1,25(OH)2D3 provides protection against diabetic kidney disease by downregulating the TLR4-MyD88-NF-κB pathway.

The over-activation of Toll-like receptors (TLRs) is a typical immune response to injury. Previous work has suggested that controlling the over-activation of TLR4-MyD88-NF-κB may represent a new therapeutic option for diabetic kidney disease (DKD). 1,25(OH)2D3 has also been shown to exert a protective effect on DKD, although the mechanism involved has yet to be elucidated. The aim of this study was to investigate whether 1,25(OH)2D3 protects against DKD by down-regulating the innate immune TLR-NF-κB pathway. NRK-52E cells were cultured under normal or high-glucose conditions. We then used siRNA to knock down TLR4 expression under high-glucose conditions. NRK-52E cells cultured under high-glucose conditions, and streptozotocin (STZ)-induced diabetic rats, were treated with different doses of 1,25(OH)2D3 and used as in vitro and in vivo models, respectively. Renal biochemical indicators were then measured to evaluate the influence of 1,25(OH)2D3 treatment on DKD in diabetic rats. Histological analysis was also performed to determine the extent of infiltration by inflammatory cells and tubulointerstitial fibrosis. Using RT-qPCR, western blotting, immunohistochemistry and immunofluorescence, we determined the expression levels of TLR4, MyD88, NF-κB p65, MCP-1 and α-SMA to investigate whether 1,25(OH)2D3 could reduce the development of tubulointerstitial fibrosis. Knocking down TLR4 abolished the tubulointerstitial fibrosis caused by high-glucose conditions. High doses of 1,25(OH)2D3 consistently reduced the expression of TLR4-MyD88-NF-κB in NRK-52E cells. Moreover, high doses of 1,25(OH)2D3 had an obvious protective effect on kidney injury and inhibited the infiltration of inflammatory cells and tubulointerstitial fibrosis in diabetic rats. In conclusion, high doses of 1,25(OH)2D3 protected against tubulointerstitial fibrosis both in vitro and in vivo by downregulating the expression of TLR4-MyD88-NF-κB.

[Association of vitamin D receptor ApaI and TaqI gene polymorphisms with pigmented pretibial patches in T2DM patients].

OBJECTIVE: To explore the association of vitamin D receptor ApaI and TaqI gene polymorphisms with pigmented pretibial patches (PPPs) in type 2 diabetes mellitus (T2DM) patients. METHODS: A total of 233 patients with T2DM were enrolled. Patients were assigned to PPPs group (n=106) or non-PPPs (n=127) according to the presence of PPPs. Allelic and genotypic comparisons of VDR-TaqI and VDR-ApaI between the different groups were evaluated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The genotype and allele gene frequency were similar at ApaI in two groups (P>0.05). The PPPs group showed a higher frequency of TT allele at TaqI in the T2DM (95%) when compared to that of non-PPPs group (87%), while the frequency of Tt and tt allele were lower in PPPs group than non-PPPs group in T2DM (5% vs 13%, all P<0.05). The frequency of T allele were higher at TaqI in PPPs group (98%) than non-PPPs group (93%) in T2DM, while PPPs had a lower frequency of t allele (2% vs 7%, all P<0.05). CONCLUSION: It seems that gene polymorphisms of VDR-TaqI, not VDR-ApaI, contributes to the risk of PPPs in T2DM patients in the Han nationality in Tianjin area.

[STAT signaling pathway mediates high glucose induced cardiac fibroblasts proliferation and collagen deposition in vitro].

