Zuogui Pilltreat Glucocorticoid-induced Osteoporosis Based On Mechanism Of Crosstalk Between Let-7f And Autophagy Regulating Bone Formation

Objective1.In vivo,we aim to determine the therapeutic effect of Zuogui Pill on GIOP and to analyze the expression changes of let-7f and autophagy related factors during Zuogui Pill treating GIOP.2.In vitro,we aim to explore the effect and mechanism of Zuogui Pill in promoting osteogenic differentiation of BMSCs through regulating the crosstalk of let-7f and autophagy.Methods1.Animal experimental studies(1)Therapeutic effect of Zuogui Pill(ZGW)on GIOP rats60 six-month-old SD rats were randomly divided into 3 groups:control(CON),GIOP,and ZGW group,with 20 rats in each group.After successful modeling(3 months)by injecting dexamethasone,corresponding drugs were given.The animals were euthanasia and the samples were collected at the first month(Ml),second months(M2)and third months(M3)after drug intervention.①Blood of abdominal aorta was collected,centrifuged,and the upper serum of blood was aspirated to detect ALP activity by using the Alkaline Phosphatase Test kit;②The L1-3 vertebral bodies were isolated and detected for bone density;③ After ②,the L2 vertebral bodies were separated and underwent μCT scaning and three-dimensional reconstruction;④ After③,the L2 vertebrae extracorporeal tissue(such as attachment,soft tissue and endplate cartilage)was removed,and the ends of the vertebral bodies were smoothed into a cylinder with upper and lower ends parallel,about 5mm high",and underwent lumbar compression test;⑤ The L4 vertebral bodies were separated,fixed by formaldehyde,decalcified,dehydrated,embedded,sliced,and HE staining was used to observe the morphological changes of lumbar vertebrae.(2)Effect of ZGW on the number of autophagosomes in lumbar vertebral osteoblastsThe L5 vertebral bodies were isolated from(1),and the microstructure of osteoblasts was observed under transmission electron microscope,and the number of autophagosomes were detected.(3)Expression changes of let-7f and autophagy related factors in lumbar vertebrae during ZGW treating GIOP rats① Seperated the L6 vertebral bodies from(1),removed the extracorporeal tissue and stored them in liquid nitrogen.② RT-qPCR was used to detect gene expression of let-7f,ULK2,Beclin-1,LC3,ATG5,ATG12,Runx2,Opn,Alp,and Ocn;2.Cell experimental study(1)The targeted regulatory relationship between let-7f and ULK2① Cultured 293T cells and packaged lentiviral vectors;② Bioinformatics softwares including TargetScan 7.2(http://targetscan.org),miRBase,RNA22,DIANA-mT,miRDB,PICTAR5,PITA and miRanda were used to predict the targeted regulation of let-7f and ULK2;③After constructed the reporter gene vector,cells were plated(blank vector group,let-7f mimic+wild type,let-7f mimic negative control+wild type,let-7f inhibitor+wild type,let-7f inhibitor negative control+wild type,let-7f mimic+mutant,let-7f mimic negative control+mutant,let-7f inhibitor+mutant,let-7f inhibitor negative control + mutant)respectively and transfected.Luciferase activity assays were used to verify the targeting relationship between let-7f and ULK2.(2)Effect of ZGW aqueous extract on osteogenic differentiation of BMSCs by regulating the crosstalk between let-7f and autophagy① Preparation of Zuogui Pill aqueous extract;② Extracted rat BMSCs,passaged,and put them cryopreservation.③ CCK8 was used to detect the proliferation of different concentrations of ZGW aqueous extract on 1,3,5,7,and 14 days;The optimal concentration of Zuogui Pill aqueous extract was used to intervent BMSCs;④ The let-7f overexpression and silencing vector were constructed and transfected into BMSCs.The optimal concentration of ZGW aqueous extract and osteogenic induction medium were added together.Grouping:control vector of miRNA group;let-7f overexpression group;let-7f overexpression+ZGW group;control vector of plux group;let-7f silencing group;let-7f silencing+ZGW group.The osteogenic differentiation ability was detected by ALP staining and ARS staining.The expression of let-7f was examined by RT-qPCR.The protein exoression of autophagy-related factors(ULK2,ATG5,ATG12,Beclin-1,LC3B)was examined by Western-blot.