| Osteoporosis(OP)is a metabolic bone disease characterized by low bone mass and bone tissue microstructure degeneration.In recent years,with the increasing demand for oral health and beauty,there are more and more patients with low bone density and even osteoporosis in orthodontic clinics.Tooth movement depends on the bone remodeling of osteoblasts and osteoclasts.In the process of orthodontic treatment,the osteoblast dysfunction in OP state is combined with osteoclast formation and differentiation,which aggravates the imbalance in the process of bone remodeling,leads to excessive absorption of alveolar bone,accelerates tooth movement,making teeth easy to loose or even fall off during orthodontic treatment,seriously affecting the effect and stability of orthodontic treatment.At present,drugs for clinical treatment of OP,such as bisphosphonates,will have a series of side effects in orthodontic treatment,such as restricting tooth movement and inducing jaw bone necrosis,due to their interference with bone resorption of osteoclasts.Therefore,it has become the focus of orthodontists’ attention to deeply understand the molecular mechanism of bone remodeling and adopt effective treatment to improve OP status,which is conducive to orthodontic treatment in OP patients.Eucommia ulmoides Oliver is a classic drug for the prevention and treatment of OP in traditional Chinese medicine.Studies have confirmed that many effective active components of Eucommia ulmoides Oliver,such as flavonoids,lignans,and aucubin have positive effects on the prevention and treatment of OP.Polysaccharides are macromolecular substances formed by the polymerization of multiple monosaccharides through glycosidic bonds.Studies have shown that plant polysaccharides have preventive and therapeutic effects on OP by balancing bone absorption and bone formation.Polysaccharide is an important bioactive component of Eucommia ulmoide Oliver.It has been confirmed that polysaccharide extracted from Eucommia ulmoides Oliver has antioxidant and anti-inflammatory activities.However,there is no relevant research on the anti-OP activity of polysaccharide extracted from Eucommia ulmoides Oliver.In this study,the structural characteristics and anti-OP activity of polysaccharide purified from Eucommia ulmoides Oliver were systematically studied.Combined with multi-omics analysis,the mechanism of polysaccharide purified from Eucommia ulmoides Oliver in prevention and treatment of OP was explored,which could be beneficial for the orthodontic treatment in OP patients.First,this study preliminarily analyzed the effect of crude polysaccharide extracted from Eucommia ulmoides Oliver cortex(c Eu OCP)on alveolar bone remodeling during orthodontic tooth movement in OP rats.The optimum extraction conditions(continuous extraction at 80℃ for 3 h,liquid-to-material ratio 20:1 m L/g)of Eucommia ulmoides Oliver polysaccharides were obtained through the polysaccharide extraction optimization experiment,and then c Eu OCP was obtained by hot water extraction and a series of purification operations.In the tooth movement model of OP rats,c Eu OCP administration can significantly improve the alveolar bone microstructure disorder,increase the alveolar bone mass,and promote the expression of Runx2 and Osterix in the alveolar bone tissue of OP rats.These results indicated that c Eu OCP can promote osteoblast-mediated bone formation in bone tissue of OP rats,preliminarily confirmed the effect of c Eu OCP in promoting bone formation and anti-OP activity.Next,c Eu OCP was further purified to obtain purified polysaccharide from Eucommia ulmoides Oliver cortex(Eu OCP3),and its structural characteristics were further analyzed.The results showed that Eu OCP3 was an acidic polysaccharide mainly composed of galacturonic acid,arabinose,rhamnose,and galactose.The average molecular weight of Eu OCP3 was 38.1 k Da,and the molecular weight dispersion index was 1.0049,which indicated that Eu OCP3 was homogeneous.The infrared spectrum detection results showed that there was a characteristic peak of sugar in Eu OCP3 sample.The characteristic absorption peak of C-H,C-O,uronic acid,and α-type I glycoside bond were also observed.The results of polysaccharide binding structure analysis showed that Eu OCP3 was mainly composed of eight glycoside fragments,including 4-Gal(p)-UA,5-Ara(f),2-Rha(p),T-Ara(f),2,3,5-Ara(f),4-Gal(p),2,4-Rha(p),and T-Gal(p).According to the results of polysaccharide binding structure analysis combined with NMR signal analysis,it was speculated that the main chain of Eu OCP3 was predominantly composed of →4)-α-Galp A-(1→4)-α-Galp A-(1→,→4)-α-Galp A-(1→5)-α-Araf-(1→,→4)-α-Galp A-(1→2)-α-Rhap-(1→,and →4)-α-Galp A-(1→5)-α-Araf-(1→2)-α-Rhap-(1→ repeating blocks,which were connected by→2,3,5)-α-Araf-(1→.The side chains,substituted at C-2 and C-5 of →2,3,5)-α-Araf-(1→,contained T-β-Araf→ and T-β-Araf→4)-α-Galp A-(1→ residues.Then,the OP mice model was established to explore the effect of Eu OCP3 on bone metabolism and related mechanisms.The results showed that the administration of Eu OCP3 could significantly improve the microstructure damage of bone tissue,increase the thickness of cortical bone and bone trabecula,promote the expression of osteoblast differentiation protein and inhibit the expression of osteoclast differentiation protein,indicating that the administration of Eu OCP3 could reverse the imbalance between bone formation and bone resorption in bone tissue of OP mice.The results of gut microflora combined with metabonomics analysis showed that Eu OCP3 could regulate the metabolism level of OP mice by changing the abundance of several bacteria in gut microflora.Combined with serum ELISA and bone tissue western blot detection,it was further found that Eu OCP3 might improve the oxidative stress state through ERK/JNK/Nrf2 signal cascade,and promote bone formation through BMP-2/Smad signal pathway,thus improving the bone metabolism in OP mice.Finally,the osteoblast differentiation model was established by MC3T3-E1 cells to explore the effect of Eu OCP3 on osteoblasts.The results showed that Eu OCP3 had no significant effect on the proliferation activity of MC3T3-E1 cells.Eu OCP3 significantly promoted the expression of osteogenic differentiation markers in MC3T3-E1 cells,and promoted the phosphorylation of ERK1/2 and Smad1/5/8,as well as the expression of BMP-2,indicating that Eu OCP3 can promote the differentiation of osteoblasts.The mechanism of Eu OCP3 promoting the differentiation of MC3T3-E1 cells was further explored.The intervention of ERK inhibitors partially blocked the phosphorylation of Smad1/5/8,the expression of BMP-2,and other markers of osteogenic differentiation in MC3T3-E1 cells induced by Eu OCP3,indicating that the ERK/BMP-2/Smad signal pathway played a key role in the process of Eu OCP3 inducing the differentiation of osteoblasts.The results showed that:(1)c Eu OCP(extracted from Eucommia ulmoides Oliver cortex)has anti-OP activity by promoting bone formation during orthodontic tooth movement of OP rats.(2)Eu OCP3(purified from c Eu OCP)is a homogeneous acidic polysaccharide mainly composed of galactouronic acid,arabinose,rhamnose,and galactose,with a molecular weight of 38.1 k Da.(3)In OP mice model,Eu OCP3 can improve the oxidative stress state through ERK/JNK/Nrf2 signal cascade,and promote bone formation through BMP-2/Smad signal pathway,thus improving the bone metabolism in OP mice.(4)ERK/BMP-2/Smad signaling pathway plays a key role in the process of Eu OCP3-induced osteoblast differentiation. |
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