| Backgrounds:Fracture is a common clinical condition that refers to a complete or partial break in the continuity of bone and can occur at all ages.However,as we age,the incidence of fractures increases,the time required for fracture healing lengthens and the number of fracture-related complications increases leading to a reduced quality of life for older people.It is therefore important to find ways to speed up the rehabilitation of elderly fracture patients and reduce the incidence of complications.Bone marrow mesenchymal stem cells are non-haematopoietic stromal stem cells that exhibit pluripotent differentiation into chondrocytes and osteoblasts and play a large role in fracture healing.A number of studies have demonstrated the feasibility of using bone marrow stem cells therapy to promote fracture healing.The delay in fracture healing with age has been attributed to a decrease in the number and function of MSCs that regenerate bone,as well as the low performance of endothelial progenitor cells that direct angiogenesis.Icariin,a traditional Chinese medicine,possesses the ability to tonify kidney energy and promote fracture healing.Icariin,the main component of Icariin,has an estrogen-like structure and can exert estrogen-like effects,binding to estrogen receptors in the body to exert corresponding effects,promoting bone formation and inhibiting bone resorption.There are many studies on the relationship between Icariin and BMSCs,and the two are closely related.In this study,we investigated the effect of Icariin on the senescence indexes of senescent BMSCs and the possible mechanism of action.Objectives:BMSCs are closely associated with fracture healing and play a large role in fracture healing.However,the number,proliferation,migration capacity and differentiation potential of BMSCs are adversely altered with age,resulting in impaired fracture healing in the elderly.Conventional treatments for orthopaedic conditions such as osteoporosis,femoral head necrosis,bone loss and osteointegr potential ative diseases are clinically ineffective due to their inability to improve bone loss,and the focus is now on tissue regeneration using BMSCs.Bone marrow stem cells transplantation is currently being investigated,but this is associated with adverse effects such as thrombosis and immune rejection.It is therefore a good idea to maintain the activity of BMSCs by delaying their own ageing.Studies have found that oestrogen is ideal for treatment because of its ability to delay ageing,antioxidant effects,promote osteogenesis and inhibit osteolysis.However,estrogen does not only act on bone marrow mesenchymal stem cells,but also acts in other systems and between other cells,affecting its own estrogen metabolism or producing adverse effects in vivo.Icariin（ICA）has an estrogen-like structure and can exert estrogen-like effects,but is not itself an estrogen and does not affect its own estrogen production.Therefore,icariin was selected as the subject of this experiment to investigate its mechanism of action and efficacy in affecting senescent BMSCs,which was the main focus of this study.The experiments were conducted using 16-month-old senescent C57 mice and 8w young C57 mice.It was found that the bone density of the 16m senescent mice decreased and fracture healing was relatively slow.In order to obtain sufficient numbers of BMSCs,the present study was carried out to create a model of senescent BMSCs by treating young mice with hydrogen peroxide and using different concentrations of icariin,estrogen and estrogen receptor antagonist to investigate whether icariin could delay the senescence of BMSCs in vitro.We investigated in vitro whether Icariin could delay the senescence of bone marrow stem cells and whether it exerted its effect on delaying the senescence of bone marrow stem cells through estrogen receptors.Methods:1.Comparison of aged C57 mice and young C57 miceA 16m aged C57 mouse and an 8w young C57 mouse were taken from the same side of the femur and Mirco-CT was performed to examine the differences in their indexes（bone density,bone surface area,etc.）and bone marrow mesenchymal stem cells were taken from both mice for culture and comparison.A fracture model was also taken from both groups of mice to observe the healing process and the difference in parameters between the two.2.Extraction and identification of BMSCs and senescence treatmentBMSCs from C57/BL mice were cultured by the applanation method.When the bone marrow stem cells reached 80%-90%in the culture dish,trypsin digestion was taken,passaged culture was performed and third generation BMSCs were taken for immunofluorescence identification.After identification,the cells were treated with hydrogen peroxide intervention to cause bone marrow stem cells senescence and grouped into control as well as hydrogen peroxide intervention groups.BMSCs from both groups were stained with senescence-associated-β-galactosidase（SA-β-Gal）and the effect on their proliferative activity was measured with CCK-8.The changes of P53,P21 and P16 in the control group and the peroxisomal intervention group of C57 mice were identified by protein blotting（Western Blot,WB）and PCR（Polymerase Chain Reaction）,and the changes of bone indexes（Runx2,OPG）in the two groups were observed.3.Icariin Effects on senescence and osteogenesis of aging BMSCs in miceBone marrow