Serum matrix metallopeptidase-9 levels in infantile epileptic spasms syndrome of unknown etiology.

PURPOSE: Epileptic spasms are the primary symptom of infantile epileptic spasms syndrome (IESS); however, their direct impact on blood-brain barrier (BBB) function is unknown. Matrix metallopeptidase-9 (MMP-9), degrades type IV collagen, a key component of the blood-brain barrier, while tissue inhibitor of metalloproteinase-1 (TIMP-1) suppresses its activity, protecting BBB integrity. This study aimed to assess serum MMP-9 and TIMP-1 levels in patients with IESS of unknown etiology. METHODS: We prospectively assessed serum MMP-9 and TIMP-1 levels prior to administering vigabatrin or adrenocorticotropic hormone therapy in patients with IESS of unknown etiology at Saitama Children's Medical Center between February 2012 and December 2023. We compared these biomarkers between patients with epileptic spasms and age-matched controls and performed a curve regression analysis between the biomarkers and the frequency of epileptic spasms. Additionally, we assessed whether MMP-9 and TIMP-1 levels were diagnostic predictors of IESS. RESULTS: This study included 22 patients with IESS (11 males) and 12 controls. Serum MMP-9 and MMP-9/TIMP-1 ratios were higher in patients with IESS than in controls (p < 0.001 and p = 0.002, respectively). A high frequency of epileptic spasms also led to higher serum MMP-9 levels (y = 0.0871x2 + 0.195x + 195.15, R²â€¯= 0.77, p < 0.001). Using MMP >188 ng/mL as the cutoff level, the sensitivity for diagnosing IESS was 95.5 %, the specificity was 75.0 %, the positive likelihood ratio was 3.82 (95 % confidence interval (CI) 1.43-10.22), and the relative risk was 8.75 (95 % CI 1.36-56.5). CONCLUSION: Patients with IESS had elevated serum MMP-9 levels, suggesting an association between epileptic spasms and blood-brain barrier dysfunction. MMP-9 level measurement may be useful for diagnosing suspected patients.

Silencing SPP1 in M2 macrophages inhibits the progression of castration-resistant prostate cancer via the MMP9/TGFß1 axis.

Background: M2 macrophages can promote the progression of castration-resistant prostate cancer (CRPC), but the specific mechanism is still unclear. Therefore, we are preliminarily exploring the molecular mechanism by which M2 macrophages regulate the progression of CRPC. Methods: The genes positively correlated with CRPC and with the most significant differences in the GEO32269 dataset were obtained. Database and immunofluorescence experiments were used to validate the localization of secreted phosphoprotein 1 (SPP1) in localized prostate cancer (PCa), hormone-sensitive prostate cancer (HSPC), and CRPC tumor tissues. The function of SPP1 in M2 macrophages was verified through cell scratch, Transwell, and an orthotopic PCa model. PCa database and Western blot were used to verify the relationship between SPP1 and matrix metallopeptidase 9 (MMP9), as well as the ability of MMP9 in M2 macrophages to promote epithelial-mesenchymal transition (EMT) in PCa cells. Results: The primary localization of SPP1 in prostate and CRPC tissues is in macrophages. Silencing SPP1 expression in M2 macrophages promotes their polarization towards the M1 phenotype and significantly inhibits the malignant progression of PCa in vitro and in vivo. SPP1 promotes the expression of MMP9 through the PI3K/AKT signaling pathway in M2 macrophages. Furthermore, MMP9 enhances the EMT and migratory capabilities of PC3 cells by activating the TGFß signaling pathway. Conclusions: We have found that the high expression of SPP1 in M2 macrophages promotes the progression of CRPC through cell-cell interactions. These findings can contribute to the development of novel therapeutic approaches for combating this deadly disease.

Effect of human epididymis protein 4 on hyperoxia-induced bronchial dysplasia in newborn rats.

