Abstract: OBJECTIVE: To investigate the efficacy of Sijunzi decoction( 四君子汤) on limb weakness in a rat model of spleen Qi deficiency(SQD), and to study its effect on mitophagy in skeletal muscle through adenosine monophosphate-activated protein kinase(AMPK)/unc-51 like autophagy activating kinase 1(ULK1) signaling.METHODS: SQD model rats were produced by fasting combined with forced swimming method for15 d. After model assessment, rats were randomly divided into four groups of 10 [low/middle/high(L/M/H) Sijunzi decoction dose groups and a normal saline(S) group]. Limb holding power(HP) and body mass(BM) were measured after 2 weeks of treatment. Following euthanasia, quadriceps femoris were dissected and myofiber and mitochondrial morphology were observed by transmission electron microscopy(TEM). Mitochondrial membrane potential(MMP), adenosine triphosphatase(ATP)and reactive oxygen species(ROS) levels were determined using colorimetric methods, and immunoblot analysis of Microtubule-associated protein light chain 3(LC3) and Sequestosome 1(p62) was performed to monitor mitophagy and AMPK/ULK1 signaling.RESULTS: Compared with control(C) group rats, in the S group, HP was reduced, the myofiber Z line was disordered, mitochondria were scattered, and numerous vacuoles and mitophagy were observed.MMP and ATP levels were reduced, ROS levels were elevated, and LC3B expression, and p-AMPKα(Thr172)/AMPKα, p-ULK1(Ser555)/ULK1, and p-Raptor(Ser792)/Raptor ratios were increased,while p62 expression and p-mT OR(Ser2448)/mT OR and p-ULK1(Ser757)/ULK1 ratios were decreased.After treatment, compared with the S group, HP was improved in M and H groups but not in the L group. Mitophagy was reduced in M, H and L groups but the Z line was disordered and vacuolization remained in the L group. ATP levels were elevated in M, H and L groups, and MMPs were elevated in M and H groups but not in the L group. ROS levels were decreased in M, H and L groups, as were LC3B expression and p-Raptor(Ser792)/Raptor ratios, while p62 expression and p-mT OR(Ser2448)/mT OR and p-ULK1(Ser757)/ULK1 ratios were increased in M and H groups but not in the L group.p-AMPKα(Thr172)/AMPKα and p-ULK1(Ser555)/ULK1 ratios were decreased in M, H and L groups.CONCLUSIONS: Sijunzi decoction improved HP,possibly by inhibiting mitophagy via suppression of AMPK/ULK1 signaling. This restored mitochondrial morphology and improved oxidative phosphorylation, which contributed to recovery of limb weakness in SQD model rats.

Key words: spleen-Qi deficiency, muscle,skeletal, mitochondria, oxidative phosphorylation, mitophagy, AMP-activated protein kinases, autophagy-related protein-1 homolog, Sijunzi decoction