OBJECTIVE: To observe the signal transducers and activator of transcriptions (STATs) protein expression changes and investigate the functional role of STATs pathway in case of high glucose-induced cardiac fibroblasts (CFs) proliferation and collagen deposition in vitro. METHODS: Rat cardiac fibroblasts were isolated from 1- to 3-day-old SD rats, cells from the second to fourth passages were used for the experiment. CFs were cultured in Dulbecco's modified Eagle's medium, supplemented with 5.5 mmol/L glucose (NG), 5.5 mmol/L glucose plus 19.4 mmol/L mannose (OC) or 25 mmol/L glucose (HG) in the presence of absence of STAT1 inhibitor (fludarabine, FLU) and STAT3 inhibitor (S3I-201). After 24 h and 48 h culture in vitro, the proliferation of CFs was measured by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. After 12 h and 24 h culture in vitro, the production of type I and III collagen was evaluated using real-time quantitative PCR and ELISA. After 0, 30, 60 and 120 min culture in vitro, the phosphorylated expression of STAT1 and STAT3 was analyzed by Western blot. RESULTS: CFs proliferation was significantly enhanced post 24 h and 48 h HG stimulation, and procollagen I and III mRNA expression was significantly upregulated post 12 h and 24 h HG stimulation. Deposition of collagen I and III was also significantly increased post 24 h and 72 h HG stimulation. STAT1 phosphorylation in CFs was increased after 120 min HG stimulation and STAT3 phosphorylation in CFs was increased post 60 min and 120 min HG stimulation. FLU and S3I-201 could inhibit HG-induced CFs proliferation and suppress of which was stimulated by FLU and S3I-201 could both suppress upregulated procollagen I and III mRNA expression and the deposition of collagen types I and III post HG stimulation. STAT1 phosphorylation inhibition resulted in less mRNA downregulation of procollagen type III than that of procollagen type I post 12 h HG stimulation. The STAT3 phosphorylation inhibition resulted in more significantly upregulated procollagen type III mRNA expression than procollagen type I mRNA expression at 12 h post HG stimulation. CONCLUSION: HG could enhance the protein expression of phosphorylated STAT1 and STAT3 in CFs, which are responsible for HG-induced increased CFs proliferation and collagen deposition in vitro.

Symptomatic Cushing syndrome and hyperandrogenemia revealing steroid cell ovarian neoplasm with late intra-abdominal metastasis.

BACKGROUND: Steroid cell tumors of ovary account for less than 0.1% of all ovarian tumors and these tumours may present at any age in association with interesting presentations related to hormonal activities. The subtype, not otherwise specified (NOS), is associated with androgenic changes in 56-77% and Cushing syndrome in 6-10%. Due to the rarity of available data regarding these tumors, little is known about their malignant potential and metastatic behaviour. We hereby report an unusual metastasis of steroid cell ovarian neoplasm presented with both Cushing syndrome and hyperandrogenemia. CASE PRESENTATION: A 31-year-old woman, who had a past medical history of ovarian tumor resection (left ovarian thecoma was initially diagnosed at that time), presented with hirsutism, hypertension and menstrual disorder. Also, laboratory work-up revealed hypercortisolism and androgen excess. Computerized tomography (CT) of the abdomen showed abdominal paraaortic masses, multiple intrahepatic nodules and retroperitoneal lymph nodes enlargement. Positron emission tomography/computed tomography (PET/CT) scan demonstrated metastatic lesions. Her ovarian tumor sections were re-examined and pathology result was corrected to steroid cell tumor (NOS) associated with active cell growth and necrosis. Subsequent excision of metastatic lesions yielded clinical improvement promptly and metastasis of steroid cell tumor was confirmed by postoperative pathological studies. However, one year after the surgical management of metastasis, recurrence happened while radiotherapy was ineffective. The patient finally died of tumor metastatic recurrence. CONCLUSION: This case reports a rare coexistence of Cushing syndrome and hyperandrogenemia which occurs based on metastasis of steroid cell ovarian neoplasm. It presents a real diagnostic challenge to both clinicians and pathologists. Therefore, it is very important to establish a final diagnosis by pathological studies along with clinical manifestations and imaging findings. Besides, it is necessary to improve follow-up of patients with this kind of tumors.

STAT1/3 and ERK1/2 synergistically regulate cardiac fibrosis induced by high glucose.

BACKGROUND: Hyperglycaemia promotes the proliferation of cardiac fibroblasts (CFs) and collagen synthesis in CFs. However, the molecular mechanism underlying the effects of HG on proliferation and collagen synthesis of CF, is not completely understood. OBJECTIVES: The objectives of the present study were to determine whether the STAT proteins has a functional role in high glucose-induced proliferation of CFs and collagen synthesis in vitro and whether the STAT signaling pathway and MAPK signaling pathway have synergetical effects on high glucose-mediated cardiac fibroblasts proliferation and collagen synthesis. METHODS: Rat CFs were cultured in Dulbecco's modified Eagle's medium, supplemented with 5.5 or 25 mmol/L D-glucose, in the presence of absence of STAT1 inhibitor Fludarabine, STAT3 inhibitor S31-201 and ERK1/2 inhibitor PD98059. Proliferation were measured by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, the production of Type I and III collagen was evaluated using real-time quantitative RT-PCR and ELISA, and the phosphorylation expression of STAT1 and STAT3 were analyzed by Western blot. RESULTS: High glucose treatment promoted the proliferation of cardiac fibroblasts and collagen types I and III synthesis. High glucose treatment induced STAT1 and STAT3 phosphorylation in cardiac fibroblasts, the mode and level of STAT1 and STAT3 phosphorylation were significantly different. Fludarabine and S31-201 could both inhibited high glucose stimulated proliferation of cardiac fibroblasts and collagen types I and III synthesis with different effects. Combination of Fludarabine and PD98059 or combination of S31-201 and PD98059 both exhibited stronger inhibitions on proliferation of cardiac fibroblasts and collagen types I synthesis, but the effects and functional modes are different. CONCLUSION: Both STAT1 and STAT3 mediate the proliferation of cardiac fibroblasts and collagen synthesis induced by high glucose. STAT1 and STAT3 both have synergetic effects with ERK1/2 on regulating proliferation of cardiac fibroblasts and collagen types I synthesis.

