| Background:Patients with exogenous or endogenous hypercortisolemia have different degrees of osteoporosis and are prone to fracture.Patients with Cushing’s syndrome suffer from a high risk of fracture,followed by the slow recovery of bone density after remission,putting patients at high risk of fracture.Therefore,it is imperative to explore the causes and practical prevention and treatment of abnormal bone metabolism in patients with Cushing’s syndrome.Previous studies have suggested that osteoporosis caused by Cushing’s syndrome is due to glucocorticoid acting on glucocorticoid receptors,affecting bone metabolism.Mineralocorticoid receptor distribution studies have shown that it is expressed in osteoblasts,osteoclasts,and osteocytes,and primary and clinical studies have shown that mineralocorticoid receptor plays a role in abnormal bone metabolism.Due to the high homology of glucocorticoid receptor and mineralocorticoid receptor,when blood cortisol levels rise,11β-HSD2 in local tissues fail to fully convert a large amount of glucocorticoid into inactive cortisone,under that circumstance,a large number of glucocorticoid can bind to mineralocorticoid receptor of the target cell,resulting in clinical manifestations of increased mineralocorticoid such as hypertension,hypokalemia,and metabolic alkalosis.It is unclear that whether inappropriate activation of target cell mineralocorticoid receptor is involved in the development and progression of osteoporosis associated with chronic hyperglucocorticosteremia disease.Our study involved the hospitalized patients with endogenous Cushing’s syndrome and primary aldosteronism in our hospital.The retrospective study method was adopted to collect the bone mineral density and related bone metabolism index of these two groups of patients,to understand the incidence of osteopenia/osteoporosis in patients and analyze the related risk factors.On this basis,in order to determine whether inappropriate activation of mineralocorticoid receptor is involved in abnormal bone metabolism caused by hyperglucocorticoidemia and to understand its pathophysiological mechanism,the exogenous chronic hyperglucocorticoidemia rat model was established to explore the effect of hyperglucocorticoidemia on bone metabolism of rats after blocking the effect of mineralocorticoid receptor.This study may provide a new idea for the pathogenesis,prevention and treatment of glucocorticoid-induced osteoporosis.Methods:Part Ⅰ:By retrospective analysis,125 patients with Cushing syndrome diagnosed in the Department of Endocrinology,West China Hospital of Sichuan University from January 2012 to September 2020 were included.We collected the general clinical data of these patients,including genders,age,height,weight,blood pressure,duration,present illness histories,past medical histories,menstrual histories,marital histories and laboratory test results such as routine blood,liver function,kidney function,coagulation function,blood lipid,blood glucose,stool and urine routine examination,blood and urine electrolyte examination,plasma cortisol levels at 8 a.m and 24 p.m,adrenocorticotropic hormone,24 hours urinary free cortisol,parathyroid hormone,25 hydroxy vitamin D,bone alkaline phosphatase,type Ⅰ collagen carboxyl terminal peptide,bone mineral density,imaging and pathological data.Functional tests include dexamethasone inhibition test results,DDAVP stimulation test results and sublithic sinus blood sampling+DDAVP stimulation test results.According to the diagnostic criteria,we assessed the prevalence of osteopenia/osteoporosis and analyzed the risk factors of bone metabolism abnormalities in Cushing syndrome patients.At the same time,we also analyzed whether there are differences in abnormal bone metabolism among patients with different types of endogenous Cushing syndrome.Part Ⅱ: By retrospective analysis,360 patients with primary aldosteronism admitted to the Department of Endocrinology,West China Hospital of Sichuan University from January 2012 to September 2020 were included.We collected the general clinical data of these patients,including gender,age,height,weight,blood pressure,duration,present illness history,past medical history,menstrual history,marital history and laboratory test results such as routine blood,liver function,kidney function and urine electrolyte examination,plasma aldosterone concentrations,plasma renin activity,parathyroid hormone,25 hydroxy vitamin D,bone alkaline phosphatase,type Ⅰ collagen carboxyl terminal peptide,bone mineral density,imaging and pathological data.The functional test results included an intravenous saline load test and a captopril challenge test.We analyzed the bone metabolism indexes and bone mineral density and evaluated the prevalence of osteoporosis and low bone mass in primary aldosteronism patients.Correlation analysis of related bone metabolism indexes was carried out to confirm the effect of increased mineralocorticoid on bone metabolism and bone mass,providing clinical evidence for our next animal experiments.Part Ⅲ: In this study,30 8-week-old male Sprague-Dawlay rats were randomly divided into 3 groups with 10 rats in each group.After 12 weeks of continuous intragastric administration with normal