Relative rigidity of cell-substrate effects on hepatic and hepatocellular carcinoma cell migration.

Polyacrylamide gels with different stiffness and glass were employed as substrates to investigate how substrate stiffness affects the cellular stiffness of adherent hepatocellular carcinoma (HCCLM3) and hepatic (L02) cells. The interaction of how cell-substrate stiffness influences cell migration was also explored. An atom force microscope measured the stiffness of HCCLM3 and L02 cells on different substrates. Further, F-actin assembly was analyzed using immunofluorescence and Western blot. Finally, cell-surface expression of integrin ß1 was quantified by flow cytometry. The results show that, while both HCCLM3 and L02 cells adjusted their cell stiffness to comply with the stiffness of the substrate they were adhered to, their tuning capabilities were different. HCCLM3 cell stiffness complied when substrate stiffness was between 1.1 and 33.7 kPa, whereas the analogous stiffness for L02 cells occurred at a higher substrate stiffness, 3.6 kPa up to glass. These ranges correlated with F-actin filament assembly and integrin ß1 expression. In a migration assay, HCCLM3 cells migrated faster on a relatively soft substrate, while L02 cells migrated faster on substrates that were relatively rigid. These findings indicate that different tuning capabilities of HCCLM3 and L02 cells may influence cell migration velocity on substrates with different stiffness by regulating cy- toskeleton remodeling and integrin ß1 expression.

Novel multi-biotin grafted poly(lactic acid) and its self-assembling nanoparticles capable of binding to streptavidin.

Targeted drug delivery requires novel biodegradable, specific binding systems with longer circulation time. The aim of this study was to prepare biotinylated poly(lactic acid) (PLA) nanoparticles (NPs) which can meet regular requirements as well conjugate more biotins in the polymer to provide better binding with streptavidin. A biotin-graft-PLA was synthesized based on previously published biodegradable poly(ethylene glycol) (PEG)-graft-PLA, with one polymer molecule containing three PEG molecules. Newly synthesized biotin-graft-PLA had three biotins per polymer molecule, higher than the previous biotinylated PLA (≤1 biotin per polymer molecule). A PEG with a much lower molecular weight (MW ~1900) than the previous biotinylated PLA (PEG MW ≥ 3800), and thus more biocompatible, was used which supplied good nonspecific protein-resistant property compatible to PEG-graft-PLA, suggesting its possible longer stay in the bloodstream. Biotin-graft-PLA specifically bound to streptavidin and self-assembled into NPs, during which naproxen, a model small molecule (MW 230 Da) and hydrophobic drug, was encapsulated (encapsulation efficiency 51.88%). The naproxen-loaded NPs with particle size and zeta potential of 175 nm and -27.35 mV realized controlled release within 170 hours, comparable to previous studies. The biotin-graft-PLA NPs adhered approximately two-fold more on streptavidin film and on biotin film via a streptavidin arm both in static and dynamic conditions compared with PEG-graft-PLA NPs, the proven nonspecific protein-resistant NPs. The specific binding of biotin-graft-PLA NPs with streptavidin and with biotin using streptavidin arm, as well as its entrapment and controlled release for naproxen, suggest potential applications in targeted drug delivery.

Differential response to CoCl2-stimulated hypoxia on HIF-1α, VEGF, and MMP-2 expression in ligament cells.

The adult human anterior cruciate ligament (ACL) has a poor functional healing response, whereas the medial collateral ligament (MCL) does not. The difference in intrinsic properties of these ligament cells can be due to their different response to their located microenvironment. Hypoxia is a key environmental regulator after ligament injury. In this study, we investigated the differential response of ACL and MCL fibroblasts to hypoxia on hypoxia-inducible factor-1α, vascular endothelial growth factor, and matrix metalloproteinase-2 (MMP-2) expression. Our results show that ACL cells responded to hypoxia by up-regulating the HIF-1α expression significantly as compared to MCL cells. We also observed that in MCL fibroblasts response to hypoxia resulted in increase in expression of VEGF as compared to ACL fibroblasts. After hypoxia treatment, mRNA and protein levels of MMP-2 increased in both ACL and MCL. Furthermore we found in ACL pro-MMP-2 was converted more into active form. However, hypoxia decreased the percentage of wound closure for both ligament cells and had a greater effect on ACL fibroblasts. These results demonstrate that ACL and MCL fibroblasts respond differently under the hypoxic conditions suggesting that these differences in intrinsic properties may contribute to their different healing responses and abilities.

