OBJECTIVE: To investigate the mechanism by which arecoline regulates the level of miR-155-5p in macrophage-secreted exosomes to induce the transformation of human oral mucosal fibroblasts (HOMFs) into fibroblast phenotype. METHODS: Exosomes were harvested from human monocytic cell line THP-1 with or without arecoline treatment. The effects of arecoline-treated THP-1 cell culture supernatant (CS), THP-1-derived exosomes (EXO), exosome-depleted THP-1 cell supernatant (NES), miR-155-5p overexpression, and miR-155-5p inhibitor on migration ability of arecoline-treated HOMF cells were examined using Transwell migration assay. The polarization of THP-1 cells was detected using flow cytometry. DCFH-DA was used to detect the level of oxidative stress in the cells with different treatments. The mRNA and protein expressions of α- SMA, type I collagen and SOCS1 in the cells were detected with qRT-PCR and Western blotting. RESULTS: Flow cytometry showed that arecoline-treated THP-1 cells exhibited obvious polarization from M₀ to M₁. Both the supernatant and exosomes from arecoline-treated THP-1 cells significantly enhanced the migration ability of HOMF cells, increased intracellular oxidative stress, up-regulated the expressions of miR-155- 5p and the mRNA and protein levels of α-SMA and type I collagen, and lowered the mRNA and protein expressions of SOCS1. In HOMF cells treated with exosomes from arecoline- treated THP-1 cells, overexpression of miR-155-5p significantly enhanced cell migration ability and increased cellular expressions of α-SMA and type I collagen, and miR-155-5p inhibitor caused the opposite changes. CONCLUSION Arecoline can up-regulate miR-155-5p expression in THP-1 cells and inhibit the expression of SOCS1 protein in HOMF cells <i>viai> the exosome pathway, thus promoting the fibrotic phenotype transformation of HOMF cells.
Humans ; Exosomes ; Arecoline/pharmacology* ; Collagen Type I ; Fibroblasts ; Macrophages ; MicroRNAs

OBJECTIVE: To investigate the inhibitory effect of genistein on activation of hepatic stellate cells (HSCs) and the role of the autophagy pathway regulated by PPAR-γ in mediating this effect. METHODS: Cultured HSC-T6 cells were exposed to different concentrations of genistein for 48 h, and HSC activation was verified by detecting the expressions of -SMA and 1(I) collagen; autophagy activation in the cells was determined by detecting the expressions of LC3-II and p62 using Western blotting. The autophagy inhibitor 3-MA was used to confirm the role of autophagy in genistein-induced inhibition of HSC activation. A PPAR-γ inhibitor was used to explore the role of PPAR-γ in activating autophagy in the HSCs. RESULTS: Genistein at concentrations of 5 and 50 μmol/L significantly inhibited the expressions of -SMA and 1(I) collagen ( < 0.05), markedly upregulated the expressions of PPAR-γ and the autophagy-related protein LC3-II ( < 0.05) and significantly down-regulated the expression of the ubiqutin-binding protein p62 ( < 0.05) in HSC-T6 cells. The cells pretreated with 3-MA prior to genistein treatment showed significantly increased protein expressions of -SMA and 1(I) collagen compared with the cells treated with genistein only ( < 0.05). Treatment with the PPAR-γ inhibitor obviously lowered the expression of LC3-II and enhanced the expression p62 in genistein-treated HSC-T6 cells, suggesting the activation of the autophagy pathway. CONCLUSIONS PPAR-γ- regulated autophagy plays an important role in mediating genistein-induced inhibition of HSC activation .
Anticarcinogenic Agents ; pharmacology ; Autophagy ; Collagen Type I ; Genistein ; pharmacology ; Hepatic Stellate Cells ; Humans ; PPAR gamma ; physiology

The collagen I was made with the dialysis method of enzymolysising the pig skin, and the static deposition in vitro of calcium phosphate was comparative studied by X-ray diffraction (XRD) and infrared spectroscopy (FTIR) under the condition of pH7. 4, Ca/P 1.67 and whether adding the collagen I into the system. Then the chemical composition of the sedimentary product and the diversification of the collagen I 's IR and Raman spectra (RS) before and after the mineralization were analyzed. The results showed that,under the physiological pH condition that there was not any collagen I, though Ca/P reached up to 1.67, the sedimentary product was CaHPO4 x 2H2O yet, however, after adding collagen I into the system, the bone-like apatite was deposited, which proved that collagen I indeed had the effects on the inducing of the bone-like apatite's mineralization in vitro; there was obviously mutual coordination action between collagen I and its mineralization product--bone-like apatite, which caused that amide peak I red-shifted, amide peak II and amide peak III decreased significantly or disappeared on the IR of collagen I, which maybe was the mechanism that how collagen I induced the depositing of the bone-like apatite.
Animals ; Apatites ; metabolism ; Collagen Type I ; pharmacology ; Osteogenesis ; drug effects ; Skin ; chemistry ; Swine

