| Purpose : Based on the theory of "spleen and kidney related",to explore the mechanism of Bushen Jianpi Prescription and its disassembled prescriptions in the prevention and treatment of bone loss in tail suspension rats from the perspective of miRNAs regulating Wnt signaling pathway and promoting osteogenesis.Material and method:Eighty healthy Wistar rats,male,six weeks of age,weight of(200±20)g,were randomly divided into five groups: normal group,model group,Jianpi Prescription group,Bushen Prescription group,Bushen Jianpi Prescription group,with 10 rats in each group,fed in a single cage.Rats were given intragastric administration according to the equivalent dose ratio of human and animal body surface area:The volume of Jianpi Prescription was 2.43g/kg,the volume of Bushen Prescription was 3.24g/kg,the volume of Bushen Jianpi Prescription was5.67g/kg.Rats in the Bushen Prescription group,Jianpi Prescription group and Bushen Jianpi Prescription were given the same volume of liquid(all 2m L)by gavage daily,while normal group and model group were given 2m L distilled water.All groups except blank control group were modeled by tail hanging method.On the day of modeling,rats in each group were given orally 2ml,once a day,for 21 days.On the22 nd day,10 rats in each group were anesthetized by intraperitoneal injection of chloral hydrate.The main venous blood was collected from the abdomen,placed at room temperature and centrifuged.The upper serum was collected and labeled in an EP tube,and stored in a refrigerator at-20℃ for future use.The other rats in each group were sacrificed by cervical dislocation.The femoral soft tissue and fascia were separated on the ultra-clean workbench,and the pulp cavity was rinsed.The rinsed liquid was made into single-cell suspension,which was blown,centrifuged,and the upper fat layer was discarded.The culture medium was re-suspended into single cell suspension,and the culture was carried out until P3 generation for osteogenesis induction.Serum osteocalcin(OC)content was detected by Elisa.The ALP activity of BMSCs was detected by p-nitrophenyl phosphate(PNPP).Alizarin red staining was used to detect the formation of calcium nodules in BMSCs of each group.In addition,32 male Wistar rats weighing 200± 20g(6 weeks of age)were randomly divided into blank group,Jianpi Prescription group,Bushen Prescription group,Bushen Jianpi Prescription group.After adaptive feeding for 1 week,The dose of Jianpi Prescription was 2.43g/kg,the dose of Bushen Prescription was 3.24g/kg,and the dose of Bushen Prescription was 5.67g/kg.The rats in the invigorating kidney group,invigorating spleen group and invigorating kidney and invigorating spleen group were given 2ml liquid for 5 days.The blank group was given 2ml distilled water intragastric administration for 5 days.At the end of the time to fill the stomach after 1 h,anesthesia with chloral hydrate intraperitoneal injection,ventral venous blood was taken.After standing at room temperature and centrifuging,the upper serum was taken,mixed together with the same group,and inactivated by water bath at 56℃,filter with filter membrane,separate packaging,stored in-80℃ ultra-low temperature refrigerator for standby.The purchased Ori Cell Wistar rat bone marrow mesenchymal stem cells(BMSCS)were transferred from P2 generation to P3 generation,and divided into 5groups.Bone marrow mesenchymal stem cells were cultured with drug serum of normal group,osteogenic induction fluid,drug serum of Jianpi Prescription group plus osteogenic induction fluid,drug serum of Bushen Prescription group plus osteogenic induction fluid,drug serum of Bushen Jianpi Prescription group plus osteogenic induction fluid.The expression of β-catenin,GSK3 β and GSK3 βphosphorylated proteins was detected by Western-blot.The mRNA expressions of Mir-214-3p,Mir-10a-5p,Wnt3 a and Runx2 were detected by real-time fluorescence quantitative PCR(RT-PCR).Results:1.Effects of Bushen Jianpi Prescription and its disassembled prescriptions osteogenic differentiation ability of tail suspension rats.1.1 Effects of Bushen Jianpi Prescription and its disassembled prescriptions on serum osteocalcin of suspension rats.Compared with normal group,serum OC content in model group was significantly decreased(P<0.01);Compared with model group,serum OC content in Jianpi Prescription group,Bushen Prescription group,Bushen Jianpi Prescription group was significantly increased(P<0.01);Compared with Jianpi Prescription group,serum OC content in Bushen Jianpi Prescription group was significantly increased(P< 0.01).1.2 Effects of Bushen Jianpi Prescription and its disassembled prescriptions on alkaline phosphatase(ALP)of suspension rats.Compared with normal group,the ALP activity of BMSCs in model group was significantly decreased(P<0.01).Compared with model group,the ALP activity of BMSCs in Jianpi Prescription group,Bushen Prescription group,Bushen Jianpi Prescription group was significantly increased(P<0.01);Compared with Jianpi Prescription group,the ALP activity of BMSCs in Bushen Prescription group and Bushen Jianpi Prescription group was significantly increased(P<0.01).Compared with Bushen Prescription group,the activity of BMSCs ALP in Bushen Jianpi Prescription group had no significant change(P > 0.05).1.3 Effects of Bushen Jianpi Prescription and its disassembled prescriptions on number of calcium nodules of suspension rats Compared with the normal group,the staining area of the model group was significantly reduced.Compared with model group,the staining area of Jianpi Prescription group,Bushen Prescription group,Bushen Jianpi Prescription group was significantly increased;Compared with Jianpi Prescription group,Bushen Prescription group,the staining area of Bushen Jianpi Prescription group was significantly increased.2.Study on the mechanism of Bushen Jianpi Prescription and its disassembled prescriptions serum regulating Wnt signaling pathway through miRNAs to promote bone formation2.1The effects of Bushen Jianpi Prescription and its disassembled prescriptions medicated serum of β-catenin,GSK3β and P-GSK3 β proteins in bone regulated by miRNAs on Wnt signaling pathway Compared with normal group,β-catenin protein expression in osteogenic induction group was significantly increased(P<0.01);Compared with osteogenic induction group,the expression of β-catenin protein in Jianpi Prescription group,Bushen Prescription group and Bushen Jianpi Prescription group was significantly increased(P<0.01);Compared with Jianpi Prescription group,the expression of β-catenin protein in Bushen Prescription group and Bushen Jianpi Prescription group was significantly increased(P<0.01);Compared Bushen Prescription group,the expression of β-catenin protein in Bushen Jianpi Prescription group was significantly increased(P<0.01).Compared with normal group,GSK3βprotein expression in osteogenic induction group was significantly increased(P<0.01);Compared with osteogenic induction group,the expression of GSK3βprotein in Jianpi Prescription group was significantly increased(P<0.01),and that in Bushen Jianpi Prescription group was significantly decreased(P<0.01);Compared with Jianpi Prescription group,the expression of GSK3βprotein in Bushen Prescription group and Bushen Jianpi Prescription group was significantly decreased(P<0.01);Compared with Bushen Prescription group,the expression of GSK3βprotein in Bushen Jianpi Prescription group was significantly decreased(P<0.01).Compared with normal group,p-GSK3βprotein expression in osteogenic induction group was significantly increased(P<0.01);Compared with osteogenic induction group,p-GSK3 β protein expression in Jianpi Prescription,Bushen Prescription group and Bushen Jianpi Prescription group was significantly increased(P<0.01);Compared with Bushen Jianpi Prescription group,p-GSK3 β protein expression in Jianpi Prescription group and Bushen Prescription groups was significantly decreased(P < 0.01).2.2Bushen Jianpi prescription and its disassembled drug-containing serum regulate Wnt signaling pathway through miRNAs to promote the mRNA expression of Mir-214-3p,Mir-10a-5p,Wnt3 a and osteogenic related gene Runx2 in bone Compared with the normal group,the expression of Mir-214-3p in osteogenic induction group was significantly decreased(P<0.01);Compared with osteogenic induction group,the expression level of Mir-214-3p in Bushen Jianpi Prescription group was significantly decreased(P<0.01).Compared with the normal group,the expression of Mir-10a-5p in osteogenic induction group was significantly decreased(P<0.01);Compared with osteogenic induction group,the expression of Mir-10a-5p in Bushen Jianpi Prescription group was significantly decreased(P<0.01).Compared with normal group,the expression of Wnt3 a in osteogenic induction group was significantly increased(P<0.01).Compared with osteogenic induction group,the expression of Wnt3 a in Jianpi Prescription group,Bushen Prescription group and Bushen Jianpi Prescription group was significantly increased(P<0.01);Compared with Bushen Jianpi Prescription group,the expression of Wnt3 a in Jianpi Prescription group and Bushen Prescription groups was significantly decreased(P<0.01).Compared with normal group,Runx2 expression was significantly increased in osteogenic induction group(P<0.01).Compared with osteogenic induction group,the expression of Runx2 in Jianpi Prescription group,Bushen Prescription group and Bushen Jianpi Prescription group was significantly increased(P<0.01);Compared with Bushen Jianpi Prescription group,the expression of Runx2 in Jianpi Prescription group and Bushen Prescription groups was significantly decreased(P<0.01).Conclusion:1.Bushen Jianpi Prescription and its disassembled prescriptions can enhance the serum OC level.The ALP level of BMSCs was increased 14 days after osteogenesis induction and calcium nodules were promoted.It is speculated that the prescription of Bushen Jianpi Prescription and its disassembled prescriptions can promote osteogenic differentiation in tail suspension rats.The bone loss of tail hanging rats was improved,and the effect of Bushen Jianpi prescription was the most significant,and the effect of Bushen Prescription was stronger than Jianpi Prescription.2.Bushen Jianpi Prescription can reduce the expression of Mir-214-3p and Mir-10a-5p,increase the expression of Wnt3 a,β-catenin,P-GSK3βand Runx2,and decrease the expression of GSK3 β.It is speculated that the prescription for Bushen Jianpi Prescription can inhibit the GSK3 β phosphorylation of β-catenin by inhibiting miRNAs expression,promote the accumulation of β-catenin,promote the expression of Wnt signaling pathway,and promote the osteogenic differentiation of BMSCs.Bushen Prescription and Jianpi Prescription could not reduce the expressions of Mir-214-3p and Mir-10a-5p,but could increase the expressions of Wnt3 a,β-catenin,P-GSK3β and Runx2,and reduce the expression of GSK3β,suggesting that bushen prescription and Jianpi prescription could also promote BMSCs osteogenic differentiation through Wnt signaling pathway.But not through miRNAs. |
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