Drynariae Rhizoma has great significance in the clinical practice of osteoporosis treatment. Based on the perspective of integrative pharmacology, the study explored the mechanism of action of Drynariae Rhizoma in the treatment of osteoporosis. Six active components in Drynariae Rhizoma were obtained, mainly including glycosides and sterols. Taking the median of 2 times of "node connectivity" as the card value, the core node of the Chinese medicine target disease gene interaction network was selected. Based on this, three topological structural eigenvalues, such as "node connectivity" "node tightness" and "node connectivity" were calculated, thereby screening out four core targets of Drynariae Rhizoma treatment for osteoporosis, including thyroid parathyroid hormone 1 receptor (PTH1R), parathyroid hormone 2 receptor (PTH2R), calcitonin receptor gene (CALCR), and SPTBN1 gene (SPTBN1). Based on the gene ontology database GO and KEGG pathway database, the molecular function, intracellular localization, and biological reactions and pathways of proteins encoded by drug target genes were determined. Combined with enrichment calculation, it is predicted that osteoporosis may play a role in biosynthetic processes, such as circulatory system, nervous system, energy metabolism, prolactin signal pathway, GnRH signaling pathway, neurotrophic factor signaling pathway and other pathway. The conclusion of this study is certain with the existing research results, and the new target and new pathway could also be used as a theoretical basis for the further verification of osteoporosis.
Drugs, Chinese Herbal ; pharmacology ; Humans ; Osteoporosis ; drug therapy ; Polypodiaceae ; chemistry ; Receptor, Parathyroid Hormone, Type 1 ; metabolism ; Receptor, Parathyroid Hormone, Type 2 ; metabolism ; Receptors, Calcitonin ; metabolism ; Rhizome ; chemistry ; Spectrin ; metabolism

G-protein coupled receptors form a large superfamily of plasma membrane proteins which serve a variety of signal transduction roles. New receptors continue to be identified. Based on sequence homology the superfamily can currently be divided into three families, the rhodopsin family which includes the vast majority of identified receptors, and the secretin. and metabotropic glutamate receptor families which share a general architecture with each other and the rhodopsin family but no obvious sequence identity. Screening for additional members of the secretin family led to the identification of the parathyroid hormone-2 (PTH2) receptor. Ligand recognition by the PTH2 receptor partially overlaps that of the PTH/parathyroid hormone-related peptide (PTHrP) receptor. This has facilitated structure-function analysis of ligands for these receptors. The physiological role of the PTH2 receptor is under investigation but its distribution suggests that it may be a neurotransmitter receptor and could participate in modulation of a number of organ systems. The relative abundance of PTH2 receptor mRNA in the brain and the inability to detect mRNA encoding PTH, its only currently identified ligand, suggest the existence of another endogenous ligand, for which evidence has recently been obtained.
Brain ; Cell Membrane ; GTP-Binding Proteins ; Humans ; Ligands ; Mass Screening ; Neurotransmitter Agents ; Parathyroid Hormone ; Receptor, Parathyroid Hormone, Type 2 ; Receptors, Metabotropic Glutamate ; Receptors, Neurotransmitter ; Rhodopsin ; RNA, Messenger ; Secretin ; Sequence Homology ; Signal Transduction

OBJECTIVE: To establish an cell model of hyperparathyroidism by isolation, in vitro culture, and identification of parathyroid cells from patients with secondary hyperparathyroidism (SHPT). METHODS: The parathyroid gland tissues obtained from 10 patients with SHPT were dissociated by collagenase digestion for primary culture of the parathyroid cells. Morphological changes and growth characteristics of the cells were assessed by microscopic imaging and cell counting. The mRNA and protein expression levels of parathyroid hormone (PTH), calcium-sensing receptor (CaSR), and glial cells missing 2 (GCM2) in the primary and passaged cells were determined by immunofluorescence, qRT-PCR, and Western blotting. RESULTS: Primary cultures of parathyroid cells were successfully obtained. The cells exhibited a high expression of PTH shown by immunofluorescence assay and had a population doubling time of approximately 71.61 h. PTH secretion in the second-passage (P2) cells was significantly lower than that in the primary (P0) and first-passage (P1) cells (P < 0.001). Despite a significant downregulation of CaSR mRNA (P=0.017) and protein (P=0.006) in P1 cells as compared with P0 cells, no significant differences were found in mRNA and protein expressions of PTH or GCM2 between the two cell generations. CONCLUSION Primary cultures of parathyroid cells isolated from SHPT patients by collagenase digestion show similar biological properties to the cells in vivo.
Humans ; Hyperparathyroidism, Secondary/metabolism* ; Parathyroid Glands/metabolism* ; Parathyroid Hormone ; RNA, Messenger/metabolism* ; Receptors, Calcium-Sensing/metabolism*

