A Bone Metabolism Study Of Zuogui Pill Regulating FNDC5 Gene Knockout Mice

Objective:To complete the establishment of FNDC5 knockout model mice and perform bone tissue transcriptome sequencing,to elucidate the effect of FNDC5 knockout on bone metabolism in male mice,and to explore the effect of Bushenqijing Recipe Zuogui Wan on improving bone metabolism in male mice And mechanism.Methods1.Use the Talen technology to construct a knockout mouse model,breed the offspring,and then perform DNA sequencing and tissue FNDC5 mRNA content detection to determine whether the gene-knockout model has been successfully constructed.2.After successful establishment of the gene-knockout model,the 8-week-old male mice were taken for HE staining and Micro-CT detection of bone morphology,and mRNA transcriptome sequencing was performed to observe the effect of FNDC5 gene knockout on their bone metabolism.3.The experimental group of 4 week old male mice were randomly divided into wild-type group,Zuogui Pill+Wild-type group,FNDC5 knockout group,Zuogui Pill+FNDC5 knockout group,3 mice in each group.The Zuogui Pill group was orally administered with 13.65 g/Kg for 4 weeks;the wild group and the FNDC5 knockout group were orally administered with an equal amount of normal saline.After 4 weeks,3%chloral hydrate was used for anesthesia.The morphological changes of femurs were observed by HE staining in mice.Micro-CT was used to detect bone density and microstructure of mice.The effects of Zuogui Pill on bone metabolism in FNDC5 knockout model mice were explored by the above experiments.4.Using network pharmacology,select related target to the active ingredients of Zuogui Pill and bone metabolism,and perform network pharmacological analysis.The analysis results are matched with transcriptome sequencing differential genes to predict how Zuogui Pill improve the gene knockout model male mice Mechanism of Bone Metabolism.Results1.The FNDC5 knockout model was successfully established,the DNA sequence successfully knocked out the region where FNDC5 was located,and the femoral FNDC5 mRNA content in male mice is decreased(P<0.01).2.Compared with the wild group mice,the femoral HE staining shows that the number of trabecular bones of FNDC5 knockout male mice was reduced and sparse,the trabecular bones were slightly broken,discontinuous,and the arrangement was disordered;Micro-CT:compared with wild-type comparison,the trabecular spacing of the gene-knockout male mice widened slightly,bone density decreased with the mean of tBMD(P<0.05)and Tb.Th(P<0.01)decreased,and Tb.Sp increased(P<0.01).Femoral transcriptome sequencing results:A total of 33,747 differential genes were obtained,of which 291 were statistically different genes,39 were up-regulated and 252 were down-regulated.The differential gene in KEGG is mainly enriched in cardiomyopathy-related,and also includes the pathway of parathyroid hormone synthesis and action,which is closely related to bone metabolism.3.The Effects of Zuogui Pills on bone metabolism of FNDC5 knockout mice:(1)HE staining results showed that Zuogui Pill can improve bone metabolism abnormalities caused by FNDC5 gene knockout in male mice,also can promote bone formation.(2)Micro-CT showed that after treatment with Zuogui Pill,compared with the gene-knockout group,the tBMD of Zuogui Pill group was increased(P<0.01)and the Tb.Sp is decreased(P<0.05),the Tb.Th had the tendency to increase(P<0.01).4.Through network pharmacological analysis,71 Zuogui Pill target genes corresponding to chemical components and 7,986 target genes related to bone metabolism were obtained.The potential targets of the drug were mapped to the disease genes and VennDiagram was used to draw Venn.It shows that there are 257 intersection genes of active compounds and bone metabolism,of which 6 effective differential genes are obtained,namely EGFR,PYGM,CAV1,NOS3,GPT2,and CASQ1 after matching the transcriptome sequencing differential genes.Compared with the wild type group,the 6 genes in the knockout model mice were shown to be down-regulated,combined with the effect of down-regulation of MMP16 gene in sequencing,according to the sequencing KEGG analysis,Zuogui Pill improving bone metabolism abnormality of gene-knockout male mice might be related to the synthesis and function of parathyroid hormone。The P38 MAPK signaling pathway is one of the KEGG enrichment pathways.The improvement of Zuoguiwan on bone metabolism in this knockout male mice may be achieved through this pathway.ConclusionsIn this study,the FNDC5 gene-knockout model was successfully constructed.Through femoral HE staining and Micro-CT detection,it was found that male mice in this model had abnormalities in bone metabolism.Kidney-tonifying formula Zuogui Pill could improve this abnormality phenomenon.Integrating transcriptome sequencing results,through network pharmacological analysis,Zuogui Pill may improve the abnormal bone metabolism of male mice caused by FNDC5 gene-knockout through the synthesis and action of parathyroid hormone and P38 MAPK signaling pathways.