OBJECTIVE: The study aimed to elucidate the role and the underlying mechanism of human epididymis protein 4 (HE4) in the pathogenesis of hyperoxia-induced bronchial dysplasia in newborn rats. METHODS: Forty neonatal Sprague-Dawley (SD) rats were separated into two groups: a normal control group (20.8% oxygen concentration) and a hyperoxia-induced group (85% oxygen concentration). Three time intervals of 24 h, 3 days and 7 days were chosen for each group. Haematoxylin-eosin staining was used to identify the pathological alterations in the lung tissue of the SD rats. Enzyme-linked immunosorbent assay was used to evaluate plasma protein levels. Real-time reverse transcription polymerase chain reaction was used to determine messenger RNA (mRNA) expression. RESULTS: In newborn SD rats, hyperoxia intervention within 7 days may result in acute lung damage. In the plasma and tissue of newborn SD rats, hyperoxia induction may raise levels of HE4, matrix metalloproteinases (MMP) 9 and tissue inhibitors of metalloproteinases (TIMP) 1. We discovered that the HE4 protein activates the phosphorylation of extracellular regulated protein kinases (ERK) and p65, activates the downstream MMP9 signalling pathway, inhibits MMP9 mRNA expression, inhibits protein activity, reduces type I collagen degradation, increases collagen secretion and promotes matrix remodelling and fibrosis in neonatal rat primary alveolar type II epithelial cells by overexpressing and silencing the HE4 gene. CONCLUSION: Through the ERK, MMP9 and TIMP1 signalling pathways, HE4 mediates the pathophysiological process of hyperoxia-induced lung damage in rats. Lung damage and lung basal remodelling are mediated by HE4 overexpression.

The Secretome of Adult Murine Hookworms Is Shaped by Host Expression of STAT6.

Co-evolutionary adaptation of hookworms with their mammalian hosts has been selected for immunoregulatory excretory/secretory (E/S) products. However, it is not known whether, or if so, how host immunological status impacts the secreted profile of hematophagous adult worms. This study interrogated the impact of host Signal transducer and activator of transcription 6 (STAT6) expression during the experimental evolution of hookworms through the sequential passage of the life cycle in either STAT6 deficient or WT C57BL/6 mice. Proteomic analysis of E/S products by LC-MS showed increased abundance of 15 proteins, including myosin-3, related to muscle function, and aconitate hydratase, related to iron homeostasis. However, most E/S proteins (174 of 337 unique identities) were decreased, including those in the Ancylostoma-secreted protein (ASP) category, and metallopeptidases. Several identified proteins are established immune-modulators such as fatty acid-binding protein homologue, cystatin, and acetylcholinesterase. Enrichment analysis of InterPro functional categories showed down-regulation of Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP), Astacin-like metallopeptidase, Glycoside hydrolase, and Transthyretin-like protein groups in STAT6 KO-adapted worms. Taken together, these data indicate that in an environment lacking Type 2 immunity, hookworms alter their secretome by reducing immune evasion proteins- and increasing locomotor- and feeding-associated proteins.

Inactive metallopeptidase homologs: the secret lives of pseudopeptidases.

Inactive enzyme homologs, or pseudoenzymes, are proteins, found within most enzyme families, that are incapable of performing catalysis. Rather than catalysis, they are involved in protein-protein interactions, sometimes regulating the activity of their active enzyme cousins, or scaffolding protein complexes. Pseudoenzymes found within metallopeptidase families likewise perform these functions. Pseudoenzymes within the M14 carboxypeptidase family interact with collagens within the extracellular space, while pseudopeptidase members of the M12 "a disintegrin and metalloprotease" (ADAM) family either discard their pseudopeptidase domains as unnecessary for their roles in sperm maturation or utilize surface loops to enable assembly of key complexes at neuronal synapses. Other metallopeptidase families contain pseudopeptidases involved in protein synthesis at the ribosome and protein import into organelles, sometimes using their pseudo-active sites for these interactions. Although the functions of these pseudopeptidases have been challenging to study, ongoing work is teasing out the secret lives of these proteins.

Lonicera japonica Thunb. Ethanol Extract Exerts a Protective Effect on Normal Human Gastric Epithelial Cells by Modulating the Activity of Tumor-Necrosis-Factor-α-Induced Inflammatory Cyclooxygenase 2/Prostaglandin E2 and Matrix Metalloproteinase 9.

Gastric inflammation-related disorders are commonly observed digestive system illnesses characterized by the activation of proinflammatory cytokines, particularly tumor necrosis factor-α (TNF-α). This results in the induction of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PEG2) and matrix metallopeptidase-9 (MMP-9). These factors contribute to the pathogenesis of gastric inflammation disorders. We examined the preventive effects of Lonicera japonica Thunb. ethanol extract (Lj-EtOH) on gastric inflammation induced by TNF-α in normal human gastric mucosa epithelial cells (GES-1). The GES-1 cell line was used to establish a model that simulated the overexpression of COX-2/PGE2 and MMP-9 proteins induced by TNF-α to examine the anti-inflammatory properties of Lj extracts. The results indicated that Lj-EtOH exhibits significant inhibitory effects on COX-2/PEG2 and MMP-9 activity, attenuates cell migration, and provides protection against TNF-α-induced gastric inflammation. The protective effects of Lj-EtOH are associated with the modulation of COX-2/PEG2 and MMP-9 through the activation of TNFR-ERK 1/2 signaling pathways as well as the involvement of c-Fos and nuclear factor kappa B (NF-κB) signaling pathways. Based on our findings, Lj-EtOH exhibits a preventive effect on human gastric epithelial cells. Consequently, it may represent a novel treatment for the management of gastric inflammation.

Phagocytosis of aggrecan-positive perineuronal nets surrounding motor neurons by reactive microglia expressing MMP-9 in TDP-43Q331K ALS model mice.

Perineuronal nets (PNNs) are extracellular matrix structures that surround excitable neurons and their proximal dendrites. PNNs play an important role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can act as a trigger for neuronal death, and this has been implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We therefore characterised PNNs around alpha motor neurons and the possible contributing cellular factors in the mutant TDP-43Q331K transgenic mouse, a slow onset ALS mouse model. PNNs around alpha motor neurons showed significant loss at mid-stage disease in TDP-43Q331K mice compared to wild type strain control mice. PNN loss coincided with an increased expression of matrix metallopeptidase-9 (MMP-9), an endopeptidase known to cleave PNNs, within the ventral horn. During mid-stage disease, increased numbers of microglia and astrocytes expressing MMP-9 were present in the ventral horn of TDP-43Q331K mice. In addition, TDP-43Q331K mice showed increased levels of aggrecan, a PNN component, in the ventral horn by microglia and astrocytes during this period. Elevated aggrecan levels within glia were accompanied by an increase in fractalkine expression, a chemotaxic protein responsible for the recruitment of microglia, in alpha motor neurons of onset and mid-stage TDP-43Q331K mice. Following PNN loss, alpha motor neurons in mid-stage TDP-43Q331K mice showed increased 3-nitrotyrosine expression, an indicator of protein oxidation. Together, our observations along with previous PNN research provide suggests a possible model whereby microglia and astrocytes expressing MMP-9 degrade PNNs surrounding alpha motor neurons in the TDP-43Q331K mouse. This loss of nets may expose alpha-motor neurons to oxidative damage leading to degeneration of the alpha motor neurons in the TDP-43Q331K ALS mouse model.

ADAM10 Alleviates Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury by Activating the Notch Signaling Pathway.