Autoimmune hypophysitis may eventually become empty sella.

Autoimmune hypophysitis (AH) is commonly believed to be a rare chronic inflammatory condition of the pituitary gland. In clinical practice, however, the disease is often seen indeed. It typically presents with hypopituitarism and pituitary mass found by MRI. We report here unusual presentations of two females with AH followed by empty sella syndrome. The two females, aged at 64 and 57-years-old, presented with anterior pituitary dysfunction, diplopia and diabetes insipidus. By MRI the two patients shared the common characteristics with diffuse homogenous contrast enhancement of the gland and increased stalk thickness. After a long period treatment with glucocorticoids, empty sella was eventually detected by MRI.

[Clinical analysis of drug-induced Pseudo-Bartter's syndrome: a report of five cases].

OBJECTIVE: To summarize the clinical characteristics and outcomes of Pseudo-Bartter's syndrome and explore its pathogenesis. METHODS: The clinical data of 5 cases of Pseudo-Bartter's syndrome at our ward from May 2008 to December 2010 was analyzed retrospectively. RESULTS: All patients were female. Long-term regimen of purgative or diuretics was prescribed. The clinical features included normotension, hypokalemic alkalosis and activation of renin-angiotensin-aldosterone. The pathological results of 3 cases of kidney biopsy showed the hyperplasia of juxtaglomerular apparatus, thickness of arteriole, infiltration of lymphocytes and monocytes and degeneration of renal tubule. Upon a definitive diagnosis, purgative or diuretics was discontinued and supplement therapy of potassium chloride initiated. The results of laboratory tests reverted to normal ranges within 4 weeks. CONCLUSION: Purgative or diuretics should be prescribed appropriately to avoid the occurrence of Pseudo-Bartter's syndrome.

[Clinical analysis of 6 cases of Bartter syndrome].

OBJECTIVE: To summarize the clinical characteristics of Bartter syndrome and investigate its pathogenesis. METHODS: The clinical data of 6 cases of Bartter syndrome at our hospital from November 2006 to May 2010 were analyzed retrospectively. RESULTS: The onset age of Bartter syndrome was 13-35 years old. The main symptoms included weakness (6/6), paralysis (1/6), numbness (5/6) and tetany (4/6). All patients had normal blood pressure. The biochemical tests showed persistent hypokalemia, metabolic alkalosis (6/6) and hyperreninemia. The pathological examination of deltoid muscle biopsy showed the swelling, degeneration and necrosis of myocytes and the deposition of immunocomplex in myolemma. And the pathological examination of renal biopsy showed the hyperplasia of juxtaglomerular apparatus (5/6) and the deposition of immunocomplex. All symptoms were relieved after a therapy of potassium supplementation or a combination of indomethacin, spironolactone and immunosuppressant. CONCLUSION: When such clinical features as weakness, paralysis, tetany, hypokalemic alkalosis and normotension are encountered, Bartter syndrome should be suspected. Serum electrolytes, blood gas analysis and activation of the renin-angiotensin-aldosterone system should be examined for a definite diagnosis. The treatment of choice includes potassium and magnesium supplementation or in combination with prostaglandin synthetase inhibitor, aldosterone antagonist and immunosuppressant. Immunologic mechanism may participate in the course of Bartter syndrome.

Effects of gamma radiation on bone-marrow stromal cells.

