| Postmenopausal osteoporosis(PMOP)is the most common age-related bone mass loss disease associated with estrogen deficiency.It is characterized by increased bon eturnover rate,reduced bone mass,bone microstructural destru-ction,increased bone fragility,and prone to fracture.Like hypertension,hyp-ercholesterolemia,it has become acommon health problem in the world.Growth hormone(GH)has a significant impact on people,mainly manifested in the growth of bone length in youth and the maintenance of bone mass in a-dulthood.While insulin-like growth factor-1(IGF-1)has equally important for bone metabolism which was producted in liver by GH stimulated.GH and IGF-1 bind to their GH receptors(GHR)and IGF-1 receptors(IGF-1R)on osteob 1a-sts,respectively,to stimulate osteoblast proliferation and differentiatio-n,thereby increasing bone mineral density(BMD).Estrogen(E)can cause proli-feration and differentiation of GH cells during pituitary development and maintain the number of pituitary GH cells in adulthood.Studies have found that hormone replacement therapy(ERT)can improve femoral BMD and serum GH and IGF-1 levels in ovariectomized osteoporosis rats.On the one hand,E may dire-ctly bind to hypothalamic estrogen receptor(ER),indirectly increase the secr-etion of GH by increasing the release of growth hormone releasing hormone(GHRH),on the other hand,it may directly affect the growth hormone cells in the anterior pituitary gland and thus increase the secretion of the GH.E2 S-timulates the osteoblast producting IGF-1,cell proliferation by the resul-ting of E2 can be abolished by IGF-1 antibodies,so,E2 effects on osteoblasts may be mediated by IGF-1.The correlation between estrogen and estrogen rece-ptor with osteoporosis has been a hot topic in scholars all over the world.It-is well known that the causes of postmenopausal osteoporosis are mostly a-ttributed to the decline of estrogen,but long-term estrogen replacement t-herapy is not routinely used in clinical practice because the disadvantages outweigh the benefits.T herefore,it is particularly important to study the specific signaling pathway mechanism of estrogen affecting bone metabolism in the prevention and treatment of osteoporosis.while the GH/IGF-1 axis may be an important signaling pathway for estrogen to affect bone metabolism,the specific role of the GH/IGF-1 axis in PMOP development remain controversia-1.The study of GH/IGF-1 axis in postmenopausal women is significant based on the important role of IGF-1 and GH in bone metabolism.Traditional Chinese medicine has certain advantages in the treatment of osteoporosis,Bushen Zhuanggu granule is the hospital preparation of the the General Hospital of Southern Theatre Command.It is obtained by Professor Deng Weimin according to the experience of clinical syndrome for many years,acc-ording to the pathogenesis of osteoporosis,according to the theory of TCM syndrome differentiation and treatment.In the treatment of postmenopausal os-teoporosis with Bushen Zhuanggu granule,our team conducted a lot of research from cell to animal experiment to clinical trial.The study confirmed that Bus-hen Zhuanggu granule can significantly relieve the clinical symptoms of pos-tmenopausal osteoporosis patients,and increase the bone mineral content(BMC)and BMD,reduce the occurrence of new fracture rate in patients with postmen-opausal osteoporosis.but the specific mechanism of its action still needs further study.If we can enrich the understanding of the pathogenesis of PM-OP,deepenthe understanding of the mechanism of action of Bushen Zhuanggu gr-anules,provide molecular mechanism support for the modernization of Bushen Zhuanggu granules,Which can provide more basis for the clinical application and promotion of Bushen Zhuanggu granules.The objectives,methods,results and conclusions of this research subject are described as follows:ObjectiveTo analyze the correlation