Osteoporosis is a metabolic condition distinguished by the degradation of bone microstructure and mechanical characteristics. Traditional Chinese medicine (TCM) has been employed in China for the treatment of various illnesses. Naringin, an ingredient found in Drynariae TCM, is known to have a significant impact on bone metabolism. For this research, we studied the precise potential effect of Drynaria Naringin on protecting against bone loss caused by stress deficiency. In this study, a tail-suspension (TS) test was performed to establish a mouse model with hind leg bone loss. Some mice received subcutaneous injections of Drynaria Naringin for 30 d. Trabecular bone microarchitecture was evaluated using micro-computed tomography analysis and bone histological analysis. Bone formation and resorption markers were quantified in blood samples from mice or in the supernatant of MC3T3-E1 cells by ELISA analysis, Western blotting, and PCR. Immunofluorescence was utilized to visualize the location of β-catenin. Additionally, siRNA was employed to knockdown-specific genes in the cells. Our findings highlight the efficacy of Drynaria Naringin in protecting against the deterioration of bone loss and promoting bone formation and Rspo1 expression in a mouse model following the TS test. Specifically, in vitro experiments also indicated that Drynaria Naringin may promote osteogenesis through the Wnt/β-catenin signalling pathway. Moreover, our results suggest that Drynaria Naringin upregulates the expression of Rspo1/Lgr4, leading to the promotion of osteogenesis via the Wnt/β-catenin signalling pathway. Therefore, Drynaria Naringin holds potential as a therapeutic medication for osteoporosis. Drynaria Naringin alleviates bone loss deterioration caused by mechanical stress deficiency through the Rspo1/Lgr4-mediated Wnt/β-catenin signalling pathway.

Chinese soft-shelled turtle Pelodiscus sinensis is an important aquaculture species in China, the male individual being more valuable in aquaculture because of its larger body size, higher growth rate and less fat compared with females. Understanding the mechanism of ovarian differentiation and development is crucial for the production of mono-sex male offspring. However, little is known about the molecular mechanism underlying turtle ovarian differentiation. Here, we characterized the Rspo1 gene, an upstream regulator of vertebrate female sexual differentiation, in P. sinensis. The messenger RNA of Rspo1 was initially expressed at stage 14, preceding gonadal sex differentiation, and exhibited a sexually dimorphic expression pattern throughout the sex determination and gonadal differentiation periods. Rspo1 was rapidly downregulated during aromatase inhibitor-induced female-to-male sex reversal, which occurred prior to gonadal differentiation. Rspo1 loss of function by RNA interference led to partial female-to-male sex reversal, with masculinized changes in the phenotype of gonads, the distribution of germ cells and the expression of testicular regulators. Collectively, these findings suggest that Rspo1 is necessary for primary female sexual differentiation in P. sinensis. This study demonstrates for the first time the functional role of Rspo1 in reptilian sex determination, and is of fundamental significance for the production of fertile pseudo-female parents and mono-sex male offspring of P.sinensis.

With a well-understood function in mammals, R-spondin1 (Rspo1) is an important regulator of ovarian development via the Wnt/β-catenin pathway. Rspo1 deficiency causes retardation of ovarian development in XX fish, and increases Rspo1 function induces femininity and sex reversal in XY fish. In this study, Rspo1 was successfully cloned from loach (Misgurnus anguillicaudatus), and its expression profile was analyzed. The full-length cDNA of Misgurnus anguillicaudatus Rspo1 (MaRspo1) comprised 1322 bp and included an open reading frame (ORF) of 795 bp, which encoded a predicted polypeptide measuring 264 amino acids in length. Phylogenetic and gene structure analyses showed a highly conserved sequence of MaRspo1 (identical to the Rspo1 genes of other species), consisting of an N-terminal signal peptide (SP), two furin-like cysteine-rich domains (FU1 and FU2), a thrombospondin type 1 repeat (TSP1) and a C-terminal region. Real-time PCR revealed the female-biased expression profile of MaRspo1, with the highest expression level among tested tissues detected in ovary. Investigation of MaRspo1 expression levels throughout the early development stage (10-60 days post hatching) under three temperature treatments (25 °C, 28 °C, and 31 °C) revealed significantly differential expression of MaRspo1 among the three temperature groups, with decreased MaRspo1 expression in the high-temperature (31 °C) group. The results of DNA methylation analysis indicated that exposure to high temperature during early development can increase the average promoter methylation level of MaRspo1 in both females and males. Taken together, the results of this study provide the basis for the further investigation of the molecular mechanism of Rspo1 in response to temperature.