The occurrence of myocardial ischemia/reperfusion injury is commonly observed during cardiac surgery; however, there remains a dearth of effective therapeutic strategies to mitigate this injury. The a disintegrin and metallopeptidase domain 10 (ADAM10) is a transmembrane protein anchored on the cell membrane surface, and its precise mechanism of action in myocardial ischemia/reperfusion injury remains incompletely understood. This study aims to investigate the impact of ADAM10 on cardiomyocyte injury induced by hypoxia/reoxygenation (H/R) and elucidate the underlying mechanisms. The ADAM10 overexpression plasmid was transfected into H9c2 cells, which were subsequently treated with the Notch signaling pathway inhibitor DAPT and cultured under H/R conditions. Cell proliferation activity was assessed using the CCK-8 assay. The levels of LDH, SOD, and MDA were quantified through colorimetric analysis. The levels of ROS and the rate of apoptosis were measured using flow cytometry. The morphological changes in the nucleus of H9c2 cells were observed by employing Hoechst 33258 staining. The mRNA expression levels of ADAM10, Notch1, NICD, and Hes1 in H9c2 cells were determined using qRT-PCR. The expressions of Notch signaling pathway and apoptosis-related proteins were analyzed by Western blot. Overexpression of ADAM10 provided protection to H9c2 cells against injury induced by H/R, leading to an increase in SOD levels and alleviation of oxidative stress caused by the accumulation of ROS and the decrease of SOD activity. Meanwhile, overexpression of ADAM10 inhibited apoptosis in H9c2 cells exposed to H/R by regulating the expression of apoptosis-related proteins, such as Bax, Bcl-2 and Cleaved-caspase-3. Additionally, overexpression of ADAM10 facilitated the activation of the Notch1 signaling pathway in H9c2 cells exposed to H/R by upregulating the protein expression of Notch1, NICD, and Hes1. However, the protective effect of ADAM10 on H/R-induced H9c2 cells was partially reversed by DAPT. Our findings demonstrate that ADAM10 exerts protective effects in H/R-induced H9c2 cells by suppressing oxidative stress and apoptosis via the activation of the Notch signaling pathway.

Perineuronal nets are phagocytosed by MMP-9 expressing microglia and astrocytes in the SOD1G93A ALS mouse model.

AIMS: Perineuronal nets (PNNs) are an extracellular matrix structure that encases excitable neurons. PNNs play a role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can trigger neuronal death, which has been implicated in amyotrophic lateral sclerosis (ALS). We investigated the spatio-temporal timeline of PNN breakdown and the contributing cellular factors in the SOD1G93A strain, a fast-onset ALS mouse model. METHODS: This was conducted at the presymptomatic (P30), onset (P70), mid-stage (P130), and end-stage disease (P150) using immunofluorescent microscopy, as this characterisation has not been conducted in the SOD1G93A strain. RESULTS: We observed a significant breakdown of PNNs around α-motor neurons in the ventral horn of onset and mid-stage disease SOD1G93A mice compared with wild-type controls. This was observed with increased numbers of microglia expressing matrix metallopeptidase-9 (MMP-9), an endopeptidase that degrades PNNs. Microglia also engulfed PNN components in the SOD1G93A mouse. Further increases in microglia and astrocyte number, MMP-9 expression, and engulfment of PNN components by glia were observed in mid-stage SOD1G93A mice. This was observed with increased expression of fractalkine, a signal for microglia engulfment, within α-motor neurons of SOD1G93A mice. Following PNN breakdown, α-motor neurons of onset and mid-stage SOD1G93A mice showed increased expression of 3-nitrotyrosine, a marker for protein oxidation, which could render them vulnerable to death. CONCLUSIONS: Our observations suggest that increased numbers of MMP-9 expressing glia and their subsequent engulfment of PNNs around α-motor neurons render these neurons sensitive to oxidative damage and eventual death in the SOD1G93A ALS model mouse.

Introducing critical proteins related to liver ischemia/reperfusion injury.

Aim: The current study aimed to introduce the key proteins involved in liver ischemia/reperfusion (I/R) injury through protein-protein interaction (PPI) analysis. Background: Liver transplantation (LT) is a well-known treatment for liver diseases that threaten patients with mortality. LT is a complex operation, and several risks, including liver I/R injury, affect its success. Improving LT requires detection of its molecular mechanism. Experiments have revealed that high throughput methods such as proteomics in combination with bioinformatics are useful tools for analyzing the molecular mechanism of disease. Methods: The differentially expressed proteins (DEPs) involved in liver I/R injury were extracted from the literature. The queried DEPs plus the first 100 neighbors were included in a network through STRING database using Cytoscape software. Degree, betweenness centrality, closeness centrality, and stress were considered to determine the central nodes. The queried DEPs were assessed by action map analysis using the CluePedia application of Cytoscape software. The key proteins were identified by comparing network analysis and action map evaluation results. Results: Six proteins, namely ALB, INS, GAPDH, CAT, IL6, and TNF, among the added first neighbors were determined as the central first neighbors. MPO, CRP, MMP9, and HMOX1 were selected as central DEPs among the queried proteins. Action map analysis confirmed the PPI findings. The final evaluation revealed that MMP9 in combination with CRP and HMOX1 plays a critical role in liver I/R injury. Conclusion: The significant role of MMP9 in liver I/R injury was detected in this study. Two central proteins (CRP and HMOX1) were shown to have a regulatory effect on MMP9; CRP activated MMP9, while HMXO1 downregulated it.