⑤ Together with let-7f overexpression vector transfection into BMSCs,autophagy activator(rapamycin)and ZGW aqueous extract were added into the medium.Grouping:control vector of miRNA group;rapamycin group;let-7f overexpression+rapamycin group;let-7f overexpression+rapamycin+ZGW group.The osteogenic differentiation ability was detected by ALP staining and ARS staining.The expression of let-7f was examined by RT-qPCR.The protein exoression of autophagy-related factors(ULK2 and Beclin-1)was examined by Western-blot.Results1.Animal experimental studies(1)ZGW effectively improved bone damage in GIOP rats① ZGW improved lumbar vertebrae bone density in GIOP ratsCompared with CON group,BMD and BMC in GIOP group were significantly lower in M1,M2 and M3(P<0.05),and BMD and BMC in ZGW group were significantly higher than those in GIOP group at M1,M2 and M3(P<0.05).② ZGW improved the microstructure of lumbar vertebrae in GIOP ratsAt Ml,M2 and M3,compared with CON group,BV/TV,Tb.N,Tb.Th and vBMD in GIOP group were significantly decreased(P<0.05),and SMI and Tb.Sp were significantly increased(P<0.05).Compared with the GIOP group,BV/TV,Tb.N and vBMD in the ZGW group were more higher than those in GIOP group at M1,M2 and M3(P<0.05),and SMI was significantly lower in the M1M2 and M3 than in the GIOP group(P<0.05),Tb.Th was significantly higher in M3 than in GIOP group(P<0.05),and Tb.Sp was significantly lower in M3 than in GIOP group(P<0.05).The μCT results showed that the microstructure of the GIOP group was severely damaged,and the ZGW group.could significantly improve the bone microstructure.③ ZGW improved lumbar vertebrae strength in GIOP ratsAt M1,M2 and M3,the compression strength of the GIOP group was significantly lower than that of the CON group(P<0.05),and the energy absorption value was significantly decreased at M2 and M3(P<0.05).Compared with the GIOP group,the compressive strength at different time points,the compression stiffness at M3 and the energy absorption were significantly increased in the ZGW group(P<0.05).④ ZGW improved HE staining of lumbar vertebrae in GIOP ratsAt different time points,the GIOP group had different degrees of damage to the trabecular bone,including a significant reduction in the number of trabecular bone,trabecular fracture,and widening of the trabecular space.The osteoclasts and osteoblasts in surface of the trabecular bone were reduced.The fat foam of medullary cavity increased significantly.The ZGW group could effectively improve the trabecular bone damage at different time points.⑤ZGW improved serum ALP activity in GIOP ratsAt M2 and M3,ALP activity in GIOP group was significantly lower than that in CON group.P<0.05),while ZGW group significantly reversed ALP activity(P<0.05).(2)ZGW improved the number of autophagosomes in lumbar vertebral osteoblasts in GIOP ratsAt different time points,the GIOP group significantly increased the number of autophagosomes in osteoblasts,while the ZGW group restored the number of autophagosomes in osteoblasts in some degrees.(3)ZGW promoted the expression of let-7f and osteogenic markers in vertebral body of GIOP rats and inhibited the expression of autophagy related factorsAt M1,GC significantly promoted mRNA expression of ULK2,Beclin-1,LC3B,and ATG5(P<0.01,P<0.001).At M2,GC significantly inhibited let-7f expression and promoted mRNA expression of Beclin-1,ATG5,and ATG12(P<0.01,P<0.001.At M3,GC significantly inhibited let-7f expression and promoted mRNA expression of ULK2,Beclin-1,LC3B,ATG5,and ATG12(P<0.05,P<0.001).These results suggested that GC could inhibit let-7f expression and increase autophagy level in vertebral bone tissue.During ZGW treating GIOP,at M1,ZGW significantly inhibited mRNA expression of ULK2 and ATG5(P<0.05,P<0.001).At M2,GC significantly promoted let-7f expression and inhibited mRNA expression of ULK2,Beclin-1,ATG5,and ATG12(P<0.05,P<0.01,P<0.001).At M3,GC significantly promoted let-7f expression and inhibited mRNA expression of ULK2,Beclin-1,LC3B,and ATG5(P<0.05,P<0.01,P<0.001).These results suggested that ZGW could rescue the effect