Liuwei Dihuang Pills-elicited inhibition of MMP-2/MMP-9 via RAGE on tight junction protein of Aβ1-40-injured bEnd.3 cells / 中成药

AIM To investigate the protective effects and the mechanism of the Liuwei Dihuang Pills on mouse brain microvascular endothelial(bEnd.3)cells damaged by β-Amyloid protein1-40(Aβ1-40).METHODS CCK8 method was used to detect the effects of Aβ1-40 and medicated serum of Liuwei Dihuang Pills(MSLDP)on cell activity,and to screen the appropriate concentration.bEnd.3 cells of the control group,the Aβ1-40 group,the MSLDP+Aβ1-40 group and the MSLDP group had their low density lipoprotein-associated protein 1(LRP1),receptor for advanced glycation end products(RAGE),matrix metalloproteinase-2(MMP-2),MMP-9,scaffold protein zonule protein-1(ZO-1)detected by Western blot.bEnd.3 cells assigned into the control group,the Aβ1-40 group,the FPS-ZM1(RAGE inhibitor)+Aβ1-40 group and the FPS-ZM1+Aβ1-40+MSLDP group had their expressions of RAGE,MMP-9,MMP-2 and ZO-1 detected by Western blot as well.RESULTS The cell activity of bEnd.3,was dose-dependently decreased by Aβ1-40(P<0.01),but was protected by MSLDP(P<0.05,P<0.01).And 10 μmol/L Aβ1-40 and 10%MSLDP were selected for subsequent experiments.Compared with the control group,the Aβ1-40 group displayed increased protein expressions of RAGE,MMP-2 and MMP-9(P<0.01),decreased protein expressions of LRP1,ZO-1 and BDNF(P<0.05,P<0.01),and decreased fluorescence intensities of LRP1 and ZO-1(P<0.01).Compared with the Aβ1-40 group,the MSLDP group shared decreased expressions of RAGE,MMP-2,MMP-9 proteins and RAGE fluorescence intensity(P<0.05,P<0.01),and increased expressions of LRP1,ZO-1 and BDNF proteins,and the fluorescence intensity of LRP1 and ZO-1(P<0.05,P<0.01);the Aβ1-40+FPS-ZM1 group displayed decreased protein expressions of MMP-2,MMP9 and RAGE(P<0.05,P<0.01),and increased ZO-1 protein expression(P<0.05);and the Aβ1-40+FPS-ZM1+ MSLDP group displayed an even more decreased protein expressions of MMP-2,MMP9 and RAGE(P<0.01),increased ZO-1 protein expression(P<0.01)due to the the combination use of FPS-ZM1 and MSLDP.CONCLUSION Liuwei Dihuang Pills can protect the tight junction of bEnd.3 injured by Aβ1-40 and neurovascular units from Alzheimer's disease by alleviating the dysfunction of the blood-brain barrier via RAGE-mediated MMP-2/MMP-9 pathway inhibition.

Relationship between matrix metalloproteinases and the occurrence and development of ovarian cancer

Ovarian cancer is one of the most malignant genital cancers, with a high mortality rate. Many researchers have suggested that matrix metalloproteinases (MMPs) have remarkably high expression in ovarian cancer tissues. MMPs are considered to be related to the occurrence, development, invasion and metastasis of ovarian cancer. Moreover, some studies have discovered that the unbalance between MMPs and tissue inhibitor of metalloproteinases (TIMPs) are associated with the malignant phenotype of tumors. This review summarizes the latest research progress of MMPs in ovarian cancer. The investigation of MMP mechanism in ovarian cancer will facilitate the development of effective anti-tumor drugs, and thereby improve the survival rate of patients with ovarian cancer.

