An ursolic acid-enriched cynomorii herba extract induces mitochondrial uncoupling : antioxidant and anti-obesity effects

Abstract

Earlier studies in our laboratory have demonstrated that Cynomorii Herba can increase mitochondrial adenosine triphosphate (ATP) generation capacity (ATP-GC), presumably by enhancing mitochondrial electron transport, in both H9c2 cells and rat hearts, which is characteristic of ‘Yang-invigoration’ in Chinese medicine. This thesis study aimed at (1) identifying the active ingredient(s) in Cynomorii Herba and (2) further investigating its biological activities and the underlying mechanism(s). Utilizing the ATP-GC assay as an activity monitor, an ursolic acid (UA) -enriched active fraction isolated from Cynomorii Herba ethanol extract, HCY2, was subjected to further investigation. Pre-incubation of HCY2 or UA was found to increase ATP-GC, which may be caused by the stimulation of mitochondrial electron transport secondary to increased fluidity of mitochondrial inner membrane. It was hypothesized that the increase in mitochondrial reactive oxygen species (ROS) production associated with the stimulation of mitochondrial electron transport chain could trigger cellular responses including mitochondrial uncoupling and glutathione reductase-catalyzed glutathione redox cycling, with resultant protection against oxidant injury. To test this hypothesis, the effect of HCY2 or UA on mitochondrial ROS production was examined. HCY2 or UA pre-incubation was found to increase mitochondrial ROS generation in H9c2 cells. The suppression of mitochondrial ROS by antioxidant (dimethylthiourea, DMTU) abrogated the HCY2 or UA-induced cellular responses and the associated cytoprotective effect. Studies using specific inhibitors of reduced glutathione regeneration and uncoupling proteins suggested that the HCY2/UA-induced mitochondrial uncoupling and glutathione redox cycling play a determinant role in cytoprotection against oxidant injury. Results obtained from animal studies indicated that long-term HCY2 or UA treatment invariably protected against oxidative tissue damage in the rat models of myocardial ischemia/reperfusion injury, carbon tetrachloride (CCl₄) hepatotoxicity and gentamicin nephrotoxicity. The tissue protection was associated with the improvement in mitochondrial functional capacity, as assessed by the measurement of ATP-GC, as well as the enhancement in glutathione redox status, with the possible involvement of mitochondrial uncoupling. As mitochondrial uncoupling has been considered as an attractive drug target for combating obesity, the anti-obesity effect of HCY2 was also examined. Co-treatment with HCY2 significantly prevented body weight gain and reduced white fat pads in the mouse model of high fat diet (HFD)-induced obesity. The weight reduction effect was associated with the enhancement in mitochondrial uncoupling in skeletal muscle tissue. HCY2 treatment was also found to ameliorate the HFD-induced hyperglycemia and dyslipidemia, suggestive of preventive effect on metabolic syndrome and the associated health consequences. The signaling pathway underlying HCY2/UA-induced weight reduction may be associated with the adenosine monophosphate-activated protein kinase (AMPK)/ peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) activation in skeletal muscle. The activation of AMPK/ PGC-1α further increased the mitochondrial content and uncoupling protein 3 (UCP3) expression, and thus regulated energy metabolism and mitochondrial biogenesis, eventually leading to weight loss in mice. In conclusion, HCY2, an UA-enriched fraction derived from Cynomorii Herba and its major component, UA, could increase mitochondrial functional capacity and up-regulate cellular/mitochondrial antioxidant status, thereby protecting against oxidant-induced cellular/tissue injury. In addition, HCY2 treatment was found to prevent HFD-induced obesity and the related metabolic syndrome. These beneficial effects produced by Cynomorii Herba are likely mediated by mitochondrial uncoupling. The long-term intake of Cynomorii Herba therefore offers a prospect for the prevention of aging and obesity.

Date of Award	2014
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology