Objective To investigate the effect of estrogen on expression of matrix GLA protein (MGP)in ovariectomized Sprague-Dawley(SD)rats and the role of estrogen in improving postmenopausal osteoporosis.Methods Thirty-six SD female rats were allocated into 3 groups randomly,every 12 rats in ovariectomized group(OVX group),estrogen group(E group)and control group(sham group).Rats in OVX and E group all underwent bilateral ovariectomy,those rats in E group were given by estradiol benzoate intramuscularly after 3 weeks of ovariectomy.Rats in sham group underwent bilateral lipectomy near the ovary.All rats were kept the urine and the serum every three weeks and were sacrificed after 15 weeks.The pathology changes of uterus,lumbar vertebral bones were observed by immunohistochemistry.Bone mineral density(BMD)of lumbar vertebra of rats was determined by dual energy X ray absorptiometry(DEXA).The content of MGP in serum and urine was determined by ELISA.Expression of underearboxylated matrix GLA Protein(MGP)was detected by immunohistochemistry.Relative quantification of MGP mRNA expression in lumbar vertebra bone was detected by Fluorescent real-time quantitative polymerase chain reaction.Results(1)After 15 weeks of ovariectomized,the endometrium of uterus and lumbar vertebra exhibit remarkable pathologic changes in OVX group.The serum estrogen of(454±66)pmol/L in OVX group were lower than in(527 ±77)pmol/L in sham group and(556 ±80)pmol/L in E group significantly (P ＜ 0.05).The BMD of lumbar vertebra of(0.263 ± 0.030)g/cm2 in OVX group were lower than (0.295 ±0.024)g/cm2 in sham group and(0.279 ±0.024)g/cm2 in E group significantly(P ＜0.01).(2)The serum MPG protein in OVX group and E group showed decreased trends after ovariectomized,which were(104 ±64)ng/L in OVX group and(134 ±6)ng/L in E group at 9 weeks,which reached statistical difference(P ＜ 0.05).However,MGP in urine in sham group did not exhibit significant difference after 15 weeks of surgery(P ＞0.05).The MGP in urine of E group showed increased trends after 12 weeks of surgery,which reached(110.0 ±3.4)ng/L at 15 weeks,in the mean time,it was found that(86.5 ±2.5)ng/L of MGP in urine in OVX group,which showed significant difference(P ＜ 0.05).(3)MGP could be observed in lumbar vertebra in OVX group by immunochemistry staining.In the other two groups,the expression of MGP was not dominant.(4)Relative quantification of MGP mRNA expression in lumbar vertebra was defined as 1 in OVX group,when compared with 0.289 ±0.260 in E group and 0.103 ±0.098 in sham group,the difference showed statistically significant(P ＜ 0.01).Conclusion Estrogen could increase the expression of MGP mRNA and protein in ovariectomized rats and might play an important role in improving postmenopausal osteoporosis.

Objective To analyze the differences with the expression of glucocorticoids receptor isoforms ( GRα, GRβ, GRγand GR-P ) and cytokines [ IL-6, macrophage migration inhibitory factor (MIF), IFN-γand IL-10] between patients with systemic lupus erythematosus (SLE) and rheumatoid ar-thritis ( RA) , and to further understand their correlations with disease activities.Methods Fifty-five pa-tients with SLE, forty-nine patients with RA and thirty-eight healthy subjects were recruited in this study. All patients were steroid-naive.The expression of GRα, GRβ, GRγ, and GR-P in peripheral blood mononu-clear cells at transcript levels were determined by real-time PCR.Enzyme-linked immunosorbent assay was used to detect the expression of IL-6, MIF, IFN-γand IL-10 in serum samples.Results The percentages of GRαin all subjects were the highest among four isoforms of GR, followed by GR-P, GRγand GRβ.Com-pared with healthy subjects, patients with SLE or RA showed significantly decreased expression of GRα( P<0.05), but increased expression of GR-P (P<0.05).The percentages of GR-P in patients with RA were higher than those in patients with SLE (P<0.05).The expression of GRαwas negatively correlated with SLE disease activity index (SLEDAI) and disease activity score 28 (DAS28).SLE or RA patients with high disease activity showed lower expression of GRαthan those with low disease activity.The levels of IL-6, IFN-γand MIF in patients with SLE or RA were significantly higher than those in healthy subjects ( P<0.05).A negative correlation was observed between the expression of IL-6 and GRαin patients with SLE (P<0.05).The expression of IFN-γwas negatively correlated with GRαin patients with RA (P<0.05). Conclusion There were significant differences with the expression of GR isoforms among patients with SLE, patients with RA and healthy subjects, indicating the change of internal environment in patients might be in-volved in GR alternative splicing.GRαwas the predominant isoform and was negatively correlated with dis-ease activities.Oversecretion of cytokines resulted in a decreased expression of GRα.This study would be useful for the diagnosis of the disease status and for monitoring clinical treatment.

