BACKGROUND: Diabetic nephropathy is one of the most serious vascular complications of diabetes mellitus. Compound preparation of huangqi and dahuang, a traditional Chinese medicine, has been used to preventing or treating diabetic nephropathy for several years, and has a certain protective effect on the kidney of diabetes mellitus patients. But its exact mechanism remains unknown and needs to be studied more.OBJECTIVE: To investigate the effect of compound preparation shenkang wan on the proliferation and secretion of extracellular matrix in cultured rat mesangial cells induced by high glucose.DESIGN: Randomized and controlled study.SETTING: Center of Integrated Traditional and Western Nephrology of Zhujiang Hospital and Medicine Department of Nanfang Hospital, Southern Medical University.MATERIALS: The serum pharmacological experiment was performed in Animal Experimental Center of Southern Medical University in A pril 2005.The cell culture experiment was conducted in Cell culture room of Southern Medical University from April 2005 to July 2005. Totally 16 normal Wistar male rats, weighted varied from 190 g to 220 g, were used in the study.METHODS: Sixteen normal Wistar male rats were randomly divided into 4 groups: normal serum group, capoten group, shenkang wan group (high dose and low dose); shenkang wan was mainly constituted of huangqi,dahuang, leech, gordon guryale seed and corn stigma and made in Pharmacy Department of Zhujiang Hospital of Nanfang Medical University, agent number: 20031214). ① The rats in capoten group and high and low dose shenkang wan group were given the corresponding drugs respectively according to 5 mg/kg, 2.4 g/kg, 1.2 g/kg weight. The rats in normal serum group were given the same volume water. After treated 7 days, all rats were hocused and separated medication serum. ② Mesangial cell was cultured in vitro with different concentrations of glucose (10, 20, 30 and 40 mmol/L).The proliferation of mesangial cell was observed with the methyl-thiazoltelrazolium colorimetric assay at 24, 48, 72 hours and 96 hours. ③ Then the cultured mesangial cells were divided into six subgroups :Low glucose control group (10 mmol/L glucose), high glucose group (30 mmol/L glucose);normal serum group (30 mmol/L glucose); capoten group (30 mmol/L glucose); shenkang wan group (high dose and low dose, 30 mmol/L glucose).After cultured 72 hours, the proliferation of mesangial cell was detected with the methyl-thiazol-telrazolium colorimetric assay, the secretion and mRNA gene expression of fibronetin levels in mesangial cell were respectively detected by enzyme linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) method.MAIN OUTCOME MEASURES: ①Proliferation of mesangial cell induced by different concentrations glucose. ② Proliferation and secretion and mRNA gene expression of fibronectin in every group.RUSULTS: ① Effect of different concentrations glucose on the prolifera-tion of mesangial cell: Compared with low concentrations glucose(10 mmol/L), 20 mmol/L glucose could accelerate the proliferation ofmesangial cell during 96 hours experiment period, but only had a statisti-cally significant difference at 72 and 96 hours (P ＜ 0.05). 30 mmol/L glu-cose could significantly accelerate the proliferation of mesangial cell thanthat of 10 mmol/L glucose from 24 hours to 96 hours (P ＜ 0.05 or P ＜ 0.01),and this effect was increasing with time in 72 hours and reduced after 72hours. 40 mmol/L glucose could significantly increase the proliferation ofmesangial cell than of low concentrations glucose in 48 hours (P ＜ 0.05),and this effect was reduced after 48 hours and even conversed to restraineffect. ② Effect of different medication serum on the proliferation ofmesangial cell: The optical density value in high glucose group is obviouslyhigher than that of low glucose control group (P ＜ 0.01). Compared withhigh glucose group, the optical density value in capoten, shenkang wangroup (high dose and low dose) was decreased markedly (P ＜ 0.01 or P＜ 0.05). While the optical density value in normal serum group was showedno difference with the high glucose group (P ＞ 0.05). ③ Effect of differentmedication serum on secretion of fibronectin in mesangial cell: Content offibronectin in high glucose group was increased more markedly than that oflow glucose group (P ＜ 0.01). Compared with high glucose group, contentof fibronectin in capoten and shenkang wan group (high dose and low dose)was showed a significantly decrease (P ＜ 0.01 or P ＜ 0.05), while contentof fibronectin in normal serum group was showed no difference with thehigh glucose group (P ＞ 0.05). ④ Effect of different medication serum onexpression of fibronectin mRNA in mesangial cell: The optical density val-ue of fibronectin strip in high glucose group was brighter than that in lowglucose group and the ratio of it and β-actin were increased markedly too(P ＜ 0.01). Compared with high glucose group, the optical density value offibronectin strip in capoten and shenkang wan group (high dose and lowdose) was showed a significantly decrease and the ratio of it and β-actinwas reduced distinctly too (P ＜ 0.01), while the ratio of it and β-actin innormal serum group was showed no difference (P ＞ 0.05).CONCLUSION: High glucose could accelerate proliferation, increase thesecretion and mRNA gene expression of fibronectin in mesangial cell,while shenkang wan could inhibit proliferation and secretion of the extra-cellular matrix in mesangial cell induced by high glucose.

AIM: To investigate the expression of the nephrin in podocyte of the diabetic nephropathy(DN) rats and the mechanism of irbesartan-induced renal protection.METHODS: The DN model was established by a single injection of streptozotocin(STZ),and DN rats were randomly divided into 2 groups: model group and irbesartan treatment group.In addition,the normal rats served as a normal control group. All the rats were received daily gavage respectively for 8 weeks. The urinary protein quality in 24 hours,body weight(BW),kidney weight (KW),KW/BW,glucemia,urea nitrogen,creatinine,total cholesterol, triacylglycerol were detected with correlative methods and the pathological changes of kidney were also detected with optic microscope and transmission electron microscope.The expression of nephrin in podocyte were detected by immunohistochemistry. RESULTS: In DN rats, irbesartan reduced the urinary protein quality in 24 hours (P

AIM: To investigate the clinical effect of Maiditong for injection(Herba Erigerontis) on coagulative and fibrionlytic system in diabetic nephropathy(DN). METHODS: 59 patients definitely diagnosed as diabetes type 2 DN were randomly divided into treatment and control groups.The treatment group were treated with Maiditong for injection,and the control group were treated with of Compound Danshen Injection.The 24 h proteinuria and vWF:Ag、Fbg、PF_(1+2)、FPA、D-dimer、AT-Ⅲ:A、tPA、PAI-1、PIC、PLG were tested before and after the therapies. RESULTS: After one therapy course(2 weeks),in the treatment group the 24 h proteinuria decreased obviously(P

BACKGROUND:There is lack of effective diagnostic methods for early knee osteoarthritis. Proteomics refers to the large-scale experimental analysis of proteomes from the overal level of intracel ular protein compositioon, expression and modification, based on which, we can understand protein-protein interaction and relationship, thereby revealing protein functions and cel activity patterns. OBJECTIVE:To apply the isobaric tags for relative and absolute quantitation (iTRAQ) to identify proteins differential y in serums of osteoarthritis Kel gren-Lawrence classification at each stage, and to find out potential molecular markers at each stage of osteoarthritis. METHODS:Sixty patients with knee osteoarthritis were enrol ed, and according to Kel gren-Lawrence (K-L) grading, they were subdivided into K-L 0, II and IV groups. There were 10 males and 10 females randomly selected in each group. Proteins differential y regulated in serums were identified by the stable isotopes 116, 117, 118 of the iTRAQ labeled, reversed-phase column separation, mass spectrometry and Swissport database search, after the serums were subjected to high abundance proteins depletion. Final y, we analyzed the proteins identified by using bioinformatics software. RESULTS AND CONCLUSION:Total y 169 proteins were identified through iTRAQ peptides experiments of different sample tags, Q-star mass spectrum identification and MASCOT library search. 153 proteins were identified as K-L 0 to K-L IV, 153 proteins were identified as K-L 0 to K-L II, and 145 proteins were identified as K-L II to K-L IV. iTRAQ technology may help to identify novel serum markers for early diagnosis of knee osteoarthritis, indicating iTRAQ technology for proteomics serum biomarkers of osteoarthritis study has good application prospect.

