Effect Of Proanthocyanidins On Proliferation And Differentiation Of Skeletal Muscle Satellite Cells Under Hypoxic-ischemic Condition

Objective: Limb ischemic necrosis is mainly attributed to peripheral arterial disease(PAD).Although the blood supply of the limb can be recovered by standard surgical treatment,tissue damage remains exacerbated by ischemia-reperfusion.In response to this damage,skeletal muscle satellite cells(SKMSCs),a population of stem cells,are activated and recruited to participate in muscle repair and regeneration.Reducing oxidative stress and promoting damaged skeletal muscle regeneration may be beneficial for the treatment of ischemic limbs.Proanthocyanidins(PC)have been shown to scavenge oxygen-free radicals and exhibit antioxidant effects to protect various organs during ischemia or ischemia-reperfusion injury.However,the role of PC and its underlying mechanisms still remain unclear.In this study,the effects of PC on ischemia injury muscle regeneration,SKMSCs proliferation and differentiation were investigated,the myogenic differentiation-associated molecules and the underlying mechanisms were further explored.Our study may offer a novel therapeutic approach for limb ischemic injury.Methods: Cell experiment : Hypoxic-ischemic condition(1% FBS,1%O2)was used to construct the cell model.SKMSCs were treated with saline or PC(2.5 or 5 μg/ml),and were observed at different time points.Cells proliferation were detected by cell counting kit-8 method at days 0,1,2,3 and4.Cell cycle was detected by flow cytometry at day 1.Total proteins were extracted and the expression of proliferation-related proteins,myogenic proteins and pathway-related proteins were detected by Western blot at days 1,3 and 5.Myotubes formation were observed using laser scanning confocal microscope at day 3.To further clarify the role of the P38-MAPK signaling pathway,After P38-MAPK signaling pathway was inhibited by the p38-MAPK pathway inhibitor SB203580,the expressions of myogenic proteins and pathway-related proteins were detected.Animal experiment: The mice were Double ligation of the proximal and distal end of femoral artery to construct hind limb ischemia modeling.Then,randomly treated with saline(n =12)or 20mg/kg PC(n =12)via intraperitoneal injection.Gastrocnemius was taken from the mice at day 7 after intervention,and the regenerating fibres was observed by hematoxylin-eosin staining.Results: Under hypoxic-ischemic condition for 1,2,3 and 4 days,the proliferation ability of the PC groups were significantly higher than that of the control group(P<0.05);Under hypoxic-ischemic condition for 1 day,PCNA expression in the PC groups were significantly higher than that in the control group(P<0.05);Under hypoxic-ischemic condition for 1 day,cells number in the s phase of the PC groups were significantly higher than that in the control group(P<0.05);Under hypoxic-ischemic condition for 3 or 5 days,the expression of myogenic proteins in the PC groups were significantly higher than that in the control group(P<0.05);Under hypoxic-ischemic condition for 3days,the formation of myotubes in the PC groups were significantly higher than that in the control group(P<0.05);Under hypoxic-ischemic condition for 3days,the expression of p-P38-MAPK in the PC groups were significantly higher than that in the control group(P<0.05),There was no significant difference between saline+p38-MAPK pathway inhibitor group and PC+p38-MAPK pathway inhibitor group(P>0.05);After lower extremity ischemia for 7 days,PC treated mice contained a higher number of regenerating fibres than that in the control group(P<0.05).Conclusion: 1.PC promotes proliferation of SKMSCs under hypoxic-ischemic condition;2.PC induces expression of myogenic proteins and myotubes by activating p38-MAPK signaling pathway,so as to promote ischemic damaged skeletal muscle regeneration.