The effects of in vitro irradiation on proliferation and hematopoietic supportive functions of stromal cells were studied. To assess the effects of radiation on stromal cells proliferation, marrow cells were exposed to a single dose of gamma radiation. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that stromal cell proliferation was significantly suppressed after radiation in a dose-dependent manner. Stromal layers obtained from irradiated marrow cells failed to establish adherent layers after 6, 8, or 10 Gy of radiation. To assess the functions of stromal cells that survived radiation, stromal layers derived from irradiated marrow cells were cocultured with freshly isolated autologous hematopoietic cells and assayed for their capacity to support prolonged granulocyte-macrophage progenitors (CFU-GM) production. Stromal layers derived from 2-Gy-irradiated marrow cells resulted in similar CFU-GM production as control cells, while stromal layers derived from 4- to 10-Gy-irradiated marrow cells significantly decreased CFU-GM production. To study the influence of radiation on hematopoietic supportive capacity in established stromal layers, stromal layers generated from non-irradiated marrow cells were irradiated and cocultured with freshly isolated autologous hematopoietic cells. Established stromal layers irradiated up to 10 Gy sustained prolonged CFU-GM production, suggesting that hematopoietic stromal supportive functions remained intact at this dose of radiation. In conclusion, our results indicated that proliferation of stromal cells and bone-marrow stromal layer formation from stromal cells are sensitive to radiation in vitro, while established bone-marrow stromal layer originating from stromal cells is relatively resistant to radiation. Data generated may have implications in future bone-marrow transplantation research.

[The protective effects of cyclosporine A on aortic immunological injuries in STZ-induced diabetic rats].

OBJECTIVE: To investigate the autoimmune injuries of diabetic macrovascular disease (aorta) and the protective effects of immunosuppressive agent (cyclosporine A, CsA) on aortic injuries in streptozotocin (STZ)-induced diabetic rats. METHODS: STZ-induced diabetic rats were assigned randomly to 6 groups which received low (BML or AML, 1 mgxkg(-1)xd(-1)), middle (BMM or AMM, 4 mgxkg(-1)xd(-1)) or high (BMH or AMH, 8 mgxkg(-1)xd(-1)) dose of CsA from 1 week before or after STZ for 8 weeks. Diabetic rats without any treatment, insulin-treated diabetic rats and normal rats were also monitored simultaneously and served as control groups. The pathologic abnormalities of the aorta were verified by HE, Masson staining and electronmicroscopy. The depositions of immunoglobulins (IgG, IgM and IgA) were determined by immunohistochemistry and immunofluorescence methods. RESULTS: At the end of study, lymphocytes infiltration and collagen content (26 582 +/- 6901) were significantly higher in diabetic aorta than those in non-diabetic aorta (Collagen: 7482 +/- 3491, P < 0.01). The deposited IgG and IgA were also significantly increased in diabetic aorta compared with non-diabetic aorta (IgG: 11 789 +/- 2491 vs. 2518 +/- 1066, P < 0.01; IgA: 17 430 +/- 3159 vs. 1135 +/- 758, P < 0.01). These changes were not affected by insulin while CsA intervention significantly reduced aortic collagen content (BMH: 13 518 +/- 5440, P < 0.01 vs. STZ) and immunoglobulin deposition (BMH: IgG: 7584 +/- 4462; IgA: 6176 +/- 1900, all P < 0.01 vs. STZ). These immunoglobulin deposition changes were confirmed by results of immunofluorescence. Aortic collagen accumulation was positively correlated to aortic immunoglobulin deposition (IgG, r = 0.556, P < 0.01; IgA, r = 0.661, P < 0.01). CONCLUSIONS: Our data suggest that the autoimmune injuries might be a promoting factor in the pathogenesis of the diabetic macrovascular disease which could lead to the development of macrovascular disease. Immunosuppressive agent, such as CsA, could inhibit the abnormal deposition of immunoglobulins and therefore, delay the development of diabetic macrovascular disease in this model.

Severe hypoglycemia and hyponatremia caused by hypopituitarism in a female patient with type 1 diabetes: A case report.

