Objective To explore the mechanism by which gentiopicroside (GPS) prevents macrophage-mediated hepatic fibrosis by regulating the macrophage migration inhibitory factor (MIF)-secreted phosphoprotein 1 (SPP1) signaling pathway in hepatic stellate cells. Methods LX-2 cells were divided into control group, transforming growth factor β(TGF-β) group, and TGF-β combined with GPS (25, 50, 100, 150 μmol/mL) groups. Cell proliferation was detected by EDU assay, cell invasion was assessed by Transwell^TM assay, and the protein expressions of α-smooth muscle actin (α-SMA) and type I collagen (COL1A1) were measured by Western blot. M1-type macrophage-conditioned medium (M1-CM) was used to treat LX-2 cells in the TGF-β group and TGF-β combined with GPS group. The concentrations of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) in the cell supernatant, as well as cell proliferation, invasion ability, and the expressions of α-SMA and COL1A1 were detected. Bioinformatics analysis was performed to identify the target intersections of GPS, hepatic fibrosis, and macrophage-related genes. Drug affinity responsive target stability (DARTS) experiments and Western blot were used to verify the regulatory effect of GPS on MIF. Furthermore, LX-2 cells were divided into control group, TGF-β group, TGF-β combined with M2-CM group, TGF-β and oe-NC combined with M2-CM group, and TGF-β and oe-MIF combined with M2-CM group to analyze the concentrations of iNOS and Arg1 in the cell supernatant, as well as changes in cell proliferation, invasion, and the expressions of α-SMA and COL1A1. LX-2 cells were also divided into control group, TGF-β group, TGF-β combined with oe-NC group, TGF-β combined with oe-MIF group, and TGF-β and oe-MIF combined with GPS group to determine the protein expressions of MIF and SPP1 by Western blot. A rat model of hepatic fibrosis was constructed to explore the potential therapeutic effects of GPS on hepatic fibrosis in vivo. Results Compared with the control group, the proliferation and invasion abilities of LX-2 cells in the TGF-β group were increased, and the protein expressions of α-SMA and COL1A1 were enhanced. GPS intervention inhibited the proliferation and invasion of LX-2 cells under TGF-β conditions and reduced the expressions of α-SMA and COL1A1. Compared with the control group, the concentration of iNOS in the cell supernatant of the TGF-β group was upregulated, while the concentration of Arg1 was decreased. M1-CM treatment further increased the concentration of iNOS, decreased the concentration of Arg1, and promoted cell proliferation and invasion, as well as upregulated the expressions of α-SMA and COL1A1 on the basis of TGF-β intervention. However, GPS could reverse the effects of M1-CM intervention. Bioinformatics analysis revealed that MIF was one of the target intersections of GPS, hepatic fibrosis, and macrophage-related genes, and GPS could target and inhibit its expression. Compared with the TGF-β group, after M2-CM intervention, the concentration of iNOS in the cell supernatant decreased, the concentration of Arg1 increased, the proliferation and invasion abilities of LX-2 cells were reduced, and the expressions of α-SMA and COL1A1 were weakened. However, overexpression of MIF reversed the effects of M2-CM intervention. Western blot results showed that compared with the control group, the protein expressions of MIF and SPP1 were enhanced in the TGF-β group. Overexpression of MIF further enhanced the expressions of MIF and SPP1, while GPS intervention inhibited the expressions of MIF and SPP1. In the animal experiment, GPS intervention treatment alleviated liver injury in rats with hepatic fibrosis and inhibited the expressions of MIF and SPP1, as well as α-SMA and COL1A1 in liver tissue. Conclusion GPS may prevent macrophage-mediated hepatic fibrosis by inhibiting the MIF-SPP1 signaling pathway in hepatic stellate cells.
Hepatic Stellate Cells/metabolism* ; Signal Transduction/drug effects* ; Macrophage Migration-Inhibitory Factors/genetics* ; Liver Cirrhosis/prevention & control* ; Macrophages/drug effects* ; Iridoid Glucosides/pharmacology* ; Humans ; Cell Proliferation/drug effects* ; Animals ; Cell Line ; Collagen Type I/metabolism* ; Collagen Type I, alpha 1 Chain ; Intramolecular Oxidoreductases/genetics* ; Rats ; Transforming Growth Factor beta/pharmacology* ; Actins/metabolism*

