Study On The Regulation Of Tsp43 Protein Of Trichinella Spiralis Muscle Larvae On The Proliferation And Differentiation Of Mouse Myoblasts

Trichinella spiralis is a zoonotic nematode parasitic on mammalian skeletal muscle cells.It is distributed globally and can cause Trichinosis.Trichinosis has caused serious harm to human health and animal husbandry.It is one of the food borne parasitic diseases.The newborn larvae of T.spiralis invade host skeletal muscle cells from intestinal mucosa with systemic blood circulation and form nurse cell,which provides a suitable living environment for T.spiralis muscle larvae.The formation of nurse cells after T.spiralis infection has always been a research hotspot.However,there are few studies on the mechanism of muscle cell remodeling and the mechanism of promoting nurse cells formation after T.spiralis infection.The study of muscle cell remodeling by T.spiralis muscle larvae is helpful for us to further clarify the formation mechanism of nurse cells and the mechanism of T.spiralis interfering with skeletal muscle repair.In this study,T.spiralis muscle larvae crude worm extract(Trichinella spiralis muscle larvae crude worm extract,TWCWE),excretory secretory antigen(excretory secretory,ES)and Tsp43 recombinant protein were collected to stimulate C2C12 cells,and the cell model of infection in vitro was established.CCK-8 method was used to detect the cell survival of C2C12 cells at different stimulation concentrations and different stimulation time.The results showed that the C2C12 cells survival increased with the increase of stimulation concentration,and the three antigens all induced the proliferation of C2C12 cells,and the optimal induction time was 6 h.In this study,the result of RT-q PCR and Western blot showed that TWCWE,ML ES and Tsp43 proteins stimulated the up regulation of PI3 K,AKT and NF-κB expression(P<0.05),the phosphorylation levels of AKT and NF-κB increased significantly(P<0.05),and the results of IF showed that compared with the control group,the fluorescence intensity of AKT in Tsp43 protein stimulation group increased with the increase of stimulation concentration.Western blot showed that ATA inhibited the up regulation of PI3 K,AKT and NF-κB and the up regulation of AKT and NF-κB phosphorylation levels in C2C12 cells stimulated by TWCWE and ML ES(P<0.01).Tsp43 protein activity was inhibited by ATA.Western Blot results showed that ATA inhibited the upregulation of PI3 K,AKT and NF-κB and the phosphorylation of AKT and NF-κB in C2C12 cells by TWCWE and ML ES(P<0.01).In this study,RT-q PCR and Western blot were used to evaluate the expression changes of myoblast regulatory factors(Myo D,Myf5),paired nuclear transcription factor 7(Pax7)and myosin heavy chain(MYH)in C2C12 cells stimulated by TWCWE,ML ES and Tsp43 proteins.The results showed that TWCWE,ML ES and Tsp43 proteins significantly inhibited the expression of Myo D,Myf5,Pax7 and MYH genes and proteins(P<0.05).Western blot showed that ATA inhibited the down-regulation of Myo D,Myf5,Pax7 and MYH in C2C12 cells stimulated by TWCWE and ML ES(P<0.01).In this study,the effects of TWCWE,MLES and Tsp43 proteins on cyclin dependent kinase inhibitor(p21)and cell cycle promoter(Cyclin D1)were detected by RT-q PCR and Western blot.The results showed that the above three antigens up-regulated the expression of Cyclin D1(P<0.01)and down regulated the expression of p21(P<0.05).In order to further explore the effect of Tsp43 protein on cell cycle,the cell cycle of C2C12 cells stimulated by different concentrations of Tsp43 protein for 6 hours was detected by flow cytometry.The results showed that the stimulation of different concentrations of Tsp43 protein caused the increase of G1 and G2 phase cells and the decrease of S phase cells in a concentration dependent manner,indicating that tsp43 protein caused the G2/M phase arrest of C2C12 cells.Pretreatment of C2C12 cells with NF-κB inhibitor Urolithin B was used to detect the changes in the expression of cell cycle related factors.The results of Western blot showed that after Urolithin B treated C2C12 cells,the up-regulated expression of Cyclin D1 protein and the down-regulated expression of p21 protein were inhibited,indicating the inhibition of PI3K/AKT/NF-κB signaling pathway can interfere with the effect of Tsp43 protein on cell cycle related factors.In conclusion,this study shows that TWCWE,ML ES and Tsp43 proteins can interfere with myoblast cycle and block G2/M phase by activating PI3K/AKT/NF-κB pathway in myoblasts,so as to regulate the proliferation and differentiation process of myoblasts and provide favorable conditions for the formation of nanny cells.Tsp43 protein plays a major role in regulating the whole process of myoblast proliferation and differentiation,and is likely to participate in NC formation.It can be used as a main candidate protein to study the mechanism of NC formation.From the perspective of the interference of T.spiralis muscle larvae with the proliferation and differentiation of myoblasts,this study expounds the reconstruction of myocytes after T.spiralis infection,so as to provide theoretical basis and basic data for the further study of the molecular mechanism of NC formation in the future.