Granulathiazole A protects 6-OHDA-induced Parkinson's disease from ferroptosis via activating Nrf2/HO-1 pathway.

Two pairs of enantiomers (1a-2b), namely (±)-alterpyrone F and (±)-alterpyrone G, along with a rare benzothiazole meroterpenoid granulathiazole A (3, GA), and two undescribed compounds called respectively granulahydeoate (4) and granulaone (5), were obtained from the co-cultivation of Alternaria brassicicola and Penicillium sp. HUBU0120. Exhaustive analyses of NMR, single crystal XRD, Mo2(OAc)4-induced circular dichroism data, and a modified Mosher's method distinguished the absolute configurations of isolates. Bioactive evaluations exhibited that GA possessed promising anti-PD activity in both in vitro and in vivo PD models viz. 6-OHDA-induced SH-SY5Y cells and 6-OHDA-induced zebrafish, respectively. Moreover, our research demonstrated that ferroptosis activated by 6-OHDA was mitigated in PD models after treated with GA. Extensive molecular mechanism studies in PD-modelled cells manifested that GA attenuated the decreased expressions of SLC7A11, GPX4, and FSP-1, and the increased level of ACSL4 via activating Nrf2/HO-1 pathway as well as ameliorated the accumulation of α-synuclein.

A study on the impact of marital status on the survival status of prostate cancer patients based on propensity score matching.

Marital status is an independent prognostic factor for survival in many types of cancers, but its prognostic impact on patients with prostate cancer (PCa) has not been established. The aim of this study was to explore the independent prognostic factors of PCa and to investigate the effect of marital status on survival outcomes in patients with different stratified by PCa. Using the surveillance, epidemiology, and end results (SEER) database, we collected data on 584,655 PCa patients diagnosed between 1975 and 2019. Marital status was classified as married, divorced, widowed, and single. We used the Kaplan-Meier analysis and single multivariate Cox proportional hazards regression analysis to determine the effect of marital status on overall survival (OS) and cancer-specific survival (CSS). In addition, we performed subgroup analyses for different ages, Gleason score and PSA values, and performed a 1:1 propensity score matching (PSM) to reduce the impact of confounding factors to obtain more accurate matching results. According to our findings, marital status was an independent prognostic factor for the survival of PCa patients and a better prognosis of married patients. Moreover, we also found that factors such as age, TNM stage, Gleason score, and PSA concentration were also considered as important predictors for the prognosis of PCa. The above findings can facilitate early detection and treatment of high-risk PCa patients, prolong their life and reduce family burden.

Studying the impact of marital status on diagnosis and survival prediction in pancreatic ductal carcinoma using machine learning methods.

Pancreatic cancer is a commonly occurring malignant tumor, with pancreatic ductal carcinoma (PDAC) accounting for approximately 95% of cases. According of its poor prognosis, identifying prognostic factors of pancreatic ductal carcinoma can provide physicians with a reliable theoretical foundation when predicting patient survival. This study aimed to analyze the impact of marital status on survival outcomes of PDAC patients using propensity score matching and machine learning. The goal was to develop a prognosis prediction model specific to married patients with PDAC. We extracted a total of 206,968 patient records of pancreatic cancer from the SEER database. To ensure the baseline characteristics of married and unmarried individuals were balanced, we used a 1:1 propensity matching score. We then conducted Kaplan-Meier analysis and Cox proportional-hazards regression to examine the impact of marital status on PDAC survival before and after matching. Additionally, we developed machine learning models to predict 5-year CSS and OS for married patients with PDAC specifically. In total, 24,044 PDAC patients were included in this study. After 1:1 propensity matching, 8043 married patients and 8,043 unmarried patients were successfully enrolled. Multivariate analysis and the Kaplan-Meier curves demonstrated that unmarried individuals had a poorer survival rate than their married counterparts. Among the algorithms tested, the random forest performed the best, with 0.734 5-year CSS and 0.795 5-year OS AUC. This study found a significant association between marital status and survival in PDAC patients. Married patients had the best prognosis, while widowed patients had the worst. The random forest is a reliable model for predicting survival in married patients with PDAC.

