OBJECTIVEThe aim of this study was to investigate the expression of collagen in the process of masseter muscle reattachment to the cortical and cancellous bones of mandible.

METHODSA total of nine adult goats were used in the study. One was the control. The other eight were treated with bilateral detachment of the masseter muscles. The biopsies of bone and muscle were taken at 2, 4, 8 and 12 weeks after the operation. The characteristics of the healing muscle-bone interfaces were examined using immunohistochemical techniques.

RESULTSImmunohistochemical analysis illustrated that the locations of collagen type I, II and III were different during the healing process, but similar in the cortical and cancellous bones.

CONCLUSIONThis study demonstrates that the distribution of the three types of collagens at the muscle-bone interfaces is associated with time, but not related with their locations.

Animals ; Collagen ; biosynthesis ; genetics ; Collagen Type I ; biosynthesis ; genetics ; Collagen Type II ; biosynthesis ; genetics ; Collagen Type III ; biosynthesis ; genetics ; Female ; Goats ; Male ; Mandible ; metabolism ; pathology ; surgery ; Masseter Muscle ; metabolism ; pathology ; surgery ; Wound Healing ; physiology

AIMTo observe the effects of hypoxia on DNA synthesis and the expression of collagen type I and III mRNA in cultured adult rat cardiac fibroblasts.

METHODSCardiac fibroblasts(CFs) were isolated from adult Wistar rat ventricule and cultured in vitro either in normoxic or hypoxic condition. Studies were conducted with the second passage of CFs. The changes of DNA synthesis was determined by measuring the incorporation of 3H-TdR into DNA and the changes of expression of pro-alpha1 (I) collagen, pro-alpha1(III) mRNA were measured by in situ hybridization respectively.

RESULTSThe 3H-TdR incorporation of CFs was increased by 34% (P < 0.05) and 36% (P < 0.01) after 6 h, 12 h hypoxia (2% O2) exposure respectively. The level of pro-alpha1(I) collagen mRNA expression was significantly elevated in the cells under hypoxia for 4 h, 8 h, and 12 h. The expression of pro-alpha1(III) mRNA increased when cells were cultured under hypoxia for 2 h.

CONCLUSIONThese results suggest that hypoxia alone can upregulate DNA synthesis and expression of collagen type I and III mRNA in adult rat cardiac fibroblasts. It may be one of the important mechanisms by which hypoxic myocardial fibrosis occur.

Animals ; Cell Hypoxia ; Cells, Cultured ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; DNA ; biosynthesis ; Fibroblasts ; metabolism ; Male ; Myocytes, Cardiac ; cytology ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar

BACKGROUNDPressure therapy improves hypertrophic scar healing, but the mechanisms for this process are not well understood. We sought to investigate the differential expression of matrix metalloproteinases (Mmps) and collagen in posttraumatic hypertrophic scar tissue with mechanical pressure and delineate the molecular mechanisms of pressure therapy for hypertrophic scars.

METHODSFibroblast lines of normal skin and scar tissue were established and a mechanical pressure system was devised to simulate pressure therapy. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assays were used to compare differences in the mRNA and protein expression of Mmps and collagen in scar fibroblasts before and after pressure therapy.

RESULTSThe expression differed between the hypertrophic scar cell line and the normal cell line. RT-PCR assays showed that Collagen I, highly expressed in the hypertrophic scar cell line, decreased significantly after pressure therapy. Mmp2, Mmp9, and Mmp12 expression in the hypertrophic scar tissue increased significantly after pressure therapy (P < 0.05). Western blotting assays further revealed that Mmp9 and Mmp12 expression increased significantly in the hypertrophic scar tissue after pressure therapy (P < 0.05) but not Mmp2 expression (P > 0.05).

CONCLUSIONMechanical pressure induces degradation of Collagen I in hypertrophic scar tissue by affecting the expression of Mmp9 and Mmp12.

Cell Line ; Cicatrix, Hypertrophic ; enzymology ; metabolism ; Collagen Type I ; genetics ; metabolism ; Humans ; Matrix Metalloproteinase 12 ; genetics ; metabolism ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism

OBJECTIVETo investigate the feasibility of transfection of recombinant human endothelial nitric oxide synthase (eNOS) into human hypertrophic scar fibroblasts (HSFbs), and to observe NO secretion and the synthesis of collagen I and III.