OBJECTIVE: The effects of the staurosporine on contraction of self-assembled constructs and extracellular matrix syntheses of goat temporomandibular joint discs were investigated. METHODS: Goat temporomandibular joint disc cells were isolated and cultured to P3, and 5.5×10⁶ cells were combined with different concentrations of staurosporine (0, 0.1, 1, 10, 100 nmol·L⁻¹) in agarose wells and cultured for one week. The samples were frozen and sectioned. Safranin-O,  Picro-sirius red and immunohistochemical staining were performed to observe the distributions of the extracellular matrix and the expression of alpha-smooth muscle actin (α-SMA). Enzyme linked immunosorbent assay (ELISA) and Blyscan kits were utilized to quan--titatively detect the contents of type Ⅰ collagen (ColⅠ) and glycosaminoglycans (GAGs). RESULTS: Each group of goat temporo-mandibular joint disc cells in the agarose wells were gathered to self-assemble into a disc-shaped base for 4 hours and then to gradually contract into a round shape. The Picro-sirius red staining was strong and indicated collagen distribution. The Safranin-O staining observed GAGs throughout the entire construct. The expression of ColⅠ was strongly posi-tive in the staurosporine groups; however, the expression of α-SMA was weak. ColⅠ and GAGs contents in the stau-rosporine groups were greater than that of the control group, especially in the 10 nmol·L⁻¹ group (P<0.01). CONCLUSIONS Staurosporine has a certain effect on the shrinkage of self-assembled constructs; however, such effect is not prominent. Staurosporine contributes to the construction synthesis of extracellular matrix.
Animals ; Collagen Type I ; Glycosaminoglycans ; Goats ; Staurosporine ; pharmacology ; Temporomandibular Joint ; Temporomandibular Joint Disc ; cytology ; drug effects

OBJECTIVE: To observe the effect of exosomes secreted by lipopolysaccharides (LPS)-stimulated macrophages on hepatic stellate cell activation and migration and explore the underlying molecular mechanism. METHODS: Human monocyte THP-1 cells were induced to differentiate into macrophages using propylene glycol methyl ether acetic acid (PMA, 100 ng/mL, 24 h) followed by stimulation with LPS, and the culture supernatant of macrophages was collected for extraction of the exosomes by ultracentrifugation. The expression of miR-155-5p in the exosomes was detected using qRT-PCR. A Transwell co-culture system was used to observe the effects of the macrophage-derived exosomes on LX2 cell (a hepatic stellate cell line) proliferation, migration, oxidative stress and the expression of fibrosis biomarkers, which were also observed in LX2 cells transfected with miR-155-5p-mimics or miR-155-5p-inhibitors. Western blotting was used to detect the expressions of SOCS1 and its downstream signal pathway proteins. RESULTS: Treatment with the exosomes from LPS-stimulated macrophages significantly enhanced the proliferation and migration ability of LX2 cells and increased the levels of oxidative stress and expressions of the fibrosis markers such as type Ⅰ collagen (<i>Pi> < 0.05). The expression of miR-155-5p in the exosomes secreted by macrophages was significantly increased after LPS treatment (<i>Pi> < 0.01). LX2 cells overexpressing miR-155-5p also exhibited significantly enhanced proliferation and migration with increased oxidative stress levels and expression of type Ⅰ collagen (<i>Pi> < 0.05), and interference of miR-155-5p expression produced the opposite effects. Western blotting showed that miR-155-5p overexpression obviously inhibited SOCS1 expression and promoted p-Smad2/3, Smad2/3 and RhoA protein expressions in LX2 cells (<i>Pi> < 0.05). CONCLUSION LPS stimulation of the macrophages increases miR-155-5p expression in the exosomes to promote activation and migration and increase oxidative stress and collagen production in hepatic stellate cells.
Humans ; Hepatic Stellate Cells ; Lipopolysaccharides/pharmacology* ; Collagen Type I ; Exosomes ; Macrophages ; MicroRNAs

OBJECTIVETo investigate the effects of spironolactone on the concentration of collagen type I, III in the myocardium of spontaneous hypertension rats (SHR).

