| Objective1.Based on network pharmacology and molecular docking technology,to explore the mechanism of Bushen Jianpi Huoxue Recipe(BSF)in the prevention and treatment of Osteosarcopenia(OS),and to predict the relationship among Pi3k/Akt,apoptosis signaling pathway and OS.2.A rat model of OS was established and the phenotype was identified to make sure that Pi3k/Akt and apoptosis signaling pathway are involved in the pathogenesis of OS.3.To clarify the role of BSF in the prevention and treatment of OS in vivo,and to explore the role of Pi3k/Akt/Bad signaling pathway in BSF in the prevention and treatment of OS.Methods1.Network pharmacology analysis of Bushen Jianpi Huoxue Recipe in the prevention and treatment of OsteosarcopeniaFirstly,TCMSP database,BATMAN-TCM database,ETCM database and TCMID database were searched respectively to screen the active ingredients and targets of BSF;the disease-related targets of GeneCards database,OMIM database,PharmGkb database,TTD database and DrugBank database were searched respectively to screen osteoporosis(OP)targets,sarcopenia(SP)targets and OS targets,the disease targets of OP,SP and OS in five databases were combined.Then,the targets of OP and SP were intersected,the intersection targets of OP-SP were combined with the targets of OS,the repetitive targets were excluded and the related targets of OS were obtained.The targets of BSF were intersected with the targets of OS to obtain the TCM-disease targets and construct the TCM-disease-target regulatory network.Then,through the construction of protein interaction network between TCM and disease targets,the network topology was analyzed and the core targets were screened.Finally,GO function enrichment analysis and KEGG pathway enrichment analysis were carried out on the common targets of TCM and disease,and the signaling pathways related to the common targets of TCM and disease were obtained,and the molecular docking of interested ingredients and targets was carried out.2.Establishment of OS rat model(1)Establishment and identification of a rat model of OSForty healthy female SD rats were randomly divided into five groups according to body weight,namely Control group(Control),Sham group(Sham),Sham+DXM group(Sham+DXM),OVX group(OVX),OVX+DXM group(OVX+DXM),with 8 rats in each group.In OVX and OVX+DXM,bilateral ovaries were removed by bilateral ovariectomy via dorsal approach.In Sham and Sham+DXM,the same volume of adipose tissue near the ovary was removed and the muscle and skin were sutured layer by layer.Except the Control,rats in each group were given intramuscular injection of penicillin sodium 80000 units once a day for 3 consecutive days.After one week rest,rats in Sham+DXM and OVX+DXM were intraperitoneally injected with dexamethasone sodium phosphate injection(DXM)lmg/kg/d for 2 weeks.Two months after operation,the relevant indexes were detected:?body weight of rats;?forelimb grip and all limb grip of rats;?DXA of rats;.micro CT of distal femur of rats;(?HE staining and TRAP staining of femur and gastrocnemius of rats.(2)Serological changes in OS ratsIn the above experiments,after anesthesia,blood was collected from abdominal aorta and serum was separated.Serum AKP,Ca and PI levels were detected by alkaline phosphatase,calcium and inorganic phosphorus test kits.Serum PINP,?-CTx,IGF-1 and E2 levels were detected by ELISA.(3)Expression changes of osteogenic and myogenic related proteinsIn the above experiments,after anesthesia,the right femur and right gastrocnemius were quickly separated,and the surrounding soft tissue was removed.After the pre-cooled PBS was rinsed,it was put into the cryopreservation tube and cryopreserved in an ultra-low temperature refrigerator.The expression of OPG and Runx2 in femur and the expression of MyoD 1 in gastrocnemius were detected by Western blotting.(4)Expression changes of Pi3k/Akt signaling pathway proteins in OS ratsThe expression of Pi3k,p-Akt and Akt in femur and gastrocnemius were detected by Western blotting.