saline,hydrocortisone,hydrocortisone + spironolactone,the rats were sacrificed and their femurs were separated.We used DXA absorptiometry to detect bone mineral density,Micro CT to detect bone microstructure,and three-point bending test to evaluate the femoral bone strength of rats,so as to explore the effects of hyperglucocorticoid on bone mineral density and bone microstructure after blocking mineralocorticoid receptor of bone.The trabecular structure of bone was observed by HE staining.The expressions of osteocalcin,osteoprotegerin,Wnt5 a and Wnt10 b were observed by immunohistochemical staining.Glucocorticoid receptor,mineralocorticoid receptor and important molecules of Wnt pathway and OPG-RANKL-RANK pathway were detected by quantitative polymerase chain reaction such as Wnt5 a,Wnt10b,β-catenin,Low density lipoprotein receptor-associated protein antibody 6,Lymphoid enhancement factor binding factor1,T cell factor,runt-related transcription factor 2,cyclin D,osteocalcin,osteoprotegerin(OPG)and RANKL ligand.We also detected the protein expression levels of important molecules in the Wnt pathway and OPG-RANKL-RANK pathway such as β-catenin,Wnt5 a,Wnt10b,osteocalcin,OPG and peroxidase body proliferation agent activated receptorγby Western blot methods,so as to explore whether the over-activation of mineralocorticoid receptor plays an effect on bone metabolism through the Wnt and OPG-RANKL-RANK pathway.Results:Part Ⅰ: The prevalence of lower than normal bone mineral density in patients with Cushing syndrome was 40%,which was significantly higher than that of healthy people.Among them,85.7% of postmenopausal women and men over 50 years old had subnormal bone mass,and 17.5% of premenopausal women and men under 50 years old had subnormal bone mass.Gender,24 hours urinary free cortisol and course of the disease may be the main factors affecting bone mineral density in patients with Cushing syndrome.Cushing’s syndrome had apparent abnormal bone metabolism.Elevated levels of parathyroid hormone and cross-linked carboxy-terminal peptide of type Ⅰ collagen and decreased levels of 25(OH)D in patients with Cushing syndrome suggested that the decrease of bone mass in patients with Cushing’s syndrome was related to active bone resorption.Part Ⅱ:The proportion of lower bone mass in primary aldosteronism patients was 43.6%,which was significantly higher than that in healthy people.Gender,plasma aldosterone concentration and course of the disease may be the main factors affecting bone mineral density in primary aldosteronism patients.Elevated levels of parathyroid hormone and cross-linked carboxy-terminal peptide of type Ⅰ collagen and decreased levels of 25(OH)D in patients with primary aldosteronism suggest that there is obvious abnormal bone metabolism in patients with primary aldosteronism,and active bone resorption may be one of the reasons for the decrease of bone mass in patients with primary aldosteronism.Part Ⅲ:The bone mineral density and bone strength of glucocorticoid rats were decreased,and the bone microstructure was damaged,showing obvious characteristics of osteoporosis,suggesting that hydrocortisone intragastric administration for 12 weeks could successfully establish the rat model of glucocorticoid osteoporosis.The bone mineral density,bone strength and bone microstructure of the femur of rats were improved after blocking the mineralocorticoid receptor of bone,which confirmed that the improper activation of the mineralocorticoid receptor induced by high glucocorticoid was involved in the development of glucocorticoid-induced osteoporosis.In addition,Wnt5 a,Wnt10b,β-catenin,Lymphoid enhancement factor binding factor1,runt-related transcription factor 2,cyclin D,osteocalcin and osteoprotegerin(OPG)mRNA expression and Wnt5 a,Wnt10b,β-catenin,osteocalcin and OPG protein were significantly increased and RANKL mRNA expression were decreased after blocking mineralocorticoid receptor which confirmed that the inappropriate activation of mineralocorticoid receptor was involved in the occurrence of glucocorticoidassociated bone diseases through the Wnt and OPG-RANKL-RANK pathway.Conclusion:The constituent ratio of low bone mass in patients with Cushing’s syndrome and primary aldosteronism was significantly higher than that in the general population.The serum parathyroid hormone level and bone resorption marker were higher than the upper limit of the normal reference value.It suggested that secondary hyperparathyroidism might be one of the causes of osteopenia in patients with Cushing’s syndrome and primary aldosteronism,and its mechanism might be related to the excessive activation of mineralocorticoid receptor.In vivo studies have found that inappropriate activation of mineralocorticoid receptor of bone is associated with the development of glucocorticoid-associated bone disease.Inappropriate activation of mineralocorticoid receptor of bone is involved in the development of glucocorticoid-associated bone disease by changing the activity of signaling molecules in the Wnt pathway and the OPG-RANKLRANK pathway.The conclusions of this study need to be confirmed by multi-center,large-sample prospective clinical trials. |
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