Nanoparticles functionalized with stem cell secretome and CXCR4-overexpressing endothelial membrane for targeted osteoporosis therapy

Background Osteoporosis is a chronic condition affecting patients’ morbidity and mortality and represents a big socioeconomic burden. Because stem cells can proliferate and differentiate into bone-forming cells, stem cell therapy for osteoporosis has been widely studied. However, cells as a live drug face multiple challenges because of their instability during preservation and transportation. In addition, cell therapy has potential adverse effects such as embolism, tumorigenicity, and immunogenicity. Results Herein, we sought to use cell-mimicking and targeted therapeutic nanoparticles to replace stem cells. We fabricated nanoparticles (NPs) using polylactic-co-glycolic acid (PLGA) loaded with the secretome (Sec) from mesenchymal stem cells (MSCs) to form MSC-Sec NPs. Furthermore, we cloaked the nanoparticles with the membranes from C–X–C chemokine receptor type 4 (CXCR4)-expressing human microvascular endothelial cells (HMECs) to generate MSC-Sec/CXCR4 NP. CXCR4 can target the nanoparticles to the bone microenvironment under osteoporosis based on the CXCR4/SDF-1 axis. Conclusions In a rat model of osteoporosis, MSC-Sec/CXCR4 NP were found to accumulate in bone, and such treatment inhibited osteoclast differentiation while promoting osteogenic proliferation. In addition, our results showed that MSC-Sec/CXCR4 NPs reduce OVX-induced bone mass attenuation in OVX rats. Graphical Abstract

Analysis and Validation of Hub Genes in Blood Monocytes of Postmenopausal Osteoporosis Patients

Osteoporosis is a common systemic bone disease caused by the imbalance between osteogenic activity and osteoclastic activity. Aged women are at higher risk of osteoporosis, partly because of estrogen deficiency. However, the underlying mechanism of how estrogen deficiency affects osteoclast activity has not yet been well elucidated. In this study, GSE2208 and GSE56815 datasets were downloaded from GEO database with 25 PreH BMD women and 25 PostL BMD women in total. The RRA algorithm determined 38 downregulated DEGs and 30 upregulated DEGs. Through GO analysis, we found that downregulated DEGs were mainly enriched in myeloid cell differentiation, cytokine-related functions while upregulated DEGs enriched in immune-related biological processes; pathways like Notch signaling and MAPK activation were found in KEGG/Rectome pathway database; a PPI network which contains 66 nodes and 91 edges was constructed and three Modules were obtained by Mcode; Correlation analysis helped us to find highly correlated genes in each module. Moreover, three hub genes FOS, PTPN6, and CTSD were captured by Cytohubba. Finally, the hub genes were further confirmed in blood monocytes of ovariectomy (OVX) rats by real-time PCR assay. In conclusion, the integrative bioinformatics analysis and real-time PCR analysis were utilized to offer fresh light into the role of monocytes in premenopausal osteoporosis and identified FOS, PTPN6, and CTSD as potential biomarkers for postmenopausal osteoporosis.

Tilapia nilotica Head Lipids Improved Bone Loss by Regulating Inflammation and Serum Metabolism Through Gut Microbiota in Ovariectomized Rats

Osteoporosis is a global health problem, and it is of great significance to replace the drugs with natural functional factors. In this study, we investigated the antiosteoporotic activity of lipids prepared from Tilapia nilotica fish head lipids (THLs) in the ovariectomized osteoporosis rats. THLs are composed of neutral lipids (NL, 77.84%), phospholipids (PL, 11.86%), and glycolipids (GL, 6.47%). There were apparent differences in the fatty acid composition of disparate components, and PL contains the most abundant Ω-3 polyunsaturated fatty acids. The results proved that THLs could improve bone microstructure, increase bone mineral density, and decrease bone resorption. To illustrate the antiosteoporotic mechanism, we analyzed the changes in gut microbial communities, proinflammation factors, serum metabolites, and metabolic pathways. Further study on gut microbiota showed that THLs significantly decreased the content of Alistipes in the gut and dramatically increased the beneficial bacteria such as Oscillospira, Roseburia, and Dubosiella. Meanwhile, proinflammation factors of serum in OVX rats decreased significantly, and metabolites were changed. Therefore, we speculated that THLs improved bone loss through reducing inflammation and changing the metabolites and metabolic pathways such as arachidonic acid metabolism and primary bile acid metabolism, etc., by altering gut microbiota. The results indicated that THLs could be a functional factor with antiosteoporotic activity.

Relationship Between Circulating Metabolic Hormones and Their Central Receptors During Ovariectomy-Induced Weight Gain in Rats

Although increasing research focuses on the phenomenon of body weight gain in women after menopause, the complexity of body weight regulation and the array of models used to investigate it has proven to be challenging. Here, we used ovariectomized (OVX) rats, which rapidly gain weight, to determine if receptors for ghrelin, insulin, or leptin in the dorsal vagal complex (DVC), arcuate nucleus (ARC), or paraventricular nucleus (PVN) change during post-ovariectomy weight gain. Female Sprague-Dawley rats with ad libitum access to standard laboratory chow were bilaterally OVX or sham OVX. Subgroups were weighed and then terminated on day 5, 33, or 54 post-operatively; blood and brains were collected. ELISA kits were used to measure receptors for ghrelin, insulin, and leptin in the DVC, ARC, and PVN, as well as plasma ghrelin, insulin, and leptin. As expected, body weight increased rapidly after ovariectomy. However, ghrelin receptors did not change in any of the areas for either group, nor did circulating ghrelin. Thus, the receptor:hormone ratio indicated comparable ghrelin signaling in these CNS areas for both groups. Insulin receptors in the DVC and PVN decreased in the OVX group over time, increased in the PVN of the Sham group, and were unchanged in the ARC. These changes were accompanied by elevated circulating insulin in the OVX group. Thus, the receptor:hormone ratio indicated reduced insulin signaling in the DVC and PVN of OVX rats. Leptin receptors were unchanged in the DVC and ARC, but increased over time in the PVN of the Sham group. These changes were accompanied by elevated circulating leptin in both groups that was more pronounced in the OVX group. Thus, the receptor:hormone ratio indicated reduced leptin signaling in the DVC and PVN of both groups, but only in the OVX group for the ARC. Together, these data suggest that weight gain that occurs after removal of ovarian hormones by ovariectomy is associated with selective changes in metabolic hormone signaling in the CNS. While these changes may reflect behavioral or physiological alterations, it remains to be determined whether they cause post-ovariectomy weight gain or result from it.

Trifolium pratense ethanolic extract alters the gut microbiota composition and regulates serum lipid profile in the ovariectomized rats

Background Trifolium pratense (red clover) ethanolic extract (TPEE) has been used as a popular over-the-counter remedy for the management of menopausal symptoms. Prolonged consumption of herbal extract has been shown to regulate the composition of gut microbiota. This study was designed to elucidate the influence of TPEE on the gut microbiota composition in the ovariectomized (OVX) rats. Methods OVX rats were treated with TPEE at 125, 250, 500 mg/kg/day, or controls (pomegranate extract, 500 mg/kg/day; estradiol, 25 μg/kg/day) for 12 weeks. Gut microbiota analysis was conducted by extracting the microbial DNA from fecal samples and microbiome taxonomic profiling was carried out by using next-generation sequencing. The levels of serum biomarkers were analyzed using enzyme-linked immunosorbent assay (ELISA) kit. The prediction of functional biomarker of microbiota was performed using PICRUSt to investigate the potential pathways associated with gut health and serum lipid profile regulation. To study the correlation between gut microbiota composition and serum lipid levels, Spearman’s correlation coefficients were defined and analyzed. Additionally, gas chromatography–mass spectrometry analysis was conducted to uncover additional physiologically active ingredients. Results TPEE-treated OVX rats showed significant reduction in serum triglycerides (TG), total cholesterols (TCHOL), and LDL/VLDL levels but increase in HDL level. The alteration in the pathways involve in metabolism was the most common among the other KEGG categories. Particularly, TPEE also significantly reduced the relative abundance of sequences read associated with inflammatory bowel disease (IBD) and the peroxisome proliferator-activated receptor (PPAR) signalling pathway. TPEE intervention was seen to reduce the Firmicutes to Bacteroidetes (F/B) ratio in the OVX rats, denoting a reduction in microbial dysbiosis in the OVX rats. Correlation analysis at the phylum level revealed that Bacteriodetes and Proteobacteria were strongly correlated with serum TG, TCHOL and HDL levels. At the species level, Bifidobacterium pseudolongum group was seen to positively correlate with serum HDL level and negatively correlated with serum AST, ALT, LDL/VLDL, TCHOL, and TG levels. Conclusions TPEE treatment showed therapeutic benefits by improving the intestinal microbiota composition which strongly correlated with the serum lipid and cholesterol levels in the OVX rats.

Anti- inflammatory effect of boric acid on cytokines in ovariectomy-induced rats

The increase in the rate of inflammation in the post-menopause period also leads to a significant increase in the use of anti-inflammatory agents. This study aimed to investigate the effect of BA supplementation on pro-and anti-inflammatory cytokines in ovariectomy (OVX) induced rats. A total of 48 nonpregnant female Wistar albino rats (80-100 g) were used in the experiment. Forty-eight rats were divided into six equal groups (n=8): Control, OVX, OVX+5 mg/kg BA (OVX+BA5), OVX+10 mg/kg BA (OVX+BA10), 5 mg/kg BA (BA5), 10 mg/kg BA (BA10). Serum TNF-α cytokine levels of rats in the OVX group were higher than in control rats (P<0.05). TNF-α levels were significantly reduced in the OVX-induced rats with 5 mg/kg BA and 10 mg/kg BA supplementation (P<0.05). While serum IL-1α and IL-6 levels were not different between OVX and control rats, serum IL-3 levels were low (P<0.05) and not affected by 5 mg/kg and 10 mg/kg BA supplementation. Serum IL-11 levels increased significantly in the OVX rats with 5 mg/kg and 10 mg/kg BA supplementation (P<0.05). As far as we know, certain doses (5 and 10 mg/kg) of BA are the first study on the prevention of increased inflammation in rats induced by OVX. Results suggest that the supplementation of BA regulates the inflammatory changes associated with OVX and thus has beneficial for menopause management.

Comparison of Necroptosis With Apoptosis for OVX-Induced Osteoporosis

As one common kind of osteoporosis, postmenopausal osteoporosis (PMOP) is associated with the death and excessive loss of osteocytes. Estrogen deficiency of PMOP can cause osteocyte death by regulating necroptosis and apoptosis, but their roles in POMP have not been compared. In the present study, ovariectomy (OVX)-induced rat and murine long bone osteocyte Y4 (MLO-Y4) cells were used to compare the influence of necroptosis and apoptosis on osteocyte death and bone loss. Benzyloxycarbonyl-Val-Ala-Asp (zVAD) and necrostatin-1 (Nec-1) were used to specifically block cell apoptosis and necroptosis, respectively. OVX rats and MLO-Y4 cells were divided into zVAD group, Nec-1 group, zVAD + Nec-1 group, vehicle, and control group. The tibial plateaus of the rat model were harvested at 8 weeks after OVX and were analyzed by micro–computed tomography, transmission electron microscopy (TEM), the transferase dUTP nick end labeling assay, and western blot. The death of MLO-Y4 was stimulated by TNF-α and was measured by flow cytometry and TEM. The results found that necroptosis and apoptosis were both responsible for the death and excessive loss of osteocytes, as well as bone loss in OVX-induced osteoporosis, and furthermore necroptosis may generate greater impact on the death of osteocytes than apoptosis. Necroptotic death of osteocytes was mainly regulated by the receptor-interacting protein kinase 3 signaling pathway. Collectively, inhibition of necroptosis may produce better efficacy in reducing osteocyte loss than that of apoptosis, and combined blockade of necroptosis and apoptosis provide new insights into preventing and treating PMOP.

Integrated Network Pharmacology and GC-MS–Based Metabolomics to Investigate the Effect of Xiang-Su Volatile Oil Against Menopausal Depression

Menopausal depression perplexes a great number of women in later life. Xiangfu-Zisu (Xiang-Su), a traditional Chinese herbal pair composed of rhizomes of Cyperus rotundus L. (Xiangfu) and leaves of Perilla frutescens (L.) Britt. (Zisu), is frequently reported with antidepressant-like effects. The volatile oil from Xiangfu and Zisu has shown good antidepressant action, but its mechanism is still unclear. This study aimed to investigate the pharmacological mechanism of Xiang-Su (XS) volatile oil against menopausal depression through gas chromatography–mass spectrometry (GC-MS)-based network pharmacology and metabolomics. First, ADME screening was performed on actual detected components of XS volatile oil to obtain active constituents, and then duplicates of active constituent–related targets and menopausal depression–related targets were collected. These duplicates were considered as targets for XS volatile oil against menopausal depression, followed by GO and KEGG enrichment analyses. It showed that a total of 64 compounds were identified in XS volatile oil, and 38 active compounds were screened out. 42 overlapping genes between 144 compound-related genes and 780 menopausal depression–related genes were obtained. Results showed that targets of SLC6A4 and SLC6A3, regulation of serotonergic and dopaminergic synapses, were involved in the antidepressant mechanism of XS volatile oil. Next, antidepressant-like effect of XS volatile oil was validated in menopausal rats by ovariectomy (OVX) combined with chronic unpredictable mild stress (CUMS). Behavioral tests, biochemical analysis, and GC-MS–based non-targeted plasma metabolomics were employed to validate the antidepressant effect of XS volatile oil. Experimental evidence demonstrated that XS volatile oil reversed behavioral parameters in the sucrose preference test (SPT), open-field test (OFT), forced swim test (FST), and serum estradiol levels in OVX rats. Furthermore, results of metabolomics indicated that XS volatile oil mainly acts on regulating metabolic pathways of phenylalanine, tyrosine and tryptophan biosynthesis, tyrosine metabolism, and tryptophan metabolism, which were corresponding with the above-predicted results. These data suggest that network pharmacology combined with metabolomics provides deep insight into the antidepressant effect of XS volatile oil, which includes regulating key targets like SLC6A4 and SLC6A3, and pathways of serotonergic and dopaminergic synapses.

Rhizoma drynariae total flavonoids combined with calcium carbonate ameliorates bone loss in experimentally induced Osteoporosis in rats via the regulation of Wnt3a/β-catenin pathway

Background Rhizoma drynariae, a traditional Chinese herb, is commonly used in treatment of bone healing in osteoporotic fractures. However, whether the Rhizoma drynariae total flavonoids (RDTF) can promote the absorption of calcium and enhance the bone formation is unclear. The aim of the present study was to investigate the preventive effects of RDTF combined with calcium carbonate (CaCO3) on estrogen deficiency-induced bone loss. Methods Three-month-old Sprague–Dawley rats were ovariectomized (OVX) and then treated with CaCO3, RDTF, and their admixtures for ten weeks, respectively. The bone trabecular microstructure, bone histopathological examination, and serum biomarkers of bone formation and resorption were determined in the rat femur tissue. The contents of osteoprotegerin (OPG), receptor activator of the NF-κB (RANK), and its ligand (RANKL) in marrow were analyzed by ELISA, and the protein expressions of Wnt3a, β-catenin, and phosphorylated β-catenin (p-β-catenin) were analyzed by Western blot. Statistical analysis was conducted by using one-way analysis of variance (ANOVA) followed by LSD post hoc analysis or independent samples t test using the scientific statistic software SPSS version 20.0 Results RDTF combined with CaCO3 could promote osteosis and ameliorate bone loss to improve the repair of cracked bone trabeculae of OVX rats. Furthermore, RDTF combined with CaCO3 also could prevent OVX-induced decrease in collagen fibers in the femoral tissue of ovariectomized rats and promote the regeneration of new bone or cartilage tissue, while CaCO3 supplementation promoted the increase in bone mineral content. Nevertheless, there was no difference in the expression of Wnt3a, β-catenin and p-β-catenin between osteopenic rats and RDTF treated rats, but RDTF combined with CaCO3 could activate the Wnt3a/β-catenin pathway. Conclusions RDTF combined with CaCO3 could ameliorate estrogen deficiency-induced bone loss via the regulation of Wnt3a/β-catenin pathway.

Anti-osteoporotic effect of Terminalia arjuna (Roxb.) Wight & Arn. In bilateral ovariectomized induced post-menopausal osteoporosis in experimental rats

Objectives In ancient times Terminalia arjuna (Roxb.) Wight & Arn. (TA) was used for fast healing of fracture and to strengthen the bone. However, no scientific study has been done to validate its usefulness in the alleviation of osteoporosis. To investigate the efficacy of stem bark TA against post-menopausal osteoporosis using bilateral ovariectomized rat model. Methods Aqueous (TAA) and methanolic (TAM) extracts of TA was evaluated for its anti-osteoporotic activity. Sham control rats were allotted as Group I (Normal control); Group II animals acted as OVX control (Disease control); Group III OVX rats were treated with estrogen (Standard group – 2 mg/kg) Group IV and V OVX rats give treatment to TAA (250 and 500 mg/kg, p.o.), respectively. This treatment is continue for the four weeks and at the end, serum biochemical parameters such as serum calcium and alkaline phosphate were evaluated. Femoral bone parameters (Compression of vertebrae, femoral neck load testing, Three point bending of tibia, Femur length and weight), histology, body weight, and fifth lumbar vertebra breaking strength were also assessed after the sacrificing the animal. Results In OVX rats, atrophy of uterus and descent of BMD were suppressed by treatment with TAA and TAM. In addition, TAM 500 completely corrected the decreased serum concentration of Calcium, Phosphorus, ALP and TRAP observed in OVX rats. TAA and TAM both increased biomechanical strength significantly in comparison to the sham group. Histological results also revealed its protective action through elevation of bone formation. TAM significantly increase the uterine and femoral bone weight The TAM showed maximum anti-osteoporotic activity in in vivo study as compare to TAA. Conclusions The results, evaluated on the basis of biochemical, bone mineral density, biomechanical, and histopathological parameters, presented that TAA and TAM has a definite antiosteoporotic effect, like to estrogen, especially effective for inhibition bone fracture induced by estrogen deficiency.