Resveratrol induces proliferation in preosteoblast cell MC3T3-E1 via GATA-1 activating autophagy.

Resveratrol (RSV) could promote osteogenic activity, but its clinical application has been hampered in view of its poor bioavailability. Therefore, it is desirable to identify with certainty the molecular target of its bone mass boosting function, which is crucial to the design of an effective therapeutic strategy for the optimal treatment of osteoporosis. Emerging evidence has indicated that GATA-1, an important transcription factor in megakaryocyte and erythrocyte differentiation, can directly activate autophagy in erythrocytes, alluding to its impact on bone metabolism. In light of this, we sought to determine whether GATA-1 would be a putative target by which RSV would act on osteoblast proliferation and, if so, to explore the underlying mechanism involved in the process. We examined the cell viability, colony formation, cell cyclin expression, autophagy level, and the expression levels of GATA-1 and adenosine 5'-monophosphate (AMP)-activated protein kinase α (AMPKα) in osteoblastic cell strain MC3T3-E1. The results showed that RSV promoted the proliferation process in MC3T3-E1 coupled with increased expression of GATA-1 and phosphorylated AMPKα and activated autophagy. When GATA-1 was interfered with siRNA, both autophagy and proliferation were decreased. Administration of the agonist of phosphorylated AMPKα1 (Thr172) promoted the translocation of GATA-1 into the nucleus. Based on the above results, we concluded that RSV induces the proliferation of MC3T3-E1 by increasing GATA-1 expression, which thence activates autophagy; and of note, AMPKα is one of the upstream regulators of GATA-1.

Synergistic anti-inflammatory and osteogenic n-HA/resveratrol/chitosan composite microspheres for osteoporotic bone regeneration.

The development of functional materials for osteoporosis is ultimately required for bone remodeling. However, grafts were accompanied by increasing pro-inflammatory cytokines that impaired bone formation. In this work, nano-hydroxyapatite (n-HA)/resveratrol (Res)/chitosan (CS) composite microspheres were designed to create a beneficial microenvironment and help improve the osteogenesis by local sustained release of Res. Study of in vitro release confirmed the feasibility of n-HA/Res/CS microspheres for controlled Res release. Notably, microspheres had anti-inflammatory activity evidenced by the decreased expression of pro-inflammatory cytokines TNF-α, IL-1ß and iNOS in RAW264.7 cells in a dose dependent manner. Further, enhanced adhesion and proliferation of BMSCs seeded onto microspheres demonstrated that composite microspheres were conducive to cell growth. The ability to enhance osteo-differentiation was supported by up-regulation of Runx2, ALP, Col-1 and OCN, and substantial mineralization in osteogenic medium. When implanted into bone defects in the osteoporotic rat femoral condyles, enhanced entochondrostosis and bone regeneration suggested that the n-HA/Res/CS composite microspheres were more favorable for impaired fracture healing. The results indicated that optimized n-HA/Res/CS composite microspheres could serve as promising multifunctional fillers for osteoporotic bone defect/fracture treatment.

Syntheses and characterization of anti-thrombotic and anti-oxidative Gastrodin-modified polyurethane for vascular tissue engineering.

Vascular grafts must avoid negative inflammatory responses and thrombogenesis to prohibit fibrotic deposition immediately upon implantation and promote the regeneration of small diameter blood vessels (<6 mm inner diameter). Here, polyurethane (PU) elastomers incorporating anti-coagulative and anti-inflammatory Gastrodin were fabricated. The films had inter-connected pores with porosities equal to or greater than 86% and pore sizes ranging from 250 to 400 µm. Incorporation of Gastrodin into PU films resulted in desirable mechanical properties, hydrophilicity, swelling ratios and degradation rates without collapse. The released Gastrodin maintained bioactivity over 21 days as assessed by its anti-oxidative capability. The Gastrodin/PU had better anti-coagulation response (less observable BSA, fibrinogen and platelet adhesion/activation and suppressed clotting in whole blood). Red blood cell compatibility, measured by hemolysis, was greatly improved with 2Gastrodin/PU compared to other Gastrodin/PU groups. Notably, Gastrodin/PU upregulated anti-oxidant factors Nrf2 and HO-1 expression in H2O2 treated HUVECs, correlated with decreasing pro-inflammatory cytokines TNF-α and IL-1ß in RAW 264.7 cells. Upon implantation in a subcutaneous pocket, PU was encapsulated by an obvious fibrous capsule, concurrent with a large amount of inflammatory cell infiltration, while Gastrodin/PU induced a thinner fibrous capsule, especially 2Gastrodin/PU. Further, enhanced adhesion and proliferation of HUVECs seeded onto films in vitro demonstrated that 2Gastrodin/PU could help cell recruitment, as evidenced by rapid host cell infiltration and substantial blood vessel formation in vivo. These results indicate that 2Gastrodin/PU has the potential to facilitate blood vessel regeneration, thus providing new insight into the development of clinically effective vascular grafts.

