Sildenafil-Induced Revascularization of Rat Hindlimb Involves Arteriogenesis through PI3K/AKT and eNOS Activation.

Hypoxia and inflammation play a major role in revascularization following ischemia. Sildenafil inhibits phosphodiesterase-5, increases intracellular cGMP and induces revascularization through a pathway which remains incompletely understood. Thus, we investigated the effect of sildenafil on post-ischemic revascularization. The left femoral artery was ligated in control and sildenafil-treated (25 mg/kg per day) rats. Vascular density was evaluated and expressed as the left/right leg (L/R) ratio. In control rats, L/R ratio was 33 ± 2% and 54 ± 9%, at 7- and 21-days post-ligation, respectively, and was significantly increased in sildenafil-treated rats to 47 ± 4% and 128 ± 11%, respectively. A neutralizing anti-VEGF antibody significantly decreased vascular density (by 0.48-fold) in control without effect in sildenafil-treated animals. Blood flow and arteriolar density followed the same pattern. In the ischemic leg, HIF-1α and VEGF expression levels increased in control, but not in sildenafil-treated rats, suggesting that sildenafil did not induce angiogenesis. PI3-kinase, Akt and eNOS increased after 7 days, with down-regulation after 21 days. Sildenafil induced outward remodeling or arteriogenesis in mesenteric resistance arteries in association with eNOS protein activation. We conclude that sildenafil treatment increased tissue blood flow and arteriogenesis independently of VEGF, but in association with PI3-kinase, Akt and eNOS activation.

Nano-Sized Extracellular Matrix Particles Lead to Therapeutic Improvement for Cutaneous Wound and Hindlimb Ischemia.

Cell-derived matrix (CDM) has proven its therapeutic potential and been utilized as a promising resource in tissue regeneration. In this study, we prepared a human fibroblast-derived matrix (FDM) by decellularization of in vitro cultured cells and transformed the FDM into a nano-sized suspended formulation (sFDM) using ultrasonication. The sFDM was then homogeneously mixed with Pluronic F127 and hyaluronic acid (HA), to effectively administer sFDM into target sites. Both sFDM and sFDM containing hydrogel (PH/sFDM) were characterized via immunofluorescence, sol-gel transition, rheological analysis, and biochemical factors array. We found that PH/sFDM hydrogel has biocompatible, mechanically stable, injectable properties and can be easily administered into the external and internal target regions. sFDM itself holds diverse bioactive molecules. Interestingly, sFDM-containing serum-free media helped maintain the metabolic activity of endothelial cells significantly better than those in serum-free condition. PH/sFDM also promoted vascular endothelial growth factor (VEGF) secretion from monocytes in vitro. Moreover, when we evaluated therapeutic effects of PH/sFDM via the murine full-thickness skin wound model, regenerative potential of PH/sFDM was supported by epidermal thickness, significantly more neovessel formation, and enhanced mature collagen deposition. The hindlimb ischemia model also found some therapeutic improvements, as assessed by accelerated blood reperfusion and substantially diminished necrosis and fibrosis in the gastrocnemius and tibialis muscles. Together, based on sFDM holding a strong therapeutic potential, our engineered hydrogel (PH/sFDM) should be a promising candidate in tissue engineering and regenerative medicine.

Accumulation of formaldehyde causes motor deficits in an in vivo model of hindlimb unloading.

During duration spaceflight, or after their return to earth, astronauts have often suffered from gait instability and cerebellar ataxia. Here, we use a mouse model of hindlimb unloading (HU) to explore a mechanism of how reduced hindlimb burden may contribute to motor deficits. The results showed that these mice which have experienced HU for 2 weeks exhibit a rapid accumulation of formaldehyde in the gastrocnemius muscle and fastigial nucleus of cerebellum. The activation of semicarbazide-sensitive amine oxidase and sarcosine dehydrogenase induced by HU-stress contributed to formaldehyde generation and loss of the abilities to maintain balance and coordinate motor activities. Further, knockout of formaldehyde dehydrogenase (FDH-/-) in mice caused formaldehyde accumulation in the muscle and cerebellum that was associated with motor deficits. Remarkably, formaldehyde injection into the gastrocnemius muscle led to gait instability; especially, microinfusion of formaldehyde into the fastigial nucleus directly induced the same symptoms as HU-induced acute ataxia. Hence, excessive formaldehyde damages motor functions of the muscle and cerebellum.

