BACKGROUND:Forskolin,a diterpenoid natural compound extracted from Coleus forskohlii,has a crucial regulatory role in skeletal muscle repair.However,the regulatory role of forskolin on myogenic differentiation of C2C12 skeletal muscle cells has not been fully explored.OBJECTIVE:To explore the effects of forskolin on the differentiation of C2C12 myoblast cell line and probe into the underlying molecular mechanisms.METHODS:C2C12 cells were treated with 0,0.1,0.25,0.5,1,5,10 and 20 μmol/L forskolin during growth,and cell proliferation was detected by cell counting kit-8 and qRT-PCR.C2C12 cells were treated with 0,0.25,0.5 and 1 μmol/L forskolin during the induction of myogenic differentiation.Immunofluorescence staining and qRT-PCR were used to detect C2C12 cells differentiation.Western blot was used to detect the expression level of myogenic differentiation-related signaling pathway proteins.RESULTS AND CONCLUSION:(1)The viability of C2C12 cells was decreased and cell proliferation was inhibited after treatment with high concentrations(＞1 μmol/L)of forskolin.(2)The qRT-PCR results showed that forskolin up-regulated the expression of Myh2,Myh4,Myomaker,but down-regulated the expression of Myh7 compared with the 0 μmol/L group,when C2C12 cells were differentiated for 4 days.Immunofluorescence staining results showed that the fusion index and myotube diameter of C2C12 cells were increased after forskolin treatment,and the number of myotubes was also increased.(3)Western blot results showed that the phosphorylated extracellular signal-regulated kinase 1/2 expression was inhibited;however,the phosphorylated protein kinase B was promoted after treatment with forskolin.The protein expression level of the myogenic differentiation transcription factor Myogenin was significantly up-regulated after treatment with forskolin.The above results demonstrate that forskolin may promote myogenic differentiation of C2C12 skeletal muscle cells through the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathway.

BACKGROUND:Forskolin,a diterpenoid natural compound extracted from Coleus forskohlii,has a crucial regulatory role in skeletal muscle repair.However,the regulatory role of forskolin on myogenic differentiation of C2C12 skeletal muscle cells has not been fully explored.OBJECTIVE:To explore the effects of forskolin on the differentiation of C2C12 myoblast cell line and probe into the underlying molecular mechanisms.METHODS:C2C12 cells were treated with 0,0.1,0.25,0.5,1,5,10 and 20 μmol/L forskolin during growth,and cell proliferation was detected by cell counting kit-8 and qRT-PCR.C2C12 cells were treated with 0,0.25,0.5 and 1 μmol/L forskolin during the induction of myogenic differentiation.Immunofluorescence staining and qRT-PCR were used to detect C2C12 cells differentiation.Western blot was used to detect the expression level of myogenic differentiation-related signaling pathway proteins.RESULTS AND CONCLUSION:(1)The viability of C2C12 cells was decreased and cell proliferation was inhibited after treatment with high concentrations(＞1 μmol/L)of forskolin.(2)The qRT-PCR results showed that forskolin up-regulated the expression of Myh2,Myh4,Myomaker,but down-regulated the expression of Myh7 compared with the 0 μmol/L group,when C2C12 cells were differentiated for 4 days.Immunofluorescence staining results showed that the fusion index and myotube diameter of C2C12 cells were increased after forskolin treatment,and the number of myotubes was also increased.(3)Western blot results showed that the phosphorylated extracellular signal-regulated kinase 1/2 expression was inhibited;however,the phosphorylated protein kinase B was promoted after treatment with forskolin.The protein expression level of the myogenic differentiation transcription factor Myogenin was significantly up-regulated after treatment with forskolin.The above results demonstrate that forskolin may promote myogenic differentiation of C2C12 skeletal muscle cells through the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathway.

