Crown Ether Host-Guest Molecular Ferroelectrics.

In recent years, molecular ferroelectrics have received great attention due to their low weight, mechanical flexibility, easy preparation and excellent ferroelectric properties. Among them, crown-ether-based molecular ferroelectrics, which are typically composed of the host crown ethers, the guest cations anchored in the crown ethers, and the counterions, are of great interest because of the host-guest structure. Such a structure allows the components to occur order-disorder transition easily, which is beneficial for inducing ferroelectric phase transition. Herein, we summarized the research progress of crown ether host-guest molecular ferroelectrics, focusing on their crystal structure, phase transition, ferroelectric-related properties. In view of the small spontaneous polarization and uniaxial nature, we outlook the chemical design strategies for obtaining high-performance crown-ether-based molecular ferroelectrics. This minireview will be of guiding significance for the future exploration of crown ether host-guest molecular ferroelectrics.

TRIM21-regulated Annexin A2 plasma membrane trafficking facilitates osteosarcoma cell differentiation through the TFEB-mediated autophagy.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, which is characterized by dysfunctional autophagy and poor differentiation. Our recent studies have suggested that the tripartite motif containing-21 (TRIM21) plays a crucial role in regulating OS cell senescence and proliferation via interactions with several proteins. Yet, its implication in autophagy and differentiation in OS is largely unknown. In the present study, we first showed that TRIM21 could promote OS cell autophagy, as determined by the accumulation of LC3-II, and the degradation of cargo receptor p62. Further, we were able to identify that Annexin A2 (ANXA2), as a novel interacting partner of TRIM21, was critical for TIRM21-induced OS cell autophagy. Although TRIM21 had a negligible effect on the mRNA and protein expressions of ANXA2, we did find that TRIM21 facilitated the translocation of ANXA2 toward plasma membrane (PM) in OS cells through a manner relying on TRIM21-mediated cell autophagy. This functional link has been confirmed by observing a nice co-expression of TRIM21 and ANXA2 (at the PM) in the OS tissues. Mechanistically, we demonstrated that TRIM21, via facilitating the ANXA2 trafficking at the PM, enabled to release the transcription factor EB (TFEB, a master regulator of autophagy) from the ANXA2-TFEB complex, which in turn entered into the nucleus for the regulation of OS cell autophagy. In accord with previous findings that autophagy plays a critical role in the control of differentiation, we also demonstrated that autophagy inhibited OS cell differentiation, and that the TRIM21/ANXA2/TFEB axis is implicated in OS cell differentiation through the coordination with autophagy. Taken together, our results suggest that the TRIM21/ANXA2/TFEB axis is involved in OS cell autophagy and subsequent differentiation, indicating that targeting this signaling axis might lead to a new clue for OS treatment.

Astragaloside IV attenuates high glucose-induced EMT by inhibiting the TGF-ß/Smad pathway in renal proximal tubular epithelial cells.

In the present study, we examined the molecular mechanism of astragaloside IV (AS-IV) in high glucose (HG)-induced epithelial-to-mesenchymal transition (EMT) in renal proximal tubular epithelial cells (PTCs). NRK-52E cell viability and apoptosis were determined by the cell counting kit-8 (CCK-8) assay and flow cytometric analysis, respectively. Expressions of E-cadherin, N-cadherin, vimentin, and occludin were measured by Western blot, and those of E-cadherin and N-cadherin were additionally measured by immunofluorescence analysis. Transforming growth factor-ß1 (TGF-ß1) and α-smooth muscle actin (α-SMA) expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The expressions of Smad2, Smad3, phosphorylated-Smad2 (p-Smad2), and p-Smad3 were measured using Western blot. We found that AS-IV could recover NRK-52E cell viability and inhibit HG-induced cell apoptosis. TGF-ß1, α-SMA, Smad2, Smad3, p-Smad2, and p-Smad3 expressions were decreased in the AS-IV-treated groups compared with the HG group. Moreover, the expressions of E-cadherin and occludin were remarkably up-regulated and those of N-cadherin and vimentin were down-regulated in the AS-IV-treated groups compared with the HG group. Interestingly, the TGF-ß1 activator SRI-011381 hydrochloride had an antagonistic effect to AS-IV on HG-induced EMT behavior. In conclusion, AS-IV attenuates HG-induced EMT by inhibiting the TGF-ß/Smad pathway in renal PTCs.

