The Role Of Proanthocyanidins In The Regeneration Of Ischemic Muscle Of Mouse Hind Limb And The Expression Change And Mechanism Of MiR-133b

objective:The myogenic differentiation of myosatellite cells?SCs?is involved in skeletal muscle regeneration after limb ischemia.Oxidative stress after limb ischemia is a major factor in skeletal muscle damage and repair process.Proanthocyanidins?PC?have anti-oxidative stress effects and protect muscle against ischemia injury.However,the protection mechanisms of PC are not yet clear.MicroRNAs?miRNAs?are small non-coding RNAs that bind in targetmRNAfordown-regulatingtheproteintranslation.Myo-microRNA-133b is closely related to muscle regeneration after ischemia injury,but the function of miR-133b in the relationship between PC and muscle damage is unknown.Hind limb ischemia?HLI?mouse model was established to investigate the role of miR-133b in the impact of proanthocyanidins?PC?on ischemic muscle.Part one,methods:The experiment is set in two parts.The first part compares different HLI models:male C57BL/6 mice?n=32?were divided into four groups by randomization.Following with protocol,the listed left femoral artery points were ligated:proximal and distal end of femoral artery?model-1?,distal end of femoral artery?model-2?,femoral artery between the superficial circumflex iliac artery and profunda femoral artery?model-3?,and both ligation points of model-2 and model-3 for a new method?model-4?.Laser Doppler Imager measured the ischemic/nonischemic perfusion ratio of hind limbs,and the functional movement score was used for motor recovery.Hematoxylin-eosin?H&E?staining was used to count central nuclei myofibers in the entire cross-sectional areas for muscle regeneration,and immunofluorescence?CD31⁺signals?assessed the angiogenesis.Results:For the first part of the experiment,the perfusion ratios of these four methods immediately decremented more than 75%,and had no differences among the methods?P<0.05?.Blood flow restorations of model-2 and model-3 were faster than model-1?P<0.05?,but no differences were reported between model-4 and model-1.Similarly,muscle regeneration in model-2 and model-3 showed limited singles to model-1?P<0.05?,as well as angiogenesis in model-2?P<0.01?,but model-4 resembled results found in model-1.Conclusion:The method of model-4 gets similar results to model-1 with simply operation.Part two,methods:The model-1 method was selected as the optimal HLI mouse model for the second part?the effects of PC on ischemic limb?:thirty-six male C57/BL6 mice followed the model-1 method to establish HLI models.The model mice were then divided into 3 groups:control group?H₂O?,low dose?1 mg/kg?PC treatment group,and high dose?20 mg/kg?PC group.Functionalmovement scores were recorded for each mouse,and mice were sacrificed at 7 days post-surgery to collect plasma and ischemic gastrocnemius muscle.The malondialdehyde?MDA?was measured by thiobarbituric acid reactive substance?TBARS?assay.The percentages of regeneration myofibers were counted by stained H&E.The ischemic gastrocnemius muscle of control group and high dose PC group at 7 days post-surgery were used for miRNA microarray analysis.RT-qPCR evaluated the expression level of miR-133b-3p,and then predicted the target genes of miR-133b-3p by bioinformatic analysis and processed GO,KEGG,and STRING analysis.Western Blot was used to evaluate Pax7,MyoD,MKP1,P38-MAPK,and Phospho-P38-MAPK expression?GAPDH as a reference protein?.Results:The recovery of functional movement in the high dose PC group was faster than the other two groups?P<0.05?,though no differences were found between the control and low dose group.In the high dose group,the MDA levels of plasma and muscles were significantly decreased compared to the other two groups at 7 days?P<0.05?,but there was no significant difference among the three groups at 21days.The percentage of regenerated myofibers in the high dose group was significantly higher than the control and low dose group at 7 days and 21 days.Using the mi RNAs microarray analysis,eight differentially expressed genes were found.The expression miR-133b-3p was up-regulated.By bioinformatics analysis,it was found that PPP2CA,PPP2CB,and MKP1 might be target genes of miR-133b-3p.Then the expression of protein showed that Pax7 and MKP1were decreased,but p-P38-MAPK,and MyoD were increased after 7 days treatment of high dose PC.Conclusion:PC could reduce the oxidative stress and promote the myofibers regeneration.The MKP1/P38-MAPK signaling pathway may be involved in this progress by up-regulate the miR-133b-3p expression.