Objective To investigate the incidence and influencing factors of aldosterone breakthrough during therapy with angiotensin Ⅱ receptor blockers (ARB) alone,or combined with angiotensin-converting enzyme inhibitors (ACEI) in Chinese patients with non-diabetic nephropathy.Methods A total of 144 patients with non-diabetic nephropathy were treated with ARB or combination therapy of ACEI and ARB for a mean follow-up period of 12 months.Aldosterone breakthrough was determined according to the change of plasma aldosterone concentration before and after treatment during 6-month and 12-month ACEI/ARB treatment.Results In 6 months,aldosterone breakthrough occurred in 21 patients,corresponding to 14.58％,while in 12 months,occurred in 39 patients,corresponding to 27.08％.Although the overall urinary protein excretion (UPE) decreased after treatment in both groups (P＜0.05),non-breakthrough group had a more remarkable reduction in UPE (P＜0.05).Univariate Logistic regression demonstrated that risk factors of aldosterone breakthrough included pre-treatment values of UPE (OR=3.643,P=0.073) and eGFR (OR=0.980,P=0.025).Multivariate Logistic model revealed pre-treatment values of eGFR was positively associated with aldosterone breakthrough (OR=0.980,P=0.025).Conclusions The incidence of the aldosterone breakthrough increases with duration of treatment.The patients with aldosterone breathrough have higher level of UPE,and enhanced decline in eGFR.Pretreatment value of eGFR is independent risk factor of aldosterone breakthrough.

Aim To investigate the effect of gelsemium alkaloids on chloride channels and cell volume in he-patic carcinoma cells. Methods The time-lapse live cell imaging and whole-cell patch clamp techniques were used respectively to detect the volume changes and currents induced by gelsemium alkaloids in HepG2 cells. Results It was found that the cell volume was decreased by (12. 48 ± 2. 2) % (P<0. 01) when ex-posed to gelsemium alkaloids for 50 min and this phe-nomenon could be inhibited by the chloride channel blocker tamoxifen. It was shown by whole-cell patch clamping that a chloride current could be evoked by extracellular application of gelsemium alkaloids ( 2μmol·L-1 ) . The current was outward-rectified with-out obvious voltage- and time-dependent inactivation. The reversal potential of the current was ( -3. 21 ± 0. 67) mV ,which was close to the equilibrium poten-tial of chloride. The extracellular application of the chloride blockers, tamoxifen and 5-notro-2-(3-phenyl-propylamino)benzoic acid (NPPB), and 47% hyper-tonic solution inhibited the current significantly ( P <0. 01 ) . Conclusion Gelsemium alkaloids could acti-vate chloride channels and induce a volume decrease ( named apoptotic volume decrease, AVD) , and these effect could be inhibited by chloride channel blockers. The results suggest that the chloride channel can be one of the targets of gelsemium alkaloids in their anti-cancer action.

Aim To clarify the effect of Borneol on the chloride channels and cell volume in poorly differentia-ted nasopharyngeal carcinoma CNE-2Z cells.Methods The technique of whole-cell patch clamp was used to detect the chloride currents and analyze the character-istics of the currents in CNE-2Z cells.The volume changes caused by Borneol were measured by the meth-od of time-lapse live cell imaging.Results The chlo-ride currents were induced by extracellular application of Borneol (20 μmol·L -1 )isotonic condition.The currents showed a characteristic of outward rectification and did not show voltage-dependent or time-dependent inactivation.The reversal potential of the currents was close to the CI-equilibrium potential. The currents were inhibited by the chloride channel blocker tamox-ifen.The currents were also inhibited by 47% hyper-tonic solution.Borneol decreased the cell volume by 9.4% in 30 min.Tamoxifen completely inhibited the Borneol-induced cell volume decrease.Conclusion Borneol can activate volume-sensitive chloride channels and induce volume decrease in CNE-2Z cells.Chloride channels play a pivotal role in the process of volume decrease caused by Borneol.

The Cathepsin L-like cysteine proteinase genes (cpls) are multifunction genes related to the parasitic abilities of plant parasitic nematodes. A new cathepsin L-like cysteine proteinase gene (Dd-cpl-1) (GenBank Accession GQ 180107) was cloned from Ditylenchus destructor by RT-PCR and RACE. The cDNA sequence consisted of a 1 131 bp open reading frame (ORF) encoding 376 amino acid residues that were franked by a 29 bp 5'-untranslated region (UTR) and a 159 bp 3'-UTR. Genomic sequence analysis showed that Dd-cpl-1 contained 7 introns, obeyed the GT/AG rule in the splice-site junctions. Homology analysis showed that the identity was 77% between Dd-cpl-1 deduced protein Dd-CPL-1 and cathepsin L-like cysteine proteinase of Bursaphelenchus xylophilus. Multi-sequence alignment indicated that there were the catalytic triad (Cys183, His322 and Asn343) and two motifs ERFNIN motif and GNFD motif in deduced protein Dd-CPL-1. Cysteine proteinases phylogenetic analysis showed that Dd-cpl-1 belonged to the sub-clade of cathepsin L-like cysteine proteinases.
Amino Acid Sequence ; Animals ; Cathepsin L ; genetics ; Cloning, Molecular ; Cysteine Proteases ; genetics ; Genes, Helminth ; genetics ; Molecular Sequence Data ; Nematoda ; enzymology ; genetics ; Phylogeny ; Sequence Alignment ; Sequence Analysis, Protein ; Sequence Homology, Amino Acid ; Solanum tuberosum ; parasitology