METHODSRecombinant human eNOS with karyocyte expressive vector was constructed in vitro, then was transfected into HSFbs which was isolated from hypertrophic scar tissues and cultured in vitro (T group). The HSFbs untransfected (normal culture) or transfected with empty-vector was used as control group and empty-vector group respectively. The mRNA expression of eNOS, collagen I and III was determined by Realtime PCR. The content of NO was determined by NO assay kit.

RESULTSThe expression of eNOS mRNA in T group was 5.92 +/- 0.21, which was obviously higher than that in empty-vector group (0.98 +/- 0.13, P < 0.05). The expression of collagen I mRNA (0.76 +/- 0.15), and collagen III (0.79 +/- 0.08) in T group was significantly lower than those in empty-vector group (0.98 +/- 0.15, 1.02 +/- 0.12, P < 0.05, respectively). The content of NO in T group (36.1 +/- 0.8 micromol/L) was obviously higher than that in empty-vector group (28.4 +/- 1.0 micromol/L, P < 0.01) and control group (27.7 +/- 1.3 micromol/L, P < 0.01).

CONCLUSIONHSFbs can be the target cells for eNOS gene transfection. The transfected cells can express eNOS and produce NO, which inhibit the synthesis of collagen.

Cicatrix, Hypertrophic ; metabolism ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; metabolism ; Fibroblasts ; metabolism ; Humans ; In Vitro Techniques ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; RNA, Messenger ; metabolism ; Transfection

Objective: To explore the pathogenic mechanism of the miR-340/high mobility group box 1 (HMGB1) axis in the formation of liver fibrosis. Methods: A rat liver fibrosis model was established by injecting CCl(4) intraperitoneally. miRNAs targeting and validating HMGB1 were selected with gene microarrays after screening the differentially expressed miRNAs in rats with normal and hepatic fibrosis. The effect of miRNA expressional changes on HMGB1 levels was detected by qPCR. Dual luciferase gene reporter assays (LUC) was used to verify the targeting relationship between miR-340 and HMGB1. The proliferative activity of the hepatic stellate cell line HSC-T6 was detected by thiazolyl blue tetrazolium bromide (MTT) assay after co-transfection of miRNA mimics and HMGB1 overexpression vector, and the expression of extracellular matrix (ECM) proteins type I collagen and α-smooth muscle actin (SMA) was detected by western blot. Statistical analysis was performed by analysis of variance and the LSD-t test. Results: Hematoxylin-eosin and Masson staining results showed that the rat model of liver fibrosis was successfully established. Gene microarray analysis and bioinformatics prediction had detected eight miRNAs possibly targeting HMGB1, and animal model validation had detected miR-340. qPCR detection results showed that miR-340 had inhibited the expression of HMGB1, and a luciferase complementation assay suggested that miR-340 had targeted HMGB1. Functional experiments results showed that HMGB1 overexpression had enhanced cell proliferation activity and the expression of type I collagen and α-SMA, while miR-340 mimics had not only inhibited cell proliferation activity and the expression of HMGB1, type I collagen, and α-SMA, but also partially reversed the promoting effect of HMGB1 on cell proliferation and ECM synthesis. Conclusion: miR-340 targets HMGB1 to inhibit the proliferation and ECM deposition in hepatic stellate cells and plays a protective role during the process of liver fibrosis.
Animals ; Rats ; Cell Proliferation ; Collagen Type I/metabolism* ; Fibrosis ; Hepatic Stellate Cells ; HMGB1 Protein/genetics* ; Liver Cirrhosis/pathology* ; MicroRNAs/metabolism*

OBJECTIVETo characterize the role of Smads proteins in alpha 2 (I) collagen (COL1A2) gene expression induced by bone morphogenetic protein-2 (BMP-2) in odontoblast cell line MDPC-23.

METHODSEndogenous Smad protein expression was determined by immunocytochemistry. Smads function and their role in COL1A2 gene expression were investigated in cotransfection experiments using promoter-luciferase reporter gene construct.

RESULTSMDPC-23 cells expressed Smad1, Smad5 and Smad6. BMP-2 promoted the activation of COL1A2 promoter reporter construct. Transient overexpression of Smad1 or Smad5 was enhanced, while overexpression of Smad6 inhibited BMP-2-induced COL1A2 promoter activity. BMP-2 inducibility could be blocked by overexpression of Smad1 or Smad5 dominant negative mutant.