RATIONALE: The misdiagnosis of hypopituitarism is common due to its rarity and its nonspecific clinical manifestations. Our case report highlights the importance of critical evaluation regarding hypopituitarism as a cause of recurrent hypoglycemia, hyponatremia, and gastrointestinal symptoms in patients with T1DM, as misdiagnosis might be fatal to the patient. PATIENT CONCERNS: We herein report the case of 35-year-old female patient who presented with 18 years of history of well-controlled type 1 diabetes mellitus and a 6-month history of recurrent nausea and vomiting, generalized weakness, hyponatremia, and severe hypoglycemia, despite a reduction in the dose of insulin. She was considered as having "type 1 diabetes and gastroparesis." Four months later, she was diagnosed with hypothyroidism, and 25 µg/d of levothyroxine was prescribed. However, the levothyroxine had to be discontinued 1 week later because of frequent vomiting by the patient. DIAGNOSIS: Further evaluation in our hospital revealed low-normal adrenocorticotropic hormone, low-normal serum cortisol, and low 24-hours urinary cortisol excretion. Secondary hypothyroidism and hypogonadotropic hypogonadism were also demonstrated. Based on the endocrinological findings, she was diagnosed with hypopituitarism possibly due to lymphocytic hypophysitis. Diabetic nephropathy was another diagnosis made after kidney biopsy. INTERVENTIONS: The patient was treated with 100 mg/d of hydrocortisone intravenously for 2 weeks. After that, she continued on 15 mg/d of prednisone, and then 25 µg/d of levothyroxine was administered. OUTCOMES: The patient's insulin requirement increased to a premorbid level, the severe hypoglycemia resolved, the physical discomforts were alleviated, and blood electrolytes returned to normal. LESSONS: This uncommon case reinforced the significance of a timely diagnosis and appropriate treatment of hypopituitarism. We recommend that physicians focus their awareness on this potentially life-threatening disease, as it is a condition potentially fatal to the patient if not recognized and treated.

[The effects of cyclosporine A on immunoglobulins deposition in retina of streptozotocin-induced diabetic rats].

OBJECTIVE: To study the autoimmune injuries on diabetic retinopathy (DR) and the protective effects of immunosuppressive therapy with cyclosporine A (CsA) on the DR of streptozotocin (STZ)-induced diabetic rats. METHODS: STZ-induced diabetic rats were randomly divided into 3 groups: group 1: diabetic group without any treatment; group 2: insulin-treated group; group 3: CsA-treated group which was further divided into 2 subgroups: subgroup A: CsA was given 1 week before hyperglycemia appeared and subgroup B: CsA was given one week after hyperglycemia appeared. Subgroup A and subgroup B were further subdivided into 3 groups respectively, based on the dose of CsA (1, 4 and 8 mg x kg(-1) x d(-1)). As a control group, normal rats were also simultaneously monitored. The pathologic changes in the retina were investigated with HE stain and the deposition of immunoglobulins was detected with immunohistochemistry and immunofluorescent microscopy. RESULTS: After 8 week, the deposition of IgG, IgA and IgM was quite significant in the retina of diabetic rats. The data also suggested that insulin treatment had no effects on the DR. In contrast,with CsA intervention, the deposition of immunoglobulins on the retina of diabetic rats vanished. CONCLUSIONS: Autoimmune injuries were shown, in the present study, to play a critical role in the pathogenesis of diabetic retinopathy. Immunosuppressive treatment with CsA showed protective effects by inhibiting the deposition of immunoglobulins in the retina of diabetic rats.

[Regulation of receptor activator of nuclear factor kappaB ligand and osteoprotegerin expression by prostaglandin E2 in osteoblastic-like cells].

OBJECTIVE: To investigate the regulation of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) mRNA expression by prostaglandin E2 (PGE2) in osteoblastic-like cells and involved signaling pathways. METHODS: Rat UMR106 osteoblast-like cells were cultured and treated with various dose of PGE2 or regulators of different signaling pathways such as protein kinase A, protein kinase C, ERK-MAPK and calcium/calmodulin pathways for different period of time,the cells were then harvested at indicated time points, total RNA were isolated and RANKL/OPG mRNA expression were studied by real-time PCR. RESULTS: PGE2, Forskolin and db-cAMP increased RANKL mRNA by 2.8 times (P = 0.002), 2.2 times (P = 0.006) and 2.1 times (P = 0.005) respectively, and inhibited OPG mRNA expression by 12% (P < 0.01), 85% (P = 0.005) and 70% (P = 0.013) respectively, while RANKL and OPG mRNA expression were down-regulated by A23187 by 58% (P = 0.002) and 53% (P = 0.017) respectively. As for inhibitory experiments, the stimulatory effects of PGE2 on RANKL mRNA expression could be inhibited only by KT-5720 by 53% (P < 0.01), while the other inhibitors did not have any effect at all. The inhibitory effects of PGE2 on OPG mRNA expression were partially blocked by KT-5720 by 47% (P = 0.01), verapamil by 38% (P = 0.029) and W7 by 43% (P < 0.01) respectively, while KN-62, chelerythrine and PD98059 had no effects. CONCLUSION: PGE2 can up-regulate the expression of RANKL but down-regulate the expression of OPG. The up-regulation of RANKL mRNA expression by PGE2 was mediated through the activation of PKA signaling pathway while PGE2 induced down-regulation of OPG mRNA expression was predominantly mediated via PKA as well as the Ca2+ /Calmodulin signaling pathways.

