Investigation of the molecular mechanisms of Fructus Ligustri Lucidi on calcium balance and vitamin D metabolism

PolyU Library Call No.: [THS] LG51 .H577P ABCT 2017 Caoxv, 156 pages :color illustrationsOsteoporosis affects over 200 million people worldwide and results in 687,000 reported hip fractures each year in China. Current drug therapies for osteoporosis have been linked to severe adverse effects while reducing fracture risks. Fructus Ligustri Lucidi (FLL) is a kidney-tonifying traditional Chinese medicine. The osteoprotective effect of FLL has been demonstrated by our laboratory, which makes FLL a potential candidate for prevention and treatment of osteoporosis. The current study is aiming at characterizing the efficacy of FLL and its active components on bone protection through regulating calcium balance and vitamin D metabolism in vivo and the underlying molecular mechanisms involved in modulating vitamin D metabolic enzymes in vitro. Oleanolic acid (OA) and ursolic acid (UA) are the major chemical constituents in FLL. The first part of the study was designed to investigate if OA is the active compound in FLL accounting for its osteoprotective effects in vivo using mature ovariectomized mice. After oral administration of OA for six weeks, OA at both doses significantly improved OVX-induced bone deterioration at tibia, femur, and lumbar vertebra L4 in OVX mice. OA treatments increased bone calcium but did not alter the circulating levels of bone turnover markers. Treatments of OA also significantly reduced urinary calcium loss and induced serum 1,25(OH)₂D₃ level through increasing renal CYP27B1 mRNA expression in OVX mice. The results suggested that the osteoprotective effect of OA might act through modulation of calcium-vitamin D axis in mature OVX mice. Previous study demonstrated the bone protective effect of FLL through modulation of calcium and 1,25(OH)₂D₃ in aged rats and such actions are independent of estrogen status in female rats. The second part of the study was aiming at identifying if OA and UA are the active ingredients of FLL using aged female rats. The micro-CT results showed that FLL, OA, and OA+UA groups increased bone mineral density and bone micro-architecture parameters at tibia, femur, and lumbar vertebra L2 after drug treatment for 12 weeks. Both OA and OA+UA group enhanced net calcium balance by suppressing urinary calcium loss. OA and OA+UA treatments were also found to significantly suppress renal CYP24A1 mRNA and protein expressions while FLL and OA+UA showed induction of renal CYP27B1 protein expression in aged female rats. The results indicated that OA and UA are active constituents of FLL that exerts bone protective effects. The bone protective actions might be associated with regulating calcium balance and vitamin D metabolism in aged female rats.In the final part of the study, the direct effects of FLL, OA, UA, OA+UA on CYP27B1 and CYP24A1 expressions in HKC-8 cells were characterized. The result showed that FLL and OA+UA significantly induced CYP27B1 mRNA and protein expressions through inducing CYP27B1 promoter activity, but did not alter CYP24A1 expressions in HKC-8 cells. OA appeared to suppress CYP24A1 protein expression while UA significantly induced CYP27B1 protein expression level in HKC-8 cells. However, both OA and UA unexpectedly induced CYP24A1 mRNA expression through activating CYP24A1 promoter activity. The present study indicated that OA and UA are the active compounds in FLL that play a role in modulating the regulation of renal CYP27B1 and CYP24A1 expressions in vitro. Furthermore, HKC-8 could serve as an in vitro screening platform for identifying the potential active constituents that modulate renal vitamin D metabolism. In summary, OA and UA are found to be the active constituents in FLL that exert bone protective effects. Apart from their reported direct actions on bone, the bone protective effects are shown to be associated with their actions in enhancing calcium balance and regulating vitamin D metabolism in vivo in mature OVX mice and aged female rats. OA and UA appeared to regulate vitamin D via modulation of CYP27B1 and CYP24A1 expressions at both transcriptional and translational levels in vitro in HKC-8 cells. The studies presented in this thesis provide strong pre-clinical evidence for the use of FLL as the potential botanical therapies for osteoporosis.Department of Applied Biology and Chemical TechnologyPh.D., Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, 2017Doctorat