Targeted-anti-osteoporosis Effects And Mechanisms Of NSC-OST Based On "Mutual Reinforcement" Theory

Background:Targeted drug therapy for osteoporosis(OP)has received much concern,and more and more attention has been paid to the advantage of TCM on osteoblast(OB)-osteoclast(OC)system conversion.In view of the low absorption of Osthole(OST),our previous research has successfully finished the synthesis of the water-soluble NSC-OST micelle based on the theory of"homology of herbs and food" and "mutual reinforcement".It should have a strong affinity for bone and has been proved to contribute to OB’ s differentiation.Obj ect i ve:To further optimize the preparation process of NSC-OST micelle and verify its bone-targeted effect;to investigate the biological effect of NSC-OST from bone formation mediated by OB and bone resorption mediated by OC;combining its intervention effect on castrated rats(mineral density,microstructure,metabolism,and biological mechanics index of the bone),to analysis NSC-OST’s intervention in bone formation and bone resorption from multiple perspectives,and discuss its possible mechanisms for treating OP;to reveal the connotation of"mutual reinforcement”theory of TCM,and to provide experimental basis for further research on new targeted-anti-osteoporosis drugs.Methods:(1)Bone targeting experiments:the adsorption rate of hydroxyapatite on NSC-OST was detected by adsorption method,and the metabolic tissue distribution of NSC-OST was detected by RP-HPLC,thus,the bone targeting was evaluated in vitro and in vivo.(2)Synthesis process optimization of NSC-OST:Aiming at the problems existing in the previous protocol,the preparation scheme of NSC and preparation process of micellar were optimized based on the chitosan with lower molecular weight.(3)Effect of NSC-OST on rats OB and OC in vitro:Primary osteoblasts from the skull of SD rats were isolated and identified by Giemsa staining,ALP staining and alizarin red staining.The group was divided into control,estradiol,OST and NSC-OST group.CCK-8 method was used to detect the effect on OB proliferation;the expression and activity of ALP was done by staining and the ALP kit;Western Blot,immunofluorescence were applied to detect BMP and collal.OC was obtained by separating and inducing rat bone marrow mononuclear cells,which were divided into blank,RANKL,OST and NSC-OST group.CCK 8 method was used to detect the influence on OC vitality,the bone absorption board experiment,TRAP staining,Western Blot and qPCR were applied to detect the expression of TRAP and the influence on OC differentiation,OC apoptosis was observed by acridine orange staining.The above results were combined to analyze the bone-targeted-effect and mechanism of NSC-OST in vitro.(4)The prevention and treatment effect and mechanism of NSC-OST on ovariectomized rats:rats were divided into sham group(sham),model group(Ovx),nilestradiol group(Nil),OST group(OST)and NSC-OST group(NSC-OST).The changes of bone metabolism indexes in serum were detected by ELISA.Biomechanical test(three-point bending test)was used to observe the mechanical changes in bones;Micro-CT was used to observe and analyze the changes of bone microstructure in rats;Pathological staining was used to observe the morphological and pathological changes of bones;Immunohistochemistry and q-PCR were used to detect the changes of osteoclast specific genes such as transcription factor NFATcl,CTSK and c-fos.Results:(1)Bone targeting experiments:when NSC-OST was added to hydroxyapatite solution,the release rate of OST was accelerated,which were statistically different at 5min,10min,15min,25min 35min and 120min(P<0.05).30min after intravenous inj ection,the OST content in femur of NSC-OST group was higher than that of OST group(P<0.05),while there was no significant difference in other tissues.2h after intragastric administration,the OST distribution in femur was consistent with that of intravenous administration.(2)Synthesis process optimization of NSC-OST:The drug load of NSC-OST increased to 10%with the new chitosan(molecular weight:50kD,deacetylation degree:95%),and the encapsulation rate increased from 80%to about 85%too.At the same time,with the optimized process of synthesis,micellar stability was also improved.