mRNA Levels Of Matrix Metalloproteinase Research Articles

Effects of Porphyromonas endodontalis lipopolysaccharides on the expression of matrix metalloproteinase-9 in mouse osteoblasts

Objective: To evaluate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of matrix metalloproteinase-9 (MMP-9) mRNA and protein as well as enzyme activity in MC3T3-E1 cells and the role of nuclear factor-κB (NF-κB) in the process, so as to investigate the expression of MMP-9 dependent signaling pathways in mouse osteoblasts induced by Pe LPS. Methods: The experiment was conducted in 3 sessions: MC3T3-E1 cells were treated with various concentrations of Pe LPS (0-20 mg/L) and 10 mg/L Pe LPS for different time intervals (0-48 h). The expression of MMP-9 mRNA and protein were detected by real-time reverse transcription-PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), while the enzyme activity was detected by gelatin zymography method. The expression of MMP-9 mRNA was also detected in 10 mg/L Pe LPS treated MC3T3-El cells after pretreated with specific NF-κB inhibitor BAY 11-7082 for l h. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. Results: The levels of MMP-9 mRNA and protein increased significantly after the treatment with various concentrations of Pe LPS (0-20 mg/L), which indicated that Pe LPS induced osteoblasts to express MMP-9 in dose dependent manners. The expression of MMP-9 protein increased from (5 395±362) ng/L (blank control group) to (12 684±375) ng/L (20 mg/L group). Maximal induction of MMP-9 mRNA expression was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 24 h. The expression of MMP-9 mRNA in the 20 mg/L group was about 7 times than that in the blank control group. After 24 h, the expression of MMP-9 mRNA decreased. Maximal expression of MMP-9 protein was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 48 h ([35 055±2 346] ng/L) showing the highest enzyme activity. The mRNA of MMP-9 decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. Conclusions: Pe LPS might induce the expression of MMP-9 in MC3T3-E1 cells through the signaling of NF-κB.

Fructose-rich diet differently affects angiotensin II receptor content in the nucleus and a plasma membrane fraction of visceral adipose tissue.

The adipose tissue renin-angiotensin system (RAS) is proposed to be a pathophysiological link between adipose tissue dysregulation and metabolic disorders induced by a fructose-rich diet (FRD). RAS can act intracellularly. We hypothesized that adipocyte nuclear membranes possess angiotensin receptor types 1 and 2 (AT1R and AT2R), which couple to nuclear signaling pathways and regulate oxidative gene expression under FRD conditions. We analyzed the effect of consumption of 10% fructose solution for 9 weeks on biochemical parameters, adipocyte morphology, and expression of AT1R, AT2R, AT1R-associated protein (ATRAP), NADPH oxidase 4 (NOX4), matrix metalloproteinase-9 (MMP-9), and manganese superoxide dismutase (MnSOD) in adipose tissue of Wistar rats. We detected AT1R and AT2R in the nuclear fraction. FRD reduced the level of angiotensin receptors in the nucleus, while increased AT1R and decreased AT2R levels were observed in the plasma membrane. FRD increased the ATRAP mRNA level and decreased MnSOD mRNA and protein levels. No significant differences were observed for MMP-9 and NOX4 mRNA levels. These findings coincided with hyperleptinemia, elevated blood pressure and triglycerides, and unchanged visceral adipose tissue mass and morphology in FRD rats. Besides providing evidence for nuclear localization of angiotensin receptors in visceral adipose tissue, this study demonstrates the different effects of FRD on AT1R expression in different cellular compartments. Elevated blood pressure and decreased antioxidant capacity in visceral fat of fructose-fed rats were accompanied by an increased AT1R level in the plasma membrane, while upregulation of ATRAP and a decrease of nuclear membrane AT1R suggest an increased capacity for attenuation of excessive AT1R signaling and visceral adiposity.

Upregulation of Matrix Metalloproteinase-9 in Primary Cultured Rat Astrocytes Induced by 2-Chloroethanol Via MAPK Signal Pathways.