YAP mediates the positive regulation of hnRNPK on the lung adenocarcinoma H1299 cell growth.

Lung cancer is the leading cause of cancer death worldwide, and non-small cell lung cancer (NSCLC) accounts for 80%-85% of diagnostic cases. The molecular mechanisms of NSCLC pathogenesis are not well understood. Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a multifunctional protein that regulates gene expression and signal transduction and closely associated with tumorigenesis, but its mechanism of action in the pathogenesis of NSCLC is unclear. In this study, we observed that the expression pattern of hnRNPK in H1299 lung adenocarcinoma cells varied depending on the cell density in culture. Moreover, hnRNPK stimulated the ability of proliferation and colony formation of H1299 cells, which is important for the multilayered cell growth in culture. We further investigated whether there is an association between hnRNPK and the elements involved in the cell contact inhibition pathway. By using quantitative reverse transcriptase-polymerase chain reaction assay and a YAP activity reporter system, we found that hnRNPK upregulated the mRNA and protein levels and transcriptional activity of Yes-associated protein 1 (YAP), a master negative regulator of Hippo contact inhibition pathway. Furthermore, YAP knockdown with siRNA abolished the stimulatory effect of hnRNPK on H1299 cell proliferation. These results suggested that YAP could be one of the effectors of hnRNPK. Our data may provide new clues for further understanding the biological functions of hnRNPK, particularly in the context of lung adenocarcinoma oncogenesis.

Regulation and related mechanism of GSN mRNA level by hnRNPK in lung adenocarcinoma cells.

Gelsolin (GSN) is an actin filament-capping protein that plays a key role in cell migration. Here we show that heterogeneous nuclear ribonucleoprotein K (hnRNPK) regulates GSN expression level by binding to the 3'-untranslated region (3'UTR) of GSN mRNA in non-small cell lung cancers (NSCLC) H1299 cells which are highly metastatic and express high level of GSN. We found that hnRNPK overexpression increased the mRNA and protein level of GSN, whereas hnRNPK knockdown by siRNA decreased the mRNA and protein level of GSN in both H1299 and A549 cells, indicating a positive role of hnRNPK in the regulation of GSN expression. Furthermore, hnRNPK knockdown affected the migration ability of H1299 and A549 cells which could be rescued by ectopic expression of GSN in those cells. Conversely, GSN knockdown in hnRNPK-overexpressing cells could abort the stimulatory effect of hnRNPK on the cell migration. These results suggest that hnRNPK function in the regulation of cell migration is GSN-dependent. Taken together, these data unveiled a new mechanism of regulation of the GSN expression by hnRNPK and provides new clues for the discovery of new anti-metastatic therapy.

Absorption and efflux characteristics of CP-25 in plasma and peripheral blood mononuclear cells of rats by UPLC-MS/MS.

Paeoniflorin-6'-O-benzene sulfonate (code: CP-25), is an active monomer obtained by modifying the structure of paeoniflorin (Pae). CP-25 can alleviate the course of adjuvant arthritis (AA) rats by regulating immune inflammatory response and reducing bone damage. In addition, our research has found that immune cells are important target cells for its anti-inflammatory and immunomodulatory effects. Therefore, it is of great significance to study the pharmacokinetics of CP-25 in immune cells. The aim of this study was to investigate the absorption and efflux of CP-25 in plasma and peripheral blood mononuclear cells (PBMCs) of rats. We established a sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to rapidly determine CP-25 in plasma and PBMC of rat. We found that the transport amount of CP-25 in PBMC gradually decreased with the increase of time, and reached equilibrium after 1 h. Moreover, there is a certain correlation between the concentration of CP-25 in plasma and the concentration of CP-25 in PBMC. In addition, we used several transporter inhibitors to study their effects on the efflux of CP-25 in PBMC. The efflux of CP-25 in PBMC increased with the increase of time in the first 30 min, and the efflux of CP-25 decreased gradually after 30 min. Furthermore, after multiple administration of 50 mg/kg in rats, concentration of CP-25 in PBMC is similar to the change of concentration of CP-25 in plasma. Our results suggest that CP-25 may enter PBMC by passive transport, and P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) may be involved in the efflux of CP-25 in PBMC. This research provides a basis and guidance for further study of the clinical application of CP-25.

YWHAZ Binds to TRIM21 but Is Not Involved in TRIM21-stimulated Osteosarcoma Cell Proliferation.

OBJECTIVE: Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21 (TRIM21) in osteosarcoma cell proliferation. METHODS: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation (BIFC) were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ. RESULTS: TRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However, overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21. CONCLUSION: Our results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma.

PPAR­Î³ agonist rosiglitazone protects rat peritoneal mesothelial cells against peritoneal dialysis solution­induced damage.

Long-term peritoneal dialysis (PD) leads to ultrafiltration failure (UFF). Peritoneal mesothelial cells, which form the innermost monolayer of the peritoneal cavity, have been shown to regulate various responses, including inflammation, in UFF. The present study was designed to investigate the effect of the peroxisome proliferator­activated receptor­Î³ (PPAR­Î³) agonist, rosiglitazone, on peritoneal dialysis solution (PDS)­induced injuries in rat peritoneal mesothelial cells (RPMCs). RPMCs were cultured for different durations and with different concentrations of PDS. The gene expression levels of aquaporin­1 (AQP­1) and zonula occluden­1 (ZO­1) were determined using reverse transcription­quantitative polymerase chain reaction analysis. The protein levels of AQP­1, ZO­1 and PPAR­Î³ were measured using western blot analysis. Interleukin (IL)­6 and IL­8 were detected using ELISA. The RPMCs were damaged by stimulation with 4.25% PDS for 72 h. The expression levels of AQP­1 and ZO­1 were increased, and the secretion of IL­6 and IL­8 were decreased by rosiglitazone. The use of the PPAR­Î³ inhibitor, GW­9662, completely prevented the effects of rosiglitazone. These results indicated that PDS exposure stimulated an inflammatory response in the RPMCs. The PPAR­Î³ activator, rosiglitazone, appeared to relieve the injury by inhibiting inflammation, and regulating the expression of AQP­1 and ZO­1, however further investigations are required to elucidate the potential underlying mechanism.

Atorvastatin alleviates renal ischemia-reperfusion injury in rats by promoting M1-M2 transition.

Acute kidney injury (AKI) often occurs as a result of ischemia-reperfusion (IR). Previous studies have demonstrated that inflammation is an important contributor to AKI. Atorvastatin (ATO) possesses anti­inflammatory properties and has been demonstrated to exert protective effects against renal IR injury (IRI). However, the underlying mechanism requires further study. In the present study, a rat model of renal IRI was successfully established. Consistent with the results of a previous study, ATO significantly attenuated IRI, which was supported by a decrease in serum creatinine and an increase in creatinine clearance rate, as well as alleviated pathological alterations in renal tubular cells. There are two types of activated macrophages: Proinflammatory M1 and anti­inflammatory M2 macrophages, which have been demonstrated to exert contributory and protective effects on IRI, respectively. The present study demonstrated that treatment with ATO significantly decreased M1 macrophage density and increased M2 macrophage density, as compared with the IR group. In addition, it is well known that M1 macrophages can be induced by T helper 1 cytokines, including tumor necrosis factor (TNF)­α and interferon (IFN)­Î³, whereas M2 macrophages can be induced by peroxisome proliferator-activated receptor (PPAR)­Î³. The present study indicated that ATO treatment significantly decreased the expression levels of TNF­α and IFN­Î³, and increased PPAR­Î³ expression. In conclusion, ATO may ameliorate renal IRI by promoting M1­M2 transition. Furthermore, ATO­mediated macrophage polarization in rats with renal IRI may be associated with the downregulation of TNF­α and IFN­Î³, and the upregulation of PPAR-γ.

Involvement of peroxisome proliferator activated receptor-γ in the anti-inflammatory effects of atorvastatin in oxygen-glucose deprivation/reperfusion-stimulated RAW264.7 murine macrophages.