(3)Effects of NSC-OST on OB and OC in vitro:OST inhibited OB proliferation on the 2nd day(P<0.05),and the inhibitory effect was stronger than that of beta-estradiol(P<0.05).OB proliferation in three groups were all inhibited on the 3rd day(P<0.05),and the effect of OST and NSC-OST was stronger than that of bata-estradiol(P<0.05).NSC-OST,OST and bata-estradiol all inhibited OB proliferation(P<0.05)on the 4th day.NSC-OST enhanced ALP activity and promoted type I collagen and BMP2 protein expression on the 7th day(P<0.05),but the ALP activity of OST group was lower than beta-estradiol and NSC-OST group(P<0.05),and the expression of type I collagen was the highest in NSC-OST group(P<0.05).After 5-6 days of induction on bone marrow mononuclear cells,typical OCs with TRAP positive were observed under the microscope,and bone resorption pits were o’bserved on the bone slices under the scanning electron microscope.The cells were stained with toluidine blue and showed a purple green color.Both OST and NSC-OST could significantly inhibit RANKL-induced monocyte differentiation into OC,and inhibit TRAP expression,fusion index,pits numbers,area,average area and other parameters,and the inhibitory effect was more obvious in NSC-OST group(P<0.05).Compared with the control group,OST group and NSC-OST group can significantly promote the apoptosis of OC(P<0.01),and NSC-OST has a more significant effect(P<0.01).(4)Effect and mechanism of NSC-OST on OVX rats with OP:12 weeks after modeling,the weight of all OVX rats increased significantly,while the uterine and surrounding tissues showed kind of atrophy,and the uterine index was significantly inhibited(P<0.05).The uterine index of Nil group was higher than that of the other 3 OVX groups(P<0.05).Serum biochemistry:the contents of BGP,B-ALP,TRAP and beta-CTX in OVX group were significantly increased(P<0.01).Nil and NSC-OST can reduce the content of BGP?B-ALP TRAP and beta-CTX(P<0.01).Serum beta-CTX levels in the OST and NSC-OST groups were lower than those in the model group(P<0.05).Biomechanics:the maximum load,maximum deflection and stiffness of femur in OVX group were significantly inhibited(P<0.01),maximum load and maximum deflection could be increased after Nil intervention,and NSC-OST could increase the maximum load(P<0.05),but the difference of three indicators between OST group and model group was not statistically significant(P>0.05).Micro-CT:NSC-OST could significantly prevent bone loss induced by OVX model and significantly improve the bone microenvironment of trabecular bone structure.BMD,BV/TV,Tb.N and Tb.Th in OVX group were significantly inhibited(P<0.01),while Tb.SP was significantly increased,compared with the sham group(P<0.01).The five indicators in Nil group could all improve the effect of OVX to different degrees,and the difference was statistically significant compared with OVX(P<0.01).In addition to the Tb.Th in OVX group,there were statistically significant differences in all indicators between the other groups and the model group(P<0.05).Bone tissue pathology:the number of OC in OVX groups was obviously increased,which is relatively less in observed group.N.OC/BS and OC.S/BS in the model was significantly inhibited in the OVX group(P<0.01),while the drugs could reverse the two parameters at different levels,which the is better in Nil group(P<0.01),and the NSC-OST group is superior to the OST group(P<0.05).The expressions of NFATcl,CTSK,c-fos,MMP-9 and TRAP in OVX group were significantly higher than those in the sham group,and the expressions were inhibited after drug intervention among which the Nil group had the best inhibitory effect.Conclusion:The optimized NSC-OST micelle has stable physical and chemical properties,which are bone targeting.NSC-OST can promote bone formation mediated by OB through BMP signaling,and inhibit bone resorption by regulation OC on NFATcl and some other specific gene expression,thus to improve the bone microstructure and mechanical properties through dual-directional regulation on OB-OC system.