2-Chloroethanol (2-CE) is one of the reactive metabolites of 1,2-DCE in vivo, which might contribute to brain edema formation induced by 1,2-dichloroethane (1,2-DCE) poisoning. Thus, the purpose of this study was to explore the roles of mitogen-activated protein kinase (MAPK) signal pathways in upregulation of matrix metalloproteinase-9 (MMP-9) in 2-CE exposed rat astrocytes. Expression of p38 MAPK (p38), extracellular signal regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and MMP-9 at both protein and gene levels in rat astrocytes were determined using western blot and real-time RT-PCR methods. The results showed that both protein and mRNA levels of MMP-9 in 2-CE exposed astrocytes significantly increased. Meanwhile, protein levels of phosphorylated p38 (p-p38), ERK1/2 (p-ERK1/2) and JNK1/2 (p-JNK1/2) in 2-CE exposed astrocytes also significantly increased. In addition, both protein and mRNA levels of MMP-9 significantly decreased in response to reduced protein levels of p-p38, p-ERK1/2 and p-JNK1/2 achieved by supplement with their specific inhibitors, indicating that activation of MAPK signal pathways might play an important role in upregulation of MMP-9 expression at the transcriptional level in 2-CE exposed astrocytes. Furthermore, since pretreatment of n-acetyl-l-cysteine (NAC), a powerful antioxidant amino acid, could attenuate the elevated levels of MMP-9, p-p38, p-ERK2 and p-JNK1/2 in 2-CE exposed astrocytes, activation of MAPK signal pathways in 2-CE exposed astrocytes could be mediated partially by reactive oxygen species (ROS), which was most likely generated in the metabolism of 2-CE.

Electric field stimulation protects injured spinal cord from secondary inflammatory response in rats.

To investigate acute beneficial effects of electrical field stimulation (EFS) on secondary inflammatory response in spinal cord injury (SCI) rats. Sprague-Dawley (SD) rats were divided into three groups,sham group rats received laminectomy only, control group rats were subjected to SCI only, and EFS group rats received EFS immediately after the injury. During the 30-min-stimulation, the injury potential modulated to 0 ± 0.5 mV by EFS. At 12h, 24h and 48h after the surgery, the rats in each group were sacrificed. Immunofluorescence staining for macrophage marker (ED-1), the tautomerase activity of macrophage inhibitory factor (MIF) assay and real-time PCR analysis for interleukin-1β (IL-1β) and matrix metalloproteinase-9 (MMP-9) were performed. Compared to the rats in control group, the rats treated with EFS presented less ED-1 positive cells 12h (P <; 0.05), 24h (P <; 0.01) and 48h (P <; 0.05) after the surgery and showed a lower MIF tautomerase activity 12h (P <; 0.01), 24h (P <; 0.01) and 48h (P <; 0.01) after the surgery. Moreover, EFS significantly decreased the mRNA levels of IL-β (P <; 0.05) and MMP-9 at 48h (P <; 0.01) after the injury. EFS could attenuate secondary inflammatory response of injured spinal cord shortly after SCI, and EFS treatment could be a candidate for SCI therapy.

T140 blocks the SDF-1/CXCR4 signaling pathway and prevents cartilage degeneration in an osteoarthritis disease model.

Osteoarthritis (OA) is one of the most common diseases affecting older people; however, there remains no effective targeted drug to combat OA. The aims of this study were (1) to explore the effect of T140 in regulating degeneration of articular cartilage in vivo by targeted blocking of the SDF-1/CXCR4 signaling pathway, and (2) to provide experimental evidence for the development of a novel OA-targeted pharmacotherapy. Thirty-six healthy Hartley guinea pigs were randomly divided into three groups: a T140-treated group (n = 12), a phosphate buffer saline control group (n = 12) and an untreated control group (n = 12). At 2, 4, 6, 8, 10 and 12 weeks of treatment, SDF-1 in serum was quantified by enzyme-linked immunosorbent assay. At 12 weeks of treatment, the cartilage from knee tibial plateau in the knee joint was collected for H&E, Safranin-O staining and Mankin grading; measurement for mRNA levels of matrix metalloproteinases (MMP-3, MMP-9 and MMP-13), aggrecan (ACAN) and collagen II (Col II) using RT-PCR; and measurement for Col II protein levels by western blot. Results showed that SDF-1 in serum increased in the T140 group and increased in the control groups. H&E and Safranin-O staining revealed less cartilage loss in T140-treated animals compared to controls. The mRNA levels of MMP-3, MMP-9 and MMP-13 in cartilage were much lower in the T140 group than other groups, but mRNA levels of ACAN and Col II in cartilage were higher in the T140-treated group. Col II protein levels in the T140 group and control groups were different. T140 can downregulate the expression of matrix-degrading enzyme and lessen the degeneration of cartilage by blocking the SDF-1/CRCR4 signaling pathway in vivo. This mechanism may present a pharmacological target for the treatment of OA.

