| Objective: Osteoporosis is a chronic,systemic bone disease characterized by decreased bone mass,destruction of bone microstructure and increased bone fragility,thus increasing the risk of fracture.As a metabolic bone disease,osteoporosis affects the health of hundreds of millions of people all over the world.It is estimated that by2050,the population of osteoporosis in China alone will increase to 212 million.Previous studies have shown that osteoporosis is related to many factors,including age,gender,race,heredity,reproductive status,low calcium intake and exercise.In recent years,the viewpoint of osteoporosis caused by iron overload has gradually attracted our attention.Iron is an essential trace element for human body.The stability of iron metabolism is very important to maintain the normal physiological activities of human body.Iron participates in hematopoiesis,hemoglobin and myoglobin formation,oxygen transport and storage,and many enzymatic reactions,including DNA biosynthesis and ATP production.Iron deficiency can lead to iron deficiency anemia,which leads to insufficient oxygen supply to cells and tissues in human body.Therefore,it is necessary to maintain an adequate supply of iron in the body,but iron overload can also bring a lot of harm.Excessive iron can be deposited in liver,heart,pancreas,central nervous system and bone marrow.Iron depositied in bone marrow can lead to osteopenia and even osteoporosis.The disorder of bone metabolism caused by iron overload can occur in hematological system diseases such as hereditary hemochromatosis,thalassemia and sickle cell disease.In addition,it is also very common in healthy postmenopausal women.Recent studies have shown that iron overload is considered to be an independent risk factor for osteoporosis.The balance between osteoblastic bone formation and osteoclastic bone resorption is required to maintain bone homeostasis.Dysregulation between osteoblast and osteoclast often leads to disorder of bone metabolism.Studies have shown that iron overload can induce osteoblast apoptosis and decrease its viability.Iron overload can produce hydroxyl radicals and other reactive oxygen species through Fenton reaction and Haber – Weiss reacton.The release of reactive oxygen species exceeds the antioxidant capacity of cells,leading to oxidative stress,which can damage DNA,protein and lipid in cells and cause cell damage.However,the mechanism of osteoblast apoptosis induced by iron overload and the relationship between reactive oxygen species and apoptosis still need to be further explored.In the first part of this article,we studied the effect of reactive oxygen species on osteoblast apoptosis induced by iron overload.Autophagy is an evolutionarily highly conserved process,also known as type II programmed cell death.Autophagy is characterized by the formation of double membrane vesicles called autophagosomes,which seal organelles and then transport them to lysosomes for proteolysis.Autophagy can eliminate potentially dangerous organelles and play an important role in maintaining cell homeostasis.However,the relationship among autophagy,apoptosis and reactive oxygen species is complex,and the role of autophagy in apoptosis is controversial.In the second part of this article,we investigated the effect of endogenous autophagy on the increase of reactive oxygen species and apoptosis induced by iron overload.Endogenous autophagy has limited ability to protect osteoblasts from apoptosis induced by iron overload.We hope to find a drug that can resist iron overload and explore its molecular mechanism.Salidroside is the root tuber extract of Rhodiola,which is a traditional Chinese herbal medicine with antioxidant capacity.In the third part of this article,we studied the role of Nrf2 / HO-1 pathway in the iron overload induced reactive oxygen species elevation and apoptosis of osteoblasts.Methods: MC3T3-E1 osteoblasts were cultured.Iron citrate(FAC)was used as an iron donor to induce iron overload condition in osteoblasts.Osteoblasts were treated with FAC(0,100,200,400,800,1600 μM)for 24 or 48 hours,and cell viability was detected by CCK-8.Osteoblasts were treated with FAC(0,400,800,1600 μM)for 48 hours.The apoptosis of osteoblasts was detected by Hoechst staining,Annexin V / PI staining flow cytometry and Western blot.Subsequently,the experiment was divided into four groups:(1)Control group;(2)FAC alone