Sanguis Draxonis Compound Facilitates Steroid-induced Osteonecrosis Renovation Through Balancing The System Of Osteoblast And Osteoclast

Objective(1)To elucidate the pathological features and renovation mechanisms of steroid-induced osteonecrosis(SION)through the equilibrium system of bone remodeling maintained by osteoblasts and osteoclasts.(2)To screen some Sanguis Draxonis compounds that can enhance the mineralization activity of osteoblasts or attenuate the bone resorbing activity of osteoclasts.(3)To detect the effect of selected compound(s)on osteoblast and osteoclast activities,and elucidate the molecular mechanisms of promoting SION renovation through measuring the gene and protein expression in osteoblast and osteoclast-related signaling pathways.(4)To observe the effect of selected compound(s)on SION rats through determining the gene and protein expression in osteoblast and osteoclast-related signaling pathways.MethodsIn Chapter One,we included three human femur heads with steroid-induced osteonecrosis(SIONFH)at pericollapse period,observed the specific pathological manifestations among different lesions of femur heads via radiological images and pathological staining,then measured the transcription and expression levels of osteoblastic and osteoclastic genes.We clarified the regeneration mechanisms through elucidating the pathological features of SION related to osteoblast and osteoclast activities.In Chapter Two,we isolated and purified primary osteoblasts and osteoclasts,then treated them with Sanguis Draxonis extractions of same concentration during the differentiation processes respectively in vitro,to select some compounds which can enhance the mineralization function of osteoblasts using alkaline phosphatase(ALP)staining and alizarin red staining,or block bone resorbing function of osteoclasts using tartrate-resistant acid phosphatase(TRAcP)staining,actin ring staining and bone resorption assays.Then we measured the osteoblastic and osteoclastic gene and protein expression in primary cells simulated with selected compound of optimizing concentration through fluorescent staining,immunofluorescence staining,western blot,RT-qPCR and other techniques.That is to say,we confirmed the action mechanisms of selected compound on osteoblast and osteoclast differentiation.In Chapter Three,we used selected compound to intervene SION rats established with imiquimod and methylprednisolone,then comparatively analyze the regeneration situation of rat femur heads using bone histomorphometry techniques such as H&E staining and micro-CT scanning.We also measured the gene and protein expression in famous signaling pathways related to osteoblast and osteoclast differentiation,using fluorescent imaging,Western blot and RT-qPCR.That is to say,we described the regulation mechanism of selected compound on promoting the renovation of femur head necrosis in SION rats.Results(1)The gross and X-ray manifestations of healthy area,necrotic area and sclerotic area in SIONFH human were consistent with those under the microscopy.The bone mass of necrotic bone was reduced dramatically,and the expression of osteoclastic proteins,such as RANKL,NFATcl and CTSK was upregulated,and the expression of osteoblastic protein(Runx2)was downregulated.Conversely,the above proteins associated with osteoclastogenesis were decreased in sclerotic bone,Runx2 protein was overexpressed.(2)The six chalcone compounds extracted from Sanguis Draxonis showed little effect on osteoblast mineralization,but inhibited RANKL-induced osteoclast differentiation to some extent,and Loureirin B(LrB)exhibited the strongest inhibitory effect among them.Furthermore,during the process of inhibiting osteoclast formation and function,LrB can not only repress NFATcl activity through p38/MAPK,JNK/MAPK and calcium signaling pathways,but also attenuate ROS production via Keapl/Nrf2/ARE signaling pathway.(3)LrB can remarkly reduce ROS activity and osteoclast number in femur heads of SION rats,downregulate the expression of CTSK protein relating to osteoclast function and upregulate the expression of TNFRSF11IB gene relating to osteoblast function,resulting in increasing the trabecular bone mass.Through the above biological function,LrB facilitated the necrotic bone renovation in SION rats.ConclusionsSanguis Draxonis extractions,especially Loureirin B,can facilitate steroid-induced osteonecrosis renovation through balancing the bone remodeling system maintained by osteoblast and osteoclast.