Effect Of 1,2,3,4,6-penta-O-galloyl-?-d-glucose On Bone Formation And Its Correlation With Estrogen Receptors

Osteoporosis(osteoporosis;OP)is a systemic bone metabolism disease with the third highest incidence of chronic diseases in China.It is characterized by systemic damage to bone mass and microstructure,leading to fragile fractures.OP is mainly divided into primary OP,secondary OP and idiopathic OP.Among them,the incidence of primary OP is the highest.Primary OP is mainly caused by aging and degeneration of bone tissue function and reduction of sex hormone secretion.Primary OP is divided into postmenopausal OP(PMOP)and senile OP.Since the incidence of women is much higher than that of men,the treatment of postmenopausal OP has become a research hotspot.Studies have suggested that oxidative stress caused by decreased estrogen levels is an important cause of OP.Excessive levels of reactive oxygen species(ROS)in the body couldcause mitochondria to swell,increase mitochondrial membrane permeability,and reduce function,further exacerbating ROS production and inducing apoptosis.Osteoblasts(OBs)are the main cells involved in bone formation,and their excessive apoptosis will affect bone repair and lead to PMOP.Traditional Chinese medicine treats PMOP mainly to nourish the liver and kidney,nourish the kidney and strengthen the spleen,and promote blood circulation and stasis.Danggui Shaoyao San(DSS)is one of the effective formula to treat PMOP.Paeonia lactiflora is a junior medicine in DSS,which has the functions of nourishing blood,softening the liver,nourishing yin and so on.It is one of the important single-flavor medicines that make up the OP traditional Chinese medicine compound preparation and its use frequency couldreach about 1/3.The main chemical constituents of Paeonia lactiflora are terpenes and phenolic compounds.Studies have confirmed that the active ingredients of Paeonia lactiflora such as paeoniflorin has effective function on treating OP.However,the phenolic active ingredients represented by 1,2,3,4,6-pentagalloylglucose(PGG)have not been obtained enough attention.Studies have shown that phenolic compounds could promote the growth and differentiation of osteoblasts,and this effect may be related to their antioxidant properties.However,it has not been reported whether PGG has a protective effect on bone.The research aims to observe whether PGG has bone protection and further explore its effect on OBs under oxidative stress and its relationship with estrogen receptor(ER)The research focuses on the following points1.Intervention effect of PGG on zebrafish bone formation inhibition modelA bone formation inhibition model for zebrafish juveniles was established,grouped as follows:blank subdivision(Control group),25 ?M prednisolone(prednisone;Pred)model group,10-8 M 17?-estradiol(E2)Positive drug group,10-10,10-9,10-8 mol·L-1 PGG group.The zebrafish were fertilized for 3 days and then modeled with Pred for 2 days.Each group was then administered for 4 days while modeling.Calcein staining showed that the number of zebrafish vertebrae in the model group was significantly reduced.E2 and After 10-9,10-8 mol·L-1 PGG,the bone mass of zebrafish increased significantly2.Effect of PGG on osteoblast differentiationOsteoblasts(OBs)differentiation experiments were carried out on the pre-osteoblast cell line MC3T3-E1 cells and bone marrow mesenchymal stem cells(BMSCs).OB-induction medium was used to stimulate cell differentiation.Alkaline phosphatase(ALP)staining and ALP activity test results show that PGG could significantly promote ALP expression and increase ALP vitality on day 3 of MC3T3-E1 cell differentiation.Alizarin red staining showed that on the 14th day of differentiation,PGG significantly promoted the formation of calcified nodules in MC3T3-E1 cells and BMSCs;qRT-PCR results showed that PGG significantly promoted expression of ALP,osteocalcin(OCN),and Runt-related transcription factor 2(Runx2)mRNA level3.Effect of PGG on osteoblasts under oxidative stressAn oxidative stress model of MC3T3-E1 cells induced by H2O2 was constructed in vitro.The effects of PGG on MC3T3-E1 cell apoptosis and mitochondrial function under oxidative stress were observed.The results of MTT,Western blot,PI/Annexin V-FITC flow cytometry showed