Expression and effect of glucocorticoid receptor β on children with idiopathic nephrotic syndrome

AIM: To elucidate the significance of glucocorticoid(GC) receptor isoform β(GRβ)in children with idiopathic nephrotic syndrome(INS), and to evaluate the effect of sera from GC-resistant INS on the expression of GRβ. METHODS: The percentage of GRβ positive staining peripheral blood mononuclear cells(PBMC) and the quantity of nuclear protein of GRβ in PBMC were detected by immunocytochemistry and Western blotting assay, respectively. The effect of sera isolated from children with GC-resistant INS on GRβ expression was examined by cell culture in vitro. RESULTS: The number of GRβ positive staining PBMC and the quantity of nuclear protein of GRβ in children with GC-resistant INS were significantly higher than those in patients with GC-sensitive INS(P<0 01). The quantity of GRβ protein in nuclei of PBMC in children with GC-resistant INS was significantly higher than that in normal control and children with GC-sensitive INS. After 24 hours incubation with sera from children with GC-resistant INS, the percentage of GRβ positive PBMC and the quantity of nuclear GRβ protein in children with GC-sensitive INS and normal control increased significantly(P<0.05). CONCLUSION: Increased expression of GRβ is closely related with the GC resistance in children with INS, and high expression of GRβ is mediated, at least in part, by certain humoral factors in the serum.     

Role of glucocorticoid receptor in hepatic injury in early stage after severe multiple injury

AIM: To investigate the functions of GR in the course of hepatic secondary injury after severe multiple injury. METHODS: Rat model was produced by adopting severe thoracic impact injury accompanied with mono-side femur fracture, and glucocorticoid receptor was blocked before severe multiple injury. Hepatic macropathology and alterations under light microscope were examined. Maximal binding volume of glucocorticoid receptor (GR) in hepatic tissue was assayed by radio-ligand binding assay and protein content was assayed by Western blot. RESULTS: Maximal binding volume and protein content of GR were gradually decreased in hepatic tissue after severe multiple injury, obviously lower than that in normal control at 4 h after trauma (P＜0. 01), and the lowest value was at 12 h after trauma. Maximal binding volume was 12. 9% of normal control (P＜0. 01), and protein content was 21. 9% of normal control (P＜0. 01). In addition, no significant pathological alteration in liver was found after trauma. However, the pathological analysis showed that the administration of GR blocker before severe multiple injury could cause severe hepatic congestion and infiltration of inflammatory cells in hepatic sinusoid in a dose-dependent manner. CONCLUSION: GR insufficiency could cause secondary hepatic injury in early stage after severe multiple injury, implying an important role of GR in injury and anti-injury mechanism of hepatic tissue cells.     

Effect of nitric oxide in ocular inflammatory response after intraocular lens implantation

AIM: To investigate the effect of nitric oxide (NO) in ocular inflammation after intraocular lens implantation. METHODS: All New Zealand rabbits were divided randomly into three groups: 1. control group, 2. L-arginine (L-Arg) group, 3. N-nitro-L-arginine (L-NNA) group. Extracapsular cataract extraction (ECCE) and posterior chamber intraocular lens implantation (IOL) were operated in all animals of each groups. The inflammatory response of all rabbit eyes, including cornea edema and anterior chamber exudation were investigated 1, 3, 7, 14, 30 days afteroperation. Meanwhile, aqueous humor was drawn for white blood cell (WBC) counting and classifying, as well as for NO₂^-/NO₃^- measurement. RESULTS:NO₂^-/NO₃^- contents, total WBC and anterior chamber exudation in aqueous humor of L-Arg group were higher than that in control group. While that of L-NNA group were lower than that in control group.CONCLUSION:NO plays a role in intraocular inflammatory response after intraocular lens implantation. L-NNA, a nitric oxide synthase exhibitor, decreased NO contents, therefore it may reduce intraocular inflammatory response after intraocular lens implantation.     

Effects of burn serum on the membrane of intestinal epithelial cell in rats

AIM:To explore the mechanism and significance of the intestinal epithelial cellular membrane damage following burn serum. METHODS:The intestinal epithelial cell(IEC-6) were cultured. The changes of total membranous phospholipid contents fluidity of the IEC membrane were dynamically examined with fluorescence polarization technique and HPCE. RESULTS:In the early stage after stimulation by 20% burn serum, the membranous fluidity obviously decreased. The total phospholipid contents decreased, the content of PLA₂ markedly increased. CONCLUSION:The serial changes in IEC after burned could result in the damages of IEC membrane structure, the integrity of cell membrane and function.     

