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

The aim of this study was to determine the role of neutrophil collagenase in the pathogenesis of acute lung injury induced by endotoxin. 28 Sprague-Dawley were randomized into control group and LPS-enduced groups. Samples of left lung were obtained in 2 h (group L1), 6 h (group L2), 12 h (group L3) after intravenous LPS. Immunohistochemsitry was employed for detection of expression of neutrophil collagenase. Pathological scores, lung wet/dry weight ratio and the number of neutrophils were measured. The results showed that the concentration of neutrophil collagenase in LPS-enduced groups (group L1, L2, L3) were significantly higher than that of control group (P<0.01). Pathological scores, lung wet/dry weight ratio and the number of neutrophils in LPS-enduced groups (group L1, L2, L3) were also significantly higher than that of control group (P<0.01). Moreover, among group L1, L2 and L3, there were significant correlations in concentration of neutrophil collagenase and pathological scores, lung wet/dry weight ratio, the number of neutrophils (P<0.05). The present study showed that neutrophil collagenase play an important role in the pathogenesis and progress of endotoxic acute lung injury.
Endotoxins ; Lung/pathology ; Matrix Metalloproteinase 8/*metabolism ; Random Allocation ; Rats, Sprague-Dawley ; Respiratory Distress Syndrome, Adult/chemically

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*

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

OBJECTIVE: To investigate the expressions of matrix metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8) in nasopharyngeal carcinoma and their association with Epstein-Barr virus latent membrane protein-1 (LMP-1). METHOD: The expressions of MMP-9, IL-8 and LMP-1 were immunohistochemical studied in 53 nasopharyngeal carcinoma sections. We statistically analyzed the correlation of these data and also the relationship between the clinical features and the experimental data of these patients. RESULT: The positive expression rate of MMP-9, IL-8 and LMP-1 were all 66.04% (35/53) and their average expression score were (33.19 +/- 29.73)%, (33.46 +/- 30.23)%, (35.49 +/- 29.63)% respectively. The expressions of MMP-9 and IL-8 both showed positive correlation with the expression of LMP-1 (r = 0.792, 0.786 respectively). The expressions of MMP-9, IL-8 and LMP-1 showed significant relationship with lymph nodes metastasis (P < 0.05), but no-significant association with age, gender, pathological classification, and clinical stage. CONCLUSION The expressions of MMP-9, IL-8 and LMP-1 were significantly associated with neck lymph nodes metastasis in nasopharyngeal carcinoma. The correlation between LMP-1 and MMP-9, IL-8 showed LMP-1 might enhance neck lymph nodes metastasis by up-regulating the expressions of MMP-9 and IL-8.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Interleukin-8 ; metabolism ; Lymphatic Metastasis ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Middle Aged ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Neck ; Prognosis ; Viral Matrix Proteins ; metabolism ; Young Adult

The aim of this study was to determine the role of neutrophil collagenase in the pathogenesis of acute lung injury induced by endotoxin. 28 Sprague-Dawley were randomized into control group and LPS-enduced groups. Samples of left lung were obtained in 2 h (group L1), 6 h (group L2), 12 h (group L3) after intravenous LPS. Immunohistochemsitry was employed for detection of expression of neutrophil collagenase. Pathological scores, lung wet/dry weight ratio and the number of neutrophils were measured. The results showed that the concentration of neutrophil collagenase in LPS-enduced groups (group L1, L2, L3) were significantly higher than that of control group (P<0.01). Pathological scores, lung wet/dry weight ratio and the number of neutrophils in LPS-enduced groups (group L1, L2, L3) were also significantly higher than that of control group (P<0.01). Moreover, among group L1, L2 and L3, there were significant correlations in concentration of neutrophil collagenase and pathological scores, lung wet/dry weight ratio, the number of neutrophils (P<0.05). The present study showed that neutrophil collagenase play an important role in the pathogenesis and progress of endotoxic acute lung injury.
Animals ; Endotoxins ; Female ; Lung ; pathology ; Male ; Matrix Metalloproteinase 8 ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Respiratory Distress Syndrome, Adult ; chemically induced ; enzymology ; etiology ; pathology

OBJECTIVETo study the metastasis-associated molecules differentially expressed in highly and poorly metastatic sublines and the mechanism of metastasis in lung giant cell carcinoma.

METHODSHighly and poorly metastatic sublines (PLA801D and PLA801C)were used as metastasis model. Cell motility and invasion assay in vitro were first compared between the two sublines. Then, gelatin zymography analysis was used to determine the MMP-2 and MMP-9 activity. The protein expression level of secreted MMP-2, MMP-9, TIMP-1, TIMP-2 and intracellular expression level of p53, p16, PCNA, CD44(V6) isomeride, E-cadherin, CK18, nm23-H1 as well as the mRNA expression level of MMP-2, MMP-9, TIMP-1, TIMP-2, VEGF were compared through Western blot. Semi-quantitative RT-PCR analysis was used to determine the intracellular mRNA expression of MMP-2, MMP-9, TIMP-1, TIMP-2 and VEGF.

