Objective To explore the effect of overwork (OW) on extracellular matrix of arterial vessel wall in rats. Methods Random number grouping method was employed to assign 18 Sprague-Dawley rats into three groups(n=6):the control group(no special treatment),group OW(forced swimming twice a day for 15 days),and sleep deficiency(SD)+OW group(in addition to forced swimming twice a day,the rats were put on the platforms in water to limit sleep for 15 days).On the 16th day,the abdominal aorta and common carotid artery were collected after blood sampling from heart under deep anesthesia.A part of the abdominal aorta sample was taken for Masson staining of collagen fiber,and Verhoeff-Van Gieson staining was carried out for the elastic fiber of common carotid artery.Image J was employed for the quantitative analysis of collagen fiber and elastic fiber content.The expression of collagen 1(Col-1) protein was quantified by immunohistochemistry and the ultrastructure of vascular matrix was examined by transmission electron microscopy.The other part of the abdominal aorta sample was used to determine the mRNA levels of matrix metalloproteinase(MMP)-1,MMP-2,MMP-9,tissue inhibitor of metalloproteinases-1(TIMP-1),and Col-1 by quantitative real-time polymerase chain reaction. Results Compared with that in control group,the content of collagen fiber in groups OW and SD+OW had no significant change(all P>0.05);the content of elastic fiber in groups OW and SD+OW decreased(all P<0.001) and had no significant difference between each other(P>0.05).The vascular vessel wall of group OW showed slight fiber breakage,while that of group SD+OW presented wormhole-like or spongy fiber fragmentation.The mRNA levels of MMP-1 and MMP-2 in groups OW and SD+OW had no significant difference between each other(P>0.05) but were higher than that in control group(all P<0.001).The mRNA levels of MMP-9 and TIMP-1 had no significant difference among the three groups(all P>0.05).Groups OW and SD+OW had lower mRNA level(all P<0.001) and protein level(all P<0.001) of Col-1 than control group,while the mRNA and protein levels of Col-1 had no significant difference between groups OW and SD+OW(P>0.05). Conclusion OW can reduce the content of Col-1 and elastic fibers in the extracellular matrix of arterial vessels,destroy the elastic lamina of vascular wall,up-regulate the expression of MMP-1 and MMP-2,thereby injuring arterial vessels.
Animals ; Collagen Type I ; Extracellular Matrix/metabolism* ; Matrix Metalloproteinase 1/metabolism* ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; RNA, Messenger/genetics* ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1/metabolism*

Biomarkers ; urine ; Cross-Sectional Studies ; Diabetes Mellitus, Type 2 ; pathology ; urine ; Enzyme-Linked Immunosorbent Assay ; Hepatitis A Virus Cellular Receptor 1 ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; urine ; Kidney Diseases ; pathology ; urine ; Lipocalin-2 ; urine ; Membrane Proteins ; urine ; Sialoglycoproteins ; urine ; Tissue Inhibitor of Metalloproteinase-2 ; urine

OBJECTIVETo explore the effect of Modified Hangqi Chifeng Decoction (MHCD) on levels of collagen type IV (Col IV), matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2) in extracellular matrix (ECM) of glomerular mesangial cells (GMCs) in LPS induced mice.

METHODSNormal serum and telmisartan, high, medium, low dose MHCD containing serums were prepared by using serum pharmacology method. GMCs were cultured in vitro. The proliferation of mesangial cells were induced using LPS as stimulating factor. GMCs were divided into six groups, i.e., the normal group, the model group, the telmisartan group, high, medium and low dose MHCD groups. Col IV content in the supernatant of mesangial cells was detected using ELISA. Protein expressions of MMP-2 and TIMP-2 were detected using Western blot.