Surface functionalization of exosomes for chondrocyte-targeted siRNA delivery and cartilage regeneration.

Osteoarthritis (OA) is the most prevalent degenerative cartilage disease, but no effective treatment is currently available to ameliorate the dysregulation of cartilage catabolism. Cartilage degeneration is closely related to the change in the physiology of chondrocytes: for example, chondrocytes of the OA patients overexpress matrix metallopeptidase 13 (MMP13), a.k.a. collagenase 3, which damages the extracellular matrix (ECM) of the cartilage and deteriorate the disease progression. Inhibiting MMP13 has shown to be beneficial for OA treatments, but delivering therapeutics to the chondrocytes embedded in the dense cartilage is a challenge. Here, we engineered the exosome surface with the cartilage affinity peptide (CAP) through lipid insertion to give chondrocyte-targeting exosomes, CAP-Exo, which was then loaded with siRNA against MMP13 (siMMP13) in the interior to give CAP-Exo/siMMP13. Intra-articular administration of CAP-Exo/siMMP13 reduced the MMP13 level and increased collagen COL2A1 and proteoglycan in cartilage in a rat model of anterior cruciate ligament transection (ACLT)-induced OA. Proteomic analysis showed that CAP-Exo/siMMP13 treatment restored the altered protein levels in the IL-1ß-treated chondrocytes. Taken together, a facile exosome engineering method enabled targeted delivery of siRNA to chondrocytes and chondrocyte-specific silencing of MMP13 to attenuate cartilage degeneration.

Identification of Circulating Inflammatory Proteins Associated with Calcific Aortic Valve Stenosis by Multiplex Analysis.

Calcific aortic valve stenosis (CAVS) is characterized by increasing inflammation and progressive calcification in the aortic valve leaflets and is a major cause of death in the aging population. This study aimed to identify the inflammatory proteins involved in CAVS and provide potential therapeutic targets. We investigated the observational and causal associations of 92 inflammatory proteins, which were measured using affinity-based proteomic assays. Firstly, the case-control cohort identified differential proteins associated with the occurrence and progression of CAVS. Subsequently, we delved into exploring the causal impacts of these associated proteins through Mendelian randomization. This involved utilizing genetic instruments derived from cis-protein quantitative loci identified in genome-wide association studies, encompassing a cohort of over 400,000 individuals. Finally, we investigated the gene transcription and protein expression levels of inflammatory proteins by single-cell and immunohistochemistry analysis. Multivariate logistic regression and spearman's correlation analysis showed that five proteins showed a significant positive correlation with disease severity. Mendelian randomization showed that elevated levels of two proteins, namely, matrix metallopeptidase-1 (MMP1) and sirtuin 2 (SIRT2), were associated with an increased risk of CAVS. Immunohistochemistry and single-cell transcriptomes showed that expression levels of MMP1 and SIRT2 at the tissue and cell levels were significantly higher in calcified valves than in non-calcified control valves. These findings indicate that MMP1 and SIRT2 are causally related to CAVS and open up the possibility for identifying novel therapeutic targets.

Matrix metallopeptidase 9 contributes to the beginning of plaque and is a potential biomarker for the early identification of atherosclerosis in asymptomatic patients with diabetes.

