Effect of breviscapin on Ito and IK1 channel current in isolated ventricular myocytes of rats

AIM: The effects of breviscapin on transient outward potassium current (I_to) and inward rectifier potassium current (I_K1) channel in isolated ventricular myocytes of rats were determined. The mechanism of breviscapin at the ionic channel level was explored. METHODS: Single ventricular myocyte of rats was isolated by enzymatic dissociation. The whole-cell patch-clamp recording technique was used to record the change of I_to and I_K1 channel current influenced by breviscapin. RESULTS: Breviscapin decreased the I_to channel current in a dose-dependent and voltage-dependent manner in ventricular myocytes of rats. (1) The current-voltage curve was significantly decreased. Breviscapin at concentrations of 0.02, 0.05, 0.08, 0.10 g·L^-1 respectively decreased I_to current density (10.07±0.50)%, (27.47±2.36)%, (42.72%±2.50)% and (56.09±5.60)%. I_to was inhibited in a voltage-dependent manner, showing a significant attenuating effect at test potentials from 0 to + 50 mV. (2) The I_to activation curve, the activation curve and the recovery curve were not altered. (3) Breviscapin did not affect I_K1. CONCLUSION: Breviscapin concentration-dependently decreases I_to channel current in ventricular myocytes of rat.     

Effects of atorvastatin on cardiac myocyte hypertrophy of neonatal rat induced by angiotensinⅡ in vitro and TLR4 expression

AIM: To assess effects of atorvastatin (Ator) on cardiac myocytes (CM) hypertrophy of neonatal rat induced by angiotensinⅡ (AngⅡ) in vitro and Toll like receptor 4 (TLR4) expression for theoretical bases of preventing and treating myocardial hypertrophy.METHODS: CM of neonatal Sprague-Dawley (SD) rats were isolated with trypsin digestion method and those growth-arrested cells were stimulated with 10^-7 mol/L AngⅡ in the presence of various concentrations of Ator.The method of coomassie brilliant blue was adopted to evaluate the protein contents of CM.The changes in β-MHC,AT₁ receptor and TLR4 mRNA expression were observed by RT-PCR.RESULTS: ① AngⅡ increased the protein content of CM and β-MHC mRNA expression significantly,upregulated AT₁ receptor and TLR4 gene expression.② In a dose-dependent manner,Ator inhibited the increases in the protein contents of CM and β-MHC mRNA expression induced by AngⅡ.③ In a dose-dependent manner,Ator downregulated the AT₁ receptor and TLR4 mRNA expression of CM hypertrophy of neonatal rat induced by AngⅡ.CONCLUSION: Ator inhibits CM hypertrophy,downregulates the AT₁ receptor and TLR4 gene expression.     

Effects of resveratrol on the L-type calcium current by protein kinase G pathway in isolated guinea pig ventricular myocytes

AIM: To investigate the effects of resveratrol on the L-type calcium current in isolated guinea pig ventricular myocytes. METHODS: The whole cell patch clamp method was used. RESULTS: （1）Resveratrol (1, 50, 100 μmol/L) reduced the I_Ca-L by 18.31%±3.15%, 56.20%±2.50% and 84.51%±4.01% in a concentration-dependent manner (n=5, P<0.05). But it has no change on I-V shape of I_Ca-L. (2) 8Br-cGMP (100 μmol/L), an activator of protein kinase G(PKG), deduced the density of I_Ca-L by 10.50%±1.11%. Applying resveratrol and 8Br-cGMP simultaneously decreased the I_Ca-L significantly by 87.58%±3.49％ (n=6, P<0.05). (3) 5 μmol/L H8, a PKG inhibitor, inhibited the decrease in I_Ca-L caused by resveratrol. CONCLUSION: Resveratrol inhibits I_Ca-L in guinea pig ventricular myocytes, and this inhibitory effect involves the PKG pathway.     

