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: In vitro 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 ( P<0.05). In the in vivo 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 ( P<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

Objective To explore the impact of sinomenine on bleomycin A5-induced pulmonary fibrosis (PF) in rats and the underlying mechanism. Methods MRC-5 cells were cultured and treated with sinomenine to determine its optimal concentration and time through the MTT assay. Subsequently, MRC-5 cells were incubated with 80 μmol/L sinomenine for 48 hours or transfected with miR-21 mimic/a disintegrin-like and metalloproteinase with thrombospondin type 1 motif (ADAMTS-1) siRNA prior to sinomenine treatment. The expression of miR-21, ADAMTS-1, collagen type 1 (Col1) and collagen type 3 (Col3) was detected by quantitative real-time PCR (qRT-PCR) and/or Western blot analysis. Thirty SD rats were randomly divided into control group, sinomenine group and sinomenine combined with miR-21 agomir group, with 10 animals in each group. Bleomycin A5 were intratracheally administered to establish the PF model. Then, rats in control group, sinomenine group and sinomenine +miR-21 agomir group were treated with 9 g/L sodium chloride solution, sinomenine and sinomenine+miR-21 agomir, respectively. On day 28, all rats were sacrificed. HE and Masson staining was performed in pulmonary tissue. The expression of ADAMTS-1, Col1 and Col3 in pulmonary tissue were detected by qRT-PCR and/or Western blot analysis. ELISA was used to measure serum procollagen type 1 carboxyterminal propeptide (P1CP) and procollagen type 3 aminoterminal propeptide (P3NP) levels. Results Administration of sinomenine decreased miR-21 levels, up-regulated ADAMTS-1 expression, and promoted Col1 and Col3 degradation in MRC-5 cells. Importantly, interfering with the miR-21/ADAMTS-1 signaling pathway partially reversed the promotive effect of sinomenine on Col1 and Col3 degradation. Treatment of SD rats with sinomenine reduced alveolitis and PF scores, decreased serum P1CP and P3NP levels, up-regulated pulmonary ADAMTS-1 expression, and down-regulated Col1 and Col3 expression. However, these effects were reversed by miR-21 agomir. Conclusion Sinomenine promotes Col1 and Col3 degradation and inhibits PF in rats by miR-21/ADAMTS-1 pathway.
Rats ; Animals ; Pulmonary Fibrosis/genetics* ; Procollagen/metabolism* ; Rats, Sprague-Dawley ; Signal Transduction ; Bleomycin/adverse effects* ; Collagen Type III/metabolism* ; MicroRNAs/metabolism*

The traditional Chinese medicine Shensong Yangxin (SSYX) can improve the clinical symptoms of arrhythmia in an integrated manner. This study aimed to investigate the electrophysiological effect of SSYX on the hearts of myocardial-infarcted rabbits and further explore the mechanism by which SSYX alleviates myocardial fibrosis. Myocardial infarction (MI) was established in rabbits by ligation of the left circumflex coronary. The rabbits were treated with SSYX (0.5 g/kg/d) or saline for 8 weeks by oral administration. Microelectrode array (MEA) technology was used in vivo for extracellular electrophysiological recordings of the infarct border zone. Masson's trichrome staining was used to observe myocardial fibrosis. Western blotting was performed to evaluate the protein expression levels of collagen I (COL I) and collagen III (COL III). Quantitative real-time polymerase chain reaction (real-time PCR) was performed to evaluate the TGF-β1 and MMP-2 mRNA expression levels. The results showed that the total activation time (TAT) and the dispersion of TAT were significantly increased and the excitation propagation markedly disordered after MI. SSYX could significantly decrease TAT and the dispersion of TAT, and significantly ameliorate the chaotic spread pattern of excitation. Furthermore, SSYX treatment could significantly decrease COL I and COL III protein levels and down-regulate TGF-β1 and MMP-2 mRNA expression levels in MI rabbits. It was concluded that SSYX may ameliorate cardiac electrophysiological abnormalities in infarcted hearts by decreasing the protein levels of COL I and COL III, down-regulating the mRNA expression levels of TGF-β1 and MMP2, and thereby reducing adverse cardiac remodeling.
Animals ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Female ; Heart Rate ; drug effects ; Hyperplasia ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Myocardial Infarction ; drug therapy ; pathology ; Myocardium ; metabolism ; pathology ; Rabbits ; Transforming Growth Factor beta ; genetics ; metabolism

OBJECTIVETo investigate the effects of Biejia Ruangan Tablet ([symbol in text], BRT)-containing serum on the expression of matrix metalloproteinase (MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) in cultured renal interstitial fibroblasts.