Comparative study of normal and rheumatoid arthritis fibroblast-like synoviocytes proliferation under cyclic mechanical stretch: role of prostaglandin E2.

Fibroblast-like synoviocytes (FLSs) are one of the main contributors of prostaglandin E(2) (PGE(2)) in the hyperplastic synovium of rheumatoid arthritis (RA) patients. cyclooxygenase-2 (COX-2)/PGE(2) pathway is involved in the proliferation of several cell types. We have previously shown that mechanical stretch affects COX-2 and PGE(2) production in human RA FLSs; however, its role in cell proliferation remains to be elucidated. In this study, a comparison is drawn between human RA and normal FLSs to understand the role of mechanical stretch and PGE(2) on the proliferation of FLSs. The results showed that physiological level (6%, 1 Hz) of cyclic mechanical stretch significantly decreased the proliferation of RA FLSs but not normal FLSs, while the induction of apoptosis was not observed by stretch in either RA or normal FLSs. IL-1ß (5 ng/ml)-induced COX-2/PGE(2) levels are downregulated by stretch in RA FLSs only. Further investigation showed that high concentration (100 and 500 ng/ml) of PGE(2) significantly induced cell proliferation only in RA FLSs, and this induction failed to be suppressed by stretch. In conclusion, this study demonstrated that elevated levels of PGE(2) in the synovial cavity are involved in the proliferation of RA FLSs, and cyclic mechanical stretch regulates the RA synovial hyperplasia.

Novel PEG-graft-PLA nanoparticles with the potential for encapsulation and controlled release of hydrophobic and hydrophilic medications in aqueous medium.

This study concerns the encapsulation and controlled release of both hydrophobic and hydrophilic medications with one polymer, which are delivered together as a combined therapy to treat diseased tissue. To test our hypothesis that the novel PEG-graft-PLA (PEG, polyethylene glycol; PLA, polylactic acid) can deliver both the hydrophobic and hydrophilic medications on account of its amphiphility, charge, and graft structure, PEG-graft-PLA (molecular weight of PEG = 1900) with very low critical micelle concentration was synthesized. One hydrophilic (insulin) and one hydrophobic (naproxen) model medication were loaded in separately during its self-assembly in aqueous solution. The resulting nanoparticles (NPs) were narrowly distributed and spherical, with average particle size around 200 nm, zeta potential >-10 mV, and encapsulation efficiency >50%. The NPs realized controlled release of insulin and naproxen for over 24 and 160 hours, respectively. Specifically, the bioactivity of the insulin released from the NPs was maintained. Owing to encapsulation, both for hydrophobic and hydrophilic medicines, and NPs obtained with similar size and zeta potential, as well as maintenance of bioactivity of loaded protein, we expect the applications of PEG-graft-PLA NPs in combination therapy.

Electrospun poly (ɛ-caprolactone)/silk fibroin core-sheath nanofibers and their potential applications in tissue engineering and drug release.

One of the key tenets of tissue engineering is to develop scaffold materials with favorable biodegradability, surface properties, outstanding mechanical strength and controlled drug release property. In this study, we generated core-sheath nanofibers composed of poly (ɛ-caprolactone) (PCL) and silk fibroin (SF) blends via emulsion electrospinning. Nanofibrous scaffolds were characterized by combined techniques of scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), contact angle and tensile measurements. An in vitro FITC release study was conducted to evaluate sustained release potential of the core-sheath structured nanofibers. We found that the conformation of SF contained in PCL/SF composite nanofibers was transformed from random coil to ß-sheet when treated with methanol, leading to improved crystallinity and tensile strength of nanofibrous scaffolds. The hydrophobicity and diameter of nanofibers decreased when we increased the content of SF in PCL/SF composite nanofibers. Furthermore, we evaluated the potential of fabricated PCL/SF composite nanofibers as scaffold in vitro. The results confirmed that fabricated PCL/SF scaffolds improved cell attachment and proliferation. Our results demonstrated the feasibility to generate core-sheath nanofibers composed of PCL and SF using a single-nozzle technique. The produced nanofibrous scaffolds with sustained drug release have potential application in tissue engineering.

Evaluation of Lysimachia christinae Hance extracts as anticholecystitis and cholagogic agents in animals.