Brassisterol A, a new ergosterol from co-cultivation of fungi attenuates neuroinflammation via targeting NLRP3/caspase-1/GSDMD pathway.

Three new ergosterol derivatives brassisterol A-C (1-3) and two new epimeric bicycle-lactones brassictones A and B (4 and 5), were isolated from the co-cultivation of Alternaria brassicicola and Penicillium granulatum. The absolute configurations of these isolates were confirmed by extensive NMR spectra, TD-DFT ECD calculation, and the single crystal XRD data analysis. Amongst the metabolites, compound 1 exhibited potential anti-Parkinson's disease activity in both MPTP-induced zebrafish and MPP+-induced SH-SY5Y cells. Molecular mechanism studies in vitro showed that 1 attenuated the increase of α-synuclein, NLRP3, ASC, caspase-1, IL-1ß, IL-18, and GSDMD expression in the MPP+ induced PD model. Molecular docking in silico simulations exhibited that 1 was well accommodated to one of the binding pockets of NLRP3 8ETR in an appropriate conformation via forming typical hydrogen bonds as well as possessing a high negative binding affinity (-8.97 kcal/mol). Thus, our work suggested that 1 protected dopaminergic cell from neuroinflammation via targeting NLRP3/caspase-1/GSDMD signaling pathway.

Macrophage polarization in tissue fibrosis.

Fibrosis can occur in all major organs with relentless progress, ultimately leading to organ failure and potentially death. Unfortunately, current clinical treatments cannot prevent or reverse tissue fibrosis. Thus, new and effective antifibrotic therapeutics are urgently needed. In recent years, a growing body of research shows that macrophages are involved in fibrosis. Macrophages are highly heterogeneous, polarizing into different phenotypes. Some studies have found that regulating macrophage polarization can inhibit the development of inflammation and cancer. However, the exact mechanism of macrophage polarization in different tissue fibrosis has not been fully elucidated. This review will discuss the major signaling pathways relevant to macrophage-driven fibrosis and profibrotic macrophage polarization, the role of macrophage polarization in fibrosis of lung, kidney, liver, skin, and heart, potential therapeutics targets, and investigational drugs currently in development, and hopefully, provide a useful review for the future treatment of fibrosis.

Study of efficacy and antibody duration to fourth-dose booster of Ad5-nCoV or inactivated SARS-CoV-2 vaccine in Chinese adults: a prospective cohort study.

Introduction: China experienced a record surge of coronavirus disease 2019 cases in December 2022, during the pandemic. Methods: We conducted a randomized, parallel-controlled prospective cohort study to evaluate efficacy and antibody duration after a fourth-dose booster with Ad5-nCoV or inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. Results: A total of 191 participants aged ≥18 years who had completed a three-dose regimen of the inactivated SARS-CoV-2 vaccine 6 months earlier were recruited to receive the intramuscular Ad5-nCoV booster or the inactivated SARS-CoV-2 vaccine. The Ad5-nCoV group had significantly higher antibody levels compared with the inactivated vaccine group at 6 months after the fourth vaccination dose. After the pandemic, the breakthrough infection rate for the Ad5-nCoV and the inactivated vaccine groups was 77.89% and 78.13%, respectively. Survival curve analysis (p = 0.872) and multivariable logistic regression analysis (p = 0.956) showed no statistically significant differences in breakthrough infection between the two groups. Discussion: Compared with a homologous fourth dose, a heterologous fourth dose of Ad5-nCoV elicited a higher immunogenic response in healthy adults who had been immunized with three doses of inactivated vaccine. Nevertheless, the efficacy of the two vaccine types was equivalent after the pandemic.

Safety and immunogenicity of Ad5-nCoV immunization after three-dose priming with inactivated SARS-CoV-2 vaccine in Chinese adults.

