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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.
RÉSUMÉ
Avermectins can affect biological processes of multiple tumor, including tumor cell proliferation and metastasis, cell cycle arrest, induction of apoptosis and autophagy, regulation of tumor microenvironment and tumor stem cells. Avermectins can be administered alone or combined with chemotherapeutic drugs to reverse multidrug resistance. To further explore the anti-tumor mechanism of avermectins will provide reliable experimental and theoretical guidance for future clinical application.
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Objective To evaluate the protective effect of pterostilbene against ultraviolet B (UVB)-induced acute damage in HaCaT cells,and to explore related mechanisms.Methods The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo1ium (MTS) assay and flow cytometry were performed to estimate the proliferative activity and the apoptosis and necrosis rate of HaCaT cells treated with different concentrations of pterostilbene respectively,so as to screen the non-toxic concentration of pterostilbene.HaCaT cells were randomly divided into several groups:normal control group receiving no treatment,UVB group irradiated with 57 mJ/cm2 UVB,3 pterostilbene groups treated with 2.44,4.88 and 9.75 μmol/L pterostilbene respectively for 24 hours,3 pterostilbene + UVB groups treated with 2.44,4.88 and 9.75 μmol/L pterostilbene respectively for 24 hours followed by UVB radiation.Western blot analysis was conducted to detect changes of the transcription factor NF-E2-related factor 2 (Nrf2) expression in cell nuclei and cytoplasm before and after the treatment with pterostilbene and UVB,quantitative PCR to determine the mRNA expression of catalase and superoxide dismutase in the HaCaT cells,and enzyme-linked immunosorbent assay (ELISA) to evaluate the activity of catalase and superoxide dismutase.Results MTS assay and flow cytometry showed that 2.44,4.88 and 9.75 μmol/L pterostilbene had non-toxic effect on HaCaT cells.The protein expression of Nrf2 in the nuclei and cytoplasm in the normal control group was 1.03 ± 0.08 and 1.04 ± 0.11 respectively.Compared with the normal control group,the protein expression of Nrf2 in the nuclei and cytoplasm experienced no significant changes in the 2.44-,4.88-and 9.75-μmol/L pterostilbene groups,and the UVB group showed similar protein expression of Nrf2 in the cytoplasm,but significantly increased protein expression of Nrf2 in the nuclei (1.77 ± 0.08,q =17.24,P ＜ 0.01).Compared with the normal control group and UVB group,the 2.44-,4.88-and 9.75-μmol/L pterostilbene + UVB groups all showed significantly lower protein expression of Nrf2 in the cytoplasm (0.86 ± 0.10,0.87 ± 0.11 and 0.46 ± 0.11 respectively,all P ＜ 0.05),but significantly higher protein expression of Nrf2 in the nuclei (2.38 ± 0.11,2.57 ± 0.11 and 2.07 ± 0.13,all P ＜ 0.01).As qPCR showed,UVB radiation could significantly inhibit the mRNA expression of CAT (P ＜ 0.05),but had no obvious effect on the mRNA expression of SOD (P ＞ 0.05).The mRNA expression of CAT and SOD experienced no significant changes in the 2.44-,4.88-and 9.75-μmol/L pterostilbene groups compared with the normal control group (P ＞ 0.05).However,2.44,4.88 and 9.75 μmol/L pterostilbene could significantly reduce the inhibitory effect of UVB radiation on the mRNA expression of CAT (P ＜ 0.05) and up-regulate the mRNA expression of SOD in the pterostilbene + UVB groups (P ＜ 0.05).ELISA revealed that UVB radiation could inhibit the activity of CAT and SOD in the HaCaT cells (both P ＜ 0.001),while 2.44,4.88 and 9.75 μmol/L pterostilbene could reduce the inhibitory effect of UVB radiation on the activity of CAT and SOD (all P ＜ 0.05).However,the activity of CAT and SOD were still lower in the 2.44-,4.88-and 9.75-μmol/L pterostilbene + UVB groups than in the normal control group (P ＜ 0.05).Conclusion Pterostilbene can prevent UVB-induced acute damage in HaCaT cells by activating the Nrf2 pathway and up-regulating the expression of the downstream antioxidant enzymes CAT and SOD.
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Objective To explore the factors associated with the rehabilitation outcome of lower limbs training with MOTOmed, such as the height and distance of seats, the crank length and the speed, as well as the height of patients. Methods The human lower limb model was simplified into a model of the bars. The activity of knee was as a dependent variable, and the individual body height, distance (S) and height (H) of seats, the crank length (L1) and speed (ω1 ) were as the independent variables, and analysed with control variate method and MAT-LAB simulation analysis. Results Controlling the individual body height, all the other factors influenced the knee joint angle, angular veloci-ty and angular acceleration value (initial or the end value). Among these factors, L1 impacted the mobility mostly, and less as S and H; ω1 mainly impacted the angular velocity and acceleration. Controlling the other factors as S=550 mm, H=165 mm, L1=180 mm, ω1=10 r/s, the higher the body height would be, the less the knee joint activities, angular velocity and angular acceleration are. Conclusion It is necessary to adjust the height and distance of seats, crank length and the speed to meet the individual body height when training with MOTOmed for lower limbs rehabilitation.