Downregulation of aortic angiotensin II type 1 receptor mRNA expression by simvastatin in hyperlipidemic rats

AIM:To study the impact of hyperlipidemia on aortic AT₁ mRNA expression and vasoactive substances, and investigate the potential mechanism on reversion of endothelial dysfunction during the statin therapy.METHODS:The investigation included control, hyperlipidemic and simvastatin-treated groups. Hyperlipidemic model was set up on the 4-week atherogenic diet, followed by a 16-week treatment in the simvastatin treated group (simvastatin 10 mg·kg^-1·d^-1) and without treatment in the hyperlipidemic group. Serum lipid level, the expression of AT₁mRNA of aorta and level of serum AngⅡ and nitric oxide (NO) were measured. RESULTS: Compared with the control group, hyperlipidemic rats showed a stronger expression of AT₁ mRNA and lower level of NO. No significant difference in systolic blood pressure and AngⅡ was showed in this group. In contrast, in simvastatin treated group, expression of AT₁ mRNA as well as lipid(TC, TG, LDL-C) levels were significantly decreased and NO level increased which associated with improvement of endothelial dysfunction. CONCLUSION:By regulated the lipid level, downregulated AT₁ mRNA expresstion and increased the NO activity, simvastatin restored endothelial function and inhibited atherogenesis.     

Long-term effects of TCV116 on left ventricular remodeling and heart function after myocardial infarction in rats

AIM: To investigate the effects of long-term TCV116 on left ventricular remodeling and heart function after myocardial infarction. METHODS: Myocardial infarction (MI) was caused by ligation of the left anterior descending coronary artery in rats. One week after the surgical performance, the surviving rats were randomly assigned to the following treatment protocols: (1) MI rats with no therapy; (2) MI rats treated with TCV116 2 mg/kg per day; (3) Sham-operated control; (4) Sham-operated rats, treated with TCV116 2 mg/kg per day. At 22 weeks, cardiac hemodynamic parameters such as MAP, LVSP, dp/dt_max and LVEDP, and histomorphometric parameters such as LVW/BW and LVCA/BW were measured, mRNA of cardiac genes such as βMHC, BNP, TGF-β₁, collagen I and III were quantified, and survival rates were calculated. RESULTS: Compared with sham-operated rats, MI rats without therapy showed significant increases in histomorphometric parameters as well as in mRAN expressions of cardiac genes (P<0.01); While their hemodynamic parameters were significantly impaired (P<0.01), and survival duration shortened (P<0.05). Compared with MI rats without therapy, MI rats treated with TCV116 showed significant attenuation of mRAN expression of cardiac genes (P<0.01); While their hemodynamic parameters were significantly improved (P<0.05 or P＜0.01), and survival duration extended (P<0.05). CONCLUSION: Treatment with long-term angiotensin II type 1 receptor antagonist may improve left ventricular remodeling and cardiac function after MI in rats.     

Differential regulating effect on estrogen receptor α and β expression in female rat hippocampus and cortex regions between different types of estrogen regents

AIM: To study the regulatory effect two different estrogen reagents on expressions of estrogen receptor α and β in female rat hippocampus and cortex regions. METHODS: 12 cycles after ovariectomy, female rats were orally injected with premarin or progynova for 3 cycles before sacrificed. Semiquantitative RT-PCR was used to detect the ERs mRNA expression and SP immunohistochemistry was performed to measure the ERs protein distribution and expression. RESULTS: In premarin group, ER α mRNA levels in both hippocampus and cortex tissues decreased significantly compared with control. ER α protein level in hippocampus was lower than that in the control. However, ER α protein level in cortex had no statistical difference. ER β mRNA in the two regions and ER β protein in cortex had no statistical differences compared with control, while ER β protein level in hippocampus was higher than that in the control. In progynova group, both mRNA and protein levels of ER β increased significantly in the two regions compared with the control, and ER α mRNA level also increased in hippocampus, but ER α mRNA level in cortex and ER α protein levels in the above two regions showed no statistical differences. CONCLUSION: There were differential regulatory effects on ER α and ER β expression in female rat cognitive regions between the two different types of estrogen reagents, which may be one of the mechanisms of varied effects in different estrogen replacement therapy reagents.     