All-trans-retinoic acid suppresses rat embryo hindlimb bud mesenchymal chondrogenesis by modulating HoxD9 expression.

In vertebrates, 5'-Hoxd genes (Hoxd9), which are expressed in the hindlimb bud mesenchyme, participate in limb growth and patterning in early embryonic development. In the present study, We investigated the mechanisms by which ATRA regulates cultured E12.5 rat embryo hindlimb bud mesenchymal cells (rEHBMCs). Following exposure to ATRA over 24 h, mRNA and protein expression levels of HoxD9 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR (qPCR), and western blotting. Flow cytometry was used to detect apoptosis. ATRA inhibited the condensation and proliferation, and promoted the apoptosis rate of the rEHBMCs in a dose-dependent manner. Sox9 and Col2a1 in rEHBMCs were downregulated by ATRA in a dose-dependent manner at both mRNA and protein levels. Similarly, HoxD9 was downregulated by ATRA in a dose-dependent manner, in parallel with the cartilage-specific molecules Sox9 and Col2a1. Both qPCR and western blotting showed that both Shh and Gli3 were downregulated. Overexpression of HoxD9 reversed the effects of ATRA. These results demonstrate that ATRA suppresses chondrogenesis in rEHBMCs by inhibiting the expression of HoxD9 and its downstream protein targets, including Sox9 and Col2a1. This effect may also be correlated with inhibition of the Shh-Gli3 signaling pathway.

Chronic Ouabain Prevents Na,K-ATPase Dysfunction and Targets AMPK and IL-6 in Disused Rat Soleus Muscle.

Sustained sarcolemma depolarization due to loss of the Na,K-ATPase function is characteristic for skeletal muscle motor dysfunction. Ouabain, a specific ligand of the Na,K-ATPase, has a circulating endogenous analogue. We hypothesized that the Na,K-ATPase targeted by the elevated level of circulating ouabain modulates skeletal muscle electrogenesis and prevents its disuse-induced disturbances. Isolated soleus muscles from rats intraperitoneally injected with ouabain alone or subsequently exposed to muscle disuse by 6-h hindlimb suspension (HS) were studied. Conventional electrophysiology, Western blotting, and confocal microscopy with cytochemistry were used. Acutely applied 10 nM ouabain hyperpolarized the membrane. However, a single injection of ouabain (1 µg/kg) prior HS was unable to prevent the HS-induced membrane depolarization. Chronic administration of ouabain for four days did not change the α1 and α2 Na,K-ATPase protein content, however it partially prevented the HS-induced loss of the Na,K-ATPase electrogenic activity and sarcolemma depolarization. These changes were associated with increased phosphorylation levels of AMP-activated protein kinase (AMPK), its substrate acetyl-CoA carboxylase and p70 protein, accompanied with increased mRNA expression of interleikin-6 (IL-6) and IL-6 receptor. Considering the role of AMPK in regulation of the Na,K-ATPase, we suggest an IL-6/AMPK contribution to prevent the effects of chronic ouabain under skeletal muscle disuse.

'Methyl palmitate attenuates adjuvant induced arthritis in rats by decrease of CD68 synovial macrophages.

The study was designed to investigate the potential anti-arthritic effects of methyl palmitate in an adjuvant arthritis model in rats that shares many histopathological similarities with human RA. The underlying mechanism and its effect on CD68 macrophages were investigated, as a further argument to its possible efficacy in RA treatment. A normal control group was injected only with saline, arthritic group, and three treatment groups with CFA induced arthritis received methyl palmitate (MP) at three different doses (75, 150, 300 mg/kg/week for 3 weeks, intraperitoneal). The degree of ipsilateral paw swelling, ankle diameter, spleen index, thymus index and the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß were measured. In addition, the underlying molecular mechanism was investigated using CD68 expression. Methyl palpitate significantly and dose dependently decreased the arthritic symptoms as measured by ipsilateral paw volume and ankle diameter. It showed no effect on body weight but significantly decreased splenic, thymus index, serum TNF-α and IL-1ß. CD68 macrophages expression and the overall synovial inflammatory cellularity were halted. Methyl palmitate exhibits significant anti-inflammatory and exerts a potential anti-arthritic effect in a rat model of adjuvant induced arthritis. Furthermore, it inhibits expression of synovial CD68 macrophage that validate its therapeutic potential adjuvant arthritis.