(5)Changes of apoptosis signaling pathway in OS ratsIn the above experiments,after anesthesia,the left femur and gastrocnemius were quickly and completely separated,and the surrounding soft tissues were removed.After rinsing with pre-cooled PBS,the left femur and gastrocnemius were placed in a 10 ml centrifuge tube and fixed with paraformaldehyde.The left femur and gastrocnemius were stained with TUNEL.The expression of Bcl2,Bcl-xl and Bak proteins in femur and gastrocnemius were detected by Western blotting.3.Bushen Jianpi Huoxue Recipe can prevent and treat OS by regulating Pi3k/Akt/Bad signaling pathway(1)Bushen Jianpi Huoxue Recipe can prevent and treat OS72 healthy female SD rats were randomly divided into six groups according to their body weight,namely Control group(Control),Sham operation group(Sham),OS model group(OS),OS Bushen Jianpi Huoxue Recipe group(OS+BSF),OS estradiol group(OS+E2),OS alendronate sodium group(OS+ALN),with 12 rats in each group.In OS,OS+BSF,OS+E2 and OS+ALN,bilateral ovaries were removed by bilateral ovariectomy via dorsal approach.In Sham,the same volume of adipose tissue near the ovary was removed and the muscle and skin were sutured layer by layer.Except the Control,rats in each group were given intramuscular injection of penicillin sodium 80000 units once a day for 3 consecutive days.After one week rest,rats in OS,OS+BSF,OS+E2 and OS+ALN were intraperitoneally injected with DXM lmg/kg/d for 2 weeks.The modeling time was 3 months.After 3 months,drug intervention was started.Starting from the fourth month,each group was given corresponding drugs every day.The Control,Sham and OS were given 10ml/kg normal saline every day.The OS+BSF group was given 2.979g/kg Recipe by gavage every day.The OS+E2 group was given 0.104mg/kg estradiol by gavage once a day.The OS+ALN group was given 1.042 mg/kg alendronate once a day.The treatment lasted for 3 months.Three months later,the relevant indexes were detected:?body weight,?forelimb grip,?DXA,?micro CT of distal femur,?HE staining and TRAP staining of femur and gastrocnemius.(2)Bushen Jianpi Huoxue Recipe improves serum indexes of OS ratsIn the above experiments,after anesthesia,blood was collected from abdominal aorta and serum was separated.Serum AKP,Ca and PI levels were detected by alkaline phosphatase,calcium and inorganic phosphorus test kits.Serum PINP,?-CTx,IGF-1 and E2 levels were detected by ELISA.(3)Bushen Jianpi Huoxue Recipe enhances the expression of osteogenic and myogenic related genes in rats with OSIn the above experiments,after anesthesia,the right femur and right gastrocnemius were quickly separated,and the surrounding soft tissues were removed.After the pre-cooled PBS was rinsed,it was put into the cryopreservation tube and cryopreserved in the ultra-low temperature refrigerator.RT-PCR was used to detect the expression of osteogenic genes OPG and Runx2 in femur and MyoD1 and Myogenin in gastrocnemius of rats with OS.(4)Bushen Jianpi Huoxue Recipe prevents and treats OS by regulating Pi3k/Akt/Bad signaling pathway? The right gastrocnemius of rats in the above experiments was taken.RT-PCR was used to detect the expression of Pi3k and Akt mRNA in the Pi3k/Akt signaling pathway of gastrocnemius in rats with OS.?The right gastrocnemius of rats in the above experiments was taken.Western blotting was used to detect the expression of Pi3k,p-Akt and Akt proteins in the Pi3k/Akt signaling pathway of gastrocnemius of rats with OS.?In the above experiments,after anesthesia,the left femur and gastrocnemius were quickly and completely separated,and the surrounding soft tissues were removed.After rinsing with pre-cooled PBS,the left femur and gastrocnemius were placed in a 10 ml centrifuge tube and fixed with paraformaldehyde.The left femur and gastrocnemius were stained with TUNEL.?The right gastrocnemius of rats in the above experiments was taken.RT-PCR was used to detect the expression of Bcl2,Bcl-x1 and Bad mRNA in the apoptotic signaling pathway of gastrocnemius in rats with OS.?The right gastrocnemius of rats in the above experiments was taken.Western blotting was used to detect the expression of Bcl2,Bcl-x1 and Bad proteins in the apoptotic signaling pathway of gastrocnemius of rats with OS.Results1.Network pharmacology analysis of Bushen Jianpi Huoxue Recipe in the prevention and treatment of OSThe 54 active ingredients of BSF regulate the occurrence of OS by acting on 19 intersection targets.Among them,quercetin,kaempferol,luteolin and isorhamnetin were the most active ingredients(target number? 4).The key targets of network topology analysis are AKT1,AR,ESR1,FOS,TP53 and CAV1.Go enrichment analysis showed that RNA polymerase ? basic transcription factor binding,steroid binding,nuclear receptor activity,transcription factor activity,RNA polymerase ?transcription factor binding,steroid hormone receptor activity,basic transcription mechanical binding,basic RNA polymerase ? transcription mechanical binding,ATPase binding,protein phosphatase binding,etc,may be related to the effect of Bushen Jianpi Huoxue Recipe in the prevention and treatment of OS.KEGG pathway enrichment analysis showed that MAPK signaling pathway,prolactin signaling pathway,TNF signaling pathway,GnRH secretion,estrogen signaling pathway,synthesis and secretion of growth hormone,thyroid hormone signaling pathway,insulin resistance,insulin signaling pathway,apoptosis,cell aging and so on may be involved in the pathogenesis of OS.2.Establishment of OS rat model(1)Establishment and identification of a rat model of OSAfter OVX and OVX combined with DXM intervention,uterine atrophy,increased fat content,decreased bone mineral content,decreased SMI and RSMI,decreased forelimb grip,decreased BMD of the whole body and femur,aggravated bone microstructure damage of the distal femur,increased osteoclasts of the distal femur,thinned trabecular bone,severe bone marrow adiposis,aggravated muscle fat infiltration,and shrinkage of muscle nucleus were observed in rats.The effect of OVX combined with DXM was more obvious.(2)Serological changes in OS ratsCompared with the Control,Sham and Sham+DXM group,the serum AKP,PINP and ?-CTx contents of OVX+DXM group were significantly increased,while the IGF-1,E2 and Ca contents were significantly decreased,and PI had a downward trend.(3)Expression changes of osteogenic and myogenic related proteinsAfter OVX combined with DXM intervention,the expression of osteogenic proteins OPG and Runx2 in femur decreased,and the expression of MyoD1 in gastrocnemius also decreased.(4)Expression changes of Pi3k/Akt signaling pathway proteins in OS ratsAfter OVX combined with DXM intervention,the protein expression of Pi3k,p-Akt and Akt decreased in femur and gastrocnemius.(5)Changes of apoptosis signaling pathway in OS ratsAfter OVX combined with DXM intervention,the expression of Bcl2 and Bcl-x1 proteins in femur and gastrocnemius decreased,whereas Bak protein increased.3.Bushen Jianpi Huoxue Recipe can prevent and treat OS by regulating Pi3k/Akt/Bad signaling pathway(1)Bushen Jianpi Huoxue Recipe can prevent and treat OSAfter intervention,in OS rats the wet weight of uterus decreased,fat content decreased,bone mineral content increased,SMI and RSMI increased,grip of forelimb increased,BMD of whole body and femur increased,bone microstructure of distal femur improved,osteoclast of distal femur decreased,bone trabecular number increased,bone marrow adiposis reduced and muscle fat infiltration reduced.The effect of BSF is similar to that of E2 and ALN.(2)Bushen Jianpi Huoxue Recipe improves serum indexes of OS ratsCompared with Control group,AKP,IGF-1,E2,PI in OS group decreased significantly,Ca has a downward trend,PINP and ?-CTx increased significantly.Compared with OS group,BSF,E2 and ALN significantly increased serum AKP,IGF-1,E2 and PI levels,Ca has an increasing trend,BSF,E2 and ALN significantly decreased serum PINP levels,and ?-CTx tended to decrease.(3)Bushen Jianpi Huoxue Recipe enhances the expression of osteogenic and myogenic related genes in rats with OSAfter the intervention of BSF,the expression of OPG and Runx2 genes in femur and MyoD1 and Myogenin genes in gastrocnemius were increased.(4)Bushen Jianpi Huoxue Recipe can prevent and treat OS by regulating Pi3k/Akt/Bad signaling pathway?By RT-PCR detection of gastrocnemius,it was found that compared with Control group and Sham group,the expression of Pi3k and Akt mRNA in gastrocnemius of OS group was significantly decreased;compared with OS group,BSF,E2 and ALN significantly increased the expression of Pi3k and Akt mRNA.?Western blotting of gastrocnemius muscle showed that compared with Control group and Sham group,the expression of Pi3k and Akt proteins in gastrocnemius of OS group was significantly decreased;compared with OS group,BSF significantly increased the expression of Pi3k and Akt proteins;compared with OS group,E2 significantly decreased the expression of Akt protein;compared with OS group,ALN significantly up-regulated the expression of Pi3k protein,down-regulated p-Akt and Akt proteins expression.?TUNEL staining and apoptotic rate of femur were detected,the apoptotic rate of TUNEL staining in OS group,OS+BSF group and OS+E2 group was significantly higher than that in Control group and Sham group.The apoptotic rate of TUNEL staining in OS+BSF group was significantly lower than that in OS group.The results showed that the apoptotic cells in femur of OS rats were increased.BSF significantly reduced the apoptotic rate of femur cells in OS rats and inhibited the apoptosis of femur cells in OS rats.?TUNEL staining of gastrocnemius and determination of apoptosis rate showed that the apoptosis rate of gastrocnemius TUNEL staining in OS group was significantly higher than that in Control group and Sham group.The apoptosis rate of gastrocnemius TUNEL staining in OS+BSF group,OS+E2 group and OS+ALN group was significantly lower than that in OS group.The results showed that the apoptosis rate of gastrocnemius in OS group was increased.BSF,E2 and ALN significantly reduced the apoptosis rate of gastrocnemius in OS group and inhibited the apoptosis of gastrocnemius in OS group.?RT-PCR detection of gastrocnemius showed that compared with Control group and Sham group,the mRNA expression of anti-apoptotic proteins Bcl2 and Bcl-xl in gastrocnemius of OS rats decreased significantly,and the mRNA expression of pro-apoptotic protein Bad increased significantly.Compared with OS group,BSF,E2 and ALN significantly increased the mRNA expression of anti-apoptotic protein Bcl2 and Bcl-xl in gastrocnemius,and decreased the mRNA expression of pro-apoptotic protein Bad.?Western blotting of gastrocnemius showed that compared with the Control group and Sham group,the gastrocnemius anti-apoptotic protein Bcl-xl of OS rats was significantly reduced,and the pro-apoptotic protein Bad was significantly increased.Compared with OS group,Bushen Jianpi Huoxue Recipe and estrogen significantly increased the expression of anti-apoptotic proteins Bcl2 and Bcl-xl,and down-regulated the expression of pro-apoptotic protein Bad,which was consistent with gene expression.Conclusion1.Bushen Jianpi Huoxue Recipe can prevent and treat OS through multi-ingredient,multi-target and multi-pathway.Pi3k/Akt/Bad signaling pathway may be involved in regulating the physiological and pathological process of OS.2.Ovariectomy combined with Dexamethasone can successfully construct the rat model of OS,Pi3k/Akt and apoptosis signaling pathway are involved in the pathogenesis of OS.3.Bushen Jianpi Huoxue Recipe can prevent and treat OS in rats by regulating Pi3k/Akt/Bad signaling pathway in vivo. |
PDF Full Text Request