Effects of angiotensionⅡon metalloproteinases and matrix in hypertrophied myocardium from infarcted rats

AIM: To investigate the roles of angiotensionⅡ (AngⅡ) receptors (AT₁, AT₂) antagonists on matrix metalloproteinases (MMPs) and extracellular matrix (ECM) system in septal myocardium from infarcted rats.METHODS: The model of rat myocardium infarction (MI) was established by permanent ligation of the left coronary artery. The treatments of the AT₁ receptor antagonist valsartan (10 mg·kg^-1·d^-1) or AT₂ receptor antagonist PD123319 (30 mg·kg^-1·d^-1) were started 7 days prior to surgery. On day 14 after MI, protein levels of MMP-2, 3, 9, fibronectin (FN), tenascin-C (TN-C) in interventricular septum (IS) were determined. The distributions of FN and TN-C were also determined by immunofluorescence.RESULTS: Pathological changes of IS on day 14 after MI showed typical myocardial hypertrophy. Protein expressions of MMP-2, 3, 9 and TN-C of IS in banding group were higher than those in sham-operation group (P<0.01). The expressions of TIMP-1 and FN were lower than those in sham-operation group (P<0.01). Protein expressions of MMP-2, 3, 9 and TN-C in valsartan group were obviously lower than those in banding and PD123319 groups (P<0.01). TIMP-1 and FN protein expressions in valsartan group were higher than those in banding and PD123319 groups (P<0.01). No difference between banding and PD123319 groups was observed (P>0.05).CONCLUSION: AngⅡis involved in myocardium remodeling in infarcted rats, which is mediated via AT₁ receptor to degrade matrix by MMPs. The heart protection of AT₁ receptor antagonists may relate to inhibition of MMPs.     

Effect of platelet activating factor on the action potential and potassium currents in guinea-pig ventricular myocytes

AIM: To investigate the effects of platelet activating factor (PAF) on the action potential and potassium currents in guinea-pig ventricular myocytes. METHODS: By using whole-cell patch clamp technique, the effects of PAF on APD₉₀, I_K1 and I_K were investigated in enzymatically dispersed single guinea-pig ventricular myocytes. RESULTS: With 5 mmol/L ATP in the pipette electrode, 1 μmol/L PAF increased APD₉₀ from (225.8±23.3) ms to (352.8±29.8) ms (n=5, P<0.05), decreased I_K1 and I_K tail currents from (-6.1±1.3) nA to (-5.6±1.1) nA (n=5, P<0.05) at -120 mV and from (173.5±16.7) pA to (152.1±11.5) pA (P<0.05, n=4) at +30 mV, respectively. In contract, without ATP in the pipette electrode, 1 μmol/L PAF shortened APD₉₀ from (153.0±24.6) ms to (88.2±19.4) ms (n=5, P<0.01). Incubation of myocytes with 1 μmol/L glibenclamide, a blocker of I_KATP restored prolongation of APD induced by PAF. CONCLUSION: In guinea-pig ventricular myocytes, with 5 mmol/L ATP in the pipette, PAF prolonged APD partly due to the inhibition of I_K and I_K1, while with 0 mmol/L ATP in the pipette, PAF induced an activation of I_KATP, hence a decrease in APD was observed. Therefore, PAF might amplify the heterogeneity between ischemia and normal cardiac myocytes during ischemic reperfusion, which might play a vital role in the pathogenesis of the arrhythmias induced by ischemia/reperfusion.     

Electrophysiological characteristics of the membrane current of cardiomyocytes-like cells derived from rat bone marrow mesenchymal stem cells

AIM: To study the electrophysiological characteristics of the membrane currents of cardiomyocytes-like cells derived from rat bone marrow mesenchymal stem cells (MSCs).METHODS: MSCs were induced,cultured and identified according to the reference.At the fourth week after treatment with 5-azacytidine(5-aza),cardiomyocytes-like cells were detected for the membrane current with the whole-cell patch-clamp technique and compared with the undifferentiated MSCs.RESULTS: The undifferentiated MSCs only expressed potassium currents.MSCs were stained positive for troponin T after treatment with 5-aza,and two kinds of inward currents and three kinds of outward currents were expressed.They respectively were the fast inward sodium current (I_Na),the L-type calcium current (I_Ca),the transient outward potassium current (I_to),the ultra-rapid delayed rectifier potassium current (I_kur) and the slow delayed rectifier potassium current (I_ks).Compared with the undifferentiated MSCs,the potassium currents of cardiomyocytes-like cells derived from MSCs were mainly made up of I_kur and I_ks.CONCLUSION: After treatment with 5-azacytidine,MSCs are differentiated into cardiomyocytes-like cells,which express the current of I_Na,I_Ca,I_to,I_kur and I_ks.     

Blunted response of L-type calcium current to simulated ischemia and reperfusion in ventricular myocytes from diabetic rabbits

AIM: To evaluate the effects of simulated acute ischemia and reperfusion on L-type calcium current (I_{Ca, L}) in ventricular myocytes from diabetic and non-diabetic rabbits.METHODS: Using whole-cell patch clamp techniques, I_{Ca, L} was measured in left ventricular myocytes isolated from 6-week alloxan-induced diabetic rabbits and age-matched control ones at baseline, 5 min of simulated ischemia, and 5 min of reperfusion.RESULTS: There were no significant differences on baseline maximum I_{Ca, L} densities between diabetic and control ventricular myocytes. In control cells (n=11), maximal I_{Ca, L} densities of baseline, ischemia and reperfusion were (-8.36±1.63)pA/pF, (-5.90±1.75)pA/pF and (-4.22±1.02)pA/pF, respectively. The I_{Ca, L} of ischemia was less than that of baseline (P<0.01), while the I_{Ca, L} of reperfusion was less than those of baseline (P<0.01) and ischemia (P<0.05). In diabetic cells (n=9), the I_{Ca, L} of baseline, ischemia and reperfusion were (-7.55±1.62)pA/pF, (-6.05±1.58)pA/pF and (-5.12±1.13)pA/pF, respectively. Only I_{Ca, L} of reperfusion was less than that of baseline (P<0.01), while I_{Ca, L} of ischemia was not significantly different from that of baseline (P>0.05) or reperfusion (P>0.05).CONCLUSION: I_{Ca, L} in diabetic ventricular myocytes represents blunted response to acute ischemic injury, being decreased more slowly than that in control cells. Post-ischemic reperfusion is still a potent inhibitor against I_{Ca, L} in both diabetic and non-diabetic cells. This study may be indicative of the mechanism about ischemia-reperfusion injury to diabetic myocardium and the therapy for diabetic patients with ischemic heart disease.     

Effect of hypercholesterolemia on the ionic currents in cardiac ventricular myocytes of rats

AIM: To determine whether chronic hypercholesterolemia affects ionic currents on cardiac ventricular myocytes of rats. METHODS: Whole-cell patch-clamp technique was used to record the ionic currents in single cardiac myocytes isolated from normal cholesterolemia and hypercholesterolemia rats. RESULTS: In the hypercholesterol group (group Ⅱ), serum total-cholesterol level was significantly higher than that of normal group (group Ⅰ) ［(3.10±0.62)mmol·L-1 vs (1.18±0.37)mmol·L-1, P<0.01, n=20］. The serum triglyceride content of group II was remarkably higher than that of group Ⅰ ［(1.51±0.30)mmol·L-1 vs (0.43±0.15)mmol·L-1, P<0.01, n=20］. In ventricular myocytes of rats, 50% repolarization of action potential duration (APD50) prolonged from (70.86±8.12)ms (group Ⅰ) to (116.16±6.90)ms (group Ⅱ) (n=10 in each group, P<0.01); APD90 prolonged from (95.10±7.27)ms (group Ⅰ) to (144.04±7.39)ms (group Ⅱ) (n=10 in each group, P<0.01); at the test potential of -120 mV, Ik1 increased from (-16.98±4.54) pA/pF(group Ⅰ) to (-19.92±4.08) pA/pF (group Ⅱ) (n=12 in each group, P<0.05); at the test potential of 0 mV, ICa-L decreased from (-8.56±1.29) pA/pF (group Ⅰ) to (-5.24±0.90) pA/pF (group Ⅱ) (n=10 in each group, P<0.01); at the test potential of +60 mV, Ito decreased from (13.20±1.97) pA/pF (group Ⅰ) to (10.30±1.97) pA/pF (group Ⅱ) (n=8 in each group, P<0.05). CONCLUSION: Hypercholesterolemia affects the ionic currents on cardiomyocytes of rats greatly, which may be the ionic mechanism of cardiac toxicity induced by hypercholesterolemia.     

