Study On The Effect And Mechanism Of Icariin On Lipid Metabolism In FNDC5 Gene Knockout Mice

ObjectiveFNDC5 knockout(KO)worsen the fat accumulation induced by age in female mice.The purpose of this study was to explore the therapeutic effects and mechanisms of icariin(ICA)on fat accumulation induced by KO in middle-aged mice.Methods1.KO mice were produced by TALEN-mediated DNA targeting technique.DNA was extracted from tails,PCR was amplified by polymerase chain reaction and first-generation gene sequencing was used to determine whether KO mice were homozygotes.The RNA of subcutaneous white fat(s WAT),muscle and bone was extracted and detected the expression of FNDC5 to verify whether KO mice were successfully constructed by real-time quantitative polymerase chain reaction(RT-q PCR).2.To investigate the effect of FNDC5 gene on the body,we measured body weight of FNDC5-KO mice at 4 week,8 week,18 week-old and of age and sex-matched wild-type(WT)mice.3.The content of serum triglyceride(TG),total cholesterol(TC)were measured at 8week,18 week-old female WT mice and KO mice.s WAT was taken for hematoxylin-eosin(H&E)staining to observe the morphological changes of mice in each group,and RNA was extracted to detect the expression of thermogenesis-related genes,such as FNDC5,PGC1-αand UCP1.4.Intervention of traditional Chinese medicine: 50-week-old WT mice and KO mice,half male and half female,were randomly divided into WT mice control group(saline,WT-CON,n= 6),WT mice icariin treatment group(WT-ICA,40 mg/kg/day,n = 6);KO mice control group(saline,KO-CON,n = 6)and KO mice icariin treatment group(KO-ICA,40 mg/kg/day,n = 6).Icariin(ICA)was dissolved in dimethyl sulfoxide and diluted with normal saline.The drug was given intragastric administration once a day for 21 days.The body weight was recorded every 3 days.After the last administration,the mice were anesthetized with 3% chloral hydrate,and the serum was extracted to detect TG,TC and s WAT were taken for HE staining.The expression of UCP1 was observed by immunohistochemistry and RNA was extracted to detect the expression of thermogenic gene FNDC5,PGC1-,UCP1 and the core factor of Hippo signal pathway,such as YAP,TAZ,Tead3.Results1.FNDC5 gene deletion mice were successfully constructed and DNA generation sequences showed that the double strands were base deletion sequences.Compared with WT mice,the expression of FNDC5 m RNA in skeletal muscle,s WAT and bone in KO mice was significantly decreased.There was no significant difference in body weight between male and female in 4 and 8 weeks mice,but the female KO mice at 18 and 50 weeks old was significantly higher.However,there was no difference in body weight between 18 and50-week-old male KO mice.2.Compared with WT mice,the serum TG in 8 and 18-week-old female KO mice increased significantly,but the serum TC in 8-week-old mice had no difference and the serum TC increased significantly in 18-week-old KO mice.The results of HE staining showed that there was no significant difference in adipocyte area and fat quantity per unit area between 8-week-old female KO and WT mice,but there was significant difference in adipocyte area and fat quantity per unit area between 18-week-old female KO and WT mice of the same age.3.Compared with WT,the expression of thermogenic genes FNDC5,PGC1-α and UCP1 m RNA decreased significantly in 8 and 18-week-old female KO mice.4.After the treatment of icariin for 21 days,the body weight of both male and female mice in WT-ICA group decreased significantly,while the body weight of KO-ICA female mice was not significantly different from that of KO-CON,while the body weight of KO-ICA male mice decreased significantly.The serum TC content of female WT-ICA mice was significantly lower than that of WT-CON,while the serum TC content of KO-ICA female mice was not significantly different from that of KO-CON and there was no significantly difference in TG content among the three groups.HE staining showed that there were significant differences in adipocyte area and number of adipocytes per unit area between female WT-ICA mice and WT-Con mice,while there was no significant difference in adipocyte area and fat number per unit area between female KO-ICA mice and KO-CON mice.UCP1 immunohistochemistry in s WAT showed that the expression of UCP1 in female WT-ICA mice was significantly higher than that in WT-CON mice,while there was no significant difference in UCP1 expression between KO-ICA female mice and KO-CON mice.5.The expression of thermogenic genes FNDC5,PGC1-α and UCP1 m RNA in female WT-ICA mice was significantly higher than that in WT-CON mice,while the expression of FNDC5,PGC1-αand UCP1 m RNA in female KO-ICA mice was not significantly different from that in KO-CON mice.However,the expression of thermogenic genes FNDC5,PGC1-αand UCP1 m RNA in male WT-ICA mice was significantly higher than that in WT-CON mice,and the expression of thermogenic genes FNDC5,UCP1 m RNA and KO-CON in male KO-ICA mice was significantly higher than that in male KO-ICA mice.6.The expression of Hippo signal pathway genes TAZ,YAP,TEAD3 in female WT-ICA mice was significantly higher than that in WT-CON mice,while the expression of TAZ,YAP in KO-ICA female mice was not significantly different from that in KO-CON mice.There was no significant difference in TAZ,YAP,TEAD3 expression between KO-ICA and KO-CON mice in both female and male mice.Conclusions1.FNDC5 deficiency had no effect on the body weight of young and male mice,but there was significant difference in the body weight in 18 weeds female mice.2.NDC5 deficiency accelerated fat accumulation in female mice with age,which was related to the decrease of WAT browning and the increase of adipocyte size caused by the decrease of UCP1.3.ICA significantly reduced the body weight of WT mice both male and female and FNDC5 deficiency attenuated this effect of ICA only in female mice,suggesting that FNDC5 is essential for the effect of icariin only in female mice.4.Sex differences of icariin are accounted for the differences on FNDC5 and Hippo signaling pathway.