AIM: To observe the effect of Retinervus luffae fructus (RLF) on the myocardial ischemia in mice. METHODS: Mice were supplied with RLF (166.7,333.3, 666.7 g/L) twice daily (0.01 mL/g, ig). One week later, 30 U/kg pituitrin was injected intraperitoneally. The electrocardiogram was recorded, and the content of lactose dehydrogenase (LDH) in the serum and the activities of superoxide dismutase (SOD), maleicdialdehyde (MDA) in the myocardium were examined. RESULTS: RLF reduced the height of T wave in electrocardigram in myocardial ischemia mice and inhibited the decrease in heart rate. RLF also reduced the content of LDH in serum and the content of MDA in myocardium. The increase in the activity of SOD in myocardium was also observed. CONCLUSION: RLF may protect myocardium against ischemia injury. The mechanism may be related to the increase in activity of SOD and the suppression of the lipid peroxidation.

Objective To study the association of bone metabolism with the degree of proteinuria in patients of chronic kidney diseases (CKD). Methods A total of 71 CKD patients diagnosed as primary glomerulopathy were randomly selected from 2008.1-2009.5 in the First People's Hospital of Shanghai. They were classified into three groups according to proteinuria:group A of 25 patients, proteinuria ＜1.0 g/24 h; group B of 16 patients, proteinuria 1.0-＜3.5 g/24 h;group C of 30 patients, proteinuria ≥ 3.5 g/24 h. Fifty-eight healthy persons were selected from our medical examination center at the same time as control. Serum albumin, calcium, phosphorus,PTH, 25 hydroxy vitamin D3, bone gla protein (BGP), degradation products of C-terminal telopeptides of type I collagen (CTx), 24-h urinary protein excretion, and the ratio of urinary calcium to creatinine (UCa/Cr) were measured. Bone mineral density (BMD) was detected by dualenergy X-ray absorptiometry. Results Compared with control group, serum levels of calcium [(2.23±0.08), (2.13±0.09), (2.04±0.06)vs (2.37±0.12)mmol/L], 25-(OH)D3 [(50.19±6.58), (47.78±6.69), (42.42±10.85) vs (56.34±8.34) nmol/L] were significantly lower and UCa/Cr was significantly higher in A, B, C groups respectively (all P＜0.05). In group B and C, BGP was lower [(18.69±7.35), (16.13±5.76) vs (22.88±6.21) μg/L] and CTx was higher [(413.59±114.93),(516.21±314.25) vs (304.53±234.15) ng/L] (all P＜0.05). BMD was lower only in group C [(1.028±0.090) vs (1.090±0.062) g/cm2, P＜0.05]. Pearson analysis showed that 24-h urinary protein excretion was negatively correlated with serum calcium and 25 hydroxy vitamin D3, and positively correlated with UCa/Cr. UCa/Cr was positively correlated with serum CTx and negatively correlated with BGP. 25-(OH) D3 was positively correlated with BGP and negatively correlated with CTx. Conclusion Bone metabolism disorder exists in CKD patients, presenting the decrease of bone formation and the increase of bone resorption, which is associated with as the degree of proteinuria, especially in patients with nephrotic syndrome.

Objective To study the change of serum insulin-like growth factor 1(IGF-1)in primary nephrotic syndrome(PNS)patients and its relationship with bone metabolism, and to investigate the clinical significance of IGF-1 in the mechanism of bone metabolic disorders in PNS patients. Methods A total of 30 PNS patients with chronic kidney disease(CKD)stage 1 and 2 were randomly selected from 2008.1 to 2009.5 in our hospital. Serum IGF-1, albumin, calcium, phosphorus, PTH,25 hydroxy vitamin D3, bone gla protein(BGP), degradation products of C-terminal telopeptides of type I collagen(CTx), 24-hour urinary protein excretion, and ratio of urinary calcium to creatinine(UCa/Cr)were measured. Healthy control group of 61 persons were randomly selected from our medical examination center at the same time. Results Serum levels of calcium, 25 hydroxy vitamin D3 and BGP were significantly lower;CTx and UCa/Cr were significantly higher in PNS patients(P<0.05)as compared to healthy control group. BMD of PNS patients was lower but without significant difference compared with healthy control group[(1.078± 0.090)g/cm2 vs(1.090±0.062)g/cm2, P>0.05]. Serum level of IGF-1 was significantly lower in PNS patients and was positively correlated with BMD and BGP,and negatively correlated with 24-hour urinary protein excretion and CTx. Conclusions Bone metabolic disorder exists in PNS patients with the appearance of decreased bone formation and increased bone absorption.Serum level of IGF-1 has good correlations with bone biochemical markers.which may be used as a new bone biochemical marker of bone metabolism in kidney disease.

