The Mechanism Of Codonopsis Pilosula Polysaccharides Affecting Osteoporosis In Ovariectomized Rats

Objective:The study aimed to investigate the impact of Codonopsis ginseng polysaccharide（CPPs）on osteoporosis in ovariectomized rats,and explore the effect and mechanism of CPPs in the osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells（BMSCs）and H₂O₂-induced oxidative damage of mouse osteoblast precursor cells（MC3T3-E1）.Methods:1.The osteoporosis（OP）model was established in SPF female Sprague-Dawley（SD）rats through bilateral ovariectomy（OVX）.The experiments were divided into several groups,including the Sham group（sham operation group）,the OVX group,the E2 group（OVX+50μg/kg/d estradiol）,the CPPs-L group（OVX+100 mg/kg/d CPPs）,the CPPs-M group（OVX+200 mg/kg/d CPPs）,and the CPPs-H group（OVX+400 mg/kg/d CPPs）.After a 12-week feeding period,samples were collected.Serum biochemical indicators were detected using enzyme-linked immunosorbent assay（ELISA）.The histomorphology of the liver,kidney,and distal femur was detected using Hematoxylin-eosin（H&E）staining.While micro-computed tomography（Micro-CT）was utilized to observe the microstructure of the femur and measure bone mineral density（BMD）as well as trabecular bone related parameters including bone tissue volume fraction（BV/TV）,trabecular number（Tb.N）,trabecular thickness（Tb.Th）,and trabecular separation（Tb.Sp）.The number of osteoclasts in the femur was detected using tartrate-resistant acid phosphatase staining（TRAP）.The protein expressions of Runx2,β-catenin,and Pumilio 2（PUM2）in femur were assessed through immunohistochemistry（IHC）.2.We also investigated the effects of CPPs on the osteogenic/adipogenic differentiation of rat BMSCs and the Wnt/β-catenin signaling pathway.In this study,BMSCs were extracted from SD rats using the whole bone marrow adherent method.The phenotype of the BMSCs was identified using flow cytometry.The effects of different concentrations of CPPs on the proliferation of BMSCs were evaluated at different time points using the CCK-8 method to determine the optimal concentration and time for subsequent experiments.（1）BMSCs were subjected to varying concentrations of CPPs（0,25,50,100μg/m L）to evaluate the impact on osteogenic and adipogenic differentiation,as well as the expression ofβ-catenin.（2）To further investigate the Wnt/β-catenin signaling pathway,the inhibitor DKK1 was added and BMSCs were divided into three groups:control,CPPs group（100μg/m L CPPs）,and CPPs+DKK1 group（100μg/m L CPPs+500 ng/m L DKK1）.The related indexes of osteogenic and adipogenic differentiation were then measured in each group.We utilized real-time fluorescent quantitative reverse transcription polymerase chain reaction（q RT-PCR）to detect the m RNA expression of osteogenic and adipogenic differentiation-related factors.Additionally,we used Western blot（WB）to detect the protein expression of these factors.BMSCs were cultured in osteogenic differentiation induction medium for 14 days,and alkaline phosphatase（ALP）staining was used to observe ALP activity at the early stage of osteogenic differentiation.After 28 days,alizarin red staining（ARS）was used to observe the formation of mineralized nodules in the late stage of osteogenic differentiation.BMSCs were cultured in adipogenic differentiation induction medium for 21 days,oil red O staining（ORO）was used to observe the adipogenic differentiation.Additionally,β-catenin protein expression in BMSCs was detected using Western blotting and immunofluorescence.3.This study investigates the impact of CPPs on the function of MC3T3-E1 cells under oxidative damage caused by H₂O₂.The study involved the examination of the effects of various concentrations of H₂O₂ and CPPs on the proliferation of MC3T3-E1cells at different time points.The experiment’s concentration and time were screened to ensure accurate results.（1）To investigate the impact of CPPs on H₂O₂-induced oxidative damage and mitophagy in MC3T3-E1 cells.（1）The experiment involved dividing MC3T3-E1 cells into three groups:a control group,a H₂O₂（200μM）group,and a H₂O₂+CPPs（25,50,100μg/m L）group.The study measured cell proliferation,osteogenic differentiation,apoptosis,ROS,and the expression of mitophagy-related proteins in each group.