| Osteoporosis is a worldwide health problem,which is a metabolic disease caused by the imbalance between bone resorption and bone formation.Osteoporosis is characterized by decreased bone density and changes in bone structure caused by bone loss,and increased brittleness and the risk of fracture in severe cases.Arecanut is one of the most important cash crops in Hainan of China and parts of East Africa.Arecanut contains a variety of bioactive components,including areca,polysaccharide,crude fiber and phenolic substances.As an important component of arecanut,areca seed polyphenols have many biological activities,such as antibacterial,antioxidant,hypoglycemic,antithrombotic and so on.Here,hainan arecanut(Areca catechu L.)was used as raw material to prepare arecanut seed polyphenol(ACP),and the composition of ACP was analyzed.Bilateral ovaries osteoporosis rat model was established to evaluate anti-osteoporotic activity of ACP in vivo,and its improving mechanism was clarified by lucubrating intestinal microecology,serum metabolites and Wnt/?-catenin pathway.The main research results were as follows:(1)ACP composition spectrum analysisArecanut was used as raw material to prepare ACP by water extraction method,and its composition spectrum was analyzed by HPLC-MS/MS.It was found that ACP was mainly composed of 10 phenolic substances,including procyanidin B2,procyanidin B1,catechin,quinic acid,guaverin,trans/cis spruce neoside,naringin+naringin Chalcone,trans/cis resveratrol,leucochrome and eugenic acid,among which the content of catechin reached the highest of 28.28 mg/g.(2)ACP improved osteoporosis in ovariectomized ratsBilateral ovariectomized osteoporosis rats were used as a model to evaluate the effect of ACP on osteoporosis.The bone metabolism indexes in serum and urine were determined by ELISA.The bone structure was detected by H&E staining and micro-CT.The results showed that compared with OVX group,ACP significantly increased bone density,bone volume fraction,trabecular number and trabecular thickness,reduced trabecular separation,and improved the bone microstructure of ovariectomized rats.ACP significantly decreased the contents of deoxypyridine(DPD),urinary phosphorus(P),urinary calcium(Ca)and serum TRACP,inhibited bone resorption.Meanwhile,ACP significantly increased the levels of osteocalcin(OCN),osteogenic alkaline phosphatase(BALP),osteoprotectin(OPG)and Runx2 in serum,promoted bone formation.These results indicated that ACP could significantly improve osteoporosis in rats.(3)Anti-osteoporosis mechanism of ACP by regulating intestinal microecology in ovariectomized ratsThe diversity and difference of intestinal microbiota were determined by 16 SrDNA high-throughput sequencing.H&E staining and immunohistochemistry were used to observe the histological morphology and intestinal microenvironment of the digactyl.The relationships among Paneth cells,regulatory microbial protein(LYZ),pro-inflammatory factors,macrophage infiltration levels and intestinal microecology were analyzed.Studies showed that in the OVX group,Paneth cells decreased,Lyz expression decreased,and inflammation level increased.In the phylum level,Bacteroidetes,Firmicutes and Proteobacteria increased(P<0.001).ACP significantly increased the number of Paneth cells,promoted the expression of LYZ,decreased the level of inflammation,and decreased the content of Alistipes(P<0.001).These results indicate that ACP can increase the expression of LYZ by maintaining the number of Paneth cells in osteoporosis rats,and participate in the regulation of intestinal microbiota by controlling inflammatory response and improving osteoporosis.(4)Improving mechanism of ACP on osteogenesis by regulating metabolic pathways in ovariectomized ratsSerum biomarkers of osteoporosis was screened by serum metabolomics.The expression of LDL receptor related protein 5(LRP5),5-hydroxy initial synthetase 1(Tph1)and enterogenous tryptophan serotonin(5-HT),bone alkaline phosphatase(BALP),osteocalcin(OCN),Runt related transcription factor 2(RUNX2)in the bone tissue was determined by H&E staining,immunohistochemical,q RT-PCR and Western blotting.Results showed that compared with OVX group,ACP significantly enhances the content of7-ketone deoxycholic acid,indoles and 15-DNA-?12,14-prostaglandin,significantly improved the expression of Lrp5,Htr1 b,BMP2,Wnt-10?,Smad4,?-catenin,suggesting that ACP decreased the expression of enterogenous 5-HT through the LRP5/TPH1/5-HT signaling pathway,thus significantly increased the activation of Wnt/?-catenin pathway,promoted bone formation and improved osteoporosis. |
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