between serum GH/IGF-1 and BMD in postmenopa-usal women.To observed the effects of Bushen Zhuanggu granule on serum IGF-1 and GH expression,the expression IGF-1R and GHR in bone tissue,immunohistoche mical optical density and GH immuse positive cell number in ovariectomized rats.MethodsNol.Clinical research:To collect and record the results of peripheral blood examination of GH,IGF-1,25(OH)·D3,PTH,PINP,β-CTX,BGP,Ca,and E2,and BMD with its T-score from clinical subjest,to study the correlation between GH and IGF-1 and BMD of postmenopausal women,and to do the ROC diagnostic test curve to analyze the cut-off value and significance of GH,IGF-1 as the diagn-osis of osteoporosis and osteopenia.No2.Animal research:using ovariectomized rats as a model,using Bushen Zhuang gugranule as a tool drug,using DEXA to measure BMD,ELISA to detect serum IGF-1 and GH concentration,qPCR to determine the relative expression levels of GHR and IGF-1R in bone tissue,pituitary GH immunohistochemical staining to analyze the optical density value and positive cell count,the study will clarify the effect of Bushen Zhuanggu granule on BMD of ovariectomized rat model maybe by the GH/IGF-1 axis.Results1 Relationship between bone mineral density and serum IGF-1 and GH expression in postmenopausal women1.1 Comparison of general clinical data① The age comparison showed that:the normal group was lower than the osteopenia group and the osteoporosis group(P<0.05 for all),and there was no statistical difference between the osteopenia and the osteoporosis group(P>0.05).② BMI,25(OH)D3 and PTH comparison showed that:there was no statistical difference between the normal group and the osteopenia group(P>0.05 for all);compared with the normal group and the osteopenia group,the BMI and 25(OH)D3 of the osteoporosis group decreased and the PTH increased(P<0.05 for all).③ β-CTX and P Ⅰ NP comparison showed that:there was no statistical di-fference between the normal group and the osteopenia group(P>0.05 for all);compared with the normal group,the β-CTX and P Ⅰ NP of the osteoporosis group increased(P<0.05 for all);and there was no statistical difference between the osteopenia group and the osteoporosis group(P<0.05 for all).④ BGP and Ca comparison showed that:there was no statistical difference between the three groups(P>0.05 for all).⑤The comparison of duration since menopause,E2,IGF-1,GH,lumbar spine,f-emoral neck,Proximal femur’s BMD and T-score showed that the normal group was higher than the osteopenia group(P<0.05 for all),and the osteopenia group was higher than the osteoporosis group(P<0.05 for all).1 2 Correlation analysis of IGF-1 and GH expression levels and related vari-ables in serum① Serum IGF-1 levels were negatively correlated with age,duration since menopause,PINP,PTH(r=-0.249,P=0.002;r=-0.271,P<0.001;r=-0.117,P=0.029;r=-0.129,P=0.017,respectively).and positively correlated with BMI,E2,G-H,the BMD of lumbar spine(L1-4),femoral neck,Proximal femur and their T-sco-re(r=0.130,P=0.016;r=0.286,P<0.001;r=0.128,P=0.018;r=0.484,P<0.001;r=0.479,P<0.001;r=0.4 09,P<0.001;r=0.403,P<0.001;r=0.430,P<0.001;r=0.432,P<0.001,respectively).② Serum GH levels were negatively correlated with age,duration since menopause,BMI,PTH(r=-0.153,P=0.004;r=-0.16,P=0.003;r=-0.124,P=0.022;r=-0.120,P=0.025,respectively).and positively correlated with with E2,IGF-1,1-umbar spine(L1-4),femoral neck,Proximal femur and their T-score(r=0.110,P=0.041;r-0.128,P=0.018;r=0.222,P<0.001;r=0.216,P<0.001;r=0.187,P<0.001;r=0.188,P<0.001;r=0.143,P<0.001;r=0.138,P<0.001,respectively).1.3 Partial correlation of IGF-1 and GH expression in serum after adjust age,BMI and duration since menopause with related variables① Serum IGF-1 expression level was negatively correlated with PTH,PIN-P(r=-0.139,P=0.010;r=-0.339,P<0.001,respectively),and positively corre-lated with E2,GH,lumbar spine(L1-4),femoral neck,Proximal femur and their T-s-core(r=0.234,P<0.001;r=0.108,P=0.047;r=0.416,P<0.001;r=0.412,P<0.001;r=0.323,P<0.001;r=0.317,P<0.001;r=0.348,P<0.001;r=0.353,P<0.001,re-spectively).