RESULTSCompared with the normal group, Col IV content obviously increased in the model group after 72-h LPS stimulation; protein expressions of MMP-2 and TIMP-2 were obviously up-regulated, and MMP-2/TIMP-2 ratio was down-regulated in the model group (P < 0.01). Compared with the model group, Col IV content obviously decreased in high and medium dose MHCD groups and the telmisartan group (P < 0.01); protein expressions of MMP-2 were obviously down-regulated in medium and low dose MHCD groups (P < 0.01, P < 0.05); the protein expression of TIMP-2 was obviously down-regulated in high, medium, low dose MHCD groups and the telmisartan group (P < 0.01). The pro- tein expression of TIMP-2 was obviously lower in the high dose MHCD group than in the low dose MHCD group (P < 0.01). MMP-2/TIMP-2 ratio was obviously up-regulated in the telmisartan group, high and medium dose MHCD groups (P < 0.01).

CONCLUSIONMHCD could regulate disordered MMP-2/TIMP-2 ratio in LPS induced ECM, inhibit excessive production of Col IV in ECM, promote the degradation of ECM, reduce the accumulation of ECM, thereby, delaying the process of glomerular sclerosis.

Animals ; Cells, Cultured ; Collagen Type IV ; metabolism ; Extracellular Matrix ; metabolism ; Kidney Glomerulus ; cytology ; Matrix Metalloproteinase 2 ; metabolism ; Mesangial Cells ; drug effects ; Mice ; RNA, Messenger ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism

OBJECTIVETo investigate the expressions of inflammation- and fibrosis-related genes in perinephric and subcutaneous adipose tissues in patients with adrenocorticotropic hormone (ACTH)-independent Cushing's syndrome.

METHODSThe perinephric and subcutaneous adipose tissues adipose tissues were obtained from 8 patients with ACTH-independent Cushing's syndrome undergoing laparoscopic retroperitoneal adrenalectomy. Real-time PCR was used to detect the mRNA expression levels of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metallopeptidase 2 (MMP-2), TIMP metallopeptidase inhibitor 1 (TIMP-1), early growth response 1 (EGR1), CCAAT/enhancer binding protein β(CEBPβ), uncoupling protein 1(UCP-1), PPARγ coactivator 1 alpha (PGC1α) and cell death-inducing DFFA-like effector a (CIDEA).

RESULTSThe mRNA level of CIDEA was significantly higher in the perinephric adipose tissue (peri-N) than in the subcutaneous adipose tissue (subQ) (P<0.05). The expressions of CEBPβ, UCP-1, and PGC1α mRNA in the peri-N were similar with those in the subQ. The expressions of IL-6, TIMP1 and EGR1 mRNA in the subQ were significantly higher than those in the peri-N (P<0.05). No significant difference in TNF-α and MMP-2 mRNA levels was found between peri-N and subQ.

CONCLUSIONThe expression levels of the inflammation- and fibrosis-related genes are higher in the subQ than in the peri-N of patients with ACTH-independent Cushing's syndrome, suggesting that chronic exposure to endogenous hypercortisolism may cause adipose tissue dysfunction.

Adrenalectomy ; Adrenocorticotropic Hormone ; CCAAT-Enhancer-Binding Protein-beta ; metabolism ; Cushing Syndrome ; metabolism ; surgery ; Early Growth Response Protein 1 ; metabolism ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; metabolism ; Real-Time Polymerase Chain Reaction ; Subcutaneous Fat ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Uncoupling Protein 1 ; metabolism

BACKGROUNDAs a novel molecular markerof non-small cell lung cancer (NSCLC), PRDI-BF1 and RIZ homology domain containing protein 14 (PRDM14) is over-expressed in NSCLC tumor tissues. Extracellular matrix degradation mediated by the balance between matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) is one of the most important mechanism in lung cancer metastasis. This study aimed to determine if PRDM14 promoted the migration of NSCLC cells through extracellular matrix degradation mediated by change of MMP/TIMP expression.

METHODSThe expression of PRDM14 was down-regulated in human cell line A 549 after transfection with lentiviral vector-mediated short-hairpin ribonucleic acids (shRNAs) which targeted the PRDM14 promoter. Cellular migration of shRNA-infected cells was detected by a scratch wound healing assay and transwell cell migration assay. Expression levels of MMP1, MMP2, TIMP1, and TIMP2 were measured by quantitative real-time polymerase chain reaction (RT-PCR).