OBJECTIVE: To investigate the anti-adhesive effect and underlying mechanism of dynamic and static stress stimulation on the early healing process of rat Achilles tendon injury. METHODS: Achilles tendon tissues of 15 male Sprague Dawley (SD) rats aged 4-6 weeks were isolated and cultured by enzyme digestion method. Rat Achilles tendon cells were treated with tumor necrosis factor α to construct the Achilles tendon injury cell model, and dynamic stress stimulation (dynamic group) and static stress stimulation (static group) were applied respectively, while the control group was not treated. Live/dead cell double staining was used to detect cell activity, ELISA assay was used to detect the expression of α smooth muscle actin (α-SMA), and real-time fluorescence quantitative PCR was used to detect the mRNA expression of collagen type Ⅰ (COL1A1), collagen type Ⅲ (COL3A1), and Scleraxis (SCX). Thirty male SD rats aged 4-6 weeks underwent Achilles tendon suture and were randomly divided into dynamic group (treated by dynamic stress stimulation), static group (treated by static stress stimulation), and control group (untreated), with 10 rats in each group. HE staining and scoring were performed to evaluate the healing of Achilles tendon at 8 days after operation. COL1A1 and COL3A1 protein expressions were detected by immunohistochemical staining, α-SMA and SCX protein expressions were detected by Western blot, and maximum tendon breaking force and tendon stiffness were detected by biomechanical stretching test. RESULTS: <i>In vitroi> cell experiment, when compared to the static group, the number of living cells in the dynamic group was higher, the expression of α-SMA protein was decreased, the relative expression of COL3A1 mRNA was decreased, and the relative expression of SCX mRNA was increased, and the differences were all significant ( <i>Pi><0.05). In the <i>in vivoi> animal experiment, when compared to the static group, the tendon healing in the dynamic group was better, the HE staining score was lower, the expression of COL1A1 protein was increased, the expression of COL3A1 protein was decreased, the relative expression of SCX protein was increased, the relative expression of α-SMA protein was decreased, and the tendon stiffness was increased, the differences were all significant ( <i>Pi><0.05). CONCLUSION Compared with static stress stimulation, the dynamic stress stimulation improves the fibrosis of the scar tissue of the rat Achilles tendon, promote the recovery of the biomechanical property of the Achilles tendon, and has obvious anti-adhesion effect.
Animals ; Achilles Tendon/injuries* ; Male ; Rats ; Rats, Sprague-Dawley ; Collagen Type I/metabolism* ; Collagen Type III/metabolism* ; Tendon Injuries/therapy* ; Wound Healing ; Stress, Mechanical ; Actins/metabolism* ; Cells, Cultured ; Tissue Adhesions/prevention & control* ; Tumor Necrosis Factor-alpha/metabolism* ; RNA, Messenger/genetics* ; Disease Models, Animal ; Collagen Type I, alpha 1 Chain/metabolism* ; Biomechanical Phenomena ; Basic Helix-Loop-Helix Transcription Factors

Amelogenin (AMG) is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG (rhAMG) on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1, 1, 10, 100, 1000, 10,000, 100,000 ng · mL) of recombinant human AMG (rhAMG) and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type I (COL I), osteopontin (OPN), runt-related transcription factor 2 (Runx2), cementum attachment protein (CAP), and alkaline phosphatase (ALP) genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer. Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomal-associated membrane protein-1 (LAMP-1), which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100,000 ng · mL increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG (100,000 ng · mL) upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100,000 ng · mL remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10,000 and 100,000 ng · mL rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue.
Alkaline Phosphatase ; metabolism ; Amelogenin ; physiology ; Animals ; Biomarkers ; metabolism ; Calcification, Physiologic ; Cell Adhesion Molecules ; metabolism ; Cell Proliferation ; Cementogenesis ; physiology ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Gene Expression Regulation ; In Vitro Techniques ; Integrin-Binding Sialoprotein ; metabolism ; Mice ; Microscopy, Confocal ; Osteocalcin ; metabolism ; Osteopontin ; metabolism ; Real-Time Polymerase Chain Reaction

This study is to investigate effects of genistein on rat femoral bone metabolic in vitro. Rat femoral tissues was isolated and randomly divided into two groups including control group and genistein (1 x 10(-5) mol x(-1)) group. Determinations of alkaline phosphatase (ALP) activity, calcium content and osteoprotegerin (OPG), type I-collagen (Collagen-I), RANKL, Runx-2 and bone morphogenetic protein (BMP-2) mRNA expression were done by real-time PCR. The results showed that 1 x 10(-5) mol x L(-1) genistein could increase the activity of ALP and contents of Ca, regulate bone metabolism activity of OPG, RANKL, BMP-2, Collagen-I and Runx-2 mRNA expression level. Genistein can significantly modulate bone metabolism related gene expression level of rat femoral tissue in vitro, and can increase calcium content and the activity of ALP.
Alkaline Phosphatase ; metabolism ; Animals ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Calcium ; metabolism ; Collagen Type I ; genetics ; metabolism ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Enzyme Activation ; drug effects ; Femur ; metabolism ; Gene Expression Regulation ; Genistein ; pharmacology ; Osteoprotegerin ; genetics ; metabolism ; Phytoestrogens ; pharmacology ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction

OBJECTIVETo find an ideal method inducing dental pulp stem cells (DPSC) osteogenic differentiation. To compare the effect of co-culture method and that of mineralizing culture medium.

METHODSDPSC were co-cultured with osteoblasts using cell culture inserts system as experiment group, and DPSC were cultured in mineralizing culture medium as control group. The cell morphology and ultrastructure and mineralized nodes were analyzed under phase contrast microscope, transmission electron microscope, and alizarin red S staning. Bone sialoprotein (BSP), Runx-2, osteocalcin, and collagen-1 (Col-1) osteoblastic genes expressions of DPSC cultivated in special niche of osteoblasts were assayed by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThe mineralization nudoles of experiment group were more than control group. Fifteen days later, BSP and Col-1 genes in the DPSC of co-cultures were 9.807 ± 1.135 and 2.913 ± 0.310, respectively. And those in the DPSC of mineralizing culture medium were 6.478 ± 0.781 and 1.703 ± 0.184, respectively. Co-cultures and mineralizing were significantly different (P < 0.05).

CONCLUSIONSAs osteoblasts can secret lots of osteogenic cell cytokines, they have more significant effect than mineralizing culture medium on osteogenesis of DPSC.

Cell Differentiation ; Coculture Techniques ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Dental Pulp ; cytology ; Gene Expression Regulation, Developmental ; Humans ; Integrin-Binding Sialoprotein ; metabolism ; Microscopy, Electron, Transmission ; Microscopy, Phase-Contrast ; Osteoblasts ; cytology ; Osteocalcin ; metabolism ; Osteogenesis ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism ; ultrastructure