The Alleviating Effect And Application Of Melatonin On Compact Bone-Derived Mesenchymal Stem Cells Injured By Gamma Radiation

Radiotherapy is a common method of using ionizing radiation to treat malignant tumors.While killing tumor cells,radiation inevitably damages the surrounding normal tissues and cells.Ionizing radiation affects bone metabolic activity and increases the risk of fracture,osteoporosis and poor bone healing.Stem cells are more vulnerable to radiation than highly differentiated or non proliferating cells.As an important seed cell of bone tissue engineering,compact bone-derived mesenchymal stem cells may be the key mechanism of bone loss caused by radiation.Radiation-related drugs are generally divided into three categories:protective drugs,radiomitigators and therapeutic drugs.It is of great significance to find radiation radiomitigators used shortly after radiation exposure to promote the timely repair of radiation bone injury.At present,only several radiation-related drugs have been approved for clinical treatment,among which amifostine has many side effects,and the production cost of granulocyte colony stimulating factor is high and difficult to obtain.Melatonin,as an indole heterocyclic compound,widely exists in animals and plants.It possesses good antioxidant,anti apoptotic,anti-inflammatory and anti-tumor properties.It has been proved to have radiation protective effect on multiple organs in a variety of animal radiation models.Melatonin also plays the role of radiation sensitization in the treatment of some tumors.It also affects bone reconstruction and repair by regulating the dual effects of osteogenic and osteoclastic activity.Therefore,melatonin is expected to become a radiomitigator to deal with radiation induced bone injury.Objective:To investigate the gamma radiation-induced damage of compact bone-derived mesenchymal stem cells in vitro and the effect of melatonin treatment on radiation-induced damage.To explore the mechanism of melatonin in response to radiation injury.To establish the animal model of bone defect after local radiation,construct the local administration method of melatonin,and explore its effect and preliminary mechanism on the repair of bone defect after radiation in vivo.To establish the animal model of bone defect after local radiation,construct the systemic administration system of melatonin,and explore its effect and preliminary mechanism on the repair of bone defect after radiation in vivo.Methods:Section 1 The alleviating effect of melatonin on compact bone-derived mesenchymal stem cells injured by gamma radiationCompact bone-derived mesenchymal stem cells from normal C57BL/6 mice were isolated,cultured and irradiated with 6 Gy gamma rays.Then different concentrations of melatonin were added for 24 hours.The cells were divided into five groups:control,radiation group(IR),1 μM melatonin treatment post radiation group(IR+LMT),10 μM melatonin treatment post radiation group(IR+MMT)and 100 μM melatonin treatment post radiation group(IR+HMT).The control group did not receive radiation.CCK-8 experiment was used to explore the effects of radiation and melatonin on cell proliferation.Apoptosis was detected by flow cytometry.The self-renewal ability of cells in each group was detected by CFU-F experiment.The osteogenic and adipogenic differentiation of cells in each group was induced,and then stained with alkaline phosphatase,alizarin red and oil red to detect the osteogenic and adipogenic differentiation activity of cells in each group.Through the above experiments,the effects of gamma ray radiation and melatonin on the biological characteristics of compact bone-derived mesenchymal stem cells were explored.Section 2 The mechanisms of melatonin alleviating the injury of compact bone-derived mesenchymal stem cells induced by gamma radiationThe cells were divided into five groups:control,radiation group(IR),1 μM melatonin treatment post radiation group(IR+LMT),10 μM melatonin treatment post radiation group(IR+MMT)and 100 μM melatonin treatment post radiation group(IR+HMT).The control group did not receive radiation.γH2AX immunofluorescence staining was used to explore the degree of DNA damage of cells in each group.The expression levels of apoptosis-related transcription factors Bcl-2 and Bax were detected by Western blot.The expression levels of genes related to cell self-renewal and multi-directional differentiation were analyzed by qRT-PCR.The cells in each group were labeled with DCFH-DA fluorescent probe to qualitatively analyze the oxidative stress of cells.Then,the labeled cells in each group were put on flow cytometry to quantitatively detect the level of reactive oxygen species in cells.After the cells were lysed,the levels of several oxidative stress markers in each group were analyzed,including malondialdehyde,superoxide dismutase and glutathione.In