RESULTSMigration of PRDM14-shRNA-infected cells was significantly inhibited relative to control cells as measured by the scratch wound healing (P < 0.05) and transwell cell migration assays (P < 0.01). The expression of MMP1 in A549 cells infected by PRDM14-shRNA was down-regulated significantly (P < 0.01), whereas the expression of TIMP1 and TIMP2 was up-regulated significantly (P < 0.01).

CONCLUSIONSPRDM14 accelerates A549 cells migration in vitro through extracellular matrix degradation. PRDM14 is considered as a potential therapeutic target in metastatic NSCLC.

Carcinoma, Non-Small-Cell Lung ; metabolism ; Cell Line, Tumor ; Cell Movement ; genetics ; physiology ; Extracellular Matrix ; metabolism ; Humans ; Matrix Metalloproteinase 1 ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Neoplasm Metastasis ; genetics ; Repressor Proteins ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism

OBJECTIVETo investigate the influence of siRNA-mediated down-regulation of CD147 on growth, proliferation and movement of human breast cancer cell line MDA-MB-231.

METHODSThe protein expression of CD147, MMP-2 and TIMP-2 of the MDA-MB-231 cells were analyzed by ABC. Lentiviral expression vector of CD147 gene was constructed and transfected into MDA-MB-231 cells. RT-PCR and Western blot were used to detect the mRNA and protein level changes of CD147 genes to identify the optimal time point, followed by detection of changes of mRNA and protein expression of MMP-2 and TIMP-2 genes. CCK-8 reagent method and cell scratch test were used to detect the proliferation and migration change of MDA-MB-231 cells. The nude mouse model of breast cancer by hypodermic injection with MDA-MB-231 cells was established to document the effect of CD147 siRNA on the tumor transplants.

RESULTSAfter transfection of lentiviral expression vector of CD147 gene, protein of CD147, MMP-2 and TIMP-2 were weakly or negative expressed, significantly weaker than those of control group (P < 0.01). After 72 hours of transfection, average down-regulation rate of CD147 and MMP-2 were 96.03% ± 0.84% and 96.03% ± 0.84%, respectively. Both CD147 mRNA and MMP-2 mRNA expression were down-regulated (P < 0.05), while TIMP-2 mRNA expression showed no significant deference (P > 0.05). No less than 2 days after transfection, cell growth of MDA-MB-231 cell line was found significantly inhibited (P < 0.05). After 24 hours of transection, average migration distance of MDA-MB-231 cell line and control group were (0.64 ± 0.12) mm and (4.69 ± 0.85) mm, respectively, which indicated a lower migrate speed. Down regulation of CD147 led to reduction of volume and mass of nude mouses. The growth of the carcinoma transplant was inhibited upon siRNA-mediated down-regulation of CD147 (P < 0.05), with an average tumor mass of (1.85 ± 0.98) g and both reduction of tumor size and tumor mass.

CONCLUSIONSCD147 may alter the MMP-2/TIMP-2 balance in MDA-MB-231 cells. CD147 gene silencing inhibits the proliferation and migration of MDA-MB-231 cells and the growth of carcinoma transplants in nude mice.

Animals ; Basigin ; metabolism ; Blotting, Western ; Breast Neoplasms ; genetics ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Mice ; Mice, Nude ; Neoplasm Transplantation ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Transfection

OBJECTIVETo investigate the effect of RbAp48 knockdown on the migration and invasion of human cervical cancer cells and explore the mechanism.

METHODSA small interference RNA (siRNA) was used to knock down the expression of RbAp48 in MS751 cells. The changes in cell migration and invasion were evaluated using wound healing assay and Transwell assay, respectively, and the expressions of RbAp48, vimentin, N-cadherin, E-cadherin, Snail, Twist, MMP-2 and TIMP-2 were determined with Western blotting.

