Effect Of Sulforaphane On Activity And Mineralization Of The Osteoblast Under Hypoxic Environment

1.Research BackgroundOsteoporosis is an important public health problem,especially primary osteoporosis,which has become one of the chronic diseases that seriously affects the health of middle-aged and elderly people,but the diagnosis and treatment is not ideal.Fracture is a serious consequence of osteoporosis,which significantly increases the disability and mortality of patients,and brings great family and socioeconomic burden.Effective anti-osteoporosis treatment can reduce the occurrence of fractures.Patients can also effectively avoid re-fracture,and identifying and actively treating osteoporosis has very important clinical and social significance.At present,osteoporosis drugs on the market regulate calcium absorption,inhibit the biological activity of osteoclasts,reduce the number of osteoclasts,reduce bone loss,and promote the formation of osteoblasts,but also exist as increased incidence of fractures,risk of tumors and thrombosis,etc.So we consider whether there is a suitable Chinese herbal medicine or plant for anti-osteoporosis treatment.There are many researches on the mechanism of osteoporosis,and oxidative stress is one of the more in-depth mechanisms.Sulforaphane(SFN)is derived from various cruciferous plants and has antioxidant,mitochondrial protection and anticancer effects.Antioxidant stress is the most prominent function.At present,there is little literature on the role of SFN in osteoblasts.We intend to understand the effects of SFN on osteoblasts under oxidative stress.Cruciferous vegetables are easily obtained and supplemented in our daily diet.It is hoped that its antioxidant function can make it as one of the daily health care and even herbal medicines to prevent osteoporosis.2.Materials and MethodsFirstly,hFOB1.19 was divided into normoxia groups(20%O2)and hypoxia groups(1%O2)for 24h,48h,72h,96h,120h.The proliferation,apoptosis and reactive oxygen species levels were observed.Secondly,we added Oumol/L,5umol/L,10umol/L,20umol/L SFN to cells for 24h and 48h under normoxic conditions,the effect of SFN on osteoblasts was observed.Finally,we divided the cells into 4 groups,namely,normoxia group,hypoxia 48h reoxygenation group,SFN retreatment for 24h,hypoxia-reoxygenation group,SFN retreatment for 48h,hypoxia-reoxygenation group and cultured to 21d,respectively,observed cells growth,antioxidant indicators and mineralization indicators at 7d,14d and 21d.3.ResultThe first experiment,we found after 48 hours of hypoxia,proliferation was significantly reduced and apoptosis was significantly increased.The second experiment,it was found that 10umol/L SFN stimulation can inhibit oxidation,promote cells growth and reduce apoptosis,and more than 10umol/L of SFN may had a negative effect on the proliferation of osteoblasts.So we used 48h as the experimental time and 10umol/L as the SFN pretreatment concentration.When osteoblasts were cultured in hypoxic and reoxygenated environment,oxidative stress would occur.The intracellular ROS would increase,the proportion of apoptosis would increase,the activity of caspase-3 would increase,and the oxidative indexes HIF-1a,VEGF and MMP-9 would increase.Osteogenic indicators OPN,RUNX2,OSTERIX,ALP,and COL1 would decrease,affecting the osteogenesis of osteoblasts.Pretreatment with 10umol/L SFN could reduce the apoptosis of osteoblasts,reduce the oxidation index of cells,increase the osteogenesis index of cells,promote osteogenic mineralization,and the effect of 48h pretreatment would be better than that of 24h pretreatment.4.ConclusionOsteoblasts may undergo oxidative stress on the condition of hypoxia and reoxygenation,which may affect the proliferation and mineralization of osteoblasts.The pretreatment of proper amount of SFN can mitigate stress status and improve osteogenesis of osteoblasts under oxidative stress.