The Effect Of Saussurea Involucrata Cell Culture On Anti-osteoporosis In Vitro

Osteoporosis, considered as a global public health issue, is caused by the imbalance between bone formation regulated by osteoblasts and bone resorption conducted by osteoclasts. Saussurea involucrata (S.involucrata) is a rare and precious traditional medicinal herb in China, which can enhance bone formation, anti-inflammation, and the blood circulation etc. S.involucrata cell culture is a kind of callus clumps or cells, which obtained by modern biotechnology. To date, pharmacology and efficacy study of S.involucrata cell culture is a hot spot in current research of S.involucrata. The present thesis evaluated the influence of S.involucrata cell culture on osteoblast proliferation, development and its mechanism by using human osteoblast MG-63 and on the effects of the osteoclast formation and the mature osteoclast by using mouse macrophage RAW264.7, osteoclast progenitor cells.RAW264.7 cells were induced by receptor activator of NF-κB ligand (RANKL) to differentiate into osteoclasts. By addition of S.involucrata cell culture, obvious inhibitions of multinucleated osteoclast formation and dose-depending decreasing of TRAP activity were detected. Lactate dehydrogenase (LDH) and flow cytometry assays of mature osteoclast with S.involucrata cell culture indicated that S.involucrata cell culture destroyed the cell membrane of osteoclast, significantly improved the extracellular LDH activity and induced apoptosis. The results of RT-PCR further illustrated that S. involucrata cell culture potentially decreased the ability of bone resorption by down-regulating TRAP and matrix metalloproteinase-9 (MMP-9) mRNA expression.On using human osteoblast MG-63 cells, MTT results showed that S.involucrata cell culture was non-toxic and promoted its proliferation at different concentrations of 31.25 μg/mL and 62.5μg/mL, but toxic at 125μg/mL. By addition of S.involucrata cell culture, increased alkaline phosphatase activity (ALP) improved the early differentiation of MG-63. In addition,4-hydroxyproline content in collagens and the amount of mineralized nodules were also increased by addition of S.involucrata cell culture, indicating an advanced mineralization of MG-63. Moreover, higher ratio of osteoprotegerin(OPG)/RANKL induced an excessive bone formation. The p38 inhibitor SB203580 inhibited the acceleration effect of S.involucrata cell culture on proliferation, differentiation, mineralization and OPG/RANKL expression of MG-63. These results proved that S.involucrata cell culture facilitated the regulation of p38 MAPK signaling pathway to promote osteoblastic bone formation activity.In conclusion, S.involucrata cell culture can inhibit osteoclast differentiation, induce mature osteoclast apoptosis and decrease the activity of bone resorption. On the other hand, S.involucrata cell culture could promote osteoblast proliferation, differentiation and mineralization. Altogether, S.involucrata cell culture has potential anti-osteoporosis effect at the cell level.