The Effect Of Lycopene On Osteoclasts' Bone Resorption Function Mediated By Reactive Oxygen Species(ROS)

Objective Osteoclasts have been shown to produce reactive oxygen species (ROS) thatcan stimulate bone resorption. In this investigation We explored whether lycopene, a kindof antioxidant, can inhibit bone resorption by inhibiting osteoclasts' ability to produceROS.Methods Osteoclasts isolated from the long bones of neonatal Wistar rats were cultured forfurther study. The cells were planted into 24-well plates(coverslips or bone flaps wereplaced in the wells already) and divided randomly into 5 groups(A, B, C, D and E), andcultured in a-minimal essential medium supplemented With varying doses of lycopene orvitamin C at the start of culture and at each medium change. The morphology of osteoclastswas observered with inverted microscope and the osteoclasts were counted every day. On day 2, 4and 6, some coverslips with cells were stained according to the kit' instruction to accesstartrate-resistant acid phosphatase (TRAP) activity. On day 6, the medium in some wellswas aspirated, and the cells on coverslips and bone flaps were respectively stained fornitroblue tetrazolium (NBT) reduction assays. On day 7, the bone flaps were processedfor observation under the scanning electron microscope. The resorption pits were analyzed by theimage-pro plus5.0, well-known image analyzing software.Results (1)The osteoclasts' adherence was observed after cultured for 30 minutes.Under the inverted microscope, there is no obvious difference in osteocalsts' morphologyamong the 5 groups. The difference of osteoclasts' amounts were not obvious eitheramong 5 groups at the same time or in any group at different time, P＞0.05. (2)In theTRAP+ multinucleated cells, the active area with tartrate-resistant acid phosphatase wasstained mauve, however the cell nucleuses stained negatively or stained lightly. The parapodium ofosteoclasts is distinct. The difference of TRAP+ multinucleated cells' amounts weere notobvious either among 5 groups at the same time or in any group at different time, P＞0.05.(3) Nitroblue tetrazolium can be reduced to purple-colored formazan by the ROSproduced by osteoclasts, so it can be used as an indicator for the level of ROS in the cells.The results showed the formazan-staining cells were decreased by treatment with10^-5mol/L lycopene, indicating that lycopene inhibited the production of ROS inosteoclasts. (4) The bone flaps were resorbed by osteoclasts and formed lots of pits withdifferent appearances. Lycopene at a concentration of 10^-7mol/L partially reduced the area of resorption pits, whereas 10^-5mol/L lycopene almost completely inhibited the resorptionfunction. The results from the analysis of image-pro plus5.0 showed that the mean area ofpits was different between B, C, D and A, P＜0.05; the difference among B, C and D wasalso obvious; however the difference between E and A was not obvious.Conclusion Lecopene can inhibit the resorption function of osteoclasts in vitro bydecreasing the production of reactive oxygen species, and it is not a kind of fatal drug forosteoclasts.