Data on the safety and immunity of a heterologous booster (fourth dose) after three-doses of inactivated SARS-CoV-2 vaccine in Chinese adults are limited. We evaluate the safety and immunogenicity of Ad5-nCoV in a randomized, double-blind, parallel-controlled phase 4 clinical trial in Zhejiang, China (NCT05373030). Participants aged 18-80 years (100 per group), administered three doses of inactivated SARS-CoV-2 vaccine ≥6 months earlier, are enrolled and randomized 1:1 into two groups, which are administered intramuscular Ad5-nCoV or inactivated SARS-CoV-2 vaccine (CoronaVac or Covilo). All observed adverse reactions are predictable and manageable. Ad5-nCoV elicits significantly higher RBD-specific IgG levels, with a geometric mean concentration of 2924.0 on day 14 post-booster, 7.8-fold that of the inactivated vaccine. Pseudovirus-neutralizing antibodies to Omicron BA.4/5 show a similar pattern, with geometric mean titers of 228.9 in Ad5-nCoV group and 65.5 in inactivated vaccine group. Ad5-nCoV booster maintains high antibody levels on day 90, with seroconversion of 71.4%, while that of inactivated vaccine is 5.2%, almost pre-booster levels. A fourth Ad5-nCoV vaccination following three-doses of inactivated SARS-CoV-2 vaccine is immunogenic, tolerable, and more efficient than inactivated SARS-CoV-2 vaccine. Ad5-nCoV elicits a stronger humoral response against Omicron BA.4/5 and maintains antibody levels for longer than homologous boosting.

Effects of ulinastatin therapy in emergency severe multiple trauma: A single-center randomized controlled trial.

BACKGROUND: Severe multiple traumas are one of the most common diseases and carry a significant financial burden with high disability and mortality. There are no effective drugs in the clinical management of severe multiple traumas, and there is an absence of evidence-based medicine concerning the treatment of severe multiple traumas. METHODS: The present study explored whether ulinastatin (UTI) can improve the outcome of severe multiple traumas. The present research included patients who were hospitalized in intensive care units after being diagnosed with severe multiple trauma. Patients received UTIs (400,000 IU) or placebos utilizing computer-based random sequencing (in a 1:1 ratio). The primary outcome measures were 30-day mortality, multiple organ dysfunction syndrome, inflammatory response, coagulation function, infection, liver function, renal function, and drug-related adverse effects. RESULTS: A total of 239 individuals were classified into 2 groups, namely, the placebo group (n = 120) and the UTI group (n = 119). There were no statistically significant differences in baseline clinical data between the 2 groups. The 30-day mortality and multiple organ dysfunction syndrome in the UTI group were remarkably improved compared with those in the placebo group. UTI can protect against hyperinflammation and improve coagulation dysfunction, infection, liver function, and renal function. UTI patients had markedly decreased hospitalization expenditures compared with the placebo group. CONCLUSION: The findings from the present research indicated that UTIs can improve the clinical outcomes of patients with severe multiple traumas and have fewer adverse reactions.

Improving Accuracy in Mass Spectrometry-Based Mass Determination of Intact Heterogeneous Protein Utilizing the Universal Benefits of Charge Reduction and Alternative Gas-Phase Reactions.

In mass analysis of proteins, mass spectrometry directly measures the mass to charge ratios of ionized proteins and promises higher accuracy than that of indirect approaches measuring other physicochemical properties, provided that the charge states of detected ions are determined. Accurate mass determination of heterogeneously glycosylated proteins is often hindered by unreliable charge determination due to the insufficient resolution of signals from different charge states and inconsistency among mass profiles of ions in individual charge states. Limited charge reduction of a subpopulation of proteoforms using electron transfer/capture reactions (ETnoD/ETnoD) solves this problem by narrowing the mass distribution of examined proteoforms and preserving the mass profile of the precursor charge state in the reduced charge states. However, the limited availability of ETnoD/ETnoD function in commercial instruments limits the application of this approach. Here, utilizing a range of charge-dependent and accuracy-affecting spectral features revealed by a systematic evaluation at levels of both the ensemble and subpopulation of proteoforms based on theoretical models and experiments, we developed a limited charge reduction workflow that enables using collision-induced dissociation and higher energy collisional dissociation, two widely available reactions, as alternatives to ETnoD/ETnoD while providing adequate accuracy. Alternatively, substituting proton transfer charge reduction for ETnoD/ETnoD provides higher accuracy of mass determination. Performing mass selection in a window-sliding manner improves the accuracy and allows profiling of the whole proteoform distribution. The proposed workflow may facilitate the development of universal characterization strategies for more complex and heterogeneous protein systems.