Aims: To determine the roles of matrix metallopeptidase-9 (MMP9) on human coronary artery smooth muscle cells (HCASMCs) in vitro, early beginning of atherosclerosis in vivo in diabetic mice, and drug naïve patients with diabetes. Methods: Active human MMP9 (act-hMMP9) was added to HCASMCs and the expressions of MCP-1, ICAM-1, and VCAM-1 were measured. Act-hMMP9 (n=16) or placebo (n=15) was administered to diabetic KK.Cg-Ay/J (KK) mice. Carotid artery inflammation and atherosclerosis measurements were made at 2 and 10 weeks after treatment. An observational study of newly diagnosed drug naïve patients with type 2 diabetes mellitus (T2DM n=234) and healthy matched controls (n=41) was performed and patients had ultrasound of carotid arteries and some had coronary computed tomography angiogram for the assessment of atherosclerosis. Serum MMP9 was measured and its correlation with carotid artery or coronary artery plaques was determined. Results: In vitro, act-hMMP9 increased gene and protein expressions of MCP-1, ICAM-1, VCAM-1, and enhanced macrophage adhesion. Exogenous act-hMMP9 increased inflammation and initiated atherosclerosis in KK mice at 2 and 10 weeks: increased vessel wall thickness, lipid accumulation, and Galectin-3+ macrophage infiltration into the carotid arteries. In newly diagnosed T2DM patients, serum MMP9 correlated with carotid artery plaque size with a possible threshold cutoff point. In addition, serum MMP9 correlated with number of mixed plaques and grade of lumen stenosis in coronary arteries of patients with drug naïve T2DM. Conclusion: MMP9 may contribute to the initiation of atherosclerosis and may be a potential biomarker for the early identification of atherosclerosis in patients with diabetes. Clinical trial registration: https://clinicaltrials.gov, identifier NCT04424706.

Selected markers of ovarian cancer and their relation to targeted therapy (Review).

Despite advances in surgical treatment techniques and chemotherapy-including anti-angiogenic and immune poly (ADP-ribose) polymerase inhibitors, the 5-year survival rate in ovarian cancer (OC) remains low. The reasons for this are the diagnosis of cancer in advanced clinical stages, chemoresistance and cancer recurrence. New therapeutic approaches are being developed, including the search for new biomarkers that are also targets for targeted therapy. The present review describes new molecular markers with relevance to targeted therapy, which to date have been studied only in experimental research. These include the angiogenic protein angiopoietin-2, the transmembrane glycoprotein ectonucleotide pyrophosphatase/phosphodiesterase 1, the adhesion protein E-cadherin, the TIMP metallopeptidase inhibitor 1 and Kruppel-like factor 7. Drugs affecting cancer stem cells (CSCs) in OC, such as metformin and salinomycin, as well as inhibitors of CSCs markers aldehyde dehydrogenase 1 (with the drug ATRA) and the transcription factor Nanog homeobox (microRNA) are also discussed. A new approach to prevention and possible therapies under investigation such as development of vaccines containing a subpopulation of CD117(+) and CD44(+) stem cells with a promising option for use in women with OC was described.

Inhibition of HIV-1 release by ADAM metalloproteinase inhibitors.

HIV-1 gp120 glycan binding to C-type lectin adhesion receptor L-selectin/CD62L on CD4 T cells facilitates viral attachment and entry. Paradoxically, the adhesion receptor impedes HIV-1 budding from infected T cells and the viral release requires the shedding of CD62L. To systematically investigate CD62L-shedding mediated viral release and its potential inhibition, we screened compounds specific for serine-, cysteine-, aspartyl-, and Zn-dependent proteases for CD62L shedding inhibition and found that a subclass of Zn-metalloproteinase inhibitors, including BB-94, TAPI, prinomastat, GM6001, and GI25423X, suppressed CD62L shedding. Their inhibition of HIV-1 infections correlated with enzymatic suppression of both ADAM10 and 17 activities and expressions of these ADAMs were transiently induced during the viral infection. These metalloproteinase inhibitors are distinct from the current antiretroviral drug compounds. Using immunogold labeling of CD62L, we observed association between budding HIV-1 virions and CD62L by transmission electron microscope, and the extent of CD62L-tethering of budding virions increased when the receptor shedding is inhibited. Finally, these CD62L shedding inhibitors suppressed the release of HIV-1 virions by CD4 T cells of infected individuals and their virion release inhibitions correlated with their CD62L shedding inhibitions. Our finding reveals a new therapeutic approach targeted at HIV-1 viral release.

Recombinant protein production and functional analysis of a M60-like-2 metallopeptidase enzyme from the carcinogenic liver fluke Opisthorchis viverrini.

