| Background and Aim:The classic role of the renin-angiotensin system（RAS）is to keep blood pressure stable.Recently,the existence of RAS has been discovered in bone tissue,and it has been discovered that angiotensin II（Ang II）,the main component of RAS,has the effect of stimulating the formation of osteoclasts and inhibiting the activity of osteoblasts.In animal experiments,RAS blockers can reduce osteoporosis related to the angiotensin receptor 1（AT1）-osteoprotectin（OPG）/nuclear factor-κB ligand（RANKL）signaling pathway.Our previous studies have found that the angiotensin converting enzyme 2/angiotensin（1-7）/Mas receptor（ACE2/Ang-（1-7）/Mas）axis can antagonize the effect of Ang II on systemic blood pressure and local inflammation.effect.Recently,it has been found that both osteoblasts and osteoclasts express ACE2 and Mas.Activating the ACE2/Ang-（1-7）/Ma axis can stimulate the proliferation of osteoblasts and inhibit the activity of osteoclasts,thereby reducing bone resorption and promoting bone remodeling,and improving osteoporosis.In the state of uremic high transformation bone disease,the incidence of osteoporosis and fractures increases significantly.The mechanism is related to secondary hyperparathyroidism,and the activation of local RAS also plays an important role in it.However,the role of angiotensin（1-7）/Mas receptor axis in it is not yet clear.In this study,in vitro and in vivo experiments were conducted to observe the changes of RAS components and the expression of ACE2/Ang（1-7）/Ma in osteoblasts and osteoclasts of uremic rats in the state of secondary hyperparathyroidism.ACE2 activator Diminazene aceturate（DIZE）and Ang1-7receptor Mas inhibitor A779 interfere with the effects of ACE2/Ang-（1-7）/Ma axis on the high transforming bone disease state and results of uremic rats The influence of the biological behavior of bone cells and osteoclasts.Part 1:The effect and mechanism of angiotensin（1-7）on the high turnover bone disease in uremic ratsMethods:5-week-old male S-D rats（body weight 150±10 g）were used 5/6nephrectomy to make a rat model of uremia,and were fed with high-phosphorus diet（1.2%phosphorus,1.0%calcium）after the operation.Rats of the same age were used as the sham group and received a normal phosphorus diet（0.9%phosphorus and1.0%calcium）.At 12 weeks of age,uremic rats were randomly divided into model control group,valsartan group（5 mg/kg/d,gavage）,Ang（1-7）group（200 ng/kg/min,subcutaneously Micro-osmotic pump infusion）,DIZE group（1mg/kg/d,gavage）and A779 group（400 ng/kg/min,infusion via subcutaneous micro-osmotic pump）,a total of 6 groupwith sham group（n=6）.Each group of rats took blood every 2 weeks and kept urine for 24 hours to detect the levels of calcium,phosphorus,PTH and creatinine in blood,and the levels of urine protein in 24 hours.ELISA measures the levels of serum osteocalcin,type I collagen cross-linked C-terminal peptide（NTX）and anti-tartrate acid phosphatase 5b（TRAP-5b）.Tetracycline was injected intraperitoneally once at the age of 17 weeks,and the rats were sacrificed at the age of 18 weeks.The renal pathological changes of the rats in each group were observed and compared.Take the femur and use a micro-CT scanning system to check the bone mineral density（BMD）of the trabecular bone in the proximal region of the femur.Fix and embed the femur,slice it with a heavy-duty microtome and stain with Giemsa and Von Kossa,observe the morphological changes of trabecular bone and cortical bone under a microscope,take pictures and calculate the trabecular bone volume（TBV）,osteoid volume（OV/TV）,Osteoblast and osteoclast index（OBI and OCI）,fluorescence microscope observation of bone mineralization rate（MAR）changes.Results:（1）At 18 weeks of age,none of the rats in each group of uremia died,but the weight was significantly lower than that of the sham operation group（P<0.001）.18-week-old uremic rats showed obvious proteinuria,increased blood creatinine and PTH,abnormal blood calcium,phosphorus,osteocalcin,NTX,and TRAP-5b levels（all P<0.05）,and changes in rats above 18 weeks More serious（P<0.05）.Among the five groups of uremic rats,there was no significant difference in the levels of blood PTH,calcium,phosphorus,osteocalcin,and TRAP-5b（P>0.05）;compared with the uremic model group,the valsartan group,Ang（1-7）The levels of proteinuria,serum creatinine,NTX,and TRAP-5b in group 1-7)and DIZE group were lower（P<0.05）.On the contrary,the levels of proteinuria,serum creatinine,NTX and TRAP-5b in group A779 were lower Higher than the model group（P<0.05）.（2）Micro-CT examination showed that the BMD values of trabecular bone in the proximal femur of the five groups of uremic rats were significantly lower than those in the sham operation group（P<0.05）.Among them,the Ang（1-7）group and the DIZE group The BMD value of rats was the highest,and the BMD value of rats in the A779group was the lowest,and the differences were statistically significant（P<0.05）.（3）Bone pathological examination showed that the values of TBV,OBI,OCI and MAR of trabecular bone in the model group were 8.0±3.2,25.1±5.4,14.3±4.4,and5.3±2.1,respectively,while in the Ang（1-7）group The values of rats in the and DIZE groups were 6.3±3.1,7.3±2.2,30.8±5.0,28.2±3.9,8.1±2.7,9.2±3.3,6.7±3.8,and 7.2±1.9.In the A779 group,their values were 8.1±1.3,25.5±6.1,23.1±8.1,1.93±0.13.Compared with the model group,the OCI of the Ang（1-7）group and the DIZE group was significantly decreased（all P<0.05）,and the A779 group was significantly increased（P<0.01）,but the differences in other indicators were not statistically significant（P>0.05）).Conclusions:Ang（1-7）or DIZE can inhibit the excessive activation of trabecular osteoclasts in uremic rats with highly transforming bone disease.This effect can be inhibited by the Mas receptor antagonist A779.The part 2:The effect and mechanisms of angiotensin（1-7）on the function of osteoblasts and osteoclasts cultured in vitro under high PTH stateMethods:6-week-old male SD rats received 5/6 nephrectomy and a high-phosphorus diet（1.2%phosphorus,1.0%calcium）for 6 weeks to make a uremic high-transformation bone disease group model.Sham rats of the same age fed with normal phosphorus diet（0.9%phosphorus and 1.0%calcium）served as the control group.Rats were sacrificed and tibias were taken,and BM-MSCs were cultured to induce differentiation into osteoblasts using bone fragment adherence separation and screening method;bone marrow cells were taken to culture bone marrow macrophages by density gradient centrifugation,M-CSF（40 ng/ml）and RANKL stimulated to differentiate into mature osteoclasts.At the same time,osteoblast MC3T3-E1 cell line and osteoclast precursor RAW 264.7 cell line were cultured in vitro.After PTH(10-10M)stimulation,angiotensin II(10-7M),Ang II+valsartan(10-6M),Ang（1-7）(10-7M),Ang（1-7）+A779 were adminstrted to observe the expression of RUNX2,ALP,COL-1,OCN,and the activity of Akt,P38 MAPK,JNK and ERK signaling molecules.Results:（1）In the absence of PTH,Ang II and Ang（1-7）respectively reduced and increased the number of calcification nodules and alkaline phosphatase activity during the differentiation of primary BM-MSCs,and stimulated and inhibited,respectively RANKL-induced osteoclast formation in bone marrow macrophages can be completely antagonized by valsartan or A779,respectively.Ang II and Ang（1-7）down-regulated and up-regulated the expression of RUNX2,ALP,COL-1,OCN in MC3T3-E1 cells,respectively,and promoted and inhibited the expression of cathepsin K and TRAP in RAW 264.7 cells,respectively.Ang II rapidly increased the phosphorylation of Akt and p38 in RAW 264.7 cells,while Ang（1-7）significantly reduced the phosphorylation of p38 and ERK.（2）In the presence of PTH,the differentiation of primary BM-MSCs into osteoblasts and the differentiation of bone marrow macrophages into osteoclasts were significantly enhanced.Although Ang II and Ang（1-7）did not affect the differentiation of BM-MSC and the expression of RUNX2,ALP,COL-1,and OCN in MC3T3-E1 cells,Ang II also failed to stimulate bone marrow macrophages to osteoclasts.The differentiation process is further accelerated,or the expression of cathepsin K and TRAP in RAW 264.7 cells is stimulated to further increase,but Ang（1-7）shows a significant inhibitory effect on the activation of the osteoclast ERK signaling pathway,which has a significant impact on the above process.And the effect of Ang（1-7）can be antagonized by A779or ERK signal agonist C16-PAF,but cannot be antagonized by P38 MAPK,JNK agonist Hesperetin and Anisomycin.Conclusion:Ang II/AT1R and Ang（1-7）/Mas have opposite effects on osteoblasts and osteoclasts cultured in vitro;in the presence of high PTH,The effect of Ang II/AT1R and Ang（1-7）/Mas on osteoblasts was significantly weakened,but Ang（1-7）/Mas and its downstream ERK signal retained its inhibitory effect on osteoclasts. |
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