[Relationship between gastrointestinal dyskinesis and gastrointestinal neurons in diabetic mellitus: experiment with rats].

OBJECTIVE: To investigate the relationship between gastrointestinal dyskinesis and histologic changes of gastrointestinal myenteric plexus cholinergic and nitrergic neurons in STZ-induced diabetic rats. METHODS: 45 SD rats were randomly divided into control group, diabetic group and insulin group. 16 weeks after diabetic model established, gastrointestinal motility of rats was measured and histologic changes of myenteric plexus cholinergic neuron and nitrergic neuron was observed. RESULTS: Compared with control group, gastrointestinal motility of diabetic group was markedly slow (P < 0.01), the myenteric plexus cholinergic neuron counting of gastric antrum and small intestine were significantly decreased (6.6 +/- 2.9 vs 15.7 +/- 3.8 15.6 +/- 10.3 vs 22.6 +/- 7.4, P < 0.01), yet the number of nitrergic neuron only markedly reduced in gastric antrum (5.3 +/- 1.2 vs 11.8 +/- 2.2, P < 0.01). The gastrointestinal mobility, gastric antrum nitrergic neuron and small intestine cholinergic neuron counting of insulin group were markedly higher than that of diabetic group (P < 0.05), yet lower than that of control group (P < 0.01). CONCLUSION: The gastrointestinal dyskinesis of STZ-induced diabetic rats might be associated with lesions of gastrointestinal myenteric plexus cholinergic neuron and nitrergic neuron. Insulin intensive therapy can partly ameliorate diabetic gastrointesternal dyskinesis.

[Significance of ultrasound bone measurement in diagnosis of hyperthyroidism].

OBJECTIVE: To evaluate the role of ultrasound in bone density measurement in patients with hyperthyroidism. METHODS: The speed of sound (SOS) through distal radius and midshaft tibia of 118 patients with hyperthyroidism 138 males and 80 females, aged 20 approximately 73, were measured with ultrasonic apparatus, the bone mass density (BMD) was measured at the lumbar spine and proximal femur with dual energy X-ray absorptiometry, the serum levels of total thyroxine (TT(4)), total triiodothyronine (TT(3)), free thyroxine (FT(4)), free triiodothyronine (FT(3)), and thyroid stimulating hormone (TSH) were measured by radioimmunoassay, and alkaline phosphatase (ALP) was measured by routine method. RESULTS: The SOS of the patients with hyperthyroidism was significantly lower than that of the healthy subjects (P < 0.05). A strong positive correlation was found between SOS and BMD. The prevalence of SOS (Z < -1, Z < -2.5) through distal radius and midshaft tibia was significantly higher than that of BMD (Z < -1, Z < -2.5) at lumbar spine and proximal femur in the patients with hyperthyroidism. A negative linear correlation was found between SOS and ALP. CONCLUSION: SOS may be one of the significant markers for abnormal bone metabolism for hyperthyroidism.

[Effects of OGP(10-14) and its derivative G38I on proliferation and differentiation of rat osteoblasts in vitro].

OBJECTIVE: To investigate the effects of the carboxyl end of osteogenic growth peptide (OGP)-OGP((10-14)) and its derivative G38I on the proliferation and differentiation of osteoblasts (OBs). METHODS: Osteoblasts were isolated from the calvariae of newborn SD rats and cultured to G3. OGP((10-14)) or G38I of the concentrations of 10(-15) to 10(-7) mol/L were added to culture medium for 48 hours respectively. The number of cells was counted and MTT analysis was used to examine the proliferation of the cells. The ultrastructure of cells was investigated by electron microscopy. The osteoblasts of G3 were divided into experimental groups, treated with OGP((10-14)) or G38I of the concentration of 10(-11) mol/L for 48 hours, and control group. The alkaline phosphatase activity in the culture medium was measured. The protein expression level of type-I collagen was evaluated by immunohistochemistry. The core binding factor 1 (Cbfa1) and type-I collagen mRNA level of osteoblasts were determined by RT-PCR. RESULTS: With a biphasic effect on, OGP((10-14)) and G38I stimulated the number enhancement of OBs dose-dependently at low concentration and inhibited it at high concentration. The numbers of OB were the highest (37 x 10(4)/ml +/- 7 x 10(4)/ml and 30 x 10(4)/ml +/- 5 x 10(4)/ml respectively) when treated by OGP((10-14)) or G38I of the concentration of 10(-11)mol/L. The rough endoplasm net was flourishing and the secreting vesicle was abounding in the experimental cells. There was calcium crystal in the control cells. The activity of alkaline phosphatase in the culture medium of the OGP (10(-14)) and G38I groups were higher than that in the control group (4.47 U/g and 3.82 U/g vs 2.21 U/g). The protein expression level of type-I collagen was higher and the mRNA levels of Cbfa1 and type-I collagen were higher in the OGP((10-14)) and G38I groups were increased in the experimental groups in comparison with the control group (P < 0.05, P < 0.01, and P < 0.05). CONCLUSION: They stimulated cell number enhancement dose dependently at low concentration and followed by inhibition at high concentration. Just as the native OGP, OGP((10-14)) and its derivative G38I stimulate the proliferation of osteoblasts, and improve their activity, up-regulate the Cbfa1 and type-I collagen mRNA expression levels and increase the collagen synthesis, thus promoting the differentiation and osteogenic effect of osteoblasts.

[Impacts of osteoclast-like cells cultured on bone wafer and their sub-cellular structures on osteoblast].

OBJECTIVE: To investigate the effects of osteoclast-like cells (OLC) and its sub-cellular structures on the osteoblast (OB) differentiation and function. METHODS: Spleen cells from C57 mice administrated with 5-fluorouracil were induced by IL-3, 6 and granulocyte-macrophage colony stimulating factor (GM-CSF), and 1alpha, 25-(OH)(2)D(3) to obtain massive OLCs. These OLC cells were cultured in culture fluid and on bone wafers (called bolcs). Osteoblasts were cultured and added with NaF, OLCs of two kinds, culture fluid free of OLC, and sub-unit structures such as nucleus, mitochondria, and cytoplasma from OLCs for 5 days. The proliferation rate of OBs was measured by MTT method and the alkaline phophatase (ALP) activity was measured by PNPP method. Immunochemistry was used to detect the core-binding factor alpha1 (Cbfalpha1) in the OBs, Enzyme linked immunosorbent assay was used to measure the osteocalcin. RESULTS: The OB number was lower in the OLC (1.288 +/- 0.039), OLC cytoplasm (1.138 +/- 0.024), 50% OLC culture fluid (1.203 +/- 0.033), 50% OLC culture medium of OLCs cultured on bone wafer (1.128 +/- 0.028) in comparison with the pure OB group (1.393 +/- 0.016, all P < 0.05). The increase functions of OBs by OLC cultured on bone wafer and their nucleus and mitochondria were all more significant than those of the OLCs not cultured on bone wafer. The ALP activity was increased in the NaF (1.027 +/- 0.024), OCL cytoplasm (1.850 +/- 0.033), 50% OLC medium (2.074 +/- 0.065), 50% OLC medium of OLCs cultured on bone wafer (1.718 +/- 0.048), and mitochondria and cytoplasm of the OLC cultured on bone wafer groups (1.246 +/- 0.037, all P < 0.05). NaF (0.0825 +/- 0.0025), OLCs (0.0775 +/- 0.0025), nucleus (0.0775 +/- 0.0025), mitochondria (0.0875 +/- 0.0025), and cytoplasm of OLCs (0.1100 +/- 0.0007), 50% OLC medium (0.0900 +/- 0.0000), 50% OLC medium of OLCs cultured on bone wafer (0.1200 +/- 0.0041), OLCs cultured on bone wafer and nucleus, mitochondria, and cytoplasm of OLCs cultured on bone wafer all significantly increase the oeteocalcin activity of OBs (0.525 +/- 0.0063, all P < 0.05). NaF (57.6% +/- 2.6%), OLC cytoplasm (45.3% +/- 4.7%), 50% OLC medium (46.6% +/- 3.3%), 50% medium of OLCs cultured on bone wafer (54.0% +/- 2.1%), OLCs cultured on bone wafer (44.8% +/- 3.0%), and cytoplasm of OLCs cultured on bone wafer (48.7% +/- 3.5%) all significantly increased the Cbfalpha1 protein in the OBs (32.8% +/- 4.5%, all P < 0.05). CONCLUSION: The sub-cellular elements of OLC and the supernatant of OLC culture media free of OLC promote the functions of OB, especially the OLCs cultured on bone wafer.

Effects of Cordyceps sinensis on the Expressions of NF-κB and TGF-ß1 in Myocardium of Diabetic Rats.

Objective. To investigate the effect of Cordyceps sinensis (CS) on the expressions of NF-κB and TGF-ß1 in myocardium of streptozotocin-induced diabetic rats. Methods. A total of 53 healthy male SD rats, mice age of 8 weeks and weight of 220 ± 20 g, were randomly divided into five groups by randomized block design: normal control group (n = 10), diabetic group (n = 10), low dose of CS group (n = 12; CS 0.6 g·kg(-1)·d(-1)), middle dose of CS group (n = 11; CS 2.5 g·kg(-1)·d(-1)), and high dose of CS group (n = 10; CS 5 g·kg(-1)·d(-1)). The diabetic models with tail intravenous injection by streptozotocin (45 mg·kg(-1)). Diabetic rats were sacrificed after 8 weeks; the expressions of NF-κB and TGF-ß1 proteins and mRNA in the cardiac muscle were determined by using immunohistochemistry staining and reverse transcription polymerase chain reaction (RT-PCR) method. The data were analyzed using one factor analysis of variance. Result. The expressions of NF-κB and TGF-ß1 proteins and mRNA in the cardiac muscle of diabetic rats were significantly raised (P < 0.05), which could be decreased by CS (P < 0.05). Conclusions. The changes on the expressions of NF-κB and TGF-ß1 in myocardium may be involved in the occurrence of diabetic cardiomyopathy (DC). CS may play its role on myocardial protection by regulating the expressions of NF-κB and TGF-ß1 in myocardium.

Efficacy and safety of avandamet or uptitrated metformin treatment in patients with type 2 diabetes inadequately controlled with metformin alone: a multicenter, randomized, controlled trial.

BACKGROUND: At present, China has listed the compound tablet containing a fixed dose of rosiglitazone and metformin, Avandamet, which may improve patient compliance. The aim of this study was to evaluate the efficacy and safety of Avandamet or uptitrated metformin treatment in patients with type 2 diabetes inadequately controlled with metformin alone. METHODS: This study was a 48-week, multicenter, randomized, open-labeled, active-controlled trial. Patients with inadequate glycaemic control (glycated hemoglobin [HbA1c] 7.5-9.5%) receiving a stable dose of metformin (≥1500 mg) were recruited from 21 centers in China (from 19 November, 2009 to 15 March, 2011). The primary objective was to compare the proportion of patients who reached the target of HbA1c ≤7% between Avandamet and metformin treatment. RESULTS: At week 48, 83.33% of patients reached the target of HbA1c ≤7% in Avandamet treatment and 70.00% in uptitrated metformin treatment, with significantly difference between groups. The target of HbA1c ≤6.5% was reached in 66.03% of patients in Avandamet treatment and 46.88% in uptitrated metformin treatment. The target of fasting plasma glucose (FPG) ≤6.1 mmol/L was reached in 26.97% of patients in Avandamet treatment and 19.33% in uptitrated metformin treatment. The target of FPG ≤7.0 mmol/L was reached in 63.16% of patients in Avandamet treatment and 43.33% in uptitrated metformin treatment. Fasting insulin decreased 3.24 ± 0.98 µU/ml from baseline in Avandamet treatment and 0.72 ± 1.10 µU/ml in uptitrated metformin treatment. Overall adverse event (AE) rates and serious AE rates were similar between groups. Hypoglycaemia occurred rarely in both groups. CONCLUSIONS: Compared with uptitrated metformin, Avandamet treatment provided significant improvements in key parameters of glycemic control and was generally well tolerated. REGISTRATION NUMBER: ChiCTR-TRC-13003776.

Expression, purification and bioactivity characterization of extracellular domain of murine osteoprotegerin ligand.

Osteoprotegerin ligand (OPGL) is a key regulator of formation and activation of osteoclasts. In the present study, the cDNA encoding the extracellular domain of murine OPGL (sOPGL) was synthesized by RT-PCR and cloned into fusion expression vector pET-42a(+) in a certain strategy on purpose that the fusion tag could be completely removed by factor Xa from the expressed fusion protein without any vector-encoded sequence left. Induced with IPTG, the recombinant E. Coli cells produced a 47 kD protein in high level that could be recognized, through Western blotting analysis, by the antibody against OPGL. The expressed products were purified through Glutathione-sepharose 4B affinity chromatography. Along with the fusion molecule, a protein about 30 kD was also specifically bound to the resin. The 30 kD molecule could be recognized by polyclonal antibody against GST-IGF-1, but not by antibody against OPGL. It suggested that the 30 kD molecule was derived from the degradation of the fusion protein. After the cleavage with factor Xa and further purification, the fusion tag was removed and the recombinant sOPGL was obtained. Finally, we confirmed that the recombinant sOPGL could promote osteoclast formation from mouse bone marrow cells in a dose dependent manner.