RESULTSAfter siRNA-mediated RbAp48 knockdown, MS751 cells showed a significantly reduced expression of RbAp48 with significantly suppressed cell migration and invasion (P<0.01). RbAp48 knockdown induced obvious down-regulation of the expressions of interstitial cell phenotype proteins vimentin, N-cadherin, and MMP-2 and up-regulation of epithelial cell phenotype proteins E-cadherin and TIMP-2, suggesting the inhibition of epithelial- mesenchymal transition of the cells. The expressions of Snail and Twist were significantly down-regulated in the cells following RbAp48 knockdown.

CONCLUSIONKnockdown of RbAp48 can significantly inhibit epithelial-mesenchymal transition and suppress the migration and invasion of cervical cancer cell line MS751, the mechanism of which may involve the down-regulation of Snail and Twist expressions.

Antigens, CD ; metabolism ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Movement ; Down-Regulation ; Epithelial-Mesenchymal Transition ; Female ; Gene Knockdown Techniques ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Neoplasm Invasiveness ; Nuclear Proteins ; metabolism ; RNA, Small Interfering ; Retinoblastoma-Binding Protein 4 ; genetics ; Snail Family Transcription Factors ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Transcription Factors ; metabolism ; Twist-Related Protein 1 ; metabolism ; Up-Regulation ; Uterine Cervical Neoplasms ; pathology ; Vimentin ; metabolism

OBJECTIVETo explore the active ingredients in the Chinese yellow wine could inhibit the proliferation and migration of rat vascular smooth muscle cells induced by homocysteine (Hcy).

METHODSThe primary culture and identification of rat vascular smooth muscle cells (VSMCs) was conducted, and the VSMCs in passage 4-7 were used in the following experiments. The VSMCs were divided into 7 groups: control, Hcy (1 mmol/L), Hcy + oligosaccharide, Hcy + polypeptides, Hcy + polyphenols, Hcy + alcohol, Hcy + Chinese yellow wine and were given the corresponding treatment. The proliferation of VSMCs was determined by MTT. Transwell chambers and would healing were employed to test the migratory ability of VSMCs. Wester blot and gelatin zymography were used to investigate the expressions and activities of metal matrix proteinase 2/9 (MMP-2/9) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in VSMCs of each group.

RESULTSCompared with control group, the proliferation, migration and the expression and activity of MMP-2/9 of VSMCs were significantly increased in the VSMCs of Hcy group (P < 0.01). Compared with Hcy group, the proliferation, migration and the expression and activity of MMP-2/9 of VSMCs were significantly decreases in the VSMCs of polypeptides group, polyphenols group and Chinese yellow wine group. However, the expression of TIMP-2 among each group had no significant difference.

CONCLUSIONPolypeptides and polyphenols in the Chinese yellow wine could inhibit the proliferation and migration of VSMCs induced by Hcy.

Animals ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Homocysteine ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Peptides ; chemistry ; Polyphenols ; chemistry ; Rats ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Wine