Objective To observe the change of insulin-like growth factor 1 (IGF-1)before and after glucocorticoid (GC) therapy and to explore the effect of its change on bone metabolism in primary nephrotic syndrome (PNS) patients.Methods A total of 39 PNS patients with mean age of (36.73±12.15) years received GC therapy were selected from January 2008 to August 2009 in our hospital.Serum IGF-1,albumin,calcium,phosphorus,parathormone (PTH),25hydroxy vitamin D3,bone gla protein (BGP),degradation products of C-terminal telopeptides of type I collagen (CTx),24-hour urinary protein excretion and the ratio of urinary calcium to creatinine were measured at five time points-before GC therapy,4 weeks,8 weeks,12 weeks and 24 weeks after the use of GC.BMD was also detected at the same time points.Correlations among indexes were analyzed by Pearson.Results Thirty-six PNS patients fulfilled the follow-up and had complete clinical data,while other 3 patients lost.After GC treatment,serum calcium and 25hydroxy vitamin D3 were significantly increased in a time-dependent manner and were negatively correlated with 24-hour urinary protein excretion (r=-0.749,r=-0.831,P＜0.05,respectively).Serum BGP and IGF-1 were decreased after GC therapy in a time-dependent manner while CTx was significantly increased until week 12 after treatment (P＜0.05).Compared with pre-treatment,BMD of various parts had no significant difference at week 4; BMD of lumbar spine (L1-L4) was significantly decreased until week 8 (P＜0.05); BMD of femoral neck and femoral shaft was significantly decreased at week 24 (P＜0.05).IGF-1 was positively correlated with BGP and BMD (r=0.896,r=0.495,P＜0.05) and negatively correlated with serum CTx (r=-0.697,P＜0.05 ).Conclusions Serum IGF-1 level decreases in a time-dependent manner after GC treatment,which is correlated to BGP,CTx and BMD.Glucocorticoid treatment affects bone metabolism through IGF1 pathway possably in patients with PNS.IGF-1 may be used as a new bone biochemical marker of glucocoritcoid - induced osteoporosis.

Objective To explore the impact of rituximab on Th17 cells and related cytokines in patients with diffuse large B-cell lymphoma (DLBCL) in vitro and its significance.Methods 20 cases of DLBCL untreated patients and 20 healthy subjects were enrolled in the name of DLBCL group and health control group, respectively.4 peripheral blood samples were collected from every case to separate peripheral blood mononuclear cells (PBMCs), which were assigned to 4 subgroups according to different culture conditions: blank subgroup(subgroup A), rituximab subgroup (subgroup B), rituximab and serum subgroup (subgroup C) and polarization subgroup (subgroup D) (added IL-6 and TGF-β).After cultured in vitro, the percentage of Th17 cells in each subgroup was tested by flow cytometry, and the cytokine IL-17 in the abovementioned culture fluid was measured by enzyme-linked immunosorbent assay (ELISA).Results In health control group, the percentage of Th17 cells and the level of IL-17 in subgroup D [(17.12 ± 4.90) ％ and (45.735±10.012) pg/ml] were significantly higher than those in subgroup A, B, C (P ＜ 0.05), and there was no difference in each other subgroup A, B, C (P ＞ 0.05).The percentage of Th17 cells and the level of IL-17 in the DLBCL subgroup A were significantly lower than those in health control subgroup A [(0.69±0.24) ％ and (6.012±1.312) pg/ml vs (2.43±0.61) ％ and (8.217±1.681) pg/ml (P ＜ 0.05)].In DLBCL group, after cultured with rituximab, the percentages of Th17 cells in subgroup B, C, D were (2.34±0.48) ％, (2.31±0.53) ％ and (16.92±4.81) ％, and the levels of IL-17 were (7.944±1.538) pg/ml, (7.957±1.533) pg/ml and (44.417±9.881) pg/ml, respectively, which were all significantly higher than those in subgroup A.Besides, the percentage of Th17 cells and the level of IL-17 in DLBCL subgroup D were significantly higher than those in subgroup B, C (P ＜ 0.05), while there was no difference between subgroup B and subgroup C.Conclusion Experiments in vitro confirmed that the percentage of Th17 cells in PBMCs of DLBCL patients was lower than that in healthy persons, and rituximab could elevate the percentage of Th17 cells in PBMCs of DLBCL patients.

Objective To study skeletal muscle atrophy and the change of autophagy in skeletal muscle of patients with chronic kidney disease.Methods Mean muscle cross sectional area,mRNA and protein expression of autophagy markers Bcl-2-adenovirus E1B interacting protein 3 (LC3B),Bcl-2-adenovirus E1B interacting protein 3 (Bnip3),Beclin-1 were measured in rectus abdominis biopsies obtained from 22 consecutive patients with stage 5 CKD scheduled for peritoneal dialysis from 4 hospitals in Shanghai.Control biopsies were obtained from another 8 healthy subjects during elective surgery for adenomyosis and 6 subjects during elective surgery for abdominal wall hernias.Rectus abdominis muscles were obtained at the beginning of surgery.HE staining was performed and mean cross sectional area (CSA) was calculated.Electron microscopy was used to confirm the changes of autophagy.mRNA levels of LC3B,Beclin-1,Bnip3 were evaluated by RT-PCR and protein levels of those parameters were evaluated by Western blotting.Results Compared with control group,mean CSA of muscle fibers was decreased and the transcript levels of LC3B,Beclin-1,Bnip3 were up-regulated in CKD group.Similarly,protein levels of LC3BⅠ,LC3B Ⅱ,Beclin-1 and Bnip3 were increased in CKD group.Additionally,activation of autophagy was confirmed by the appearance of autophagosomes by electron microscopy.Conclusion Chronic kidney disease may cause skeletal muscle atrophy and lead to activation of autophagy,which may contribute to muscle atrophy.

Objective To investigate the expression of Notch 1 receptor in renal tissues of patients with hepatitis B virus associated-glomerulonephritis (HBV-GN) and its role in the pathogenesis of HBV-GN.Methods A total of 48 patients with HBV-GN confirmed by renal biopsy during 2008-2010 were enrolled in the study.Distribution of Notch1 receptor in renal tissue of HBV-GN was detected by immunohistochemistry and the association between the distribution of Notch1 receptor and HBsAg was examined by double-label immunofluorescence assays.Correlations of Notch1 receptor expression with renal pathology and clinical parameters of HBV-GN were analyzed.Results Notch1 receptor distributed mainly in renal tubular epithelial cells and interstitial area as brownish red granules,and a few expression in glomerulus was also found.The positive score of Notch1 receptor expression in HBV-GN patients was significantly higher as compared to primary glomerulonephritis patients with serum HBsAg positive or negative and normal renal tissue controls.Notch1 receptor expression was more obvious in membrano-proliferative glomerulonephritis (MPGN) and mesangial proliferative nephritis (MsPGN) patients,but there was no significant difference among the different pathology groups.Distribution of Notch1 receptor was consistent with the distribution of HBsAg and its intensity was positively correlated with renal interstitial fibrosis (r=0.473,P=0.001),tubular atrophy (r=0.690,P=0.000),inflammatory cell infiltration (r=0.616,P=0.000).Negative correlation was found between renal function and the intensity of Notch1 receptor (r=-0.393,P=0.006).Conclusions Notch1 receptor expression increases in the renal tissues of HBV-GN patients and distributes mainly in renal tubular epithelial cells and interstitium,which is consistent with the distribution of HBsAg.Its intensity is closely correlated with renal interstitial lesions and renal function.Abnormal expression of Notchl receptor in renal tissue of HBV-GN may be involved in the progress of HBV-GN.