Study on mutations of exon BH of the androgen receptor gene in 45 cases of patients with prostate cancer

AIM and METHODS: To learn more about the mechanism of prostate cancer (PC) development and progression to androgen independence, the exons BH of the androgen receptor (AR) gene of forty-five patients with prostate cancer, six puncture tissues and thirty-nine slide tissues, were analyzed by polymerase chain reaction-single strand conformation technique (PCR-SSCP). RESULTS: Seven abnormal mobility shifts were found in five patients by PCR-SSCP. Combining the method with direct DNA cycle sequencing, two distinct missense (Glu872Gln, Met886Ile) point mutations were identified in puncture tissues from two patients of advanced prostate cancer with distant metastasis. These two point mutations represented two novel mutations. CONCLUSION: AR gene mutations might play an important role in the development and progression of prostate cancer.     

Changes in the expression of endothelin system in rats with chronic heart failing

AIM: To study the changes of endothelin system during chronic heart failure (CHF) and imply the relationship between endothelin system and the course of CHF by observing the mRNA expression of endothelin receptors (ET_AR and ET_BR) and PreproET₁ in early stage and later stage of CHF caused by left coronary artery ligation. METHODS: The mRNA expression of ET_A, ET_B receptors and PreproET₁ were detected by RT-PCR technique. The plasma concentrations of ET₁ and ANP were determined by RIA method. RESULTS: The plasma concentrations of ET₁ and ANP, and the mRNA expression of ET receptors and PreproET₁ in the lefe ventricle increased significantly in early stage (myocardial infarction 10 d). While the plasma concentrations of ET₁ and ANP in later stage (myocardial infarction 70 d) were higher than those in the early stage. However, the mRNA expression of ET_AR, ET_BR and PreproET₁ decreased significantly. The mRNA expression of ET_AR in myocardial infarction (MI) 70 d rats had no difference with those in sham-operated rats and the mRNA expression of ET_BR and PreproET₁ in MI 70 d rats was lower significantly than those in MI 10 d rats, but significantly higher than those in sham-operated rats. CONCLUSION: The changes of ET receptors and PreproET₁ mRNA expression are involved in the cardiac function modulation during the different stages in chronic heart failure.     

Effects of burn sera on IκBα degradation and NF-κB activation in monocytes in vitro

AIM: To investigate the effects of burn sera on IκBα degradation, NF-κB activation in peripheral blood monocytes (PBMCs) in order to explore the role of burn sera on activation of monocytes. METHODS: PBMCs isolated from healthy volunteers were stimulated by sera from healthy volunteers and burn patients and by burn sera together with PDTC (pyrrolidine dithioncarbamate). Activation of monocytic NF-κB was tested by electrophoretic mobility shift assay (EMSA) and the degradation of monocytic IκBα was determined by Western blotting. RESULTS: When compared to that in control group, cytosolic IκBα degradation occurred within 30 min after PBMCs stimulated by burn sera, and peaked at 60 min. But IκBα gradually recovered in the cytoplasm after 2 h of stimulation. Meanwhile, activity of monocytic NF-κB was markedly increased, reached the peak at 30 min to 60 min after stimulation, and gradually decreased after 2 h of stimulation. PDTC (an antioxidants) effectively inhibited the monocytic IκBα degradation and activation of NF-κB induced by burn sera. CONCLUSION: Burn sera might induce the degradation of IκBα, then activate NF-κB, which ultimately lead to the secretion of cytokines from the monocytes.     

