| Chapter Ⅰ Effect of iron accumulation on coagulation state in ratsObjective:To investigate the changes of coagulation function related indexes in iron accumulation rat model,and to preliminarily explore the effect of iron accumulation on coagulation status and its potential mechanism.Methods:Twelve male SPF SD rats aged 6 months selected,which with the average body weight 300±20g,were divided into control(Ctrl)and iron accumulation groups(FAC)(n=12)according to random number table,six rats in each group.The iron accumulation group was intervened by intraperitoneal injection of ferric ammonium citrate 90 mg/kg,and the control group was intraperitoneally injected with equal volume of normal saline twice a week for 9 weeks.After the intervention,serum ferritin,D-dimer,blood lipid,peripheral blood cells,coagulation function parameters and serum nitric oxide(NO)content were measured in both groups.H&E and MSB(Martius scalet blue)staining were used to observe the microthrombosis in the distal femur bone.The parameters of the two groups were statistically analyzed by t-test.P<0.05 showed that there was significant difference,with statistical significance.Results:Serum ferritin levels in FAC group and Ctrl group were 136.36±35.41 ng/ml and 68.44±16.86 ng/ml,respectively,which were significantly higher in FAC group.The fibrinogen in the FAC group was 2.03±0.13 g/L,which was significantly higher than that in the control group(1.78±0.46 g/L).Meanwhile,D-dimer contents(534.95±31.81 ng/ml)increased compared to control group(329.02±84.99 ng/ml),whereas the thrombin time(TT:39.64±2.18 s)and prothrombin time(PT:8.7±0.39 s)were significantly shorter in the FAC group.There were no significant changes in peripheral blood cells,TG,HDL and LDL in the FAC group,but there was an increasing trend of cholesterol in the FAC group.H&E and MSB staining showed microthrombus in the bone marrow of the iron accumulation rats,as well as microthrombus in the myocardium.The level of NO in control group and FAC group was 21.851±1.77 μmol/L and 9.13±1.55 μmol/L,respectively,which was significantly lower in FAC group.Conclusions:In the rat model of iron accumulation,iron accumulation had a significant effect on blood coagulation.The effect on the coagulation status is manifested in the hypercoagulability of blood,and the existence of microthrombosis in bone microvessels.The bioavailability of NO was decreased in the iron accumulation group.Excess iron may affect the dysfunction of microvascular endothelial cells and promote the formation of microblood through the NO-mediated signaling pathway.Chapter Ⅱ Correlation between abnormal blood status and decreased bone mass under iron accumulation in ratsObjective:To further investigate the correlation between abnormal blood coagulation and decreased bone mass in rats with iron accumulation.Methods:Twelve male SPF SD rats aged 6 months selected,which with the average body weight 300±20g,were divided into control(Ctrl)and iron accumulation groups(FAC)(n=12)according to random number table,six rats in each group.The iron accumulation group was intervened by intraperitoneal injection of ferric ammonium citrate 90 mg/kg,and the control group was intraperitoneally injected with equal volume of normal saline twice a week for 9 weeks.After the intervention,serum ferritin,CTX-I,PINP and coagulation parameters were measured.Vessel density was determined by ink staining,and iron deposition in liver sections was observed by H&E staining.Micro-CT was used to reconstruct the three-dimensional shape of the trabecular bone of the distal femur,and the spatial structure parameters were measured.The parameters of the two groups were statistically analyzed by t-test.P<0.05 showed that there was significant difference,with statistical significance.Results:Serum ferritin levels in control group and FAC group were 71.76±11.32 ng/ml and 128.6±28.32 ng/ml,respectively,which were significantly higher in FAC group.In terms of bone mineral density,the above two groups were 0.400±0.030 g/cm3 and 0.167±0.024 g/cm3,respectively,with the latter being significantly lower.In FAC group,PINP index was significantly lower than that in control group,and CTX-I was significantly higher than that in control