addition,the protein expression levels of pro-inflammatory factors TNF-α and IL-6,and markers of ERK/Nrf2 signal pathway in each group was detected by Western blot to explore the changes of cellular inflammation and oxidative stress.Through the above experiments,we preliminarily explore the mechanisms of melatonin on compact bone-derived mesenchymal stem cells damaged by gamma radiation.Section 3 The effect and mechanisms of local melatonin application on bone defect regeneration after local radiationThirty two C57BL/6 mice were divided into four groups:control,radiation group(IR),matrix gel post radiation group(IR+Gel),matrix gel combined with melatonin post radiation group(IR+Gel+MT).The control group did not receive radiation,and the other groups received 6 Gy gamma radiation in the femoral region.The local melatonin administration system was constructed by mixing melatonin with Matrigel.After radiation,bone defects with a diameter of 1.5 mm and a depth of 1 mm were formed at the distal end of the femur of mice in each group with a dental slow drill,and the bone defects were injected with Matrigel or Matrigel combined with melatonin.After 1 month,the osteometric parameters of new bone formation were analyzed by Micro-CT tomography.Bone tissue specific staining and immunohistochemical staining were used to analyze the histology and levels of TNF-α and IL-6 in the new bone area.The expression level of Nrf2 protein in the new bone area was analyzed by immunofluorescence staining of bone tissue.Through the above experiments,the effect of local melatonin administration on the regeneration of bone defects after radiation in vivo and its mechanisms were explored.Section 4 The effect and mechanisms of systemic melatonin application on bone defect regeneration after local radiationThirty two C57BL/6 mice were divided into four groups:control,radiation group(IR),high concentration melatonin treatment group(IR+HMT)and low concentration melatonin treatment group(IR+LMT).The control group did not receive radiation,and the other groups only received 6 Gy gamma radiation in the femoral region.The systemic melatonin administration system was constructed by intraperitoneal injection.After radiation,bone defects with a diameter of 1.5 mm and a depth of 1 mm were formed at one-third of the distal end of the femur of mice in each group with a dental slow drill.Then the drug treatment groups were injected with melatonin once a day for consecutive 28 days.The osteometric parameters of new bone formation were analyzed by Micro-CT tomography.Bone tissue specific staining and immunohistochemical staining were used to analyze the histology and the levels of osteogenic and osteoclastic markers in the new bone area.The serum levels of inflammatory and oxidative stress markers were analyzed by ELISA.Through the above experiments,the effect of systemic melatonin administration on the regeneration of bone defects after radiation in vivo and its mechanisms were explored.Results:Section 1 The alleviating effect of melatonin on compact bone-derived mesenchymal stem cells injured by gamma radiationCCK-8 results showed that 1 mM melatonin was cytotoxic,while the cytotoxicity 1 to 100μM melatonin was less than 10%.Gamma rays destroyed the cell proliferation activity of compact bone-derived mesenchymal stem cells,while melatonin treatment partially saved the cell proliferation activity,and the effect of 100 μM melatonin was better than other melatonin treatment groups.Flow cytometry showed that radiation significantly increased the apoptosis rate of stem cells,while 100 μM and 10 μM melatonin treatment reduced the apoptosis rate.CFU-F results showed that the colony formation of radiation group was significantly inhibited by gamma rays,while the colony formation of IR+HMT group was significantly better than radiation group,but still lower than control group.The results of multidirectional differentiation experiment showed that radiation significantly inhibited the osteogenic and adipogenic staining positive rate of stem cells,100 μM and 10 μM melatonin treatment increased the positive rates of ALP,alizarin red and oil red.Section 2 The mechanisms of melatonin alleviating the injury of compact bone-derived mesenchymal stem cells induced by gamma radiationIt could be seen from the results of cellular immunofluorescence that gamma radiation significantly increased the positive staining rate of yH2AX,while 100 μM and 10 μM melatonin decreased the positive staining rate.The results of Western blot showed that the expression level of anti-apoptotic protein Bcl-2 was significantly downregulated after radiation,but increased significantly after 100 μM melatonin treatment,while the expression trend of Pro-apoptotic protein Bax was almost the opposite.qRT-PCR results showed that Klf4 and Nanog genes were significantly downregulated after