② Serum GH expression levels were negatively correlated with BGP(r=0.111,P=O.031)and positively correlated with IGF-1,lumbar spine(L1-4),femoral neck,Proimal femur and their T-score(r=0.108,P=0.047;r=0.203,P<0.001;r=0.199,P=0.001;r=0.171,P=0.001;r=0.174,P=0.001;r=0.132,P=0.014;r=0.127,P=0.019,respectively).1.4 ROC Curve Assessment① ROC curves assess the value of serum IGF-1 in the diagnosis of osteope-nia or osteoporosis:the critical value of serum IGF-1 levels for osteopenia was 73.35ng/mL,the sensitivity and specificity were 69.5%and 83.8%,respect-ively,and the area under the ROC curve was 0.822,P<0.001 indicates that the diagnostic indicator is available.The critical values of serum IGF-1 levels in osteoporosis were 69.74 ng/ml,sensitivity and specificity were 86%and 72.6%,respectively.The area under the roccurve was 0.826,and P<0.001 indic-ated that the diagnostic index was valid.② ROC curves assess the value of serum GH in the diagnosis of osteopenia or osteoporosis:the critical value of serum IGF-1 levels for osteopenia was 0.647ng/mL,the sensitivity and specificity were 95.8%and 13.7%,respective-ly,and the area under the ROC curve was 0.563,P>0.05 indicated that the diagn-ostic index was.The critical values of serum GH levels in osteoporosis were 0.517ng/ml,sensitivity and specificity were 97.8%and 16,3%,respectively,and the area under the roccurve was 0.563,P>0.05,indicating that the diagnostic index was invalid.2 Effect of Bushen Zhuanggu Granules on BMD of ovariectomized rat by GH/IGF-1 axis2.1 Weight comparisonDuring the experiment,no rats died,and the weight of rats in each group increased b-efore treatment.At 3 months and 6 months,there was no statistical difference in weight(P>0.05 for all).2.2 BMD comparisonComparison the BMD of femur in rats:①After 3 months intervention:the OVX group decreased compared with SHAM group(P<0.05),while BSZG group increased compared with OVX group and E2 group(P<0.05 for all),and there was no statistical difference when compared with SHAM group(P>0.05).②After 6 mon-ths:compared with SHAM group,OVX group decreased(P<0.05);BSZY group and E2 group were higher than the SHAM group(P<0.05 for all),and there was no st-atistical difference between the two drugs intervention groups(P>0.05).Comparison the BMD of vertebral in rats:①After 3 months intervention:OVX group decreased compared with SHAM group(P<0.05);Compared with the OVX grou-p,BSZG group and E2 increased(P<0.05 for all),and there was no statistical difference when compared with the SHAM group(P>0.05).After 6 months int-ervention:0-VX group decreased compared with SHAM group(P<0.05);BSZG group and E2 group were higher than the OVX group(P<0.05 for all),but there was no statistical difference between the two drug groups(P>0.05).2.3 Comparison of expression levels of GHR and IGF-1RThe relative expression of GHR in the left tibia of rats was compared:①after 3 months intervention,the VOX group decreased compared with the SHAM group(P<0.0 5),and the two drug intervention groups were higher than the SHAM group and the OVX group(P<0.05 for all),but there was no statistical diff-erence between the two drug intervention groups(P<0.05).②After 6 months,t-he OVX group decreased compared with SHAM group(P<0.05),two drug interve-ntion groups were higher than SHAM group and OVX group(P<0.05 for all),but BSZG group was higher than E2 group(P<0.05).The relative expression of IGF-1R in the left tibia of rats was compared:①after 3 months intervention,the OVX group decreased compared with the SHAM group(P<0.05),the two drug groups increased compared with OVX group(P<0.05 for all),and there was no statistical difference between the two drug interv-ention groups(P<0.05).