METHODSDifferent BRT-containing sera were prepared by gastric gavages to rats with the high-dose (7 g/kg), mid-dose (3.5 g/kg), and low-dose (1.75 g/kg) BRT respectively. The expression of extracellular matrix in NRK-49F cells was induced by treatment with human transforming growth factor-β1 (recombined human TGF-β1), and BRT-containing serum. Western blotting and Northern blotting were used to measure type I and III procollagen, MMP-9, and TIMP-1.

RESULTSThe high dose BRT-containing serum could decrease the type I and III procollagen gene expression which boosted by TGF-β1, at the same time cut down TIMP-1 protein and gene expression which increased by TGF-β1 (P <0.05). Treatment of cells with recombined human TGF-β1 had no significant effect on MMP-9 expression and BRT-containing serum also had no effect on MMP-9 expression.

CONCLUSIONSHigh dose BRT has anti-fibrosis effects in NRK-49F cells, as indicated by its inhibition of type I and III procollagen and TIMP-1 expression.

Animals ; Cells, Cultured ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; Kidney ; cytology ; Male ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Serum ; metabolism ; Tablets ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transforming Growth Factor beta1 ; pharmacology

This study aims to investigate the effect of total flavones of Fructus Chorspondiatis (TFFC) on the mRNA and protein expression of collagen type I and III of rat cardiac fibroblasts (CFs) induced by angiotensin II (Ang II), and explore its anti-myocardial fibrosis molecular mechanism. Neonatal rat CFs were prepared from Sprague-Dawley rats (1-3 d after birth). The expression of collagen type I and III mRNA and protein were measured by RT-PCR and Western blotting, respectively. The study showed that stimulation of neonatal rat CFs with 100 nmol.L-1 of Ang II for 72 h resulted in a significant increase of the expression of collagen type I and III mRNA and protein. The changes on the expression level were blocked by TFFC. The results demonstrated that TFFC can inhibit myocardial fibrosis induced by Ang II in rats, which is probably associated with the collagen type I and III mRNA and protein levels up-regulated by Ang II, and TFFC was shown to decrease the expression levels of collagen type I and III mRNA and protein.
Anacardiaceae ; chemistry ; Angiotensin II ; pharmacology ; Animals ; Animals, Newborn ; Cells, Cultured ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Fibroblasts ; cytology ; metabolism ; Flavones ; administration & dosage ; isolation & purification ; pharmacology ; Fruit ; chemistry ; Myocardium ; cytology ; metabolism ; Plants, Medicinal ; chemistry ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the clinicopathologic features of collagen III glomerulopathy and its cause, pathogenesis and prognosis.

METHODSFive cases of collagen III glomerulopathy that collected from 2005 to 2014 were observed by renal biopsy. The morphologic characteristics were studied by light microscopy, immunofluorescence, immunohistochemical and electron microscopy.

RESULTSThe glomerular mesangium became expansion but no hypercellularity, basement membrane appeared thickened. The glomeruli showed collagen type III deposit by immunohistochemistry method, and collagen fibers increased by electron microscopy. The patients often show serious proteinuria, nephrotic syndrome and renal function damage.

CONCLUSIONSCollagen III glomerulopathy is an idiopathic glomerular disease, characterized by massive accumulation of collagen type III within the glomerular mesangial areas and basement membrane. Collagen III glomerulopathy is extremely rare. The etiology and pathogenesis may relate to the abnormality of collagen III gene. There is no specific treatment for it and its prognosis is poor.

