基于PI3K/Akt介导的糖酵解研究益气解毒方缓解支持细胞能量供应功能放射损伤机制OA

Objective To investigate the effects of ionizing radiation on Sertoli cell glycolytic energy supply function and the protective mechanism of Yiqi Jiedu Formula(YQJDF).Methods Sixty Sprague-Dawley rats were randomly assigned to the blank(n=24),Anduolin(n=12),YQJDF high-dose(n=12),and YQJDF low-dose(n=12)groups using the random number table method.The groups received intragastric administration of deionized water,Anduolin suspension(0.35 g/kg),YQJDF high-dose(10.44 g/kg),and YQJDF low-dose(5.22 g/kg)at 10 mL/kg once daily for 5 days,respectively.Prepared corresponding serum,and used high-performance liquid chromatography-mass spectrometry(HPLC-MS)to detect the drug components in the drug-containing serum.Subsequently,cellular experiments were performed.(1)Used the GC-2 spermatocytes to establish normal control group,model control group,and lactic acid intervention series(2,4,5,6,8,10,16,and 20 mmol/L).All groups were cultured in medium containing 10%blank serum.After pretreatment with the corresponding serum,all groups except the normal control group were irradiated with 12 Gy 60 Co γ-rays to establish a GC-2 radiation injury model.Cell viability was assessed using the CCK-8 assay at 24 h post-irradiation.(2)Used mouse testicular Sertoli cell line TM4 to establish normal control(10%blank serum),model control(10%blank serum),Anduolin-containing serum(10%Anduolin-containing serum),YQJDF high-dose-containing serum(10%YQJDF high-dose-containing serum),and YQJDF low-dose-containing serum groups(10%YQJDF low-dose-containing serum).After pretreatment with the corresponding serum,all groups except the normal control group were irradiated with 12 Gy 60 Co γ-rays,with analyses at 24 h post-irradiation.Used colorimetry to detect lactic acid content,lactate dehydrogenase(LDH)activity,pyruvate,phosphofructokinase(PFK)activity,and adenosine triphosphate(ATP)content.The glycolysis rate and maximum glycolysis capacity were measured using an Aligent Seahorse XF Pro XFe 96 Extracellular Flux Analyzer.Monocarboxylate transporter 4(MCT4)and glucose transporter 1(GLUT1)expression was determined using immunofluorescence.(3)Based on the experiment(2)group,set up an additional wortmannin group(10%blank serum).Used colorimetry to detect lactic acid content of TM4 cells.Phosphatidylinositol 3-kinase 85 kDa regulatory subunit alpha(PI3K p85α),protein kinase B(Akt),and phosphorylated Akt(p-Akt)expressions were detected using western blotting,with that of p-Akt further confirmed using immunofluorescence.Results Chlorogenic acid,albiflorin,and ginsenosides(Re,Rg1,Rg6)were detected as the main active components in the YQJDF-containing seurm.In the experiment(1),compared with the normal control group,cell viability was reduced in the model control group(P<0.01).Compared with the model control group,cell viability was increased in the 4,5,6,8,and 10 mmol/L lactic acid intervention groups(P<0.05,P<0.01);however,cell viability decreased in the 20 mmol/L lactic acid group(P<0.01).In the experiment(2),compared with the normal control group,the lactic acid content,LDH activity,MCT4 expression,glycolysis rate,GLUT1 expression,PFK activity,pyruvate content,and ATP content were all decreased in the model control group(P<0.05,P<0.01).Compared with the model control group,the lactic acid content,LDH activity,MCT4 expression,glycolysis rate,GLUT1 expression,PFK activity,pyruvate content,and ATP content were all increased in YQJDF high-dose-containing serum group(P<0.05,P<0.01).In the experiment(3),compared with the normal control group,model control group and wortmannin group showed reductions in lactic acid content and p-Akt expression,model control group showed reductions in PI3K p85α expression and p-Akt/Akt ratio(P<0.05,P<0.01).Compared with the model control group,YQJDF low-dose-containing serum group showed increased lactic acid content and p-Akt expression(P<0.01).Conclusion Ionizing radiation impairs the glycolytic capacity and lactate secretion of Sertoli cells by inhibiting the phosphoinositide 3-kinase(PI3K)/Akt signaling pathway and downregulating the expression and activity of key glycolytic enzymes/proteins,including GLUT1,PFK,LDH,and MCT4,resulting in an insufficient energy supply to spermatogenic cells.In contrast,YQJDF restores glucose uptake,glycolytic capacity,and energy supply in Sertoli cells by activating the PI3K/Akt pathway.