Tenuigenin down-regulates the release of nitric oxide, matrix metalloproteinase-9 and cytokines from lipopolysaccharide-stimulated microglia

Tenuigenin (TEN), an active component of Polygala tenuifolia root extracts, has been shown to provide neuroprotection in neurodegenerative disorders. To date, most of these studies have focused on the effect that TEN has on neurons. Because activated microglia can release neurotoxic factors that cause neuronal damage, the present study was designed to investigate the effects of TEN on activated microglia. The results showed that TEN can significantly decrease the release of nitric oxide (NO) from lipopolysaccharide (LPS)-activated rat microglia in a dose-dependent manner. The western blotting results showed that TEN did not inhibit iNOS expression at protein level. However, the electron paramagnetic resonance (EPR) technique revealed that TEN directly scavenged the NO radical. Additionally, TEN can significantly decrease the secretion and mRNA levels of matrix metalloproteinase-9 (MMP-9) and pro-inflammatory cytokines (TNF-α/IL-1β) in activated microglia. At a high dose (10−4M), TEN can significantly inhibit the secretion of another gelatinolytic MMP, MMP-2, but it had no effect on the mRNA level of MMP-2. In conclusion, these results suggest that TEN exerts an anti-inflammatory effect by down-regulating the release of NO, MMP-9 and cytokines.

Female Hormone 17β-Estradiol Downregulated MMP-2 Expression and Upregulated A1PI Expression in Human Corneal Stromal Cells.

Collagens are essential for cornea functions. In non-ocular tissues, it has been demonstrated that sex hormones modulate the collagen remodeling. In this study, we investigated whether the primary female hormone 17β-estradiol plays a role in the expressions of matrix metalloproteinases and proteinase inhibitors in cultured human corneal stromal cells. We found that 17β-estradiol treatment significantly reduced the matrix metalloproteinase-2 mRNA in human corneal stromal cells as well as the matrix metalloproteinase-2 proteins, while the matrix metalloproteinase-9 mRNA level was not significantly altered. 17β-estradiol also upregulated the expression of proteinase inhibitor, alpha1-proteinase inhibitor. The expression of transcription factor specificity protein 1 was reduced by 17β-estradiol. Furthermore, 17β-estradiol did not change the viability and apoptosis of the corneal stromal cells. The downregulation of matrix metalloproteinase-2 and upregulation of alpha1-proteinase inhibitor by 17β-estradiol possibly serve as protective factor for the normal tomography in antagonizing the extracellular matrix degeneration in many cornea diseases.

Solasodine inhibits invasion of human lung cancer cell through downregulation of miR-21 and MMPs expression

Solasodine, a naturally occurring aglycone of glycoalkaloid in eggplant (Solanum melongena), was found to inhibit proliferation in various tumor cells. However, the effect of solasodine on cancer cell metastasis remains unclear. This study investigates the suppression mechanism of solasodine on motility of human lung cancer cell A549 in vitro. Results show that solasodine reduces viability of A549 cells. Treatment with non-toxic doses of solasodine suppresses markedly cell invasion. Solasodine reduces the mRNA level of matrix metalloproteinase-2 (MMP-2), MMP-9 and extracellular inducer of matrix metalloproteinase (EMMPRIN), but increases the expression of reversion-inducing cysteine-rich protein with kazal motifs (RECK), as well as tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. Immunoblotting assays indicate that solasodine is effective in suppressing PI3K and Akt phosphorylation. Moreover, solasodine downregulates oncogenic microRNA-21 (miR-21), which has been known to target RECK. Downregulation of miR-21 by miR-21 inhibitor increases RECK expression and decreases cell invasion, suggesting that downregulation of miR-21 by solasodine may contribute to elevate RECK expression and subsequently inhibiting cell invasion. Taken together, the results reveal that inhibition of A549 cell invasion by solasodine may be, at least in part, through blocking MMP expression. Solasodine also reduces PI3K/Akt signaling pathways and downregulates expression of miR-21. These findings demonstrate an attractive therapeutic potential for solasodine in lung cancer anti-metastatic therapy.

Anti-EndometrioticEffectsofPuerariaFlowerExtract inHumanEndometrioticCellsandMice.

Pueraria flowers have been used as a vegetable and an ingredient for tea and jelly. In this study, we investigated the effects of Pueraria flower extract (PFE) on endometriosis, a common gynaecological disease characterised by local sterile inflammation of peritoneal cavity. PFE suppressed the adhesion of human endometriotic cells 11Z and 12Z to human mesothelial Met5A cells. In addition, PFE significantly inhibited the migration of 11Z and 12Z cells as shown by wound-healing and transwell migration assays. PFE reduced the protein and mRNA levels of matrix metalloproteinase (MMP)-2 and MMP-9 in endometriotic cells. Moreover, extracellular signal-regulated kinase (ERK)1/2 was activated by PFE treatment, and an ERK1/2 inhibitor, PD98059, significantly inhibited PFE-inhibited cell migration in endometriotic cells. Furthermore, PFE significantly suppressed endometriotic lesion formation in a mouse model. These data suggest that Pueraria flower is a potential anti-endometriotic agent for the inhibition of endometriotic cell adhesion, migration, and MMP expression.

