Objective: To investigate the effects of colony-stimulating factor 1 receptor (CSF-1R) inhibitor pexidartinib (PLX3397) on the senescence of bone marrow-derived macrophages (BMDM) stimulated by lipopolysaccharide (LPS). Methods: BMDM were isolated and cultured from femurs and tibiae of 10 male C57BL/6 mice aged 6-8 weeks (obtained from Laboratory Animal Center of Guizhou Medical University). They were divided into blank control group, LPS group (treated with 1 μg/ml LPS for 24 h) as well as low, medium and high concentration PLX3397 pretreatment groups (treated with 100, 500 and 1 000 nmol/L PLX3397 for 4 h respectively followed by 1 μg/ml LPS for 24 h). The corresponding markers of macrophages were detected by flow cytometry. Cell viability was detected by cell counting kit-8 and cellular senescence was detected by senescence-associated-β-galactosidase (SA-β-gal) staining. Meanwhile, protein expressions of cycle-dependent kinase inhibitor p16, p21 and CSF-1R were detected by Western blotting, and the expressions of p16 and p21 were detected by intracellular immunofluorescence. Real-time fluorescence quantitative PCR (RT-qPCR) was used to investigate the mRNA levels of senescence-associated secretory phenotype (SASP) genes including interleukin (IL), IL-1β, chemokine-1/10 (CXCL-1/10), matrix metalloproteinase-8 (MMP-8), and transforming growth factor-β (TGF-β). Results: The rate of SA-β-gal positive staining in medium and high concentration PLX3397 pretreatment groups [(39.33±4.93)% and (36.33±3.06)% respectively] were significantly downregulated compared with LPS group [(52.00±3.00)%] (P=0.020, P=0.005). The expression of CSF-1R protein in low, medium and high concentration PLX3397 pretreatment groups were (0.74±0.18, 0.61±0.07, 0.54±0.06), all of which were significantly lower than that in LPS group (1.16±0.08) (P=0.013, P=0.002, P<0.001). The expression levels of CSF-1R mRNA in low, medium and high concentration PLX3397 pretreatment groups (1.04±0.06, 0.90±0.05, 1.18±0.08) showed similar trend (2.90±0.25) (P<0.001). The average fluorescence intensity of p16 in all PLX3397 pretreatment groups were 49.76±3.65, 48.21±1.72, 47.99±1.26 respectively, which were significantly lower than that in LPS group (66.88±5.85) (P=0.001, P<0.001, P<0.001). The average fluorescence intensity of p21 in medium and high concentration PLX3397 pretreatment groups were (34.43±3.62, 30.13±0.86), significantly lower than that in LPS group (46.82±5.33) (P=0.043, P=0.007). The expression of p16 protein in low, medium and high concentration PLX3397 pretreatment groups (0.56±0.04, 0.55±0.04, 0.35±0.19) were significantly lower than that in LPS group (0.98±0.10) (P=0.003, P=0.002, P<0.001), as well the expression of p21 protein (0.69±0.20, 0.42±0.08, 0.26±0.14) (P=0.032, P=0.002, P<0.001). According to the results of RT-qPCR, the expressions of IL-6, IL-1β, CXCL-1, CXCL-10 and MMP-8 in PLX3397 pretreatment groups were significantly lower than those in LPS group (P<0.001), while the expression of TGF-β increased (P<0.001). Conclusions: LPS could induce the cell senescence, increase the secretion of SASP and aggravate local inflammation by activating the CSF-1R on the cell surface of bone marrow-derived macrophages. CSF-1R inhibitor PLX3397 might attenuate CSF-1R activation associated with LPS and inhibit the senescence of bone marrow-derived macrophages induced by LPS.
Mice ; Animals ; Male ; Lipopolysaccharides/pharmacology* ; Macrophage Colony-Stimulating Factor/metabolism* ; Matrix Metalloproteinase 8/metabolism* ; Mice, Inbred C57BL ; Macrophages ; Transforming Growth Factor beta/metabolism* ; RNA, Messenger/metabolism*

