BACKGROUND:At present,hydroxyapatite has been used more and more widely in the field of facial fillers due to its good biocompatibility and low immunity.The main sources are divided into natural extraction and artificial synthesis.However,there are few comparative studies on natural extraction and artificial synthesis of hydroxyapatite,especially the difference in stimulating collagen secretion between the two.OBJECTIVE:To investigate the physicochemical differences between naturally derived and commercially available synthetic calcium hydroxyapatite particles and promoting collagen secretion.METHODS:(1)Natural hydroxyapatite particles from pig bones and two kinds of commercially available synthetic hydroxyapatite particles(denoted as SYN1 and SYN2)were used to characterize the microstructure and surface element content of the three kinds of materials.(2)The three kinds of materials with different mass concentrations(1,5,and 10 mg/mL)were co-cultured with human skin fibroblasts.Cell proliferation was detected by CCK-8 assay.The three kinds of materials at 5 mg/mL were co-cultured with human skin fibroblasts.Cell adhesion was observed by CCK-8 assay and scanning electron microscopy.The expression of type Ⅰ collagen was detected by RT-PCR,ELISA,and western blot assay.(3)Twelve New Zealand rabbits were selected,of which six were subcutaneously injected with a mixture of natural hydroxyapatite and 2％sodium carboxymethylcellulose gel on the back.The remaining six rabbits were subcutaneously injected with a mixture of SYN2 and 2％sodium carboxymethylcellulose gel on the back.The samples were collected 1 and 3 months after injection and stained with Masson and Sirius red.RESULTS AND CONCLUSION:(1)Scanning electron microscope showed that the crystal grains of natural hydroxyapatite particles were more regular and uniform,with holes evenly distributed between particles.The SYN1 grains were smaller and densely arranged,while the SYN2 grains were irregular.The median particle sizes of natural hydroxyapatite particles,SYN1,and SYN2 were 38,24,and 40 pm,respectively.Natural hydroxyapatite contained Mg and Zn,SYN1 did not contain Mg and Zn,and SYN2 did not contain Zn.(2)CCK-8 assay showed that 1 and 5 mg/mL of natural hydroxyapatite and SYN2 promoted the proliferation of human skin fibroblasts,while 5 and 10 mg/mL of SYN1 inhibited the proliferation of human skin fibroblasts.The number of cells adhered to the surface of natural hydroxyapatite particles was more than that of SYN1 and SYN2,and the cells on the surface of natural hydroxyapatite particles spread well,with visible filopodia.RT-PCR,ELISA,and western blot assay results showed that the expression of type Ⅰ collagen in the natural hydroxyapatite particle group was higher than that in the SYN1 group and the SYN2 group.(3)The results of Masson and Sirius red staining showed that the amount of type Ⅰ collagen in the subcutaneous tissue of the natural hydroxyapatite particle group was greater than that of the SYN2 group 1 and 3 months after injection.(4)The results show that compared with synthetic hydroxyapatite particles,natural hydroxyapatite particles have a richer distribution of trace elements and can better promote fibroblast adhesion,proliferation and collagen regeneration.

BACKGROUND:At present,hydroxyapatite has been used more and more widely in the field of facial fillers due to its good biocompatibility and low immunity.The main sources are divided into natural extraction and artificial synthesis.However,there are few comparative studies on natural extraction and artificial synthesis of hydroxyapatite,especially the difference in stimulating collagen secretion between the two.OBJECTIVE:To investigate the physicochemical differences between naturally derived and commercially available synthetic calcium hydroxyapatite particles and promoting collagen secretion.METHODS:(1)Natural hydroxyapatite particles from pig bones and two kinds of commercially available synthetic hydroxyapatite particles(denoted as SYN1 and SYN2)were used to characterize the microstructure and surface element content of the three kinds of materials.(2)The three kinds of materials with different mass concentrations(1,5,and 10 mg/mL)were co-cultured with human skin fibroblasts.Cell proliferation was detected by CCK-8 assay.The three kinds of materials at 5 mg/mL were co-cultured with human skin fibroblasts.Cell adhesion was observed by CCK-8 assay and scanning electron microscopy.The expression of type Ⅰ collagen was detected by RT-PCR,ELISA,and western blot assay.(3)Twelve New Zealand rabbits were selected,of which six were subcutaneously injected with a mixture of natural hydroxyapatite and 2％sodium carboxymethylcellulose gel on the back.The remaining six rabbits were subcutaneously injected with a mixture of SYN2 and 2％sodium carboxymethylcellulose gel on the back.The samples were collected 1 and 3 months after injection and stained with Masson and Sirius red.RESULTS AND CONCLUSION:(1)Scanning electron microscope showed that the crystal grains of natural hydroxyapatite particles were more regular and uniform,with holes evenly distributed between particles.The SYN1 grains were smaller and densely arranged,while the SYN2 grains were irregular.The median particle sizes of natural hydroxyapatite particles,SYN1,and SYN2 were 38,24,and 40 pm,respectively.Natural hydroxyapatite contained Mg and Zn,SYN1 did not contain Mg and Zn,and SYN2 did not contain Zn.(2)CCK-8 assay showed that 1 and 5 mg/mL of natural hydroxyapatite and SYN2 promoted the proliferation of human skin fibroblasts,while 5 and 10 mg/mL of SYN1 inhibited the proliferation of human skin fibroblasts.The number of cells adhered to the surface of natural hydroxyapatite particles was more than that of SYN1 and SYN2,and the cells on the surface of natural hydroxyapatite particles spread well,with visible filopodia.RT-PCR,ELISA,and western blot assay results showed that the expression of type Ⅰ collagen in the natural hydroxyapatite particle group was higher than that in the SYN1 group and the SYN2 group.(3)The results of Masson and Sirius red staining showed that the amount of type Ⅰ collagen in the subcutaneous tissue of the natural hydroxyapatite particle group was greater than that of the SYN2 group 1 and 3 months after injection.(4)The results show that compared with synthetic hydroxyapatite particles,natural hydroxyapatite particles have a richer distribution of trace elements and can better promote fibroblast adhesion,proliferation and collagen regeneration.

