ObjectiveTo investigate the protective effect of Linggui Zhugantang （LGZGT）-medicated serum against H₂O₂-induced injury in H9c2 cells and its relationship with the phosphatidylinositol 3- kinase/protein kinase B （PI3K/Akt） signaling pathway. MethodThe LGZGT-medicated serum and blank serum were prepared based on serum pharmacology. H9c2 cells were cultured in vitro and divided into a normal group， an H₂O₂ group， a 20% blank serum group， and a 20% LGZGT-medicated serum group. The cells were treated with corresponding drugs for 12 h and cultured with 100 μmol·L^-1 H₂O₂ for another 6 h. The effect of 20% LGZGT-medicated serum on the proliferation activity of H9c2 cells induced by H₂O₂ was detected by cell counting kit-8 （CCK-8） assay. Mitochondrial reactive oxygen species （ROS） level was detected by the fluorescence probe. The levels of malondialdehyde （MDA）， lactate dehydrogenase （LDH）， catalase （CAT）， and glutathione peroxidase （GSH-Px） were detected by colorimetry. Western blot was used to detect the protein expression levels of phosphoinositide 3-kinase （PI3K）， phosphorylated-PI3K （p-PI3K）， protein kinase B （Akt）， and phosphorylated-Akt （p-Akt）. Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） was used to detect mRNA expression of PI3K and Akt. Flow cytometry was used to detect the apoptosis rate. After the addition of PI3K inhibitor LY294002， the levels of mitochondrial ROS， LDH， and GSH-Px， protein expression of PI3K， p-PI3K， Akt， and p-Akt， and cell apoptosis rate were detected. ResultCompared with the normal group， the H₂O₂ group showed blunted cell viability （P<0.01）， increased levels of mitochondrial ROS， MDA， and LDH （P<0.01）， decreased levels of CAT and GSH-Px （P<0.01）， reduced phosphorylation and mRNA expression of PI3K and Akt （P<0.05， P<0.01）， and increased apoptosis rate （P<0.01）. Compared with the H₂O₂ group， the 20% LGZGT-medicated serum group showed potentiated cell viability， reduced levels of mitochondrial ROS， MDA， and LDH （P<0.01）， increased levels of CAT and GSH-Px （P<0.01）， up-regulated phosphorylation and mRNA expression of PI3K and Akt （P<0.05， P<0.01）， and decreased apoptosis rate （P<0.01）. The combined use of LGZGT-medicated serum and inhibitor LY294002 reversed the above-mentioned effects of LGZGT-medicated serum on H9c2 cells （P<0.05， P<0.01）. ConclusionThe protective effect of LGZGT-medicated serum on H₂O₂-induced H9c2 cell injury may be related to the regulation of the PI3K/Akt signaling pathway to reduce oxidative stress and apoptosis.