group.The D-dimer in the FAC group was 549.11±48.74 g/L,which was significantly higher than that in the control group(370.85±54.45 g/L).After ink staining,the microvascular density in the iron accumulation group(17.46±2.07%)was significantly reduced compared with that in the control group(21.81±2.98%).Through the first part,it was found that fibrinogen and D-dimer in FAC group were higher than those in control group,and thrombin time and prothrombin time were shorter than those in control group.Conclusions:In the iron accumulation rat model,iron accumulation had a significant effect not only on blood coagulation function,but also on bone metabolism.The bone mass was negatively correlated with hypercoagulability and vascular density in iron accumulation ratsChapter Ⅲ Effect of anticoagulant intervention on bone mass of iron accumulated ratsObjective:To investigate the effect of anticoagulation on microthrombus,micro vascular bed and bone mineral density in iron accumulated rats.Methods:Four groups of maleratmodels were established:Ctrl+NS group,Ctrl+fondaparinux group,FAC+NS group,and FAC+fondaparinux group.Twelve rats in each group were fed for 9 weeks.Fondaparinux is an anticoagulant.In FAC+fondaparinux group,90 mg/kg ferric ammonium citrate was injected intraperitoneally twice a week,and fondaparinux was injected subcutaneously 0.2 mg/kg once a day;in FAC+NS group,90 mg/kg ferric ammonium citrate was injected intraperitoneally twice a week,and the same volume of NS was injected subcutaneously once a day;in Ctrl+fondaparinux group,fondaparinux was injected subcutaneously 0.2 mg/kg once a day;in Ctrl+NS group,the same volume of NS was injected subcutaneously once a day.The intervention lasted for 9 weeks.After the intervention,D-dimer content,serum PINP and CTX-I of each group were determined by ELISA,microthrombosis in the bone of the distal femur was observed by H&E and MSB staining,vascular density was determined by ink staining,and three-dimensional morphological reconstruction of the bone trabecula of the distal femur was performed by Micro-CT.Comparison between the two groups was conducted through t-test for statistical analysis,and comparison between multiple groups was conducted through one-way ANOVA and Bonferroni test.Results:The vascular density of Ctrl+NS group and Ctrl+ fondaparinux group were 22.08±1.56%and 23.29±1.60%,respectively.In terms of bone mineral density,the above two groups were 0.397 ± 0.018 g/cm3 and 0.415 ± 0.022 g/cm3,respectively.There was no significant difference between the two groups.Fondaparinux had no significant effect on bone metabolism and vascular density.D-dimer content in FAC+ fondaparinux group was 374.70±53.00 ng/ml,which was significantly lower than that in FAC+NS group(527.62±36.93 ng/ml).The microvessel density and bone mineral density in FAC+fondaparinux group were significantly higher than those in FAC+NS group.Compared with the above two groups in terms of the osteoblast activity index PINP and the osteoclast activity index CTX-I,the FAC+fondaparinux group(PINP:12.03+0.94 ng/ml,CTX-I:79.21+4.88 ng/ml)was significantly improved compared with the FAC+NS group(8.47+0.76 ng/ml,CTX-I:89.89+3.60 ng/ml).By H&E staining and MSB staining,no microthrombosis was found in bone marrow of rats in FAC+fondaparinux group,but microthrombosis was found in FAC+NS group.The Micro-CT parameters of BV/TV,Tb.Th,Tb.N in FAC+NS group were 8.55±1.98%,0.145±0.005 mm,0.610 0.152/mm,respectively,which were significantly lower than those in the FAC+fondaparinux group(BV/TV:15.68±3.32%,Tb.Th:0.197±0.012 mm,Tb.N:0.953±0.096/mm).The separation parameters of bone trabeculae in FAC+NS group(Tb.SP:0.908±0.083 mm)was significantly higher than those in FAC+fondaparinux group(Tb.SP:0.802±0.034 mm).Conclusion:Anticoagulation can reduce bone loss in rats with iron accumulation.Anticoagulants may increase bone formation,reduce bone absorption and improve bone mass by improving microcirculation,increasing microvascular density and inhibiting microthrombosis in rats with iron accumulation.Hypercoagulable status and microthrombosis are important influencing factors of iron accumulation leading to osteoporosis. |
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