The major objective was to explore the effect of early hyperbaric oxygen (HBO) therapy on the tissue structure, apoptosis, and metalloproteinases of kidney cells in Goto-Kakizaki (GK) rats with type 2 diabetes mellitus. GK rats (n = 24) were divided randomly and evenly into model, metformin hydrochloride (MH), and hyperbaric oxygen (HBO) groups, while healthy Wistar rats (n = 8) were used as normal control group. The healthy rats in the normal control group and the GK rats in the model group were both intragastrically administered with purified water (5 mL/kg) once per day. Meanwhile, the rats in the MH group received intragastric administration of MH (250 mg/kg) once daily, while the rats in the HBO group inhaled pure oxygen under a constant pressure (0.15 MPa) for 30 min. After 3 weeks of treatment, the body weight of each rat was measured, and the blood samples were collected from tails. Subsequently, the kidneys of all rats were excised for weighing mass and further examination. For each renal sample, the sections were firstly embedded with paraffin and sliced to prepare histopathologic sections stained using HE, PAS and Masson, respectively, for subsequent observation with optical microscopy. Later, the apoptosis of kidney cells was examined using the TUNEL method by computing the apoptotic index. Furthermore, the histopathologic sections were also examined using the immunohistochemistry approach with Caspase-3, MMP-2, and TIMP-2 antibodies, respectively. At the same time, the plasma concentration of TGF-β1 of the rats in each group was detected using ELISA method. These resultant data showed that the pathological changes of the HBO group were less than those of the model group with respect to increased glomerular volume density of mesangial cells, broadening mesangial matrix and thickening basement membrane as well as swelling renal tubular epithelial cells. The index of cell apoptosis and Caspase-3 expression in the HBO group showed no significant differences (P > 0.05) compared with those in the normal control and MH groups respectively, but demonstrated significant decrease compared with that in the model group (P < 0.01). Meanwhile, the MMP-2 and TIMP-2 expressions of the HBO group were stronger than those in the model and MH groups, but weaker than those in the normal control group (P < 0.05). Although the plasma concentration of TGF-β1 in HBO, MH and model groups was greater than that in the normal control group, no significant statistical difference was distinguished among these four groups (P > 0.05). These results indicate that the HBO treatment can inhibit the apoptosis and Caspase-3 expression of renal cells of GK rats, adjust the activity of MMP-2 and its inhibitors, and reduce the accumulation of extracellular matrix. This implies that the HBO treatment might protect renal tissues, thus delaying occurrence and retaining development of diabetic nephropathy.
Animals ; Apoptosis ; Caspase 3 ; metabolism ; Diabetes Mellitus, Experimental ; physiopathology ; Diabetes Mellitus, Type 2 ; physiopathology ; Diabetic Nephropathies ; therapy ; Hyperbaric Oxygenation ; Kidney ; cytology ; Matrix Metalloproteinase 2 ; metabolism ; Oxygen ; administration & dosage ; Rats ; Rats, Wistar ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Transforming Growth Factor beta1

OBJECTIVETo study the influence of CD147 gene silencing on the expression of ANXA2, MMP-2 and TIMP-2 of thyroid medullary carcinoma TT cells and related biological characteristics.

METHODSProtein expression of CD147, ANXA2, MMP-2 and TIMP-2 was detected by immunocytochemistry.RNAi technology was used to identify the specific siRNA sequences and the optimal time point of effective inhibition of CD147 gene. The expression of ANXA2, MMP-2 and TIMP-2 at mRNA and protein levels was detected with RT-PCR and Western blot, respectively. MTT method was used to detect the proliferation of the TT cells, flow cytometry (FCM) to detect the cell cycle and apoptosis changes of TT cells and transwell chamber assays to document the influence of CD147 gene silencing on migration and invasion of the TT cells.

RESULTSThe protein expression of CD147, ANXA2, MMP-2 and TIMP-2 proteins was variable in the TT cells. Two siRNA sequences were identified to effectively silence CD147 gene in the TT cells, in which relative expression of MMP-2 was reduced at both mRNA and protein levels; although the expression of ANXA2 mRNA and protein did not change significantly. TIMP-2 protein expression markedly decreased in an absence of its mRNA expression. The proliferation of the TT cells was inhibited upon the CD147 gene silencing along with a significant increase of G(0)/G(1) phase cells and a decrease of G(2)/M phase cells.However, the proportion of the apoptotic cells in all experimental groups did not change. The number of the penetrating cells through the membrane filters did not show significant changes in all experimental groups in the Transwell chamber assays.

CONCLUSIONSThrough RNAi technology, two CD147 siRNA sequences are identified and shown to effectively inhibit CD147 gene expression of the TT cells. CD147 gene silencing leads to growth inhibition of the TT cells and alteration of the cell cycle. However, silencing CD147 does not significantly affect the apoptosis, migration and invasion of the TT cells.

Annexin A2 ; genetics ; metabolism ; Basigin ; genetics ; metabolism ; Carcinoma, Medullary ; metabolism ; pathology ; Cell Cycle ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Humans ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Thyroid Neoplasms ; metabolism ; pathology ; Tissue Inhibitor of Metalloproteinase-2 ; genetics ; metabolism ; Transfection ; Tumor Cells, Cultured