of GC on let-7f inhibition and over autophagy.At M1,GC significantly inhibited mRNA expression of Alp(P<0.01).At M2,GC significantly inhibited Opn,Alp,and Ocn(P<0.05,P<0.01).At M3,GC significantly inhibited Runx2,Opn,Alp,and Ocn(P<0.01,P<0.001).These results suggested that GC could inhibit osteogenesis.During ZGW treating GIOP,at M2,ZGW significantly promoted mRNA expression of Alp and Ocn(P<0.05,P<0.01).At M3,GC significantly promoted Runx2,Opn,Alp,and Ocn.These results suggested that ZGW could rescue the inhibition effect of GC on osteogenesis.2.Cell experimental study(1)let-7f targeted regulation of ULK2① Bioinformatics predicted that let-7f has a potential binding site with ULK2;② Luciferase activity assay showed that let-7f mimic+wild type significantly reduced the expression of ULK2 compared with the blank vector group(P<0.01)and ompared with let-7f mimic+wild type,let-7f inhibitor+wild type significantly increased the expression of ULK2(P<0.01),while in the transfected mutant vector,there was no statistical difference in ULK2 expression,indicating that let-7f and ULK2 have targeted regulation.(2)ZGW aqueous extract up-regulated expression,inhibited autophagy,and then promoted osteogenic differentiation of BMSCs① BMSCs proliferation increased by dose-dependent before 7 days and decreased dose-dependent after 7 days.10 ug/ml concentration may be the best concentration of ZGW;② Let-7f overexpression and let-7f overexpression+ZGW significantly promoted ALP positive staining and calcified nodules formation.Let-7f silencing significantly inhibited ALP positive staining and calcified nodules formation,while ZGW could rescue the silencing effect of let-7f.Rapamycin significantly inhibited ALP positive staining and calcified nodules formation in control vector of miRNA group and let-7f overexpression group,while ZGW showed the rescued effect.③ Let-7f overexpression or silencing significantly promoted(P<0.01)or inhibited(P<0.001)the expression of let-7f,indicating that the overexpression and silencing construction were successful.Rapamycin significantly inhibited let-7f expression(P<0.01),while ZGW rescue the inhibition effect(P<0.01).④Let-7f overexpression significantly inhibited the protein expression of autophagy related factors ULK2,ATG5,ATG12,Becinl,and LC3B.And ZGW treatment showed the similar inhibition effct,though the inhibition effect was lower than let-7f overexpression.And let-7f overexpression combined with ZGW treatment also significantly inhibited the protein expression of autophagy related factors,and the inhibition effct was stronger than let-7f overexpression alone.Similar,in control vector of plux group,ZGW treatment significantly inhibited the protein expression of autophagy related factors.Let-7f silencing could promote the protein expression of autophagy related factors,while let-7f silencing combined with ZGW treatment could rescue the promotion effect.Rapamycin significantly promoted the protein expression-of autophagy related factors ULK2 and Beclin-1,while ZGW treatment showed the significant rescued effect.Conclusion1.Animal experimental study:(1)ZGW can effectively improve GIOP bone damage(increase bone mass,improve bone microstructure,increase bone strength,improve bone tissue morphological damage,increase serum ALP activity,promote the mRNA expression of osteogenic markers Runx2,Opn,Alp,and Ocn);(2)ZGW can reverse the effect of GC in inhibiting the expression of let-7f and inducing excessive autophagy in osteoblasts(the expression of let-7f in bone tissue was significantly increased;the number of autophagosomes in osteoblasts was significantly decreased,and autophagy-associated factors ULK2,Beclin-1,LC3,ATG5,and ATG12 were significantly down-regulated).2.Cell experimental study:(1)Bioinformatics prediction and dual luciferase reporter gene assay verify that let-7f targets binding to ULK2 and negatively regulates ULK2 expression;(2)By let-7f overexpression/silencing,the intervention of autophagy activator and other techniques,we indicate that ZGW may promote osteoblast differentiation of BMSCs by regulating the crosstalk of let-7f and autophagy.