Objective To study the expression and transcription of PKC? in renal tissue of streptozotocin-induced diabetic rats. Methods Hyperglycemia was induced with streptozotocin(55 mg/kg) in Sprague-Dawley rats. After 5 weeks, the expression of PKC? protein and mRNA was measured by immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Results In diabetic animals, the expression of PKC? was greatly enhanced especially in the proximal tubules and the glomeruler mesangial areas with upregulating of the membrane-associated PKC?. RT-PCR analysis showed that mRNA level of PKC? increased by 1.67 folds in diabetic rats as compared with the normal ones. Conclusion The expression of PKC? protein and mRNA is upregulated in early diabetic nephropathy, which suggests an interaction between PKC? and the pathogenesis of diabetic nephropathy.

Objective To investigate the expression and translocation of protein kinase C (PKC) ?Ⅱ isoform in renal tissue of diabetic rats and the effects of blocking renin-angiotensin system (RAS) on them. Methods Diabetic rats induced with streptozotocin were randomized to 4 groups. (1) Diabetic control without treatment. (2) Treatment with irbesartan (40 mg?kg -1 ?d -1 ). (3)Treatment with fosinopril (40 mg?kg -1 ?d -1 ) and (4) Treatment with a combination of irbesartan and fosinopril (20 mg?kg -1 ?d -1 each). Six normoglycemic rats served as normal control. After 4 weeks, blood glucose and insulin were measured and expression and translocation of PKC?Ⅱ in renal cortex and medulla were assessed by immunohistochemistry and Western blot. Results The expressions of total and membrane fraction in renal cortex of diabetic control rats weredecreasedto66.0%and50.0% of the values of normal control rats respectively (both P

Objective To study the inhibitive effect and mechanism of PPAR?1 on the extracellular matrix (ECM) accumulation of mesangial cells induced by Ang Ⅱ .Methods The plasmid of PPAR?1/WT (wild type) was transfected into mesangial cells. After 48 hours of Ang Ⅱ stimulation, the gene expression of TGF-?1, PAI-1, c-fos and c-jun was examined by RT-PCR. Protein levels of p-ERK, I-?B and nucleus/cytosol ratio of NF-?B were estimated by Westen-blot. The concentrations of FN and TGF-?1 were estimated by ELISA. The activity of PPAR?1 was examined by specific PPRE binding activity. Plasmid expressing non-functional dominant negative type of mPPAR?1, pIRES2-EGFP-mPPAR?1/DN (DN), and blank plasmid, pIRES2-EGFP (Blank) were used as controls. Effects of 6 ?mol/L PPAR? agonist pioglitazone (Pio) were also studied. Results The expression of TGF-?1 and PAI-1 mRNA in mesangial cells induced by Ang Ⅱ was inhibited by PPAR?1(P

Objective To investigate the effects of antisense Smad2 oligodeoxynudeotides(ODN) on fibronetin(FN) and collagen Ⅳ(ColⅣ) secretion of rat mesangial cells cultured with high glucose, explore the action of Smad2 in the glomerulosclerosis and to find a new method to retard the progress of glomerular fibrosis. Methods 20-mer antisense, sense and random ODNs were designed and synthesized that were phosphorothioate modified to increase stability. The antisense ODN encompassed the ATG of the rat Smad2 gene. ODN was tranferred transiently into rat mesangial cells through liposome. Rat cells were treated with high glucose. mRNA and protein of Smad2 were detected by RT-PCR and cytochemistry. FN and ColⅣ were examined by ELISA. Results Antisense ODN significantly decreased mRNA and protein expression of Smad2 in rat mesangial cells treated with high glucose(P

0 05).Among them,WT but neither DN, Blank, nor Pio significantly ameliorated the increased concentrations of TGF-a1 and fibro nectin in culture medium induced by HG. Compared with DN and Blank, WT transfect ion significantly attenuated high glucose-caused elevation in c-fos, c-jun and p-ERK expression, reduction in Ⅰ-?B level, and incretio n in nucleus/cytosol ratio of NF-?B, while Pio demonstrated similarly signific ant changes only in p-ERK and NF-?B. No significant difference of GLUT-1 mRN A level was detected between HG groups. Conclusions Overexpressed PPAR?1 can su ppress increased ECM production from glomerular mesangial cells cultured in HG c ondition. Its inhibitory effects on activation of ERK/AP-1 and NF-?B pathways are involved. The further increase of PPAR?activity may have direct anti-fibr otic effect in diabetic kidney.