Mucin plays a crucial role in safeguarding mucosal tissues by obstructing the translocation of microorganisms. Mucosal tissue-dwelling parasites must devise a strategy to surmount this mucin barrier in order to establish colonization. In a recent discovery, it was observed that the liver fluke Opisthorchis viverrini secretes two mucinases, namely Ov-M60-like-1 and Ov-M60-like-2. Ov-M60-like-1 was previously characterized. Here, we study the Ov-M60-like-2 by utilizing the wheat germ expression system to produce recombinant proteins and conducted a functional analysis of its enzymatic activity on bovine submaxillary mucin (BSM). Subsequently, we delved deeper into understanding the role of this enzyme in host-parasite interactions by evaluating its mucinase activity on mucins from the bile duct of O. viverrini-infected hamsters. Through successful production of recombinant proteins using the wheat germ expression system, we observed that this enzyme displayed mucinase activity over a wide pH range (pH 2 to pH 10) against BSM. Our investigations revealed it ability to digest mucin from the bile duct. These findings suggest that Ov-M60-like-2 possess a mucinase activity, together with Ov-M60-like-1, enabling the liver fluke to successful colonization of the host's bile duct.

Cornus officinalis var. koreana Kitam extracts alleviate cadmium-induced renal fibrosis by targeting matrix metallopeptidase 9.

ETHNOPHARMACOLOGICAL RELEVANCE: Cornus officinalis var. koreana Kitam (Cornus officinalis) is a commonly used Chinese herbal medicine and has a good clinical efficacy in kidney and liver diseases. Recent years, a number of studies reported the significant effects of Cornus officinalis on renal fibrosis. However, it is still unclear about the underlying specific mechanism, the bioactive ingredients, and the target gene regulatory network. AIM OF THE STUDY: We investigated the impact of Cornus officinalis extract on cadmium-induced renal fibrosis, screened the bioactive ingredients of Cornus officinalis using a pharmacological sub-network analysis, and explored the regulatory effects of Cornus officinalis extracts on target gene matrix metallopeptidase 9 (MMP9). METHODS: Male C57BL/6N mice were treated with single or combinatorial agents such as saline, cadmium chloride, Cornus officinalis, Isoginkgetin and FSL-1. Isoginkgetin is a compound with anti-MMP9 activity. FSL-1 can induce MMP9 expression. Masson staining and Western blot and immunohistochemistry analyses were used for assessing renal fibrosis. In addition, wound healing model was established using BUMPT (Boston university mouse proximal tubular) cells to investigate how Cornus officinalis affected cadmium-induced cell migration. The main Cornus officinalis bioactive compounds were identified by UHPLC-MS (Ultra-high-performance liquid chromatography - mass spectrometry). The MMP9 target for Cornus officinalis active ingredients were confirmed through a pharmacological sub-network analysis. RESULTS: Aqueous extracts of Cornus officinalis protected from renal dysfunction and kidney fibrosis induced by cadmium chloride in mice. In vitro experiments validated that Cornus officinalis extracts inhibited cell migration ability especially in cadmium chloride condition. The sub-network analysis and chemical components profiling technique revealed the active compounds of Cornus officinalis. Cellular thermal shift assay verified the binding abilities of three active components Daidzein, N-Acetyl-L-tyrosine or Swertisin with matrix metalloproteinase-9. Gelatin zymography assay revealed that the activity of MMP9 was inhibited by the three active components. We further confirmed that MMP9 was involved in the process of Cornus officinalis extracts reducing renal fibrosis. Cornus officinalis attenuated the cadmium-induced renal fibrosis was correlated with decreased expression of MMP9, collagen I, α-SMA (alpha-smooth muscle actin) and vimentin. CONCLUSIONS: This study demonstrated that Cornus officinalis extracts could alleviate the cadmium chloride-induced renal fibrosis by targeting MMP9, and might provide new insights into the mechanism of treating renal fibrosis by Cornus officinalis.

Glibenclamide pretreatment attenuates early hematoma expansion of warfarin-associated intracerebral hemorrhage in rats by alleviating perihematomal blood-brain barrier dysfunction.