radiation.Compared with the radiation group,Klf4,Nanog,Oct4 and Sox2 in the IR+HMT group were significantly upregulated.The expression levels of Runx2 and OPN genes were significantly downregulated in radiation group cells,but were significantly upregulated after 100 μM and 10 μM melatonin treatment.The expression changes of Cebpa and Pparg,two genes related to lipogenesis,were almost opposite to Runx2 and Opn.In addition,the detection results of reactive oxygen species showed that with the increase of melatonin treatment concentration,the content of cellular reactive oxygen species gradually decreased and the activity of antioxidant enzymes gradually increased.The results of Western blot also showed that radiation increased the expression levels of pro-inflammatory factors TNF-α and IL-6 in compact bone-derived mesenchymal stem cells,but significantly downregulated after 100 μM melatonin treatment.Similarly,radiation decreased the expression levels of ERK,p-ERK,Nrf2 and HO-1 protein markers in ERK/Nrf2 signaling pathway,but significantly upregulated after 100 μM melatonin treatment.Section 3 The effect and mechanisms of local melatonin application on bone defect regeneration after local radiationFrom the Micro-CT results,it could be seen that the new bone formation in the radiation group was worse than that the other groups,and the new bone cortical line was not successfully connected.BV/TV,BMD and Tb.Th values in the IR+gel+MT group were not significantly different with the control,showing a good effect of bone regeneration.According to the histological staining results,the new bone formation in the radiation group was less and disordered,and there were obvious depressions in the bone cortex.The control group and IR+gel+MT group had the best new bone formation,and the cortical bone had been completely connected and leveled.In addition,TNF-α and IL-6 levels were significantly upregulated in bone defect area in radiation group,were decreased slightly in IR+Gel group,and were significantly decreased in IR+Gel+MT group.As a key transcription factor of anti oxidation,the expression level of Nrf2 in radiation group was the lowest,followed by IR+gel group,while were relatively high in the IR+Gel+MT group and control group.Section 4 The effect and mechanisms of systemic melatonin on bone defect regeneration after local radiationThe results of Micro-CT and histological staining showed that the new bone formation in the IR group was the worst,followed by IR+LMT group,while IR+HMT group was better.BV/TV,BMD and Tb.Th values in IR+HMT group were significantly higher than those in the IR group but still lower than the control.Radiation severely inhibited the osteogenic and osteoclastic activity in bone defect area,but it rebounded after melatonin treatment.In addition,the serum levels of IL-1β,IL-6 and TNF-α were increased significantly in the IR group,but were decreased significantly in IR+HMT group.The serum MDA level in the radiation group was the highest,while the levels of SOD and CAT were the lowest.The treatment of high concentration melatonin in IR+HMT group significantly reduced the level of oxidative stress.Conclusion:1.Gamma radiation seriously damages the biological characteristics of compact bone derived mesenchymal stem cells.It reduces the activity of cell proliferation and self-renewal and increases cell apoptosis,and induces the differentiation of stem cells from osteogenesis to adipogenesis.100 μM melatonin exerts palliative effect on irradiated compact bone derived mesenchymal stem cells,and can successfully save some lost stem cell characteristics;2.Melatonin exerts its palliative effect through a variety of mechanisms.It can enhance the DNA stability of compact bone derived mesenchymal stem cells after radiation,promote the expression of anti-apoptotic proteins,inhibit the expression of pro-apoptotic proteins,promote the transcription of self-renewal and osteogenic differentiation marker genes,inhibit the transcription of adipogenic marker genes,and reduce the level of oxidative stress and inflammation of stem cells after radiation;3.The local administration of melatonin combined with matrix gel can promote the new bone formation of bone defect after radiation and achieve good bone healing effect.It may be related to melatonin regulating the level of oxidative stress and inflammation in the new bone area;4.Systemic melatonin administration can promote the new bone formation of bone defect after radiation and achieve good bone healing effect.It may be related to the regulation by melatonin on the osteogenic and osteoclastic activity in the new bone area,and the level of systemic inflammation and oxidative stress;5.Melatonin possesses good biosafety,less side effects and is quite available.It is expected to be applied in the alleviating treatment of radiation bone injury in clinic in the future.