stem cells were further treated with hydrogen peroxide and divided into four groups.On the third day after hydrogen peroxide treatment,different drugs were added to the culture medium according to the groups:control group（Control）:a certain amount of PBS was added to the culture medium,Icariin group（ICA）:equal amounts of different concentrations of Icariin were added to the culture medium,Icariin plus estrogen group（ICA+E）:the highest concentration of Icariin was selected Icariin plus a certain amount of estrogen,Icariin plus estrogen receptor antagonist group（ICA+ICI）:the highest concentration of Icariin was selected to add a certain amount of estrogen receptor antagonist.The effect of CCK-8 on the viability of senescent BMSCs was measured at 24,48 and 72h.The cells were cultured for 3 days,and the changes in senescence proteins such as P53,P21 and P16,as well as osteogenic parameters（Runx2 and OPG）were identified by Western Blot（WB）and PCR（Polymerase Chain Reaction）.The effect ofβ-galactosidase staining on the rate of staining positive cells was also observed for three days of treatment with Icariin.4.Effect of Icariin on fracture healing in senescent miceThe aging mice were randomly divided into aging control group and aging patchouli glycoside-treated group,and a fracture model was made.2 weeks later,the ipsilateral fracture was taken from the molded femur and Mirco-CT and HE slides were performed to compare the healing status between the two groups.Results:1.Significantly lower bone density and reduced number of trabeculae in aged C57mice at 16m compared to young C57 mice at 8w.2.16m aging C57 mice and 8w young C57 mice were studied together to create a fracture model,and the fracture healing rate of aging mice was relatively slow.3.16m aged C57 mice and 8w young C57 mice were cultured with bone marrow mesenchymal stem cells and found that the number of bone marrow mesenchymal stem cells in aged mice was low,their morphology was abnormal and their proliferation capacity was weak.4.Bone marrow mesenchymal stem cells grow close to the wall and are irregular in shape.5.Immunofluorescence was used to identify mouse bone marrow stem cells,and CD29 and CD44 were found to be positively expressed;CD45 was found to be negatively expressed and identified as bone marrow stem cells.6.Treatment of bone marrow stem cells with hydrogen peroxide resulted in senescence,altered morphology and a significantly higher percentage ofβ-galactosidase staining positive cells,and reduced proliferative activity,similar to that of bone marrow stem cells extracted from 16m mice.7.ICA promotes the proliferation of senescent BMSCs and enhances their cell viability.8.WB of senescence indexes in the hydrogen peroxide-treated group and the control group showed that P53,P21 and P16 protein indexes were significantly increased.9.After 7 days and 21 days of osteogenesis induction in the senescence-treated and control groups,it was found that the osteogenic ability of the senescence-treated group decreased,and the differences in alizarin red and ALP staining were obvious.10.In the control group,ICA group,ICA+E group and ICA+ICI group,it was found that the ICA group decreased the aging index of BMSCs and increased with increasing concentration within a certain range(10-7mmol/L-10-3mmol/L);the effect of ICA+E group was slightly better compared with the optimal concentration of ICA;the effect of ICA+ICI group was poorer and close to the control group,and epimedium may delay the aging of BMSCs through ER-mediated aging pathway.11.The osteogenic ability was found in the control,ICA,ICA+E and ICA+ICI groups,and the osteogenic indexes were detected using alizarin red,ALP staining and WB,and the osteogenic ability was found to be promoted:ICA+E≥ICA group>ICA+ICI group≈control group12.Aging mice gavaged with 200 mg/kg of ICA solution can promote fracture healing.Conclusions:1.Aging mice showed decreased bone mass and prolonged fracture healing time compared to younger mice.2.Bone marrow stem cells from senescent mice had altered cell morphology and reduced proliferation viability compared to those from young mice.3.Two hours of treatment of bone marrow stem cells with hydrogen peroxide and replacement with complete medium resulted in a significant increase in the positive rate of galactose stemase stained cells in BMSCs and an increase in P53,P21 and P16 protein indices,suggesting the feasibility of the method that hydrogen peroxide treatment can induce senescence in bone marrow stem cells.4.Hydrogen peroxide-induced senescence leads to a decrease in osteogenic capacity of bone marrow mesenchymal stem cells5.The use of ICA can promote a decrease in senescence-related indices of senescent BMSCs and recovery of proliferative capacity,while improving osteogenic capacity.6.The results of the group treatment of senescent BMSCs showed that the cell viability and osteogenic capacity of BMSCs increased with the increase of ICA concentration within a certain range(10-7mmol/L-10-3mmol/L).Among the groups,ICA+E had the most pronounced effect,while ICA+ICI had almost no effect compared to the control group,suggesting that ICA may exert its effect of delaying the senescence of BMSCs through ER mediation.7.Gavage of 200 mg/kg of ICA solution promotes fracture healing in aging mice. |
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