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Objective To detect the role of miR-34a on macrophage inflammation and polarization under high glucose conditions.Methods Mouse macrophages were collected and cultured during high glucose for 3－28 days.RT-qPCR was used to detect the expression of miR-34a,iNOS,MCP-1 and Arg-1 mRNA.Then miR-34a was over-expressed or silenced,ELISA was used to detect the expression of IL-6,IL-1β,TNF-α and qRT-PCR was used to detect the expression of iNOS and MCP-1 mRNA.Western blot was used to detect the expres-sion of Notch1.Results Expression of miR-34a increased under high glucose conditions in RAW264.7 cells continuously.Over-expression of miR-34a promoted the expression of MCP-1 and iNOS observed by RT-qPCR and increased the expression of IL-6,IL-1β and TNF-α detected by ELISA.Further studies showed that siRNA-Notch1 down-regulated the expression of miR-34a,MCP-1,iNOS,IL-6,IL-1β and TNF-α.Conclusions Chronic high glucose condition stimulates the expression of miR-34a which promotes M1 macrophage polarization and releasing of pro-inflammatory factors.
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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.
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ABSTRACT:Objective To investigate the effects of ω-3 polyunsaturated fatty acids on the proliferation of human gastric cancer cell line AGS and the possible mechanisms.Methods Human gastric cancer line AGS and human microvascular epithelial cell HMEC-1 were treated with different concentrations of docosdhexaenoic acid (DHA)and eicosapentaenoic acid (EPA).The inhibition of cell proliferation was evaluated by MTT assay and cell morphology.Flow cytometry was used to detect the cell cycle change.The expressions of mitochondrial respiratory membrane protein complex Ⅰ,Ⅱ and V were analyzed with Western blot.Results DHA and EPA could markedly inhibit the proliferation of AGS in significant time-dependent and concentration-dependent manners (P 0.05).Conclusion ω-3 PUFAs can selectively inhibit the growth and proliferation of human gastric cancer cell line AGS.These effects may be as-sociated with arresting cell cycle in G0/G1 phase and inhibiting the energy metabolism of AGS cells.
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Objective To investigate the inhibitory effects of docosahexaenoic acid (DHA)and 5-fluorouracil (5-FU)in combination on human gastric cancer cell line AGS in vitro .Methods Human gastric cancer line AGS was treated with different concentrations of DHA and 5-FU alone or in combination.The inhibition of cell proliferation was evaluated by MTT assay.Dose of median (IC50 )of drugs (alone or in combination)and the combination index (CI)were calculated using the median-effect equation and the combination index equation of Chou-Talalay.Flow cytometry was used to detect the cell cycle distribution.The expression of mitochondrial respiratory membrane protein complex in AGS cells was analyzed with Western blot.Results DHA and 5-FU alone or in combination could markedly suppress the proliferation of AGS in significantly time-dependent and dose-dependent manners (P <0.05).IC50 values with DHA or 5-FU administered for 24 h and 48 h were 5 1.60 μg/mL (DHA:24 h),34.82 μg/mL (DHA:48 h),45.90 μg/mL (5-FU:24 h),and 1 6.86 μg/mL (5-FU:48 h), respectively.DHA remarkably strengthened the inhibitory effect of 5-FU and decreased IC50 of 5-FU by 3.56 -2.1 5 folds.The combination of DHA and 5-FU showed synergism.Flow cytometry showed that AGS cells treated with DHA and 5-FU were arrested in G0/G1 phase and the proportion of AGS cells in G0/G1 phase increased compared with that in the control group,DHA group and 5-FU group,while the proportion of the cells in S phase decreased significantly (P < 0.05 ).Western blot showed after treatment with DHA and 5-FU for 48 h,the expression of mitochondrial respiratory membrane protein complex was significantly decreased compared with control group,DHA group and 5-FU group (P <0.05).Conclusion DHA could act synergistically with 5-FU in inhibiting the growth of gastric carcinoma cells,and meanwhile decrease the dose of 5-FU.The mechanism may be associated with cell cycle arrest in G0/G1 phase and interference in the energy metabolism of AGS cells due to inhibition of the expression of mitochondrial oxidative respiratory chain complexes by the two compounds.