Protective effects of KLF4 on blood-brain barrier and oxidative stress after cerebral ischemia-reperfusion in rats through the Nrf2/Trx1 pathway.

PURPOSE: To investigate the role of KLF4 in CI/R injury and whether Nrf2/Trx1 axis acted as a downstream pathway of KLF4 to exert the protective role in blood-brain barrier destruction after CI/R. METHODS: The tMCAO rat model in vivo was constructed and received the intracerebroventricular injection of 5 µg/kg and 10 µg/kg rhKLF4 before operation. TTC, brain water content, neurological function, ELISA, behavioral tests, HE, TUNEL, and qRT-PCR were performed to detect the protective role of KLF4 on CIR. Double-fluorescence staining and western blot were performed to determine the localization and spatiotemporal expression in brain tissues. Furthermore, we also analyzed the effect of KLF4 on the blood-brain barrier (BBB) and related mechanisms in vivo and in vitro. Nrf2 inhibitor tretinoin was applied, which was intraperitoneally injected into CIR rat. Evans blue staining was conducted. In vitro OGD/R models of bEnd.3 cells were also established, and received KLF4 overexpressed transfection and 12.5 µM tretinoin incubation. The permeability of bEnd.3 cells was evaluated by TEER and FITC-dextran leakage. BBB-related factors and oxidative stress were also analyzed, respectively. The tubular ability of KLF4 on OGD/R bEnd3 cells was also evaluated. RESULTS: In vivo study confirmed that KLF4 was expressed in astrocyte, and its content increased with time. KLF4 protected against brain injury caused by cerebral ischemia-reperfusion, reduced cerebral infarction area and oxidative stress levels, and promoted the recovery of behavioral ability in rats. Simultaneously, mechanism experiments confirmed that the repair effect of KLF4 on cerebral ischemia-reperfusion injury was closely related to the Nrf2/Trx1 pathway. KLF4 exerted the neuroprotective effect through upregulating Nrf2/Trx1 pathway. Consistent with in vivo animal study, in vitro study also confirmed the effect of KLF4 on the permeability of bEnd.3 cells after OGD/R injury through Nrf2/Trx1 pathway. CONCLUSION: Collectively, KLF4 played neuroprotective role in CIR induced MCAO and OGD/R, and the beneficial effects of KLF4 was partly linked to Nrf2/Trx1 pathway.

Epithelial sodium channel enhanced osteogenesis via cGMP/PKGII/ENaC signaling in rat osteoblast.

The amiloride-sensitive epithelial sodium channel (ENaC) is a major contributor to intracellular sodium homeostasis. In addition to epithelial cells, osteoblasts (Obs) express functional ENaCs. Moreover, a correlation between bone Na content and bone disease has been reported, suggesting that ENaC-mediated Na(+) regulation may influence osteogenesis. Obs were isolated and cultured by enzyme digestion. Cell proliferation and differentiation were evaluated by WST-8 assay kit and AKP assay kit respectively. PKGII expression was silenced by siRNA. The mRNA expression was investigated by semi-quantitative PCR and the protein expression was determined by Western-blot. The cell-permeable cGMP analog 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) increased α-ENaC channel expression in primary rat Obs as indicated by RT-PCR. In addition, 8-pCPT-cGMP stimulation enhanced expression of the mRNA encoding cGMP-dependent protein kinases II (PKGII). The cGMP analog also promoted osteoblast proliferation, differentiation and induced the expression of several osteogenic genes, including core binding factor al, osteocalcin, alkaline phosphatase, collagen type I, and osteopontin. Furthermore, the expression of α-ENaC, the main functional subunit of ENaC, was reduced when a small interfering RNA specific for PKGII was introduced into Obs. Treatment with 8-pCPT-cGMP in cells transfected with the siRNA for PKGII partially reversed downregulated α-ENaC mRNA expression. Our results suggest that 8-pCPT-cGMP stimulates proliferation, differentiation, and osteogenic gene expression in Obs through cGMP/PKGII-dependent regulation of ENaC channel expression. The cGMP/PKGII signaling pathway is a potential target for pharmaceutical interventions to treat metabolic bone diseases.

miR-26a-5p restoration via EZH2 silencing blocks the IL-6/STAT3 axis to repress the growth of prostate cancer.

BACKGROUND: Interleukin-6 (IL-6) is involved in the activation of several oncogenic pathways in prostate cancer. However, its upstream trans-signaling pathway remains largely unknown. This work proposes a mechanistic explanation of IL-6's upstream effectors in prostate carcinogenesis. RESEARCH DESIGN & METHODS: Samples were harvested to validate the expression of EZH2, miR-26a-5p, and IL-6. Moreover, the protein and its phosphorylation of STAT3 (signal transducer and transcription activator 3) were assessed in prostate cancer cells. We explored the effects of these effectors on malignant phenotypes in vitro and tumor growth in vivo using functional assays. Bioinformatics analysis, dual-luciferase reporter gene assays, and chromatin immunoprecipitation (ChIP) assays were used to determine their binding relationships. RESULTS: Overexpression of EZH2 and IL-6, and under expression of miR-26a-5p was observed in prostate cancer. Silencing IL-6 repressed STAT3 to suppress the malignant phenotypes of prostate cancer cells. Mechanistically, EZH2 inhibited miR-26a-5p expression by promoting H3K27 histone methylation, and miR-26a-5p restricted the malignant phenotypes of prostate cancer by targeting IL-6. Ectopic EZH2 expression reduced xenograft growth by inhibiting miR-26a-5p and activating the IL-6/STAT3 axis. CONCLUSION: EZH2 May potentially be involved in regulating its expression by recruiting H3K27me3 to the miR-26a-5p promoter region, which could further impact the IL6/STAT3 pathway.

Effects of lysophosphatidic acid receptor 5 on NLRC4 inflammasome in brain tissues of transient cerebral ischemia/reperfusion rat.

AIM: To explore whether LPA5 was involved in the inflammatory responses in CI/R injury by regulation of NLRC4. METHOD: The cerebral I/R model in rats was constructed with ischemia of 2h and different time points of reperfusion. After that, western blot was used to determine protein expression (LPA5, NLRC4, AIM2, caspase-1, cleaved-caspase-1, mature IL-1ß, and precursor IL-1ß). And LPA5 and NLRC4 expression were also detected by using immunofluorescence experiment. Afterward, two sequence of LPA5-siRNA were injected into rats via intracerebroventricular administration. TTC staining and HE staining were performed. RESULT: As the reperfusion time was prolonged, LPA5 content was continuously increased, and the highest expression of NLRC4 was found at 4h of reperfusion. And protein expression of AIM2, cleaved-caspase-1, and mature IL-1ß was also at highest level at 4h. And after reperfusion of 4h, LPA5 siRNA1# or 2# was injected into lateral ventricles. LPA5 silence markedly reduced the infract volume and improved the histological change of ischemic zone. And LPA5 silence significantly downregulated NLRC4, AIM2, and the ratio of cleaved-caspase-1/caspase-1 and mature IL-1ß/precursor IL-1ß. And compared with LPA5-siRNA2#, LPA5-siRNA1# exerted a more significant effect. CONCLUSION: Low expression of LPA5 can protect against the inflammatory responses in CI/R model of rats through inhibiting NLRC4 inflammasomes.

Confined Crystallization of Pigment Red 146 in Emulsion Droplets and Its Mechanism.

In this work, the effect of confined space on crystallization processes of pigments was investigated by using C.I. Pigment Red 146 (PR 146) as a model compound. The colloidal system (i.e., emulsion droplets) was used as a nanoreactor to prepare nanoscale PR 146 for the inkjet printer. The effects of the space confinement were investigated by comparing the products of PR 146 prepared from bulk solution, macroemulsion, and miniemulsion. The results showed that PR 146 crystallized in mini-emulsion had the narrowest particle size distribution and the average particle size can be as small as 172.5 nm, one order of magnitude smaller than the one obtained from the bulk solution. X-ray diffraction (XRD) data revealed that PR 146 crystallized in all three solutions where the crystalline state and had similar crystallite sizes. The process mechanism of crystallization confined in the miniemulsion droplets was proposed and explained. The function mechanism of the co-stabilizer during the crystallization of PR 146 in emulsion was also explained. It was found that sodium chloride could counteract the pressure difference as an osmotic pressure agent and prevent the migrating of water from small droplets into big droplets. The influences of dosages of emulsifiers and co-stabilizers on droplet size and the size of the obtained PR 146 particles were evaluated and the optimal conditions were determined. Furthermore, the disparity of PR 146 products prepared by different methods was investigated by UV⁻Vis spectra. The aqueous dispersion of PR 146 crystallized in miniemulsion had the highest absorbance and darkest color.