The effect and mechanism of allicin on the migration and invasion properties of human osteosarcoma U87 cells / 西安交通大学学报(医学版)

ABSTRACT:Objective To investigate the anti-metastatic effect of allicin on glioma cell line U87 and related mechanisms.Methods In this study,we employed MTT assay to test the anti-proliferative effect of allicin. Transwell assay was used to test the anti-metastatic ability of allicin.Real-time PCR and Western blotting were employed to test the effect of allicin on the expressions of matrix metalloproteinase-2 (MMP-2 ) and matrix metalloproteinase-9 (MMP-9).Western blotting was employed to test the phosphorylated level of p38.Results Allicin could significantly inhibit the proliferation and invasion of U87 cells (concentration>8 μg/mL,P <0.05). Meanwhile allicin (concentration<8μg/mL)could inhibit the invasion of U87 cells.After treatment with allicin for 24 hours,the expressions of MMP-2 and MMP-9 were decreased significantly (P < 0.05 ).Moreover,allicin treatment decreased the phosphorylated level of p38 obviously (P < 0.05 ).Conclusion Allicin inhibits the invasion and migration of glioma cell line U87 by reducing the expressions of MMP-2 and MMP-9 via suppressing the activity of p38 signal pathway,suggesting that allicin is a potential therapeutic agent for glioma.

Effects of mechanical stretch on gene expression of extracellular matrix in rabbit corneal fibroblasts / 医用生物力学

Objective To explore the combined effects of mechanical stretch and interleukin-1β (IL-1β) on gene expression of extracellular matrix in rabbit corneal fibroblasts. Methods Isolated rabbit corneal fibroblasts were subjected to 15% equibiaxial stretch at frequency of 0.1 Hz for 12 h, 24 h and 36 h, respectively, in presence of IL-1β. The gene expressions of matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases 1 (TIMP-1) and collagen type I alpha 1 (Collagen Iα1) were detected by real-time quantitative PCR. Results The mRNA levels of MMP-1, MMP-3 and MMP-9 could be up-regulated by IL-1β alone. However, MMP-1 and MMP-3 mRNA levels decreased with time, while MMP-9, TIMP-1 and collagen Iα1 increased with time. Compared with corresponding IL-1β treatment with mechanical stretch groups, the mRNA levels of MMP-1, MMP-3 and MMP-9 were increased and the mRNA levels of TIMP-1 and collagen Iα1 were decreased in a time-dependent manner. The mRNA level of Collagen Iα1 was decreased by loading mechanical stretch alone, and would further decrease time-dependently in combination with IL-1β treatment. Conclusions Mechanical stretch combined with IL-1β may facilitate the corneal tissue damage, thereby contribute to the development of keratectasia.

The effect of tumor necrosis factor (TNF)-alpha to induce matrix metalloproteinase (MMPs) from the human dental pulp, gingival, and periodontal ligament cells / 대한치과보존학회지

OBJECTIVES: In the present study, three kinds of tissues cells (pulp, gingiva, and periodontal ligament) were investigated if those cells express MMP and TIMP when they were stimulated with neuropeptides (substance P, CGRP) or proinflammatory cytokine, TNF-alpha. MATERIALS AND METHODS: The cells cultured from human dental pulp (PF), gingiva (GF) and periodontal ligament were (PDLF) stimulated with Mock, SP, TNF-alpha, and CGRP for 24 hrs and 48 hrs. for an RNase protection assay and Enzyme Linked Immunosorbent Assay. Cells (PF, GF and PDLF) seeded in 100 mm culture dish were stimulated with SP (10(-5), 10(-8) M) or only with medium (Mock stimulation) for 4hrs and for 24 hrs for RNase Protection Assay, and they were stimulated with CGRP (10(-5) M) and TNF-alpha (2 ng/mL) for 24 hrs and with various concentraion of TNF-alpha (2, 10, and 100 ng/mL) for Rnase Protection Assay with a human MMP-1 probe set including MMP 1, 2, 8, 7, 8, 9, 12, and TIMP 2, 3. In addition, cells (PF, GF and PDLF) were stimulated with Mock and various concentraion of TNF-alpha(2, 10, and 100 ng/mL) for 24 hrs and with TNF-alpha(10 ng/mL) for 48 hrs, and the supernatents from the cells were collected for Enzyme Linked Immunosorbent Assay (ELISA) for MMP-1 and MMP-13. RESULTS: The expression of MMPs in PF, GF, PDLF after stimulation with SP and CGRP were not changed compared with Mock stimulation for 4 hrs and 24 hrs. The expression of MMP-1, -12, -13 24 hrs after stimulation with TNF-alpha were upregulated, however the expression of TIMP-3 in PF, GF, PDLF after stimulation with TNF-alpha were downregulated. TNF-alpha (2 ng/mL, 10 ng/mL, 100 ng/mL) increased MMP-1 and MMP-12 expression in PF dose dependently for 24 hrs. CONCLUSIONS: TNF-alpha in the area of inflammation may play an important role in regulating the remodeling of dentin, cementum, and alveolar bone.

Reacción de la dentina a los sistemas adhesivos resinosos: aspectos biológicos relacionados y biodegradación de la capa híbrida / Reaction of the dentin adhesive resin system: issues related biological and bio-degradation of the hybrid layer

La adhesión con sistemas adhesivos resinosos en dentina está siendo cuestionada, los estudios longitudinales in vivo y de envejecimiento in vitro al respecto demuestran que existe una degradación de la capa híbrida a nivel de las paredes pulpares del diseño cavitario. El presente artículo plantea una síntesis de numerosas conclusiones obtenidas de diversas investigaciones, para dar a entender la realidad de la adhesión en dentina y despertar una actitud restauradora diferente más allá del empleo único de sistemas adhesivos resinosos para pretender una unión adhesiva longeva en la dentina.

The adhesion with resinous adhesive systems in dentine has been questioned. Longitudinal studies in vivo and in vitro have demonstrated degradation of the hybrid layer at the level of the pulpar walls. The present article summarizes numerous conclusions obtained from different investigations, to explain the updates of adhesion in dentine and to promote a different restoring attitude that goes beyond the resinous adhesive systems alone.

Expression of collagenase in mice with hyperoxia-induced acute lung injury / 中华急诊医学杂志

Objective To investigate the role of interstitial collagenase in the pathogenesis of acute lung injury induced by hyperoxia outside of sealed cages and breath room air,and to study the mechanism of The severity of lung injury.Methods Seventy-two C57BL/6 mice were divided into normal control group,hyperoxia for 24 hours group,hyperoxia for 48 hours and hyperoxia for 72 hours group randomly,18 mice in each group.The hyperoxia group exposedin sealed cages with＞95%oxygen,and the control group were put in the inspiratory room.The expression of interstitial collagenase mRNA and protein in lung tissues was studied by reverse transcript-polymerase chain reaction(RT-PCR)and immunohistochemistry.Results Hyperoxia caused acute lung injury in mice.by The expression of interstitial collagenase mRNA in lung tissues was increased after 24 hours of hyperoxia compared with their control group[0.59±0.11 vs 0.07±0.01,q=3.t5 P＜0.01],the expression was higher at 72 hours of hyperoxia(0.68±0.12,q=3.78 P＜0.01).Immunohistochemistry study showed interstitial collagenase protein was mainly expressed in cytoplasm of airway epithelial cells,while Ⅱ type alveolar epithelial cells mainly and vascular smooth muscle cells in hyperoxia mice.The expression of interstitial collagenase protein in airway epithelium significantly increased at 24 hours of hyperoxia compared with their control group[(28.54±9.60) vs (13.48±4.32)q=2.62 P＜0.05],and the expression level was lower after 48 and 72 hours of hyperoxia(20.32±5.68) vs, (15.24±4.65).Conclusion Hyperoxia cause acute lung injury in mice;interstitial collagenase play an important role in the development of hyperoxia-induced lung injury in mice.

Effect of Heparin on Expression of mRNA of MMP 1,2,9 in Adult Rat Wound

PURPOSE: This study was to investigate how the heparin, which has been known to induce neovascularization by MMP in the infarcted tissue of the myocardium, had influence on the expression of mRNA of MMP 1,2,9 of the skin wound of rat. METHODS: Full depth skin wounds were created on the dorsum of Sprague-Dawley 60 rats. The experimental rats were divided into two groups according to the concentration of heparin(100microgram/ml in 20, 300microgram/ml in 20). Heparin soaked gelatin sponges in different concentration were inserted into the pocket of experimental rats and the wounds were closed. Normal saline soaked gelatin sponges were used in control rats. Wounds were harvested at 48 and 72 hours after closure. We performed histologic study in H-E stain. RNA was isolated from the harvested tissue and then real time polymerase chain reaction was performed to determine the gene expression of MMP-1,2,9. RESULTS: We observed that inflammatory cell decreased in heparin soaked group and heparin increased the expression of MMP-1,9 mRNA of dorsal wound of rat at 72 hours (p<0.05). CONCLUSION: This result suggest that heparin may be used inducing another factor inducing scarless wound healing by increasing MMP.

EXPRESSION OF MATRIX METALLOPROTEINASE-9 IN HUMAN ABDOMINAL AORTIC ANEURYSMAL TISSUES / 药物分析学报

Objective To study the effects of MMP-9 (Matrix Metalloproteinase-9, MMP-9) in the pathogenesis of abdominal aortic aneurysms (AAAs) by localizing the expression of MMP-9 in the aneurysmal tissues. Methods By means of immunohistochemistry, the frozen sections (5 μm) with aneurysmal tissues (n = 10) were incubated with MMP-9 antibody-added agents, then the sections were stained and observed under the microscope to localize the expression of MMP-9, which displayed a brown precipitate within the arterial walls. The normal arterial wall tissues(n= 10)and the diseased arterial wall tissues from the arterial occlusive diseases (AODs) (n= 15) were also immunized exactly the same way as control. Results A quantity of positive granules which appeared within the aortic media showed the strong expression of MMP-9 in the AAAs, with the positive rate reaching 95%(19/20), while no expression of MMP-9 was observed in the normal artery. However, the scattered distributed positive granules were scen within the arterial wall of some cases of the AODs, implying the weak positive expression of MMP-9 in this disease with the positive rate of 26.7%(4/15). There was a significant difference of the expression of MMP-9 within the arterial wall between the AAAs and AODs(P＜0. 01). Conclusion High expression of MMP-9 within the aortic media faciliatates the degradation of collagen and elastin fibres and subsequent dilation of the aortic artery , thus playing an important role in the pathogenesis of AAAs. To refrain MMP-9 from enhanced expressing within the aortic wall is of clinical significance in the prevention and treatment of AAAs.

Immunolocalization of Matrix Metalloproteinase-2, -9 and Tissue Inhibitor of Metalloproteinase-1, -2 in Suture-induced Corneal Neovascularization

PURPOSE: Metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been linked to the angiogenic process in general. In order to understand the potential roles of MMPs and TIMPs in corneal neovascularization process, we examined the expression and activities of MMP-2, MMP-9, TIMP-1 and TIMP-2 during the course of suture-induced corneal neovascularization in rat model. METHODS: Corneal neovascularization of rat cornea was induced by suturing. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in sutured corneas was examined by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The activities of MMP-2 and MMP-9 were measured before and after suture by gelatin zymography. RESULTS: MMP-2 proenzyme, and TIMP-1, -2 were expressed in normal corneas, predominantly in corneal epithelium. After injury, expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 increased, notably in healing corneal epithelium, infiltrating inflammatory cells, stromal fibroblasts, and ingrowing vascular endothelial cells. The intensity of immunostaining and enzymatic activities of MMP-2 and MMP-9 paralleled the magnitude of inflammatory cell infiltration, which peaked around day 7 after suture. Immunoreactivity of MMP/TIMP decreased significantly two weeks after suturing. At day 35 after suture, staining of MMP-2, TIMP-1, -2 remained visible only in corneal epithelium and vascular endothelial cells. CONCLUSIONS: MMPs as well as TIMPs were upregulated during suture-induced corneal neo-vascularization, suggesting that both may take part in extracellular matrix remodeling in the corneal wound healing, inflammatory, and neovascularization processes.