Palmitic acid increases invasiveness of pancreatic cancer cells AsPC-1 through TLR4/ROS/NF-κB/MMP-9 signaling pathway

Pancreatic cancer (PC) is an aggressive malady with proclivity for early metastasis. Overexpression of toll-like receptor 4 (TLR4) in pancreatic ductal adenocarcinoma, the most common type of pancreatic malignancy, correlates to tumor size, lymph node involvement, venous invasion and pathological stage. Lipopolysaccharides (LPS) are natural TLR4 ligands that have been shown to increase the invasive ability of PC cells. However, rapid inactivation of circulating LPS and low systemic absorption of inhaled LPS from the bronchoalveolar compartment make other agonists such as saturated fatty acids more suitable to be considered for TLR4-related cell invasiveness. Interestingly, PC risk was strongly associated to intake of saturated fat from animal food sources, in particular to consumption of saturated palmitic acid (PA). In the present study, we investigated the influence of PA on the invasive capacity of human PC cells AsPC-1. Using specific inhibitors, we found that PA stimulation of these tumor cells induced a TLR4-mediated cell invasion. Our results also indicate that the signaling events downstream of TLR4 involved generation of reactive oxygen species, activation of nuclear factor-kappa beta, and secretion and activation of matrix metalloproteinase 9 (MMP-9). Furthermore, PA stimulation decreased the levels of the micro RNA 29c (miR-29c). Of note, while inhibition of miR-29c increased MMP-9 mRNA levels, MMP-9 secretion and activation, and invasiveness, miR-29c mimic abrogated all these PA-stimulated effects. These results strongly suggest that miR-29c could be an attractive potential pharmacological agent for antitumoral therapy in PC.

2-Chloroethanol Induced Upregulation of Matrix Metalloproteinase-2 in Primary Cultured Rat Astrocytes Via MAPK Signal Pathways.

This study was to explore the mechanisms underlying 1,2-dichloroethane (1,2-DCE) induced brain edema by focusing on alteration of matrix metalloproteinase-2 (MMP-2) in rat astrocytes induced by 2-chloroethanol (2-CE), an intermediate metabolite of 1,2-DCE in vivo. Protein and mRNA levels of MMP-2, and the phosphorylated protein levels of p38 MAPK (p-p38), extracellular signal regulated protein kinase (p-ERK1/2) and c-Jun N-terminal kinase (p-JNK1/2) in astrocytes were examined by immunostaining, western blot or real-time RT-PCR analysis. Findings from this study disclosed that protein levels of MMP-2 were upregulated by 2-CE in astrocytes. Meanwhile, protein levels of p-p38, p-ERK1/2 and p-JNK1/2 were also increased apparently in the cells treated with 2-CE. Moreover, pretreatment of astrocytes with SB202190 (inhibitor of p38 MAPK), U0126 (inhibitor of ERK1/2) or SP600125 (inhibitor of JNK1/2) could suppress the upregulated expression of p-p38, p-ERK1/2, and p-JNK1/2. In response to suppressed protein levels of p-p38 and p-JNK1/2, the protein levels of MMP-2 also decreased significantly, indicating that activation of MAPK signal pathways were involved in the mechanisms underlying 2-CE-induced upregulation of MMP-2 expression.

Role of matrix metalloproteinase 13 gene expression in the evaluation of radiation response in oral squamous cell carcinoma.

BACKGROUND:Matrix Metalloproteinase 13 (MMP13) is a member of collagenase family and it is involved in the degradation of extracellular matrix and basement membrane protein. It is thought to be associated with tumor invasion and metastasis. Elevated MMP13 expression has been found in carcinoma of the breast, urinary bladder, head and neck and others. It is observed that MMP13 gene is also correlated with radiation response in OSCC (Oral squamous cell carcinoma) cell line based study. The present study correlates the MMP13 expressions with clinicopathological parameters and radiation response in OSCC patients.MATERIALS AND METHODS:The MMP13 mRNA levels were determined by employing qRT-PCR (real-time quantitative reverse transcriptase-polymerase chain reaction).RESULTS:We observed high expression of MMP13 mRNA in OSCC patients when compared with matched controls. Statistically significant up regulation of MMP13 mRNA expression was found in tobacco chewers, advanced T-stage (p < 0.001) and lymph node metastasis (p < 0.01). MMP13 mRNA levels were also elevated in non responders as compared to responders to radiation treatment.CONCLUSIONS:To the best of our knowledge, this is the first report that indicates role of MMP13 in radiation response in OSCC patients and could be used as potential bio-marker for radiotherapy treatment in OSCC patients.

