Production of Antioxidant, Angiotensin-Converting Enzyme Inhibitory and Osteogenic Gelatin Hydrolysate from Labeo rohita Swim Bladder.

Optimization of antioxidants and angiotensin-converting enzyme (ACE) inhibitory potential gelatin hydrolysate production from Labeo rohita (rohu) swim bladder (SBGH) by alcalase using central composite design (CCD) of response surface methodology (RSM) was investigated. The maximum degree of hydrolysis (DH), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), total antioxidants (TAO), and ACE inhibitory activity were achieved at 0.1:1.0 (w/w) enzyme to substrate ratio, 61 °C hydrolysis temperature, and 94-min hydrolysis time. The resulting SBGH obtained at 19.92% DH exhibited the DPPH (24.28 µM TE/mg protein), ABTS (34.47 µM TE/mg protein), TAO (12.01 µg AAE/mg protein), and ACE inhibitory (4.91 µg/mg protein) activity. Furthermore, SBGH at 100 µg/ml displayed osteogenic property without any toxic effects on MC3T3-E1 cells. Besides, the protein content of rohu swim bladder gelatin (SBG) and SBGH was 93.68% and 94.98%, respectively. Both SBG and SBGH were rich in glycine, proline, glutamic acid, alanine, arginine, and hydroxyproline amino acids. Therefore, SBGH could be an effective nutraceutical in functional food development.

Effect of probiotics on postmenopausal bone health: a preclinical meta-analysis.

Postmenopausal osteoporosis is a major concern for women worldwide due to increased risk of fractures and diminished bone quality. Recent research on gut microbiota has suggested that probiotics can combat various diseases, including postmenopausal bone loss. Although several preclinical studies have explored the potential of probiotics in improving postmenopausal bone loss, the results have been inconsistent and the mechanism of action remains unclear. To address this, a meta-analysis was conducted to determine the effect of probiotics on animal models of postmenopausal osteoporosis. The bone parameters studied were bone mineral density (BMD), bone volume fractions (BV/TV), and hallmarks of bone formation and resorption. Pooled analysis showed that probiotic treatment significantly improves BMD and BV/TV of the ovariectomised animals. Probiotics, while not statistically significant, exhibited a tendency towards enhancing bone formation and reducing bone resorption. Next, we compared the effects of Lactobacillus sp. and Bifidobacterium sp. on osteoporotic bone. Both probiotics improved BMD and BV/TV compared with control, but Lactobacillus sp. had a larger effect size. In conclusion, our findings suggest that probiotics have the potential to improve bone health and prevent postmenopausal osteoporosis. However, further studies are required to investigate the effect of probiotics on postmenopausal bone health in humans.

Taurine deficiency as a driver of aging.

Aging is associated with changes in circulating levels of various molecules, some of which remain undefined. We find that concentrations of circulating taurine decline with aging in mice, monkeys, and humans. A reversal of this decline through taurine supplementation increased the health span (the period of healthy living) and life span in mice and health span in monkeys. Mechanistically, taurine reduced cellular senescence, protected against telomerase deficiency, suppressed mitochondrial dysfunction, decreased DNA damage, and attenuated inflammaging. In humans, lower taurine concentrations correlated with several age-related diseases and taurine concentrations increased after acute endurance exercise. Thus, taurine deficiency may be a driver of aging because its reversal increases health span in worms, rodents, and primates and life span in worms and rodents. Clinical trials in humans seem warranted to test whether taurine deficiency might drive aging in humans.

Sesamol improves bone mass in ovary intact growing and adult rats but accelerates bone deterioration in the ovariectomized rats.

Sesamol, an active component in sesame seeds, is known for its health benefits. However, its effect on bone metabolism remains unexplored. The present study aims to investigate the effect of sesamol on growing, adult and osteoporotic skeleton and its mechanism of action. Sesamol at various doses were administered orally to growing, ovariectomized, and ovary-intact rats. Alterations in bone parameters were examined using micro-CT and histological studies. Western blot and mRNA expression from long bones were performed. We further evaluated the effect of sesamol on osteoblast and osteoclast function and its mode of action in the cell culture system. These data showed that sesamol was able to promote peak bone mass in growing rats. However, sesamol had the opposite effect in ovariectomized rats, evident from gross deterioration of trabecular and cortical microarchitecture. Concurrently, it improved the bone mass in adult rats. In vitro results revealed that sesamol enhances the bone formation by stimulating osteoblast differentiation through MAPK, AKT, and BMP-2 signaling. In contrast, it enhances osteoclast differentiation and expression of osteoclast-specific genes in osteoclast differentiation medium. Interestingly, in presence of estrogen, the effect reversed and sesamol decreased osteoclast differentiation, in vitro. Sesamol improves bone microarchitecture in growing and ovary-intact rats, whereas it enhances the bone deterioration in ovariectomized rats. While sesamol promotes bone formation, its opposing effect on the skeleton can be attributed to its dual effect on osteoclastogenesis in presence and absence of estrogen. These preclinical findings suggest a special attention towards the detrimental effect of sesamol in postmenopausal women.

Maternal omega-3 LC-PUFA supplementation programs an improved bone mass in the offspring with a more pronounced effect in females than males at adulthood.

Early balanced nutrition is vital in achieving optimal skeletal mass and its maintenance. Although a lower omega-6 (n-6): omega-3 (n-3) long-chain polyunsaturated fatty acid (LC-PUFA) ratio is strongly linked with bone health, its maternal effect in the programming of the offspring's skeleton remains to be elucidated. Plugged C57BL/6 mice were fed either n-3 LC-PUFA Enriched Diet (LED) or a control diet (C) throughout their gestation and lactation. Offspring born to both the groups were weaned onto C till 6, 12, and 24 weeks of their age. Offspring's skeleton metabolism and serum fatty acid composition was studied. In humans, seventy-five mother-female newborns pairs from term gestation were tested for their maternal LC-PUFA status relationships to venous cord blood bone biomarkers. Offspring of maternal LED supplemented mice exhibited a superior bone phenotype over C, more prominent in females than males. A lower serum n-6/n-3 LC-PUFA in the LED group offspring was strongly associated with blood biomarkers of bone metabolism. Sexual dimorphism evidenced had a strong correlation between offspring's LC-PUFA levels and bone turnover markers in serum. A higher potential for osteoblastic differentiation in both LED offspring genders and reduced osteoclastogenesis in females was cell-autonomous effect. The human cross-sectional study also showed a positive correlation between maternal n-3 PUFA and cord blood markers of bone formation in female newborns at birth. Maternal dietary n-6/ n-3 fat quality determines offspring's bone growth and development. Our data suggest that the skeleton of female offspring is likely to be more sensitive to this early exposure.

Maternal Plasma Glycerophospholipids LC-PUFA Levels Have a Sex-Specific Association with the Offspring's Cord Plasma Glycerophospholipids-Fatty Acid Desaturation Indices at Birth.

Fatty acid desaturases, the enzymes responsible for the production of unsaturated fatty acids (FA) in fetal tissues, are known to be influenced by maternal-placental supply of nutrients and hormones for their function. We hypothesize that there could be a gender-specific regulation of unsaturated FA metabolism at birth, dependent on the maternal fatty acid levels. In this study, 153 mother-newborn pairs of uncomplicated and 'full-term' pregnancies were selected and the FA composition of plasma glycerophospholipids (GP) was quantified by gas chromatography. The FA composition of mother blood plasma (MB) was compared with the respective cord blood plasma (CB) of male newborns or female newborns. Product to substrate ratios were estimated to calculate delta 5 desaturase (D5D), delta 6 desaturase (D6D) and delta 9 stearoyl-CoA-desaturase (D9D/SCD) indices. Pearson correlations and linear regression analyses were employed to determine the associations between MB and CB pairs. In the results, the male infant's MB-CB association was positively correlated with the SCD index of carbon-16 FA, while no correlation was seen for the SCD index of carbon-18 FA. Unlike for males, the CB-D5D index of female neonates presented a strong positive association with the maternal n-6 long chain-polyunsaturated FA (LC-PUFA), arachidonic acid. In addition, the lipogenic desaturation index of SCD18 in the CB of female new-borns was negatively correlated with their MB n-3 DHA. In conclusion, sex-related differences in new-borns' CB desaturation indices are associated with maternal LC-PUFA status at the time of the birth. This examined relationship appears to predict the origin of sex-specific unsaturated FA metabolism seen in later life.

Maternal high-cholesterol diet negatively programs offspring bone development and downregulates hedgehog signaling in osteoblasts.

Cholesterol is one of the essential intrauterine factors required for fetal growth and development. Maternal high cholesterol levels are known to be detrimental for offspring health. However, its long-term effect on offspring skeletal development remains to be elucidated. We performed our studies in two strains of mice (C57BL6/J and Swiss Albino) and human subjects (65 mother-female newborn dyads) to understand the regulation of offspring skeletal growth by maternal high cholesterol. We found that mice offspring from high-cholesterol-fed dams had low birth weight, smaller body length, and delayed skeletal ossification at the E18.5 embryonic stage. Moreover, we observed that the offspring did not recover from the reduced skeletal mass and exhibited a low bone mass phenotype throughout their life. We attributed this effect to reduced osteoblast cell activity with a concomitant increase in the osteoclast cell population. Our investigation of the molecular mechanism revealed that offspring from high-cholesterol-fed dams had a decrease in the expression of ligands and proteins involved in hedgehog signaling. Further, our cross-sectional study of human subjects showed a significant inverse correlation between maternal blood cholesterol levels and cord blood bone formation markers. Moreover, the bone formation markers were significantly lower in the female newborns of hypercholesterolemic mothers compared with mothers with normal cholesterolemic levels. Together, our results suggest that maternal high cholesterol levels deleteriously program offspring bone mass and bone quality and downregulate the hedgehog signaling pathway in their osteoblasts.

Ethanolic extract from the root and leaf of Sida cordifolia promotes osteoblast activity and prevents ovariectomy-induced bone loss in mice.

BACKGROUND: Sida cordifolia is traditionally found in the Indian system of medicine, well known for its medicinal and nutritional properties among local natives. PURPOSE: The present study aims to investigate the osteo-protective effect of root and leaf ethanolic extract of S. cordifolia (RE and LE) and its underlying mechanism. METHODS: Antioxidant activity of RE and LE was assessed. Total phenolic and flavonoid content were determined. HPLC profiling of RE and LE was performed to examine the polyphenol content. The effect of RE and LE on osteoblast cells proliferation, differentiation, mineralization, and expression of the protein associated with osteogenesis were evaluated using primary calvarial osteoblast culture. Skeletal effects of RE and LE of S. cordifolia were investigated in C57BL/6J ovariectomized mice. Micro CT was employed to evaluate the alteration in trabecular and cortical bone microarchitecture. Histology studies were performed on the isolated vertebra. qPCR analysis and western blotting was done to check the key bone markers. RESULTS: RE and LE showed a potent antioxidant activity, owing to a notable polyphenol content. Both RE and LE did not alter the cell viability but significantly increased the osteoblast cell proliferation, differentiation, and mineralization. Moreover, they enhanced the mRNA expression of osteogenic genes. Both RE and LE stimulated the activation of ERK, AKT, and CREB. Both RE and LE had no direct effect on osteoclastogenesis, but both increased Opg/Rankl ratio expression in osteoblast cells. Both RE and LE at 750 mg/kg/day significantly improved the trabecular and cortical microarchitecture of femur and tibia by increasing bone mineral density, bone volume fraction, trabecular number, and trabecular thickness, and decreasing trabecular separation and structural model index in ovariectomized mice. Furthermore, vertebral histology of lumbar vertebrae revealed that RE and LE significantly enhance the vertebral bone mass and exert osteo-protective effects by stimulating osteoblast function and inhibiting osteoclast function. CONCLUSION: In conclusion, both RE and LE stimulate osteoblast differentiation through activating ERK, AKT, and CREB signalling pathways and indirectly inhibits osteoclast differentiation. RE and LE also improve the trabecular and cortical microarchitecture of ovariectomized mice, making it a promising agent to prevent postmenopausal bone loss.

Lutein activates downstream signaling pathways of unfolded protein response in hyperglycemic ARPE-19 cells.

We have earlier demonstrated that lutein effectively prevents hyperglycemia generated sustained oxidative stress in ARPE-19 cells by activating Nrf2 (nuclear factor erythroid 2-related factor 2) signaling. Since evidence portrays an intricate connection between ER (endoplasmic reticulum) stress and hyperglycemia-mediated oxidative stress, we aimed to explore the protective mechanism of lutein on hyperglycemia-induced ER stress in ARPE-19 cells. To determine the effect of lutein, we probed three major downstream branches of unfolded protein response (UPR) signaling pathways using western blot, immunofluorescent and RT-PCR techniques. The data showed a reduction (38%) in protein expression of an imperative ER chaperon, BiP (binding immunoglobulin protein), in glucose-treated ARPE-19 cells. At the same time, lutein pretreatment blocked this glucose-mediated effect, leading to a significant increase in BiP expression. Lutein promoted the phosphorylation of IRE1 (inositol requiring enzyme 1) and subsequent splicing of XBP1 (X-box binding protein 1), leading to enhanced nuclear translocation. Likewise, lutein activated the expression and translocation of transcription factors, ATF6 (activating transcription factor 6) and ATF4 (activating transcription factor 4) suppressed by hyperglycemia. Lutein also increased CHOP (C/EBP-homologous protein) levels in ARPE-19 cultured under high glucose conditions. The mRNA expression study showed that lutein pretreatment upregulates downstream UPR genes HRD1 (ERAD-associated E3 ubiquitin-protein ligase HRD1), p58IPK (protein kinase inhibitor p58) compared to high glucose treatment alone. From our study, it is clear that lutein show protection against hyperglycemia-mediated ER stress in ARPE-19 cells by activating IRE1-XBP1, ATF6, and ATF4 pathways and their downstream activators. Thus, lutein may have the pharmacological potential for protection against widespread disease conditions of ER stress.

Maternal high protein-diet programs impairment of offspring's bone mass through miR-24-1-5p mediated targeting of SMAD5 in osteoblasts.

Maternal nutrition is crucial for the offspring's skeleton development and the onset of osteoporosis later in life. While maternal low protein diet has been shown to regulate bone mass negatively, the effect of a high protein diet (HP) remains unexplored. Here, we found that C57BL/6 mice fed with HP delivered offspring with decreased skeletal mineralization at birth and reduced bone mass throughout their life due to a decline in their osteoblast maturation. A small RNA sequencing study revealed that miR-24-1-5p was highly upregulated in HP group osteoblasts. Target prediction and validation studies identified SMAD-5 as a direct target of miR-24-1-5p. Furthermore, mimic and inhibitor studies showed a negative correlation between miR-24-1-5p expression and osteoblast function. Moreover, ex vivo inhibition of miR-24-1-5p reversed the reduced maturation and SMAD-5 expression in the HP group osteoblasts. Together, we show that maternal HP diminishes the bone mass of the offspring through miR-24-1-5p.

Hyperglycemia impairs osteoblast cell migration and chemotaxis due to a decrease in mitochondrial biogenesis.

Diabetes is associated with an increase in skeletal fragility and risk of fracture. However, the underlying mechanism for the same is not well understood. Specifically, the results from osteoblast cell culture studies are ambiguous due to contradicting reports. The use of supraphysiological concentrations in these studies, unachievable in vivo, might be the reason for the same. Therefore, here, we studied the effect of physiologically relevant levels of high glucose during diabetes (11.1 mM) on MC3T3-E1 osteoblast cell functions. The results showed that high glucose exposure to osteoblast cells increases their differentiation and mineralization without any effect on the proliferation. However, high glucose decreases their migratory potential and chemotaxis with a decrease in the associated cell signaling. Notably, this decrease in cell migration in high glucose conditions was accompanied by aberrant localization of Dynamin 2 in osteoblast cells. Besides, high glucose also caused a shift in mitochondrial dynamics towards the appearance of more fused and lesser fragmented mitochondria, with a concomitant decrease in the expression of DRP1, suggesting decreased mitochondrial biogenesis. In conclusion, here we are reporting for the first time that hyperglycemia causes a reduction in osteoblast cell migration and chemotaxis. This decrease might lead to an inefficient movement of osteoblasts to the erosion site resulting in uneven mineralization and skeletal fragility found in type 2 diabetes patients, in spite of having normal bone mineral density (BMD).

Glycosaminoglycans from fresh water fish processing discard - Isolation, structural characterization, and osteogenic activity.

Glycosaminoglycans (GAGs) play an important role in various biological activities. A lot of them are present in fish processing discards from abattoirs and fish processing industries which can serve as a valuable source of GAGs. We have, in this paper, isolated and characterized GAGs from fish processing discard (head) generated from the processing of Labeo rohita (L. rohita) and Piaractus brachypomus (P. brachypomus) and have determined their ability to promote osteogenic activity. Isolated GAGs showed higher amounts of chondroitin sulfate/dermatan sulfate (CS/DS) than heparan sulfate (HS). CS/DS from both the fish have a distinct disaccharide composition indicating differences in their structure. Biological activity, in terms of promoting osteogenesis, evaluated in MC3T3-E1 cells and primary cells of the calvaria showed that early mineralization, characterized by alkaline phosphatase staining and activity, and late mineralization, was supported by both the GAGs.

Chlorpyrifos Exposure Induces Parkinsonian Symptoms and Associated Bone Loss in Adult Swiss Albino Mice.

Prenatal and early life exposure of chlorpyrifos (CPF), a widely used pesticide, is known to cause neuronal deficits and Parkinson's disease (PD). However, data about the effect of its exposure at adult stages on PD-like symptoms and associated bone loss is scanty. In the present study, we investigated the impact of CPF on the behavioral alterations seen in PD using adult Swiss albino mice. PD is often associated with bone loss. Hence, skeletal changes were also evaluated using micro-computed tomography and histology. MPTP was used as a positive control. Cell culture studies using MC3T3E-1, SHSY5Y, and primary osteoclast cultures were done to understand the cellular mechanism for the behavioral and skeletal changes. Our results showed that CPF treatment leads to PD-like symptoms due to the loss of dopaminergic neurons. Moreover, CPF has a deleterious effect on the trabecular bone through both indirect changes in circulating factors and direct stimulation of multinucleate osteoclast cell formation. The impact on the bone mass was even stronger than MPTP. In conclusion, this is the first report demonstrating that CPF induces parkinsonian features in adult Swiss albino mice and it is accompanied by loss of trabecular bone.

Bone loss in MPTP mouse model of Parkinson's disease is triggered by decreased osteoblastogenesis and increased osteoclastogenesis.

Bone loss is a non-motor symptom of Parkinson's disease (PD). It is unclear whether a patient's immobility or the endocrine changes in the body causes bone deterioration. To address this issue, we used an animal model of the disease where Swiss albino mice were injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on day 1 and were left untreated for eight weeks. Behavioral phenotypes of PD, and striatal acetylcholinesterase and dopamine levels were measured. Cortical and trabecular bones were assessed by µ-CT and histology. Gene expression studies were done through quantitative real-time PCR. Effect of MPP+ and MPTP-treated mice serum on MC3T3E-1, SH-SY5Y, and primary osteoclast cells were also studied. Our results demonstrated that MPTP treatment leads to PD like symptoms. It shows a loss of trabecular bone mass and quality by decreasing osteoblast and increased osteoclast number and activity. This effect was accompanied by reduced osteogenic and elevated osteoclastogenic genes expression. While MPP+ had a cytotoxic effect on dopaminergic neurons, it did not affect bone cells. However, ex-vivo treatment of the serum from MPTP-treated mice decreased osteoblastogenesis and increased osteoclastogenesis in cell culture. In conclusion, our study suggests that MPTP-induced parkinsonian features in mice leads to trabecular bone loss by decreased bone formation and increased bone resorption due to changes in the serum circulating factors. This study characterizes the microarchitectural and cellular changes in the skeleton of a mouse model of PD that can be further utilized to investigate therapeutic avenues to treat bone loss in PD patients.

Influence of antifertility agents Dutasteride and Nifedipine on CatSper gene level in epididymis during sperm maturation in BALB/c mice.

Calcium (Ca2+) signaling is critical for successful fertilization. In spermatozoa, capacitation, hyperactivation of motility and the acrosome reaction are all mediated by increases in intracellular Ca2+ through CatSper (sperm-specific cation channel). The CatSper channel complex contains four pore-forming α subunits (CatSper1-4) and five accessory subunits called ß, δ, ε, γ and ζ. Genetic deletion of any of the four CatSper genes in mice results in loss of hyperactivated motility and male infertility. Despite their vital role in male fertility, almost very little is known about influence of antifertility agents on CatSper gene expression in epididymis and epididymal spermatozoa. Therefore, we performed quantitative real-time qPCR analysis for CatSper expression in the epididymis and epididymal sperm of BALB/c mice after treatment with Dutasteride (DS), a dual 5-α reductase inhibitor and Nifedipine (NF) a calcium channel blocker as positive control. We observed that treatment with antifertility agents Dutasteride and Nifedipine induced significant decreases in the caput and cauda epididymal sperm counts, motility and fertility which could partly be attributed to alteration in the normal morphology of the sperm associated with downregulation/upregulation of CatSper mRNAs in epididymis and epididymal spermatozoa of male BALB/c mice. These can be explained on the basis of interference with mechanisms affecting calcium ion signaling resulting in changes in intracellular calcium required for sperm activity, finally affecting sperm maturation and fertility of male BALB/c mice. These studies provide some novel avenues for developing new male contraceptives in future.

Stimulation of liver IGF-1 expression promotes peak bone mass achievement in growing rats: a study with pomegranate seed oil.

Peak bone mass (PBM) achieved at adulthood is a strong determinant of future onset of osteoporosis, and maximizing it is one of the strategies to combat the disease. Recently, pomegranate seed oil (PSO) has been shown to have bone-sparing effect in ovariectomized mice. However, its effect on growing skeleton and its molecular mechanism remain unclear. In the present study, we evaluated the effect of PSO on PBM in growing rats and associated mechanism of action. PSO was given at various doses to 21-day-old growing rats for 90 days by oral gavage. The changes in bone parameters were assessed by micro-computed tomography and histology. Enzyme-linked immunosorbent assay was performed to analyze the levels of serum insulin-like growth factor type 1 (IGF-1). Western blotting from bone and liver tissues was done. Chromatin immunoprecipitation assay was performed to study the histone acetylation levels at IGF-1 gene. The results of the study show that PSO treatment significantly increases bone length, bone formation rate, biomechanical parameters, bone mineral density and bone microarchitecture along with enhancing muscle and brown fat mass. This effect was due to the increased serum levels of IGF-1 and stimulation of its signaling in the bones. Studies focusing on acetylation of histones in the liver, the major site of IGF-1 synthesis, showed enrichment of acetylated H3K9 and H3K14 at IGF-1 gene promoter and body. Further, the increased acetylation at H3K9 and H3K14 was associated with a reduced HDAC1 protein level. Together, our data suggest that PSO promotes the PBM achievement via increased IGF-1 expression in liver and IGF-1 signaling in bone.

Skeletal Site-specific Changes in Bone Mass in a Genetic Mouse Model for Human 15q11-13 Duplication Seen in Autism.

Children suffering from autism have been reported to have low bone mineral density and increased risk for fracture, yet the cellular origin of the bone phenotype remains unknown. Here we have utilized a mouse model of autism that duplicates 6.3 Mb region of chromosome 7 (Dp/+) corresponding to a region of chromosome 15q11-13, duplication of which is recurrent in humans to characterize the bone phenotype. Paternally inherited Dp/+ (patDp/+) mice showed expected increases in the gene expression in bone, normal postnatal growth and body weight acquisition compared to the littermate controls. Four weeks-old patDp/+ mice develop a low bone mass phenotype in the appendicular but not the axial skeleton compared to the littermate controls. This low bone mass in the mutant mice was secondary to a decrease in the number of osteoblasts and bone formation rate while the osteoclasts remained relatively unaffected. Further in vitro cell culture experiments and gene expression analysis revealed a major defect in the proliferation, differentiation and mineralization abilities of patDp/+ osteoblasts while osteoclast differentiation remained unchanged compared to controls. This study therefore characterizes the structural and cellular bone phenotype in a mouse model of autism that can be further utilized to investigate therapeutic avenues to treat bone fractures in children with autism.