The aim of this study is to develop the Tripterygium glycosides nanoemulsion gels and investigate its pharmacodynamics. Oleic acid was used as oil phase, polyoxyethylene castor oil as surfaetant, and 1,2-propanediol as cosurfactant to screen the formula of Tripterygium glycoside nanoemulsion using the pseudo-temary phase diagrams. Then the nanoemulsion gels was prepared. The ICR mouse ears were sensitazated by 7% DNCB, and then were excited by 0.3% DNCB to stimulate the model of mouse chronic dermatitis and eczema. The concentrations of IFN-γ, IL-4 and IL-8 in mouse blood were determined by ELISA. The results showed that Tripterygium glycosides nanoemulsion gels could significantly inhibit the swelling of mouse ears(P < 0.01) and ameliorate the edama and erythema of model mouse ears skin. Also it could significantly decrease the expression of IFN-γ and IL-4 in model mouse blood. Tripterygium glycosides nanoemulsion gels had a good therapeutic effect on mouse model of dermatitis and eczema. It was expected to provide a new and long-acting exterernal preparation for the treatment of dermatitis and eczema.
Animals ; Chemistry, Pharmaceutical ; instrumentation ; methods ; Dermatitis ; drug therapy ; immunology ; Drug Carriers ; chemistry ; Drugs, Chinese Herbal ; chemistry ; pharmacokinetics ; Emulsions ; chemistry ; Female ; Glycosides ; chemistry ; pharmacokinetics ; Humans ; Interleukin-4 ; immunology ; Interleukin-8 ; immunology ; Mice ; Mice, Inbred ICR ; Nanoparticles ; chemistry ; Tripterygium ; chemistry

OBJECTIVES: This study aimed to demonstrate the activities and phosphorylation changes induced by acute ethanol treatment and withdrawal conditions in the intracellular signal transduction molecules [such as extracellular signal-regulated kinase (ERK), glycogen synthase kinase 3beta (GSK3beta), and Akt] of the SH-SY5Y neuroblastoma cell line. METHODS: The acute treatment exposed SH-SY5Y cells to 100 mM ethanol, and we took samples 30 minutes, 60 minutes, and 24 hours after initiating this treatment. After 24 hours' continuous ethanol treatment, we initiated ethanol withdrawal, taking samples at 30 minutes and 60 minutes. We assayed the kinase phosphorylations via an immunoblot analysis using phosphorspecific antibodies, quantified by optical densitometry. RESULTS: Ethanol treatment induced a transient increase in phosphorylation of GSK3beta and Akt at 30 minutes but failed to change the phosphorylation level of ERK. Ethanol withdrawal induced a transient ERK phosphorylation increase at 30 minutes, but it had no effect on the phosphorylation of GSK3beta or Akt. CONCLUSION: The results indicate that the ethanol-induced cellular response includes the ERK, GSK3beta, and Akt systems. In particular, the ERK pathway may play a role in the acute withdrawal response. This also suggests that a relatively short exposure to ethanol, such as the 24-hour exposure in this study, can induce functional adaptation within a cell.
Antibodies ; Cell Line ; Densitometry ; Ethanol ; Glycogen Synthase Kinase 3 ; Glycogen Synthase Kinases ; Humans ; MAP Kinase Signaling System ; Neuroblastoma ; Phosphorylation ; Phosphotransferases ; Signal Transduction

