Objective:To explore the effect of hairy and enhancer of split related protein 2 (Hey2) on osteoclast differentiation through the activation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1).Methods:RAW264.7 cells were induced with receptor activator of NF-κB ligand (RANKL) to differentiate into osteoclasts. Experimental groups were divided by different concentrations of RANKL (0, 10, 20, 50 μg/L) and different processing time (0, 3, 5, 7 days). Hey2 overexpression experiment was grouped as follows: blank control group, RANKL group, empty plasmid vector control group (Hey2-NC+RANKL), Hey2 overexpression group (Hey2-OE+RANKL); similarly, groups in Hey2 knockdown experiment were as follows: blank control group, RANKL group, negative control group (si-NC+RANKL), Hey2 knockdown group (si-Hey2+RANKL). Chromatin immunoprecipitation experiment groups were divided as non-specific IgG control group (IgG control group), non-specific IgG group (IgG RANKL group), Hey2-specific antibody control group (anti-Hey2 control group), Hey2-specific antibody group (anti-Hey2-RANKL group). For the different RANKL concentration groups and different induction time groups, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the mRNA expressions of nuclear factor of NFATc1, cathepsin K (CTSK), and cellular feline osteosarcoma oncogene (c-Fos) and tartrate-resistant acid phosphatase (TRAP) staining was used to assess the formation of multinucleated osteoclasts. After Hey2 overexpression or knockdown, RT-qPCR and Western blotting were used to detect the gene and protein expressions of NFATc1, c-Fos, and CTSK. TRAP staining was used to evaluate the formation of multinucleated osteoclasts. Bioinformatics prediction (NCBI, JASPAR) and chromatin immunoprecipitation (ChIP) assay were used to validate the binding of Hey2 to the NFATc1 promoter region.Results:During the osteoclastic differentiation of RAW 264.7 cells induced by RANKL, the expression of Hey2 could be detected, and the expression level of Hey2 decreased with the increase of RANKL concentration and induction time. In the 50 μg/L RANKL group, the expression levels of Hey2 gene (0.18±0.00) and protein (0.22±0.02) were significantly lower than those in the control group (1.00±0.00, 0.52±0.01) ( t=41.67, 12.88; both P<0.001). In the 50 μg/L RANKL group inducted for 5 days, the expression levels of Hey2 gene (0.27±0.02) and protein (0.79±0.01) were significantly lower than those in the control group (1.00±0.00, 1.15±0.02) ( t=11.47, 108.60; both P<0.001). Hey2 overexpression significantly reduced the gene and protein expressions of NFATc1, c-Fos, and CTSK, as well as the production of TRAP-positive cells (all P<0.05). Hey2 knockdown significantly increased the gene and protein expressions of NFATc1, c-Fos, and CTSK, as well as the production of TRAP-positive cells (all P<0.05). After inducing RAW264.7 cells with 50 μg/L RANKL for 1 day, ChIP results showed that among the two sample groups treated with Hey2 antibody, the detection level of the NFATc1 promoter region (-400 to -200 bp) in the anti-Hey2-RANKL group (18.06±0.06) was significantly higher than that in the anti-Hey2 control group (13.37±0.36) ( t=12.56, P<0.001). Conclusions:Hey2 can bind to the downstream target gene NFATc1 at -400 to -200 bp region of the promoter. As a transcriptional repressor, Hey2 inhibits osteoclast differentiation.