Objective:To investigate the mechanism of neurofibromin 1 (NF1) in gallbla-dder cancer.Methods:The experimental study was conducted. Human gallbladder cancer cell lines, including GBC-SD, NOZ, SGC996, EH-GB1, ZJU0428, human embryonic kidneys cell line 293T and human cervical cancer cell line HELA, were cultured. The recombinant plasmids (mRFP-YAP1 FL-FLAG and eGFP-MYC-NF1 2650?2750-HA) were constructed for co-immunoprecipitation experiment. The truncated Yes associated protein 1(YAP1) and NF1 recombinant proteins were purified in vitro. The interaction between NF1 and YAP1 in vitro or in vivo were verified by isothermal titration calori-metry (ITC) assay, GST pull-down experiment, co-immunoprecipitation, immunofluorescence, laser confocal microscopy, and the expression of NF1 protein in different gallbladder cancer cell lines was verified by Western blot experiments. Observation indicators: (1) interaction between NF1 and YAP1 in vitro; (2) interaction between NF1 and YAP1 in cells; (3) expression of NF1 protein in different human gallbladder cancer cell lines. The dissociation constants were exported from ITC 200 software and represented as Mean± SD. Count data were represented as absolute numbers. Results:(1) Interaction between NF1 and YAP1 in vitro. ① Results of ITC assay showed that there was interac-tion between PPQY and YAP1-WW1, between PPQY and YAP1 (Amino acid residues 162?275), and the dissociation constants between PPQY and YAP1-WW1, between PPQY and YAP1(Amino acid residues 162?275) were (0.42±0.06)mmol/L, (0.69±0.14)mmol/L, respectively. ② GST pull-down results indicated that the target protein His-Sumo-YAP1 WW1 was obviouly observed in protein lane of reaction system between GST-PPQY recombinant protein and His-Sumo-YAP1 WW1, relative to the reaction system between GST protein and His-Sumo-YAP1 WW1. The target protein His-Sumo-YAP1 WW2 was obviouly observed in protein lane of reaction system between GST-PPQY recombinant protein and His-Sumo-YAP1 WW2, relative to the reaction system between GST protein and His-Sumo-YAP1 WW2. (2) Interaction between NF1 and YAP1 in cells. ① Co-immunoprecipitation results indica-ted that NF1 protein was observed in cell lysis solution which was incubated by FLAG gel beads and cotransfected with mRFP-YAP1 FL-FLAG and eGFP-MYC-NF1 2650?2750-HA. ② Immuno-fluorescence and laser confocal microscopy results indicated that YAP1 and NF1 with obvious fluorescence were co-localized in the cytoplasm of human gallbladder cancer NOZ cells. However, YAP1 with obvious fluorescence was localized in the nucleus of human gallbladder SGC996 cells and NF1 showed weak fluorescence. (3) Expression of NF1 protein in different human gallbladder cancer cell lines. Western blot results showed that with the expression level of NF1 protein in HELA cell line as the standard, the relative expression levels of NF1 protein in EH-GB1, GBC-SD, NOZ, SGC996, ZJU0428 cell lines were 1.28, 0, 1.01, 0, 0, respectively. Conclusion:NF1 affects the gallbladder cancer by directly acting on YAP1 protein.

Objective:To investigate the correlation between activities of daily living and hemoglobin level in patients with acute ischemic stroke (AIS).Methods:A retrospective analysis was performed in 190 patients with acute ischemic stroke from August 2015 to April 2017. The hemoglobin (Hb) levels at admission were analyzed . According to the Barthel index (BI) score, the patients were divided into good improvement group ( n=119) and poor improvement group ( n=71). Spearman correlation analysis was used to study the relationship between hemoglobin level and short-term activities of daily living in patients with acute ischemic stroke. Results:(1)The short-term activities of daily living was improved in 119 patients. Among them, 62 (52.1%) were under 65 years old and 57 (47.9%) were over 65 years old, 9 (7.56%) male patients with hemoglobin <120 g/L and 39 (32.77%) male patients with hemoglobin > 150 g/L. There were 71 patients with poor short-term activities of daily living. Among them, 23(32.39%)were under 65 years old and 48(67.61%)were over 65 years old, 1(1.41%)male patients with hemoglobin <120 g/L and 12(16.9%) male patients with hemoglobin > 150 g/L. There were significant differences in age (χ 2= 6.985, P=0.008) and male hemoglobin level (χ 2= 8.069, P=0.005) between patients with good activities of daily living and patients with poor activities of daily living.(2)Regression analysis showed that age > 65 years ( β =1.386, OR=4.000, 95% CI=1.189-3.461, P=0.025) and abnormal hemoglobin levels in men (Hb<120 g/L or > 150 g/L) ( β =1.089, OR=2.972, 95% CI=1.383-6.388, P=0.005) were the influencing factors of poor short-term quality of life in patients with acute ischemic stroke.(3)Spearman correlation analysis showed that abnormal hemoglobin levels in men (Hb<120 g/L or>150 g/L) were positively correlated with poor short-term activities of daily living in patients with acute ischemic stroke( r=0.244, P=0.004). Conclusion:Abnormal hemoglobin level is associated with poor short-term activities of daily living improvement in patients with acute ischemic stroke.