Objective To explore the glomerular change of renin-angiotensin system (RAS) expression in ATIaR gene knockout mice and its effects on extracellular matrix (ECM) remodeling under diabetic condition. Methods ATlaR knockout mice were generated previously. Hyperglycemia was induced by peritoneal injection of streptozotocin in ATIaR knockout mice and wild type mice. Normal AT1aR knockout mice and wild type mice were used as control group. Twelve weeks later, kidneys were harvested and frozen quickly in dry ice-acetone. Glomendi were collected by laser capture microdissection and total RNA was extracted, mRNA expression of AT1aR, AT1bR, AT2R, angiotensinogen, ACE, renin, and CYP11B2 was assessed by real-time PCR. ECM accumulation was evaluated by PAS staining. Protein levels of transforming growth factor β1(TGF-β1), type 1 plasminogen activator inhibitor(PAI-1), monocyte chemotactie protein 1(MCP-1) and renin were semi-quantitated by immunostaining. Results Compared to the wild type, mRNA expression of AT1bR, angiotensinogen, renin, CYP11B2 within glomeruli was upregulated significantly in ATlaR knockout mice (P<0.05), but no change of ACE expression was found in these two groups. AT2R protein was poorly detected in AT1aR knockout glomeruli and downregulated in wild type glomemli. ECM accumulation was significanfly increased associated with the parallel increase in TGF-β1, PAI-1, MCP-1 and renin within glomendi (P <0.05). Conclusions AT1aR gene knockout cannot improve ECM deposition in diabetic nephropathy. The compensate change of RAS components may be involved in this scenario: upregulation of AT1bR, downregulation of AT2R. CYP11B2 and renin may function in a novel pathway.

OBJECTIVETo investigate the effects of small interfering RNA (siRNA) targeting YAP on the proliferation and apoptosis of human periodontal ligament stem cells (hPDLSCs).

METHODSSynthesized sequences of siRNA were transfected into hPDLSCs by Lipofectamine™ 2000. The expression of YAP was identified by using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Proliferation activity was detected by using cell counting kit-8 (CCK-8). Changes in the cell cycle and apoptosis rate were detected by using flow cytometry. Results were analyzed by using SPSS 19.0, and P < 0.05 was considered statistically significant.

RESULTSExpression of YAP mRNA and protein were significantly downregulated after 48 h of transfection (P < 0.001). No obvious difference was found in the expression levels of YAP protein between 48 and 72 h, thus indicating that siRNA could inhibit the expression of YAP persistently and effectively. Proliferation activity was inhibited, and apoptosis rate was increased. Cell cycle was changed as the proportion of G₁and S phases increased (P < 0.01) and G₂ phase decreased (P < 0.05).

CONCLUSIONKnocking down YAP gene by siRNA could inhibit proliferation activity, induce apoptosis, and change the cell cycle of hPDLSCs. Thus, YAP could regulate the proliferation and apoptosis of hPDLSCs.

Adaptor Proteins, Signal Transducing ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; Humans ; Periodontal Ligament ; drug effects ; Phosphoproteins ; RNA, Messenger ; RNA, Small Interfering ; pharmacology ; Stem Cells ; drug effects ; Transfection

Objective To study the neuroprotective role of TFP5 in a MPTP-induced mouse model of Parkinson's disease (PD).Methods C57BL/6 mice were used as experimental animals.Briefly, 5 consecutive days of intraperitoneal injection of 25 mg/Kg 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was applied to induce mouse PD model.The mice were randomized into 5 groups including control group,model group, scrambled TFP5 peptide (Scb) group, TFP5 group and roscovitine group.On the 7th day after the first injection of MPTP,behavior tests were performed, and then western blot method was employed to detect the expression of p25 and phosphorylated MEF2D in substantia nigra.Tyrosine hydroxylase (TH) immunohistochemical staining was performed to observe the apoptosis of dopaminergic neurons in substantia nigra pars compacta (SNpc) 28 days after the first injection of MPTP.Results MPTP increased the expression of p25 (0.48±0.10 vs 0.26±0.02, P＜0.05) and phosphorylated MEF2D (0.81±0.10 vs 0.22±0.02, P＜0.05) in substantia nigra, but decreased the number of dopaminergic neurons in SNpc (348.67±24.40 vs 463.29± 19.61, P＜0.05),resulting in motor impairment in the model mice (P＜0.05).Intraperitoneal injection of 30mg/Kg of TFP5 for 3 days effectively reduced the excessive phosphorylation of MEF2D (0.25 ± 0.12 vs 0.81 ± 0.10, P＜ 0.05) in substantia nigra, rescued dopaminergic neuron reduction of SNpc (422.92±8.41 vs 348.67±24.40, P＜0.05), and improved the motor ability of the model mice (P ＜0.05).Roscovitine exerted almost same neuroprotective role as TFP5 ,while Scb had no protective effect.Conclusion TFP5 can rescue MPTP-induced damage of dopaminergic neurons in substantia nigra, and thus improve motor impairment of model mice,which may be mediated by the inhibition of Cdk5/p25 activity.