METHODSTwenty 8-week male SHR were assigned randomly into spironolactone (SHR-SPIRO, n=10) and control groups (SHR-CON, n=10), sex-age matched Wistar Kyoto rats (WKY group, n=7) were also served as controls. The rats of SHR-SPIRO group were given 20 mg/(kg*d) of spironolactone, the rats of SHR-CON and WKY groups were given the same volume of distilled water. After 16 weeks, the concentration of collagen type I was analyzed with Western blot. The areas of collagen type I and III were observed under polarized light microscopy and the ratio of type I/III collagen was calculated through accumulation score.

RESULTSCompared with WKY group,the concentration of collagen type I in SHR-CON group was significantly higher (1.87 ±0.2 Compared with 1.21 ±0.7, P<0.05). After 16 weeks of treatment the concentration of collagen type I (1.42 ±0.05 Compared with 1.87 ±0.2, P<0.05) and I/III ratio in SHR-SPIRO group were significantly reduced (15.64 ±1.34 Compared with 20.8 ±3.04, P<0.05) compared with SHR-CON group; but there were no differences in accumulation area scores of collagen type III among three groups (368.3 ±30.2 Compared with 481.6 ±32.4 Compared with 406.2 ±45.3, P>0.05).

CONCLUSIONThe deposition of collagen type I in myocardium may be involved in myocardial fibrosis of SHR, and spironolactone can decrease the concentration of collagen type I, which may be one of the mechanisms for its therapeutic effects.

Animals ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Male ; Mineralocorticoid Receptor Antagonists ; pharmacology ; Myocardium ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Spironolactone ; pharmacology

OBJECTIVETo investigate the role of recombinant human transforming growth factor beta3 (rhTGFbeta3) on fibroblast and its possible mechanism.

METHODSNormal skin fibroblast (NSFb) and hypertrophic scar fibroblast (HSFb) were cultured in vitro, and were processed by different concentrations of rhTGFbeta3. NSFb and HSFb in DMEM solution without rhTGFbeta3 were employed as control. The changes in the protein and mRNA expression of type I and III collagen in NSFb and HSFb were observed.

RESULTS(1) The expression of type I and III procollagen in NSFb was evidently different from that of HSFb (2) The synthesis of type I and III procollagen in all test groups was increased obviously after rhTGFbeta3 process (P < 0.001) while the ratio of type I to III procollagen was decreased when compared with that in control group. (3) The effects of rhTGFbeta3 on the biological behavior exhibited an obvious dose- effects relationship. The contents of type I to III procollagen in HSFb were higher than those in NSFb when the dose of rhTGFbeta3 was same.

CONCLUSIONrhTGFbeta3 could effectively promote the synthesis of type I and III procollagen, especially type III procollagen in fibroblasts. This might be beneficial to the accelerate of wound healing and to inhibit or prevent scar formation.

Cicatrix, Hypertrophic ; metabolism ; Collagen Type I ; biosynthesis ; Collagen Type III ; biosynthesis ; Fibroblasts ; drug effects ; metabolism ; Humans ; In Vitro Techniques ; Recombinant Proteins ; pharmacology ; Transforming Growth Factor beta3 ; pharmacology

OBJECTIVE: To establish a visual reporting system for evaluating the activity of collagen Ⅰ α 1 chain (<i>COL1A1i>) gene promoter in immortalized human hepatic stellate cells, so as to estimate the activation status of the cells and provide a new cell model for the screening and study of anti-hepatic fibrosis drugs. METHODS: The promoter sequence of human <i>COL1A1i> was amplified from the genomic DNA of human hepatocarcinoma cell line HepG2. Based on the pLVX-AcGFP1-N1 plasmid, the recombinant plasmid pLVX-COL1A1-enhanced green fluorescent protein (EGFP) was constructed, in which the enhanced green fluorescent protein gene expression was regulated by the <i>COL1A1i> promoter. The monoclonal cell line was acquired by stably transfecting pLVX-COL1A1-EGFP into the immortalized human hepatic stellate cell line LX-2 by the lentivirus packaging system and screening. The cell line was treated with transforming growth factor-β1 (TGF-β1) or co-treated with TGF-β1 and drugs with potential anti-hepatic fibrosis effects. The EGFP fluorescence intensity in cells was analyzed by the fluorescence microscope and ImageJ 1.49 software using a semi-quantitative method. The <i>COL1A1i> and <i>EGFPi> mRNA were detected by reverse transcription real-time quantitative PCR (RT-qPCR), and corresponding proteins were detected by Western blot. RESULTS: The recombinant plasmid pLVX-COL1A1-EGFP with the expression of <i>EGFPi> regulated by <i>COL1A1i> promoter was successfully constructed. Kozak sequence was added to enhance the expression of <i>EGFPi>, which was identified by double digestion and sequencing. The LX-2 monoclonal cell line LX-2-CE stably transfected with pLVX-COL1A1-EGFP was obtained. After co-treatment with TGF-β1 and 5 μmol/L dihydrotanshinone Ⅰ with potential anti-hepatic fibrosis effect for 24 h, the total fluorescence intensity and the average fluorescence intensity of LX-2-CE were lower than those in TGF-β1 single treatment group (<i>Pi> < 0.05), the intracellular mRNA and protein levels of <i>COL1A1i> and <i>EGFPi> were also lower than those in the TGF-β1 single treatment group (<i>Pi> < 0.05). CONCLUSION A reporter system for estimating activation of hepatic stellate cells based on <i>COL1A1i> promoter regulated <i>EGFPi> expression is successfully constructed, which could visually report the changes in <i>COL1A1i> expression, one of the activation-related markers of hepatic stellate cells, <i>in vitroi>. It provides a new cell model for the screening and study of anti-hepatic fibrosis drugs.
Humans ; Transforming Growth Factor beta1/pharmacology* ; Hepatic Stellate Cells/pathology* ; Liver Cirrhosis/genetics* ; Collagen Type I/pharmacology* ; RNA, Messenger/metabolism*

