Study On The Anti-osteoporosis Effect And Mechanism Of Guilu Erxian Glue Through Osteoblast-derived Exosomes

Background:The primary mechanism of osteoporosis is the imbalance of bone homeostasis between osteoblasts and osteoclasts.The theory of "balance of yin and Yang" in traditional Chinese medicine has a subtle relationship with the balance of bone homeostasis between osteoblasts and osteoclasts.Based on osteoblasts' mechanism,the research group explored Guilu Erxian glue's mechanism affecting the signal interaction between osteoblasts and osteoclasts and found that the mechanism includes:?up-regulating the expression of TGF-?,Smad3,and p-smad3 in TGF-?/Smads signaling pathway.? Mmp3-opn-mapk pathway was activated to balance the coupling between osteoblast and osteoclast.Further studies have shown that exosomes are the main mediators of the interaction between osteoblasts and osteoclasts.Objective:To optimize the preparation technology of exosomes;to verify the effect of exosomes derived from osteoblasts after the intervention of Guilu Erxian glue on osteoclasts in vitro and in vivo,and to reveal that Guilu Erxian glue plays an anti-osteoporotic role by regulating TGF-? signal pathway and osteoclast differentiation signal pathway through exosome pathway using PRM and PCR technology from the aspects of bone metabolism index,bone mineral density,bone microstructure,and biomechanics.The osteoporosis mechanism provides an experimental basis for elucidating bone homeostasis's regulation mechanism under the guidance of the "Yin Yang balance" theory of traditional Chinese medicine.Methods:?Optimization of extraction technology of primary osteoblasts in vitro:given the low extraction efficiency of primary osteoblasts,the extraction steps were optimized to improve the extraction and culture efficiency of osteoblasts,and ALP and alizarin red staining identified the osteoblasts.?Preparation and validation of osteoblast-derived exosomes:exosomes were prepared by ultracentrifugation and isolated from osteoblast-derived exosomes.Transmission electron microscopy,NTA and Western blot were used to verify the successful preparation of exosomes.?Effect and mechanism of GEG-OB-EV on ovariectomized rats with osteoporosis:ovariectomized rats were divided into the normal saline group(NS),model group(OVX),alendronate sodium group(ALN),OB-EV group(OB-EV),and GEG-OB-EV group(GEG-OB-EV).The CCK-8 method was applied to determine the optimal concentration of GEG for OB.ELISA was used to detect serum bone metabolism indexes.Micro-CT was utilized to observe bone microstructure changes.HE staining was applied to observe bone histopathological changes.A three-point bending test was utilized to test bone biomechanical properties.PRM and RT-PCR were used to detect the protein and mRNA expression changes of TGF-? and rank/RANKL/OPG signaling pathway.?Effects of GEG-Mc3t3-EV on raw264.7-induced osteoclasts:Mc3t3-E1 and RAW264.7 culture systems were established.RAW264.7 cells were induced by RANKL and M-CSF as intervention objects and divided into blank control group(BC),exosome inhibition group(gw4869),GEG medicated serum group(GEG),mc3t3-EV group(Mc3t3-EV),GEG-Mc3t3-EV group(GEG-Mc3t3-EV),alendronate sodium group(ALN).The optimal concentration of GEG for Mc3t3-E1 was determined by the EDU method.The uptake of Mc3t3-EV by RAW264.7 was evaluated by the transwell chamber and PKH26 labeled EV.The apoptosis of RAW264.7 was assessed by flow cytometry.The differentiation and function of osteoclasts were evaluated by trap semi-quantitative staining,bone resorption plate,and RT-PCR.Results:?Optimization of extraction technology of primary osteoblasts in vitro:the traditional secondary enzyme digestion method was improved,which replaced the original challenging and long-time operation of stripping excess tissue on the bone surface with oscillating vortex washing,and replaced the cutting step with batch cutting in 50ml centrifuge tube so that the extraction efficiency of osteoblasts was much improved,and the identification of ALP and alizarin red staining was correct.?Preparation and validation of osteoblast derived exosomes:OB-EV transmission electron microscope images showed that EV was complete in shape,approximately spherical,and relatively uniform in size,with an average particle size of 74.75nm,the proportion of 30-150 nm particles was 97.66%,and the particle concentration was 1.67*109/ml.The quantitative concentration of OB-EV by BCA