②After 6 months intervention,the OVX group decreased compared with SHAM group(P<0.05),and the two drug intervention groups were higher than OVX group(P<0.05 for all),but the difference between the two inter-vention groups was not statistically significant(P>0.05).2.5 Optical density of pituitary immunostaining and GH immuse positive cell countThe rat pituitary immunohistochemical staining under electron microsco-py:(10 times and 20 times under electron microscopy)the anteri of pituitary is located in the outermost layer and the innermost layer is the nerve of pitu-itary,while the middle part of the pituitary is located between the anterior lobe and the nerve.(40 times under electron microscopy)Growth hormone cells were distributed in the anterior lobe,in the shape of strips or lumps,and brown to varying degrees after staining.The brown degrees show as following:OVX gr-oup<SHAM group<E2 group<BSZG group.The brown of 3 months intervention was deeper than that of the 6 months intervention.Number of positive cells in each group:OVX group<SHAM group<E2 group<BSZG group.Comparison the optical density value of pituitary immunostaining:①After 3 months intervention,compared with the SHAM group,the OVX group decreased(P<0.05);both the BSZG group and the E2 group increased(P<0.05 for all).T-here was no statistical difference between the two drug intervention groups(P>0.05).②After 6 months,the OVX group decreased compared with the SHAM gr-oup(P<0.05),the BSZG group and the E2 group were higher than that in SHAM group(P<0.05 for all)and there was no statistical difference between the two drug intervention groups(P>0.05).GH immuse positive cell number comparison:①after 3 months interventio n,the OVX group decreased compared with SHAM group(P<0.05);both BSZG group and E2 group increased compared with OVX group(P<0.05 for all),and there was no statistical difference between the two drug intervention groups(P>0.05).②After 6 months,the OVX group decreased(P<0.05)compared with the SHAM gr-oup,and the BSZY group and E2 group were higher than the OVX group(P<0.05),but there was no statistical difference between the two groups(P>0.05).2.6 Analysis the correlation of expression levels of GH,IGF-1 and its related recepto rs with BMD① Serum GH and IGF-1 concentrations,and their receptor GHR and IGF-1R in bone tissue expression levels were positively correlated with femur BMD(r=0.725,P<0.001:r=0.469,P=0.001;r=0.660,P<0.001;and r=0.648,P<0.001,resp-ectively).② Serum GH and IGF-1 concentrations,and their receptor GHR and IGF-1R in bone tissue expression levels were positively correlated with vertebral BMD(r=0.707,P<0.001;r=0.429,P=0.002;r=0.573,P<0.001;and r=0.630,P<0.001,respectively).2.7 Analysis the correlation between optical density value,positive cell count and serum GH levelSerum GH concentrations were positively correlated with the optical de-nsity values and he number of positive cells(r=0.721,P<0.001;r=0.829,P=0.001,respectively).Conclusions① From the clinical sample statistics,it was confirmed that GH and IGF-1 were significantly reduced in serum of postmenopausal osteoporosis and ost-eopenia patients,and positively correlated with BMD,indicating that they may be involved in the pathological process of PMOP patients.Besides,GH and IGF-1 had positive correlation with serum E2 level,and may play a certain role in the regulation of bone turnover level.② Through ROC curve analysis,we can draw a conclusion that serum IGF-1 may beused as a serum marker to assist in the diagnosis of postmenopausal o-steopenia and osteoporosis.③ Bushen Zhuanggu granule may improve the serum GH expression level by increasing the proliferation and secretion function of pituitary GH cells,and then improve synthesis of IGF-1 in liver.In addition,it improve the expression of GHR and IGF-1R in bone tissue,thereby increasing the bone density in ov-ariectomized rats. |
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