OBJECTIVE: To identify the miRNAs targeting vitamin D receptor (VDR) gene and their effect on parathyroid hormone (PTH) secretion in secondary hyperparathyroidism. METHODS: Primary parathyroid cells with secondary hyperparathyroidism were isolated by collagenase digestion and cultured. The miRNAs targeting VDR were screened by bioinformatics methods and full transcriptome sequencing, and dual-luciferase reporter assay was used to verify the targeting relationship between VDR and the screened miRNA. The effects of overexpression or inhibition of the candidate miRNA on VDR mRNA and protein expressions and PTH secretion were evaluated using qRT-PCR and Western blotting. The expression levels of the candidate miRNAs and VDR mRNA in clinical specimens of parathyroid tissues were verified by qRT-PCR, and the expression of VDR protein was detected by immunohistochemistry. RESULTS: We successfully isolated primary parathyroid cells. Dual-luciferase reporter assay verified the targeting relationship of hsa-miR-149-5p, hsa-miR-221-5p, hsa-miR-222-3p, hsa-miR-29a-5p, hsa-miR-301a-5p, hsa-miR-873-5p, hsa-miR-93-3p with VDR, and among them, the overexpression of hsa-miR-149-5p and hsa-miR-301a-5p significantly increased PTH secretion in the parathyroid cells. In patients with secondary hyperparathyroidism, hsa-miR-149-5p was highly expressed in the parathyroid tissues (P=0.046), where the expressions of VDR mRNA (P=0.0267) and protein were both decreased. CONCLUSION The two miRNAs, hsa-miR-149-5p and hsa-miR-301a-5p, may promote the secretion of PTH in patients with secondary hyperparathyroidism by down-regulating the expression of VDR gene.
Humans ; Hyperparathyroidism, Secondary/genetics* ; MicroRNAs/metabolism* ; Parathyroid Hormone ; RNA, Messenger ; Receptors, Calcitriol/genetics*

Familial hypocalciuric hypercalcemia (FHH) is a genetic disorder characterized by mild asymptomatic hypercalcemia, hypocalciuria, a decreased urinary calcium-creatinine ratio, and normal or elevated parathyroid hormone levels. FHH is caused by an inactivating heterozygous mutation of the calcium-sensing receptor (CaSR) gene. A 62-year-old woman was referred to our center because of inadequate glucose control. Her son had hypercalcemia. Biochemical and endocrine studies showed hypercalcemia, hypocalciuria, a decreased urinary calcium-creatinine ratio, normal parathyroid hormone levels, normal PTH-related peptide levels, decreased free T4, increased TSH, and thyroid antibody positivity. Direct sequencing analyses revealed a heterozygous mutation of G21R at exon 2 and a single nucleotide polymorphism of R990G at exon 7 of the CaSR gene. Here, we report a case of FHH associated with a heterozygous mutation and a single nucleotide polymorphism of the CaSR gene, which coexisted with autoimmune thyroiditis and hypothyroidism. To our knowledge, this is the first such case reported.
Exons ; Female ; Glucose ; Humans ; Hypercalcemia ; Hypothyroidism ; Middle Aged ; Parathyroid Hormone ; Parathyroid Hormone-Related Protein ; Polymorphism, Single Nucleotide ; Receptors, Calcium-Sensing ; Thyroid Gland ; Thyroiditis, Autoimmune

The calcium sensing receptor (CaSR) plays an important role in calcium homeostasis. Activating mutations of CaSR cause autosomal dominant hypocalcemia by affecting parathyroid hormone secretion in parathyroid gland and calcium resorption in kidney. They can also cause a type 5 Bartter syndrome by inhibiting the apical potassium channel in the thick ascending limb of the loop of Henle in the kidney. This study presents a patient who had autosomal dominant hypocalcemia with Bartter syndrome due to an activating mutation Y829C in the transmembrane domain of the CaSR. Symptoms of hypocalcemia occurred 12 days after birth and medication was started immediately. Medullary nephrocalcinosis and basal ganglia calcification were found at 7 years old and at 17 years old. Three hypercalcemic episodes occurred, one at 14 years old and two at 17 years old. The Bartter syndrome was not severe while the serum calcium concentration was controlled, but during hypercalcemic periods, the symptoms of Bartter syndrome were aggravated.
Bartter Syndrome* ; Basal Ganglia ; Calcium ; Extremities ; Homeostasis ; Humans ; Hypocalcemia* ; Kidney ; Loop of Henle ; Nephrocalcinosis ; Parathyroid Glands ; Parathyroid Hormone ; Parturition ; Potassium Channels ; Receptors, Calcium-Sensing*

Menopause is the time at which menstruation stops in women. After menopause, women are more susceptible to some diseases, especially osteoporosis and cardiovascular disease. Vitamin D has a protective effect against osteoporosis by facilitating the absorption of calcium and affecting parathyroid hormone. Vitamin D also affects cardiovascular function by lowering the blood pressure, which affects the renin–angiotensin system and alters the low-density lipoprotein receptor activity. This paper discusses supplemental vitamin D in postmenopausal women with osteoporosis and cardiovascular disease.
Absorption ; Blood Pressure ; Calcium ; Cardiovascular Diseases ; Female ; Humans ; Menopause ; Menstruation ; Osteoporosis ; Parathyroid Hormone ; Receptors, Lipoprotein ; Vitamin D* ; Vitamins*

OBJECTIVETo establish HEK293 cell lines with stable expression of human parathyroid hormone (PTH) receptors.

METHODSThe purified gene fragments of PTH-related peptide receptor (PTHR) and its mutant form (DSEL) were cloned separately into pcDNA3.1(+) vector after digestion with EcoR I and Not I, and the resulted pcDNA3.1(+)-PTHR and pcDNA3.1(+)-DSEL plasmids were verified by restriction enzyme digestion and DNA sequencing. HEK293 cells were transfected with these plasmids and the expression of PTHR and DSEL in the cells were examined by RT-PCR and ELSIA.

RESULTSSequencing and restriction enzyme digestion analysis showed that PTHR and DSEL cDNAs were correctly cloned into pcDNA3.1(+)vector. After a 48-h transfection of HEK293 cells with the recombinant plasmids and G418 selection, the positive cell clones stably expressing the constructs were obtained, which showed expressions of PTHR and DSEL mRNAs detected by RT-PCR. These positive cells showed high levels of PLC and aAMP production in response to PTH stimulation.

CONCLUSIONThe HEK293 cell lines with stable expression of PTH1R or DSEL gene established in this study provide useful cell models for studying the physiological functions of PTH peptides.

Gene Expression ; Genetic Vectors ; HEK293 Cells ; Humans ; Plasmids ; Receptors, Parathyroid Hormone ; genetics ; metabolism ; Sequence Analysis, DNA ; Signal Transduction ; genetics ; Transfection

The important factors involved in the regulation of PTH are calcium, vitamin D, and phosphorus. However, recent studies have suggested that magnesium may also play a significant role in the modulation of PTH. The aims of this study was to analyze the relationship between serum magnesium and PTH levels in the hemodialysis patients. We studied 66 stable patients under maintenance hemodialysis for more than 6 months. Calcium carbonate was used as a phosphate binder in all patients. No patient had been previously treated with vitamin D and aluminum hydroxide. Biochemical parameters were evaluated 3 times during 7 months, and the mean values were computed. The mean serum magnesium level was 2.7+/-0.4 mEq/L. Hypermagnesemia(defined as serum Mg>2.2 mEq/L) was found in 60 patients(90.1%). Serum magnesium levels were inversely correlated with serum iPTH levels(r=-0.579; p<0.001). Serum total and ionized calcium levels were inversely correlated with serum iPTH levels(r=-0.743; p<0.001, r=-0.699; p<0.001, respectively). Serum alkaline phosphatase levels positively correlated with serum iPTH levels (r=0.364; p=0.003). In lower iPTH group(serum iPTH2=0.72; p<0.001). Hypermagnesemia is frequent in hemodialysis patients. There is a significant inverse relationship between serum magnesium and PTH levels. In addition, serum magnesium concentration has an important influence on PTH regulation. These results suggest that hypermagnesemia may have a suppressive effect on PTH regulation in hemodialysis patients.
Alkaline Phosphatase ; Aluminum Hydroxide ; Calcium ; Calcium Carbonate ; Humans ; Magnesium* ; Parathyroid Hormone* ; Phosphorus ; Receptors, Calcium-Sensing ; Renal Dialysis* ; Renal Osteodystrophy ; Vitamin D

PURPOSE: It is known that secondary hyperparathyroidism in end stage renal disease (ESRD) patients is associated with vitamin D receptor (VDR) gene polymorphism, but there is no consensus on its genotype. There is lack of data in Ca, P, calcitriol and VDR polymorphism. METHODS: We measured serum Ca, P, alkaline phosphatase, parathyroid hormone (PTH), and 1,25 (OH)2D3 of the 53 hemodialysis patients. The genotypes of VDR were classified BB, Bb, bb according to restriction patterns in PCR of the patients' DNA using Bsm I restriction enzyme. RESULTS: The patients with BB, Bb, bb type were 0 (0%), 15 (28.3%), 38 (71.7%) respectively. Serum PTH levels were 70.0+/-63.3 pg/mL and 146.9+/-184.9 pg/mL in Bb, bb type respectively, and showed significant statistical difference (p<0.05). Serum 1,25 (OH)2D3 levels were 7.68+/-3.41 pg/mL and 6.59+/-2.67 pg/mL in Bb and bb genotype respectively without statistical significance. And there was no significant statistical differences among the serum levels of calcium, phosphorus, alkaline phosphatase. CONCLUSION: Vitamin D receptor gene polymorphism is associated with secondary hyperparathyroidism in hemodialysis patients, and the b allelle is suggestive of poorer bone mineral metabolism.
Alkaline Phosphatase ; Calcitriol ; Calcium ; Consensus ; DNA ; Genotype ; Humans ; Hyperparathyroidism ; Hyperparathyroidism, Secondary* ; Kidney Failure, Chronic ; Metabolism ; Parathyroid Hormone ; Phosphorus ; Polymerase Chain Reaction ; Receptors, Calcitriol* ; Renal Dialysis* ; Vitamin D* ; Vitamins*