OBJECTIVETo evaluate the effects of taurine in diet on the expression of type I and III collagen and collagen ratio at different time points in rats lung by image process technology.

METHODSWistar rats were randomly divided into three groups: the saline instilled with a control diet (the saline treated group); silica instilled with a control diet (the silica treated group); and silica instilled with a diet containing 2.5% taurine (the taurine treated group). Animal models were established by the direct tracheal instillation of silica into rat lungs exposed surgically. The taurine concentration of serum was analyzed by means of HPLC. Paraffin embedded lung sections were stained with Sirius red. Polarization microscopy and Image Pro Plus Version 4.5 for windows were used for detecting type I and III collagen.

RESULTSThe concentration of taurine in serum of the taurine treated group was significantly elevated compared to the saline treated and silica treated group (P < 0.05 or P < 0.01). Sirius red polarization microscopy showed that type I and III collagen positive area percentage were elevated in the silica treated rats compared with the saline treated group. On the 7th, 14th, 21st, 28th day after silica instillation type I collagen positive area percentage was increased by 3.84, 3.77, 3.73, 9.83 respectively (P < 0.01), and type III collagen positive area percentage were elevated by a little in the silica treated rats compared with saline treated group. The taurine treatment significantly decreased elevation of silica type I collagen positive area percentage of lung by 2.39, 1.62, 7.13 at the 7th, 21st, 28th day respectively (P < 0.05 or P < 0.01), and type III collagen positive area percentage of lung by 2.62 at the 28th day (P < 0.05) compared with the silica treated group. The ratio of type I to III collagen was increased from the 7th day to 28th day after silica instillation, and reached 1.87 at the 28th day with the maximal ratio in the silica-treated group.

CONCLUSIONTreatment with taurine can effectively attenuate type I and III collagen expression in the rat lung induced by silica particles at different time points in our study.

Animals ; Collagen Type I ; biosynthesis ; Collagen Type III ; biosynthesis ; Female ; Lung ; drug effects ; metabolism ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Silicon Dioxide ; toxicity ; Taurine ; pharmacology

OBJECTIVETo observe the effects of complement fragment C3f on expression and secretion of collagen I, III and transforming growth factor( TGF)-beta1 in human embryonic lung fibroblast (MRC-5) cells.

METHODSMRC-5 cells were cultured with C3f (the synthetic 17 peptides fragments of complement C3). The extracellular and intracellular expression levels of type I, III collagens and TGF-beta1 in MRC-5 cultures were detected by ELISA and immunohistochemistry, respectively.

RESULTSThe expression levels of type I, III collagen and TGF-beta1 in the supernatant of MRC-5 cultures decreased significantly with the concentrations of C3f as compared with controls (P < 0.05). Also the expression level of TGF-beta1 in MRC-5 cytoplasm reduced significantly as compared with controls (P < 0.05).

CONCLUSIONThe results of present in vitro study showed that the complement fragment C3f could reduce the formation of TGF-beta1 and type I, III collagens in MRC-5 cells, and inhibit the lung tissue fibrosis.

Cell Line ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Complement C3b ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; Humans ; Lung ; cytology ; drug effects ; embryology ; Transforming Growth Factor beta1 ; metabolism