银耳多糖对氧糖剥夺/复氧损伤心肌细胞的保护作用及机制研究OA

Objective To investigate the protective effect and molecular mechanism of Tremella fucifor-mis polysaccharide(TFP)on cardiomyocytes injured by oxygen-glucose deprivation/reoxygenation(OGD/R).Methods The rat cardiomyocyte cell line H9c2 was divided into control group(NC group),TFP treat-ment group(TFP group),OGD/R injury group(OGD/R group)and TFP combined with OGD/R treatment group(TFP+OGD/R group).Cell counting kit-8(CCK-8)and 5-ethynyl-2'-deoxyuridine(EdU)were used to detect the proliferation ability of cardiomyocytes;Western blot was used to detect the expression levels of apoptosis-related proteins including Bcl-2-associated X protein(Bax),B-cell leukemia/lymphoma 2 protein(Bcl-2),cleaved-poly(ADP-ribose)polymerase 1(cleaved-PARP-1),cleaved-cysteinyl aspartate-specific prote-ase 3(cleaved-caspase-3),autophagy-related proteins including microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅰ)/LC3-Ⅱ,ubiquitin-binding protein p62,and AMPK/mTOR pathway proteins including p-aden-osine monophosphate activated protein kinase(p-AMPK),adenosine monophosphate activated protein kinase(AMPK),p-mechanistic target of rapamycin(p-mTOR),and mechanistic target of rapamycin(mTOR);flow cytometry was used to detect cell apoptosis,and immunofluorescence was performed to observe the formation of autophagosomes and autolysosomes.To further explore the regulatory mechanism of TFP on cell apoptosis and autophagy,an autophagy activator rapamycin(RaPa)treatment group and an AMPK activator 5-amino-imidazole-4-carboxamide ribonucleotide(AICAR)treatment group were additionally added,which were co-treated with TFP+OGD/R respectively,and the above indicators were detected.Results Treatment with 100 μg/mL TFP alone had no significant effect on apoptosis and autophagy in normal cardiomyocytes,and only showed a very slight statistically different effect on cell viability,and had no biological toxicity.The apoptosis rate of cardiomyocytes in the OGD/R group was higher than that in the NC group(P<0.05);while the apop-tosis rate in the TFP+OGD/R group was decreased compared with that in the OGD/R group(P<0.05).Compared with the NC group,the expression level of anti-apoptotic proteins in the OGD/R group was down-regulated,and the expression levels of pro-apoptotic proteins and autophagy-related proteins were up-regulated(P<0.05);compared with the OGD/R group,the expression level of anti-apoptotic proteins in the TFP+OGD/R group was up-regulated,and the expression levels of pro-apoptotic proteins and autophagy-related proteins were down-regulated(P<0.05);compared with the TFP+OGD/R group,the expression level of an-ti-apoptotic proteins in the TFP+OGD/R+RaPa group was down-regulated,and the expression levels of pro-apoptotic proteins and autophagy-related proteins were up-regulated(P<0.05).Compared with the NC group,the expression level of p-AMPK in the OGD/R group was up-regulated,while the expression level of p-mTOR was down-regulated(P<0.05);compared with the OGD/R group,the expression level of p-AMPK in the TFP+OGD/R group was down-regulated(P<0.05),and the expression level of p-mTOR was up-regula-ted(P<0.05);compared with the TFP+OGD/R group,the expression level of p-AMPK in the TFP+OGD/R+AICAR group was up-regulated,and the expression level of p-mTOR was down-regulated(P<0.05).The expression levels of cleaved-caspase-3 and LC3-Ⅱ in both the TFP+OGD/R group and the TFP+OGD/R+AICAR group were down-regulated compared with those in the OGD/R group(P<0.05);the ex-pression levels of cleaved-caspase-3 and LC3-Ⅱ in the TFP+OGD/R+AICAR group were up-regulated com-pared with those in the TFP+OGD/R group(P<0.05).Conclusion TFP can,by regulating the AMPK/mTOR pathway,inhibit excessive autophagy and apoptosis,thereby alleviating OGD/R-induced cardiomyocyte injury.