| Objective:Taking primary osteoblasts and ovariectomized mouse models as the research objects,the preventive effects of deer antler skin extract on osteoporosis were discussed in vitro and in vivo,and the main components of deer antler skin extract were identified.Methods:The deer antler skin extract（DASE）,deer antler mesenchymal extract（DAME）,deer antler extract（DAE）,deer antler skin heat extract（HDASE）,deer antler mesenchymal heat extract（HDASME）and deer antler heat extract（HDAE）were prepared by cold extraction and hot extraction respectively.SDS-PAGE and BCA protein assays were used for qualitative and quantitative analysis,and the optimal extract was selected by CCK-8 method.The primary osteoblasts were subjected to scratch test,alizarin red staining and quantitative,alkaline phosphatase staining and quantitative and real-time fluorescent quantitative PCR analysis to explore the effect of deer antler skin extract on osteoblasts at the cellular level.After two months of gavage,OVX mice were subjected to enzyme-linked immunosorbent assay,bone mineral density and bone microstructure detection,HE staining,real-time fluorescent quantitative PCR analysis and Western Blot,to determine the preventive effect of the deer antler skin extract on osteoporosis.According to SDS-PAGE,the DASE was cut off by using an ultrafiltration tube.The main active parts of the deer antler skin extract that worked were screened out by CCK-8,cell scratch test,apoptosis test and real-time fluorescent quantitative PCR analysis,and the proteins of the main active parts were identified by LC-MS.Results:（1）The protein contents of DASE,DAME,DAE,HDASE,HDAME and HDAE were77.83%,73.8%,65.9%,46.3%,46.8%and 43.8%,respectively,in BCA.The results of CCK-8 showed that the proliferation efficiency of the hot extract was generally lower than that of the corresponding cold extract.DAME was not as efficient as DASE and DAE.However,DAE has many literature reports,so DASE was used for subsequent experiments.（2）The results of cell scratch test show that the extract of deer antler skin can significantly promote the migration of primary osteoblasts.（3）Alizarin red staining and alkaline phosphatase staining and their quantitative results showed that DASE can significantly promote the differentiation,mineralization and extracellular matrix synthesis of primary osteoblasts in a time-dependent manner.（4）Real-time fluorescence quantitative PCR analysis showed that the effects of deer antler skin extract on osteoblasts could significantly up-regulate osteogenesis-related marker genes Opn,Bmp2,Runx2,Ocn,Col1a1 and Osx.（5）Enzyme-linked immunosorbent assay showed that compared with Control group,estradiol（E2）,procollagen I-N-terminal propeptide（PINP）and BALP in OVX group decreased significantly（P<0.01）,while tartrate-resistant acid phosphatase（TRAP）increased significantly（P<0.05）.Compared with OVX group,estradiol（E2）,procollagen I-N-term I Nal propeptide（PINP）and BALP in DASE treatment group increased significantly（P<0.05）,while tartrate-resistant acid phosphatase（TRAP）decreased significantly（P<0.05）.（6）Scanning the femur of mice by Micro-CT.The number of trabecular bone in OVX group was less than that in control group,while the number of trabecular bone in OVX+DASE group was more than that in OVX group.The results of three-dimensional imaging of femur and HE staining showed that the trabecular bone in Control group was moderate in size and thickness,closely arranged and orderly,with good bone connectivity.In OVX group,the trabecular bone became shorter and thinner,and the number decreased,sparse and fractured,showing a loose shape;After DASE treatment,the bone microstructure improved obviously,and the trabecular structure was more complete than that of OVX group.（7）The deer antler skin extract acted on the ovariectomized mouse model,and the results of real-time fluorescence quantitative PCR analysis showed that the osteogenesis related marker genes Smad1,Bmp2,Runx2,Smad4 and Col1a1 could be up-regulated.（8）Western Blot results showed that the bone-related proteins Runx2,Bmp2,Smad1,Smad5 and Alp in the model group were lower than those in the control group,while those in the drug group were higher than those in the model group.（9）CCK-8 and cell scratch test of>50 k Da DASE,<50 k Da DASE and DASE showed that the proliferation and migration abilities of>50 k Da DASE and DASE were obviously improved compared with those of the control group,and the proliferation and migration abilities of>50 k Da DASE and DASE were almost the same,but the proliferation and migration abilities of<50 k Da DASE and the control group were weak.（10）The results of>50 k Da DASE,<50k Da DASE and DASE on H2O2-induced apoptosis of primary osteoblasts showed that:Compared with the blank group,the percentage of apoptotic cells in H2O2-induced group increased,while the percentage of apoptotic cells in H2O2+DASE group decreased.As in H2O2+DASE group,the percentage of apoptotic cells in H2O2+>50 k Da DASE group also decreased,while the percentage of apoptotic cells in H2O2+<50 k Da DASE group also decreased,but it was not as obvious as that in H2O2+>50 k Da DASE group and H2O2+DASE group.（11）The primary osteoblasts were treated with>50 k Da DASE,<50 k Da DASE and DASE for 7 days,respectively.Real-time fluorescence quantitative PCR analysis showed that the expressions of Runx2,Col1a1,Ocn and Osx in>50 k Da DASE,<50 k Da DASE and DASE groups were significantly up-regulated,but the up-regulation of>50 k Da DASE group was greater than that in<50 k Da DASE group.（12）According to LC-MS protein identification,the main components of DASE are keratin,serum albumin,protein containing AIG1 G domain,protein containing PKD channel domain,ubiquitin carboxy-terminal hydrolase MINDY,protein containing IF rod-like domain,protein containing BRCT domain,hydroxyl stereo dehydrogenase-like protein 2,etc.Conclusion:The extract of deer antler skin can promote the proliferation,differentiation,mineralization,extracellular matrix synthesis and cell migration of osteoblasts,and can improve bone density and bone microstructure,thus improving osteoporosis.The deer antler skin extract>50 k Da and deer antler skin extract have almost the same proliferation,migration and anti-apoptosis ability,and the main protein bands contain a variety of proteins. |