This study aims to investigate the intervention effect of the aqueous extract of Epimedium sagittatum Maxim on the mouse model of bleomycin(BLM)-induced pulmonary fibrosis, so as to provide data support for the clinical treatment of pulmonary fibrosis. Ninety male C57BL/6N mice were randomized into normal(n=10), model(BLM, n=20), pirfenidone(PFD, 270 mg·kg~(-1), n=15), and low-, medium-, and high-dose E. sagittatum extract(1.67 g·kg~(-1), n=15; 3.33 g·kg~(-1), n=15; 6.67 g·kg~(-1), n=15) groups. The model of pulmonary fibrosis was established by intratracheal instillation of BLM(5 mg·kg~(-1)) in the other five groups except the normal group, which was treated with an equal amount of normal saline. On the day following the modeling, each group was treated with the corresponding drug by gavage for 21 days. During this period, the survival rate of the mice was counted. After gavage, the lung index was calculated, and the morphology and collagen deposition of the lung tissue were observed by hematoxylin-eosin(HE) and Masson staining, respectively. The levels of reactive oxygen species(ROS) in lung cell suspensions were measured by flow cytometry. The levels of glutathione peroxidase(GSH-Px), total superoxide dismutase(T-SOD), and malondialdehyde(MDA) the in lung tissue were measured. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling(TUNEL) was employed to examine the apoptosis of lung tissue cells. The content of interleukin-6(IL-6), chemokine C-C motif ligand 2(CCL-2), matrix metalloproteinase-8(MMP-8), transforming growth factor-beta 1(TGF-β1), alpha-smooth muscle actin(α-SMA), E-cadherin, collagen Ⅰ, and fibronectin in the lung tissue was measured by enzyme-linked immunosorbent assay(ELISA). The expression levels of F4/80, Ly-6G, TGF-β1, and collagen Ⅰ in the lung tissue were determined by immunohistochemistry. The mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue were determined by qRT-PCR. The content of hydroxyproline(HYP) in the lung tissue was determined by alkaline hydrolysation. The expression of α-SMA and E-cadherin was detected by immunofluorescence, and the protein levels of α-SMA, vimentin, E-cadherin in the lung tissue were determined by Western blot. The results showed the aqueous extract of E. sagittatum increased the survival rate, decreased the lung index, alleviated the pathological injury, collagen deposition, and oxidative stress in the lung tissue, and reduced the apoptotic cells. Furthermore, the aqueous extract of E. sagittatum down-regulated the protein levels of F4/80 and Ly-6G and the mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue, reduced the content of IL-6, CCL-2, and MMP-8 in the alveolar lavage fluid. In addition, it lowered the levels of HYP, TGF-β1, α-SMA, collagen Ⅰ, fibronectin, and vimentin, and elevated the levels of E-cadherin in the lung tissue. The aqueous extract of E. sagittatum can inhibit collagen deposition, alleviate oxidative stress, and reduce inflammatory response by regulating the expression of the molecules associated with epithelial-mesenchymal transition, thus alleviating the symptoms of bleomycin-induced pulmonary fibrosis in mice.
Mice ; Male ; Animals ; Pulmonary Fibrosis/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Epimedium/metabolism* ; Fibronectins/metabolism* ; Matrix Metalloproteinase 7/therapeutic use* ; Matrix Metalloproteinase 8/therapeutic use* ; Vimentin/metabolism* ; Interleukin-6/metabolism* ; Mice, Inbred C57BL ; Lung ; Collagen/metabolism* ; Bleomycin/toxicity* ; RNA, Messenger/metabolism* ; Cadherins/metabolism*

OBJECTIVETo explore the effects of hydrogen sulfide (H(2)S) on myocardial fibrosis and expressions of MAPK1/3 and MMP-8 in diabetic rats.

