| ObjectiveWe are ready to construct FNDC5 deletion model,observing the effect of FNDC5 deficiency on bone and lipid metabolism,and exploring the function and mechanism of kidney tonifying recipe Yougui Pill can up-regulate FNDC5 to improve the abnormal of bone and lipid metabolism.Methods1.FNDC5 gene deletion model was constructed by Talen technique.The model was successfully constructed by DNA,PCR amplification and gene sequencing,and the expression of FNDC5 in muscle was detected by quantitative PCR;2.The mouse model were divided into wild group,FNDC5 deletion group and Yougui Pill aid FNDC5 deletion group(8 mice,male and female).Yougui Pill group according to the 16.65g/kg daily intragastric gavage for 4 weeks,and the wild group,FNDC5 deletion group daily gavage of saline.After 4 weeks,2%chloral hydrate anesthesia was used;3.Serum Irisin was detected by ELISA in each group of mice4.Effect of Yougui Pill on lipid metabolism in FNDC5 deficient mice:weighing of mice,serum triglyceride(TG)content,extraction of subcutaneous fat RNA,RT-qPCR to detect the expression of FNDC5,PGC-1 alpha,UCP1 mRNA,the expression of UCP1 protein was detected by immunofluorescence of fat.5.Effect of Yougui Pill on bone metabolism in FNDC5 deficient mice:analysis the serum alkaline phosphatase(ALP)activity;mice were detected in bone mineral density and bone microstructure by micro-CT,observe the morphology of mice HE staining,extraction of femoral RNA,RT-qPCR to detect the expression of FNDC5 and Runx2 mRNA.Results1.FNDC5 deletion model was successfully constructed,and the expression of FNDC5 and mRNA in muscle decreased obviously(p<0.01)2.The results of ELISA detection of Irisin in serum were compared with wild group,significantly decreased the content of Irisin protein of FNDC5 deletion mice' s blood(P<0.01);compared with the FNDC5 deletion group,Irisin protein content in blood increased significantly in Yougui Pill group(P<0.01).3.Effect of Yougui Pill on lipid metabolism in FNDC5 deficient mice:(1)compared with the wild group,no significant difference between the FNDC5 deletion group at each time point of the weight.Yougui Pill group from the beginning of the intervention of the fourth week,compared with FNDC5 deficient group,in female mice,body weight in Yougui Pill group significantly decreased from drug intervention,in fifth week,the weight has decreased but the difference was not statistically significant;In 6th,7th and 8th week,body weight decreased significantly compared with FNDC5 group,and has statistically significant difference(P<0.01);In male mice,YouguiWan group from the drug intervention began more weight loss than FNDC5 group,but the difference was not statistically significant;(2)Compared with the wild group,the content of TG in male and female mice in FNDC5 deficient mice increased in serum(P<0.01);Compared with the FNDC5 deletion group,the content of TG in serum was significantly decreased in Yougui Pill group(P<0.01);(3)Compared with the wild group,in female and male mice' s subcutaneous fat,the expression of FNDC5 mRNA in FNDC5 deletion group were significantly lower(P<0.05),the expression of PGC-1 alpha and UCP1 mRNA were significantly increased(P<0.05 And P<0.01);Compared with the FNDC5 deletion group,the expression of FNDC5 in Yougui Pill group were significantly increased(P<0.05).In female mice,the expression of UCP1 in Yougui Pill group was significantly higher than that of FNDC5 group(P<0.01);In male mice,the expression of UCP1 in Yougui Pill group was significantly higher than that group of FNDC5 deletion group(P<0.05).And Yougui Pill group of female and male mice PGC-1 alpha expression and deletion of FNDC5 group,no significant difference;(4)In immunofluorescence,wild group expression of UCP1 weak fluorescence,and the number of fat cells were less,large lipid droplets;Compared with the wild group,the expression of UCP1 fluorescence in FNDC5 deletion group were significantly increased,and the number of fat cells increased significantly;Compared with the deletion of FNDC5 group,the expression of UCP1 increased in Yougui Pill group,and fat cells increased,lipid droplets became smaller.4.Effect of Yougui Pill on bone metabolism in FNDC5 deletion mice:(1)Compared with the wild group,the activity of ALP in female and male mice in FNDC5 deletion group blood decreased significantly(P<0.01);Compared with the FNDC5 deletion group,ALP activity of Yougui Pill group in the serum increased;(2)In female mice,compared with wild group.tBMD,TbBMD,Tb.Th in FNDC5 deletion group were significantly decreased(P<0.01),Tb.Sp increased significantly(P<0.01).Compared with the FNDC5 deletion group,tBMD,TbBMD,Tb.Th in Yougui Pill group were significantly increased(P<0.05,P<0.01 and P<0.05),Tb.Sp decreased significantly(P<0.01);In male mice,compared with wild group,deletion of FNDC5 group tBMD,Tb.Sp had no significant difference while TbBMD and Tb.Th decreased,but the difference was not statistically significant;Compared with the FNDC5 deletion group,Tb.Sp had no significant difference in Yougui Pill group;Although tBMD,TbBMD and Tb.Th has an upward trend,t the difference was not statistically significant.(3)The results of HE staining showed that the wild group trabecular structure complete,dense,trabecular bone around the cell distribution;In female mice,compared with wild group,FNDC5 deletion group showed significantly reduced the number of trabecular bone,trabecular bone fracture,sparse,disordered,discontinuous,small bone beam spacing is increased,the formation of cavity;Bone cells decreased significantly;Compared with the FNDC5 deletion group,Yougui Pill group female mice bone trabeculae was neat,structural integrity,bone cells increased significantly,but with the wild group bone trabecula cracks.In male mice,compared with wild group,FNDC5 deficient group were rare trabecular bone fracture,arranged more sparse,trabecular bone around cells decreased;compared with FNDC5 deficient group,Yougui Pill group trabecular arranged closely,structural integrity,bone cells increased(4)in female mice,compared with wild group,the expression of FNDC5 and Runx2 mRNA deletion group was significantly decreased(P<0.01);Compared with the FNDC5 deletion group,expression of FNDC5 and Runx2 mRNA in Yougui Pill group increased significantly(P<0.05 and P<0.01).In male mice,compared with wild group,the expression of FNDC5 and Runx2 mRNA in FNDC5 deletion group has decreased,but the difference was not statistically significant;Compared with the FNDC5 deletion group,the expression of FNDC5 and Runx2 mRNA Yougui Pill group has a rising trend,but the difference was not statistically significant.ConclusionThis study successfully constructed FNDC5 gene deletion model,and found that the deletion of FNDC5,bone metabolism and lipid metabolism were abnormal,and Yougui Pill can improve the abnormal bone and lipid metabolism.FNDC5 may be the target of Yougui Pill... |
PDF Full Text Request