ETHNOPHARMACOLOGICAL RELEVANCE: Lysimachia christinae Hance is one of the herbs commonly used in traditional Chinese medicine for the treatment of cholecystitis and cholagogic efficiency. AIMS OF THE STUDY: The water extract of Lysimachia christinae Hance was investigated to see if it possesses cholecystitis and cholagogic effects through traditional pathways. MATERIALS AND METHODS: Lithocholic acid (LCA) and Escherichia coli were used to induce cholecystitis in adult guinea pigs. The present study evaluated the cholagogic effects of LCHE treatment on bile secretion and bile emptying in Sprague-Dawley rats and male Kunming mice. RESULTS: The results showed that LCHE not only produced excellent anticholecystitis effects but also improved lesion severity in gallbladders induced by LCA. Similarly, LCHE administered to animals in the high-dose group exhibited an antibacterial effect in acute cholecystitis, and treatment with a mid-range or a high dose of LCHE resulted in an antipyretic effect, however, three doses of LCHE treatment groups had no effect on pathological change induced by Escherichia coli in gallbladder. Treatment with a high dose of LCHE significantly promoted bile secretion (0-90min, P<0.01), and treatment with a mid-range dose also significantly promoted bile secretion (30-60min P<0.05). Furthermore, treatment with a high dose of LCHE significantly promoted bile emptying (P<0.01). CONCLUSIONS: Our results demonstrate that LCHE exhibits a marked anticholecystitis and cholagogic activity in animals, which supports previous claims of its use in traditional Chinese medicine.

Combined effects of TNF-α, IL-1ß, and HIF-1α on MMP-2 production in ACL fibroblasts under mechanical stretch: an in vitro study.

The dynamics between inflammatory factors, mechanical stress, and healing factors, in an intra-articular joint, are very complex after injury. Injury to intra-articular tissue [anterior cruciate ligament (ACL), synovium] results in hypoxia, accumulation of various pro-inflammatory factors, cytokines, and metalloproteases. Although the presence of increased amounts of matrix-metalloproteinases (MMP) in the joint fluid after knee injury is considered the key factor for ACL poor healing ability; however, the exact role of collective participants of the joint fluid on MMP-2 activity and production has not been fully studied yet. To investigate the combined effects of mechanical injury, inflammation and hypoxia induced factor-1α (HIF-1α) on induction of MMP-2; we mimicked the microenvironment of joint cavity after ACL injury. The results show that TNF-α and IL-1ß elevate the activity of MMP-2 in a dose- and time-dependent manner. In addition, mechanical stretch further enhances the MMP-2 protein levels with TNF-α, IL-1ß, and their mixture. CoCl(2) -induced HIF-1α (100 and 500 µM) also increases the levels and activity of MMP-2. Mechanical stretch has a strong additional effect on MMP-2 production with HIF-1α. Our results conclude that mechanical injury, HIF-1α and inflammatory factors collectively induce increased MMP-2 production in ACL fibroblasts, which was inhibited by NF-κB pathway inhibitor (Bay-11-7082).

Cyclic mechanical stretch downregulates IL-1ß-induced COX-2 expression and PGE(2) production in rheumatoid arthritis fibroblast-like synoviocytes.

In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) are one of the primary sources of inflammatory cytokines, including prostaglandins (PGs) and matrix metalloproteinases (MMPs) in joints that are detrimental to the bone, cartilage, and the surrounding tissue. Many studies, in recent years, have shown that physiotherapies play a beneficial effect on the maintenance of joint homeostasis in RA; however, the underlying mechanisms involved are still not fully elucidated. This study was performed to investigate cellular mechanism of mechanical strain-mediated actions in RA-FLS. RA-FLS were grown on collagen-coated silicone membranes and were exposed to 6% cyclic mechanical stretch at a frequency of 0.5 Hz for different times in the presence/absence of IL-1ß. Real-time PCR and western blotting were used to detect the mRNA and protein level of cyclooxygenase-2 (COX-2) and MMP-1. The production of prostaglandin E(2) (PGE(2)) was quantified by ELISA method. Our results showed that 6% cyclic mechanical stretch significantly inhibited IL-1ß-induced MMP-1 (gene) and COX-2 (gene and protein) expression at 15, 40, and 80 min. It also downregulated the IL-1ß-induced production of PGE(2). Further investigation of nuclear factor kappa B (NF-κB) signal pathway-related effectors IκB-α and IκB-ß revealed that 6% cyclic stretch inhibited their IL-1ß-induced degradation in cytoplasm as well as reversed their gene transcription levels. Our data suggest that gentle level of cyclic mechanical stretch exerts a protective effect on RA-FLS as it downregulates the level of MMP-1 protease, COX-2, and proinflammatory PGE(2). The underlying mechanism appears to be, in part, executed through NF-κB and its upstream effectors.

TGF-ß1 promoted MMP-2 mediated wound healing of anterior cruciate ligament fibroblasts through NF-κB.