RESULTSThe in vitro cell invasion potential of highly metastatic subline PLA801D was significantly higher than that of poorly metastatic subline PLA801C by about 4 folds, while the cell motility potential was similar. The secreted MMP-2 activity was notably higher in PLA801D, which was initiated by the higher expression of MMP-2 at protein and mRNA level. In addition, the expression level of p53, PCNA, CK18 protein and VEGF mRNA were significantly higher, while the expression level of p16, E-cadherin and nm23-H1 protein were significantly lower in PLA801D. Some molecules such as MMP-9, TIMP-1, TIMP-2, CD44(V6) isomeride, which had been reported to be associated with tumor metastasis, were not observed to change significantly between the two sublines.

CONCLUSIONThere are significant differences in metastatic potential and phenotypes between highly and poorly metastatic sublines of lung giant cell carcinoma. Some differentially expressed molecules might be playing roles in promoting or inhibiting metastasis of lung giant cell carcinoma, which may be useful to elucidate the mechanism of metastasis.

Carcinoma, Giant Cell ; metabolism ; pathology ; Cell Line, Tumor ; Humans ; Interleukin-8 ; genetics ; Lung Neoplasms ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Neoplasm Invasiveness ; Neoplasm Metastasis ; RNA, Messenger ; analysis ; Tissue Inhibitor of Metalloproteinase-1 ; analysis ; Vascular Endothelial Growth Factor A ; genetics

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 compare the effects of matrix metalloproteinase (MMP) inhibitor doxycycline, losartan, and their combination on the expression of MMP-8, 13, tissue inhibitor of MMP-1, 2 (TIMP-1, 2), and collagen remodeling in the noninfarcted myocardium after acute myocardial infarction (AMI) in rats.

METHODSTwo hundred and fifty-four AMI rats, induced by left coronary ligation, were randomly assigned to the following groups: (1) AMI controls group (n = 64); (2) doxycycline group (30 mg x kg(-1) x d(-1), n = 63); (3) losartan group (10 mg x kg(-1) x d(-1), n = 62); (4) concomitant doxycycline and losartan group (30 and 10 mg x kg(-1) x d(-1) respectively, n = 65); and (5) Sham-operated rats (n = 30), which were randomly selected to serve as noninfarction controls. Each group was further divided into three subgroups of 1, 2, and 4 weeks that received treatment. After the completion of treatment, the rats were killed. The mRNA and protein expression of MMPs and TIMPs in the noninfarcted myocardium were quantified by RT-PCR and Western blot, respectively. The type I and type III collagen volume fraction (CVF) of the noninfarced myocardium were assessed immunohistochemically.

RESULTSNo significant difference existed in myocardial infarction sizes among the 12 subgroups of AMI controls and the three treatment groups (42%-48%, all P > 0.05). Compared with sham operated rats, the mRNA and protein expression of MMP-8 and 13 significantly increased by 39%-183% in all three subgroups of AMI controls (all P < 0.05), except both of their mRNA expressions in 2-week subgroups; the mRNA and protein levels of TIMP-1 increased only in 1-week subgroup of AMI controls by 104% and 67%, respectively (both P < 0.05); the mRNA of TIMP-2 increased in all 1, 2, and 4-week subgroups by 144%-232% (all P < 0.05), but its protein expression lagged and only enhanced in 2 and 4-week subgroups of AMI controls by 231% and 332%, respectively (both P < 0.05). Meanwhile, both type I and type III CVF of noninfarcted myocardium significantly increased in all three subgroups of AMI controls (type I CVF: 3.01%-5.64% vs 1.53%-1.67%, P < 0.01-0.001; type III CVF: 2.19%-4.42% vs 1.46%-1.59%, P < 0.05-0.001), with type I CVF being higher in 4-week than in 1 and 2-week subgroups (5.64% vs 3.01% and 3.02% respectively, all P < 0.05). Compared with AMI controls, all three kinds of treatment significantly reduced the increased mRNA and protein expressions of MMP-8, 13 and TIMP-1, 2 after AMI by 14%-60% (all P < 0.05), as well as type I/III CVF in their 2 and 4-week subgroups (type I CVF: 1.56%-2.38% vs 3.02%-5.64%, P < 0.05-0.001; type III CVF: 1.92%-2.65% vs 4.19%-4.42%, P < 0.05-0.01), except for doxycycline's effect on type III CVF in any of its three subgroups (all P > 0.05). Among the three treatment groups, significant differences existed in the above mentioned indicators only at some subgroup levels (all P < 0.05).

CONCLUSIONSLike losartan, doxycycline can also suppress the enhanced mRNA and protein expression of MMP-8, 13 and TIMP-1, 2, and reduce type I collagen deposition in the noninfarcted myocardium after AMI in rats. However, it has no effect on type III collagen deposition.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Animals ; Collagen Type I ; biosynthesis ; genetics ; Collagenases ; biosynthesis ; genetics ; Doxycycline ; pharmacology ; Drug Synergism ; Female ; Losartan ; pharmacology ; Matrix Metalloproteinase 13 ; Matrix Metalloproteinase 8 ; biosynthesis ; genetics ; Matrix Metalloproteinase Inhibitors ; Myocardial Infarction ; metabolism ; Myocardium ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics ; Tissue Inhibitor of Metalloproteinase-2 ; biosynthesis ; genetics ; Tissue Inhibitor of Metalloproteinases ; biosynthesis ; genetics

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