Toward Enhanced Antioxidant and Protective Potential: Conjugation of Corn Cob Xylan with Gallic Acid as a Novel Approach.

Maize ranks as the second most widely produced crop globally, yielding approximately 1.2 billion tons, with corn cob being its primary byproduct, constituting 18 kg per 100 kg of corn. Agricultural corn production generates bioactive polysaccharide-rich byproducts, including xylan (Xyl). In this study, we used the redox method to modify corn cob xylan with gallic acid, aiming to enhance its antioxidant and protective capacity against oxidative stress. The conjugation process resulted in a new molecule termed conjugated xylan-gallic acid (Xyl-GA), exhibiting notable improvements in various antioxidant parameters, including total antioxidant capacity (1.4-fold increase), reducing power (1.2-fold increase), hydroxyl radical scavenging (1.6-fold increase), and cupric chelation (27.5-fold increase) when compared with unmodified Xyl. At a concentration of 1 mg/mL, Xyl-GA demonstrated no cytotoxicity, significantly increased fibroblast cell viability (approximately 80%), and effectively mitigated intracellular ROS levels (reduced by 100%) following oxidative damage induced by H2O2. Furthermore, Xyl-GA exhibited non-toxicity toward zebrafish embryos, offered protection against H2O2-induced stress, and reduced the rate of cells undergoing apoptosis resulting from H2O2 exposure. In conclusion, our findings suggest that Xyl-GA possesses potential therapeutic value in addressing oxidative stress-related disturbances. Further investigations are warranted to elucidate the molecular structure of this novel compound and establish correlations with its pharmacological activities.

Antioxidant Fucoidans Obtained from Tropical Seaweed Protect Pre-Osteoblastic Cells from Hydrogen Peroxide-Induced Damage.

Some antioxidant compounds decrease the amount of intracellular reactive oxygen species (ROS) and consequently reduce the deleterious effects of ROS in osteoblasts. Thus, these compounds fight against osteoporosis. Brown seaweeds are a rich source of antioxidant fucose-containing sulfated polysaccharides (fucans and fucoidans). We obtained six fucoidans (FRFs)-F0.3, F0.5, F0.7, F1.0, F1.5, and F2.1-from Dictyota mertensii by proteolytic digestion followed by sequential acetone precipitation. Except for F0.3, all FRFs showed antioxidant activity in different in vitro tests. In pre- osteoblast-like cells (MC3T3-L1) exposed to H2O2-oxidative stress, caspase-3 and caspase-9 were activated, resulting in apoptosis of the cells. We also observed a decrease in superoxide dismutase (SOD) and alkaline phosphatase (ALP) activity. The antioxidant FRFs protected the cells from the oxidative damage caused by H2O2, decreasing intracellular ROS and caspase activation, and increasing SOD activity. The most effective protection against damage was provided by F0.7, F1.5, and F2.1. At 0.5 mg/mL, these FRFs also suppressed the H2O2-mediated inhibition of ALP activity. The data indicated that FRFs F0.7, F1.5, and F2.1 from D. mertensii were antioxidants that protected bone tissue from oxidative stress and could represent possible adjuvants for the treatment of bone fragility through counteracting oxidative phenomena.

Changes in human intervertebral disc biochemical composition and bony end plates between middle and old age.

OBJECTIVE: This study evaluates molecular, nutritional and biochemical alterations in human intervertebral discs between middle and old age. METHODS: Twenty-eight human lumbar intervertebral discs from donors were evaluated and separated into two groups: Middle-aged (35-50 years old, relatively non-degenerate discs of Pfirrmann grades 1-3, n = 15) and Old-aged (≥80 years old, all degenerate Pfirrmann grade 4 or 5, n = 13). Parameters which might be expected to to be related to nutrient supply and so the health of disc cells (eg the porosity of the vertebral endplate, cell viability and cell density) and to disc extracellular composition (ie quantification of glycosaminoglycan disaccharides and hyaluronic acid molecular weight) and collagen organization, were analyzed. Three regions of the intervertebral disc (anterior annulus fibrosus, nucleus pulposus, and posterior annulus fibrosus) were examined. RESULTS: The old-aged group showed a decrease in content of sulphated and non-sulphated glycosaminoglycans relative to middle-aged and there were also alterations in the proportion of GAG disaccharides and a decrease of collagen fiber size. Hyaluronic acid molecular weight was around 200 kDa in all regions and ages studied. The anterior annulus differed from the posterior annulus particularly in relation to cell density and GAG content. Additionally, there were changes in the bony endplate, with fewer openings observed in the caudal than cranial endplates of all discs in both groups. CONCLUSIONS: Results show the cranial vertebral endplate is the main vascular source for the intervertebral discs. Hylauronic acid molecular weight is the same through the intervertebral disc after age of 50 years.

Effects of Training and Overtraining on Intervertebral Disc Proteoglycans.

STUDY DESIGN: Animal experimental study. OBJECTIVE: Evaluate the effect of physical activity and overtraining condition on glycosaminoglycan concentration on the intervertebral disc (IVD) using a rat running model. SUMMARY OF BACKGROUND DATA: Some guidelines recommend the implementation of a physical exercise program as treatment for low back pain; however, cyclic loading impact on the health of the IVD and whether there is a dose-response relationship is still incompletely understood. METHODS: Thirty-two rats ages 8 weeks were divided into four groups with eight animals each. The first 8 weeks were the adaptive phase, the overtraining phase was from the ninth to the eleventh week, which consisted of increasing the number of daily training sessions from 1 to 4 and the recovery phase was represented by the 12th and 13th weeks without training. Control group 1 (CG1) did not undergo any kind of training. Control group 2 (CG2) completed just the adaptive phase. Overtraining group 1 (OT1) completed the overtraining phase. Overtraining group 2 (OT2) completed the recovery phase. Running performance tests were used to assess the "overtraining" status of the animals. IVD glycosaminoglycans were extracted and quantified, and identified by electrophoresis. RESULTS: Glycosaminoglycans showed a distribution between chondroitin sulfate and dermatan sulfate. Glycosaminoglycans quantification showed decreasing concentration at the following order: OT1 > CG2 > OT2 > CG1. Increased expression of dermatan sulfate was verified at the groups submitted to any training. CONCLUSION: Overtraining condition, as assessed by muscle and cardiovascular endurance did not lessen glycosaminoglycan concentration in the IVD. In fact, physical exercise increased glycosaminoglycan concentration in the IVD in proportion to the training load, even at overtraining condition, returning to normal levels after the recovery phase and glycosaminoglycan production is a reversible acute positive response for mechanical stimulation of the IVD. LEVEL OF EVIDENCE: N/A.

Ionic and biochemical characterization of bovine intervertebral disk.

INTRODUCTION: Intervertebral disks have been associated with low back pain, and many therapies have been proposed for its treatment. The cellular and molecular knowledge of intervertebral disks composition and precise methods to quantify disk components are important for any type of proposed therapy. Thus, the aim of this study was to correlate glycosaminoglycans presence with the quantitation of cells, ions and collagen fiber distributions in different intervertebral disk sections. METHODS: In total, 14 intervertebral disks were used from cattle. All of the disks were dehydrated, separated in seven sections and digested in sodium-free papain buffer. Glycosaminoglycan measurements were performed in the samples according to agarose electrophoresis method; total cells were measured using the PicoGreen® technique, ions were quantified, and collagen fiber birefringence was analyzed with polarized light. RESULTS: Cations Na+ and K+ are more concentrate in the nucleus (Na(+) = 1688.50 ± 110 mmol/L; K(+) = 111.9 ± 28 mmol/L) of intervertebral disks than the annulus (Na(+) = 652.80 ± 75 mmol/L; K(+) = 55.6 ± 8 mmol/L). A negative correlation between cells number and sodium/potassium was observed (p < 0.001) Additionally, thin collagen fibers were largest in the nucleus, similar to hyaluronate distribution. CONCLUSIONS: The results suggest that annulus fibrosus cells are also sensitive to changes in ionic concentrations such as nucleus pulposus cells. Additionally, hyaluronate is related to thin collagen fibers type II.

Effects of shock wave therapy on glycosaminoglycan expression during bone healing.

BACKGROUND: Several cases of delayed bone consolidation have been treated with extracorporeal shock wave therapy (ESWT) to improve bone healing and a key role of the extracellular matrix glycosaminoglycans in osteogenesis has been suggested. OBJECTIVE: In this study, we aimed to identify and quantify the amount of sulfated glycosaminoglycans (GAG) and hyaluronic acid (HA) within rat femurs following bone drilling and treatment with shock waves. METHODS: To identify and quantify the sulfated glycosaminoglycans (GAG) and hyaluronic acid (HA) within rat femurs following bone drilling and ESWT, 50 male Wistar rats were evaluated. The animals were divided into two groups, both of which were subjected to bone drilling. One of the groups was treated with ESWT. The rats were sacrificed on the 3rd, 7th, 14th, 21st, and 28th day. GAG presence was analyzed by agarose gel electrophoresis with subsequent densitometry and ELISA. RESULTS AND DISCUSSION: The content of sulfated GAGs increased significantly from the 3rd to the 28th day (p = 0.002). Chondroitin sulfate was expressed more highly than the other GAGs. HA content increased significantly at the 3rd day in animals treated with ESWT compared to the control group (p = 0.003). CONCLUSION: ESWT stimulates of sulfated glycosaminoglycans during bone healing and enhanced early expression of HA compared to the control group.

Modifications in bone matrix of estrogen-deficient rats treated with intermittent PTH.

Bone matrix dictates strength, elasticity, and stiffness to the bone. Intermittent parathyroid hormone (iPTH), a bone-forming treatment, is widely used as a therapy for osteoporosis. We investigate whether low doses of intermittent PTH (1-34) change the profile of organic components in the bone matrix after 30 days of treatment. Forty 6-month-old female Wistar rats underwent ovariectomy and after 3 months received low doses of iPTH administered for 30 days: daily at 0.3 µg/kg/day (PTH03) or 5 µg/kg/day (PTH5); or 3 times per week at 0.25 µg/kg/day (PTH025). After euthanasia, distal femora were processed for bone histomorphometry, histochemistry for collagen and glycosaminoglycans, biochemical quantification of sulfated glycosaminoglycans, and hyaluronan by ELISA and TUNEL staining. Whole tibiae were used to estimate the bone mineral density (BMD). Histomorphometric analysis showed that PTH5 increased cancellous bone volume by 6% over vehicle-treated rats. In addition, PTH5 and PTH03 increased cortical thickness by 21% and 20%, respectively. Tibial BMD increased in PTH5-treated rats and this group exhibited lower levels of chondroitin sulfate; on the other hand, hyaluronan expression was increased. Hormonal administration in the PTH5 group led to decreased collagen maturity. Further, TUNEL-positive osteocytes were decreased in the cortical compartment of PTH5 whereas administration of PTH025 increased the osteocyte death. Our findings suggest that daily injections of PTH at low doses alter the pattern of organic components from the bone matrix, favoring the increase of bone mass.

The expression of glycosaminoglycans and proteoglycans in the uterine cervix of albino rats after local hyaluronidase infusion.

OBJECTIVE: To assess the local effect of hyaluronidase injection on the expression of glycosaminoglycans (GAGs) and proteoglycans (PGs) in the extracellular matrix of the uterine cervix from pregnant albino rats. METHODS: Ten pregnant rats were divided into two groups on day 18 of pregnancy. The experimental group (Gexp) of rats received an intracervical infusion of 0.02 mL of hyaluronidase diluted to 1 mL with distilled water, whereas the control group (Gc) received 1 mL of distilled water. On day 20 of pregnancy, the pregnant rats were sacrificed and the uterine cervixes from all rats were then dissected. The qualitative expression of hyaluronic acid (HA) was assessed by immunohistochemistry and quantified by sandwich ELISA. To compare the quantitative GAG values between groups, a Student's t-test for independent samples was performed. PGs were also assessed by immunohistochemical analysis. RESULTS: The electrophoretic profile of newly synthesized radioactively labeled GAGs degraded by specific enzymes showed that there were two predominant GAGs in both Gc and Gexp, i.e. heparan sulfate (HS) and a mixture of hondroitin sulfate (CS) and dermatan sulfate (DS). The concentrations of GAGs showed a significant reduction of CS/DS (p < 0.004) and HS (p < 0.005) relative to Gc. HA staining was less intense in the lamina propria and area surrounding the blood vessels in Gexp compared to Gc. The HA contents were also significantly reduced (p < 0.012). CONCLUSIONS: Intracervical hyaluronidase infusion promoted a significant reduction in the concentration of sulfated GAGs as assessed by both qualitative (histochemical) and quantitative (fluorometric) measurements of HA.

Fucan effect on CHO cell proliferation and migration.

Fucan is a term used to denominate sulfated L-fucose rich polysaccharides. Here, a heterofucan, named fucan B, was extracted from the Spatoglossum schröederi seaweed. This 21.5 kDa galactofucan inhibited CHO-K1 proliferation and migration when fibronectin was the substrate. Fucan B derivatives revealed that such effects depend on their degree of sulfation. Fucan B did not induce cell death, but promoted G1 cell cycle arrest. Western blotting and flow cytometry analysis suggest that fucan B binds to fibronectin and activates integrin, mainly integrin α5ß1, which induces FAK/RAS/MEK/ERK activation. FAK activation inhibits CHO-K1 migration on fibronectin and ERK blocks cell cycle progression. This study indicates that fucan B could be applied in developing new antitumor drugs.

The low level laser therapy effect on the remodeling of bone extracellular matrix.

The low level laser therapy (LLLT) has been used as an option to accelerate the regeneration of bone tissue. In this study, both femurs of male Wistar rats (30 animals) were injured with a drill and the effect of LLLT using a laser diode (100 mW at 660 nm) in the bone matrix on the left paw measured. LLLT effect on the healing bone tissue matrix was evaluated by a combination of immunohistochemical histomorphometry, confocal immunofluorescence microscopy and isolation and characterization of glycosaminoglycans. Histomorphometric analysis showed that LLLT increased bone matrix and showing more organized. Alcian Blue and PAS staining seems to suggest differential glycosaminoglycans and glycoproteins. The data showed increased expression of chondroitin sulfate and hyaluronic acid, after reduction as the LLLT and mature bone, resembling the expression of osteonectin and biglycan. The difference in expression of siblings (DMP-1, OPN and BSP) is in accordance with the repair accelerated bone formation after the application of LLLT as compared with control. The expression of osteonectin and osteocalcin supports their role in bone mineralization protein, indicating that LLLT accelerates this process. The overall data show that LLLT bone changes dynamic array, shortening the time period involved in the bone repair.

Sulfated fucans extracted from algae Padina gymnospora have anti-inflammatory effect

Sulfated polysaccharides were extracted with acetone from brown algae Padina gymnospora. The fraction precipitated with 1.5 volumes of acetone (F1.5) purified in Sephadex G-75 was characterized by infrared and nuclear magnetic resonance of 13C and ¹H, through which the presence of sulfate groups on the C4 of α-L-fucose could be observed. This polysaccharide showed that an MW of 25,000 Da was effective in reducing leukocyte influx into the peritoneal cavity in mice at 10 mg/kg and 25 mg/kg body weight, causing a decrease of 60 and 39 percent, respectively. In the present study, it was observed that this fucan has anti-inflammatory properties but no cytotoxic action, indicating its potential use in the pharmaceutical industry.

Heparin-integrin interaction in endothelial cells: downstream signaling and heparan sulfate expression.

Endothelial cells (ECs) are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up-regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, Western blotting analyses, and [(35) S]-sulfate metabolically labeling of the cells. We observed that the up-regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine-phosphorylation of focal adhesion-associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin-dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca(2+) release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca(2+) chelator, Ca(2+) signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin-induced up-regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca(2+) /NO pathways.

Chemical reduction of carboxyl groups in heparin abolishes its vasodilatory activity.

Previous studies have shown that heparin induces vascular relaxation via integrin-dependent nitric oxide (NO)-mediated activation of the muscarinic receptor. The aim of this study was to identify the structural features of heparin that are necessary for the induction of vasodilatation. To address this issue, we tested heparin from various sources for their vasodilatation activities in the rat aorta ring. Structural and chemical characteristics of heparin, such as its molecular weight and substitution pattern, did not show a direct correlation with the vasodilation activity. Principal component analysis (PCA) of circular dichroism (CD), (1)H-nuclear magnetic resonance (NMR) and vasodilation activity measurements confirmed that there is no direct relationship between the physico-chemical nature and vasodilation activity of the tested heparin samples. To further understand these observations, unfractionated heparin (UFH) from bovine intestinal mucosa, which showed the highest relaxation effect, was chemically modified. Interestingly, non-specific O- and N-desulfation of heparin reduced its anticoagulant, antithrombotic, and antihemostatic activities, but had no effect on its ability to induce vasodilation. On the other hand, chemical reduction of the carboxyl groups abolished heparin-induced vasodilation and reduced the affinity of heparin toward the extracellular matrix (ECM). In addition, dextran and dextran sulfate (linear non-sulfated and highly sulfated polysaccharides, respectively) did not induce significant relaxation, showing that the vasodilation activity of polysaccharides is neither charge-dependent nor backbone unspecific. Our results suggest that desulfated heparin molecules may be used as vasoactive agents due to their low side effects.

Glycosaminoglycans of abdominal skin after massive weight loss in post-bariatric female patients.

