Metalloproteinase 9 (MMP-9) is able to degrade collagen IV, an important component of blood-brain barrier (BBB). Expression of MMPs, especially MMP-9, correlates with BBB disruption during central nervous system inflammation. Propofol has been reported to have anti-inflammation effects. In this study, we investigated the effects of propofol on TNF-α-induced MMP-9 expression in human cerebral microvascular endothelial cells (hCMEC/D3 cells) and explored the underlying mechanisms. The hCMEC/D3 cells were treated with propofol (25 μM), followed by TNF-α (25 ng/mL). We showed that TNF-α treatment markedly increased MMP-9 expression and decreased collagen IV expression in hCMEC/D3 cells, which was blocked by pretreatment with propofol. TNF-αinduced downregulation of collagen IV was also reversed by MMP-9 knockdown with siRNA. We revealed that TNF-α upregulated MMP-9 expression in hCMEC/D3 cells through activation of Ca 2+ /CAMK II/ERK/NF-κB signaling pathway; co-treatment with inhibitors of CaMK II (KN93), ERK (LY3214996), NF-κB (PDTC) or Ca 2+ chelator (BAPTA-AM) abrogated the effect of TNF-α on MMP-9 expression. We further established an in vitro BBB model by co-culturing of hCMEC/D3 cells and human astrocytes for 6 days and measuring trans-endothelial electrical resistance (TEER) to reflect the BBB permeability. TNF-α treatment markedly decreased TEER value, which was attenuated by pretreatment with propofol (25 μM) or MMP-9 knockdown with siRNA. In conclusion, propofol inhibits TNF-α-induced MMP-9 expression in hCMEC/D3 cells via repressing the Ca 2+ /CAMKII/ERK/NF-κB signaling pathway. TNF-αimpaired BBB integrity could be reversed by propofol, and propofol attenuates the inhibitory effect of TNF-α on collagen IV.
Microtubule affinity regulating kinase 4 (MARK4) plays a crucial role in the regulation of NOD-like receptor pyrin domain 3 (NLRP3) inflammasome activation, which leads to the generation of bioactive interleukin (IL)-1β and IL-18. E74-like ETS transcription factor 3 (ELF3) participates in endothelial inflammatory processes. We hypothesized that ELF3 modulates MARK4 expression in vascular endothelial cells, thus contributing to high glucose-mediated NLRP3 inflammasome activation. Plasma IL-1β, IL-18, NLRP3 inflammasome and MARK4 expression was increased in diabetic patients and rats. An in vitro study indicated that high glucose increased IL-1β and IL-18 expression and activated the NLRP3 inflammasome via upregulation of MARK4 in human umbilical vein endothelial cells (HUVECs). Furthermore, high glucose increased ELF3 expression. ELF3 downregulation reversed the effects of high glucose treatment. Accordingly, the effects of ELF3 overexpression were similar to those of high glucose treatment and were counteracted by siMARK4. Furthermore, ELF3 was found to interact with SET8. High glucose inhibited SET8 expression and histone H4 lysine 20 methylation (H4K20me1), a downstream target of SET8. Overexpression of SET8 inhibited high glucose-induced MARK4 expression and NLRP3 inflammasome activation. The effects of shSET8 were similar to those of high glucose treatment and were counteracted by siMARK4. A mechanistic study found that ELF3 and H4K20me1 were enriched in the MARK4 promoter region. si-ELF3 attenuated MARK4 promoter activity and augmented the inhibitory effect of SET8 on MARK4 promoter activity. Furthermore, SET8 downregulation and ELF3 upregulation were confirmed in diabetic patients and rats. In conclusion, ELF3 interacted with SET8 to modulate MARK4 expression, which participated in hyperglycaemia-mediated endothelial NLRP3 inflammasome activation.
Elucidation of the metabolic pathways determining pigmentation and their underlying regulatory mechanisms in maize kernels is of high importance in attempts to improve the nutritional composition of our food. In this study, we compared dynamics in the transcriptome and metabolome between colored SW93 and white SW48 by integrating RNA-Seq and non-targeted metabolomics. Our data revealed that expression of enzyme coding genes and levels of primary metabolites decreased gradually from 11 to 21 DAP, corresponding well with the physiological change of developing maize kernels from differentiation through reserve accumulation to maturation, which was cultivar independent. A remarkable up-regulation of anthocyanin and phlobaphene pathway distinguished SW93 from SW48, in which anthocyanin regulating transcriptional factors (R1 and C1), enzyme encoding genes involved in both pathways and corresponding metabolic intermediates were up-regulated concurrently in SW93 but not in SW48. The shift from the shikimate pathway of primary metabolism to the flavonoid pathway of secondary metabolism, however, appears to be under posttranscriptional regulation. This study revealed the link between primary metabolism and kernel coloration, which facilitate further study to explore fundamental questions regarding the evolution of seed metabolic capabilities as well as their potential applications in maize improvement regarding both staple and functional foods.
Kallikrein-related peptidase 8 (KLK8) acts as an oncogene or anti-oncogene in various tumours, and the abnormal expression of KLK8 is involved in the carcinogenesis of several tumours. However, the role of KLK8 in colorectal cancer (CRC) and the underlying mechanism remain largely unclear. In this study, the carcinogenic effect of KLK8 was determined via CCK-8 and colony formation assays in vitro and a xenograft model in nude mice in vivo. The metastasis-promoting effect of KLK8 was investigated with transwell migration and invasion assays and wound-healing assay in vitro and a metastasis model in nude mice in vivo. Bioinformatics analyses and mechanistic experiments were conducted to elucidate the molecular mechanism. Herein, we reported that KLK8 had a promotive effect on the proliferation, migration and invasion of RKO and SW480 cells. Epithelial−mesenchymal transition (EMT) played an important role in the promotive effects of KLK8 on CRC. In addition, protease-activated receptor-1 (PAR-1) antagonist SCH79797 but not protease-activated receptor-2 (PAR-2) antagonist FSLLRY-NH2 attenuated the proliferation, migration and invasion of KLK8-upregulated RKO and SW480 cells. PAR-1 antagonist SCH79797 reduced the tumour volume of xenograft model and decreased the metastatic nodules in the livers of metastasis model. Furthermore, SCH79797 could reverse the positive impact of KLK8 on the EMT process in CRC both in vitro and in vivo. Taken together, these findings demonstrated for the first time that KLK8 promoted EMT and CRC progression, and this effect might be, at least partly mediated by PAR1-dependent pathway.
Hyperglycemia-mediated endothelial inflammation participates in the pathogenesis of cardiovascular complications in subjects with diabetes. Previous studies reported that phosphatase and tensin homolog deleted on chromosome ten (PTEN) and SET8 participate in high glucose-mediated endothelial inflammation. In this study, we hypothesize that SET8 regulates PTEN expression, thus contributing to high glucose-mediated vascular endothelial inflammation. Our data indicated that plasma soluble intercellular adhesion molecule-1 (sICAM-1) and endothelial selectin (e-selectin) were increased in patients with diabetes and diabetic rats. PTEN expression was augmented in the peripheral blood mononuclear cells of patients with diabetes and in the aortic tissues of diabetic rats. Our in vitro study indicated that high glucose increased monocyte/endothelial adhesion, endothelial adhesion molecule expression and p65 phosphorylation in human umbilical vein endothelial cells (HUVECs). Moreover, high glucose led to endothelial inflammation via upregulation of PTEN. Furthermore, high glucose inhibited SET8 expression and histone H4 lysine 20 methylation (H4K20me1), a downstream target of SET8. SET8 overexpression reversed the effects of high-glucose treatment. shSET8-mediated endothelial inflammation was counteracted by siPTEN. Furthermore, SET8 was found to interact with FOXO1. siFOXO1 attenuated high glucose-mediated endothelial inflammation. FOXO1 overexpression-mediated endothelial inflammation was counteracted by siPTEN. H4K20me1 and FOXO1 were enriched in the PTEN promoter region. shSET8 increased PTEN promoter activity and augmented the positive effect of FOXO1 overexpression on PTEN promoter activity. Our in vivo study indicated that SET8 was downregulated and FOXO1 was upregulated in the peripheral blood mononuclear cells of patients with diabetes and the aortic tissues of diabetic rats. In conclusion, SET8 interacted with FOXO1 to modulate PTEN expression in vascular endothelial cells, thus contributing to hyperglycemia-mediated endothelial inflammation.
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