Objective:To explore the effect of hairy and enhancer of split related protein 2 (Hey2) on osteoclast differentiation through the activation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1).Methods:RAW264.7 cells were induced with receptor activator of NF-κB ligand (RANKL) to differentiate into osteoclasts. Experimental groups were divided by different concentrations of RANKL (0, 10, 20, 50 μg/L) and different processing time (0, 3, 5, 7 days). Hey2 overexpression experiment was grouped as follows: blank control group, RANKL group, empty plasmid vector control group (Hey2-NC+RANKL), Hey2 overexpression group (Hey2-OE+RANKL); similarly, groups in Hey2 knockdown experiment were as follows: blank control group, RANKL group, negative control group (si-NC+RANKL), Hey2 knockdown group (si-Hey2+RANKL). Chromatin immunoprecipitation experiment groups were divided as non-specific IgG control group (IgG control group), non-specific IgG group (IgG RANKL group), Hey2-specific antibody control group (anti-Hey2 control group), Hey2-specific antibody group (anti-Hey2-RANKL group). For the different RANKL concentration groups and different induction time groups, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the mRNA expressions of nuclear factor of NFATc1, cathepsin K (CTSK), and cellular feline osteosarcoma oncogene (c-Fos) and tartrate-resistant acid phosphatase (TRAP) staining was used to assess the formation of multinucleated osteoclasts. After Hey2 overexpression or knockdown, RT-qPCR and Western blotting were used to detect the gene and protein expressions of NFATc1, c-Fos, and CTSK. TRAP staining was used to evaluate the formation of multinucleated osteoclasts. Bioinformatics prediction (NCBI, JASPAR) and chromatin immunoprecipitation (ChIP) assay were used to validate the binding of Hey2 to the NFATc1 promoter region.Results:During the osteoclastic differentiation of RAW 264.7 cells induced by RANKL, the expression of Hey2 could be detected, and the expression level of Hey2 decreased with the increase of RANKL concentration and induction time. In the 50 μg/L RANKL group, the expression levels of Hey2 gene (0.18±0.00) and protein (0.22±0.02) were significantly lower than those in the control group (1.00±0.00, 0.52±0.01) ( t=41.67, 12.88; both P<0.001). In the 50 μg/L RANKL group inducted for 5 days, the expression levels of Hey2 gene (0.27±0.02) and protein (0.79±0.01) were significantly lower than those in the control group (1.00±0.00, 1.15±0.02) ( t=11.47, 108.60; both P<0.001). Hey2 overexpression significantly reduced the gene and protein expressions of NFATc1, c-Fos, and CTSK, as well as the production of TRAP-positive cells (all P<0.05). Hey2 knockdown significantly increased the gene and protein expressions of NFATc1, c-Fos, and CTSK, as well as the production of TRAP-positive cells (all P<0.05). After inducing RAW264.7 cells with 50 μg/L RANKL for 1 day, ChIP results showed that among the two sample groups treated with Hey2 antibody, the detection level of the NFATc1 promoter region (-400 to -200 bp) in the anti-Hey2-RANKL group (18.06±0.06) was significantly higher than that in the anti-Hey2 control group (13.37±0.36) ( t=12.56, P<0.001). Conclusions:Hey2 can bind to the downstream target gene NFATc1 at -400 to -200 bp region of the promoter. As a transcriptional repressor, Hey2 inhibits osteoclast differentiation.

Benign biliary stricture (BBS) refers to complete or incomplete stricture of the biliary tract caused by a series of non-malignant diseases. BBS often has complex and diverse etiologies, and severe complications may occur if it is not adequately treated. Diagnostic methods currently used in clinical practice include imaging, endoscopic retrograde cholangiopancreatography, endoscopic ultrasonography, and choledochoscopy, and treatment methods include balloon dilatation, stent implantation, percutaneous transhepatic biliary drainage, and surgical treatment. At present, endoscopic diagnosis and treatment of BBS has become the preferred method. However, there is still no clear classification of BBS, which needs further investigation. By consulting related literature in China and globally, this article summarizes the issues associated with the endoscopic diagnosis and treatment of BBS.

OBJECTIVE To observe the regulation effect of chidamide on energy metabolism in HCT-8 and HT-29 cells. METHODS HCT-8 and HT-29 cells were treated with chidamide 5,10 and 20 μmol · L-1. Morphological changes of these cells were observed under an ordinary optical microscope. Cell proliferation was detected by MTT. ATP production was determined by CellTiter-Glo? assay kit. Metabolic changes were tested by glycolytic stress kit. The mRNA level of lactate dehydrogenase A (LDH-A)was analyzed by real-time quantitative PCR,whereas the protein level of LDH-A was analyzed by Western blotting. RESULTS Compared with control group,cell morphology of HCT-8 and HT-29 cells in chidamide treated group was irregular,accompanied by deformation,shrinkage and cell debris, and the inhibitory rate of proliferation increased(P<0.05). There was no significant difference in ATP total content between chidamide 5 and 10 μmol · L-1 16 h treatment groups,but in chidamide 20 μmol · L-1 treatment group it was decreased(P<0.05). Chidamide 20μmol · L-1 had no effect on oxygen consumption rate, but glycolysis ATP generation rate was reduced by 30.7% and 37.9%(P<0.05),respectively. Chidamide 20μmol · L-1 had no effect on LDH-A mRNA level,but it decreased the protein level of LDH-A(P<0.01). CONCLUSION Chidamide can abate the respiratory metabolic ability of HCT-8 and HT-29 cells. The mechanism may be related to the down-regulation of LDH-A.

