Protection And Mechanisms Of β-Hydroxybutyrate On Stroke-Related Sarcopenia In Mice

BackgroundStroke is the single greatest cause of physical disability in the adult population.Research showed that the decrease in the number of motor units of hemiplegic limbs could be observed from 4 hours to 30 hours after stroke.The incidence of stroke-related sarcopenia was 14.0%-53.5%.Compared with stroke patients without sarcopenia,sarcopenia increased the risk of poor recovery after stroke by 2.71 times.At present,there is still a lack of specific treatment methods for stroke-related sarcopenia after nerve injury.Reasonable adjustment of nutrition structure,positive regulation of catabolic-anabolic and nutritional support is expected to reduce the incidence of strokerelated sarcopenia.Ketogenic diet(KD)is mainly composed of high fat/low carbohydrate,and has been proved to play an obvious protective role in many neurodegenerative diseases.However,it has certain limitations in clinical application because it needs to be strictly evaluated before use to eliminate contraindications and reduce the occurrence of adverse reactions.Among them,β-hydroxybutyrate(BHB),as the main active component of KD,has been proven in clinical trials to be safe in supplementing ketogenic preparations for patients with acute brain injury.In the mice sepsis model,BHB inhibited gene expression related to ubiquitin proteasome system(UPS),regulated the synthesis and proteolysis,then significantly improved the muscle weakness.Based on this point,the study intended to construct mice model of transient ischemia/reperfusion to explore the role and potential mechanism of BHB in improving stroke-related sarcopenia by regulating protein synthesis and proteolysis.ObjectiveTo explore whether BHB improved stroke-related sarcopenia by regulating protein synthesis and proteolysis,independent of its brain protective effect,and tried to reveal its possible mechanisms.MethodsThe temporary middle cerebral artery occlusion(tMCAO)model of adult male C57BL/6J mice was established.The infarct volume,muscle mass,and muscle fibers cross-sectional area(CSA)in the paretic and nonparetic sides of mice were measured for 3 days and 7 days after tMCAO.The mRNA expression of UPS related genesmuscle specific ring finger protein 1(MuRF1),muscle atrophy F-box(MAFbx),protein synthesis related genes--4E-binding protein 1(4EBP1),and S6 kinase(S6K)and muscle regeneration and proliferation related genes--myogenic differentiation 1(Myod1),myogenin(Myog)and proliferating cell nuclear antigen(Pcna)in the paretic and nonparetic gastrocnemius(GTN)muscle were detected to evaluate the effect of BHB on stroke-related sarcopenia.To evaluate whether the muscle protective effect of BHB directly acted on local muscle,further,we delayed BHB administration 10 hours or 24 hours after tMCAO for 3 days,7 days and 14 days.Under the condition that the infarct volume was basically stable,the above sarcopenia indexes were observed in both groups.In addition,the expression of Foxo3a and H3K9bhb were detected,and the enrichment of H3K9bhb in the Foxo3a promoter region was detected by ChIPqPCR.Results1.Immediate administration of BHB improved stroke-related sarcopenia and reduced infarct volumeWe found that BHB immediately administrated for 3 and 7 days,the infarct volume was significantly reduced,and the muscle mass and muscle fibers CSA of the paretic and nonparetic sides were increased,the expression of 4EBP1,S6K,Myodl,Myog and Pcna were up-regulated,while the expression of MuRF1 and MAFbx were down-regulated after tMCAO in mice.2.Delayed administration of BHB improved stroke-related sarcopenia independent of its brain protectionAt 10 hours after tMCAO,when the infarct volume was stable,BHB or Glucose was administrated for 3,7 and 14 days,which improved the muscle strength score and the above sarcopenia indexes of mice.The time window of BHB administration was further expanded.BHB delayed administration for 24 hours for 3 days,7 days and 14 days.The results showed that BHB still improved the above indicators of sarcopenia in mice.3.BHB may induce H3K9 β-hydroxybutylation to inhibit proteolysis mediated by Foxo3aBHB significantly inhibited mRNA and protein expression of Foxo3a at each time point in both paretic and nonparetic GTN muscles,and increased the total expression of H3K9bhb.At the same time,ChIP-qPCR confirmed the endogenous binding of H3K9bhb with Foxo3a promoter in response to the transcription inhibition of Foxo3a.Moreover,BHB enhanced the enrichment efficiency of H3K9bhb protein in Foxo3a gene promoter.ConclusionPathological changes occurred in skeletal muscle after ischemic stroke,that is,stroke-related sarcopenia.BHB inhibited the expression of Foxo3a by inducing H3K9bhb to enrich in the Foxo3a promoter region,and then decreased protein breakdown.At the same time,BHB promoted protein synthesis and regeneration and proliferation of muscle,thus alleviating stroke-related sarcopenia.