Dachengqi Decoction Attenuates Intestinal Vascular Endothelial Injury in Severe Acute Pancreatitis in Vitro and in Vivo

Background/Aims: Dachengqi decoction (DCQD) is a well-known traditional Chinese herbal drug with strong anti-inflammatory effects. Angiopoietin-1 (Ang-1) plays a vital role in maintaining the stability and integrity of the vascular wall and prevents vascular leakage due to inflammatory mediators. Our previous work found that DCQD protects against pancreatic injury in rats with severe acute pancreatitis (SAP). This study aims to investigate the effects of DCQD on intestinal endothelial damage in both damaged human umbilical vein endothelial cells (HUVECs) and SAP rats. Methods: HUVECs were randomly divided into four groups: control group, TNF-α group, TNF-α plus Ang-1 group (Ang-1 group), and TNF-α plus DCQD group (DCQD group). Cells were incubated for 6 h, 12 h, and 24 h, before collection. The treatment concentration of DCQD was decided based on a Cell Counting Kit-8 (CCK-8) assay. The monolayer permeability of the HUVECs was assessed by measuring the transendothelial electrical resistance (TEER). Apoptosis was analyzed by flow cytometry. mRNA and protein expression of aquaporin 1 (AQP-1), matrix metalloproteinase 9 (MMP9), and junctional adhesion molecule-C (JAM-C) was evaluated by RT-PCR, immunocytofluorescence, and western blot. Forty male Sprague-Dawley rats were randomized into a control group, SAP group, SAP plus Ang-1 group (Ang-1 group), and SAP plus DCQD group (DCQD group). SAP was induced by intraperitoneal injection of cerulein and lipopolysaccharide (LPS), while the control group received 0.9% saline solution. Evans blue was injected through the penile vein and the rats were then sacrificed 12 h after modeling. Levels of serum amylase, TNF-α, IL-1β, IL-2, and IL-6 were determined by using ELISA. Intestinal tissue was analysed by histology, and capillary permeability in the tissues was evaluated by Evans blue extravasation assay. Protein and mRNA expression of AQP-1, MMP9, and JAM-C were assessed by immunohistofluorescence, western blot, and RT-PCR. Results: DCQD reduced the permeability of HUVEC induced by TNF-α in vitro. Furthermore, DCQD altered the mRNA and protein levels of JAM-C, MMP9, and AQP-1 in HUVECs after TNF-α induction. SAP intestinal injury induced by cerulein combined with lipopolysaccharides was concomitant with increased expression of JAM-C and MMP9, and reduced expression of AQP-1 in intestinal tissue. Pretreatment with DCQD attenuated SAP intestinal injury and lowered the levels of serum amylase, TNF–α, IL-1β, IL-2, and IL-6 effectively. Our study demonstrated that DCQD decreased the expression of JAM-C and MMP9 and increased the expression of AQP-1 both in vitro and in vivo. Conclusion: DCQD can reduce capillary endothelial damage in acute pancreatitis-associated intestinal injury and the mechanism may be associated with the regulation of endothelial barrier function-associated proteins AQP-1, MMP9, and JAM-C.

Anticancer activity of Nelumbo nucifera stamen extract in human colon cancer HCT-116 cells in vitro.

The aim of the present study was to investigate the anticancer activities of Nelumbo nucifera (Ba lotus) stamen ethanol crude extract (BLSEE) in human colon carcinoma HCT-116 cells. MTT assay, flow cytometry analysis and reverse transcription-polymerase chain reaction assay were employed to investigate the anticancer mechanisms of BLSEE (100, 200 and 400 µg/ml) in HCT-116 cells. BLSEE reduced HCT-116 cell proliferation in a dose-dependent manner. BLSEE treatment also significantly increased the sub-G1 population in HCT-116 cells (P=0.0020 at 400 µg/ml), as shown by flow cytometry assay. Following treatment with BLSEE, the mRNA levels of the apoptosis-associated factors Fas, Fas ligand, tumor necrosis factor-related apoptosis-inducing ligand, death receptor 4 (DR4), death receptor 5 (DR5), caspases 3, 8 and 9, and B-cell lymphoma-2 (Bcl-2) associated X protein were increased, and the expression of anti-apoptotic Bcl-2 and Bcl-extra large was decreased in HCT-116 cells. The mRNA levels of matrix metalloproteinase (MMP)-2, MMP-9, TIMP metallopeptidase inhibitor 1 and TIMP metallopeptidase inhibitor 2 were also regulated by BLSEE treatment. In addition, BLSEE was able to modulate the expression of inflammation-associated nuclear factor-κB, inhibitory κBα, inducible nitric oxide synthase and cyclooxygenase 2 in HCT-116 cells. The present study clearly indicated the cytotoxicity of BLSEE in HCT-116 cells through induced cellular apoptosis. These results also suggested the BLSEE may be a powerful agent against colon cancer cells.