Objective:To investigate the correlation between serum 25-hydroxyvitamin D (serum 25(OH)D) and Meige syndrome.Methods:A retrospective analysis was performed on 47 patients with Meige syndrome (Meige syndrome group) treated in Yuquan Hospital of Tsinghua University admitted from August 2012 to July 2018 in our hospital.In the same period, 69 healthy people of the same age group were selected as the healthy control group.The difference of serum 25(OH)D concentration among different subtypes of Meige syndrome (type I, II, III) was compared.Logistic regression analysis was used to analyze the correlation between serum 25(OH)D level and Meige syndrome.Results:The serum 25(OH)D concentration in patients with Meige syndrome was (12.68±6.77) μg/L, which was significantly lower than that in the healthy control group ((17.93±6.93) μg/L). The difference was statistically significant ( t=4.044, P<0.001). The serum 25(OH)D concentrations of subtypes I, II and III in patients with Meige syndrome were (14.7±8.14), (11.4±5.02), (8.38±4.99) μg/L, respectively. There was no significant difference among the three types ( F=1.892, P=0.231). Logistic regression results showed that serum 25(OH)D levels were correlated with Meige syndrome ( OR=0.938, 95% CI: 0.885-0.995, P=0.034). Conclusion:The serum 25(OH)D expression level in patients with Meige syndrome is low, suggesting that vitamin D deficiency may be involved in the pathogenesis of Meige syndrome.

OBJECTIVE：To evaluate in vitro antioxidant activities of 4 different polar parts of ethanol extract from Amomum tsao-ko，and to lay a foundation for the research and development of antioxidant chemical components in the plant. METHODS ： The dried fruits of A. tsao-ko were crushed ，then were hearted and reflux extracted with 95％ ethanol. The extraction fluid was concentrated by rotary evaporation and evaporated in water bath to obtain the ethanol extract. The extract was dispersed in water ， and then extracted with petroleum ether ，ethyl acetate and n-butanol organic solvents one by one. Each solvent extract was combined and the lower water phase were collected. Finally ，the petroleum ether part ，ethyl acetate part ，n-butanol part and water part were obtained，after rotary evaporation concentration and water bath evaporation. Through in vitro antioxidant activity tests ，using 2， 6-di-tert-butyl-4-methylphenol（BHT）as positive control ，DPPH radical ，superoxide anion radical scavenging ability and Fe 3+ reducing ability of different polar parts of ethanol extract from A. tsao-ko were investigated. RESULTS ：The scavenging rates of 4 polar parts of ethanol extract from A. tsao-ko on DPPH radical were all over 80％；the order of scavenging ability was ethyl acetate part＞BHT＞n-butanol part ＞petroleum ether part ＞water part. Those of the 4 polar parts to superoxide anion radical were between about 30％-40％ mostly；the order of scavenging ability was n-butanol part ＞petroleum ether part ＞water part ＞ethyl acetate part ＞ BHT；but those were weaker than their scavenging ability to DPPH r adical. The polar parts of ethanol extract also had a certain reduction ability to Fe 3+；the order of the reduction ability was n-butanol part ＞BHT＞ethyl acetate part ＞petroleum ether part ＞ water part on the whole ，but that of water part rose to the stron- gest when its concentration was 4.0 μg/mL. CONCLUSIONS： The different polar parts of ethanol extract from A. tsao-kom have certain in vitro antioxidant capacity ，but the order of antioxidant activity of different polar parts was not the same in different antioxidant activity tests ；ethyl acetate part has the 163.com strongest scave nging effect on DPPH radical ，n-butanol part has the strongest scavenging ability on superoxide anion radical and reducing ability on Fe 3+.

Objective To explore the effects of husband and wife joint education model on marriage adjustment and birth and quality of life in female patients with infertility. Methods Totally 360 sterile females who received In Vitro Fertilization-Embryo Transfer (IVF-ET) in the Fertility Dpartment of a Class Ⅲ Grade A hospital in Wenzhou were selected using convenient sampling from June to August 2018. The study subjects were randomly divided into intervention (n=180) and control (n=180) groups. The conventional nursing method was used in the control group, and the husband and wife joint education model was applied in the intervention group after the conventional nursing. On the basis of transplantation, the dyadic adjustment scale and birth and quality of life scale were used before the patients were discharged to compare the result of marriage adjustment and birth and quality of life in the two groups. Results The martial adjustment score and birth and quality of life score in the intervention group scored (114.56±20.64) and (68.05±15.18) respectively, both higher than those of the control group, and there was statistically significant difference between the two groups (P＜0.05). Conclusions Husband and wife joint education model can not only promote the intimate relationship between patients and their spouse, but also improve their quality of life, which is worth applying in the clinical care of patients with infertility.