ObjectiveTo investigate the expression and distribution of Toll-like receptor 4 (TLR4) in renal tissue of HBV associated nephropathy (HBV-GN) and its role in the pathogenesis and clinical manifestations of HBV-GN.MethodsRenal tissues were sampled from 48 HBV-GN patients confirmed by renal biopsy and 154 non-HBV-GN patients.The distribution of TLR4 in renal tissue and the relationship between the distribution of TLR4 and HBsAg were detected by immunohistochemistry.Integrating case record,correlations between the expression of TLR4 with clinical parameters including pathology,glomeruli,kidney tubules lesions,renal interstitial inflammatory infiltration and blood serum HBV were analyzed.ResultsTLR4 mainly distributed in the renal tubular epithelial cells and interstitial areas as brownish red and granular,which was in consistent with HBsAg distribution.The TLR4 positive rate and score in HBV-GN group were higher than those in non-HBV-GN group (P ＜ 0.05 ).TLR4 positive score was slightly higher in mesangial proliferative glomerulonephritis group and focal segmental glomerulosclerosis group,which had no significant difference (P ＞ 0.05).Kidney tubules lesions were strongly associated with TLR4 expression (r =0.748,P ＜ 0.001 ) which increased with aggravation of renal interstitial fibrosis ( r =0.569,P ＜0.001 ),tubular atrophy ( r =0.577,P ＜ 0.001 ) and inflammatory cell infiltration ( r =0.684,P ＜0.001 ).No obvious correlation with glomeruli lesions was observed ( r =0.293,P =0.053 ).Negative correlation could be seen between TLR4 and the renal function ( R2 =0.784),systolic blood pressure ( R2 =0.869),high sensitivity C-reactive protein (R2 =0.979) and urinary protein (R2 =0.615 ) by regression analysis.Other clinical parameters had no statistical significances.ConclusionsThe expression of TLR4 is abnormal in the renal tissue of HBV-GN patients,mainly in renal tubular epithelial cells and interstitial,which is consistent with the distribution of HBsAg.Its intensity is closely related with renal interstitial lesions,renal function changes and inflammatory cell infiltration.A speculation,that HBV can promote abnormal expression of TLR4 in renal tissues of HBV-GN which may be involved in the lesion progress of HBV-GN,is made upon our study.

Objective To explore the expression and role of Toll receptor 4(TLR4)in human proximal tubular epithelial cell line HK-2,infected by HBV. Methods The serum of HBV DNA copies between 107-108/ml was collected. Before and after infected by HBV DNA positive serum. the HK-2 cells' morphology and the expression of α-smooth muscle actin(α-SMA)were observed by microscopy and immunofluorescence, and the effects of different concentrations of lipopolysaccharides(U)S.TLR4-stimulating factor)and CLI-095(TLR4 Inhibitor)on the proliferation rate of HK-2 cells were observed by MTT assays. After HBV serum and 10μg/ml LPS and 5μl/ml CLI-095 acted on HK-2 cells,TLR4 protein expression was measured by immunofluorescence and Western-blotting assay, and HBsAg and HBeAg in cell culture medium were detected by ELISA. and HBV DNA copies by fluorescence quantitative PCR. Results The longer HBV infected HK-2 cells, the more irregular of the cells' shape, the fewer number of the cells were left. But compared with HBV infected after 24 hours, α-SMA was more expressed after HBV infected 12 hours. After infected by HBV serum in 24 hours.HK-2 cells' proliferation rate was positively correlation in a dose range of LPS, but was negatively correlated with the CLI-095(P＜0.05＝.The levels of HBsAg and HBeAg in cell culture medium were largest when the LPS concentration was at 10μg/ml and CLI-095 at 5μg/ml.The expression of TLR4 significantly increased in HK-2 cells treated with LPS compared with those with CLI-095.but HBV DNA levels and HBsAg and HBeAg expression levels were lower. Conclusions HBV infection may promote cell transdifferentiation and cell injury. The stimulation of HK-2 infected with HBV by LPS may upregulate the expression of TLR4 and reduce the copies of HBV DNA.

Objective To investigate the effect of parathyroid hormone (PTH) on the transition and connective tissue growth factor (CTGF) expression of human renal proximal tubular epithelial cell line HK-2 . Methods The expression of CTGF mRNA and protein of HK-2 cells were measured by real time RT-PCR and Western blot respectively . The effect of PTH on the phenotypic transformation of HK-2 cells was examined by light microscopy . The expression of α-smooth muscle actin (α-SMA) in HK-2 cells was detected by immunofluorescence . Results Basal level of CTGF mRNA and the protein expression were detected in HK-2 ceils . PTH upregulated the expression of CTGF mRNA and protein with the maximal response at the concentration of 10-10 mol/L and the best stimulating time was at 72 h . After exposure to PTH (10-10tool/L) for 12 hours, the highest level of luciferase activity was 1 .96 fold as compared to control (1 .888±0 .078 vs 0 .989±0 .030, P＜0 .01 ) . Untreated cells showed negligible expression of ±-SMA,whereas ±-SMA expression was significantly increased in cells treated with PTH . Conclusion PTH up-regulates CTGF expression and induces transition of HK-2 cells .