Objective To explore the protective effects of dipeptidyl peptidase-4 inhibitor (DPP-4I) on AD-like neurodegenerative changes and its mechanism. Methods The human neuroblastoma cell line SH-SY5Y on the logarithmic phase was divided into six groups:control group (CON group, treated with PBS contained 1‰DMSO for 12 h), wortmannin intervention group (W group, treated with 0.03 μmol/L wortmannin for 12 h), DPP-4I intervention group (DPP-4I group, treated with 10μmol/L DPP-4I for 12 h), both DPP-4I and wortmannin intervention group (DPP-4I+W group, pre-treated with 10 μmol/L DPP-4I for 2 h, then 0.03 μmol/L wortmannin for 12 h), DPP-4I, wortmannin and Ex9-39 intervention group (DPP-4I+W+Ex9-39 group, pre-treated with 10μmol/L Ex9-39 for 2 h, then 10μmol/L DPP-4I for 2 h followed by 0.03μmol/L wortmannin for 12 h), and Ex9-39 intervention group (Ex9-39 group, treated with 10μmol/L Ex9-39 for 12 h). MTT assay was used to detect the cell vitality. Western blot assay was used to detect the level of total tau protein (tau-5) and phosphorylated tau at different sites (pSpS199/202, pT231 and pS396), the level of phosphorylated neurofilaments (NF-H, NF-M) and phosphorylation of critical enzyme in PI3K/Akt/GSK-3β signaling pathway. Results (1) The cell vitality decreased, the levels of pSpS199/202, pT231, pS396 and NF-H/M increased significantly in W group than those in CON group. However, comparing with CON group, the above mentioned parameters reversed in DPP-4I group. Comparing with W group, the cell vitality increased and phosphorylated levels of above mentioned indices were decreased in DPP-4I+W group. (2) The cell vitality showed a decline trend while the levels of phosphorylation tau at three different sites and NF-H/M were higher in Ex9-39 group than those in CON group. Comparing with DPP-4I+W group, the results of the phosphorylated levels showed the same changes in DPP-4I+W+Ex9-39 group. (3) Comparing with CON group, the expression levels of phosphorylated PI3K, Akt and GSK3β increased significantly in DPP-4I group, while those decreased in W group. Additionally, the expression levels of phosphorylated PI3K, Akt and GSK3β were significantly increased in DPP-4I+W group than those in W group. Conclusion DPP-4I can enhance the level of GLP-1 and activate PI3K/Akt/GSK-3βinsulin signaling pathway to improve the hyperphosphorylated tau and NFs induced by wortmannin, and to protect AD-like neurodegeneration.

Objective: To further explore TCE-induced hepatotoxicity and its mechanisms by identification of trichloroethylene (TCE) induced abnormal histone methylation in human liver cells. Methods: L-02 cells were treated with 0 and 8 mmol/L TCE for 24 h. Histones were extracted by acid. Liquid chromatography electrospray ionization tandem mass spectrometry (ESI-LC-MS/MS) were used to identify and quantify TCE related histone methylations. TCE induced abnormal methylation of H3K79 me2 and H3K79 me3 were validated by Western blot analysis. The further analysis of the function of histone abnormal methylation modifications were done by single cell gel electrophoresis (SCGE) and Western blot analysis of p53 and ɤH2AX. Results: After treatment with TCE for 24 h in L-02 cells, the 36 TCE related histone methylation sites in 28 peptide segments were identified by MS. After treatment with TCE in concentrations of 0 and 8.0 mmol/L in L-02 cells for 24 h, the relative expression level of histone H3K79 me3 were 1.00±0.06, 0.70±0.09 (t=15.01, P=0.015); the relative expression level of histone H3K79 me2 were 1.00±0.05, 0.74±0.07 (t=16.69, P=0.018); the Olive Tail Moment about DNA damage were 1.46±0.28, 3.12± 0.68 (t=15.22, P=0.018); the relative expression levels of p53 were 1.00±0.04, 1.24±0.04 (t=18.71, P= 0.012); and the relative expression levels of ɤH2AX were 1.00 ± 0.03, 1.56 ± 0.11 (t=8.32, P=0 045). Conclusion TCE can induce changes in the relative expression level of H3K79 me2 and H3K79 me3 in L-02 cell, and induce DNA damage, suggesting that TCE may induce changes in the relative expression level of H3K79 me2 and H3K79 me3 by DNA damage.