（2）To investigate the effects of mitophagy inhibition,Cyclosporin A（Cs A）was added to MC3T3-E1 cells.The cells were divided into four groups:control,H₂O₂（200μM）,H₂O₂+CPPs（100μg/m L）,and H₂O₂+CPPs+Cs A（0.4μM）.The levels of osteogenic differentiation,apoptosis,ROS,and mitophagy were measured in each group.（2）The study aims to investigate the effect of CPPs on H₂O₂-induced MC3T3-E1cell PUM2 and the role of PUM2 in CPPs-protected osteoblast oxidative damage.（1）Firstly,to determine the m RNA and protein expression of PUM2 in MC3T3-E1 cells treated with different concentrations of H₂O₂（50,100,200,400?M）.（2）Secondly,MC3T3-E1 cells were transfected with plasmid to over-express PUM2 in MC3T3-E1cells,and the cells were divided into three groups:control group（NC group）,negative plasmid group（p Ex-NC group）,and PUM2 plasmid group（p Ex-PUM2 group）.The protein expression of osteogenic differentiation related factors,the level of intracellular ROS and the expression of mitophagy-related factors in each group were detected.（3）MC3T3-E1 cells were divided into four groups:control group,H₂O₂（200μM）group,H₂O₂+CPPs（100μg/m L）group,and H₂O₂+CPPs+p Ex-PUM2 group.The osteogenic differentiation function and the expression of mitophagy-related factors were measured in each group.Cell proliferation was assessed using the cell counting kit-8（CCK-8）assay.Protein expression levels of osteogenic differentiation factors（COL I,Runx2）,autophagy-related factors（LC3,P62）,and apoptosis-related factors（Caspase-9,Caspase-3,Bax,Bcl-2）were detected using WB analysis.ALP activity was detected using ALP staining,while mineralized nodule formation was detected using ARS staining.Cell apoptosis was evaluated using flow cytometry.Intracellular ROS levels were detected using flow cytometry and fluorescence microscopy,while PUM2protein expression was observed using immunofluorescence and fluorescence microscopy.Results:1.In the OVX group,there was a significant decrease in Tb.N and Tb.Th（P<0.05）,and a significant increase in Tb.Sp and TRAP staining area（P<0.05）compared to the Sham group.However,in the E2,CPPs-L,CPPs-M,and CPPs-H groups,there was a significant decrease in Tb.N（P<0.05）,Tb.Sp（P<0.05）,and TRAP staining area（P<0.05）compared to the OVX group.Notably,there was no significant difference in the above indicators between the E2 group and the Sham group.In comparison to the Sham group,the OVX group exhibited a decrease in the expression of Runx2 andβ-catenin in the femur（all P<0.05）,along with an increase in the expression of PUM2（P<0.05）.However,the E2 group and the CPPs-L,CPPs-M,and CPPs-H groups showed an increase in the expression of Runx2 andβ-catenin when compared to the OVX group（all P<0.05）,while the expression of PUM2 decreased（P<0.05）.2.Effects of CPPs on osteogenic/adipogenic differentiation of rat BMSCs.The positive rates of CD29,CD90,CD34 and CD45 identified by flow cytometry were 96.62%,98.78%,0.27%and 0.28%,respectively.（1）Compared with the control group,CPPs（25,50,and 100?g/m L）significantly increased the m RNA and protein expression of osteogenic differentiation related factors（Runx2,COL I,ALP,and OPN）in BMSCs（P<0.05）,ALP activity,and mineralized nodule formation.The m RNA and protein expressions of adipogenic differentiation related factors（PPAR-γand C/EBP-α）in BMSCs were significantly inhibited（P<0.05）,and the formation of lipid droplets was also inhibited.（2）Compared with the CPPs group,the m RNA and protein expressions of osteogenic differentiation related factors（Runx2,COL I,ALP,OPN）in CPPs+DKK1 group were significantly decreased（all P<0.05）,ALP activity and mineralized nodule formation were decreased.The m RNA and protein expressions of adipogenic differentiation-related factors（PPAR-γand C/EBP-α）and the formation of lipid droplets were significantly increased（all P<0.05）.3.The effect of CPPs on the function of MC3T3-E1 cells under H₂O₂-induced oxidative damage.（1）CPPs could alleviate H₂O₂-induced oxidative damage in MC3T3-E1 cells by increasing mitophagy.