Effects of cardiomyopeptidin on sodium current in ventricular myocytes of guinea pigs

AIM: To determine the effect of cardiomyopeptidin on sodium current (I_Na) in ventricular myocytes of guinea pigs and to explore the mechanism of cardiomyopeptidin action at the ionic channel level. METHODS: Single ventricular myocytes of guinea pigs were obtained by enzymatic dissociation method. The whole-cell patch-clamp recording technique was used to record the change of I_Na. RESULTS: Cardiomyopeptidin (1, 5, 10, 50, 100 and 500 mg/L) decreased I_Na in a dose-dependent manner. The inhibition rates were (0±1)%, (6±2)%, (10±2)%, (15±1)%, (22±9)% and (30±6)%, respectively. The time to peak (TTP) was delayed from (2.8±0.7) ms to (3.0±0.8) ms (P<0.05) by cardiomyopeptidin (50 mg/L). In the presence of cardiomyopeptidin (50 mg/L), the current density-voltage curve of I_Na was shifted and without change of its active potential, peak potential, reversal potential, and the shape of the curve. The steady activation curve, the steady inactivation curve and the steady inactivation recovery curve of I_Na were not affected. CONCLUSION: Cardiomyopeptidin inhibits the I_Na in guinea pig ventricular myocytes, which may be one of the mechanisms of its antiarrhythmic effect.     

Alteration of cardiac M3 receptor and its relationship with arrhythmias in drug-induced and ischemia-induced arrhythmic models

AIM: To investigate the alteration of cardiac M₃ receptor and its relationship with arrhythmias in various arrhythmic models. METHODS: Forty Wistar rats were randomly divided into 4 groups: control, aconitine, BaCl₂ and ischemia. In the later three groups, arrhythmias were induced by treatment with aconitine, BaCl₂ and coronary artery occlusion, respectively. The arrhythmias were recorded for 1 h. Western blotting was then used to detect M₃ receptor contents. RESULTS: Arrhythmias were all induced in each group. In aconitine-induced arrhythmias, duration of arrhythmias and arrhythmia score were significantly increased than those in other two model groups. Western blotting showed that the expression of M₃ receptor upregulated 2.3, 1.4 and 1.3 folds respectively, more abundant in various arrhythmic groups than that in the normal control. Moreover, M3 receptor expression in aconitine group increased significantly than that in BaCl₂ and ischemia group. The arrhythmias and M₃ receptor protein expressions in myocytes were positively correlated. CONCLUSION: Arrhythmias upregulate the expression of cardiac M₃ receptor. The upregulating levels of M₃ receptor proteins diverge strikingly in different arrhythmic models. It is probably that the diversity of increase in M₃ receptor is positive related to severity of ventricular arrhythmias.     

Effect of Kang Xianling decoction on TGF-β1-Smad pathway in a unilateral ureteral obstruction rat model

AIM: To study the effect of Kang Xianling decoction,comprised of dahuang,danshen,taoren,niuxi and danggui,on TGF-β₁-Smad pathway in unilateral ureteral obstruction rat model.METHODS: Eighteen male SD rats were divided into 3 groups,sham group,model group and model group treated with Kang Xianling decoction randomly.Renal interstitial fibrosis model was established in rats by unilateral ureteral obstruction (UUO).After treatment for additional 14 d,parameters of hydroxyproline in obstructed kidney from 3 groups were analyzed.Rats were sacrificed and the pathological statuses of their kidneys were checked by HE staining and electron microscopy.Transforming growth factor-β₁ (TGF-β₁) mRNA in kidney tissue was determined by RT-PCR.TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),phosphorylated Smad2 and Smad2 protein were determined by Western blotting.RESULTS: Parameters of hydroxyproline in animals of model group were significantly increased than those in sham operation group (P<0.05).The mRNA expression of TGF-β₁ and the protein expression of TβRⅠ,TβRⅡ,phosphorylated Smad2 and Smad2 in kidney tissue of animals in model group were significantly up-regulated.After intervention with Kang Xianling decoction,the above-mentioned up-regulated parameters,except TGF-β₁,were all significantly inhibited.Compared to model group,the pathological changes in renal tissues in treatment group were remarkable improved.CONCLUSION: Kang Xianling decoction inhibits the TGF-β₁-Smad pathway and the protein expression of TβRⅠ,TβRⅡ,phosphorylated Smad2 and Smad2,so as to decrease the level of collagen in obstructed kidney and to alleviate the renal interstitial fibrosis in UUO rats.     

Prorenin activates ERK1/2 and up-regulates TGF-β expression in human renal mesangial cells

AIM: To investigate whether human prorenin can active the (pro)renin receptor leading to the phosphorylation of extracellular regulated-kinase 1 and 2 (ERK1/2) and whether the putative (P)RR blocker "handle-region" peptide (HRP) can inhibit this pathway in cultured human renal mesangial cells (HRMCs). METHODS: HRMCs were cultured in vitro and were pretreated with AT₁ blocker olmsartan and AT₂ blocker PD123319 for 30 min, then they were stimulated by prorenin, PD98059 (inhibitor of ERK1/2) and HRP, respectively. Phosphorylated ERK1/2 was evaluated in Western blotting method. The concentration of transfer growth factor-β (TGF-β) was measured using ELISA method. The mRNA of TGF-β was evaluated by RT-PCR. RESULTS: We found that prorenin induces the activation of (P)RR in cultured HRMCs, in turn, increased the phosphorylated ERK1/2. The protein level of TGF-β was up-regulated by the stimulation of prorenin. ERK1/2 inhibitor PD98059 significantly decreased the phosphorylated ERK1/2 and then down-regulated the TGF-β. HRP could inhibit neither the phosphorylation of ERK1/2 nor the increase of TGF-β.CONCLUSION: Prorenin induces the phosphorylation of ERK1/2 in cultured HRMCs owing to the combination to (P)RR. The phosphorylation of ERK1/2 leads to TGF-β increasing dramatically. It probably plays a key role in the development of kidney disease. HRP affects neither the signaling of ERK1/2 nor the TGF-β.     

Effects of arsenic trioxide on activities of MMPs and expression of TIMP-1 and TGF-β1 in skin fibroblasts and polymorphonuclear neutrophils

AIM: To observe the effects of arsenic trioxide (As₂O₃) on activities of matrix metalloproteinases (MMPs), expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor beta1 (TGF-β₁) in human fibroblast (hFb), and to discuss weather As₂O₃ promotes the healing of chronic skin ulcer through regulating collagen metabolism. METHODS: Zymography was used for testing activity of MMP-9 deriving from rat polymorphonuclear neutrophils (PMNs) and activities of MMP-1, MMP-2 secreted by hFb. Immunocytochemical method was used to determine the expressions of TIMP-1 and TGF-β₁. RESULTS: At the concentration of 50 mg/L, As₂O₃ elevated the activity of MMP-9 (P<0.01). At the concentration of 0.8 mg/L, As₂O₃ increased the activities of MMP-1 and MMP-2 (P<0.01, respectively). After hFb was cultured with As₂O₃ for 6 h, 12 h and 18 h, the expressions of TIMP-1 and TGF-β₁ decreased continuously (P<0.01). CONCLUSION: As₂O₃ elevates the activities of MMP-1, MMP -2 and MMP-9, also inhibits the expressions of TIMP-1 and TGF-β₁, suggesting that arsenic preparation may exert positive effect on healing chronic skin ulcer through regulating collagen metabolism.     

Expression of ROCK I and TGF-β1 in pulmonary arterioles of rat with chronic thromboembolism pulmonary hypertension

AIM:To investigate the dynamic expression of Rho kinase (ROCK I) and transforming growth factor β₁ (TGF-β₁) in pulmonary arterioles of rat with chronic thromboembolic pulmonary hypertension. METHODS: Sixty-four male Wister rats were randomly divided into eight groups: beginning control group, embolism for 3 d, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks groups and end control group. The pulmonary thromboembolism (PTE) model was established by injecting thrombin into jugular vein two times in two weeks and each rat underwent peritoneal injection with tranexamic acid one time a day during experiment to prevent thrombolysis. The mean pulmonary artery pressure (mPAP), right ventricular hypertrophy index (RVHI), relative medial thickness of small pulmonary arteries (PAMT) and vessel wall area/total area (WA/TA) were measured. The levels of ROCK I mRNA and TGF-β₁ protein in rat pulmonary artery were determined by in situ hybridization, immunohistochemistry and image analysis, respectively. RESULTS: mPAP, PAMT and WA/TA were higher respectively in embolism from 4 weeks group to 12 weeks group than those in beginning control group (mPAP: all P<0.01, PAMT and WA/TA: 4 weeks group P<0.05, 8 weeks group and 12 weeks group P<0.01). RVHI was elevated in 8 weeks group P<0.05, in 12 weeks group P<0.01. ROCK I mRNA and TGF-β₁ protein in pulmonary arterioles got the enhanced positive signals of in situ hybridization or immunohistochemistry staining with prolonging the time of rats with pulmonary thromboembolism. ROCKⅠ mRNA: embolism from 3 d group to 2 weeks group P<0.05, 4 weeks group to 12 weeks group P<0.01, TGF-β₁ protein: 1 week group and 2 weeks group P<0.05, 4 weeks group to 12 weeks group P<0.01. Linear correlation analysis showed that ROCK I mRNA and TGF-β₁ protein were positively correlated with mPAP, RVHI and vessel remodeling index (all P<0.01), ROCK I mRNA were positively correlated with TGF-β₁ protein (P<0.01). CONCLUSION:ROCK I and TGF-β₁ play a role in the pathogenesis of chronic thromboembolic pulmonary hypertension and pulmonary vessel remodeling. TGF-β₁ produces biological effect by active ROCK signal pathway.     