The adult human anterior cruciate ligament (ACL) has poor functional healing response. Transforming growth factor (TGF)-ß1 enhances the wound repair by stimulating matrix proteins deposition as well as the proliferation and migration of cells. However, the function of the TGF-ß1-induced matrix metalloproteinases' (MMPs) activities in the wound healing process is poorly understood. In this study, exogenous MMP-2 is added to mimic the TGF-ß1-induced MMP-2 expression. Role of NF-κB pathway is further examined. Our results show that TGF-ß1 induces dramatic elevation of MMP-2 activities and the MMP-2/tissue inhibitors of metalloproteinases ratio. Furthermore, the exogenous MMP-2 significantly promoted in vitro wound healing abilities of ACL fibroblasts that are significantly blocked with the addition of its inhibitors. TGF-ß1 also increases the proliferation of ACL fibroblasts whereas MMP-2 alone does not, indicating that MMP-2 activities are not involved in the proliferation. TGF-ß1-induced MMP-2 activity is inhibited by Bay11-7082 and Bay11-7085 (NF-κB inhibitors). Our results demonstrate that increased TGF-ß1 facilitates the ACL healing process by promoting the fibroblasts migration and proliferation. The migration process is mediated by MMP-2 and NF-κB pathway is involved in TGF-ß1-mediated MMP-2 release.

Simultaneous determination of nine flavonoids in Polygonum hydropiper L. samples using nanomagnetic powder three-phase hollow fibre-based liquid-phase microextraction combined with ultrahigh performance liquid chromatography-mass spectrometry.

A simple, inexpensive, and efficient nanomagnetic powder three-phase hollow fibre-based liquid-phase microextraction (HF-LPME) technique combined with ultrahigh performance liquid chromatography-mass spectrometry (UPLC-MS) was developed for the simultaneous analysis of nine flavonoids in Polygonum hydropiper L. samples. The final, optimised extraction conditions were as follows: an organic solvent of ethyl acetate, a donor phase of aqueous KH2PO4 at pH 3.0, an acceptor phase of aqueous NaHCO3 at pH 8.5, a stirring rate of 1000 rpm, and an extraction time of 50 min. Under these conditions, analyte calibration curves were all linear, with correlation coefficients ≥ 0.9994. The relative standard deviation for all analytes in intra-day (0.8-2.2%) and inter-day (1.7-3.5%) precision tests was well within the acceptable ranges, as were the limits of quantitation (LOQ < 0.054 µg/L) and detection (LOD < 0.170 µg/L). Recoveries for all standard compounds were between 95.17% and 99.82%, with a RSD of no more than 2.3%. Correlative analyses demonstrated that the physicochemical parameters of the compounds themselves also influenced the extraction efficiency. This technology proved to be rapid, sensitive, and reliable for the quality control of P. hydropiper L. samples.

Mechanical stretch regulates the expression of matrix metalloproteinase in rheumatoid arthritis fibroblast-like synoviocytes.

Mechanical stretch plays a crucial role in articular joints. In rheumatoid arthritis (RA), it is well known that fibroblast-like synoviocytes (FLS) produce matrix metalloproteinases (MMPs), resulting in local invasion into and degradation of bone and cartilage. We sought to examine whether mechanical stretch regulates the expression and underlying signal pathways of MMP secretion (MMP-1, -3, -13) in RA-FLS. FLS were grown on elastic silicone membrane in an equibiaxial strain apparatus and were exposed to 6% mechanical stretch (equivalent to gentle stretch exercise) for 15 min and 75 min, respectively. Semiquantitative PCR and real-time PCR were used to measure and analyze gene expression. Protein levels were determined by Western blotting. The results showed that 15 min of mechanical stretch inhibited MMP-1 and MMP-13 mRNA and protein level. However, the degree of inhibition by 75 min of stretch in expression of MMP-1 and MMP-13 was lower compared with 15 min stretch groups. The mRNA expression of ERK-1, ets-1 and citied-2 were increased by 6% mechanical stretch under both time points, however c-jun and c-fos mRNA level were affected differently after 15 min and 75 min mechanical stretch compared to control group. There were no significant changes on MMP-3 and ets-2 mRNA level under both 6% mechanical stretch time points. In the presence of pro-inflammatory cytokines (IL-1beta and TNF-alpha), the stretch also reduced the mRNA expression of MMP-1 and MMP-13. In short, our results showed that gentle mechanical strain affects MMP-1 and MMP-13 expression, potentially through the ERK-1-ets-1-cited-2-c-jun signaling pathway.