METHODSForty adult male SD rats were randomized into 4 groups, namely the control group, diabetes mellitus group (STZ group), diabetes mellitus with H(2)S treatment group (STZ+H(2)S group), and normal rats with H(2)S treatment group (H(2)S group). Diabetes was induced by intraperitoneal injections of 40 mg/kg streptozotocin (STZ). The rats in the control group received daily intraperitoneal injections of saline, and those in STZ+H(2)S group and H(2)S group were given NaHS (100 µmol/kg) injections. After 8 weeks, the pathologies of cardiac fibrosis were examined with HE staining, and the expressions of collagen I, MAPK1/3 and MMP-8 were analyzed with Western blotting.

RESULTSCompared with the control group, the diabetic rats showed increased collagen content and obvious interstitial fibrosis in the myocardial tissue with significantly increased expression levels of collagen I, MAPK1/3 and MMP-8 (P<0.05); all these changes were obviously reversed by treatment with H(2)S (P<0.05). Collagen I, MAPK1/3 and MMP-8 expression levels and the degree of myocardial fibrosis were comparable between H(2)S group and control group (P>0.05).

CONCLUSIONHydrogen sulfide can attenuate cardiac fibrosis in diabetic rats, and the mechanism may involve the inhibition of MAPK1/3/MMP-8 signal pathway.

Animals ; Collagen Type I ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; Fibrosis ; Hydrogen Sulfide ; pharmacology ; Injections, Intraperitoneal ; Male ; Matrix Metalloproteinase 8 ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Myocardium ; pathology ; Rats ; Rats, Sprague-Dawley

This study was performed to evaluate the contribution of adiponectin to the production of interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and MMP-13 in human endothelial cells and osteoblasts in arthritic joints. Cultured human umbilical vascular endothelial cells (HUVECs) and osteoblasts were stimulated with adiponectin (1 or 10 mug ml-1) or IL-1beta (0.1 ng ml-1) in the presence or absence of hypoxia for 24 h. The protein expression patterns were examined by analyzing culture supernatants using the enzyme-linked immunosorbent assay (ELISA). Adiponectin significantly stimulated the production of VEGF, MMP-1 and MMP-13 in osteoblasts but not in endothelial cells, whereas it significantly stimulated the production of IL-6 and IL-8 in both endothelial cells and osteoblasts. The increase in VEGF production induced by adiponectin was significantly greater than that induced by IL-1beta. The production of IL-6 and IL-8 in adiponectin-stimulated endothelial cells was approximately 10-fold higher than that in IL-1beta-stimulated endothelial cells; in osteoblasts, adiponectin-induced IL-6 and IL-8 secretion was approximately twofold higher than that induced by IL-1beta. In addition, IL-8 production in endothelial cells was approximately sevenfold higher than in osteoblasts. However, IL-6 levels were similar between the two cell types, suggesting that adiponectin may be involved in the production of IL-8 in endothelial cells, which may have an important role in neutrophil recruitment to arthritic joints. Furthermore, the increases in protein expression induced by adiponectin were differentially regulated by hypoxia. In conclusion, adiponectin has a more important role than does IL-1beta in the production of mediators that drive synovitis and joint destruction in endothelial cells and osteoblasts at physiological concentrations.
Adiponectin/pharmacology/*physiology ; Arthritis, Rheumatoid/metabolism ; Cell Hypoxia ; Cell Line ; Human Umbilical Vein Endothelial Cells/drug effects/*metabolism ; Humans ; Interleukin-6/genetics/*metabolism ; Interleukin-8/genetics/*metabolism ; Matrix Metalloproteinase 1/genetics/*metabolism ; Osteoblasts/drug effects/*metabolism ; Vascular Endothelial Growth Factor A/genetics/*metabolism

BACKGROUNDSepsis-induced myocardial injury (SIMI) is caused by a variety of mechanisms. The aim of the study is to investigate the effects of metalloproteinase-8 (MMP-8) on SIMI and its mechanisms in rats.