Basement Membrane ; metabolism ; Biopsy ; Collagen Type III ; genetics ; metabolism ; Female ; Fluorescent Antibody Technique ; Glomerular Mesangium ; metabolism ; Humans ; Immunohistochemistry ; Kidney Diseases ; etiology ; pathology ; Kidney Glomerulus ; pathology ; Microscopy, Electron ; Prognosis ; Proteinuria ; diagnosis

OBJECTIVETo investigate the role of the PI3K/Akt signaling pathway in hydrogen sulfide-induced alterations in expression of collagen I and collagen III in hepatic stellate cells.

METHODSIn vitro cultured rat hepatic stellate cells (HSC-T6) were treated with hydrogen sulfide, or left untreated for use as controls, and divided into groups for treatment with different inhibitors for the various factors involved in the PI3K/Akt signaling pathway. Reverse transcription-PCR was used to measure Collagen I and collagen III mRNA expression. Western blotting was used to detect the protein expression of PI3K and p-Akt, which are upstream proteins of the PI3K/Akt pathway.

RESULTSCompared with the untreated control cells, the hydrogen sulfide treated cells showed elevated expression of collagen I mRNA (F =14.635, P less than 0.05), collagen III mRNA (F =14.620, P less than 0.05), PI3K protein (F =26.672, P less than 0.05), and p-Akt (F =23.522, P less than 0.05). Compared to the cells treated with hydrogen sulfide alone, the cells treated with the various inhibitors showed lower expression of collagen I mRNA (F =14.635, P less than 0.05), collagen III mRNA (F=14.620, P less than 0.05), PI3K protein (F =26.672, P less than 0.05), and p-Akt protein (F =23.522, P less than 0.05).

CONCLUSIONHydrogen sulfide can activate the PI3K/Akt pathway and elevate the expression of collagen I and collagen III in rat hepatic stellate cells.

Animals ; Cells, Cultured ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Hepatic Stellate Cells ; drug effects ; metabolism ; Hydrogen Sulfide ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; genetics ; Rats ; Signal Transduction

OBJECTIVETo observe the effect of tetrandine on gene expression of collagen type I, collagen type III, transformation growth factor-beta1 and to investigate the inhibitory effect of tetrandine on the scar tissue hyperplasia in rabbits' ears.

METHODSAfter the scar model was formed on the rabbits' ears, the rabbits were divided into 4 groups to receive intro-lesion injection with saline, or prednisolone (Pre) or tetrandrine in low concentration (L-Tet, 1.0 mg/ml) or tetrandrine in high concentration (H-Tet, 7.5 mg/ml). The morphological changes of scar tissue were observed. The changes of fibroblasts quantity and collagen expression were observed with HE and Masson staining. Immunohistochemical study was used to observe the expression level of collagen type I and collagen type III and TGF-beta1. Collagen type I and collagen type III and TGF-beta1, and signal factor Smad 3 mRNA were detected with RT-PCR.

RESULTS(1) 24 days after injury, all the wounds healed completely with formation of red, tough and hypertrophic scar. HE and Masson staining showed significant increase of fibroblasts and collagen density with irregularly arrangement. (2) Compared with that in saline group, the scar in other groups became softer, lighter and thinner, especially in H-Tet group. (3) HE and Masson staining shows the scar in Tet and Pre groups contained less fibroblasts and lower collagen dentsity with comparatively regular arrangement than that in saline group (P < 0.01), especially in H-Tet group. (4) According to the immunohistochemical study, the expression of collage type I and III and TGF-beta was positive in all the groups, but the positive rate and the ratio of collagen density I to III decreased in the order of saline, L-Tet, H-Tet and Pre groups (P < 0.01). (5) PT-PCR detection results showed that the amplification bands brightness of collagen type I and III and TGF-beta1 and signal molecular Smad 3 mRNA in scar tissue were obviously different. Compared with that in saline group, the expression of collagen type I and III and TGF-beta1 and Smad 3 mRNA decreased in Tet and Pre groups (P < 0.01). H-Tet group showed the most obvious reduce in the expression of type I collagen and TGF-beta1 and Smad 3 mRNA. Conclusions Tetrandine can significantly suppress the expression of collagen type I and collagen type III and TGF-beta1 on hypertrophic scar of rabbit ears, and reduce signal factor Smad 3 mRNA' s expression. It may be one of the important mechanism for its inhibitory effect on scar hyperplasia.