74 - Photoprotective Role of Nrf2 in UVA-Mediated MMP-1 Via MAPK/AP-1 Signaling in Keratinocyte HaCaT Cells and Mouse Skin: The Photoprotective Effects of Sulforaphane

Ultraviolet A (UVA) irradiation plays a role in premature aging of the skin through triggering oxidative stress-associated stimulation of matrix metalloproteinase-1 (MMP-1) responsible for collagen degradation, a hallmark of photoaged skin. Compounds which can activate Nrf2, a transcription factor regulating antioxidant gene expression, could thus be developed into anti-photoaging agents. We aimed to investigate whether genetic silencing of Nrf2 affected UVA (4 J/cm2)-mediated MMP-1 upregulation through MAPK/AP-1 signaling in immortalized human keratinocyte (HaCaT) cells. Then, we explored the anti-photoaging effects of sulforaphane (SF) having abilities to activate Nrf2 on MMP-1 and collagen expressions in association with phosphorylation of MAPKs (ERK, JNK and p38), c-Jun and c-Fos in the skin of BALB/c mice subjected to repetitive UVA irradiation; three times per week for 2 weeks and the total dose was 60 J/cm2. Our findings revealed that Nrf2 knockdown promoted MMP-1 activity and mRNA levels in UVA-irradiated HaCaT cells. Treatment of Nrf2-depleted HaCaT cells with MAPK inhibitors significantly suppressed MMP-1 and AP-1 transcriptional activity following UVA exposure. Our results also demonstrates the in vivo relevance of the in vitro findings as SF capable of activating Nrf2 provided the protective effects on UVA-mediated MMP-1 induction and collagen depletion in correlation with decreased levels of phosphorylated MAPKs, c-Jun and c-Fos in mouse skin. In conclusion, Nrf2 plays a photoprotective role in UVA-mediated MMP-1 upregulation probably through an involvement of MAPK/AP-1 signaling. Additionally, SF with Nrf2 activating properties could protect against UVA-induced MMP-1 expression through MAPK/AP-1 pathway and may represent promising anti-photoaging candidates.

Gastrodia elata Blume Extract Modulates Antioxidant Activity and Ultraviolet A-Irradiated Skin Aging in Human Dermal Fibroblast Cells.

Gastrodia elata Blume (GEB), a traditional herbal medicine, has been used to treat a wide range of neurological disorders (e.g., paralysis and stroke) and skin problems (e.g., atopic dermatitis and eczema) in oriental medicine. This study was designed to investigate the antioxidant ability of GEB and its antiaging effect on human dermal fibroblast cells (HDF). The total phenolic and flavonoid contents of GEB were 21.8 and 0.43 mg/g dry weight (DW), respectively. The ergothioneine content of GEB was 0.41 mg/mL DW. The DPPH and ABTS radical scavenging activities of GEB at 5 and 10 mg/mL approximately ranged between 31% and 44%. The superoxide dismutase activity of GEB at 10 and 25 mg/mL was 57% and 76%, respectively. GEB increased procollagen type 1 (PC1) production and inhibited matrix metalloproteinase-1 (MMP-1) production and elastase-1 activity in UVA-irradiated HDF. PC1 messenger RNA (mRNA) levels decreased upon UVA irradiation, but recovered in response to high doses of GEB in HDF. On the contrary, GEB significantly decreased MMP-1 and elastase-1 mRNA levels, which were markedly induced in UVA-irradiated HDF. Collectively, these results suggest that GEB has sufficient antioxidant ability to prevent the signs of skin aging in UVA-irradiated human skin cells, suggesting its potential as a natural antiaging product.

Effects and mechanism of RhoC downregulation in suppressing ovarian cancer stem cell proliferation, drug resistance, invasion and metastasis.

