Molecular Mechanism Of Betulinic Acid Inhibits Oxidative Damage Of Lymphocytes In Mice Base On Mitochondrial Signaling Pathway

Background:The modern animal husbandry has been highly intensified in recent years which leads t o more incidences of oxidative stress in the animals. The oxidative stress not only causes damage to the animal itself, but also causes huge economical loss to the animal farming industry. Thus, there is an increasing interest in researching and developing of novel antioxidants for its application in the animal farming industry. Betulinic acid (BA), a pentacyclic lupane-type triterpene, exists widely in food, fruits, medicinal herbs and plants. BA exhibits a wide variety of biological and pharmacological activities including antioxidant, immunomodulation, anti-cancer and anti-inflammatory. Our previous research found that BA can promote lymphocyte proliferation, activate peritoneal macrophages, enhance antioxidant capacity, and improve the body's immunity. Thus, the objectives of the present study are to test the hypothesis that Dex induced lymphocytes apoptosis via oxidative stress could be ameliorated by BA and to understand its mechanism of action.Objective:1) To investigate the corelation between the protective effect of betulinic acid on lymphocyte and its anti-oxidative stress property,2) to elucidate the molecular mechanism of betulinic acid on lymphocyte against oxidative stress,3) to provide a scientific support for fur ther research and development of betulinic acid and its applications.Methods:A total of 50 male Kunming mice were randomly divided into five groups:the control group, the Dex group, the low, medium and high dose of BA with Dex group (BA at the dose of 0.25,0.5, and 1 mg/kg body weight, respectively). BA was administered orally to mice with 1% starch jelly daily for 14 days, while mice in control and Dex groups were given an equivalent amount of 1% starch jelly only. Mice were given a single dose of Dex intraperitoneal (25mg/kg body weight) 8 h after the last administration of BA, while the control received an equal volume of sterile saline. Fifteen hours after Dex administration, the mice were weighed and sacrificed by cervical dislocation. The lymphoid tissue and liver was quickly excised. The following parameters were determined using various method, include cell viability, reactive oxygen species (ROS) level, the activities of superoxide dismutase (SOD) and glutathione perioxidase (GSH-Px), and the content of malondialdehyde (MDA) in liver, thymus and spleen. Cytochrome C (Cyt C) release, mitochondrial membrane potential were measured. The mRNA expression of Bcl-2, Bax, Caspase-9 and Caspase-3 were detected by RT-PCR, and the protein expression of Bcl-2, Bax, Caspase-9 and Caspase-3 were detected by Western blot.Results:BA pretreatment enhanced splenocyte and thymocyte viability, increase the activities of SOD, GSH and GSH-Px, reduce the levels of MDA and ROS. Moreover, BA's ROS scavenging activity disrupted the mitochondrial apoptotic pathway by restoring mitochondrial function, reducing the level of Cyt C, decreasing the expression of pro-apoptotic protein Bax, preventing the decline of anti-apoptotic protein Bcl-2, inhibiting caspase-9 and caspase-3 activation, and improving cell survival.Conclusion:These findings reveal a potential protective capability of BA against Dex -induced oxidative stress via mitochondrial mediated signal pathway.