Role of androgen receptor in prostate cancer cell proliferation by use of RNAi

AIM: To study the role of androgen receptor (AR) in hormone-dependent and hormone-independent prostate cancer cell proliferation by knocking down AR expression with adenovirus-delivered siRNA. METHODS: Four well-designed siRNAs were synthesized and inserted into the adenovirus plasmid pShuttle-H1-Ri. The recombinant pShuttle-H1-Ri-AR plasmid was then co-transfected with pcDNA-AR to HEK293 cell line and Western blot was used to detect the inhibitory efficiency of different siRNAs on AR expression. Recombinant adenovirus containing more efficient siRNAs were prepared and used to infect three different humane prostate cancer cell lines including LNCap、C4-2B and CWR22Rv1. The efficiency of knocking down AR expression was detected by Western blot. The effect of AR-knocking down on cell proliferation was detected by MTT colorimetric assay. RESULTS: All of the four designed siRNAs could knock down AR expression in transient co-transfection. Infecting with recombinant adenovirus containing more efficient siRNAs in hormone-dependent and hormone-independent prostate cancer cell lines specifically knocked down AR expression with high efficiency. Knocking down AR expression significantly decreased the proliferation rate in all these prostate cancer cells. CONCLUSION: The suppressed expression of AR in prostate cell lines mediated by siRNA could efficiently inhibit the cell proliferation, and these results show that AR plays an important role in the proliferation of hormone-dependent and hormone-independent prostate cancer cells. AR is an important therapeutic target for the treatment of prostate cancer.     

Application of molecular recognition theory in thyrotropin and its receptor system

AIM: The selective recognition of the sense peptides which are located in special regions of thyrotropin receptor (TSHR) by their corresponding antisense peptides has been investigated. Three pairs of sense and antisense peptides were named TR1 (aa37-45) and RT1 (aa45-37), TR2 (aa353-366) and RT2 (aa366-353), TR3 (aa648-655) and RT3 (aa655-648). METHODS: To prepare three affinity chromatography columns, antisense peptides were immobilized, called RT1-sepharose 4B, RT2-sepharose 4B and RT3-sepharose 4B, respectively and investigate the retardative behavior for each of native peptide TR1, TR2 or TR3 on above columns with stepwise elution. RESULTS: Each of the three immobilized antisense peptides recognized and retarded its corresponding sense peptide-TR1, TR2 or TR3 instead of those non-complementary peptides. Immobilized RT1 recognized free TSHR protein molecule as well. In additional, bovine thyrotropin was recognized by immobilized TR1. CONCLUSION: The results indicate that molecular recognition theory exsits in thyrotropin receptor system. It may be useful to isolate biological molecules and to locate epitopes of TSH on TSHR molecule. Otherwise, antisense peptide may be used for treatment of experimental autoimmunolized thyroid disease (AITD) in the rat.     

Effect of insulin on the secretion of VEGF and its receptor in serum-free cultured bovine thoracic aortic endothelial cells

AIM: This study was designed to investigate the secretion of VEGF and its receptor (flt-1 or flk-1/KDR) protein by cultured bovine thoracic aortic endothelial cells treated with various insulin concentrations. METHODS: Endothelial cells was isolated from bovine thoracic aorta, and cultured in serum-free medium, then incubated with different insulin concentrations (30 mU/L, 300 mU/L, 3 000 mU/L). The level of VEGF and its receptor (flt-1 or flk-1/KDR) protein were detected by immunohistochemical staining. RESULTS: As compared with no insulin group, the expression of VEGF protein in low insulin concentration (30 mU/L and 300 mU/L) groups were significantly increased (P＜0.01). The expression of VEGF protein in high insulin concentration (3 000 mU/L) group was significantly decreased (P＜0.05). Howerer, no difference of the expression of VEGF receptor (flt-1 or flk-1/KDR) protein among all groups (P＞0.05) was observed. CONCLUSION: Low concentration insulin up-regulates the VEGF protein expression while high concentration insulin down-regulates the VEGF protein expression in bovine thoracic aortic endothelial cells, but insulin had no directly effect on the VEGF receptor (flt-1 or flk-1/KDR) protein expression in bovine thoracic aortic endothelial cells.     

Effect of fluvastatin on left ventricular remodeling after myocardial infarction in rats

