile in dermis, and fibroblasts displayed growth suppression and morphological changes of cell senescence with a permanent switch of mitotic to stably postmitotic phenotypes, of group B and group A, the positive rates of SA-?-Gal were 13.6 % and 0.00 % and the positive rates of p16 protein were 81.8 % and 42.9 % respectively, there were significant differences between group B and group A(? 2=21.412, P
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<p><b>OBJECTIVE</b>To investigate the relationships between skin photoaging and point mutations in mitochondrial DNA (mtDNA) control region for replication of dermal fibroblast.</p><p><b>METHODS</b>Cultured dermal fibroblasts were treated by 8-methoxypsora len /ultraviolet A (8-MOP/UVA). mtDNA was extracted by one-step-method and th e PCR products of D-loop and adjacent transcription promoter (DLP(6)) fragment of mtDNA control region for replication were detected by polymerase chain reaction-single strain conformation polymorphism and direct sequencing.</p><p><b>RESULTS</b>After treated by 8-MOP/UVA, point mutations of 414 T-->G of DLP(6) fragment of mtDNA control region for replication largely accumulated.</p><p><b>CONCLUSION</b>Accumulation of point mutations of DLP(6) fragment of mtDNA control region for replication may be closely associated with skin photoaging.</p>
Subject(s)
Adult , Humans , Male , Binding Sites , Cells, Cultured , DNA Replication , DNA, Mitochondrial , Radiation Effects , Dermis , Cell Biology , Fibroblasts , Cell Biology , Radiation Effects , Methoxsalen , Pharmacology , Photosensitizing Agents , Pharmacology , Point Mutation , Ultraviolet RaysABSTRACT
Objective: To investigate reversal of drug resistance in human ovarian cancer cells by c-jun antisense. Methods: A c-jun antisense was applied to treat resistant and sensitive A2780 cell lines, and to observe the expression levels of c-jun, ?-GCS and GSH in two cell lines. Results: c-jun antisense inhibit c-jun gene expression in resistance cell lines . The mRNA level of the key enzyme in GSH synthesis, ?-glutamyl cysteine synthetase, was also reduced. The GSH content in resistant cells was dropped about 75 % . MTT analysis show that the resistant cells IC_(50) to cisplatin was dropped from 40 ?mol/L to 1.0 ?mol/L after a c-jun antisense treatment. No significant effect was observed in senstive cells (0.2 ?mol/L). Conclusion: A c-jun antisense can inhibit its gene expression in cells, and GSH synthesis in resistant cell was also inhibited. The resistant cells could be reversed to the level of sensitive cells.
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Objective To determine the efficacy and safety of the 308nm excimer laser in the treatment of vitiligo. Methods A Self-controlled study was conducted. The vitiligo lesions in stable stage of 75 patients were treated twice a week by the 308 nm excimer laser for 6 weeks. The efficacy and factors related to efficacy were evaluated 3 days later after the final treatment. Results No improvement was observed in any of the untreated vitiligo lesions. However, of the treated lesions, 6 completely disappeared, 33 obtained significant improvement, 30 moderate improvement, 6 no improvement. The effective rate was 92.0% and the markedly effective rate was 52.0%. The lesions on the face and neck had a better response to the treatment than those on the trunk or limbs, and the latter responded better than those on the joints of extremities. Conclusion The 308 nm excimer laser is safe and effective in treatment of vitiligo in stable stage and the efficacy is related to the anatomic sites.
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Objective To construct recombinant adenovirus vector containing mouse CD40Ig fusion gene for the study of induction of donor-specific tolerance. Methods CD40Ig fusion gene was constructed by PCR overlapping technique, and was cloned into the shuttle plasmid pAdTtrack-CMV. The linearized shuttle plasmid was co-transfected into the E.coli strain BJ5183 with bone plasmid pAdeasy1, then the recombinant adenovirus plasmid was generated. The adenovirus was packaged and amplified in Cells 293. Results The recombinant virus AdCD40Ig was successfully constructed and its titer was 5?109 efu/ml. Conclusion AdCD40Ig can be used as an agent in experiment to induce donor-specific tolerance.
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Objective To investigate the relationship between expression of matrix metalloproteinases of dermal fibroblasts and wrinkle formation in photoaging skin.Methods In situ hybridization histochemistry and immunocytochemistry were used to detect the expression of matrix metalloproteinases MMP-1,MMP-3mRNA and the expression of tissue inhibitor of MMP-1at(TIMP-1)protein in dermal fibroblasts from non-lesional skin of psoriatic patients during and after PUVA treatment.Results The expression of MMP-1and MMP-3mRNA of dermal fibroblasts from non-lesional skin was persistently up-regulated during PUVA thera-py,and lasted more than6months after treatment;while the expression of TIMP-1protein was only slightly expressed for a short period during PUVA therapy.Conclusion Wrinkle formation in photoaging skin after PUVA therapy is correlated with the imbalance of expression of matrix metalloproteinases and their inhibitors of dermal fibroblasts.
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AIM: To examine the quality of Radix Astragali Injection from different manufacturers. METHODS: The HPLC-ELSD method for the determination of astragaloside Ⅳ in Radix Astragali Injection was established with hypersil ODS 2 C 18 column(250mm?4.6mm,5?m), column temperature at 40℃. The mobile phase was acetonitrile-water (36∶64) and flow rate was at 1.0mL?min -1. The evaporation light-scattering detector was used with its drift tube temperature at 100℃ and the gas flow rate: 3.0L?min -1. RESULTS: Within 2.4～12?g astragaioside Ⅳ had a good linearity. The average recovery was 101.6%, RSD=2.9%,n=5. By analyzing the samples of 10 batch of Radix Astragali Injection from 5 manufactuers, it showed there were significant difference in the contents of astragaloside Ⅳ. CONCLUSION: The method is simple and fast. The results are accurate and reproducible and can be used in the quality control of Radix Astragali Injection. The significant differences in the astragaloside content may influence clinical treatmem.
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Objective:To study the protective effect of Huangqi Injection on cardiotoxicity induced by doxorubicin (DOX) and its mechanism. Methods: The molders of Dox-induced myocardial mitochondria damage of rat in vitro and Dox-induced cardiotoxicity in mice were used. The protective effect of Huangqi Injection on cardiotoxicity induced by doxorubicin was determined by biochemical method.Results: Doxorubicin can increse malondialdehyde level, induce mitochondrin swelling and decrease glutathione (GSH) content of myocardial mitochondria of rat in vitro, while all these damages caused by doxorubicin were reduced significantly by Huangqi Injection. Cardiotoxicity of doxorubicin in mice as measured by increases of myocardial malondialdehyde level and serum creatine phosphokinase activity, decreases of superoxide dimutase was significantly alleviated by Huangqi Injection. Conclusions: Huangqi Injection can protect heart against Dox-induced cardiotoxicity, which provides experimental evidence for Huangqi Injection as an anti-tumor adjuvant drug in clinical application.