Objective To verify enzyme activity inhibition of a novel histone deacetylase inhibitor ( HDACi ) JZ005 using an HDACi chemiluminescence detection kit and a cell-based screening model .Methods The plasmid with p21 gene promoter elements and luciferase reporter gene was transfected into human embryonic kidney cells 293 , and the stable transfectants were established by G418 screening.Enzyme activity inhibition of JZ005 on histone deacetylases (HDACs) was verified by the HDACi chemiluminescence detection kit and the cell-based screening model .A well-known HDACi , tri-chostatin A ( TSA) was used as the positive control .MTT assay was used to detect the protection of rat H 9c2 myocardial cells suffering from CoCl 2-induced hypoxia and treated with different concentrations of JZ 005 .The expression of acetylated histone H3 protein of normal and CoCl 2-induced hypoxia H9c2 cells before and after JZ005 treatment was assayed by West-ern blotting while the effect of drug administration on apoptosis was detected by flow cytometry ( FCM) .Results An HDA-Ci cell-based screening system targeting the p21 gene promoter was ranging established .The JZ005, a HDACi, markedly suppressed the activity of HDACs by more than 50%with the concentration ranging from 50 to 400 μmol/L.JZ005 signifi-cantly protected H9c2 cells from hypoxia injury .Cell viability was increased by 38.33%,56.00% and 35.20% compared with control,accompanied by an enhanced acetylation level of histone H 3.JZ005(25,50 and 100 μmol/L) treatment sig-nificantly decreased the number of apoptotic cells (6.63%,10.56% and 8.89%) compared to control group (12.89%). Conclusion An HDACi cell-based screening system is successfully established .JZ005 effectively protects myocardial cells against hypoxia injury while enhancing the acetylation level of histone H 3.Our results indicate that JZ005 might be developed as a potential drug for hypoxia treatment .

Objective To examine the anticancer effect of a novel histone deacetylase inhibitor (HDACi), JZ004, on colon cancer cells HCT-8 and HT-29, and to investigate the molecular mechanisms of proliferation inhibition and apoptosis induction of cancer cells treated by JZ 004.Methods Colon cancer cells were treated with a series of concentrations of JZ004 .MTT assay was used to detect the proliferation of cancer cells .The cell cycle distribution and apoptosis were deter-mined by flow cytometry .Rhodamine 123 and DCFH-DA were applied to detect the mitochondrial membrane potential (ΔΨm) and reactive oxygen species ( ROS) production.The protein expressions of acetyl-histone H3, p21, cyclin-dependent kinase(CDK)4, Bcl-2, Mcl-1 and Bax were assayed by Western blotting .Results JZ004 was found to inhibit proliferation and induce apoptosis of colon cancer cells in a time-and dose-dependent manner , accompanied by a dose-dependent hyperacetylation of histone H3.JZ004 induced the cancer cell arrest in G 0/G1 phase by increasing the expres-sion level of p21 while CDK4 was downregulated .JZ004 also increased cellular ROS production and reduced ΔΨm by regu-lating the expressions of Bcl-2 family proteins .Conclusion As a novel HDACi , JZ004 effectively inhibits proliferation and increases ROS production to induce apoptosis of colon cancer cells .The results indicate that JZ004 is a potential compound to be developed as an anti-colon cancer agent for clinic application .

Objective To study the effect of pioglitazone on the differentiation and function of rat osteoclast-like cells (OLC), and to probe the relationship between activated PPARγ2 and osteoclasts. Methods On day 1 of OLC formation from nonadherent bone marrow ceils (BMC) obtained from rats induced by M-CSF and receptor activator of NF-кB ligand (RANKL), 1, 5 and 10μmol/L pioglitazone hydrochloride was added. RT- PCR was performed to determine the mRNA expressions of PPARγ2 and receptor activator of NF-кB (RANK) on day 3, 5 and 7 during incubation, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells,the number of bone resorption pits and the ratio of its area on dentin slice were counted, the activity of TRAP and the mean fluorescence intensity of integrin β3 (CD61) of OLC were also measured. Results (1) The effect on the differentiation of OLC: The addition of pioglitazone at the start of the culture period induced a dose-dependent decrease in TRAP-positive OLC and the activity of TRAP (P < 0.01 or P < 0.05) ; the mRNA expression of PPARγ2 was up-regulated by 5 and 10 μmol/L pioglitazone in the early stage of incubation and attenuated with thematuration of OLC on the contrary, however, the expression of RANK was down-regulated by 5 and 10 μmol/L piolitazone in every stage of incubation (P < 0.05 or P < 0.01), combined with decrease in TRAP-positive OLC from day 3 by 10 μmol/L pioglitazone. (2) The effect on the function of OLC: the number of bone resorption pits and the ratio of its area on dentin slice were decreased in groups of 5 and 10 μmol/L pioglitazone (P < 0.01 orP < 0.05), no obvious change was noted in the group with 1 μmol/L pioglitazone compared with the control group; the mean fluorescence intensity of CD61 were down-regulated in groups of 5 and 10 μmol/L pioglitazone (P < 0.05 or P <0.01). Conclusion Activation of PPARγ2 pathway by pioglitazone could partially inhibit differentiation and function of OLC derived from rat BMC.