AIM: To clarify the protective effect of long-term administration of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor fluvastatin on ventricular remodeling after myocardial infarction (MI) in rats and its mechanisms. METHODS: Myocardial infarction were established by ligated left coronary anterior artery in SD rats, 24 hours after the operation, the survival rats were treated by gavage fluvastatin (20 mg·kg^-1·d^-1) or distilled water for 8 weeks. Doppler echocardiography, homodynamic and cardiac histomorphometry were used to assess the ventricular remodeling and cardiac function. The plasma levels of total cholesterol (Tch), creatinine (Cr), glutamic-oxal (o) acetic transaminase (AST), lipid peroxidation (LPO), glutathione perioxidase (GSH-PX), nitrogen monoxide (NO₂^-/NO₃^-) were detected. RESULTS: The Tch, Cr and AST were not significant difference in groups. Left ventricular end-diastole pressure, right relative weight, left ventricular posterior wall thickness, collagen volume fraction and the lung weight were decreased in AMI+fluvastatin group compared to AMI group (P＜0.05, P<0.01); The levels of LPO, NO₂^-+NO₃^- in plasma and LPO in myocardium decreased, but plasma GSH-PX level increased in AMI+fluvastatin group (P＜0.05). CONCLUSION: Fluvastatin ameliorates the ventricular structural remodeling in a rat model of infarction, and delays the development of heart failure. The anti-oxidation mechanism of fluvastatin may take part in this process.     

Effects of Retinervus luffae fructus on serum lipid level in experimental hyperlipidemia rats

AIM: To investigate effects of Retinervus luffae fructus (RLF) on level of serum lipid and body weight in hyperlipidemia rats. METHODS: Thirty-two male SD rats were randomly divided into four groups: control group (A), hyperlipidemia group (B), hyperlipidemia + RLF group (C), RLF group (D). Both group A and C were fed normal diet every day, while group B and group D fed high fat diet. Meanwhile, group C and D were administered with RLF solution at the dose of 10 mL/kg, respectively for 14 days, while group A and B were administered with drinking water. RESULTS: (1) At the end of experiment, a significant reduction was found in the levels of serum total cholesterol (TC) and triglyceride (TG) of group C animals treated with RLF solution; (2) The levels of serum TC of group D was progressively decreased compared to the level of serum TC at the beginning of experiment; (3) The level of high-density lipoprotein cholesterol (HDL-C) of group C remained unaltered 8d after treatment with RLF solution; (4) The body weight in group C was obviously lower than that in group B. CONCLUSION: RLF had an obvious hypolipidemic effect on hyperlipidemia rats. It can inhibit the decrease in the HDL-C and the increase of body weight in rats.     

Effect of heterogenic corneal stroma transplantation on peripheral T cells of rats

AIM: To Compare immunogenicity of three kinds of heterogenic corneal stroma. METHODS: 36 SD rats were randomized into 4 groups, each group consisting of 9 rats. Group 1 was control group. Three kinds of heterogenic corneal stroma: porcine, rabbit and chicken corneal stroma were heterotopically transplanted to subcutaneous layer of 27 (group 2-4) SD rats, respectively. The expression of CD4⁺, CD8⁺, CD25⁺, CD71⁺ on peripheral T cells was identified and analyzed by dual fluorescence flow cytometry at 7, 14, 28 days after operation. RESULTS: Compared with control group, the expression of CD4⁺, CD8⁺, CD25⁺, CD71⁺ was no significant change in porcine corneal stroma group(P＞0.05), the expression of CD4⁺ was increased in rabbit corneal stroma (P＜0.05), CD4⁺, CD4⁺ CD71⁺ markedly higher in the chicken corneal stroma (P＜0.01) at 7 days after operation. CONCLUSION: The immunogenicity of porcine stroma is the lowest in three kinds of heterogenic corneal stroma (chicken, rabbit and porcine).     

Changes of cortisol content in plasma and hippocampus after focal cerebral ischemia-reperfusion in rats

AIM: To investigate the relationship between glucocorticoid (Gc) and injury of hippocampus neurons and the effect of Gc on dementia episode after cerebral ischemia-reperfusion. METHODS: The rat model of middle cerebral artery occlusion (MACO) was established. Cortisol contents in hippocampus and plasma of the model rats were examined by means of the radioimmunoassay at 2 h, 6 h, 12 h, 24 h after reperfusion. RESULTS: The levels of cortisol content in model group were significantly higher than those in sham group and normal group both in hippocampus and plasma. The highest cortisol content was observed at 6 hours after reperfusion. HE staining showed that the impairment of hippocampus neurons was aggravated progressively with reperfusion interval elongating. CONCLUSION: The increased cortisol in hippocampus and plasma, after 2 h cerebral ischemia and 24 h reperfusion, could aggravate the injury of hippocampus neurons and lead to dementia post stroke.