Cancer stem cells are considered to be the root cause of tumor initiation, metastasis, recurrence and therapeutic resistance. Recent studies have reported that RhoC plays a critical role in regulating cancer stem cells; however, its function in ovarian cancer stem cells (OCSCs) remains unknown. The ovarian cancer cell line A2780, and the paclitaxel-resistant A2780 cell line (A2780-PTX) were obtained. A2780 cells were used to isolate and identify the highly invasive A2780-PM cells, and A2780-PTX cells were used to isolate and identify the highly drug-resistant and highly invasive A2780-PTX-PM cells by Transwell assay. MTT, Transwell and wound healing assays were used to compare the differences in cell proliferation, invasion and migration ability among the four cell lines. Immunofluorescence was used to detect the expression of stem cell markers CD117 and CD133. OCSCs were sorted by flow cytometry. Following si-RhoC transfection of the OCSCs, cell proliferation, drug resistance, invasion and migration ability and RhoC, CD117 and CD133 expression levels were assayed. RT-PCR was used to assess RhoC, CD117, CD133 and matrix metalloproteinase9 (MMP9) mRNA expression levels. A2780-PM and A2780‑PTX-PM cells exhibited higher cell proliferation, drug resistance, and invasion and migration ability than the A2780 and A2780-PTX cell lines. Furthermore, CD133 and CD117 expression levels were higher in the A2780-PM and A2780‑PTX-PM cells than levels in the A2780 and A2780-PTX cells. Transfection of si-RhoC in OCSCs suppressed the proliferation, drug resistance, invasion, migration and CD117 and CD133 expression levels. Furthermore, the expression levels of RhoC, CD117, CD133, MDR1, and MMP9 mRNA were downregulated in the transfected population. Taken together, our results demonstrated that RhoC downregulation may inhibit the proliferation, drug resistance, invasion and migration of OCSCs, and RhoC may play an important role in the formation of OCSCs.

The role of LOX-1 on innate immunity against Aspergillus keratitis in mice.

To explore the effects of lectin-like ox-LDL receptor (LOX-1) on innate immunity against Aspergillus fumigatus (A. fumigatus ) in mice cornea. The mRNA levels of LOX-1 were tested in normal and A. fumigatus infected corneas of C57BL/6 and BALB/c mice. The expression of LOX-1, pro-inflammatory cytokines TNF-α, CXCL1 and IL-6, anti-inflammatory cytokines IL-10, and matrix metalloproteinase 9 (MMP9) were tested with treatment with LOX-1 neutralizing antibody or control IgG in A. fumigatus infected corneas of C57BL/6. Macrophages and neutrophils were extracted from susceptible C57BL/6 mice, and pretreated with LOX-1 neutralizing antibody or IgG, then stimulated with A. fumigatus. The mRNA levels of LOX-1, TNF-α, CXCL1, IL-6, IL-10 and MMP9 were evaluated by polymerase chain reaction. The expression of LOX-1 was significantly increased in C57BL/6 mice corneas after A. fumigatus infection compared with BABL/c mice. After treatment with LOX-1 neutralizing antibody, the expression of LOX-1, TNF-α, CXCL1, IL-6, MMP9 and IL-10 in C57BL/6 corneas were significantly decreased compared with treatment with control IgG; the expression of LOX-1, CXCL1, IL-6 and IL-10 were significantly decreased in macrophages, while TNF-α and MMP9 expressions had no change; LOX-1, TNF-α, CXCL1, IL-6, MMP9 and IL-10 expressions were significantly decreased in neutrophils. The expression of LOX-1 can affect the expression of pro-inflammatory and anti-inflammatory cytokines in fungal infected corneas, macrophages and neutrophils of C57BL/6. LOX-1 inhibition rebalances the inflammatory response of fungal keratitis in mice.

Chi-Ju-Di-Huang-Wan protects rats against retinal ischemia by downregulating matrix metalloproteinase-9 and inhibiting p38 mitogen-activated protein kinase.