BACKGROUND: The number of post-bariatric patients had a significant increase over the last years, and a better understanding of the consequences of massive weight loss on skin is imperative. Despite weight-loss-related changes in collagen and elastin have been reported, less is known about changes in another of the matrix components of the skin, the glycosaminoglycans. The objective of this study is to evaluate abdominal skin glycosaminoglycans concentrations and perlecan and collagen III expression in post-bariatric female patients. METHODS: Skin tissue samples from the abdomen of lean (n = 19) and post-bariatric (n = 24) female patients were compared. Sulfated glycosaminoglycans and hyaluronic acid were extracted, characterized and quantified. Perlecan and collagen III expression was assessed by immunofluorescence. RESULTS: The major glycosaminoglycans found were dermatan sultafe and hyaluronic acid; the others were found in smaller amounts. The skin of the post-bariatric patients had lower concentrations of heparan sulfate (p = 0.002) while hyaluronic acid, dermatan sulfate, and chondroitin sulfate concentrations were similar to the lean women's skin. Post-bariatric skin showed decreased expression of perlecan and increased expression of collagen III. No correlation was found among glycosaminoglycans concentrations and age, body mass index, frequency of pregnancies, or skin types, but it was observed in higher skin heparan sulfate concentrations in post-bariatric patients who had their weights stabilized for over than 24 months (p = 0.000). CONCLUSION: Abdominal skin of post-bariatric women presented decreased heparan sulfate concentrations and perlecan expression and increased expression of collagen III.

Heparin induces rat aorta relaxation via integrin-dependent activation of muscarinic M3 receptors.

Previous reports have shown that heparin may promote human hypotension and vascular relaxation by elevation of NO levels through unclear mechanisms. We hypothesized that endothelial muscarinic M(3) receptor activation mediates the heparin-induced vasodilation of rat aortic rings. The experiments were carried out using unfractionated heparin extracted from bovine intestinal mucosa, which elicited an endothelium and NO-dependent relaxation of aortic segments with maximal potency and efficacy (EC(50): 100±10 µmol/L; E(max): 41±3%). Atropine and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide inhibitors reduced the heparin-dependent relaxation, indicating that M(3) muscarinic receptor is involved in this phenomenon. However, no direct binding of heparin to muscarinic receptors was observed. More importantly, studies performed using the arginine-glycine-aspartic acid peptide and 1-(1,1-dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine, an Src family inhibitor, reduced by 51% and 73% the heparin-dependent relaxation, respectively, suggesting the coupling of heparin and M(3) receptor through extracellular matrix molecules and integrin. Furthermore, unfractionated heparin induced activation of focal adhesion protein kinase, Src, and paxillin. Finally, fluorescence resonance energy transfer approach confirmed the interaction of the M(3) receptor to integrin. Taken together, these data demonstrate the participation of M(3) receptor and integrin in heparin-dependent relaxation of vascular smooth muscle. These results provide new insights into the molecular mechanism and potential pharmacological action of heparin in vascular physiology.

Anti-inflammatory properties of a heparin-like glycosaminoglycan with reduced anti-coagulant activity isolated from a marine shrimp.

The anti-inflammatory properties of a heparin-like compound from the shrimp Litopenaeus vannamei are related. Besides reducing significantly (p<0.001) the influx of inflammatory cells to injury site in a model of acute inflammation, shrimp heparin-like compound was able to reduce the matrix metalloproteinase (MMPs) activity in the peritoneal lavage of inflamed animals. Moreover, this compound also reduced almost 90% the activity of MMP-9 secreted by human activated leukocytes. Negligible anti-coagulant activities in aPPT assay and a poor bleeding potential make this compound a better alternative than mammalian heparin as a possible anti-inflammatory drug.

A non-anticoagulant heterofucan has antithrombotic activity in vivo.

Fucan is a term used to denominate a family of sulfated L-fucose-rich polysaccharides. The brown alga Spatoglossum schröederi (Dictyotaceae) has three heterofucans namely fucan A, B and C. The 21 kDa fucan A is composed of a core of a beta (1-3) glucuronic acid-containing oligosaccharide of 4.5 kDa with branches at C4 of the fucose chains alpha (1-3) linked. The fucose is mostly substituted at C4 with a sulfate group and at C2 with chains of beta (1-4) xylose. This fucan has neither anticoagulant (from from 0.1 to 100 microg) nor hemorrhagic activities (from 50 to 800 microg/mL). The antithrombotic test in vivo showed that fucan A has no activity in any of the concentrations (from 0.2 to 20 microg/g/day) tested 1 h after polysaccharide administration. However, when fucan A was injected endovenously 24 h before the ligature of the venae cavae, we observed a dose-dependent effect, reaching saturation at around 20 microg/g of rat weight. In addition, this effect is also time-dependent, reaching saturation around 16 h after fucan administration. In addition, regardless of the administration route, fucan A displayed antithrombotic activity. The exception was the oral pathway. Of particular importance was the finding that fucan A stimulates the synthesis of an antithrombotic heparan sulfate from endothelial cells like heparin. The hypothesis has been raised that the in vivo antithrombotic activity of fucan A is related to the increased production of this heparan. Taken together with the fact that the compound is practically devoid of anticoagulant and hemorrhagic activity, the data suggest that it may be an ideal antithrombotic agent in vivo.