Objective To explore the anti-apoptotic mechanism of Roux-en-Y gastric bypass (RYGB) through exam?ine the postoperative change of adiponectin levels and expressions of pancreatic islets relative apoptotic protein. Methods Sixty SD rats were randomly allocated to RYGB group (n=20), type 2 diabetes mellitus group (T2DM, n=20) and normal con?trol group (NC, n=20). Rats in the NC group were fed with normal diet. In order to make type 2 diabetic rat models, the rats in the T2DM and RYGB groups were fed with high fat diet (22.19 kJ/g) combined with administration of intraperitoneal strep?tozotocin injection (STZ, 30 mg/kg) on the 13th day of high fat diet. RYGB operation were performed in RYGB group and sham-operation were performed in the T2DM and NC groups when diabetic model was contructed. Rats were weight preoper?atively and at the 7th, 14th, 21st days after operations. Fasting plasma glucose and adiponectin (ELISA) were measured preoper?atively and at 21st day postoperatively. Protein expressions of Bcl-2,Caspase 8 and Caspase 9 in pancreatic islets were ex?amined by immunohistochemistry at the 21st day postoperatively. Results Body weights do not vary significantly among three groups preoperatively. Compared to rats in the NC group, fast plasma glucose level was higher but adiponectin was low?er in rats in RYGB and T2DM groups. Body weights of rats in RYGB group decreased significantly compared to those of rats in NC and T2DM groups postoperatively. Compared to rats in T2DM group, fasting glucose level was lower while adiponectin concentrations was higher in rats in RYGB group but no differences of these parameters were seen in rats in NC group at the 21st day postoperatively. Expression of Bcl-2 in RYGB group was significantly elevated while expressions of Caspase 8 and Caspase 9 were significantly decreased compared to those in T2DM group postoperatively. Conclusion Adiponectin levelswas elevated;expressions of Bcl-2 was increased;expressions of Caspase 8, Caspase 9 were decreased upon RYGB opera?tion in T2DM model. RYGB might reduce pancreatic islets apoptosis through mitochondrial pathway.

OBJECTIVE: To explore the mechanisms that mediate the anti-inflammatory activity of Eurycoma longifolia. METHODS: Kunming mouse models of xylene-induced ear swelling and lipopolysaccharide (LPS)-induced acute pneumonia were used to compare the anti- inflammatory activities of aqueous and ethanol extracts of Eurycoma longifolia. UPLC-Q-TOF-MS/MS was used to identify the chemical composition in the ethanol extract of Eurycoma longifolia, based on which the potential antiinflammatory targets of Eurycoma longifolia were screened using the databases including SwissADME, SwissTargetPrediction, and Genecards. The String database was used to generate the protein-protein interaction (PPI) network, and Cytoscape was used for network topology analysis and screening the core targets. The enrichment of the core targets was analyzed using Metascape database, the core components and targets were docked with Autodock software, and the docking results were visualized using Pymol software. In a RAW264.7 cell model of LPS-induced inflammation, the Griess reagent was used to measure NO level, and Western blotting was performed to detect the expression levels of MAPK1, JAK2, and STAT3 proteins to verify the anti- inflammatory mechanism of Eurycoma longifolia. RESULTS: The ethanol extract (75%) of Eurycoma longifolia (ELE) was the active site, which contained a total of 37 chemical components. These chemical compounds and diseases had 541 targets, involving the JAK/STAT3, cAMP and other signaling pathways. Twelve indicator components were identified, which all showed good results of molecular docking with two core targets involved in the signaling pathways. In the cell validation experiment, treatment of the cells with low-, medium-, and high-dose ELE significantly reduced NO release in the cells, and ELE at the medium dose significantly decreased the cellular expressions of JAK2 and STAT3. CONCLUSION The anti-inflammatory activity of Eurycoma longifolia is attributed primarily to its active ingredients bitter lignin and alkaloids, which may regulate the JAK/STAT3 signaling pathway by targeting JAK2 and STAT3.
Animals ; Mice ; Network Pharmacology ; Eurycoma ; Lipopolysaccharides ; Molecular Docking Simulation ; Tandem Mass Spectrometry ; Anti-Inflammatory Agents/pharmacology* ; Ethanol ; Plant Extracts/pharmacology*