UNASSIGNED: R-spondins, including R-spondin 1 (RSPO1), are a family of Wnt ligands that help to activate the canonical Wnt/β-catenin pathway, which is critical for intestinal epithelial cell proliferation and maintenance of intestinal stem cells. This proliferation underpins the epithelial expansion, or intestinal adaptation (IA), that occurs following massive bowel resection and short bowel syndrome (SBS). The purpose of this study was to identify if recombinant human RSPO1 (rhRSPO1) could be serially administered to SBS zebrafish to enhance cellular proliferation and IA.
UNASSIGNED: Adult male zebrafish were assigned to four groups: sham + PBS, SBS + PBS, sham + rhRSPO1, and SBS + rhRSPO1. Sham fish had a laparotomy alone. SBS fish had a laparotomy with distal intestinal ligation and creation of a proximal stoma. Fish were weighed at initial surgery and then weekly. rhRSPO1 was administered post-operatively following either a one- or two-week dosing schedule with either 3 or 5 intraperitoneal injections, respectively. Fish were harvested at 7 or 14 days with intestinal segments collected for analysis.
UNASSIGNED: Repeated intraperitoneal injection of rhRSPO1 was feasible and well tolerated. At 7 days, intestinal epithelial proliferation was increased by rhRSPO1. At 14 days, SBS + rhRSPO1 fish lost significantly less weight than SBS + PBS fish. Measurements of intestinal surface area were not increased by rhRSPO1 administration but immunofluorescent staining for β-catenin and gene expression for cyclin D1 was increased.
UNASSIGNED: Intraperitoneal injection of rhRSPO1 decreased weight loss in SBS zebrafish with increased β-catenin + cells and cyclin D1 expression at 14 days, indicating improved weight maintenance might result from increased activation of the canonical Wnt pathway.

Leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) suppresses food intake after its activation by binding of its ligands, R-spondins. We investigated the mechanism of food intake suppression by R-spondin1 in a region-specific Lgr4 gene knockout (LGR4 cKO) mouse model, generated by deletion of the Lgr4 gene in arcuate nucleus (ARC) using Lgr4fx/fx mice combined with infection of an AAV-Cre vector. After R-spondin1 administration, LGR4 cKO mice didn\'t exhibit a suppressed appetite, compared to that in control mice, which received a vehicle. In ARC of LGR4 cKO mice, Pomc mRNA expression was reduced, leading to suppressed food intake. On the other hand, neurons-specific LGR4 KO mice exhibited no differences in Pomc expression, and no structural differences were observed in the ARC of mutant mice. These results suggest that LGR4 is an essential part of the mechanism, inducing Pomc gene expression with R-spondin1 in ARC neurons in mice, thereby regulating feeding behavior. Abbreviations: LGR4: Leucine-rich repeat-containing G-protein coupled receptor 4; RSPOs: roof plate-specific spondins; ARC: arcuate nucleus; AAV: adeno associated virus; POMC: pro-opiomelanocortin; CART: cocaine and amphetamine-regulated transcript; NPY: neuropeptide Y; AgRP: agouti-related peptide; Axin2: axis inhibition protein 2; Lef1: lymphoid enhancer binding factor 1; ccnd1: cyclin D1.

Leucine-rich repeat G protein-coupled receptors (LGRs) and their endogenous ligands R-spondin1-4 (Rspo) are critical in embryonic development and in maintenance of stem cells. The functions of the Rspo-LGR system in differentiated cells remain uncharacterized. In this study, the expression profiles of LGRs and Rspos were characterized in mature hepatocytes. A liver-specific knockout of LGR4 in mouse was generated and used to study hepatic ischemia/reperfusion-induced injury (HIRI) as well as lipopolysaccharide/ D- galactosamine (LPS/D-Gal)-induced liver injury. We have demonstrated that, in adult liver, LGR4 is expressed in hepatocytes and responds to Rspo1 with internalization. Rspo1 is responsive to various nutritional states and to mTOR signaling. Activation of LGR4 by Rspo1 significantly reduced tumor necrosis factor-α (TNFα)-induced cell death, and levels of NF-κB-p65 and caspase-3 in cultured hepatocytes. Knockdown of hepatic LGR4 rendered hepatocytes more vulnerable to TNFα-induced damage in cultured primary cells and in the setting of HIRI and LPS/D-Gal-induced liver injury. Rspo1 potentiated both basal and Wnt3a-induced stabilization of β-catenin. Disruption of β-catenin signaling reversed the protective effects of Rspo1 on TNFα-induced hepatocyte toxicity. LGR4 knockdown increased nuclear translocation of NF-κB-p65 in response to acute injury. Overexpression of IKKβ attenuated the protective effects of Rspo1 on TNFα-induced cell death. In conclusion, the Rspo1-LGR4 system represents a novel pathway for cytoprotection and modulation of stress-induced tissue damage. NEW & NOTEWORTHY Functional LGR4 is present in mature hepatocytes. R-spodin1 protects hepatocytes from tumor necrosis factor-α-induced cell death. Liver-specific knockdown of LGR4 renders liver more susceptible to acute injury. LGR4 protects hepatocytes from injury by inhibition of NF-κB signaling.

GATA4 is a well-known transcription factor of the GATA family implicated in regulation of sex determination and gonadal development in mammals. In this study, we cloned the full-length cDNA of Paralichthys olivaceus gata4 (Po-gata4). Phylogenetic, gene structure, and synteny analysis showed that Po-GATA4 is homologous to GATA4 of teleost and tetrapod. Po-gata4 transcripts were detected in Sertoli cells, spermatogonia, oogonia and oocytes, with higher transcript levels overall in the testis than the ovary. The promoter region of P. olivaceus R-spondin1was found to contain a GATA4-binding motif. Results of CBA (cleaved amplified polymorphic sequence-based binding assay) indicated that GATA4 could indeed bind to the promoter sequence of R-spondin1. Moreover, human GATA4 recombinant protein could upregulate R-spondin1 in P. olivaceus ovary cells and FBCs (flounder brain cell line). In FBCs, overexpression of Po-gata4 resulted in elevated transcript levels of R-spondin1. Taken together, our results indicate that Po-GATA4 is involved in gonadal development by regulating R-spondin1 expression.

r-spondin1 (rspo1) encodes a secreted protein that is involved in the determination and differentiation of the mammalian ovary. However, little information is yet available for teleosts. Here, we identified a homologue of rspo1 in Cynoglossus semilaevis. The full-length cDNA of rspo1 had a length of 2,703 bp with an open reading frame of 834 bp, encoding a protein with a length of 277 amino-acids. rspo1 expression was detected via qRT-PCR in various tissues, and significant sexually dimorphic expression was observed in the gonads. Furthermore, ISH located rspo1 in germ cells such as spermatogonia, spermatocytes, spermatids, spermatozoa, and oocytes, as well as in somatic cells of the gonads. Following knockdown of rspo1 in an ovarian cell line, the expressions of wnt4a, β-catenin, foxl2, and StAR were highly affected; wnt4a and β-catenin were significantly downregulated, whereas foxl2 and StAR were significantly upregulated. In summary, these data suggest that rspo1 may be involved in the regulation of ovarian development and differentiation through a conserved pathway, while the function of the gene in the testis remains elusive.

Radiation-induced intestinal injury (RIII) commonly occurs in patients who received radiotherapy for pelvic or abdominal cancer, or who suffered from whole-body irradiation during a nuclear accident. RIII can lead to intestinal disorders and even death given its integrity damage that results from intestinal stem cell (ISC) loss. Recovery from RIII relies on the intensity of supportive treatment, which can attenuate lethal infection and give surviving stem cells an opportunity to regenerate. It has been reported that RSPO1 is a cytokine with potent and specific proliferative effects on intestinal crypt cells. MSCs have multiple RIII-healing effects, including anti-inflammatory and anti-irradiation injury properties, due to its negative immune regulation and its homing ability to the damaged intestinal epithelia. To combine the comprehensive anti-injury potential of MSCs, and the potent ability of RSPO1 as a mitogenic factor for ISCs, we constructed RSPO1-modified C3H10 T1/2 cells and expected that RSPO1, the ISC-proliferative cytokine, could be delivered to the site of injury in a targeted manner. In this study, we transferred C3H10/RSPO1 intravenously via the retro-orbital sinus into mice suffering from abdominal irradiation at lethal dosages. Our findings demonstrated that C3H10/RSPO1 cells are able to directionally migrate to the injury site; enhance ISC survival, proliferation, and differentiation; and effectively repair the radiation-damaged intestinal epithelial cells. This study suggests that the directional delivery of RSPO1 by MSCs is a promising strategy to ameliorate, and even cure, RIII.

N-glycosylation is a post-translational protein modification with a wide variety of functions. It has been predicted that R-spondin1 (RSPO1) is N-glycosylated, although this remains unknown. The present study identified that RSPO1 was N-glycosylated at Asn137, and that N-glycosylation of RSPO1 negatively influenced its secretion and enhancing effect on Wnt/β-catenin signaling. In vitro treatment with peptide-N-glycosidase F increased the electrophoretic mobility of RSPO1. Furthermore, treatment of wild-type (wt) RSPO1-overexpressing HT1080 cells with tunicamycin (TM), which inhibits N-glycosylation, resulted in a significant reduction in the molecular weight of RSPO1. However, TM treatment had no effect in the RSPO1 mutant whereby the Asn137 residue was replaced by Gln (N137Q). These results demonstrated for the first time that RSPO1 is N-glycosylated at Asn137. RSPO1 is a secreted protein that has Wnt/β-catenin signaling-enhancing activity and is expected to have therapeutic applications. The role of N-glycosylation in RSPO1 was evaluated by conducting comparative experiments with wt and N137Q RSPO1, which revealed that the N137Q mutant increased the secretion and Wnt/β-catenin signaling-enhancing effect of RSPO1, compared with wt RSPO1. These results suggest that N-glycosylation of RSPO1 has a negative influence on its secretion and Wnt/β-catenin signaling-enhancing effect.