（1）Compared to the control group,the proliferation of MC3T3-E1 cells in the H₂O₂ group significantly decreased（P<0.05）.Additionally,the protein expressions of apoptosis-related factors such as Caspase-9,Caspase-3,and Bax significantly increased（all P<0.05）,while the protein expression of Bcl-2significantly decreased（P<0.05）.Moreover,the apoptosis rate significantly increased（P<0.05）.The protein expressions of osteogenic differentiation related factors Runx2and COL I were significantly reduced（all P<0.05）,which led to a decrease in the formation of ALP and mineralized nodules.Additionally,the ratio of autophagy related factor LC3 II/LC3 I protein was significantly decreased（P<0.05）,while the expression of P62 protein was significantly increased（P<0.05）.Moreover,the intracellular ROS level was found to be significantly increased（P<0.05）.Compared to H₂O₂,the intervention groups of CPPs（25,50,100?g/m L）showed significant decrease（P<0.05）in the protein expression levels of apoptosis-related factors Caspase-9,Caspase-3 and Bax,while the protein expression level of Bcl-2 was significantly increased（P<0.05）.Additionally,the apoptosis rate was significantly decreased（P<0.05）.The protein expressions of osteogenic differentiation related factors,Runx2 and COL I,were significantly increased（all P<0.05）,along with an increase in the formation of ALP and mineralized nodules.Additionally,the protein ratio of autophagy-related LC3 II/LC3 I significantly increased（P<0.05）,while the protein expression of P62 was significantly decreased（P<0.05）.The intracellular ROS level was also significantly decreased（P<0.05）.（2）Compared to the H₂O₂+CPPs group,the H₂O₂+CPPs+Cs A group showed a significant decrease in the protein ratio of LC3 II/LC3 I（P<0.05）,a significant increase in the protein expression of P62（P<0.05）,and a significant increase in the intracellular ROS level（P<0.05）.Additionally,the protein expressions of apoptosis-related factors Caspase-9,Caspase-3,and Bax were significantly increased（P<0.05）,while the protein expression of Bcl-2 was significantly decreased（P<0.05）,and the apoptosis rate was significantly increased（P<0.05）.（2）CPPs reduces oxidative damage caused by H₂O₂ in MC3T3-E1 cells by inhibiting the overexpression of PUM2.（1）Compared with the control group,the expression of PUM2 m RNA and protein in H₂O₂ group（50,100,200?M）increased significantly（all P<0.05）;Compared with the H₂O₂ group,the expression of PUM2m RNA and protein in the CPPs group was significantly decreased（both P<0.05）.（2）Compared with the NC group and the p Ex-NC group,the protein ratio of LC3 II/LC3I in the p Ex-PUM2 group was significantly decreased（P<0.05）,and the expression of P62 protein was significantly increased（P<0.05）.The protein expressions of osteogenic differentiation related factors Runx2 and COL I,ALP and mineralized nodule formation were significantly decreased（P<0.05）.There was no significant difference between the NC and p Ex-NC groups.（3）Compared with H₂O₂+CPPs group,the protein ratio of LC3 II/LC3 I in H₂O₂+CPPs+p Ex-PUM group was significantly decreased（P<0.05）,and the expression of P62 protein was significantly increased P<0.05).The protein expressions of osteogenic differentiation related factors Runx2 and COL I,ALP and mineralized nodule formation were significantly decreased（P<0.05）.Conclusions:1.Codonopsis Pilosula Polysaccharides alleviate osteoporosis in ovariectomy rats,with thickener bone cortex,tighter bone trabecular structure,and less vacuoles in the bone marrow cavity,significantly increased BMD,BV/TV,Tb.N,and no liver and kidney injury,blood glucose and lipid metabolism disorders.2.Codonopsis Pilosula Polysaccharides alleviate osteoporosis in ovariectomy rats through increased Runx2 andβ-catenin and reduced PUM2 presentation.3.Codonopsis Pilosula Polysaccharides promote osteogenic differentiation of BMSCs by increasing the expression ofβ-catenin.4.Codonopsis Pilosula Polysaccharides alleviate H₂O₂-induced oxidative damage,inhibit cell apoptosis,and improve osteogenic differentiation of MC3T3-E1cells by inhibiting PUM2 overexpression and increasing autophagy level.