Exploring The Application Of In Vivo Activation Of Myokines To Ameliorate Obesity By Promoting Adipocytes Browning

Objective: Skeletal muscle is not only critical for controlling body movement but also an essential secretory organ.Myokines,cytokines secreted by muscles,have been found to regulate lipid metabolism.Among them,Irisin and FGF21 can significantly improve obesity in vitro and animal experiments.At present,these proteins are overexpressed or directly injected as recombinant proteins to achieve the purpose of improving obesity.However,the overexpression disrupts the body’s original balance,and the high cost of recombinant protein preparation limits its clinical application.The emerging geneediting technology,CRISPR/d Cas9,provides a new solution for the clinical application of myokines.This technology can achieve gene activation or suppression in vivo without changing the genome structure.In this study,we applied the CRISPRa system to regulate the expression and secretion of endogenous myokines,thereby regulating the browning of adipocytes,making it a promising treatment for obesity and related metabolic diseases.Methods: We first in vitro infected the fibroblast NIH3T3 with lentivirus containing CRISPRa system to screen for the most efficient sg RNA to up-regulate the expression of FGF21 and Irisin.The selected sg RNA virus was then used to infect myoblast cell line C2C12 for endogenous verification.Next,the effect of the conditioned media of FGF21 and Irisin-activated C2C12 cells on adipogenic differentiation of 3T3-L1 adipose precursor cells was examined.Further,we conducted in vivo verification.First,we constructed the AAV vector of the CRISPRa system,packaged and purified the AAV virus and injected it into C57 mice via tail vein injection to detect the changes of Irisin and FGF21 expression in muscle tissue and serum.Finally,the virus will be injected into the obese model mice to observe metabolic regulation.Results: 1)C2C12 cells express and secrete FGF21 and Irisin;2)CRISPRa system can up-regulate the expression of endogenous Irisin and FGF21;3)Upregulation of FGF21 and Irisin can promote the differentiation and development of muscle cells;4)The conditioned media of Irisin and FGF21 up-regulated by CRISPRa system can promote the decomposition of adipocytes,and FGF21 can cause browning of adipocytes;5)Through the tail vein injection of mice,the AAV9-based CRISPRa system can be efficiently transduced in vivo;6)The AAV9-CRISPRa system has no apparent side effects in the body.Conclusion: Our regulation of the two myokines FGF21 and Irisin based on the CRISPRa system can promote the breakdown of adipocytes in vitro,which lays the foundation for further research on the interaction between muscle and adipose tissue,and also provides a new solution for the prevention and treatment of metabolic diseases.