| Background and Objectives:Heterotopic ossification(HO)refers to the formation of abnormal bone tissue in the soft tissue.Heterotopic ossification caused by severe injury,burn and amputation operation is common,which seriously affects the life quality of patients.At present,radiotherapy and surgical therapy are commonly used to prevent and treat heterotopic ossification,but a series of side effects largely limit the application of them.Studies have shown that the formation of heterotopic ossification is related to a variety of signal transduction pathways and biological activity regulators,in which the activation of bone morphogenetic proteins(BMPs)signaling pathway is crucial for the occurrence and development of heterotopic ossification.Therefore,we can inhibit heterotopic ossification by targeting BMP signaling pathway.If we can explore other drugs that inhibit BMP signaling pathway in clinic,it will surely provide a shortcut for prevention and treatment of heterotopic ossification.In this study,we investigated the effects of berberine and aspirin on BMP signaling pathway components,cell osteogenic differentiation and heterotopic ossification in mice.contents and methods:1 To study the effects of berberine and aspirin on the components of BMP signaling pathway.1.1 To detect the effects of berberine and aspirin on the expression of ALK2,Smad6 and Smurf1,the key components of BMP signaling pathway.Berberine(0-100 ?m),aspirin(0-10 mM)and their combination were used to treat C3H10T1/2 bone marrow mesenchymal stem cells from mice and MC3T3-E1 cells from mouse proosteoblasts.Western blot was used to detect the effects of berberine and aspirin on the expression of ALK2,Smad6 and Smurf1 proteins.1.2 Cells were pretreated with berberine and aspirin for 24 hours,then stimulated with mBMP6 for 30 minutes.The changes of BMP6-induced phosphorylation of Smad1/5 were detected by Western blot.2 To observe the effects of berberine and aspirin on the osteogenic differentiation of C3H10T1/2 and MC3T3-E1 cells.2.1 C3H10T1/2,MC3T3-E1 cells were induced to differentiate into osteoblasts by adding induction medium.Berberine,aspirin,and the combination of them were added during the culture process.Alkaline phosphatase(ALP)staining and alizarin red staining were performed on the 7th and 21 st day of culture to detect alkaline phosphatase activity and cell mineralization,and to observe the effect of berberine and aspirin on osteogenic differentiation.3 To detect the effects of berberine and aspirin on heterotopic ossification in mice.3.1 Heterotopic ossification model was established by Achilles tendon amputation/burn in mice.Berberine and aspirin were used to intervene.The Achilles tendon of 8-week-old female C57BL/6 mice was excised and the mice were burned on the back by 60?farmar.Berberine(10 mg/kg)and aspirin(10 mg/kg)were injected intraperitoneally every three days after operation.3.2 Eight weeks after operation,the effect of berberine and aspirin on the swelling degree of Achilles tendon in mice was examed by visual observation.3.3 Eight weeks after operation,the injured hind limbs of mice were taken to scan micro-CT to exam the effects of berberine and aspirin on heterotopic ossification.Results:1 In C3H10T1/2 and MC3T3-E1 cells,berberine and aspirin activate AMPK in a concentration-dependent manner;the activation of AMPK inhibits BMP6-induced phosphorylation of Smad1/5;in C3H10T1/2 cells,AMPK down-regulates the expression of ALK2 but does not change the expression of Smurf 1 and Smad6;in MC3T3-E1 cells,AMPK down-regulates the expression of ALK2 and up-regulates the expression of Smurf 1,but does not change the expression of Smad6.2 In C3H10T1/2 and MC3T3-E1 cells,berberine and aspirin could inhibit alkaline phosphatase activity and cell mineralization in a concentration-dependent manner.The combination of berberine and aspirin could enhance the inhibition of osteogenic differentiation.3 Achilles tendon amputation/burn method can successfully induce heterotopic ossification in mice.Compared with mice in heterotopic ossification model group,external observation showed that the degree of Achilles tendon swelling in berberine and aspirin intervention group was significantly reduced;Micro-CT scan showed that the formation of heterotopic bone in Achilles tendon of mice in berberine and aspirin intervention group was significantly reduced.Conclusion:Berberine and aspirin can inhibit BMP signal transduction and osteoblast differentiation,which may be mediated by AMPK.Berberine and aspirin can inhibit the progress of heterotopic ossification. |
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