OBJECTIVE: To investigate the role of Numb in regulating mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway. METHODS: Male BALB/C mouse models of acute kidney injury (AKI) were subjected to intravenous injections of Numb-siRNA or NC-siRNA with or without intraperitoneal cisplatin injections. After the treatments, the expressions and distribution of Numb and megalin in the renal tissues of the mice were detected with immunohistochemistry, and the renal expressions of Numb, S6, p-S6, S6K1, p-S6K1, 4EBP1 and p-4EBP1 were examined with Western blotting. The proximal renal tubular epithelial cells were isolated from the mice transfected with Numb-siRNA for in vitro culture. In NRK-52E cells, the effects of amino acid stimulation, Numb knockdown, and V1G1 overexpression, alone or in combination, on expressions of Numb, S6 and p-S6 were detected with Western blotting; the expressions of AMPK and p-AMPK were also detected in transfected NRK-52E cells, mouse kidneys and cultured mouse renal tubular epithelial cells. RESULTS: In BALB/C mice, injection of Numb-siRNA caused significant reductions of Numb and p-S6 expressions without affecting megalin expression in the renal proximal tubules (P < 0.05). Cisplatin treatment obviously upregulated p-S6K1 and p-4EBP1 expressions in the kidneys of the mice (P < 0.05), and this effect was significantly inhibited by treatment with Numb-siRNA (P < 0.05). In NRK-52E cells, amino acid stimulation significantly upregulated the expression of p-S6 (P < 0.05), which was strongly suppressed by transfection with Numb-siRNA (P < 0.05). Numb knockdown inhibited AMPK activation in NRK-52E cells, mouse kidneys and primary proximal tubular epithelial cells (P < 0.05). Numb knockdown significantly downregulated V1G1 expression in NRK-52E cells (P < 0.05), and V1G1 overexpression obviously reversed the inhibitory effect of Numb-siRNA on S6 phosphorylation (P < 0.05). CONCLUSION Numb promotes the activation of mTORC1 signaling in proximal tubular epithelial cells by upregulating V1G1 expression.
Animals ; Male ; Mice ; Amino Acids/pharmacology* ; AMP-Activated Protein Kinases/metabolism* ; Cisplatin/pharmacology* ; Epithelial Cells ; Low Density Lipoprotein Receptor-Related Protein-2/metabolism* ; Mammals/metabolism* ; Mechanistic Target of Rapamycin Complex 1/metabolism* ; Membrane Proteins/metabolism* ; Mice, Inbred BALB C ; Nerve Tissue Proteins/metabolism* ; RNA, Small Interfering/metabolism* ; Signal Transduction ; Vacuolar Proton-Translocating ATPases/metabolism*