Lipidomic analysis of local inflammation models shows a specific systemic acute phase response to lipopolysaccharides.

Toll-like receptors (TLR) are crucial for recognizing bacterial, viral or fungal pathogens and to orchestrate the appropriate immune response. The widely expressed TLR2 and TLR4 differentially recognize various pathogens to initiate partly overlapping immune cascades. To better understand the physiological consequences of both immune responses, we performed comparative lipidomic analyses of local paw inflammation in mice induced by the TLR2 and TLR4 agonists, zymosan and lipopolysaccharide (LPS), respectively, which are commonly used in models for inflammation and inflammatory pain. Doses for both agonists were chosen to cause mechanical hypersensitivity with identical strength and duration. Lipidomic analysis showed 5 h after LPS or zymosan injection in both models an increase of ether-phosphatidylcholines (PC O) and their corresponding lyso species with additional lipids being increased only in response to LPS. However, zymosan induced stronger immune cell recruitment and edema formation as compared to LPS. Importantly, only in LPS-induced inflammation the lipid profile in the contralateral paw was altered. Fittingly, the plasma level of various cytokines and chemokines, including IL-1ß and IL-6, were significantly increased only in LPS-treated mice. Accordingly LPS induced distinct changes in the lipid profiles of ipsilateral and contralateral paws. Here, oxydized fatty acids, phosphatidylcholines and phosphatidylethanolamines were uniquely upregulated on the contralateral side. Thus, both models cause increased levels of PC O and lyso-PC O lipids at the site of inflammation pointing at a common role in inflammation. Also, LPS initiates systemic changes, which can be detected by changes in the lipid profiles.

Triterpene saponins from Guo-gang-long attenuate collagen-induced arthritis via regulating A20 and inhibiting MAPK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The stems of Entada phaseoloides (L.) Merr commonly named "Guo-gang-long", is a traditional Chinese folk medicine that has been used clinically in China for the treatment of arthritis. Our previous study described that triterpene saponins isolated from "Guo-gang-long" could inhibit the inflammatory response. However, the potential mechanism of "Guo-gang-long" on treatment of arthritis, and whether the triterpene saponins responsible for its anti-arthritic effect are unclear. AIM: To investigate the function and mechanisms of the triterpene saponins from E. phaseoloides (ES) in collagen-induced arthritis (CIA) rats. MATERIALS AND METHODS: The chemical components of ES were analyzed by HPLC. Anti-arthritic activity of ES was investigated in CIA rats, which was established by immunization with bovine type II collagen. Three doses of ES (25, 50 and 100 mg/kg) were administrated using oral gavage to CIA rats daily for 3 weeks. The anti-arthritic activity of ES was evaluated by clinical arthritis scoring, paw swelling and mechanical sensitivity, as well as histological changes in CIA rats. The impacts of ES on the regulation of the ubiquintin-editing enzyme A20 and MAPK signaling pathway, and production of pro-inflammatory cytokines in CIA rats were examined by Western blot, quantitative real-time PCR, ELISA, and immunohistochemical staining, respectively. RESULTS: ES treatment relieved the paw swelling, hyperalgesia and joint destruction, and prevented the progression of arthritis in CIA rats. Meanwhile, ES suppressed the excessive mRNA levels and protein expression of TNF-α and IL-17 in synovial tissues and hind paw joints, and reduced the production of IL-1ß, TNF-α and IL-17 in serum. Furthermore, ES up-regulated A20 and suppressed the phosphorylation of p38 and ERK1/2 in hind paw joints, as well as inhibiting the activation of spinal p38 in CIA rats. CONCLUSION: ES could relieve rheumatic symptoms and prevent the development of rheumatoid arthritis. The effects of ES may be mediated by reducing proinflammatory cytokine levels, up-regulating A20 expression, reducing p38 and ERK1/2 activation in periphery, and inhibiting of phospho-p38 in spinal cord.

CTLA-4Ig Improves Hyperalgesia in a Mouse Model of Osteoporosis.

This study aimed to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effects of cytotoxic T lymphocyte-associated antigen-4Ig (CTLA-4Ig) administration in ovariectomized (OVX) mice. Eight-week-old female ddY mice were assigned to three groups: sham-operated mice (SHAM) treated with vehicle, OVX mice treated with vehicle (OVX), and OVX mice treated with CTLA-4Ig (CTLA-4Ig). Vehicle or CTLA-4Ig was injected intraperitoneally, starting immediately after surgery. After 4 weeks of treatment, mechanical sensitivity was examined, and the bilateral hind limbs were removed and evaluated by micro-computed tomography, immunohistochemical analyses, and messenger RNA expression analysis. Ovariectomy induced bone loss and mechanical hyperalgesia in the hindlimbs. CTLA-4Ig treatment prevented bone loss in the hindlimbs compared to vehicle administration in the OVX group. Moreover, mechanical hyperalgesia was significantly decreased in the CTLA-4Ig treatment group in comparison to the OVX group. The expression levels of tumor necrosis factor-α (TNF-α) and sclerostin (SOST), as well as the number of osteoclasts, were increased, and the expression level of Wnt-10b was decreased in the OVX group compared with the SHAM group, whereas these parameters were improved in the CTLA-4Ig group compared with the OVX group. The novelty of this research is that CTLA-4Ig administration prevented bone loss and mechanical hyperalgesia induced by ovariectomy in the hindlimbs.

Chemoradiation impairs myofiber hypertrophic growth in a pediatric tumor model.

Pediatric cancer treatment often involves chemotherapy and radiation, where off-target effects can include skeletal muscle decline. The effect of such treatments on juvenile skeletal muscle growth has yet to be investigated. We employed a small animal irradiator to administer fractionated hindlimb irradiation to juvenile mice bearing implanted rhabdomyosarcoma (RMS) tumors. Hindlimb-targeted irradiation (3 × 8.2 Gy) of 4-week-old mice successfully eliminated RMS tumors implanted one week prior. After establishment of this preclinical model, a cohort of tumor-bearing mice were injected with the chemotherapeutic drug, vincristine, alone or in combination with fractionated irradiation (5 × 4.8 Gy). Single myofiber analysis of fast-contracting extensor digitorum longus (EDL) and slow-contracting soleus (SOL) muscles was conducted 3 weeks post-treatment. Although a reduction in myofiber size was apparent, EDL and SOL myonuclear number were differentially affected by juvenile irradiation and/or vincristine treatment. In contrast, a decrease in myonuclear domain (myofiber volume/myonucleus) was observed regardless of muscle or treatment. Thus, inhibition of myofiber hypertrophic growth is a consistent feature of pediatric cancer treatment.

Noradrenergic Components of Locomotor Recovery Induced by Intraspinal Grafting of the Embryonic Brainstem in Adult Paraplegic Rats.

Intraspinal grafting of serotonergic (5-HT) neurons was shown to restore plantar stepping in paraplegic rats. Here we asked whether neurons of other phenotypes contribute to the recovery. The experiments were performed on adult rats after spinal cord total transection. Grafts were injected into the sub-lesional spinal cord. Two months later, locomotor performance was tested with electromyographic recordings from hindlimb muscles. The role of noradrenergic (NA) innervation was investigated during locomotor performance of spinal grafted and non-grafted rats using intraperitoneal application of α2 adrenergic receptor agonist (clonidine) or antagonist (yohimbine). Morphological analysis of the host spinal cords demonstrated the presence of tyrosine hydroxylase positive (NA) neurons in addition to 5-HT neurons. 5-HT fibers innervated caudal spinal cord areas in the dorsal and ventral horns, central canal, and intermediolateral zone, while the NA fiber distribution was limited to the central canal and intermediolateral zone. 5-HT and NA neurons were surrounded by each other's axons. Locomotor abilities of the spinal grafted rats, but not in control spinal rats, were facilitated by yohimbine and suppressed by clonidine. Thus, noradrenergic innervation, in addition to 5-HT innervation, plays a potent role in hindlimb movement enhanced by intraspinal grafting of brainstem embryonic tissue in paraplegic rats.

Dietary Supplemental Glutamine Enhances the Percentage of Circulating Endothelial Progenitor Cells in Mice with High-Fat Diet-Induced Obesity Subjected to Hind Limb Ischemia.

This study investigated whether glutamine (GLN) pretreatment can enhance circulating endothelial progenitor cells (EPCs) and attenuate inflammatory reaction in high-fat diet-induced obese mice with limb ischemia. Mice were assigned to a normal control (NC), high-fat control (HC), limb ischemia (HI), and GLN limb ischemia (HG) groups. The NC group provided chow diet and treated as a negative control. Mice in the HC and HI groups were fed a high-fat diet which 60% energy provided by fat for 8 weeks. Mice in the HG group were fed the same diet for 4 weeks and then transferred to a high-fat diet with 25% of total protein nitrogen provided as GLN to replace part of the casein for the subsequent 4 weeks. After feeding 8 weeks, mice in the HC group were sham-operated, while the HI and HG groups underwent an operation to induce limb ischemia. All mice except the NC group were euthanized on either day 1 or 7 after the operation. The results showed that the 8 weeks' high-fat diet feeding resulted in obesity. The HG group had higher circulating EPCs on day 1 while muscle vascular endothelial growth factor, matrix metalloproteinase-9, and hypoxia-inducible factor-1 gene expressions were higher on day 7 postischemia than those of the HI group. The superoxide dismutase activity and reduced glutathione content in affected muscles were higher, whereas mRNA expressions of interleukin-6 and tumor necrosis factor-α were lower in the HG than those in the HI group. These findings suggest that obese mice pretreated with GLN-supplemented high-fat diet increased circulating EPC percentage, enhanced the antioxidant capacity, and attenuated inflammatory reactions in response to limb ischemia.

Antiarthritic effect of chitosan nanoparticle loaded with embelin against adjuvant-induced arthritis in Wistar rats.

BACKGROUND: Rheumatoid arthritis (RA) is associated with joint damage. Effectiveness of embelin has been established in a wide variety of inflammatory disorders, but its utility as a therapeutic agent is limited by its poor absorption, rapid metabolism, and fast systemic elimination. To apprehend these limitations, we propose to use highly bioavailable embelin-loaded chitosan nanoparticles (CS-embelin NPs) for the treatment of RA. METHODS: The rats were made arthritic using a subcutaneous injection with 0.1 ml complete Freund's adjuvant (CFA) into the footpad of the left hind paw. CS-embelin NPs (25 and 50 mg/kg) was administered from day 15 to day 28 after adjuvant injection. After the experimental period, the animals were sacrificed and various biochemical markers were assessed. RESULTS: Arthritic score and paw swelling were significantly reduced after treatment with CS-embelin NPs. Arthritis-induced rats showed a significant increase in malondialdehyde (MDA) and nitric oxide (NO) with a concomitant reduction of antioxidants in the paw tissue. CS-embelin NPs (25 and 50 mg/kg) reduced MDA and NO levels and restored antioxidant levels to normalcy by mitigating oxidative stress. The arthritic rats exhibited elevated tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1beta (IL-1ß) serum concentrations, upregulated TNF- α and IL-6 protein levels and upregulated nuclear factor-kB (NF-kB) mRNA expression in paw tissues. Treatment with CS-embelin NPs (25 and 50 mg/kg) significantly reduced serum levels and down-regulated inflammatory markers to normalcy, dose-dependently. CONCLUSION: The results suggest that CS-embelin NPs displayed a protective effect against adjuvant-induced arthritis in rats mediated through antioxidant and anti-inflammatory effects.

Effect of Xuefu Zhuyu Capsule () on Angiogenesis in Hindlimb Ischemic Rats.

OBJECTIVE: To investigate the effect and mechanism of Xuefu Zhuyu Capsule (, XZC) on pro-angiogenesis in the hindlimb ischemic model rats. METHODS: A total of 100 Sprague Dawley rats were randomly divided into a model group, a regular-dose XZC group (0.48 g•kg-1•d-1) and a high-dose XZC group (0.96 g•kg-1•d-1) using random number table method. The model of hindlimb ischemic rats were made through femoral artery embolization with Bletilla microsphere agent. XZC were given on the first day after embolization surgery and lasted 5 days. Finally 72 models were obtained with 12 in each group for each time point. The lower ischemic limb was amputated on the third day after embolization surgery. Histopathological characters and the number of blood vessels of granulation tissues were observed at 36 and 48 h after amputation, respectively. The main genes were obtained from microarray analysis and were validated using real-time quantitative polymerase chain reaction. RESULTS: The vascular number of granulation tissues at both 36 and 48 h were characterized by new and fresh vessels. The number of angiogenesis in the high-dose XZC group at 36 and 48 h was greater compared with that in the regular-dose XZC and model groups (P<0.01), and high-dose XZC at 36 h increased more vessels than that at 48 h (P<0.01). Consequently, granulation tissues from the high-dose XZC group at 36 h were chosen for microarray analysis. In all, 2,085 differentially expressed genes (DEGs) were detected and 25 DEGs were determined to be directly related to angiogenesis. Four biological process terms were found including angiogenesis, regulation of angiogenesis, positive regulation of angiogenesis, and positive regulation of vascular endothelial growth factor receptor signaling pathway (P<0.05). Microarray analysis also showed 49 pathways including 11 pathways related to angiogenesis. CONCLUSION: XZC promoted angiogenesis moderately and the mechanism involved multiple DEGs and multiple pathways.

Hesperidin improves motor disability in rat spinal cord injury through anti-inflammatory and antioxidant mechanism via Nrf-2/HO-1 pathway.

Spinal cord injury (SCI) is associated with inflammation with concurrent oxidative stress and glial activation. The aim of this study was to evaluate whether hesperidin, a representative flavonoid in citrus fruits, ameliorates SCI-induced motor dysfunction and neuro-pathologic degeneration in rat model. Rats received hesperidin (100 mg/kg body weight/daily, oral administration) from 7 days prior to SCI to 7 days post SCI. Behavioral test was done on rats with SCI until 6 weeks. For the study of inflammatory molecules in SCI rats with hesperidin treatment, rats were sacrificed at day 4 post SCI, and spinal cords were collected and studied histopathologically. Behavioral tests on hind-limbs of rats with SCI revealed that treatment of hesperidin in rats with SCI significantly ameliorate the hind-limb paralysis beginning at day 21 post SCI. Hesperidin treatment in rats with SCI reduced the neuropathological changes (e.g., hemorrhage, inflammatory cell infiltration, and tissue loss) and pro-inflammatory cytokines including tumor necrotic factor-α and interleukin-1ß. In addition, oxidative stress related molecules including superoxide dismutase, catalase, nuclear factor erythroid 2-related factor-2 and heme oxygenase-1 were also increased by hesperidin treatment. Furthermore, Fe2+, bilirubin and p38 mitogen activated protein kinase, these by-product of heme catabolism in serum and spinal cord of rats with hesperidin-treatment groups were significantly increased compared with those of vehicle-treatment group. Collectively, this study implies that hesperidin accelerates recovery of locomotor function and tissue repair of damaged spinal cord, with concurrent upregulation of heme oxygenase-1 as far as rat SCI model is concerned.

Prostaglandin D2 signaling is not involved in the recovery of rat hind limb tendons from injury.

Injured tendons heal through the formation of a fibrovascular scar that has inferior mechanical properties compared to native tendon tissue. Reducing inflammation that occurs as a result of the injury could limit scar formation and improve functional recovery of tendons. Prostaglandin D2 (PGD2 ) plays an important role in promoting inflammation in some injury responses and chronic disease processes, and the inhibition of PGD2 has improved healing and reduced disease burden in animal models and early clinical trials. Based on these findings, we sought to determine the role of PGD2 signaling in the healing of injured tendon tissue. We tested the hypothesis that a potent and specific inhibitor of hematopoietic PGD synthase (HPGDS), GSK2894631A, would improve the recovery of tendons of adult male rats following an acute tenotomy and repair. To test this hypothesis, we performed a full-thickness plantaris tendon tenotomy followed by immediate repair and treated rats twice daily with either 0, 2, or 6 mg/kg of GSK2894631A. Tendons were collected either 7 or 21 days after surgical repair, and mechanical properties of tendons were assessed along with RNA sequencing and histology. While there were some differences in gene expression across groups, the targeted inhibition of HPGDS did not impact the functional repair of tendons after injury, as HPGDS expression was surprisingly low in injured tendons. These results indicate that PGD2 signaling does not appear to be important in modulating the repair of injured tendon tissue.

In Vitro and In Vivo Evaluation of the Anticancer and Anti-inflammatory Activities of 2-Himachelen-7-ol isolated from Cedrus Libani.

Cedrus libani is a majestic evergreen tree native to the Mediterranean mountains of Lebanon, Syria and Turkey. In this study, the tree heart wood was extracted using hexane to produce C. libani oil extract (CLOE) as a dark oil. GCMS analysis of CLOE identified up to 30 compounds whereby 2-himachalen-7-ol (7-HC) was the most abundant (40%). 7-HC was isolated using column chromatography and the identity of the white crystalline solid was confirmed via NMR spectroscopy and X-Ray Crystallography. 7-HC demonstrated potent cytotoxic activity against several human cancer cell lines including brain (SF-268, IC50 8.1 µg/mL) and colon (HT-29, IC50 10.1 µg/mL; Caco-2, IC50 9.9 µg/mL) with ovarian (Sk-OV-3, IC50 > 50 µg/mL) cells being the most resistant. However, while HT-29 displayed resistance to Cisplatin, 7-HC was 8-10 folds more potent. Co-treatment with 7-HC and Cisplatin showed a significant synergistic anti-proliferative effect against SF-268, HT-29 and Caco-2 cells. 7-HC also exhibited significant anti-inflammatory effect in formalin-induced paw edema in rats. Western blot analysis revealed that 7-HC displayed dose dependent inhibition of LPS-induced COX-2 protein expression in isolated rat monocytes. The present study demonstrates that 7-HC possesses promising anticancer and anti-inflammatory activities, and may serve as a lead molecule in cancer therapy.

Is Intraarticular Antibiotic Administration Effective in the Treatment of Methicillin-Resistant Staphylococcus aureus?

PURPOSE OF THE STUDY Septic arthritis is an infection of joints caused by a pathogenic microorganism. Septic arthritis has a mortality rate of 11-40% when it's not treated properly. The mortality rate with methicillin-sensitive Staphylococcus aureus (MSSA)is 5-7%, while the rate with methicillin-resistant Staphylococcus aureus (MRSA)is 13-20%. The aim of this study is to evaluate the effects of intraarticular vancomycin and teicoplanin on joint cartilage in in vivo settings and its utility in routine MRSA treatment. MATERIALS AND METHODS In our study, 35 male Sprague-Dawley rats aged 28 days were used. Rats were obtained from the Regenerative and Restorative Medicine Research Center (REMER) of Istanbul Medipol University. Rats were randomly divided into 5 groups each containing 7 rats. Joint injections were administered with isoflurane analgesia every day at 6 am. Three rats (15 rats) from each group were sacrified in seventh day and evaluated immunohistologically to evaluate acute healing in articular cartilage. All remaining rats were sacrificed on day 28 and their knees were evaluated by immunohistochemical examination. RESULTS In our study, there were no complications in any rat during injection and the study period. Hematoxylin eosin (H & E) histological staining for evaluating cartilage healing and healing levels did not show statistically significant differences between the groups at first week (p > 0.05). Matrix metalloproteinase-13 (MMP-13) staining did not show any statistically significant difference between the groups. (p > 0.05). DISCUSSION MRSAseptic arthritis, diagnosed for the first time in 1960, has recently been responsible for 6-22% of all septic arthritis and is increasing day by day. The use of systemic vancomycin or teicoplanin is the first-line treatment method in MRSA septic arthritis. Serum levels reach the desired level, especially with intravenous infusion dose. On the other hand, it has been shown that intraarticular concentration does not reach a sufficient level in studies conducted. The use of intraarticular antibiotics during treatment can lead to more effective and early disease control by turning this negative situation into favor of the patient. As a result, intraarticular vancomycin and teicoplanin maximale tolerable and maintenance doses can be safely used beside surgery and intravenous antibiotics to increase efficacy of treatment, reduction of recurrence rates and reduction of mortality in MRSAseptic arthritis. CONCLUSIONS Intraarticular vancomycin and teicoplanin maximale tolerable and maintenance doses can be safely used beside surgery and intravenous antibiotics to increase efficacy of treatment, reduction of recurrence rates and reduction of mortality in MRSA septic arthritis. Key words:arthritis, infectious; methicillin-resistant Staphylococcus aureus; mortality.