CONCLUSIONSSmad signaling is functioning and appears to be involved in BMP-2-induced COL1A2 collagen transcription in MDPC-23. Smad signaling may play an important role in odontoblast differentiation and dentin extracellular matrix formation mediated by BMP-2.

Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; genetics ; Cell Line ; Collagen ; genetics ; Collagen Type I ; Mice ; Odontoblasts ; cytology ; metabolism ; Smad Proteins ; physiology ; Transforming Growth Factor beta ; genetics

OBJECTIVE: To investigate the protective mechanisms of Chinese medicine Shexiang Tongxin Dropping Pills (STDP) on heart failure (HF). METHODS: Isoproterenol (ISO)-induced HF rat model and angiotensin II (Ang II)-induced neonatal rat cardiac fibroblast (CFs) model were used in the present study. HF rats were treated with and without STDP (3 g/kg). RNA-seq was performed to identify differentially expressed genes (DEGs). Cardiac function was evaluated by echocardiography. Hematoxylin and eosin and Masson's stainings were taken to assess cardiac fibrosis. The levels of collagen I (Col I) and collagen III (Col III) were detected by immunohistochemical staining. CCK8 kit and transwell assay were implemented to test the CFs' proliferative and migratory activity, respectively. The protein expressions of α-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), MMP-9, Col I, and Col III were detected by Western blotting. RESULTS: The results of RNA-seq analysis showed that STDP exerted its pharmacological effects on HF via multiple signaling pathways, such as the extracellular matrix (ECM)-receptor interaction, cell cycle, and B cell receptor interaction. Results from in vivo experiments demonstrated that STDP treatment reversed declines in cardiac function, inhibiting myocardial fibrosis, and reversing increases in Col I and Col III expression levels in the hearts of HF rats. Moreover, STDP (6, 9 mg/mL) inhibited the proliferation and migration of CFs exposed to Ang II in vitro (P<0.05). The activation of collagen synthesis and myofibroblast generation were markedly suppressed by STDP, also the synthesis of MMP-2 and MMP-9, as well as ECM components Col I, Col III, and α-SMA were decreased in Ang II-induced neonatal rats' CFs. CONCLUSIONS STDP had anti-fibrotic effects in HF, which might be caused by the modulation of ECM-receptor interaction pathways. Through the management of cardiac fibrosis, STDP may be a compelling candidate for improving prognosis of HF.
Rats ; Animals ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; RNA-Seq ; Transcriptome/genetics* ; Heart Failure/drug therapy* ; Collagen ; Collagen Type I/metabolism* ; Fibrosis ; Myocardium/pathology*

OBJECTIVETo investigate the effect of Qindan Capsule (QDC) on gene and protein expression of vascular adventitial collagen I and III (VAC1 and VAC3) in spontaneously hypertensive rats (SHR), for further research the possible mechanism of vascular adventitial fibroblasts remodeling.

METHODSThirty-two SHR, 40 weeks old, were equally randomized into the model group and the three treated groups treated respectively with high (750 mg/Kg x d) and low dosage QDC (150 mg/Kg x d), and losartan (30 mg/Kg x d), once a day for 12 weeks. Besides, a normal blank control group and a normal QDC (750 mg/Kg x d) medicated group were set up with same aged Wistar-Kyoto rats. Systolic blood pressures of rats were monitored, gene and protein expressions of VAC1 and VAC3 in rats' thoracic aortic adventitia were detected at the end of experiment using immune-histochemical staining and real-time quantitative fluorescent PCR respectively.

RESULTSCompared with the model group, blood pressure as well as the gene and protein expressions of VAC1 and VAC3 were all lower in the two QDC (high and low dosage) treated groups (P < 0.05).

CONCLUSIONQDC could not only effectively reduce the blood pressure in SHR, but also suppress and even reverse their thoracic aorta adventitial vascular remodeling, which is displayed by the obvious lowering of VAC1 and VAC2 expression levels.

Animals ; Aorta, Thoracic ; metabolism ; Capsules ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Hypertension ; drug therapy ; metabolism ; Male ; Phytotherapy ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY

Antisense Elements (Genetics) ; Cell Line ; Cell Proliferation ; Collagen Type I ; biosynthesis ; Genetic Therapy ; Hepatic Stellate Cells ; cytology ; metabolism ; Humans ; RNA, Messenger ; genetics ; Receptor, Angiotensin, Type 1 ; genetics ; Transfection

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*