OBJECTIVE: To investigate the effect of dihydromyricetin (DHM) on cardiac insufficiency in diabetic rats and explore the underlying mechanism. METHOD: Twenty-four male SD rats were randomized equally into normal control group, type 2 diabetes (T2DM) group fed on a high-glucose and high-fat diet for 6 weeks with low-dose streptozotocin (STZ) injection, metformin (MET) group with daily intragastric administration of MET (150 mg/kg) for 8 weeks after T2DM modeling, and dihydromyricetin (DHM) group with daily intragastric administration of DHM (250 mg/kg) for 8 weeks after modeling. The levels of fasting blood glucose, low density lipoprotein (LDL-C), triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL-C) and glycosylated hemoglobin (HbA1c) of the rats were measured, and plasma levels of insulin and high mobility group protein-1 (HMGB1) were detected with ELISA. The cardiac function of the rats was assessed using color echocardiography, ECG was measured using a biological signal acquisition system, and myocardial pathology was observed with HE staining. The protein expressions of HMGB1, nuclear factor-κB (NF-κB) p65 and phospho-NF-κB p65 (p-NF-κB p65) in the myocardial tissue were detected using Western blotting. RESULTS: Compared with the control group, the rats in T2DM group showed significant anomalies in cardiac function after modeling with significantly increased plasma HMGB1 level and expressions of HMGB1, NF-κB p65 and p-NF-κB p65 proteins in the myocardial tissue (P < 0.05 or 0.01). Treatment with DHM significantly improved the indexes of cardiac function of the diabetic rats (P < 0.05 or 0.01), decreased plasma HMGB1 level and down-regulated the protein expressions of HMGB1 and p-NF-κB p65 in the myocardial tissue (P < 0.05 or 0.01). CONCLUSION DHM treatment can improve cardiac function in diabetic rats possibly by down-regulation of HMGB1 and phospho-NF-κB p65 expressions in the myocardium.
Animals ; Diabetes Mellitus, Experimental/metabolism* ; Diabetes Mellitus, Type 2/metabolism* ; Flavonols ; HMGB1 Protein ; Heart Failure ; Male ; Metformin/therapeutic use* ; NF-kappa B/metabolism* ; Rats ; Rats, Sprague-Dawley

Objective To observe the expression of matrix gla protein(MGP) mRNA in primary osteoblasts of Sprague-Dawley (SD) rat in vitro after treatment with anti-osteoporosis agents [vitamin K2,PTH,1,25 (OH)2D3,and alendronate],and to investigate the potential role of MGP in the pathogenesis of osteoporosis.Methods Primary osteoblasts(OBs) were derived from sequential trypsin/collagenase-digested calvaria isolated from newborn SD rat (postnastal day 1-3).OBs of the second generation were identified by Van Gieson collagen staining,alkaline phosphatase(ALP) staining and calcified nodules staining.OBs of the fourth generation were selected to interfere with vitamin K2,PTH,1,25 (OH)2D3,and alendronate,then cultured for 24 h in mediums which contained various concentrations of vitamin K2 (10-7,10-6,and 10-5 mol/L),PTH (10-9,10-8,and 10-7 mol/L),1,25 (OH) 2D3(10-10,10-9,and 10-8mol/L),alendronate(10-6,10-5,and 10-4mol/L).After being cultured for 24 h,total RNA was extracted and examined by real-time quantitative RT-PCR.Results The primary cultured cells had typical morphological characters of osteoblast.van Gieson collagen staining,ALP staining,and calcified nodules staining were all positive.Vitamin K2,PTH,1,25 (OH)2D3,and alendronate could modulate the expression of MGP mRNA in osteoblasts in a dose-dependent fashion.MGP mRNA expressions were 2.56-fold,2.12-fold,and 1.57-fold with 10-5,10-6,and 10-7 mol/L of vitamin K2 treatment,respectively.The expressions were 6.78-fold,5.31-fold,and 2.23-fold with 10-7,10-8,and 10-9mol/L of PTH(1-34) treatment,8.93-fold,6.95-fold,and 3.47-fold with 10-8 10-9,and 10-10mol/L of 1,25 (OH)2D3 treatment,and 3.47-fold,2.49-fold,and 1.98-fold with 10-4,10-5,and 10-6mol/L of alendronate treatment.Conclusion Vitamin K2,PTH,1,25 (OH)2D3,and alendronate all canregulate MGP mRNA expression in calvarial osteoblasts in a dose-dependent manner.MGP seems to be a potent target of anti-osteoporosis agents,and involved in the pathogenesis of osteoporosis.