OBJECTIVETo explore the effects of parthenolide (PTL) on high glucose-stimulated cell proliferation, NF-κB activation and monocyte chemoattractant protein-1 (MCP-1) expression in rat mesangial cells (MCs).

METHODSMCs were cultured in normal glucose (5.6 mmol/L), high glucose (30 mmol/L), or high glucose with PTL. MTT assay was used to detect the cell proliferation. MCP-1 content in the supernatant was measured by ELISA, and the level of IκBα was detected by Western blotting to reflect NF-κB activity. EMSA method was used to measure the activation of NF-κB.

RESULTSMC proliferation, MCP-1 expression and NF-κB activation were significantly enhanced and the expression of NF-κB-binding protein IκBα was obviously reduced in cells cultured in high glucose. Application of PTL obviously abolished the effects of high glucose.

CONCLUSIONPTL can suppress high glucose-stimulated NF-κB activation and MCP-1 expression in rat MC. These data provide a theoretical basis for the clinical application of PTL in prevention and control of diabetic nephropathy.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; isolation & purification ; pharmacology ; Asteraceae ; chemistry ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chemokine CCL2 ; metabolism ; Dose-Response Relationship, Drug ; Glucose ; administration & dosage ; pharmacology ; I-kappa B Proteins ; metabolism ; Mesangial Cells ; cytology ; metabolism ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Plants, Medicinal ; chemistry ; Rats ; Sesquiterpenes ; isolation & purification ; pharmacology

OBJECTIVETo investigate the effect of the serum of rats fed with Shenkang pill in regulating monocyte chemoattractant protein 1 (MCP-1) expression induced by advanced oxidation protein products (AOPP) in mouse podocyte clone 5 (MPC5) and explore the underlying mechanism.

METHODSMPC5 cultured in vitro were incubated for different time lengths in the presence of different concentrations of serum of rats medicated with Shenkang pill, and the cell proliferation was assessed using MTT assay. In MPC5 treated with AOPP prior to exposure to the rat serum, the changes in the protein expressions of p38MAPK and IκBα were examined with Western blotting, NF-κB p65 nuclear translocation was analyzed with immunofluorescence assay, and MCP-1 expression in the supernatant was determined using ELISA kits.

RESULTSThe medicated rat serum time- and concentration-dependently promoted the proliferation of MPC5, with the optimal serum concentration of 5% and incubation time of 24 h. AOPP significantly increased MCP-1 expression in the cell supernatant in a time-and concentration-dependent manner; pretreatment with SB203580 (a p38 inhibitor) or parthenolide (a NF-κB inhibitor) significantly decreased MCP-1 expression, and treatment with the medicated serum significantly decreased AOPP-induced MCP-1 expression. AOPP concentration-dependently increased the protein expression of P-p38 but decreased that of IκBα. Both the medicated serum and SB203580 increased IκBα protein in AOPP-induced cells, but the effect was more obvious with the medicated serum. The medicated serum also decreased NF-κB p65 nuclear translocation in AOPP-induced MPC5.

CONCLUSIONShenkang pill-medicated serum can decrease AOPP-induced expression of MPC-1 in MPC5 by regulating p38MAPK/NF-κB to mediate its anti-inflammatory effect. This finding provides a new theoretical basis for the application of Shenkang pill to treat diabetic nephropathy.

Advanced Oxidation Protein Products ; pharmacology ; Animals ; Cell Line ; Cell Proliferation ; Chemokine CCL2 ; metabolism ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; I-kappa B Proteins ; metabolism ; Imidazoles ; Mice ; NF-KappaB Inhibitor alpha ; Pyridines ; Rats ; Serum ; chemistry ; Sesquiterpenes ; Transcription Factor RelA ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism