The epithelial and endothelial cells lining the alveolus form a barrier essential for the preservation of the lung respiratory function, which is, however, vulnerable to excessive oxidative, inflammatory, and apoptotic insults. Whereas profound breaches in this barrier function cause pulmonary edema, more subtle changes may contribute to inflammation. The mechanisms by which cigarette smoke (CS) exposure induce lung inflammation are not fully understood, but an early alteration in the epithelial barrier function has been documented. We sought to investigate the occurrence and mechanisms by which soluble components of mainstream CS disrupt the lung endothelial cell barrier function. Using cultured primary rat microvascular cell monolayers, we report that CS induces endothelial cell barrier disruption in a dose- and time-dependent manner of similar magnitude to that of the epithelial cell barrier. CS exposure triggered a mechanism of neutral sphingomyelinase-mediated ceramide upregulation and p38 MAPK and JNK activation that were oxidative stress dependent and that, along with Rho kinase activation, mediated the endothelial barrier dysfunction. The morphological changes in endothelial cell monolayers induced by CS included actin cytoskeletal rearrangement, junctional protein zonula occludens-1 loss, and intercellular gap formation, which were abolished by the glutathione modulator N-acetylcysteine and ameliorated by neutral sphingomyelinase inhibition. The direct application of ceramide recapitulated the effects of CS, by disrupting both endothelial and epithelial cells barrier, by a mechanism that was redox and apoptosis independent and required Rho kinase activation. Furthermore, ceramide induced dose-dependent alterations of alveolar microcirculatory barrier in vivo, measured by two-photon excitation microscopy in the intact rat. In conclusion, soluble components of CS have direct endothelial barrier-disruptive effects that could be ameliorated by glutathione modulators or by inhibitors of neutral sphingomyelinase, p38 MAPK, JNK, and Rho kinase. Amelioration of endothelial permeability may alleviate lung and systemic vascular dysfunction associated with smoking-related chronic obstructive lung diseases.
Imbalance in production and clearance of amyloid beta (Aβ) is the primary reason for its deposition in Alzheimer disease. Macroautophagy/autophagy is one of the important mechanisms for clearance of both intracellular and extracellular Aβ. Here, through screening, we identified alborixin, an ionophore, as a potent inducer of autophagy. We found that autophagy induced by alborixin substantially cleared Aβ in microglia and primary neuronal cells. Induction of autophagy was accompanied by up regulation of autophagy proteins BECN1/Beclin 1, ATG5, ATG7 and increased lysosomal activities. Autophagy induced by alborixin was associated with inhibition of the phosphoinositide 3-kinase (PI3K)-AKT pathway. A knock down of PTEN and consistent, constitutive activation of AKT inhibited alborixin-induced autophagy and consequent clearance of Aβ. Furthermore, clearance of Aβ by alborixin led to significant reduction of Aβ-mediated cytotoxicity in primary neurons and differentiated N2a cells. Thus, our findings put forward alborixin as a potential anti-Alzheimer therapeutic lead.
Crocus sativus
L. (family: Iridaceae)
has been documented in traditional medicine with numerous medicinal
properties. Recently, we have shown that
C. sativus
extract (IIIM-141) displays promising efficacy in a genetic mice
(5XFAD) model of Alzheimer’s disease (AD) (
ACS Chem.
Neurosci
.
2017,
16
, 1756).
To translate the available traditional knowledge and the scientifically
validated results into modern medicine, herein we aimed to carry out
its preclinical development. IIIM-141 is primarily a mixture of crocins
containing
trans
-4-GG-crocin (36 % w/w) as the principal
component. The in vitro studies show that IIIM-141 has protective
as well as therapeutic properties in assays related to AD. It induces
the expression of P-gp, thereby enhancing the amyloid-β clearance
from an AD brain. It also inhibits NLRP3 inflammasome and protects
SH-SY5Y cells against amyloid-β- and glutamate-induced neurotoxicities.
In behavioral models, it decreased the streptozotocin-induced memory
impairment in rats and recovered the scopolamine-induced memory deficit
in Swiss albino mice at 100 mg/kg dose. The acute oral toxicity study
shows that IIIM-141 is safe up to the dose of 2000 mg/kg, with no
effect on the body weight and on the biochemical/hematological parameters
of the rats. The repeated oral administration of IIIM-141 for 28 days
at 100 mg/kg dose did not cause any preterminal deaths and abnormalities
in Wistar rats. The pharmacokinetic analysis indicated that after
oral administration of IIIM-141, the majority of crocin gets hydrolyzed
to its aglycone crocetin. The sustained release (SR) capsule formulation
was developed, which showed an improved in vitro dissolution profile
and a significantly enhanced plasma exposure in the pharmacokinetic
study. The SR formulation resulted in 3.3-fold enhancement in the
area under the curve of crocetin and doubling of the crocetin/crocin
ratio in plasma compared with the extract. The data presented herein
will serve as the benchmark for further research on this botanical
candidate.
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