was about 1.106±0.082 ?g/?L.CD63,CD9,and TSG101 were positive,while calnexin was negative.?The effect and mechanism of GEG-OB-EV on ovariectomized rat osteoporosis model:CCK-8 results showed that the OB activity of 10%GEG containing serum culture at 24h was closest to the expected growth state.Bodyweight:after 12 weeks of modeling,the body weight of rats in the OVX group,ALN group,GEG-OB-EV group,and OB-EV group increased significantly at 0-2 weeks and then tended to be flat.There was no significant difference in body weight change among the four groups(P>0.05),but the body weight of rats in the four groups increased significantly compared with the NS group(P<0.05).Serum bone metabolism indexes:compared with the NS group,the OVX group had no significant difference(P>0.05).The levels of estrogen(E),PI,and OPG decreased significantly,while ALP,TRACP,?-CTX,and PINP increased significantly(P<0.05).Compared with the OVX group,GEG-OB-EV could significantly increase serum PI,OPG,E,inhibit serum ALP,TRACP,?-CTX,PINP levels,the difference was statistically significant(P<0.05);micro CT:The three-dimensional modeling analysis results revealed that trabecular bone loss was pronounced in the OVX group.Trabecular bone loss significantly improved in GEG-OB-EV,OB-EV and AIN groups compared with the OVX group,especially in GEG-OB-EV and ALN groups,but not as good as the NS group.Compared with NS group,BV/TV,TB.N,BMD,Tb.Th of GEG-OB-EV group decreased significantly,but Tb.SP increased significantly.The difference was statistically significant(P<0.01).Compared with the OVX group,the BV/TV,TB.N,BMD,Tb.Th significantly increased,Tb.SP significantly decreased.The difference was statistically significant(P<0.05).OB-EV and ALN had similar results(P<0.05).HE staining:compared with NS group,OVX group had a thinner trabecular bone,larger spacing,structural disorder,fracture phenomenon.Bone marrow edema,a large number of osteoblasts lost,bone marrow cells decreased significantly,and adipocytes increased significantly.Compared with the OVX group,the trabecular bone structure of the GEG-OB-EV group was improved,arranged slightly orderly,with more osteocytes and fewer adipocytes.Molecular mechanism results:the expressions of RANKL,CTSK,MMP14,atp6v0d2 and NFATcl in the OVX group were significantly higher than those in the NS group,and they were significantly lowered after drug intervention.The inhibition of GEG-OB-EV and ALN was the most obvious,and GEG-OB-EV could also significantly up-regulate the key molecules Smad3 and TGF-? in TGF/? signaling pathway.?Effects of GEG-Mc3t3-EV on raw264.7-induced osteoclasts:The EDU results revealed that the activity of Mc3t3-E1 cultured with 10%GEG medicated serum at 24h was the best;EV uptake results:Transwell chamber showed that the fluorescent-labeled Mc3t3-E1 cell membrane could secrete substances into the lower layer through the 1 ?m pore size filter membrane,and round or irregular fluorescent substances could be seen on the RAW264.7 cell membrane.When PKH26-Mc3t3-EV was directly added into RAW264.7,the orange,red PKH26-Mc3t3-EV tightly surrounded the blue nucleus.Trap staining and semi-quantitative analysis:RAW264.7 was induced by RANKL and M-CSF for 4-5 days and could fuse into large multinucleated cells.After trap staining,RAW264.7 showed an irregular or oval shape with a clear boundary.GEG-Mc3t3-EV could significantly inhibit the osteoclast differentiation induced by RANKL and M-CSF,and the effect was better than that of Mc3t3-EV(P<0.05).Bone resorption function:after RAW264.7 induction,obvious round bone resorption lacunae appeared on the bone resorption plate.GEG-Mc3t3-ev could significantly reduce the number of bone resorption lacunae formed by osteoclasts.Its effect was equivalent to that of GEG medicated serum,better than that of MC3T3-EV,but weaker than that of ALN(P<0.05).Molecular mechanism results:Compared with the BC group,the mRNA expressions of c-fos,CTSK,NFATcl,and trap in the GEG-Mc3t3-EV group were significantly decreased(P<0.05).Conclusion:Guilu Erxian Glue can exert an anti-osteoporosis effect on the animal body and cell level through osteoblast-derived exosomes.The mechanism may be related to up-regulating Smad3 and TGF-? in the TGF-?/Smads signaling pathway and inhibiting NFATcl and other transcription and osteoclast-specific factors RANK/RANKL/OPG,thus balancing the coupling between osteoblasts and osteoclasts,improving bone metabolism,microstructure and biomechanics.