| Objective1.To determine whether ferroptosis is involved in the pathogenesis of diabetic osteoporosis(DOP)based on animal model;2.Clarify the role of baicalein in inhibiting ferroptosis and preventing DOP in vivo;3.To explore the mechanism of baicalein inhibiting the ferroptosis of BMSCs in vitro via SLC7A11/GPX4 axis.Methods1.Animal Experiments(1)The role of ferroptosis in the pathogenesis of DOPSD rats were randomly divided into three groups: baseline group,control group and model group.The rats in the model group were fed with high-fat diet for 4 weeks,and then given a single intraperitoneal injection of STZ at a dose of 30mg/Kg body weight to establish a type 2 diabetes model.The rats' body weight,water intake and food intake,and fasting blood glucose(FBG),rats were sacrificed after 8 weeks.Indicators were examined as follows:(1)The body weight,water intake and food intakeCompare the weight,water intake and food intake of rats between the control and model groups.(2)Blood glucose and glycosylated hemoglobinFBG and glycated hemoglobin(GHb Ac1)levels in the tail vein of rats were tested at multiple time points.(3)Lumbar bone massLumbar 1-3(L1-3)vertebra were measured using dual-energy X-ray absorption method for bone mineral density(BMD).(4)The histomorphology of lumbar vertebraeAfter BMD measurement of L1-3 vertebrae,L2 vertebra in(3)were separated and stained with hematoxylin-eosin(HE)to observe histomorphological changes.(5)The microstructure of lumbar vertebraeAfter the BMD measurement of L1-3 vertebral of rat,the L3 vertebra in(3)was separated,and micro-CT was used to scan the bone microstructure to obtain the relevant parameters.(6)Bone biomechanics testAfter the BMD measurement of L1-3 vertebral of rat,the L1 vertebra in(3)was separated,and the compression test was carried out with a universal material testing machine;the rat femur was subject to the three-point bending test using a universal material testing machine.Relevant parameters were obtained respectively.(7)The level of oxidative stress and iron ionRat blood was centrifuged to obtain serum,and the serum total superoxide dismutase(t SOD)activity was determined;rat lumbar vertebral bone tissue was taken,grinded and lysed to obtain tissue lysate samples;tissue MDA and total iron ion levels were measured with kits.(8)Expression of ferroptosis-related proteinCryopreserved lumbar vertebral bone tissues of rats were taken,grinded and lysed to obtain total protein.Proteins of COX2,VDAC2,FTH1,SLC7A11,and GPX4 were detected by Western blot.(2)The preventive and therapeutic effects of baicalein on DOP of ratsSD rats were randomly divided into three groups: blank control group,model group and baicalein group.The model group and the baicalein group were fed with high-fat diet for 4weeks,and then a single intraperitoneal injection of STZ(30mg/Kg body weight)was given to establish type 2 diabetes model.The two groups were divided into 2 parts for prevention experiment and treatment experiment.(1)The effect of baicalein on blood glucoseFBG was detected at rat tail vein;oral glucose tolerance test(OGTT),namely blood glucose levels at 0h,0.5h,1h and 2h,were tested;serum GHb Ac1 level was measured by ELISA.(2)The effect of baicalein on bone massL1-3 was taken out and BMD was measured using dual-energy X-ray absorptiometry.(3)The effect of baicalein on bone biomechanicsAfter the L1-3 vertebral BMD of the rat was measured,the L1 vertebra was separated,and the compression test was carried out with the universal material testing machine;the rat femur was used for the three-point bending test using the universal material testing machine.Relevant parameters were obtained respectively.(4)The effect of baicalein on bone microstructureAfter the BMD of the L1-3 vertebral body of rat was measured,the L3 vertebral body was separated,and the micro-CT was used to scan the bone microstructure to reconstruct and analyze the relevant parameters.(5)The effect of baicalein on bone tissue histomorphologyAfter BMD measurement of L1-3 vertebral bodies in middle rats,L2 vertebral body were separated,and hematoxylin-eosin(HE)staining was performed to observe the histomorphological changes.(6)The effect of baicalein on oxidative stressThe blood was centrifuged to obtain serum,and serum total superoxide dismutase(t SOD)activity was determined;the rat lumbar vertebrae tissue was taken,lysed after grinding to obtain a tissue lysate sample and the tissue MDA level was measured with a kit respectively.(7)The effect of baicalein on osteoblast and osteoclast related factorsSerum OPG,RAKNL,TRAP5 b levels were detected by ELISA.(8)The effect of baicalein on the expression of SLC7A11/GPX4 axis proteinCryopreserved lumbar vertebra tissues of rats were taken out,grinded and lysed to obtain total protein,and the expression levels of SLC7A11 and GPX4 protein were detected by Western blot.2.Cell Experimental ResearchBone marrow mesenchymal stem cells(BMSCs)were extracted from the femurs or tibias of SD rats aged 3-4 weeks,cultured,and passaged,and P3-5 were used for the experiments.(1)The effects of baicalein and acetylcysteine on the proliferation of BMSCsBMSCs were seeded in 96-well plates,and the CCK-8 method was used to detect the proliferation of BMSCs at 1,3,5,and 7 days under different concentrations of baicalein and acetylcysteine(NAC).(2)Erastin induces lipid peroxidation and ferroptosis of BMSCsLevels of malondialdehyde(MDA)produced by BMSCs at different concentrations of Erastin were detected;CCK-8 was used to detect the survival rate(ferroptosis)of BMSCs at different concentrations of Erastin.(3)The effect of baicalein on lipid peroxidation of BMSCs and intracellular iron level under Erastin treatmentMDA produced by BMSCs and intracellular total iron levels under the intervention of baicalein was detected.(4)The effect of baicalein on the survival rate of BMSCs under Erastin treatmentThe survival rate(ferroptosis)of BMSCs under the intervention of baicalein was assayed.(5)The effect of baicalein on the proliferation of BMSCs under Erastin treatmentThe proliferation of BMSCs under the intervention of baicalein at 1,3,and 5 days were measured.(6)The effect of baicalein on the osteogenic differentiation of BMSCs under Erastin treatmentALP staining was used to observe the osteogenic differentiation of BMSCs under the intervention of baicalein.(7)The effect of baicalein on SLC7A11/GPX4 axis protein expressionThe expression of SLC7A11/GPX4 axis was detected by western blot.Results1.Animal Experimental Research(1)The role of ferroptosis in DOP pathogenesis(1)After establishing the model at rats by STZ,the FBG of the model group was significantly higher than that of the control group(P<0.01 or P<0.001)at several time points,and the body weight was significantly reduced(P<0.001).(2)BMD of the lumbar spine in the model group was significantly lower than that in the control group(P<0.01).(3)The histomorphology of lumbar vertebrae in the model group was significantly changed.The number of trabecular bone decreased,the thickness of trabecular bone became thinner,and the fat in the bone marrow increased obviously.(4)The relative bone volume(BV/TV),trabecular bone thickness,and trabecular bone number in the model group were significantly reduced(P<0.05,P<0.01 and P<0.05,respectively).(5)Compared with baseline,the maximal load and crack load of lumbar spine was were elevated(P<0.01)in control group.The maximal load,crack load and elasticity modulus of lumbar spine were decreased with an obviously trend in model group,and the maximal load of femur decreased significantly(P<0.05)compared with that in control group.(6)The serum OPG level in model group was significantly reduced(P<0.05),and RANKL and OPG/RANKL had a slight decreasing trend compared with control group.(7)The serum t SOD activity in the model group was significantly reduced(P<0.05);the bone tissue MDA and total iron ion levels were significantly increased(both P<0.05)compared with control group.(8)In model group,the expression of ferroptosis-related protein in bone tissue,including COX2 which was significantly up-regulated(P<0.01),and SLC7A11,GPX4,and FTH1 which were all significantly down-regulated(P<0.05,P<0.05,P<0.01,respectively)relative to that in control group.(2)Baicalein inhibits ferroptosis and prevents DOP(1)After modeling with STZ,the FBG of the model group and the baicalein group was significantly higher than that of the blank control group(all P<0.001),and there was no significant difference between model group and baicalein group at several timepoints.(2)In the prevention experiment,BMD of the lumbar spine in model group was significantly lower than that of blank control group(P<0.01),and the baicalein group was significantly higher than that of model group(P<0.05).In the treatment experiment,the BMD of the model group was significantly lower than that of the blank control group(P<0.05),and that in baicalein group was not statistically lower than that of the blank control group.(3)In the prevention experiment,the maximal load of the lumbar vertebra in model group was significantly lower and the compression displacement was significantly higher than that of the blank control group(both P<0.01).The maximal load of the lumbar spine in baicalein group was lower than that of blank control group with no statistical significance.The maximal stress of the femur in both model group and baicalein group were profoundly reduced(P<0.001 and P<0.05 respectively)compared with the blank control group,but that in baicalein group was notably higher than that in model group(P<0.05).In the treatment experiment,the maximal load and crack load of the lumbar vertebra in model group were significantly reduced(both P < 0.05),and baicalein group showed no significant difference from model group.The maximal load of the femur in model group was significantly lower than that in blank control group(P<0.05).(4)In the prevention experiment,BV/TV,Tb.N and Tb.Th of the lumbar spine in model group were significantly lower than those in blank control group(all P<0.01),whereas Tb.Sp was significantly increased(P < 0.05).Compared with model group,Tb.N was significantly increased at baicalein group and Tb.Sp decreased(both P<0.05)in baicalein group.In the treatment experiment,BV/TV and Tb.Th of the lumbar vertebra in model group were significantly lower than that in blank control group(all P<0.01),whereas Tb.Sp was significantly increased(P<0.01),and baicalein group showed no significant difference in all these metrics compared with model group.(5)In the prevention experiment,the trabecular bone of the lumbar vertebra in model group was obviously thinner than blank control group,and the trabecular bone in baicalein group was significantly thicker than in model group.In the treatment experiment,compared with blank control group,the lumbar trabecula in model group was significantly thinner and the bone marrow fat increased,and the bone tissue histomorphology of the baicalein group was not significantly different from that of model group.(6)In prevention experiment,serum GHb Ac1 in both model group and baicalein group were significantly elevated(P<0.05 and P<0.01 respectively)compared with blank control group.(7)In prevention experiment,the serum t SOD activity in model group was significantly lower than that in blank control group(P<0.05),and that in baicalein group was significantly higher than that in model group(P<0.001).The MDA level of bone tissue in model group was significantly higher than that in blank control group(P<0.01),and that in baicalein group was lower than that in model group with a margin significance(P=0.054).(8)In prevention experiment,the serum OPG in model group was significantly lower than that in blank control group(P<0.01),and baicalein group had a slight up-regulation trend compared with model group.The RANKL in model group has a decreasing trend compared with that in blank control group,and baicalein group has no significant difference compared with model group.Compared with blank control group,TRAP5 b in the model group has an up-regulation trend,and baicalein group has a down-regulation trend compared with model group.(9)In the prevention experiment,compared with blank control group,the expression of COX2 protein in bone tissue was profoundly up-regulated(P<0.01)in model group,but was down-regulated with an obviously trend in baicalein group relative to model group.The GPX4 expression in model group was significantly down-regulated(P<0.01)relative to blank control group,but significantly up-regulated in baicalein group relative to model group.2.Cell experiments(1)Baicalein at 1,2 and 4?M has no effect on the proliferation of BMSCs within 7 days,and can significantly inhibit the proliferation of BMSCs at 8 and 16?M(P<0.01 and P<0.001,respectively).NAC at 1,2 m M had no effect on the proliferation of BMSCs within 7days.At Day1,NAC at 8 m M can significantly promote the proliferation of BMSCs(P<0.01),and 16 m M can significantly inhibit the proliferation of BMSCs(P<0.01);at Day5,NAC at both 8 and 16 m M can significantly inhibit the proliferation of BMSCs(P<0.05 and P < 0.001,respectively);at Day7,NAC at 4,8,16 m M can significantly inhibit the proliferation of BMSCs(P<0.05,P<0.01,P<0.001,respectively).(2)Upon Erastin at 10,20,30 and 40 ?M,the MDA of BMSCs increased significantly(P<0.05,P<0.001,P<0.0001 and P<0.0001,respectively).And cell viability was notably deceased upon Erastin at 20,30 and 40 ?M(P<0.05?P<0.05 and P<0.01 respectively).(3)Upon Erastin(20?M),baicalein(4?M)and NAC(2m M)can significantly(both P<0.001)reduce MDA level of BMSCs and total iron ion levels(P<0.01 and P<0.001respectively).(4)The cell viability of BMSCs increased significantly treated with at baicalein at 4 and8?M upon Erastin at 20 ?M(P<0.05 and P<0.001,respectively).(5)Upon Erastin(10?M),baicalein(4?M)can significantly improve the proliferation of BMSCs at Day3 and Day5(P<0.05 and P<0.001 respectively).(6)Upon Erastin(4?M),baicalein can promote the osteogenic differentiation of BMSCs to some extent.(7)Erastin(10?M)significantly down-regulated SLC7A11 and GPX4(P<0.01 and P<0.01,respectively).Baicalein significantly up-regulated the expression of SLC7A11 and GPX4 inhibited by Erastin(P<0.05 and P<0.001).ConclusionThe in vivo experiments revealed that ferroptosis was involved in the pathogenesis of DOP;baicalein could effectively prevent the occurrence of DOP,and its mechanism is related to the inhibition of ferroptosis via up-regulating SLC7A11/GPX4 axis.The in vitro experiments demonstrated that baicalein can inhibit BMSCs ferroptosis via up-regulating SLC7A11/GPX4 axis,and promote the proliferation and osteogenic differentiation of BMSCs.Taken together,baicalein can prevent DOP,which is associated with the inhibition of BMSCs ferroptosis via up-regulating SLC7A11/GPX4 axis. |
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