Ischemia­reperfusion (I/R) injury is important in the pathogenesis and/or progression of various diseases, including stroke, cardiovascular disease and acute renal injury. Increasing evidence indicates that atorvastatin exerts protective effects in I/R injury­associated diseases; however, the underlying mechanisms remain to be fully elucidated. In the present study, oxygen­glucose deprivation (OGD)/reperfusion­stimulated. RAW264.7 murine macrophages served as a model of I/R injury. The knockdown of peroxisome proliferator activated receptor­Î³ (PPARγ) expression in these cells increased OGD/reperfusion­induced expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor­α (TNF­α) and interferon­Î³ (IFN­Î³), and enhanced OGD/reperfusion­induced downregulation of the expression of cluster of differentiation (CD) 206, at the mRNA and protein levels. Conversely, overexpression of PPARγ significantly attenuated OGD/reperfusion­induced alterations in the expression of iNOS, TNF­α, IFN­Î³ and CD206 at the mRNA and protein levels. Notably, atorvastatin inhibited OGD/reperfusion­induced iNOS expression and reversed OGD/reperfusion­induced downregulation of the expression of CD206 and PPARγ at the mRNA and protein levels. The results of the present study indicate that atorvastatin exhibits significant anti­inflammatory effects in OGD/reperfusion­stimulated RAW264.7 cells, possibly via PPARγ regulation. The findings of the present study may reveal a novel mechanism underlying the protective effects of atorvastatin in I/R injury­associated diseases.

Levels of vitamin D receptor and CYP24A1 in patients with end-stage renal disease.

OBJECTIVE: This study was performed to detect the expression of vitamin D receptor (VDR) and cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1) in 24 end stage renal disease (ESRD) patients and 24 healthy controls. METHOD: In this study, 24 ESRD patients and 24 healthy controls were included. RESULTS: In our study, the levels of VDR in patients with ESRD were reduced when compared with those from healthy controls (5.20±0.32 vs 8.59±1.03; P<0.01). However, the levels of CYP24A1 in ESRD patients were increased than those from healthy controls (50.18±21 vs 7.78±1.31; P<0.01). Correlation analysis showed that VDR levels were negatively correlated with CYP24A1 (r=-0.723; P<0.01). CONCLUSION: VDR levels were reduced and CYP24A1 levels were increased in patients with ESRD, and VDR levels were negatively correlated with CYP24A1.

Rosiglitazone, a Peroxisome Proliferator-Activated Receptor (PPAR)-γ Agonist, Attenuates Inflammation Via NF-κB Inhibition in Lipopolysaccharide-Induced Peritonitis.

We assessed the anti-inflammatory effect of peroxisome proliferator-activated receptor (PPAR)-γ agonist, rosiglitazone, in a lipopolysaccharide (LPS)-induced peritonitis rat model. LPS was intraperitoneally injected into rats to establish peritonitis model. Male Sprague-Dawley (SD) rats were assigned to normal saline (the solvent of LPS), LPS, rosiglitazone plus LPS, and rosiglitazone alone. A simple peritoneal equilibrium test was performed with 20 ml 4.25 % peritoneal dialysis fluid. We measured the leukocyte count in dialysate and ultrafiltration volume. Peritoneal membrane histochemical staining was performed, and peritoneal thickness was assessed. CD40 and intercellular adhesion molecule-1 messenger RNA (ICAM-1 mRNA) levels in rat visceral peritoneum were detected by reverse transcription (RT)-PCR. IL-6 in rat peritoneal dialysis effluent was measured using enzyme-linked immunosorbent assay. The phosphorylation of NF-κB-p65 and IκBα was analyzed by Western blot. LPS administration resulted in increased peritoneal thickness and decreased ultrafiltration volume. Rosiglitazone pretreatment significantly decreased peritoneal thickness. In addition to CD40 and ICAM-1 mRNA expression, the IL-6, p-p65, and p-IκBα protein expressions were enhanced in LPS-administered animals. Rosiglitazone pretreatment significantly decreased ICAM-1 mRNA upregulation, secretion of IL-6 protein, and phosphorylation of NF-κB-p65 and IκBα without decreasing CD40 mRNA expression. Rosiglitazone has a protective effect in peritonitis, simultaneously decreasing NF-κB phosphorylation, suggesting that NF-κB signaling pathway mediated peritoneal inflammation induced by LPS. PPAR-γ might be considered a potential therapeutic target against peritonitis.

Frequencies of apolipoprotein E alleles in depressed patients undergoing hemodialysis--a case-control study.

OBJECTIVE: To explore the relation between the frequencies of apolipoprotein E (ApoE) alleles and the occurrence of depression in patients undergoing hemodialysis in a Chinese population. METHODS: We examined the ApoE alleles in a sample of 288 subjects: 72 patients with depression under hemodialysis, 74 patients without depression under hemodialysis, 75 patients with depression under nondialytic treatment and 67 patients without depression under nondialytic treatment. The depression state was assessed using the Center for Epidemiological Studies Depression (CES-D) scale. Associations between the occurrence of depression and the frequencies of ApoE alleles were examined using multinomial logistic regression models with adjustment of relevant covariates. Information about sociodemographics, clinical data, vascular risk factors and cognitive function was also collected and evaluated. RESULTS: The frequencies of ApoE-ɛ2 were significantly different between depressed and non-depressed patients irrespective of dialysis (p < 0.05), but no significant difference was found in the frequencies of ApoE-ɛ4 (p > 0.05). Serum ApoE levels were significantly different between depressed and non-depressed patients in the whole sample (p < 0.05). Multinomial logistic regression models showed significant association between the frequency of ApoE-ɛ2 and the occurrence of depression in the Chinese population after control of relevant covariates, including age, sex, educational level, history of smoking and drinking, vascular risk factors and cognitive function. CONCLUSIONS: No association between the frequency of ApoE-ɛ4 and the occurrence of depression was found in patients undergoing hemodialysis. Further research is needed to find out if ApoE-ɛ2 acts as a protective factor in Chinese dialysis population since it might decrease the prevalence of depression and delay the onset age.

The von Hippel-Lindau protein pVHL inhibits ribosome biogenesis and protein synthesis.

pVHL, the product of von Hippel-Lindau (VHL) tumor suppressor gene, functions as the substrate recognition component of an E3-ubiquitin ligase complex that targets hypoxia inducible factor α (HIF-α) for ubiquitination and degradation. Besides HIF-α, pVHL also interacts with other proteins and has multiple functions. Here, we report that pVHL inhibits ribosome biogenesis and protein synthesis. We find that pVHL associates with the 40S ribosomal protein S3 (RPS3) but does not target it for destruction. Rather, the pVHL-RPS3 association interferes with the interaction between RPS3 and RPS2. Expression of pVHL also leads to nuclear retention of pre-40S ribosomal subunits, diminishing polysomes and 18S rRNA levels. We also demonstrate that pVHL suppresses both cap-dependent and cap-independent protein synthesis. Our findings unravel a novel function of pVHL and provide insight into the regulation of ribosome biogenesis by the tumor suppressor pVHL.

BF02, a recombinant TNFR2 fusion protein, alleviates adjuvant arthritis by regulating T lymphocytes in rats.

AIM: To investigate the therapeutic effects of BF02 on adjuvant arthritis (AA) in rats and the regulatory effects of BF02 on T lymphocyte function. METHODS: SD rats received a single intradermal injection of Freund's complete adjuvant emulsion into the right hind metatarsal footpad. After the onset of AA, the rats were injected BF02 (1, 3, or 9 mg/kg, sc) every 3 d for a total of 15 d. Intragastric administration of methotrexate (MTX, 0.5 mg/kg, every 3 d for a total of 15 d) was taken as the positive control drug. Arthritis index, swollen joint count, ankle joint histopathology, spleen histopathology and the paw radiography were used for evaluating the drug effects on AA rats. T lymphocyte function was assessed by measuring T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression levels, and percentage of T lymphocyte subsets. RESULTS: In the AA rats, remarkable secondary inflammatory responses exhibited, accompanied by significantly higher levels of IL-1, IL-6, TNF-α, IL-17, LTα, RANKL, and MMP-13. The expression of IL17 and TNF-α mRNAs was also substantially higher than in normal rats. The percentages of CD3(+)CD4(+) and CD4(+)CD25(+) T lymphocytes were increased, whereas the percentages of CD4(+)CD62L(+) and CD4(+)CD25(+)FoxP3(+) T lymphocytes were decreased. Treatment of the AA rats with BF02 (9 mg/kg) or MTX significantly decreased the arthritis index, swollen joint count and arthritis global assessment. Moreover, both BF02 (9 mg/kg) and MTX significantly inhibited T lymphocyte proliferation, and blocked the above mentioned aberrance in T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression, and percentages of T lymphocyte subsets. CONCLUSION: BF02 exerts therapeutic effects on AA rats via the regulation of T lymphocytes.

PAX2 protein induces expression of cyclin D1 through activating AP-1 protein and promotes proliferation of colon cancer cells.

Paired box (PAX) 2, a transcription factor, plays a critical role in embryogenesis. When aberrantly expressed in adult tissues, it generally exhibits oncogenic properties. However, the underlying mechanisms remain unclear. We reported previously that the expression of PAX2 was up-regulated in human colon cancers. However, the role of PAX2 in colon cancer cells has yet to be determined. The aim of this study is to determine the function of PAX2 in colon cancer cells and to investigate the possible mechanisms underlain. We find that knockdown of PAX2 inhibits proliferation and xenograft growth of colon cancer cells. Inhibition of PAX2 results in a decreased expression of cyclin D1. Expression of cyclin D1 is found increased in human primary colon malignant tumors, and its expression is associated with that of PAX2. These data indicate that PAX2 is a positive regulator of expression of cyclin D1. We find that knockdown of PAX2 inhibits the activity of AP-1, a transcription factor that induces cyclin D1 expression, implying that PAX2 induces cyclin D1 through AP-1. PAX2 has little effect on expression of AP-1 members including c-Jun, c-Fos, and JunB. Our data show that PAX2 prevents JunB from binding c-Jun and enhances phosphorylation of c-Jun, which may elevate the activity of AP-1. Taken together, these results suggest that PAX2 promotes proliferation of colon cancer cells through AP-1.

Glucose-based peritoneal dialysis fluids downregulate toll-like receptors and trigger hyporesponsiveness to pathogen-associated molecular patterns in human peritoneal mesothelial cells.

The objective of this study was to investigate the effects of glucose-based peritoneal dialysis (PD) fluids and icodextrin-based PD fluids on the expression of Toll-like receptor 2 (TLR2)/TLR4 and subsequent ligand-induced mitogen-activated protein kinase (MAPK) and NF-kappaB signaling and tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) mRNA expression in human peritoneal mesothelial cells (HPMCs). A human peritoneal mesothelial cell line (HMrSV5) was stimulated with glucose-based and icodextrin-based peritoneal dialysis fluids. Cell viability was assessed using MTT [3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide]. TLR2/TLR4 expression was determined by real-time PCR, Western blotting, and an immunofluorescence assay. In addition, cells were pretreated with different PD solutions and then incubated with Pam3CSK4 or lipopolysaccharide (LPS), and the degrees of MAPK and NF-kappaB activation were reflected by detecting the phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38, and p65, using a Western blot method. TNF-alpha and IL-1beta mRNA expression was measured by real-time PCR. Glucose-based peritoneal dialysis fluids suppressed the expression of TLR2 and TLR4 proteins in HPMCs. Challenge of cells with either Pam3CSK4 or LPS resulted in impaired TNF-alpha and IL-1beta production. Moreover, reduced TLR2 and TLR4 levels in glucose-based peritoneal dialysis solution-treated mesothelial cells were accompanied by reduced p42/44 (ERK1/2), JNK, p38 MAPK, and NF-kappaB p65 phosphorylation upon TLR ligand engagement. No significant changes in MAPK and NF-kappaB signaling and TNF-alpha and IL-1beta mRNA expression were observed in icodextrin-based PD solution-treated mesothelial cells. Glucose-based PD solution, but not icodextrin-based PD solution, downregulates expression of TLR2/TLR4 by human peritoneal mesothelial cells and triggers hyporesponsiveness to pathogen-associated molecular patterns.

Angiotensin II upregulates Toll-like receptor 4 and enhances lipopolysaccharide-induced CD40 expression in rat peritoneal mesothelial cells.

OBJECTIVE: Activation of Toll-like receptor 4 (TLR4) in peritoneal mesothelial cells by endotoxin contributes to peritoneal inflammation and fibrosis. Here we investigated TLR4 expression induced by angiotensin II (Ang II) and functional consequences of nuclear factor-kappaB (NF-kappaB) activation and CD40 expression in rat peritoneal mesothelial cells (RPMCs). METHODS: TLR4, CD40, tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were determined by reverse transcription polymerase chain reaction (RT-PCR) and TLR4, IkappaBalpha, phospho-IkappaBalpha, NF-kappaB p65, and phospho-NF-kappaB p65 were analyzed by Western blot. The intracellular distribution of NF-kappaB p65 was detected by immunofluorescence. RESULTS: Treatment of RPMCs with Ang II resulted in an increase in the expression of TLR4 mRNA and protein levels. Preincubation of RPMCs with Ang II significantly increased lipopolysaccharide (LPS)-induced phospho-IkappaBalpha and phospho-NF-kappaB p65 protein (P < 0.05 vs. LPS alone) and CD40, TNF-alpha, and IL-6 mRNA levels (P < 0.05 vs. LPS alone). A significantly increased nuclear staining of NF-kappaB p65 in cells treated with Ang II plus LPS was also observed. CONCLUSIONS: Ang II upregulates the expression of TLR4 by RPMCs, resulting in enhanced NF-kappaB signaling and induction of CD40, TNF-alpha, and IL-6 expression. Locally produced Ang II in the peritoneum may have an amplified role in LPS-induced peritoneal inflammation.