Elastomeric polyurethane porous film functionalized with gastrodin for peripheral nerve regeneration.

The extracellular matrix provides cells with a support structure and an attachment site in actual substrate. Its biochemical and surface properties play an important role in and have significant impact on cell attachment, proliferation, migration, differentiation, and gene expression. Leveraging the hydrophilicity and neuroprotective of gastrodin, a gastrodin/polyurethane (PU) elastomer was developed utilizing in situ polymerization and salt-leaching methods. The results showed that gastrodin/PU film had a good flexibility and supporting strength, as well as hydrophilicity. Thus film possessed highly surface area, interconnected porous structure with a pore size (10~60 µm) for cell attachment, and could provide surface cues to augment neurite extension. For PC12 cells cultured within the films, especially the 5gastrodin/PU group, presented a progressive increase with time, coupled with the upregulation of brain-derived neurotrophic factor and glial cell derived neurotrophic factor expression. This is the first report on the construction of a gastrodin/PU porous film, and the results reveal its promise as a scaffold material for neural tissue engineering.

Gastrodin Inhibits Inflammasome Through the STAT3 Signal Pathways in TNA2 Astrocytes and Reactive Astrocytes in Experimentally Induced Cerebral Ischemia in Rats.

This study was aimed to determine Gastrodin (GAS) and its underlying signaling pathway involved in suppression of inflammasome specifically in reactive astrocytes that are featured prominently in different neurological conditions or diseases including cerebral ischemia. For this purpose, TNA2 astrocytes in cultures were exposed to oxygen-glucose-deprivation (OGD) mimicking hypoxic cerebral ischemia. Separately, TNA2 cells were pretreated with GAS prior to OGD exposure. Additionally, Stattic, an inhibitor of STAT3 signaling pathway, was used to ascertain its involvement in regulating inflammasome in astrocytes exposed to OGD. In parallel to the above, adult rats subjected to middle cerebral artery occlusion (MCAO) with or without GAS pretreatment were sacrificed at different time points to determine the effects of GAS on astrocyte inflammasome. TNA2 astrocytes in different treatments as well as reactive astrocytes in MCAO were processed for immunofluorescence labeling and Western blot analysis for various protein markers. In the latter, protein expression levels of p-STAT3, NLRP3, and NLRC4 were markedly increased in TNA2 astrocytes exposed to OGD. Remarkably, the expression levels of these biomarkers were significantly suppressed by GAS. Of note, GAS especially at dose 20 µM inhibited NLRP3 and NLRC4 expression levels most substantially. Moreover, GAS inhibited the downstream proteins caspase-1 and IL-18. Concomitantly, GAS significantly suppressed the expression of STAT3 and NF-κB signaling pathway. It is noteworthy that Stattic at dose 100 µM inhibited STAT3 pathway and NF-κB activation in TNA2 astrocytes, an effect that was shared by GAS. In MCAO, GAS was found to effectively attenuate p-STAT3 immunofluorescence intensity in reactive astrocytes. Arising from the above, it is concluded that GAS is anti-inflammatory as it effectively suppresses inflammasome in OGD-stimulated astrocytes as well as in reactive astrocytes in MCAO via STAT3 and NF-κB signaling expression coupled with decreased expression of caspase-1 and IL-18.

Automatic segmentation of pulmonary fissures in computed tomography images using 3D surface features.

Pulmonary interlobar fissures are important anatomic structures in human lungs and are useful in locating and classifying lung abnormalities. Automatic segmentation of fissures is a difficult task because of their low contrast and large variability. We developed a fully automatic training-free approach for fissure segmentation based on the local bending degree (LBD) and the maximum bending index (MBI). The LBD is determined by the angle between the eigenvectors of two Hessian matrices for a pair of adjacent voxels. It is used to construct a constraint to extract the candidate surfaces in three-dimensional (3D) space. The MBI is a measure to discriminate cylindrical surfaces from planar surfaces in 3D space. Our approach for segmenting fissures consists of five steps, including lung segmentation, plane-like structure enhancement, surface extraction with LBD, initial fissure identification with MBI, and fissure extension based on local plane fitting. When applying our approach to 15 chest computed tomography (CT) scans, the mean values of the positive predictive value, the sensitivity, the root-mean square (RMS) distance, and the maximal RMS are 91 %, 88 %, 1.01 ± 0.99 mm, and 11.56 mm, respectively, which suggests that our algorithm can efficiently segment fissures in chest CT scans.