Current situation of menstruation and gynecological diseases prevalence among Chinese women: a cross-sectional study.

BACKGROUND: Gynecological diseases have been taken attention and studied worldwide. Although, no recent studies have delineated the magnitude of gynecological diseases among Chinese women. This study aims to evaluate the current situation of menstruation and gynecological diseases prevalence among Chinese women. METHODS: A cross-sectional study was conducted at a hospital affiliated with Nanjing medical university in Nanjing, China between September 2021 and February 2022. A sample size of 977 women aged 18-52 years participated in a face-to-face interview questionnaire. Logistic regression was performed to determine whether pubertal timing and menstrual characteristics were associated with gynecological diseases. RESULTS: The most prevalent gynecological disease was dysmenorrhea (45.96%), followed by polycystic ovary syndrome, PCOS (19.04%), uterine fibroids (14.23%), spontaneous abortion (13.20%), trouble conceiving (12.59%), ovarian dysfunction (11.16%) and endometriosis (4.09%). In the adjusted model, heavy bleeding with large clots was associated with an increased risk of dysmenorrhea (odds ratio, OR = 5.01, 95% Confidence interval, CI 2.26, 11.10; p = 0.000), while history of precocious puberty diagnosis was associated with a reduced risk of dysmenorrhea (OR = 0.50, 95%CI: 0.26, 0.94; p = 0.031). Regular menstrual cycle in the past 12 months and regular menstrual periods were associated with decreased risk of PCOS (OR = 0.44, 95%CI 0.30, 0.65; p = 0.000) and (OR = 0.52, 95%CI 0.36, 0.74; p = 0.000), respectively. Histories of early thelarche, early menarche, and precocious puberty diagnosis were associated with increased risk of ovarian dysfunction (OR = 1.96, 95%CI 1.25, 3.08, p = 0.004), (OR = 2.26, 95%CI 1.24, 4.13; p = 0.008) and (OR = 2.79, 95%CI 1.36, 5.74; p = 0.005), respectively. Heavy bleeding and heavy bleeding with large clots were associated with endometriosis (OR = 4.92, 95%CI 1.50, 16.15, p = 0.009) and (OR = 5.67, 95%CI 1.42, 22.56; p = 0.014), respectively. CONCLUSIONS: The prevalence of gynecological diseases is increasing among Chinese women and pubertal timing and menstrual characteristics may be associated with some gynecological diseases, specifically dysmenorrhea, PCOS, ovarian dysfunction, and endometriosis.

Microfluidic Platform for Time-Resolved Characterization of Protein Higher-Order Structures and Dynamics Using Top-Down Mass Spectrometry.

Characterization of protein higher-order structures and dynamics is essential for understanding the biological functions of proteins and revealing the underlying mechanisms. Top-down mass spectrometry (MS) accesses structural information at both the intact protein level and the peptide fragment level. Native top-down MS allows analysis of a protein complex's architecture and subunits' identity and modifications. Top-down hydrogen/deuterium exchange (HDX) MS offers high spatial resolution for conformational or binding interface analysis and enables conformer-specific characterization. A microfluidic chip can provide superior performance for front-end reactions useful for these MS workflows, such as flexibility in manipulating multiple reactant flows, integrating various functional modules, and automation. However, most microchip-MS devices are designed for bottom-up approaches or top-down proteomics. Here, we demonstrate a strategy for designing a microchip for top-down MS analysis of protein higher-order structures and dynamics. It is suitable for time-resolved native MS and HDX MS, with designs aiming for efficient ionization of intact protein complexes, flexible manipulation of multiple reactant flows, and precise control of reaction times over a broad range of flow rates on the submicroliter per minute scale. The performance of the prototype device is demonstrated by measurements of systems including monoclonal antibodies, antibody-antigen complexes, and coexisting protein conformers. This strategy may benefit elaborate structural analysis of biomacromolecules and inspire method development using the microchip-MS approach.