treatment group;(3)NAC alone treatment group;(4)FAC combined with NAC treatment group.DCFH-DA staining flow cytometry was used to detect the level of intracellular reactive oxygen species,Western blot was used to detect the expression of oxidative stress related proteins;flow cytometry and Western blot were used to detect apoptosis and the expression of apoptosis related proteins;CCK-8 was used to detect cell viability.Osteoblasts were treated with FAC(0,400,800,1600 μM)for 48 hours.The expression of autophagy related proteins was detected by Western blot and LC3 protein was detected by immunofluorescence.Subsequently,the autophagy inhibitor chloroquine(CQ)was used to divide the experiment into four groups:(1)Control group;(2)FAC alone treatment group;(3)CQ alone treatment group;(4)FAC combined with CQ treatment group.Autophagy flux was detected by Western blot;reactive oxygen species(ROS)level was detected by flow cytometry with DCFH-DA staining;oxidative stress related proteins were detected by Western blot;apoptosis and expression of apoptosis related proteins were detected by flow cytometry and Western blot;cell viability was detected by CCK-8.The osteoblasts were treated with Sal(0,0.1,1,10 μM)for 24 hours,and the cell viability was detected by CCK-8.The following experiments were divided into four groups:(1)Control group;(2)FAC alone treatment group;(3)Sal alone treatment group;(4)FAC combined with Sal treatment group.CCK-8 was used to detect cell viability;flow cytometry was used to detect apoptosis;MDA and GSH kits were used to detect the level of oxidative stress;Western blot was used to detect the expression of Nrf2 / HO-1 pathway related proteins.ML385,an inhibitor of Nrf2 / HO-1 pathway,was further used to detect the changes of Nrf2 / HO-1 pathway and cell viability.Results: 1.Iron overload can induce osteoblast apoptosis by increasing the level of reactive oxygen species.FAC was used as iron donor to induce iron overload condition in osteoblasts.MC3T3-E1 cells were treated with different concentrations of FAC,compared with the control group,the viability of osteoblasts decreased,the apoptosis rate increased and the expression of apoptosis related proteins increased,the level of intracellular reactive oxygen species increased and the expression of oxidative stress related proteins increased.When NAC and FAC were used together,compared with FAC alone group,the level of ROS decreased,the expression of oxidative stress-related protein decreased;the apoptosis rate decreased,the expression of apoptosis related protein decreased;and the cell viability increased.2.Iron overload induces osteoblast autophagy,and inhibition of autophagy increases the level of reactive oxygen species and aggravates apoptosis of osteoblasts.MC3T3-E1 cells were treated with different concentrations of FAC,the expression of autophagy related proteins increased.When autophagy inhibitor chloroquine(CQ)and FAC were used together,compared with FAC alone group,the level of reactive oxygen species increased,the expression of oxidative stress related proteins increased;the apoptosis rate increased,the expression of apoptosis related proteins increased;and the cell viability decreased.3.Salidroside alleviates the apoptosis of osteoblasts induced by iron overload.When salidroside and FAC were used together,compared with FAC alone group,the apoptosis rate of osteoblasts decreased.4.Salidroside alleviates the effect of iron overload on osteoblasts by activating Nrf2 /HO-1 pathway.Western blot results showed that FAC inhibited the expression of Nrf2 and HO-1 protein,and salidroside increased the expression of Nrf2 and HO-1 protein.FAC induced the decrease of osteoblast viability,and salidroside induced the increase of osteoblast viability,which could be inhibited by Nrf2 / HO-1 inhibitor ML385.Conclusion: 1.Iron overload can induce osteoblast apoptosis by increasing the level of reactive oxygen species.2.Iron overload induces autophagy of osteoblasts,and inhibition of autophagy increases the level of reactive oxygen species and aggravates apoptosis of osteoblasts.3.Salidroside alleviates the apoptosis of osteoblasts induced by iron overload.4.Iron overload inhibits Nrf2 / HO-1 pathway mediated by reactive oxygen species.5.Salidroside alleviates the effect of iron overload on osteoblasts by activating Nrf2 / HO-1 pathway. |
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