ObjectiveTo observe the effect of parathyroid hormone on expression of matrix GLA protein (MGP) in ovariectomized SD rats and primary osteoblast,and to study the role of MGP on the possible mechanism of postmenopausal osteoporosis.MethodsThirty-six Sprague-Dawley female rats were allocated into 3 groups,12 in each:sham operation group,ovariectomized group( OVX group),ovariectomized and parathyroid hormone treatment group.Animals in the parathyroid hormone group were injected parathyroid hormone (20 μg/kg,three times a week for 12 weeks) three weeks after ovariectomy.All rats were sacrificed after 18 weeks.Urine and serum were collected every three weeks.Lumbar vertebral bones were observed by immunohistochemistry.Bone mineral density (BMD) of lumbar vertebra of rats was determined.The content of MGP in serum and urine was determined by enzyme-linked immunosorbent assay (ELISA).Expression of undercarboxylated Matrix GLA Protein (ucMGP) was detected by immunochistochemistry.Relative quantification of MGP mRNA expression in lumbar vertebra bone was detected by Fluorescent real-time quantitative polymerause chain reaction.Results ( 1 ) 18 weeks after ovariectomy,BMD of lumbar vertebra in OVX group was lower than those in sham group and parathyroid hormone group significantly ( P＜0.05 ).(2) The content of MGP in serum and urine was dynamic variation after treatment hy parathyroid hormone,and it was significant compared with OVX group ( P＜0.05 ).( 3 ) Immunohistochemical localization of ucMGP was seen in lumbar vertebra in OVX group.(4) Relative quantification of MGP mRNA expression in lumbar vertebra in OVX group was increased significantly compared with other groups ( P＜0.01 ).( 5 ) parathyroid hormone ( 1-34 ) in 10-7mol/L,10-8mol/L,10-9 mol/L up-regulated MGP mRNA expression in primary osteoblasts about 6.78,5.31,and 2.23 times than control respectively.It was in a dose-dependent manner.ConclusionThe effect of parathyroid hormone on the expression of matrix gla protein may play an important role in mechanism of postmenopausal osteoporosis

OBJECTIVE:To determine the oil-water partition coefficients (logP) of rosiglitazone at different pH values by two kinds of methods.METHODS:A shake flask-ultraviolet spectrophotometry method was used to determine plasma concentration of rosiglitazone in water phase and organic phase at variable pH values (0.95,2.0,3.0,4.0,5.07,5.7,6.55,7.06,7.5,8.15,9.21) to calculate logP by using with n-caprylic alcohol-water as simulation system.Above logP values were compared with that calculated by Pallas software.RESULTS:The logP value was lower than 1 when pH value was lower than 4;the logP value was higher than 1 when pH value was ranged from 5 to 9.Measured logP values were in good agreement with that calculated by Pallas software.CONCLUSION:Rosiglitazone exists in the form of hydrochloride salt with good water solubility (pH

Objective To investigate the mRNA level of glucocorticoid receptor α (GRα) and heat shock protein 90 (HSP90) in peripheral blood mononuclear cells (PBMCs) and the plasma protein level of macrophage migration inhibitory factor (MIF) in patients with systemic lupus erythematosus (SLE) and to analyze their association with glucocorticoid (GC) resistance.Methods One hundred and six patients with SLE and thirty-eight healthy controls were enrolled in this study.Transcription levels of GRα and HSP90 were determined by real-time polymerase chain reaction.Enzyme-linked immunosorbent assay was used to detect the protein level of plasma MIF.The association between these parameters and GC resistance was analyzed by Spearman correlation analysis.The multivariate logistic regression model was used to analyze the risk factors for GC resistance.Results The mRNA level of GRα and HSP90 in GC resistance group was significantly lower than that in GC sensitive group [10.18(3.12,17.20) vs 16.83(12.01,24.18), P =0.001;18.46(14.77,26.45) vs 25.84 (17.97,35.90), P =0.005].MIF protein level in GC resistance group was significantly higher than that in GC sensitive group [(23.21 ±7.98) μg/L vs (18.34 ±6.29)μg/L;P =0.013].The mRNA level of HSP90 in the high MIF group was significantly lower than that in the low MIF group [23.67 (13.84,28.32) vs 26.64 (23.61,47.16);P =0.001], as well as HSP90/GRαratio(P =0.008).Additionally, the plasma protein level of MIF was negatively correlated with HSP90 (r =-0.275, P =0.004) and HSP90/GRα ratio(r =-0.341, P ＜ 0.001).SLE activity index score in GC resistance group was significantly higher than that in GC sensitive group [(12.23 ±2.86) μg./L vs (9.63 ± 3.48) μg/L;P =0.003].Logistic regression model indicated that disease activity was an independent risk factor for GC resistance (OR =17.481, 95％ CI 1.747-174.903, P =0.015).Conclusions Our preliminary findings suggest that low mRNA level of GRα and HSP90 and high protein level of MIF are associated with GC resistance.Elevated MIF level in SLE patients may play an important role in the development of GC resistance through down-regulating HSP90 and destabilizing the balance of HSP90/ Grα.Disease activity is the risk factor for GC resistance, which might be the viable evidence of therapy response.