METHODSForty male Sprague Dawley rats were randomly divided into four groups: MMP-8 inhibitor (M8I), dexamethasone (DEX), sepsis, and sham groups. The sepsis model was established by cecal ligation and puncture (CLP). Rats in the M8I group immediately received an intraperitoneal injection of M8I (0.1 mg/kg) after CLP. Rats in the DEX group immediately received an intraperitoneal (IP) injection of DEX (2 mg/kg). Rats in the sepsis and sham groups received intraperitoneal injections of normal saline. Rats were sacrificed 12 hours after CLP. Paraffin sections were stained with hematoxylin and eosin to observe the myocardium. The myocardial ultrastructure was observed with transmission electron microscopy. MMP-8, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were detected by immunohistochemistry. The expression of MMP-8 was measured by Western blotting. TNF-α and IL-1β levels in serum and myocardial tissue were determined by enzyme-linked immunosorbent assay.

RESULTSCompared with the sham group, the myocardium in the sepsis group was seriously injured. MMP-8, TNF-α and IL-1β expression was higher in the sepsis group than in the sham group. Treatment with M8I or DEX, however, attenuated sepsis induced histopathological changes in the heart, and was associated with significant reductions in serum and myocardial levels of TNF-α and IL-1β (P < 0.05). M8I significantly inhibited MMP-8 expression in myocardial tissue (P < 0.05). In addition, treatment with DEX was not associated with a change in myocardial levels of MMP-8 (P > 0.05).

CONCLUSIONMMP-8 inhibitor attenuated myocardial injury in septic rats, which might be related to reduced expression of TNF-α and IL-1β.

Animals ; Dexamethasone ; therapeutic use ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Heart Diseases ; drug therapy ; etiology ; Interleukin-1beta ; metabolism ; Male ; Matrix Metalloproteinase 8 ; metabolism ; Matrix Metalloproteinase Inhibitors ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Sepsis ; complications ; drug therapy ; Tumor Necrosis Factor-alpha ; metabolism

Previous studies suggested that polymorphisms of proinflammatory cytokine genes are important host genetic factors in Helicobacter pylori infection. The present study evaluated whether IL-8-251 polymorphism affected H. pylori eradication rate and to investigate the effect of H. pylori eradication on angiogenesis and the inflammatory process according to the IL-8-251 polymorphism. A total of 250 H. pylori-positive patients treated by endoscopic resection of the gastric neoplasm were classified into 3 groups (134 H. pylori-eradicated group, 19 H. pylori-eradication failure group, and 97 H. pylori-infected group). H. pylori status, histology, and angiogenic factor levels were evaluated at baseline, 6 months, and 18 months. H. pylori eradication rate was 92.9% in AA genotype, 85.7% in AT genotype and 88.4% in TT genotype (P value = 0.731). Elevated IL-8 and matrix metalloproteinase-9 concentrations in H. pylori-infected gastric mucosa were reversible by successful eradication of H. pylori, independent of the IL-8-251 polymorphism. It is suggested that elevated IL-8 and MMP-9 concentrations in H. pylori-infected gastric mucosa are altered significantly after successful eradication and these conditions continue for 18 months. However, IL-8-251 polymorphism does not affect H. pylori eradication rate and the sequential changes of related angiogenic factors after H. pylori eradication in Koreans.
Aged ; Alleles ; Angiopoietin-1/analysis ; Anti-Bacterial Agents/therapeutic use ; Anti-Inflammatory Agents, Non-Steroidal/therapeutic use ; Asian Continental Ancestry Group/*genetics ; Female ; Gastric Mucosa/metabolism/pathology ; Genotype ; Helicobacter Infections/*drug therapy ; *Helicobacter pylori ; Humans ; Interleukin-8/analysis/*genetics ; Male ; Matrix Metalloproteinase 9/analysis ; Middle Aged ; *Polymorphism, Single Nucleotide ; Proton Pump Inhibitors/therapeutic use ; Republic of Korea ; Retrospective Studies ; Stomach Neoplasms/pathology/surgery ; Time Factors ; Vascular Endothelial Growth Factor A/analysis

OBJECTIVETo observe the mechanism of the optimized traditional acupuncture prescription for accouchement on cervical ripening based on the molecular biology by observing related indices of cervical ripening in late-stage pregnant rats.