BACKGROUND: Hematoma expansion is a determinant of poor outcome of intracerebral hemorrhage but occurs frequently, especially in warfarin-associated intracerebral hemorrhage (W-ICH). In the present study, we employ the warfarin-associated intracerebral hemorrhage (W-ICH) rat model, to explore the efficacy and potential mechanism of glibenclamide pretreatment on hematoma expansion after intracerebral hemorrhage, hoping to provide proof of concept that glibenclamide in stroke primary and secondary prevention is also potentially beneficial for intracerebral hemorrhage patients at early stage. METHODS: In the present study, we tested whether glibenclamide, a common hypoglycemic drug, could attenuate hematoma expansion in a rat model of W-ICH. Hematoma expansion was evaluated using magnetic resonance imaging; brain injury was evaluated by brain edema and neuronal death; and functional outcome was evaluated by neurological scores. Then blood-brain barrier integrity was assessed using Evans blue extravasation and tight junction-related protein. RESULTS: The data indicated that glibenclamide pretreatment significantly attenuated hematoma expansion at 24 h after W-ICH, thus mitigating brain edema and neuronal death and promoting neurological function recovery, which may benefit from alleviating blood-brain barrier disruption by suppressing matrix metallopeptidase-9. CONCLUSIONS: The results indicate that glibenclamide pretreatment in stroke primary and secondary prevention might be a promising therapy for hematoma expansion at the early stage of W-ICH.

The cell volume-regulatory glycine transporter GLYT1 is activated following metallopeptidase-mediated detachment of the oocyte from the zona pellucida.

Independent cell volume regulation is first acquired by the oocyte in two steps that occur during meiotic maturation: (1) activation of the glycine transporter GLYT1 (Slc6a9) that mediates the intracellular accumulation of glycine to provide osmotic support in the mature egg and early preimplantation embryo, and (2) release of the oocyte from the strong attachment to its rigid extracellular matrix shell, the zona pellucida (ZP). It was recently shown that oocyte-ZP detachment requires metallopeptidase activity that is proposed to cleave transmembrane ZP proteins connecting the oocyte to the ZP. It is unknown, however, how GLYT1 is activated. We hypothesized that oocyte-ZP detachment precedes and may be required for GLYT1 activation. In identically treated pools of oocytes, oocyte-ZP detachment occurred ~20 min before GLYT1 activation. In individual oocytes, GLYT1 activity was detected only in those that were mostly or fully detached. Blocking detachment using previously validated small molecule metallopeptidase inhibitors partly suppressed GLYT1 activation. However, removal of the ZP did not accelerate GLYT1 activation. This indicates that oocyte-ZP detachment or cleavage of transmembrane ZP proteins may be required for GLYT1 to become fully activated, or alternatively that metallopeptidase activity independently affects both detachment and GLYT1 activation.

Silencing GMPPB Inhibits the Proliferation and Invasion of GBM via Hippo/MMP3 Pathways.

Glioblastoma multiforme (GBM) is a highly aggressive malignancy and represents the most common brain tumor in adults. To better understand its biology for new and effective therapies, we examined the role of GDP-mannose pyrophosphorylase B (GMPPB), a key unit of the GDP-mannose pyrophosphorylase (GDP-MP) that catalyzes the formation of GDP-mannose. Impaired GMPPB function will reduce the amount of GDP-mannose available for O-mannosylation. Abnormal O-mannosylation of alpha dystroglycan (α-DG) has been reported to be involved in cancer metastasis and arenavirus entry. Here, we found that GMPPB is highly expressed in a panel of GBM cell lines and clinical samples and that expression of GMPPB is positively correlated with the WHO grade of gliomas. Additionally, expression of GMPPB was negatively correlated with the prognosis of GBM patients. We demonstrate that silencing GMPPB inhibits the proliferation, migration, and invasion of GBM cells both in vitro and in vivo and that overexpression of GMPPB exhibits the opposite effects. Consequently, targeting GMPPB in GBM cells results in impaired GBM tumor growth and invasion. Finally, we identify that the Hippo/MMP3 axis is essential for GMPPB-promoted GBM aggressiveness. These findings indicate that GMPPB represents a potential novel target for GBM treatment.