Recombinant human defensin α1 induces proliferation of rat glomerular mesangial cells in vitro

AIM: To study the effects and mechanism of recombinant human defensin α₁ on cell proliferation in cultured rat glomerular mesangial cells.METHODS: The influences of defensin α₁ at various concentrations on rat 1097 mesangial cell line cultured in vitro were evaluated with MTT assay.The different concentrations of U0126,signal-regulated protein kinase (MEK) inhibitor,were added into the culture mediums of mesangial cells to do blocking test.Incubated with a final concentration of 3 mg/L defensin α₁,the phosphorylation of extracellular signal regulated kinase (ERK)1/2 and type IV collagen of mesangial cells in different times were evaluated by Western blotting.RESULTS: Defensin α₁ at 3-20 mg/L enhanced proliferation of rat glomerular mesangial cells.The incubation times for the maximum effect on proliferation was 12 h (P<0.01),whereas defensin α₁ concentration >20 mg/L decreased cell proliferation.The cell proliferation induced by defensin α₁ was inhibited by U0126.Stimulation of the cells with defensin α₁ at concentration of 3 mg/L for 5 minutes induced a maximum effect on a ratio of phosphorylation of ERK1/2 to total ERK.After 12 h incubation with defensin α₁,an increase in type IV collagen was observed by Western blotting and continued to increase at 24 h and 48 h (P<0.01).CONCLUSION: Defensin α₁ enhances rat glomerular mesangial cell proliferation and induces type IV collagen production by MAPK signaling pathway.     

Effects of thrombospondin 1 on rat cardiac fibroblasts induced by transforming growth factor β1

AIM:To investigate the effects of thrombospondin 1 on transforming growth factor β₁ induced rat cardiac fibroblasts (CFs). METHODS: CFs of neonatal Sprague -Dawley (SD) rats were isolated with the method of digestion and differential anchoring velocity. The proliferation and collagen synthesis of rat CFs were observed with MTT and hydroxyproline. The expression of TSP-1mRNA was analyzed by RT-PCR.RESULTS: The dose and time-dependent effects of TGF-β₁ were observed. Expression of TSP-1 was increased significantly (P<0.01). Stimulation of CFs with TGF-β₁ (20 μg/L, 24 h) significantly increased CFs proliferation and collagen synthesis (P<0.01). TSP-1 antisense oligonucleotide effectively inhibited TGF-β₁ induced CFs proliferation and collagen synthesis (P<0.01).CONCLUSION:The proliferation and collagen synthesis of CFs induced by TGF-β₁ are inhibited by TSP-1 antisense oligonucleotide, which may exert helpful effect on anti-fibrosis.     

Effects of taurine on the change of apoptosis induced by IL-1β, TNF-α and IFN-γ in rat pancreatic islet cells

AIM: To investigate the changes of apoptosis in isolated pancreatic islet cells, insulin secretion, expression of Bcl-xL and Bax induced by combination of IL-1β, TNF-α and IFN-γ, and effects of taurine on them.METHODS: Isolated pancreatic islet cells from Wistar rat were incubated in monolayer in vitro. NO^-₂/ NO^-₃ production, NOS activity, insulin secretion, the protein expression of Bcl-xL and Bax, percentage of islet cell apoptosis and DNA fragmentation in pancreatic islet cells incubated with combination of IL-1β, TNF-α and IFN-γ were measured, and the effects of taurine on the changes of them were further investigated. RESULTS: Combination of IL-1β, TNF-α and IFN-γ induced a significant increase in percentage of pancreatic islet cell apoptosis, NO^-₂/ NO^-₃ production and NOS activity, DNA ladder appearance, a decrease in insulin content, up-regulation in the protein expression of Bax and down-regulation in the protein expression of Bcl-xL (P<0.01), which were blocked by addition of taurine (P<0.01). These effects occurred in a dose dependent manner.CONCLUSION: Taurine attenuates β cell apoptosis induced by IL-1β, TNF-α and IFN-γ. The mechanism of which may be the inhibition of NOS activity and the decrease of NO production as well as the downregulation of Bax/Bcl-xL proportion.     