Objective To investigate the influence of inhibited gene expression of fisson 1 (Fis1) gene on the level of Fis1,mitofusin 1 (Mfn1) and mitochondrial membrane potential in SH-SY5Y cells with fluorine,to study the role of mitochondrial dynamic balance in the pathogenesis of chronic fluorosis.Methods SH-SY5Y cells were cultured in vitro,when adherent cells entered the logarithmic phase,using a group design,they were divided into four groups:blank control group (control),fluoride group [2 mmol/L sodium fluoride (NaF)],fluoride negative control group (2 mmol/L NaF + non-specific siRNA) and the gene-silencing group (2 mmol/L NaF + specific siRNA-Fis1).The protein expression levels of Fis1 and Mfn1 were measured by Western blotting;the mRNA expression levels of Fis1 and Mfn1 were measured by Real-time PCR;and the levels of the mitochondrial membrane potential was detected by mitochondrial membrane potential detection kit.Results Compared with control (1.37 ± 0.18,1.00 ± 0.04;1.57 ± 0.19,1.00 ± 0.04;1.00 ± 0.10),the expression levels of Fisl protein (1.72 ± 0.04) and mRNA (1.48 ± 0.13) in fluoride group were increased,the expression levels of Mfn1 protein (0.87 ± 0.02) and mRNA (0.69 ± 0.07) in fluoride group were decreased,the level of mitochondrial membrane potential (0.76 ± 0.13) was decreased (P ＜ 0.05).Compared with control,the expression levels of Fis1 protein (0.79 ± 0.07) and mRNA (0.06 ± 0.03) in gene-silencing group were decreased,the expression levels of Mfn1 protein (1.71 ± 0.04) and mRNA (1.52 ± 0.05) in gene-silencing group were increased (P ＜ 0.05),the level of mitochondrial membrane potential (0.94 ± 0.01) was decreased.Compared with fluoride group,the expression levels of Fis1 protein and mRNA in gene-silencing group were decreased,the expression levels of Mfn1 protein and mRNA in gene-silencing group were increased,the level of mitochondrial membrane potential in gene-silencing group was increased (P ＜ 0.05).Conclusion Gene expression inhibition of Fis1 gene can reduce the mitochondrial division and damage of mitochondrial membrane potential in SH-SY5Y cells induced by fluoride.

OBJECTIVE To investigate the effect and underlying mechanism of Gypsophila elegans isoorientin on the proliferation and apoptosis of human HepG2 cells. METHODS HepG2 cells were treated with isoorientin 5,10,20,40,80 and 160μmol?L-1 for 24,48 and 72 h,respectively. Cell survival was analyzed by MTT assay. HepG2 cells were treated with isoorientin 5,10 and 20μmol?L-1 for 48 h before the lactate dehydrogenase(LDH)level was detected. After treatment with isoorientin for 24 h, the variation of reactive oxygen species (ROS) was monitored by a fluorescence probe H2DCF-DA. HepG2 apoptosis and mitochondria membrane potential(MMP)were evaluated by flow cytometry. The activi?ties of caspase 3,8 and 9 were determined by colorimetry. The mRNA expression of Bcl-2 and Bax was determined by RT-PCR,and the protein expression of Bcl-2, Bax and cytochrome c was detected by Western blotting. RESULTS Isoorientin(5-160μmol?L-1)inhibited HepG2 cell survial in a concen?tration-dependent manner,the 50%inhibitory concentration(IC50)was 62.7±9.1,47.2±11.4 and(18.2± 7.5)μmol?L-1 after treatment with isoorientin for 24,48 and 72 h,respectively. Compared with the cell control group,treatment with isoorientin 5,10 and 20μmol?L-1 significantly increased the LDH level and cell apoptosis rate(P<0.05). Moreover,isoorientin 10 and 20μmol?L-1 notably increased the production of ROS,decreased the MMP(P<0.05),and increased the activities of caspase 3 and 9. RT-PCR analysis and Western blotting showed that isoorientin significantly decreased the mRNA and protein expressions of Bcl-2,decreased the mRNA and protein expressions of Bax(P<0.05),and inhibited the protein expression of cytochrome c(P<0.05). CONCLUSION Isoorientin inhibits HepG2 cell prolif?eration,but promotes cell apoptosis,which is closely related to the regulation of the mitochondrial apoptosis pathway.

Aim To investigate the protective effect and mechanism of madecassoside from hydrocotyle sibthorpioides (MHS)on learning and memory impair-ment induced by D-galactose (D-gal)in mice.Meth-ods Totally 75 SPF Kunming male mice were divided into normal control group,model group,low-,middle-and high-doses of MHS treated groups.The dementia models were induced with D-gal.The learning and memory functions were tested by Morris water maze, the level of Aβ1 -42 and its related proteins in the hip-pocampus was determined by Western blot,and the ex-pression of Aβ-related genes were determined by RT-PCR.Results Compared with model group,MHS markedly decreased the content of Aβ1 -42 ,inhibited the expression of APP,BACE1 and CatB,but promo-ted the expression of NEP and IDE.In addition,AHS significantly increased the expression of plasticity-relat-ed proteins including PSD-95,p-NMDAR1,p-CaMKII, p-PKACβ,PKCγ,p-CREB and BDNF.Conclusions MHS could remarkably ameliorate the learning and memory impairment induced by D-gal in mice,which may be due to its ability to inhibit the Aβgeneration and deposition and promote synaptic plasticity related protein expression.