that PGG significantly improved cell survival rate and inhibited OBs apoptosis.In addition,the results of JC-lflow cytometry and fluorescence staining experiments showed that PGG significantly increased mitochondrial membrane potential under oxidative stress.ATP kit test results showed that PGG significantly promoted ATP generation under oxidative stress;DCFH-DA staining and flow cytometry revealed that PPG significantly reduced ROS in MC3T3-E1 cells;Nuclear factor E2 related factor 2(Nuclear factor erythroid-2-related factor 2,Nrf2)is an important antioxidant signaling pathway that couldregulate A variety of downstream antioxidant enzymes are expressed,such as Heme oxygenase-1(HO-1).Immunofluorescence and Western blot experiments showed that PGG could promote Nrf2 nuclear translocation and up-regulate the expression of HO-1 protein.4.Study on the mechanism of the bone protection effect of PGG(1)Wether the effects of PGG on anti-osteoblast apoptosis and restoring mitochondrial function related to ER.The results of PI/Annexin V-FITC flow cytometry showed that PPG significantly inhibited the apoptosis of OBs under oxidative stress;nuclear estrogen receptor(nER)non-specific antagonist ICI182780,membrane estrogen Membrane estrogen receptor(mER)GPR30 specific antagonist G15 and another mER ERa-36 specific antagonist SNG1153 were coclutred with PGG.It was found that ICI 182780 could reverse the anti-osteoblast apoptosis effect of PGG under oxidative stress,and G15 and SNG1153 has no obvious reversal effect.In addition,the results of flow cytometry and fluorescence photography showed that ICI 182780 could reverse the effects of PGG on decresing ROS level,increasing mitochondrial membrane potential,and promoting Nrf2 nuclear translocation under oxidative stress in OBs.(2)Wether the effect of PGG on promoting OBs differentiation related to ER.Alizarin red staining results show that PGG could significantly promote the production of calcified nodules.In addition,ICI 182780 could reverse this effect after adding various ER antagonists with PGG.Then,the nuclear estrogen receptor ER-a antagonist MPP and the ER-? antagonist PHTPP were added to further explore the mechanism of PGG.As a result,it was found that MPP could significantly reverse the effect of PGG on promoting OBs differentiation,but PHTPP has no significantly reverse effect(3)The mode of action between PGG and ER.Molecular docking results show that PGG could effectively enter the active pocket of ER-a,and the binding sites are in Met343,Thr347,Ala350,and Va1534,but PGG could not enter the active pocket of ER-?.The molecular dynamics results further show that the binding free energy of PGG and ER-a is-201.40±5.13 kcal·mol-1,and the RMSD value of the protein converges to 2.5 A within 100 ns.Most RMSF values are less than 1.0A.The number of hydrogen bonds are all higher than 4Conclusions:(1)In vivo experiments show that PGG could significantly increase the bone mass of the zebrafish bone formation inhibition model induced by Pred;in vitro experiments show that PGG could promote OBs differentiation.The above results indicate that PGG has the ability of promoting bone formation.(2)PGG could increase the cell viability and inhibite OBs apoptosis under oxidative stress.(3)PGG could restore the function of mitochodria in OBs under oxidative stress,and this effect may be related to promoting the antioxidant transcription factor Nrf2 nuclear translocation.(4)ICI182780,a non-specific antagonist of nER,could reverse the effect of PGG on promoting osteogenesis and anti-OBs apoptosis,which suggests that the bone protection of PGG may be related to nER.(5)The results of molecular docking and molecular dynamics show that PGG could bind to ER-a and cannot bind to ER-?.Therefore,PGG may exert bone protection by binding to ER-?.In summary,in vitro and in vivo experiments have shown that PGG could promote bone formation.It also inhibited OBs apoptosis under oxidative stress,which may be related to improving mitochondrial function.Further research found that the effect of PGG on promoting bone formation and anti-osteoblast apoptosis may be related to ER-a.This study provides a basis for clinical application of PGG in the treatment of PMOP and a new idea for the discovery of PMOP treatment drugs.