METHODSTwenty initial pregnant Wistar rats were randomly divided into an electroacupuncture (EA) group (n = 10) and a model group (n = 10), and other 10 non-pregnancy female rats with same lot were selected as a blank control group. EA group was treated with the optimized traditional acupuncture prescription for accouchement on the 20th day of pregnant, which performed EA at bilateral "Hegu" (LI 4) for 20 min and then at bilateral "Sanyinjiao" (SP 6) for 5 min with 2 Hz/50 Hz sparse-dense wave, while the other groups without acupuncture intervention. The contents of matrix metalloproteinase 9 (MMP-9) and interleukin 8 (IL-8) in cervix tissue were detected by ELISA method.

RESULTSCompared with the blank control group, the contents of MMP-9 and IL-8 in the model group were increased significantly (both P < 0.01). Compared with the model group, the contents of MMP-9 and IL-8 in the EA group were increased significantly (P < or = 0.05).

CONCLUSIONOptimized traditional acupuncture prescription for accouchement can increase the contents of MMP-9 and IL-8 in cervix tissue of late-stage pregnant rats so as to promote cervical ripening, and the mechanism of EA in promoting cervical ripening is explained from the perspective of molecular biology.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Cervical Ripening ; metabolism ; Cervix Uteri ; enzymology ; Female ; Humans ; Interleukin-8 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Models, Animal ; Pregnancy ; Rats ; Rats, Wistar

BACKGROUNDParthenolide has been tested for anti-tumor activities, such as anti-proliferation and pro-apoptosis in recent studies. However, little is known about its role in the process of tumor angiogenesis. This study aims to investigate the effects and potential mechanisms of parthenolide on the proliferation, migration and lumen formation capacity of human umbilical vein endothelial cells.

METHODSDifferent concentrations of parthenolide were applied to the human breast cancer cell line MDA-MB-231 cells. After 24-hour incubation, the culture supernatants were harvested and used to treat human umbilical vein endothelial cells for 24 hours. Then an inverted fluorescence phase contrast microscope was used to evaluate the human umbilical vein endothelial cells. The secretion of vascular endothelial growth factor (VEGF), interleukin (IL)-8 and matrix metalloproteinases (MMP)-9 in the culture supernatant of the MDA-MB-231 cells was then measured with enzyme-linked immunosorbent assay (ELISA) assays.

RESULTSSuppression of proliferation, migration, and the lumen formation capacity of human umbilical vein endothelial cells was observed in the presence of the culture supernatants from the breast cancer cell line treated with different concentrations of parthenolide. Parthenolide decreased the levels of the angiogenic factors MMP-9, VEGF, and IL-8 secreted by the MDA-MB-231 cells.

CONCLUSIONSParthenolide may suppress angiogenesis through decreasing angiogenic factors secreted by breast cancer cells to interfere with the proliferation, migration and lumen-like structure formation of endothelial cells, thereby inhibiting tumor growth. It is a promising potential anti-angiogenic drug.

Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Female ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Interleukin-8 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Sesquiterpenes ; pharmacology ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo elucidate the relationship between collagen degradation and cervical ripening by detecting dynamic expressions of matrix metalloproteinases 2 (MMP-2) and 8 (MMP-8) in rat cervix.

METHODSPF rats were divided into 5 groups (n=6), namely non-pregnancy estrus interval group, gestational days 10, 16, and 19 groups, and immediately postpartum group. The wet weight of the cervix was measured and HE staining was used to display the general structure of the cervix. VG staining was applied to visualize the collagen fibers and muscular fibers. Immunohistochemistry was performed to observe the expressions of MMP-2 and MMP-8 in the cervix.