A new thermophilic extradiol dioxygenase promises biodegradation of catecholic pollutants.

Extradiol dioxygenases (EDOs) catalyze the meta cleavage of catechol into 2-hydroxymuconaldehyde, a critical step in the degradation of aromatic compounds in the environment. In the present work, a novel thermophilic extradiol dioxygenase from Thermomonospora curvata DSM43183 was cloned, expressed, and characterized by phylogenetic and biochemical analyses. This enzyme exhibited excellent thermo-tolerance, displaying optimal activity at 50 °C, remaining >40% activity at 70 °C. Structural modeling and molecular docking demonstrated that both active center and pocket-construction loops locate at the C-terminal domain. Site-specific mutants D285A, H205V, F301V based on a rational design were obtained to widen the entrance of substrates; resulting in significantly improved catalytic performance for all the 3 mutants. Compared to the wild-type, the mutant D285A showed remarkably improved activities with respect to the 3,4-dihydroxyphenylacetic acid, catechol, and 3-chlorocatechol, by 17.7, 6.9, and 3.7-fold, respectively. The results thus verified the effectiveness of modeling guided design; and confirmed that the C-terminal loop structure indeed plays a decisive role in determining catalytic ring-opening efficiency and substrate specificity of the enzyme. This study provided a novel thermostable dioxygenase with a broad substrate promiscuity for detoxifying environmental pollutants and provided a new thinking for further enzyme engineering of EDOs.

Bortezomib potentiates antitumor activity of mitoxantrone through dampening Wnt/ß-catenin signal pathway in prostate cancer cells.

BACKGROUND: Bortezomib (BZM), alone or in combination with other chemotherapies, has displayed strong anticancer effects in several cancers. The efficacy of the combination of BZM and mitoxantrone (MTX) in treating prostate cancer remains unknown. METHODS: Anticancer effects of combination of BZM and MTX were determined by apoptosis and proliferation assay in vivo and in vitro. Expression of ß-Catenin and its target genes were characterized by western blot and Real-time PCR. RESULTS: BZM significantly enhanced MTX-induced antiproliferation in vivo and in vitro. Mice administered a combination of BZM and MTX displayed attenuated tumor growth and prolonged survival. BZM significantly attenuated MTX-induced apoptosis. Moreover, the combination of BZM and MTX contributed to inhibition of the Wnt/ß-Catenin signaling pathway compared to monotherapy. CONCLUSIONS: This study demonstrates that BZM enhances MTX-induced anti-tumor effects by inhibiting the Wnt/ß-Catenin signaling pathway in prostate cancer cells.

Releasing Nonperipheral Subunits from Protein Complexes in the Gas Phase.

The quaternary structure is an important feature regulating protein function. Native mass spectrometry contributes to untangling quaternary structures by preserving the integrity of protein complexes in the gas phase. Tandem mass spectrometry by collision-induced dissociation (CID) can then be used to release subunits from these intact complexes, thereby providing structural information on the stoichiometry and topology. Cumulatively, such studies have revealed the preferred release of peripheral subunits during CID. In contrast, here we describe and focus on dissociation pathways that release nonperipheral subunits from hetero-complexes in CID at high collision energies. We find that nonperipheral subunits are ejected with a high propensity, as a consequence of sequential dissociation events, upon initial removal of peripheral subunits. Alternatively, nonperipheral subunits can be released directly from a charge-reduced or an elongated intact complex. As demonstrated here for a range of protein assemblies, releasing nonperipheral subunits under controlled conditions may provide unique structural information on the stoichiometry and topology of protein complexes.