Animals ; Benzylisoquinolines ; pharmacology ; Cicatrix, Hypertrophic ; drug therapy ; genetics ; pathology ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Ear ; Fibroblasts ; Gene Expression ; Male ; RNA, Messenger ; metabolism ; Rabbits ; Smad3 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

OBJECTIVETo investigate the effect of Qindan capsule (QC) on collagen synthesis and the mechanism underlying the process in spontaneously hypertensive rats (SHRs).

METHODSTwentyfour SHRs were divided into three groups: the hypertension model group, the QC treatment group, and the losartan treatment group. Eight Wistar Kyoto (WKY) rats were used as the normal control group. The systolic blood pressure (SBP) of the rats was monitored, and the thoracic aorta adventitia of the rats was segregated. The expressions of transforming growth factor 1 (TGF-β1), Smad3, and collagens I and were measured by histological staining and reverse transcription polymerase chain reaction.

RESULTSThe SBP was significantly higher in the model group than in the normal control group (P<0.01). However, a significant SBP-lowering effect was observed in QC or losartan treatment groups (P<0.05 or P<0.01) after 3 weeks of treatment. QC-treated rats showed a decrease of approximately 40 mm Hg, and the losartan-treated rats showed a decrease of approximately 50 mm Hg at the end of treatment compared with the beginning of treatment. The protein and gene levels of TGF-β1, Smad3, and collagens I and in the model group were significantly increased compared with those in the normal control group (P<0.01). However, the levels were significantly decreased in the QC or losartan treatment group compared with the model group (P<0.05 or P<0.01). However, there was no significant difference between the QC and losartan treatment groups (P<0.05).

CONCLUSIONSQC could exert its antihypertensive effect through down-regulating TGF-β1-stimulated collagen expressions. The TGF-β1/Smad3 signaling pathway may be involved in this process.

Adventitia ; drug effects ; metabolism ; pathology ; Animals ; Blood Pressure ; drug effects ; Blood Vessels ; drug effects ; metabolism ; pathology ; Capsules ; Collagen ; biosynthesis ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Losartan ; pharmacology ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Smad3 Protein ; genetics ; metabolism ; Staining and Labeling ; Systole ; drug effects ; Transforming Growth Factor beta1 ; genetics ; metabolism

OBJECTIVE: To explore the inhibitory effect of calcitonin gene-related peptide (CGRP) on cardiac fibroblast proliferation and collagen synthesis induced by isoprenaline and the underlying mechanism. METHODS: The primary cultured cardiac fibroblasts were incubated with isoprenaline (10(-5) mol/L) for 48 h after pretreatment with CGRP (10(-8) or 10(-7) mol/L) for 1 h. Cell activity was detected by MTT. The mRNA expression of collagen (types I and III) and connective tissue growth factor (CTGF) was determined by RT-PCR, and the levels of intracellular ROS were determined by DCFH-DA fluorescent probe. RESULTS: Isoprenaline significantly promoted fibroblast proliferation and up-regulated collagen (types I and III) and CTGF mRNA expression concomitantly with an increase in ROS production, which were attenuated by CGRP. The effect of CGRP on cardiac fibroblasts was inhibited by CGRP8-37, a selective antagonist of CGRP receptor. CONCLUSION CGRP is able to protect cardiac fibroblasts against isoprenaline-induced proliferation and collagen expression, which might be related to the down-regulation of CTGF expression through inhibition of ROS production.
Animals ; Animals, Newborn ; Calcitonin Gene-Related Peptide ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Connective Tissue Growth Factor ; genetics ; metabolism ; Fibroblasts ; cytology ; Isoproterenol ; antagonists & inhibitors ; pharmacology ; Myocytes, Cardiac ; cytology ; Primary Cell Culture ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism