| Objective1.Mechanism prediction,to predict the molecular mechanism of Zishen Jiangu Recipe(ZSJGR)on PMOP,and provide directions for further research based on network pharmacology and bioinformatics.2.In vitro,to explore the effect of ZSJGR on osteogenic differentiation in rat BMSCs.And further analyze the role of PI3K/AKT/mTOR signaling pathway and autophagy in the promotion of bone formation by ZSJGR.3.In vivo,to explore the bone protective effect of ZSJGR on ovariectomized rats,and further analyze the role of PI3K/AKT/mTOR signaling pathway and autophagy in the promotion of bone formation in rats by ZSJGR.Methods1.Mechanism predictionThe TCMSP and BATMAN databases were used to collect active ingredients of ZSJGR and predict its targets;the GEO database was collected OP-related microarray for further analysis of differentially expressed genes,and the GeneCards was collected PMOP genes.The overlapping of targets,differentially expressed genes and PMOP genes were considered to be the targets of ZSJGR on PMOP.Finally,pathway and biological process enrichment analysis was carried out through DVAID database.2.In vitro(1)Effects of ZSJGR on proliferation,osteogenic differentiation and mineralization of 740Y-P-induced BMSCs.Different doses of ZSJGR or normal saline were administered to rats to prepare blank,low,medium and high dose serum.BMSCs were divided into blank group(Control),model group(YP),low-dose serum group(YP+LD),medium-dose serum group(YP+MD),and high-dose serum group(YP+HD).All groups were incubated with 740Y-P for autophagy inhibition,except Control.After that,cells were cultured with 10%blank serum or drug serum.Results detection:CCK-8 kit was used to evaluate cell activity of each group after 24h incubation;②After incubation with differentiation medium,ALP staining and activity detection were performed on 7d and 14d,and alizarin red staining and calcium deposition quantification were performed on 14d and 21d.(2)Effect of ZSJGR on PI3K/AKT/mTOR signaling pathway and autophagy in 740Y-P-induced BMSCsGrouping and culturing according to the method in(1),Result detection:①MDC fluorescence staining and transmission electron microscopy were used to evaluate autophagy of BMSCs;②RT-PCR was used to detect gene expression of PI3K,AKT,mTOR,P62 and LC3;③Western-blot was used to detect protein expression of p-PI3K,p-AKT,p-mTOR,P62,LC3,OCN and Runx2.3.In vivo(1)Protective effect of ZSJGR on ovariectomized ratsForty female SD rats were divided into 5 groups equally:sham-operated group(Sham),model group(OVX),low-dose group(OVX+LD),middle-dose group(OVX+HD),high-dose group(OVX+HD).Ovariectomy was performed to create osteoporosis,the results were verified 3 months later,and samples were taken after 12 weeks of drug intervention.Result detection:①The left tibia was used for BMD,bone microstructure scanning and HE staining,and the left femur was used for biomechanical experiments.②Blood from abdominal aorta to detect the levels of E2,TRACP and PINP.(2)Effects of ZSJGR on PI3K/AKT/mTOR signaling pathway and autophagy in BMSCs of ovariectomized ratsRight femur and tibia bone marrow was used for dissociating rat BMSCs.Result detection:①RT-PCR was used to evaluate gene expression of PI3K,AKT,mTOR,P62,LC3;②Western-blot was used for p-PI3K,p-AKT,p-mTOR,P62,LC3 protein expression.Results1.Mechanism predictionWe retrieved 57 active ingredients of ZSJGR,and predicted 239 targets;and calculated 2843 differentially expressed genes,and collected 1140 PMOP-related genes in Genecards.Finally,25 therapeutic targets were confirmed after overlapping.The enrichment results showed that the genes were mainly enriched in PI3K/AKT signaling pathway,IL-17 signaling pathway,NF-κB signaling pathway,autophagy,and estrogen signaling pathway.2.In vitro(1)Effects of ZSJGR on proliferation,osteogenic differentiation and mineralization in 740Y-P-induced BMSCs.①CCK-8 suggested:Compared with Control,cell viability of YP group was significantly decreased(P<0.01);compared with YP group,cell viability of YP+MD and YP+HD groups was significantly increased(P<0.05).②ALP activity implied:at 7d and 14d,compared with Control,ALP activity of YP group was significantly decreased(P<0.01);compared with YP group,ALP activity in YP+LD,YP+MD and YP+HD groups was significantly increased(P<0.05).③Mineralization indicated:at 14d and 21d,compared with Control,calcium in YP group was significantly lower(P<0.01);compared with YP group,calcium in YP+LD,YP+MD,and YP+HD groups were significantly increased(P<0.05).(2)Effect of ZSJGR on PI3K/AKT/mTOR signaling pathway and autophagy in 740Y-P-induced BMSCs.①MDC implied:compared with Control,YP group had fewer fluorescent spots and weaker intensity;compared with YP group,YP+LD,YP+MD,YP+HD groups had more fluorescent spots and more intensity.②RT-PCR results indicated:Compared with Control,expression levels of PI3K,AKT,mTOR and P62 genes in YP group were significantly increased(P<0.01),on the contrary,expression of LC3 gene was significantly decreased(P<0.01);compared with YP group,expression of PI3K,AKT,and mTOR in YP+LD,YP+MD,and YP+HD groups were significantly decreased(P<0.05),and P62 gene in the YP+MD and YP+HD groups was significantly decreased too(P<0.01),however,expression of LC3 in YP+LD,YP+MD,and YP+HD groups increased(P<0.01).③Western-blot suggested:compared with Control,the phosphorylated protein of PI3K,AKT,and mTOR and P62 protein in YP group were significantly increased(P<0.01),the ratio of LC3Ⅱ/LC3Ⅰ was decreased(P<0.01),and the expressions of OCN and Runx2 were significantly decreased(P<0.01);compared with YP group,the protein expressions of p-PI3K,p-AKT,p-mTOR and P62 in YP+LD,YP+MD and YP+HD groups were significantly decreased(P<0.05),the OCN and LC3Ⅱ/LC3Ⅰ ratio in YP+LD,YP+MD,YP+HD groups were significantly increased(P<0.05),Runx2 was significantly increased in YP+LD,YP+MD and YP+HD groups(P<0.01).3.In vivo(1)Protective effect of ZSJGR on ovariectomized rats①BMD result:compared with Sham,the BMD of tibia in OVX group was significantly decreased(P<0.01);compared with OVX group,the BMD of tibia in OVX+MD,and OVX+HD groups was significantly increased(P<0.01).②Bone microstructure results:compared with Sham,the St.Th and St.Nu of tibia in OVX group were significantly decreased(P<0.01),while the St.Sp and SMI were significantly increased(P<0.01);compared with OVX group,the St.Th of tibia in OVX+MD and OVX+HD groups was significantly increased(P<0.01),and the St.Nu in OVX+LD,OVX+MD,and OVX+HD groups was significantly increased(P<0.05),the St.Sp in OVX+LD,OVX+MD,OVX+HD groups were significantly decreased(P<0.05),and SMI in OVX+MD,OVX+HD groups were significantly decreased(P<0.05).③HE staining:The trabecular bone in Sham group was continuous,arranged closely and orderly,the thickness of trabecular bone was wider,and there was no obvious bone lacuna.In OVX group,the trabecular bone in OVX group was less,thinned,broken,disorganized,separated,and a large number of bone lacunae were formed.With the increase of drug concentration,the number and thickness of trabecular bone gradually increased,trabecular bone separation was reduced,continuity and order were gradually restored,and bone lacuna was reduced.④Biomechanical test:Compared with Sham,the ultimate load,flexural strength and flexural modulus of femur in OVX group were significantly decreased(P<0.01);compared with OVX group,the ultimate load and flexural modulus of femur in OVX+LD,OVX+MD,and OVX+HD groups were significantly increased(P<0.05),flexural strength in OVX+MD,and OVX+HD groups were significantly increased(P<0.01).⑤Bone turnover index:Compared with Sham,serum E2 in OVX group was significantly decreased(P<0.01),while TRACP was significantly increased(P<0.05);compared with OVX group,serum E2 and PINP in OVX+MD and OVX+HD groups were significantly increased(P<0.05),whereas TRACP was significantly decreased(P<0.05).(2)Effects of ZSJGR on PI3K/AKT/mTOR signaling pathway and autophagy in BMSCs cells of ovariectomized rats①RT-PCR indicated:Compared with Sham,the gene expressions of PI3K,AKT,mTOR,and P62 in BMSCs of OVX group were significantly increased(P<0.01);the LC3 gene was significantly decreased(P<0.01);compared with OVX group,the PI3K,AKT,mTOR,and P62 mRNA in OVX+LD,OVX+MD,and OVX+HD groups were significantly decreased(P<0.05),and the LC3 was increased(P<0.01).②Western-blot indicated:compared with Sham,the phosphorylated protein of PI3K,AKT,and mTOR and P62 protein in BMSCs of OVX group were significantly increased(P<0.01);whereas LC3Ⅱ/LC3Ⅰ ratio was significantly decreased(P<0.05);compared with OVX group,the protein expressions of p-PI3K,p-AKT,p-mTOR and P62 in the OVX+LD,OVX+MD and OVX+HD groups were significantly decreased(P<0.05),and the ratio of LC3Ⅱ/LC3Ⅰ was increased(P<0.01).Conclusion1.Mechanism predictionBased on network pharmacology and bioinformatics methods,we found that 25 therapeutic targets of ZSJGR,through estrogen signaling pathway,PI3K/Akt,IL-17,NFκB signaling pathway,autophagy,etc.,to achieve multi-channel,multi-target,multisystem anti-PMOP effect.2.In vitroZSJGR could promote the proliferation,osteogenic differentiation and calcification of BMSCs.The mechanism may be that ZSJGR increases autophagy level of BMSCs through the PI3K/AKT/mTOR signaling pathway.3.In vivoZSJGR could reduce bone resorption,promote bone formation,improve bone microstructure,and increase BMD and bone strength in ovariectomized rats.The bone protective effect of ZSJGR on ovariectomized rats may be achieved by increasing autophagy level of rat BMSCs through the PI3K/AKT/mTOR signaling pathway. |
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