Rational Engineering of Hydratase from Lactobacillus acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity.

Enzymatic conversion of fatty acids (FAs) by fatty acid hydratases (FAHs) presents a green and efficient route for high-value hydroxy fatty acid (HFA) production. However, limited diversity was achieved among HFAs, to date, with respect to chain length and hydroxy position. In this study, two highly similar FAHs from Lactobacillus acidophilus were compared: FA-HY2 has a narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilizes longer chain substrates and hydrates various double-bond positions. It is revealed that three active-site residues play a remarkable role in directing substrate specificity and regioselectivity of hydration. If these residues on FA-HY2 are mutated to the corresponding ones in FA-HY1, a significant expansion of substrate scope and a distinct enhancement in hydration of double bonds towards the ω-end of FAs is observed. A three-residue mutant of FA-HY2 (TM-FA-HY2) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting the ratio of the HFA regioisomers (10-OH/13-OH) from 99:1 to 12:88. Notable changes in regioselectivity were also observed for arachidonic acid and for C18 polyunsaturated fatty acid substrates. In addition, TM-FA-HY2 converted eicosapentaenoic acid into its 12-hydroxy product with high conversion at the preparative scale. Furthermore, it is demonstrated that microalgae are a source of diverse FAs for HFA production. This study paves the way for tailor-made FAH design to enable the production of diverse HFAs for various applications from the polymer industry to medical fields.

A Physic Nut Stress-Responsive HD-Zip Transcription Factor, JcHDZ07, Confers Enhanced Sensitivity to Salinity Stress in Transgenic Arabidopsis.

Homeodomain-leucine zipper (HD-Zip) transcription factors are reported to play crucial roles in the growth, development, and stress responses of plants. However, there is little knowledge of the molecular mechanisms involved in physic nut's stress tolerance generally, or the functions of its HD-Zip genes. In the present study, a HD-Zip family transcription factor, designated JcHDZ07, was isolated from physic nut. Expression profile analysis showed that salinity stress inhibited the expression of JcHDZ07. Transient expression of JcHDZ07-YFP in Arabidopsis protoplast cells revealed that JcHDZ07 was a nuclear-localized protein. Additionally, no obvious difference in growth and development between wild-type and JcHDZ07-overexpressing plants was observed in the absence of stress. Our results further indicated that JcHDZ07 overexpressing transgenic plants had lower proline contents, lower survival rates, and activities of catalase and superoxide dismutase, but higher relative electrical leakage and malonaldehyde contents compared with wild-type plants under salinity stress conditions, suggesting that overexpression of JcHDZ07 confers enhanced sensitivity to salinity stress in transgenic Arabidopsis. Expression of salt stress-responsive genes were upregulated in leaves of transgenic plants under salinity stress, but less strongly than in wild-type plants. Collectively, our results suggest that JcHDZ07 functions as an important regulator during the process of plant responses to salinity stress.

MMP-9 Upregulation is Attenuated by the Monoclonal TLR2 Antagonist T2.5 After Oxygen-Glucose Deprivation and Reoxygenation in Rat Brain Microvascular Endothelial Cells.

BACKGROUND: Blood-brain barrier (BBB) disruption plays a key role in the pathophysiology of acute ischemic stroke. Matrix metalloproteinases-2/9 (MMP-2/9) have been shown to participate in the disruption of the BBB and hemorrhagic transformation after cerebral ischemia. Toll-like receptor 2 (TLR2) may also be correlated with endothelial cell injury during ischemia-reperfusion events. However, the correlation between MMP-2/9 and TLR2 on endothelial cells after ischemia has not yet been evaluated. The aim of the study was to evaluate the impact of TLR2 and MMP-2/9 on tight junction proteins (TJs) after oxygen-glucose deprivation and reoxygenation (OGDR). MATERIALS AND METHODS: Rat primary brain microvascular endothelial cells (BMECs) were cultured. Quantitative real-time PCR and western blotting were used to measure the mRNA and proteins expression of TLR2 and MMP-2/-9. The protein expression of TJs was detected by western blotting and immunofluorescence. RESULTS: MMP-9 significantly increased after OGDR. Protein and mRNA expression of TLR2 was also upregulated. However, claudin-5, occludin, collagen-Ⅳ, and ZO-1 were decreased after OGDR. When monoclonal anti-TLR2 antibody (T2.5) was added to BMECs after OGDR, MMP-9 was significantly downregulated, whereas occludin and collagen-Ⅳ had a tendency to increase. CONCLUSION: TLR2 antagonist T2.5 is able to downregulate the expression of MMP-9, and may constitute a therapeutic option for restoration of the BBB after OGDR.