BackgroundRetinal ischemia is a retinal disorder related to retinal vascular occlusion, glaucoma, diabetic retinopathy and age-related macular degeneration. The study aimed to evaluate the protective effects and underlying mechanisms of Chi-Ju-Di-Huang-Wan (CJDHW) against retinal ischemia in rats.MethodsHigh intraocular pressure (HIOP)-induced retinal ischemia was established in Wistar rats by raising their intraocular pressure to 120 mmHg for 60 min with in an eye whose anterior chamber was cannulated with a 30-guage needle adapted to a normal saline bottle through an intravenous line. This ischemic insult was followed by 1 or 7 days of reperfusion. The effects of CJDHW were studied by (i) electroretinogram (ERG); (ii) real-time polymerase chain reaction to determine the retinal mRNA levels of Thy-1 and matrix metalloproteinase-9 (MMP-9); (iii) Western blot analysis to determine the retinal protein levels of B cell lymphoma 2 (Bcl-2), heme oxygenase-1 (HO-1), phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and MMP-9; (iv) hematoxylin and eosin (HE) staining; (v) fluorogold retrograde labeling; and (vi) terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) apoptosis assay. Moreover, after fixation with 4 % paraformaldehyde and 30 % sucrose, the isolated retinas were sectioned and immunolabeled with goat anti-choline acetyltransferase (ChAT) polyclonal antibody, mouse anti-vimentin monoclonal antibody and rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody. The retinal sections were then incubated with rhodamine-conjugated rabbit anti-goat antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG. A daily oral intake of 3 mL of water (vehicle; Group 2) or CJDHW (2.8 or 4.2 g/kg/day; CJDHW2.8 or CJDHW4.2; Group 3 or 4) was given for 7 consecutive days either before (preischemic drug administration) or after HIOP-induced retinal ischemic injury (postischemic drug administration). In Group 5, an intravitreal injection of 4 μL of 0.5 mM SB203580 (p38 MAPK inhibitor) was performed on the ischemic eye 15 min before retinal ischemia. The control rats received a sham procedure (Group 1) where the saline reservoir was not raised.ResultsThe ischemia-induced changes (Group 2) were significantly modulated by pretreating the rats with 4.2 g/kg/day of CJDHW (Group 4; ERG: P < 0.001 on I/R day 7; HE stain: P < 0.001 on I/R day 7; TUNEL: P = 0.05 on I/R day 7; retrograde labeling: P = 0.007 on I/R day 7; Thy-1 mRNA: P = 0.02; MMP-9 mRNA: P < 0.001; Bcl-2 protein: P = 0.02; HO-1 protein: P = 0.03; P-p38 MAPK protein: P < 0.001; MMP-9 protein: P = 0.02). These modulations included the following features (Group 2 vs. 4), increased ERG b-wave amplitudes (0.38 ± 0.04 vs. 0.81 ± 0.03), increased inner retinal thickness (45.08 ± 2.85 vs. 67.98 ± 5.48 μm), increased ChAT immunolabeling, decreased vimentin/GFAP immunoreactivity, less numerous apoptotic cells in the ganglion cell layer (1.40 ± 0.55 vs. 0.60 ± 0.55), and more numerous retinal ganglion cells (887.73 ± 158.18 vs. 1389.02 ± 53.20). Moreover, increased Thy-1 (0.31 ± 0.15 vs. 0.78 ± 0.32) and decreased MMP-9 mRNA levels were found (4.44 ± 0.84 vs. 1.13 ± 0.34), respectively. Furthermore, the Bcl-2 protein level (0.78 ± 0.08 vs. 1.80 ± 0.34) was increased while the HO-1 (0.99 ± 0.20 vs. 4.15 ± 2.08), P-p38 MAPK (1.12 ± 0.18 vs. 0.57 ± 0.18) and MMP-9 levels were decreased (0.70 ± 0.23 vs. 0.39 ± 0.10). The ischemia-associated increases in P-p38 and MMP-9 protein levels were also attenuated by 0.5 mM SB203580 (P-p38 MAPK: 1.12 ± 0.18 vs. 0.18 ± 0.07, P < 0.001; MMP-9: 0.70 ± 0.23 vs. 0.21 ± 0.07, P = 0.002). This was also the case to the MMP_enzyme activity (Group 2 vs. 4: 5.03 ± 1.57 vs. 1.59 ± 0.47, P = 0.002; Group 2 vs. 5: 5.03 ± 1.57 vs. 1.35 ± 0.41, P = 0.001).ConclusionTreatment of the rats suffering from retinal ischemia with CJDHW inhibited apoptosis, increased antioxidative activity, downregulated MMP-9 and inhibited p38 MAPK.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0109-6) contains supplementary material, which is available to authorized users.

Increased receptor activator of nuclear factor κβ ligand/osteoprotegerin ratio exacerbates cartilage destruction in osteoarthritis in vitro.

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive cartilage destruction, matrix degradation and bony changes. Subchondral bone alterations in osteoarthritis are associated with cartilage destruction. It has previously been demonstrated that osteoprotegerin (OPG) and receptor activator of nuclear factor κβ ligand (RANKL) mediate this process. The RANKL/OPG ratio is altered in OA chondrocytes compared with normal chondrocytes. In the pathogenesis of OA, abnormal expression levels of matrix metalloproteinase-13 (MMP-13) are secreted by chondrocytes has a vital role in the progression of cartilage erosion. In the present study, the effect of various RANKL/OPG ratios on MMP-13 expression levels was investigated in interleukin-1β-stimulated SW1353 human chondrosarcoma cells. Cell viability was assessed by MTT assay and MMP-13 mRNA and protein expression levels were analyzed by quantitative reverse-transcription-quantitative polymerase chain reaction, ELISA and western blot analyses, respectively. The results demonstrated that an increase in MMP-13 mRNA and protein expression levels was observed with increasing RANKL/OPG ratio. These findings suggest that this mechanism may be used as a novel therapeutic strategy against OA.