Peroxisome proliferator-activated receptor-gamma is expressed by rat peritoneal mesothelial cells: its potential role in peritoneal cavity local defense.

BACKGROUND/AIMS: Peritoneal mesothelial cells (PMCs) play an important role in peritoneal inflammatory and immune response. It was reported that the peroxisomal proliferator-activated receptor-gamma (PPARgamma) ligand could effectively reduce inflammatory processes. However, the expression and function of PPARgamma in PMCs has not been reported. This study was to investigate the expression of PPARgamma in rat PMCs and the effect of PPARgamma activation on the production of CD40 and ICAM-1 induced by lipopolysaccharide (LPS). METHODS: Rat PMCs (RPMCs) were harvested from the peritoneal cavity of Sprague-Dawley rats and maintained under defined in vitro conditions. The cells were treated separately with LPS, 15d-PGJ(2), and ciglitazone at different time points. The mRNA and protein expression of PPARgamma, CD40 and ICAM-1 were detected by RT-PCR and Western blot, respectively. The intracellular distribution of PPARgamma was detected by immunocytochemistry. RESULTS: RPMCs expressed PPARgamma both at the mRNA and protein level. The specific signals for PPARgamma were mainly localized in the nucleus with weak staining in the cytoplasm. Stimulation of RPMCs with LPS resulted in a time-dependent increase in the expression of PPARgamma with the peak of mRNA at 3 h and protein at 12 h. Thereafter the expression of PPARgamma gradually attenuated. The mRNA expressions for CD40, ICAM-1 and protein expression of ICAM-1 were significantly upregulated following stimulation with LPS. Both 15d-PGJ(2) and ciglitazone decreased the expression of CD40 mRNA and ICAM-1 protein. However, ciglitazone was less effective than 15d-PGJ(2). CONCLUSIONS: There is constitutive expression of PPARgamma in cultured RPMCs and PPARgamma ligands which strongly inhibit LPS-induced CD40 and ICAM-1 production in RPMCs. It suggested that PPARgamma might play a part in the local defense of the peritoneal cavity by downregulating inflammatory mediators, which may play a potential role in preventing peritoneal fibrosis induced by peritonitis. Further in vivo study is needed to demonstrate the long-term effects.

Effect of 15d-PGJ2 on the expression of CD40 and RANTES induced by IFN-gamma and TNF-alpha on renal tubular epithelial cells (HK-2).

BACKGROUND/AIMS: Recent evidence shows that peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ameliorates a variety of inflammatory conditions. CD40 is a co-stimulatory molecule and its ligation induces production of different proinflammatory cytokines including RANTES (regulated upon activation, normal T cell expressed), which are considered as important factors in the initiation and maintenance of inflammatory response. The aim of this study was to investigate the effect of PPAR-gamma on CD40 and RANTES production on cultured human renal proximal tubular epithelial (HK-2) cells. METHODS: HK-2 cells were maintained under defined in vitro conditions and treated with either PPAR-gamma agonist 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) or 15d-PGJ2 + PPAR-gamma antagonist GW9662, and then stimulated with a combination of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). The CD40 and RANTES levels were investigated. RESULTS: HK-2 cells expressed low levels of CD40 and RANTES. Activation of HK-2 cells by combined treatment of TNF-alpha and IFN-gamma results in strong synergistic effects on the expression of CD40 and the secretion of RANTES. 15d-PGJ2 significantly decreased CD40 and RANTES expression and GW9662 partly abrogated the inhibition of 15d-PGJ2 on CD40 and RANTES. CONCLUSION: 15d-PGJ2 significantly decreased CD40 and RANTES expression in HK-2 cells, which were partially mediated by PPAR-gamma-dependent pathways. These results point to PPAR-gamma as a remarkable new target in the prevention of tubular inflammatory injury associated with renal disease.