RESULTSHE staining showed that the rat uterine cervix consisted mainly of fibroblasts and fibrous connective tissues. A small quantity of neutrophils could be seen in the cervix stroma of the rats immediately after immediately parturition, but not at the other time points. The wet weight of the antepartum cervix had increased, and a more obvious increase was seen in the wet weight of the cervix immediately after parturition. The collagen fibers of the cervix consisted of collagen fibers and smooth muscle fibers, and their proportions showed no significant variation at the time points around the parturition. Immediately after parturition, the collagen fibers and muscular fibers in the cervix became loosened as compared with that before parturition. MMP-2 expression was found in the cervical stroma but not in the squamous epithelium in nonpregnancy, term pregnancy, and immediately after parturition; the smooth muscle cells, vascular wall, and stromal fibroblasts showed positive expression of MMP-2. Enhanced intensity of MMP-2 staining was seen in term pregnancy and postpartum group in comparison with that in the other groups. MMP-8 expression was observed in the cervix of rats immediately after parturition, with scattered neutrophils positive for MMP-8 spotted in the stroma of the ripened cervix. MMP-8 expression was not detected in the other groups.

CONCLUSIONRipened cervical fibrous tissue becomes loose and broken, and cervical ripening is accompanied by infiltration of neutrophils from exogenous vessels. These changes are particularly evident after parturition. MMP-2 and MMP-8 cooperate to degrade the cervical fibers, leading to cervical softening and expansion.

Animals ; Cervical Ripening ; metabolism ; Cervix Uteri ; enzymology ; Female ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 8 ; metabolism ; Pregnancy ; Rats ; Rats, Sprague-Dawley

Matrix metalloproteinase-9 (MMP-9) secreted from macrophages plays an important role in tissue destruction and inflammation through degradation of matrix proteins and proteolytic activation of cytokines/chemokines. Whereas the MEK-ERK and PI3K-Akt pathways up-regulate MMP-9 expression, regulation of MMP-9 by JNK remains controversial. Presently, we aimed to determine the role of JNK in MMP-9 regulation in Raw 264.7 cells. Inhibition of JNK by the JNK inhibitor SP600125 induced MMP-9 in the absence of serum and suppressed the expression of TNF-alpha, IL-6 and cyclooxygenase-2 in LPS-treated Raw 264.7 cells. In a knockdown experiment with small interfering RNA, suppression of JNK1 induced MMP-9 expression. Interestingly, mouse serum suppressed SP600125-mediated MMP-9 induction, similar to IFN-gamma. However, the inhibitory activity of mouse serum was not affected by pyridone 6, which inhibits Janus kinase downstream to IFN-gamma. In addition to mouse serum, conditioned media of Raw 264.7 cells contained the inhibitory factor(s) larger than 10 kDa, which suppressed SP600125- or LPS-induced MMP-9 expression. Taken together, these data suggest that JNK1 suppresses MMP-9 expression in the absence of serum. In addition, the inhibitory factor(s) present in serum or secreted from macrophages may negatively control MMP-9 expression.
Animals ; Anthracenes/metabolism ; Cell Line ; Culture Media, Conditioned/*chemistry ; Enzyme Activation ; Enzyme Induction ; Enzyme Inhibitors/metabolism ; Extracellular Signal-Regulated MAP Kinases/genetics/metabolism ; *Gene Expression Regulation, Enzymologic ; MAP Kinase Signaling System/physiology ; Macrophages/cytology/*metabolism ; Matrix Metalloproteinase 9/genetics/*metabolism ; Mice ; Mitogen-Activated Protein Kinase 8/genetics/*metabolism ; NF-kappa B/genetics/metabolism ; Proto-Oncogene Proteins c-akt/genetics/metabolism ; Tumor Necrosis Factor-alpha/metabolism ; p38 Mitogen-Activated Protein Kinases/genetics/metabolism