Study Of Panax Notoginseng Extracts Promote The Repair Of Delayed Injury Of Skeletal Muscle By Phosphorylation Pathway Of AKT And ERK Mechanism And MR Imaging Features

Objective: Exercise-induces muscle damage(EIMD)is a very common phenomenon in sports.It often occurs after a large number of exercise unaccustomed trainings,especially after eccentric exercise.EIMD can be characterized by muscle stiffness,weakened strength,tenderness,and delayed onset muscle soreness(DOMS).There is currently a lack of effective treatment.Panax notoginseng is a Traditional Chinese Medicine(TCM)with a long history.It has various pharmacological activities such as antioxidant,anti-inflammatory,and estrogen-like biological activities.It is a commonly used TCM preparation for treating bruises,promoting blood circulation,and relieving inflammation.However,the effect of Panax notoginseng extract on the proliferation and migration of skeletal muscle cells and the efficacy of skeletal muscle injury is still unknown.In this study,both in vitro and in vivo experiments were carried out to treat rat skeletal muscle cells and EIMD models of SD rat with Panax notoginseng extract,and the non-invasive,in vivo and real time effects of EIMD model and efficacy of Panax notoginseng extract were studied by imaging MR DTI and IVIM.The purpose of this study was to evaluate the effects of Panax notoginseng extract on the proliferation and migration of skeletal muscle cells,as well as the effect for delayed injury of skeletal muscle,and the imaging parameters of MR evaluation of injury and repair of EIMD.Methods: 1.In vitro: rat skeletal muscle cells were cultured in basic medium or drug-containing medium(Panax notoginseng extract)with drug concentrations of 0,0.3125 mg/ml,0.625 mg/ml,1.25 mg/ml,and 2.5 mg/ml,respectively.Cell proliferation assay was performed using CCK-8,cell migration ability was measured by wound healing test,cell cycle was measured using a commercially available cell cycle kit,and inflammatory factors levels including IL-1β,IL-6,and TNF-α were measured by Immunohistochemical.The mechanism was further studied by adding AKT and ERK inhibitors to the culture medium.The secretion level of IGF-1 was measured by ELISA kit,immunohistochemistry,and Western Blot.The protein levels of myogenic factors including myod1 and myogenin,IGF-1R,AKT/p-AKT and ERK/p-ERK were analyzed by Western blot.2.In vivo: 40 SD rats were selected and divided into resting group(n=4),eccentric exercise+Panax notoginseng extract group(n=18),and eccentric exercise group(n=18).The rats were intragastrically administrated with the Panax notoginseng extract and and bilateral hindlimbs were performed by MR-DTI and IVIM scanning at 6 time points(Immediately,1 day,2 days,3 days,4 days,and 5 days)and then bilateral posterior quadriceps were collected.Muscles were taken to obtain the T2 value,ADC value,FA value,D value,D~* value and f value.The morphology of muscle fibers was observed by HE staining.The expression of inflammatory factors including IL-1β,IL-6 and TNF-α was detected by immunohistochemistry.Results: 1.The effects of extracts of Panax notoginseng on proliferation and migration of rat skeletal muscle cells and inhibition of inflammatory responses: the extract of Panax notoginseng at a concentration of 0.625 mg/ml can increase the expression levels of myogenic factor Myo D and Myogenin(p<0.05),and increase the number of S phase in skeletal muscle cells(p <0.05),significantly promote the proliferation and migration of skeletal muscle cells;at the same time,reduce the secretion levels of inflammatory factors including IL-1β,IL-6,TNF-α(p < 0.05),effectively suppresses excessive inflammatory responses and prevents inflammatory damage to skeletal muscle cells.2.The mechanism of Panax notoginseng extracts participating in the promotion of rat skeletal muscle cell proliferation and migration through AKT and ERK signaling pathways: Panax notoginseng extract at a concentration of 0.625 mg/ml can increase the phosphorylation levels of AKT and ERK in skeletal muscle cells(p<0.05)to activated AKT and ERK signaling pathways,and then,increased proliferation and activity of skeletal muscle cells,which could be inhibited by AKT and ERK inhibitors(p<0.05).3.Radiological evaluation of the effect of Panax notoginseng extract on the EIMD model of SD rat: Panax notoginseng extract can effectively inhibit the production of inflammatory cytokines including IL-β,IL-6 and TNF-α in skeletal muscle of EIMD model of SD rats(p<0.05),and in 2 day,the T2 value and FA value of skeletal muscle were significantly decreased(p<0.05),muscle edema and muscle fiber disorder and fracture at the injury site were relieved.At the same time,in 2 day,the D value and D~* value of the skeletal muscle in the experimental group were significantly higher than those in the control group(p<0.05),and the perfusion of the injured skeletal muscle was increased,but the f value did not change significantly(p>0.05).Conclusion: Panax notoginseng extracts can promote the proliferation and migration of skeletal muscle cells by activating AKT and ERK signaling pathways,inhibit the inflammatory reaction and accelerate the cell cycle.Through the above mechanism,Panax notoginseng extract can effectively relieve muscle fiber disruption,fracture and edema and promote blood perfusion in EIMD,and then Panax notoginseng extracts could accelerate the repair of damaged skeletal muscle,which is a potential drug to treatment the EIMD.