Therapeutic Effect And Mechanism Of Urolithin A On Duchenne Muscular Dystrophy Model Mice

Research background:Duchenne muscular dystrophy(DMD)is an X-chromosome recessive disorder that mainly occurs in boys.We believe that impaired autophagy is one of the pathogenesis of DMD.When autophagy is impaired,swelling and damaged mitochondria,protein aggregation,and sarcoplasmic reticulum expansion are often observed,which are the cytopathological characteristics of DMD.In addition,Akt(serine/threonine kinase)activation was significantly higher in the muscles of mdx mice and primary myotubes from muscular dystrophy,indicating a defect in the autophagy process.Consistent with these observations,DMD patients also exhibit a similar Akt activation pattern.3,8-dihydroxy-6H-dibenzo[B,D]pyran6-ketone,also known as urolithin A(UA),has been reported to help slow down specific aging processes and stimulate mitochondrial biogenesis by improving the function of cell mitochondria.It is a compound that can rebuild the recycling of defective mitochondria in fine cells.In addition,ingestion of this compound poses no risk to human health.Research purpose:The purpose of this study is to analyze the therapeutic effects,mechanisms,and targets of UA on DMD model mice,and explore the impact of disease progression on the therapeutic effects of urolithin A.Through a strategy combining modern analytical techniques and biological evaluation methods,the therapeutic effects of UA on DMD mice are systematically explained,providing reference for clinical treatment of DMD.Research methods:1.Study on the treatment of DMD with urolithin A via PI3K-AKT-mTOR pathwayFirstly,the effects of UA on the muscle environment of mdx mice were analyzed by evaluating the muscle capacity of the DMD disease model mice before and after treatment with UA,detecting pathological sections such as H&E staining,analyzing muscle fibrosis with Masson trichrome staining,and analyzing the expression of CD68 positive inflammatory cells in muscle with immunohistochemical staining;Mitochondrial morphology was observed using electron microscopy,VADC(voltage dependent region channel)immunofluorescence staining and ELISA were used to determine mitochondrial respiration and citrate synthase activity.Mitotic phagocytosis(Pink-1,Pdr-1)and autophagy(Bec-1)gene mRNA expression were analyzed by q-RT PCR to analyze mitochondrial changes;Finally,through the expression of autophagy biomarkers such as LC3 Ⅱ,P62,Akt,and 4E-BP1 and the direct regulation of FoxO3,autophagy related genes(LC3β,mRNA expression of Atg12,Gabarapl1,and Bnip3)to analyze whether UA can activate autophagy in mdx mice and improve damage to downstream targets of FoxO3;Finally,clarify the mechanism of action of UA.2.Relationship between therapeutic effect of urolithin A on DMD mice and disease progressionThis study analyzed the relationship between the therapeutic effect of urothelin A on DMD mice and disease progression by comparing the muscle capacity assessment,pathological section identification,muscle fibrosis,expression of CD68 positive inflammatory cells in muscle,mitochondrial changes,and expression of autophagic biomarkers in mice of different months of age before and after treatment with UA.3.Research on the therapeutic effect of UA on DMD through the target site Park14In this study,after the Park14 gene knockout in mdx mice,UA compared the muscle capacity assessment,pathological section identification,muscle fibrosis,expression of CD68 positive inflammatory cells in muscle,mitochondrial changes,and expression of autophagic biomarkers in mdx mice after treatment,and analyzed the therapeutic targets of UA for DMD.Research results:1.Study on the treatment of DMD with urolithin A via PI3K-AKT-mTOR pathwayCompared with the model group,C57BL mice in the blank group had stronger grip(P;The mitochondrial respiratory oxygen consumption and citrate synthase activity of mdx mice in the model group were significantly decreased(P<0.05),while after treatment with UA,the mitochondrial respiratory oxygen consumption and citrate synthase activity of mdx mice were significantly improved(P<0.05),indicating that UA has the ability to improve the surface type of mitochondria;Compared to wild-type C57BL mice,the mdx model group mice showed lower Pink-1,Pdr-1,and Bec-1,as well as LC3 Ⅱ,and LC3β,Expression of Atg12,Gabarapl1,and Bnip(P<0.05)and higher phosphorylation of P62 protein and Akt,and 4EBP1.After UA treatment,Pink-1,Pdr-1,and Bec-1,and LC3 Ⅱ,LC3β、The expression of Atg12,Gabarapl1,Bnip,and P62 proteins and the phosphorylation of Akt and 4E-BP1 were significantly improved(P<0.05).2.Relationship between therapeutic effect of urolithin A on DMD mice and disease progressionAs age increased,the muscle strength of mdx mice decreased(P,This indicates that the younger the month age of mdx mice,the better the muscle capacity recovery after UA treatment;Compared with mdx mice,the expression of Pink-1,Pdr-1,and Bec-1 in the 1.5-8 month old UA treatment group was significantly improved(P<0.05),while the expression of Pink-1,Pdr-1,and Bec-1 in the 12 month old UA treatment group was not significantly improved(P>0.05).This indicates that the effects of UA on mitotic-phagocytosis activation and mitochondrial function improvement in mdx mice at different months of age are influenced by mouse month of age and disease progression;In the 1.5-8 month old UA treatment group,the expression of LC3 Ⅱ protein in mdx mice was restored,with a significant decrease in P62 protein,and a decrease in phosphorylation of Akt,4E-BP1(P<0.05).At the same time,LC3β,the expressions of Atg12,Gabarapl1,and Bnip3 were significantly increased(P<0.05).3.Research on the therapeutic effect of urolithin A on DMD through the target site Park14Compared with the model group,Park14-mdx mice treated with UA did not have a greater improvement in grip(P>0.05)and a longer running distance(P>0.05),indicating that when Park14 gene was knocked out,the improvement effect of UA administration on muscle ability of mdx mice disappeared;The mdx model group mice showed lower Pink-1,Pdr-1,and Bec-1 and LC3 Ⅱ,LC3β,expression of Atg12,Gabarapl1,and Bnip(P<0.05)and higher phosphorylation of P62 protein and Akt,4E-BP1 in Park 14-mdx mice treated with UA、The expression of Atg12,Gabarapl1,Bnip,and P62 proteins and the phosphorylation of Akt,4EBP1 were not significantly improved(P<0.05).Research conclusions:UA has the effect of improving muscle capacity and fibrosis in mdx mice,reducing inflammatory infiltration in muscle,improving mitochondrial function and mitotic phagocytosis,and activating autophagy in mdx mice.It can play a therapeutic role in DMD model mice through the PI3K-AKT-mTOR pathway,but its therapeutic effect weakens or even disappears with the growth of mouse months.The therapeutic target of UA in DMD model mice is related to the Park14 gene.