TLR2 Ligand Pam3CSK4 Regulates MMP-2/9 Expression by MAPK/NF-κB Signaling Pathways in Primary Brain Microvascular Endothelial Cells.

Blood-brain barrier (BBB) destruction is associated with a variety of neurological diseases. Brain microvascular endothelial cells (BMECs) are the key constituent of BBB. Both matrix metalloproteinases-2/9 (MMP-2/9) and toll-like receptor-2 (TLR2) are coexpressed in BMECs and have been shown to play important roles in BBB breakdown. It is unknown whether TLR2 can regulate MMP-2/9 in BMECs. In this study, Pam3CSK4 was used to activate TLR2, and the expression of MMP-2/9 and tight junctions (TJs) in BBB was measured by quantitative real-time PCR and western blotting. Phosphoproteins were determined by western blotting. The inhibitors of mitogen-activated protein kinases (MAPKs) and NF-κB were used to identify the signaling pathways by which TLR2 regulates the expression of MMP-2/9 in BMECs. This study showed that Pam3CSK4 upregulated the mRNA and protein expression of MMP-9 and downregulated MMP-2 and TJ expression in BMECs simultaneously. Pam3CSK4 also induced the phosphorylation of MAPKs and NF-κB signaling pathways in BMECs. MMP-9 expression was found to decrease by pretreatment with inhibitors of ERK1/2 and JNK but not p38. However, the mRNA and protein expression of MMP-2 and MMP-9 increased after addition of a NF-κB inhibitor. Our results indicated that Pam3CSK4 was able to upregulate MMP-9 expression through ERK1/2 and JNK signaling pathways, but the NF-κB signaling pathway negatively regulated the effect of TLR2 on MMP-2 and MMP-9 expression in BMECs. The finding provides novel insight into the molecular mechanism of MMP-2/9 expression in BMECs.

Hyperthermia exacerbates the effects of cathepsin L on claudin-1 in a blood-brain barrier model in vitro.

PURPOSE: The effects of cathepsin L on claudin-1 expression were investigated under hyperthermic condition in a blood-brain barrier (BBB) model in vitro, in order to estimate the potential effects of hyperthermia on BBB dysfunction. MATERIALS AND METHODS: Brain microvascular endothelial cells (BMECs) and astrocytes were obtained from rat brain. The BBB models were randomly divided into a sham (37°C) group, a 39°C group, a 37°C+cathepsin L group and a 39°C+cathepsin L group. The permeability of BBB was judged. The expressions of cathepsin L in astrocytes and claudin-1 in BMECs were detected using immunohistochemistry method and western blot assay. RESULTS: The permeability of BBB models was higher in the 39°C group than in the sham group. The cathepsin L expression in astrocytes was higher in the 39°C group than in the sham group (P<0.01), whereas the claudin-1 expression in BMECs was lower in the 39°C group than in the sham group (P<0.01). The claudin-1 expression in BMECs was significantly lower in the 37°C+cathepsin L group than in the sham group (P<0.01). At the same time point, the claudin-1 expression in BMECs was significantly lower in the 39°C+cathepsin L group than in the 37°C+cathepsin L group (P<0.01). CONCLUSION: Hyperthermia can probably decrease claudin-1 expression in BMECs by upregulating cathepsin L expression in astrocytes in a BBB model in vitro.