Changes of dendritic cells subsets and their HLA-DR expression in peripheral blood of breast cancer patients

AIM: To explore the changes of the subsets and HLA-DR expression of dendritic cells and their concerning cytokine levels in peripheral blood of patients with breast cancer. METHODS: The subsets of the precussors of dendritic cells (pDC) in the peripheral blood of 57 cases of patients with breast cancer before operation and a week or six months after operation and 20 cases of healthy controls were analyzed by four-color FCM. The levels of IL-12p40, IL-10, IFN-γ and IL-4 in the plasmas were tested by ELISA. RESULTS: Among 57 cases of patients with breast cancer, 2 cases in Ⅲ phase and 4 cases in Ⅳphase expressed deficiency of pDC, the ratios of pDC₁/pDC₂ in the other cases inⅠ, Ⅱ, Ⅲ, Ⅳ phase were respectively 1.62±0.59, 1.41±0.63, 0.91±0.32, 0.81±0.29 before operation, which were markedly lower than those in controls (1.94±0.44). The ratios of pDC₁/pDC₂ in the cases inⅠ, Ⅱ, Ⅲ phase were 1.71±0.47, 1.52±0.54, 1.04±0.36 a week after operation, which were the same as those in pre-operation, but markedly lower than those in controls. The ratios of pDC₁/pDC₂ in the cases inⅠ, Ⅱ, Ⅲ phase were 1.92±0.72, 1.63±0.65, 1.28±0.34 six months after operation, which were markedly higher than those in pre-operation, meanwhile, to compare with controls, those were still lower for patients in Ⅱ, Ⅲ phase except in Ⅰphase. No difference between patients and controls in the expression of HLA-DR of pDCs and the levels of IL-12p40, IL-10, IFN-γ, IL-4 in plasmas and the ratios of IL-12p40/ IL-10, IFN-γ/ IL-4 was observed. CONCLUSION: The ratios of pDC₁/pDC₂ in peripheral blood of patients with breast cancer inⅠ-Ⅳ phase are decreased. Parts of patients in Ⅲ, Ⅳ phase are deficiency of pDCs. HLA-DR expression of DCs and the ability of DCs which secret the concerning cytokines do not change as pDC subsets change. pDC subsets improve markedly inⅡ, Ⅲ phase patients and recover to the normal level inⅠphase patients after operation.     

Effect of salvianolic acid B on expression of TGF-β1 receptors and Smad2 in activated mesangial cells

AIM: To study the mechanisms of salvianolic acid B (Sal B)antagonizing mesangial cell activation and kidney fibrosis through investigating the effect of Sal B on expression of transforming growth factor-β₁ (TGF-β₁) receptors and Smad2 in TGF-β₁-stimulated renal mesangial cell activation. METHODS: Mesangial cells was isolated and purified from rat kidney. TGF-β₁ was used to establish rat primary mesangial cell activation model and Smad2,Smad7 protein expression was detected. Sal B (10^-6 mol/L and 10^-5 mol/L) was employed to treat the cells; α-smooth muscle actin(α-SMA) expression was analyzed by immunofluorescence staining and Western blotting. Mesangial cells were treated with Sal B alone or additional with TGF-β₁,and TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),Smad2 phosphorylation and Smad2 protein expression was determined by Western blotting. RESULTS: Cell ular model was established by incubating with 5 μg/L TGF-β₁ for 24 h,and in early stage Smad2 was significantly phosphorylated. Sal B (10^-6 mol/L and 10^-5 mol/L) could inhibit α-SMA expression,which was the biomarker of activated mesangial cells. In addition,in Sal B group,the protein expression of TβRⅠand TβRⅡ was significantly down-regulated while Smad2 phosphorylation in mesangial cells was inhibited. CONCLUSION: Sal B inhibits the TGF-β₁-Smad pathway,the protein expression of TβRⅠ,TβRⅡ and Smad2 phosphorylation in mesangial cells,which is probably one of the mechanisms of Sal B alleviating kidney fibrosis.