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.

Objective:To investigate the related genes, signaling pathways and possible mechanisms of malignant transformation of human papillomavirus type 16 (HPV-16) immortalized cervical epithelial cell line H8.Methods:The malignant transformed H8 cell model was constructed, and the changes of cell invasion ability and cell migration ability of H8 cells after malignant transformation were detected by Transwell assay, and the changes of clone formation ability of H8 cells after malignant transformation were detected by plate clone formation assay. Total RNA was extracted from malignant transformed H8 cells and H8 cells, and the two groups of cells were sequenced by transcriptome using Illumina novaseq 6000 sequencing platform, differentially expressed genes (DEGs) were identified and analyzed, and Gene Ontology (GO) function enrichment analysis, Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis and protein-protein interaction were performed.Results:The invasion ability, migration ability and clone formation ability of malignant transformed H8 cells significantly increased as compared to H8 cells. A total of 203 differentially expressed genes were identified in H8 cells before and after malignant transformation, of which 98 were up-regulated and 105 down-regulated. GO enrichment analysis showed that DEGs were mainly involved in biological processes such as cellular processes, biological regulation, and metabolic processes. KEGG pathway enrichment analysis showed that DEGs were mainly enriched in alanine, aspartate and glutamate metabolic pathway, glycine, serine and threonine metabolism pathway, p53 signaling pathway and TGF-β signaling pathway, PI3K-Akt signaling pathway. PPI analysis screened 10 hub genes including DDIT3, TRIB3 and ASNS.Conclusions:Compared with H8 cells, malignant transformed H8 cells have a large number of differentially expressed genes and pathways at the transcriptional level, which could further provide new ideas for the mechanism of malignant transformation and carcinogenesis as well as finding new targets for the prevention of malignant transformation.

Objective:To observe the effect of observing good swallowing on the swallowing action of stroke survivors with dysphagia.Methods:Eighteen stroke survivors with dysphagia were randomly divided into a treatment group ( n=9) and a control group ( n=9). In addition to routine swallowing rehabilitation therapy, the treatment group was asked to simulate swallowing after watching a video of normal people′s swallowing action. They did so 5 times a week for 10 minutes, while the control group just watched landscape videos at the same time. The treatment lasted 8 weeks. Before and after the treatment, both groups were assessed using the eating assessment tool (EAT-10), the functional oral intake scale (FOIS) and the penetration and aspiration scale (PAS). Functional magnetic resonance imaging (fMRI) was also used to observe their swallowing action. Results:There was no significant difference between the two groups in any of the measurements before the treatment. After the 8 weeks of treatment the average EAT-10, FOIS and PAS scores of the treatment group were all significantly better than before the treatment and better than the control group′s averages at the time. fMRI showed significantly more areas activated in the precuneus, parietal lobe, posterior central gyrus, BA7, BA5, frontal lobe and paracentral lobule in the treatment group compared with before the intervention and also more than in the control group.Conclusions:Observing proper swallowing action can improve dysphagia and activation of the swallowing-related brain areas of stroke survivors.

Objective To systematically evaluate the effects of mechanical chest compression (CC) combined with manual CC and single-manual CC on the outcome indexes of cardiopulmonary resuscitation (CPR) for patients with in-hospital cardiac arrest (IHCA). Methods The relevant publicly published literatures about the effects of mechanical CC combined with manual CC and single-manual CC on the outcome of CPR were searched by using the following Chinese keywords for retrieval: "cardiac arrest, asystole, sudden death, artificial recovery, artificial press, artificial CC, unarmed CPR, unarmed resuscitation, unarmed compressions, unarmed chest compressions, unarmed, artificial, resuscitation instrument, resuscitation machine, resuscitator, CPR, LUCAS, Autopulse, Thumper, MSCPR-1A"in databases such as China Biomedical Literature (CBM), VIP, Wanfang, and China National Knowledge Internet (CNKI) from their dates of foundation to March 11, 2019, and using the following key words in English "heart arrest, cardiac arrest, cardiopulmonary arrest, Cardiopulmonary Resuscitation, Resuscitation, Cardio-Pulmonary Resuscitation, CPR, compression, mechanical, automatic, automated, load distributing band, LBD, Autopulse, LUCAS" to retrieve all the published articles especially concerning the topics on the application effects of mechanical combined with manual CC for IHCA patients' CPR in the America National Library database (PubMed), Excerpta Medica (EMbase), Web of Science, and Cochrane Library from the establishment of the databases to March 11, 2019. The indexes of outcomes included return of spontaneous circulation (ROSC) rate, survival rate after hospital discharge and incidence of complications. The literatures were extracted independently by two reviewers, the qualities of the included randomized controlled trials (RCTs) were evaluated according to the Cochrane bias risk assessment tool, and the qualities of the included observational studies were evaluated according to the literature quality assessment form (NOS). Meta analysis was performed by using RevMan 5.3 software, and publication bias was assessed by using funnel plot. Results Twenty-one studies were enrolled, including 11 RCT articles and 10 observational studies; there were 2 005 participants. The results of this Meta-analysis showed that compared with manual CC, the ROSC rate and after discharge survival rate of IHCA patients were obviously higher in combined CC group [ROSC: odds ratio (OR) = 2.50, 95% confidence interval (95%CI) = 2.03-3.09, P < 0.000 01; discharge survival rate: OR = 2.71, 95%CI = 1.91-3.85, P < 0.000 01]; the incidence of complications of combined CC was lower than that in single manual CC (OR = 0.30, 95%CI = 0.13-0.68, P = 0.004). The funnel plots indicated that there was no apparent bias in the ROSC; because the enrolled studies were relatively few, it was difficult to evaluate the symmetrical characteristics of the funnel plots for discharge survival rate and the complication rate. Conclusions For IHCA patients, combined CC can improve ROSC, discharge survival rate, and reduce the occurrence of complications. It is suggested that during the actual rescue of IHCA patients, it is better to use combined CC, that is to say, manual CC should be adopted immediately in the early stage and then replace the mechanical CC device as soon as possible.

Objective To explore the effect of tea polyphenols on the growth of human papillomavirus 16 (HPV16) subgenes-immortalized human cervical epithelial cells (H8 cells).Methods Cultured H8 cells were divided into 5 groups to be treated with 0 (control group),6.25,12.5,25 and 50 mg/L tea polyphenols respectively for 24,36,and 48 hours,and then cell counting kit-8 (CCK8)assay was performed to detect cell proliferation.After 24 hours of incubation,flow cytometry was conducted to detect cell apoptosis and cell cycle,and fluorescence microscopy to observe the morphology of apoptotic cells.Results After incubation with tea polyphenols at different concentrations for 24,36 and 48 hours,the proliferation of H8 cells was inhibited,and 12.5 mg/L tea polyphenols could inhibit the relative growth rate of H8 cells in a time-dependent manner.Flow cytometry showed that there was a significant difference in cell apoptosis rate among the 6.25-,12.5-,25-,50-mg/L tea polyphenols groups and the control group (52.62％ ± 0.62％,52.22％ ± 0.72％,42.52％ ± 0.90％,45.96％ ± 2.11％,29.96％ ± 0.70％ respectively,F =272.0,P ＜ 0.05).Moreover,all the tea polyphenol groups showed significantly increased cell apoptosis rate compared with the control group (all P ＜ 0.05).Fluorescence microscopy showed karyopyknosis,nuclear fragmentation and other typical apoptotic morphological changes in H8 cells in tea polyphenols groups.There were significant differences in the percentage of cells in G1,G2 phase and cell proliferation index among the 5 groups (all P ＜ 0.05).Compared with the control group,the 6.25-,12.5-,25-mg/L tea polyphenols groups showed significantly increased percentage of cells in G1 phase (55.96％ ± 0.72％,54.12％ ± 3.20％,65.30％ ± 1.51％ respectively,all P ＜ 0.05),but significantly decreased percentage of cells in G2 phase (3.17 ± 1.82％,4.94 ± 1.46％,4.65 ± 4.26％ respectively,all P ＜ 0.05) and lower cell proliferation index(0.44 ± 0.01,0.46 ± 0.02,0.36 ± 0.01 respectively,all P ＜ 0.05).Conclusion Tea polyphenols can inhibit the proliferation of H8 cells,induce cell apoptosis,and block cell cycle progression.

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.

Objective To explore the effect of robot-assisted therapy on the shoulder joint proprioception of convalescent stroke survivors.Methods Forty stroke survivors were enrolled and randomized into an experimental group (n =20) and a control group (n =20).Both groups received routine drug treatment and rehabilitation,including the traditional kinesitherapy,occupational therapy and physical therapy,but the experimental group was additionally provided with 20 minutes of robot-assisted upper limb therapy 6 times a week for 8 weeks.Before the intervention and at 4 and 8 weeks the multi-joint system (MJS) upper limb proprioception test system was used to evaluate the average trace error and test execution time of the upper limb.Shoulder joint proprioception was measured at 30° and 60° in intorsion and extorsion using an isokinetic dynamometer.Results Before the training there were no significant differences between the two groups in terms of any of the assessments.After 4 and 8 weeks of training,significant improvement was observed in the measurements,and those of the experimental group were significantly better than those of the control group at the same time points.Conclusion Robot-assisted therapy can facilitate the recovery of shoulder joint proprioception after a stroke.It is worthy of application in clinical practice.

Objective To investigate the protective effect of lycium barbarum polysaccharide (LBP) on DNA damage of HSF cells induced by UV.Methods We established the model of UV induced photo damage in HSF cells.We detected the viability of HSF cells by using MTT colorimetry.The UV absorption spectrum of LBP was also measured by UV spectrophotometer.The level of ROS was detected by DCFH-DA fluorescent probe method.Comet assay was employed to evaluate the DNA strand breakage damage.Results When the concentration of LBP was less than or equal to 300μg/ml,there was no significant effect on the proliferation of HSF cells (P＞0.05).When the concentration was more than 300 μg/ml,it could inhibit the cell proliferative activities (P＜0.05).Compared to the UV groups,UV+LBP groups can respectively improve the cell proliferation activity (P＜0.05).The absorbance was slight range 280 from 400 nm.Compared with the UV group,the relative fluorescence intensity and the migration distance of UV+ LBP groups were significantly decreased (P＜0.05).Conclusions Lycium barbarum polysaccharide can effectively inhibit the proliferation activity and protect the breakage of DNA strand induced by UV,which is probably due to its action of removing free radicals.

Objective To evaluate the scavenging effect of crude polysaccharides extracted from Lycium barbarum (LBP) on reactive oxygen species in ultraviolet radiation-induced HaCaT cells,and to explore its possible mechanism.Methods Cultured immortalized human keratiuocyte HaCaT cells were divided into 6 groups:blank control group receiving no treatment,LBP group treated with crude LBP alone,ultraviolet A (UVA) group treated with UVA radiation alone,ultraviolet B (UVB) group treated with UVB radiation alone,UVA + LBP group treated with crude LBP for 24 hours followed by UVA radiation,and UVB + LBP group treated with crude LBP for 24 hours followed by UVB radiation.MTT colorimetry was performed to evaluate the cellular proliferative activity,UV spectrophotometric method to measure the UVA and UVB absorption of crude LBP,dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe assay to detect the level of ROS,enzymatic-biochemical method to estimate the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px),as well as to detect the leakage of lactate dehydrogenase (LDH).Results Crude LBP at different concentrations of 0,100,200,300,400,500,600,1 500,2 000 mg/L had no obvious effects on the proliferative activity of HaCaT cells.Crude LBP had a high transmittance of ultraviolet rays at 280-400 nm.Compared with the blank control group,the UVA group and UVB group both showed significantly higher LDH leakage and ROS level,lower activities of SOD and GSH-Px (P ＜ 0.001 or 0.05).Pretreatment with crude LBP before the ultraviolet radiation could significantly increase the activities of SOD and GSH-Px,decrease the LDH leakage and ROS level in the UVA + LBP group and UVB + LBP group compared with the UVA group or UVB group (P ＜ 0.05).Conclusion Crude LBP have no effect of sunscreening agents,but can effectively scavenge ROS,decrease LDH leakage,inhibit ultraviolet radiation-induced photodamage in HaCaT cells,which may be associated with the enhancement of antioxidant enzyme activity.

Objective To evaluate effects of tea polyphenols on the mRNA and nucleoprotein expression of Nrf2/Bach1 in human skin fibroblasts (HSFs).Methods Some HSFs were incubated with tea polyphenols at different concentrations of 0,2.5,5,10,20 and 40 mg/L for 24 hours.Methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate the proliferative activity of HSFs to screen the optimal concentration of tea polyphenols.Then,some other HSFs were treated with tea polyphenols at this optimal concentration for 24 hours.Real-time quantitative PCR (RT-qPCR) was performed to determine mRNA expression of Nrf2 and Bach1,Western blot analysis to measure nuclear expression of Nrf2 and Bach1 proteins,and immunofluorescence assay to determine the distribution of Nrf2 and Bach1 protein in the cell nucleus.Results MTT assay showed that 5 mg/L tea polyphenols had no obvious effects on the proliferation of HSFs,so 5 mg/L was chosen as the optimal concentration of tea polyphenols for subsequent experiments.HSFs cultured without tea polyphenols served as control group.After the treatment,the 5-mg/L tea polyphenol group showed significantly decreased mRNA and nuclear protein expression of Bach 1 (mRNA:0.629 ± 0.077 vs.0.940 ± 0.033,t =6.397,P ＜ 0.05;protein:1.424 ± 0.171 vs.16.966 ± 1.702,t =15.730,P ＜ 0.05),but significantly increased mRNA and nuclear protein expression of Nrf2 (mRNA:1.467 ± 0.076 vs.0.977 ± 0.091,t =7.133,P ＜ 0.05;protein:6.929 ± 0.121 vs.3.537 ± 0.126,t =33.636,P ＜ 0.05) compared with the control group.Immunofluorescence assay showed increased accumulation of Nrf2 protein,but decreased accumulation of Bach1 protein in the nucleus.Conclusion Tea polyphenols can promote the mRNA and nuclear protein expression as well as nuclear distribution of Nrf2,but suppress the mRNA and nuclear protein expression as well as nuclear distribution of Bach 1,finally exerting antioxidative effects.