Serologic and histopathologic study of Chlamydia pneumoniae infection in atherosclerosis: a possible pathogenetic mechanism of atherosclerosis induced by Chlamydia pneumoniae

Chronic infection and inflammation have recently been implicated as important etiologic agents for atherosclerosis in general and, in particular, ischemic heart disease. Several agents have been suggested as possible candidates for the chronic inflammation including cytomegalovirus, Helicobacter pylori and Chlamydia pneumoniae. We hypothesized that a vascular infection with C. pneumoniae may induce a chronic inflammatory reaction in the host vascular tissue and activated inflammatory cells may express inflammatory mediators such as cyclooxygenase-2 (COX-2) and matrix metalloproteinases (MMPs). At first, we evaluated the relationship between C. pneumoniae infection and atherosclerosis indirectly by serologic study, and then, to confirm our hypothesis, we performed an immunohistochemical study of atherosclerotic plaques. The seropositive rate of anti-Chlamydia pneumoniae IgG was higher in the disease group (Group I, 59.8%, n = 254) than in the negative control group (Group III, 47.4%, n = 97) (p = 0.041), but the anti-Chlamydia pneumoniae IgA was not different in seropositivity between the two groups (Group I, 64.6%; Group III, 57.7%). The simultaneous seropositive rates of both IgG and IgA were 56.7% in Group I and 43.3% in Group III (p = 0.033). In subgroups without the conventional risk factors of atherosclerosis, these findings were more prominent. Furthermore, we performed immunohistochemical staining on the atherosclerotic aortic tissues obtained from patients that were seropositive to C. pneumoniae (n = 5), by using antibodies to C. pneumoniae, COX-2, and MMP-9. The immunoreactivity for COX-2 and MMP-9 increased in the atherosclerotic plaques itself, predominantly in the surrounding area of immunoreactive C. pneumoniae. These findings support our hypothesis and C. pneumoniae may participate in a pathogenetic mechanism for atherogenesis or progression of atherosclerosis. The present study may open a promising perspective concerning future therapeutic trials of chronic inflammation related atherogenesis under pathophysiological conditions.

Immunodetection of Metalloproteinases(MMP-2 and MMP-9) and Tissue Inhibitors of Metalloproteinases(TIMP-2) in Breast Cancer Tissues

The enhanced process of proteolysis of both the basement membrane and the stromal extracelluar matrix (ECM) contributes to the escape of breast cancer cells into the neighboring tissues, eventually leading to the formation of distant metastases. A group of enzymes thought to play a role in tumor cell invasion are the matrix metalloproteinases (MMPs). Much attention has been focused on MMP-2 and MMP-9, which are 2 members of the MMP family active against collagen of the basement membrane. The enzymatic activities of MMP-2 and MMP-9 are inhibited by the tissue inhibitors of metalloproteinases (TIMPs). TIMP-2, one member of TIMPs, inhibits MMP-2 and MMP-9. The imbalance between TIMPs and MMPs permits to tumor invasion and metastasis. Theretore, TIMPs constitute promising targets in the developmemt of anticancer terapies. Immunohistological stainings of MMP-2, MMP-9 and TIMP-2 were performed on paraffin-embedded tissue sections of 31 invasive breast carcinomas. MMP-2 and MMP-9 were associated with neoplastic cell cytoplasms in 65% of the cases and exhibited inter-tumoral variability of the staining intensity. The MMP-2 and MMP-9 stainings did not correlate with presence of metastases at time of diagnosis. TIMP-2 was detected in the peri-tumoral stroma and was present in 81% of the cases. Residual benign breast tissue was negative for TIMP-2 staining. Neoplasms with diffuse TIMP-2 staining (32%) have metastasis significantly more frequently (50% metastasis) than ceses with focal (20% metastasis) or absent (0% metastasis) TIMP-2. We conclude that the clinical outcome such as metastasis is more closely related to the presence of TIMP-2 than the corresponding MMPs. Enhanced TIMP-2 expression, therefore, may denote a stromal response to tumor invasion, indicative of aggressive behavior in the subset breast carcinoma.