Synthesis and evaluation of 2,5-furan, 2,5-thiophene and 3,4-thiophene-based derivatives as CXCR4 inhibitors.

The interaction between G-Protein coupled receptor CXCR4 and its natural ligand CXCL12 has been linked to inflammation experienced by patients with Irritable Bowel Disease (IBD). Blocking this interaction could potentially reduce inflammatory symptoms in IBD patients. In this work, several thiophene-based and furan-based compounds modeled after AMD3100 and WZ811-two known antagonists that interrupt the CXCR4-CXCL12 interaction-were synthesized and analyzed. Fifteen hit compounds were identified; these compounds exhibited effective concentrations (EC) lower than 1000 nM (AMD3100) and inhibited invasion of metastatic cells by at least 45%. Selected compounds (2d, 2j, 8a) that inhibited metastatic invasion at a higher rate than WZ811 (62%) were submitted for a carrageenan inflammation test, where both 8a and 2j reduced inflammation in the same range as WZ811 (40%) but did not reduce inflammation more than 40%. Select compounds were also modeled in silico to show key residue interactions. These preliminary results with furan-based and thiophene-based analogues contribute to the new class on heterocyclic aromatic-based CXCR4 antagonists.

IGF-1C domain-modified hydrogel enhances therapeutic potential of mesenchymal stem cells for hindlimb ischemia.

BACKGROUND: Poor cell engraftment and survival after transplantation limited the application of stem cell therapy. Synthetic biomaterials could provide an artificial microenvironment for stem cells, thereby improve cell survival and enhance the therapeutic efficiency of stem cells. METHODS: We synthesized a hydrogel by conjugating C domain peptide of insulin-like growth factor-1 (IGF-1C) onto chitosan (CS-IGF-1C hydrogel). Human placenta-derived mesenchymal stem cells (hP-MSCs), which constitutively express a red fluorescent protein (RFP) and renilla luciferase (Rluc), were co-transplanted with CS-IGF-1C hydrogel into a murine hindlimb ischemia model. Transgenic mice expressing firefly luciferase (Fluc) under the promoter of vascular endothelial growth factor receptor 2 (VEGFR2-Luc) were used. Dual bioluminescence imaging (BLI) was applied for tracking the survival of hP-MSCs by Rluc imaging and the VEGFR2 signal pathway activation by Fluc imaging. To investigate the therapeutic mechanism of CS-IGF-1C hydrogel, angiographic, real-time PCR, and histological analysis were carried out. RESULTS: CS-IGF-1C hydrogel could improve hP-MSCs survival as well as promote angiogenesis as confirmed by dual BLI. These results were consistent with accelerated skeletal muscle structural and functional recovery. Histology analysis confirmed that CS-IGF-1C hydrogel robustly prevented fibrosis as shown by reduced collagen deposition, along with increased angiogenesis. In addition, the protective effects of CS-IGF-1C hydrogel, such as inhibiting H2O2-induced apoptosis and reducing inflammatory responses, were proved by in vitro experiments. CONCLUSIONS: Taken together, IGF-1Cs provides a conducive niche for hP-MSCs to exert pro-mitogenic, anti-apoptotic, and pro-angiogenic effects, as well as to inhibit fibrosis. Thus, the incorporation of functional peptide into bioscaffolds represents a safe and feasible approach to augment the therapeutic efficacy of stem cells.