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Objective To compare hematoxylin and eosin(HE) staining,Masson's staining and picric sirius red(PSR) staining in evaluation of cardiac fibrosis.Methods Twenty adult sprague dawley (SD) rats were given subcutaneous injection of isoprenaline for 5mg/(kg · d) for 3 weeks,which induced cardiac fibrosis model successfully.In order to observing the fibrotic areas and collagen deposition,we prepared paraffin sections by using the heart specimen of rat,and carried out HE staining,Masson staining and PSR staining respectivelly for the special staining.Results All staining methods could clearly show the collagen fibers and fibrosis stage.We could clearly distinguish the type Ⅰ and type Ⅲ collagen fibers through PSR staining with immunoflurescent techniques,but not Masson and HE staining.Conclusion The results demonstrated that PSR staining is better than HE and Masson staining to be used for the evaluation of degree and type of proliferation of collagen fibers.It has important signification for treatment and prevention of cardiac fibrosis.
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Objective To compare hematoxylin and eosin(HE) staining,Masson's staining and picric sirius red(PSR) staining in evaluation of cardiac fibrosis.Methods Twenty adult sprague dawley (SD) rats were given subcutaneous injection of isoprenaline for 5mg/(kg · d) for 3 weeks,which induced cardiac fibrosis model successfully.In order to observing the fibrotic areas and collagen deposition,we prepared paraffin sections by using the heart specimen of rat,and carried out HE staining,Masson staining and PSR staining respectivelly for the special staining.Results All staining methods could clearly show the collagen fibers and fibrosis stage.We could clearly distinguish the type Ⅰ and type Ⅲ collagen fibers through PSR staining with immunoflurescent techniques,but not Masson and HE staining.Conclusion The results demonstrated that PSR staining is better than HE and Masson staining to be used for the evaluation of degree and type of proliferation of collagen fibers.It has important signification for treatment and prevention of cardiac fibrosis.
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Objective To observe the effects of large-dose ambroxol hydrochloride on lung ischemia-reperfusion injury (LIRI)and discuss the protection of ambroxol hydrochloride on Toll-like receptor 4 (TLR4)/nuclear transcription factor-kappa B (NF-κB ) after lung ischemia-reperfusion injury in rats.Methods We randomly assigned 60 healthy SD rats into four groups (n=15 for each):control group,ambroxol hydrochloride group (0.75 g/L),ischemia-reperfusion group (I/R),and I/R+ambroxol hydrochloride group.The ambroxol hydrochloride group and I/R+ambroxol hydrochloride group were injected large dose of ambroxol hydrochloride by intravenous injection.The control group and the I/R group received normal saline.The effects of ambroxol hydrochloride on lung ischemia-reperfusion (LIR)-induced pathological changes and inflammatory cytokines release level were examined.DNA ends situ labeling assay (TUNEL)was used to detect the apoptosis of cells.NF-κBp6 5 was detected by immunohistochemistry.In addition,the TLR4 signaling pathway activation in lung tissues was detected by Western blot analysis.Results Compared with those in the control group,some hemorrhage and inflammation changes of lung tissues were observed;the W/D ratio,inflammatory cytokines,apoptosis of cells,NF-κBp6 5 and TLR4 signaling pathway protein expression in I/R group was obviously increased.Compared with I/R group,some mild hemorrhage and inflammation changes of lung tissues were observed;W/D ratio,inflammatory cytokines,apoptosis of cells, NF-κBp6 5 activity, and TLR4 signaling pathway expression were all decreased significantly in I/R+ambroxol hydrochloride group.Conclusion Large dose of ambroxol hydrochloride can protect rats with lung ischemia-reperfusion injury by downregulating TLR4 signaling pathway.
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Objective To explore the relationship of the expression level of monocyte Chemotactic protein-1 and nuclear factor κB(NF κB)with atrial fibrosis of atrial fibrillation patients with rheumatic heart disease.Methods About 200 mg right atrial tissue were taken from 26 patients with rheumatic heart disease undergoing valve replacement surgery and divided into sinus rhythm (SR) group (n=12) and atrial fibrillation (AF) group (n=14).Masson staining and atrial myocardial fibrosis markers were used to determine the level of fibrosis.The mRNA levels of cytokines in atrial tissue were measured by reverse transcription polymerase chain reaction (RT-PCR)The protein level of MCP-1 and phosphorylation of NFκB in atrial tissue were detected by Western blotting.Results Compared with SR group,the AF group showed that collagen volume fraction (AF:0.42 ± 0.03;SR:0.13 ± 0.02),the mRNA levels of myocardial fibrosis markers such as transforming growth factor (TGF)-β1,type Ⅰ collagen and type Ⅲ collagen,and cytokines such as IL-1β and TNF-α,and the protein level of MCP-1 (AF:0.170±0.003;SR:0.040±0.005) and level of phosphorylation of NF-κB (AF:0.35 ± 0.02;SR:0.12 ± 0.03) were significantly increased (all P＜0.05).In addition,the expression levels of cytokines,the protein expression level of MCP1 and the phosphorylation level of NF-κB were positively correlated with collagen volume fraction of the right atrial myocardial tissue (all P＜0.05).Conclusions Activation of NF-κB may induce the expression of MCP-1 in the myocardial tissue of patients with rheumatic heart disease,and sequentially stimulate the secretion of cytokines and extracellular matrix deposition,and finally participate in the occurrence and persistence of atrial fibrillation.
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Objective To investigate the effects and possible mechanisms of platelet-activating factor (PAF) on the invasion of ovarian cancer cells and to provide a potential target for ovarian cancer therapy.Methods ( 1 ) Serous type ovarian cancer cell line OVCA429 with platelet-activating factor receptor (PAFR) positive and mucinous type cell line RMUG-L (PAFR negative) were treated with 100 nmol/L of the PAF,cell invasion ability was determined by transwell cell migration assay.(2) For determination of the optimal PAF concentration,ovarian cancer cell OVCA429 was treated by 0,0.1,1,10,100,and 1000 nmol/L of PAF for 10 minutes or 24 hour,respectively.To observe the different time point of protein changes,OVCA429 were treated by 100 nmol/L of PAF for 0,5 minutes,10 minutes,30 minutes,1 hour or 12 hours,respectively.The total proteins of treated cells were extracted according to standard protocol.The expression of p38 mitogen-activated protein kinase (p38 MAPK),phosphorylated p38 MAPK (p-p38 MAPK),transcription factor response element-binding protein (CREB),phosphorylated CREB (p-CREB) and matrix metalloproteinase-2 (MMP-2) were detected by western blot.(3) To verify the pathway involved in the PAF induction of the cancer cell invasion,we repeated the experiments by adding the inhibitors when treating cells with PAF.The inhibitors used were as follows,PAFR inhibitor-WEB2076 (50 μmol/L),pp38 MAPK inhibitor-SB203580 (10 μmol/L),CREB binding protein (CBP)-CREB interaction inhibitor217505(25 μ mol/L).All treated cells were divided into 6 groups:control group,PAF group,PAF + WEB2076 group,PAF + SB203580 group,PAF + 217505 group and PAF + SB203580 + 217505group.Results ( 1 ) By transwell assay,100 nmol/L of PAF could improve the invasion ability of OVCA429 cell significantly [ PAF:( 118 ± 23 ) cells vs.control:(36 ± 8 ) cells,P ＜ 0.0l ],while the same treatment had no effect on RMUG-L cells [PAF:(45 t 13) cells vs.control:(53 ±9) cells,P＞0.05].(2) Even a very low concentration of PAF (0.1 nmol/L) could increase the expression of p-CREB and MMP-2,while the most effective concentration of PAF was 100 nmol/L.The highest p-CREB protein expression was detected at 10 minutes after administration of 100 nmol/L PAF,as well as the expression of p-p38 MAPK protein.Even 12 hours after treatment the p-p38 MAPK protein could be detected,while there was no significant difference in the expression of CREB ( P ＞ 0.05 ).(3) As compared with PAF group,both in PAF + WEB2076 group and PAF + SB203580 group,the expressions of p-p38 MAPK,p-CREB and MMP-2 protein were decreased significantly; in PAF + 217505 group,although the expression of p-p38 MAPK and p-CREB protein was significantly higher than the control group,the expression of MMP-2 protein was significantly lower; in PAF + SB203580 + 217505 group,the expression of these three proteins were also significantly lower,but there was no significant difference as compared with that in the PAF + WEB2076 or PAF + SB203580 group.Conclusion PAF could induce MMP-2 expression and contributed to PAFR positive ovarian cancer cell invasion via activation of CREB by phosphorylating of p38 MAPK.
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Objective To investigate the effects of acidosis on the current change of transient receptor potential channel M7 (TRPM7) and collagen production in mouse cardiac fibroblast (MCFs), and to explore the pathophysiological function of TRPM7 on the cardiac fibrosis. Methods (1) The model of MCFs was established and isolated. (2) MCFs was subcuhured. (3) Patch clamp technique was used to observe the current characteristics of TRPM7 in low PH solutions. (4) The influence of acidic condition on Ca2+ influx in MCFs was recorded by calcium fluorescent indicators. Results (1) There was a high level expression of TRPM7 in MCFs and the electrophysiological characteristics of TRPM7-like (TRPM7L) was similar to that of TRPM7. (2) Ca2+ influx was increased in acidic condition, and the F340/F380 ratio was increased from 1.0 to 4. 6 at pH 4.0 compared with pH 7.0 (t=2.72, P<0.01). Conclusions (1) TRPM7 is the molecular basis of the native TRPM7L in MCFs and TRPM7L plays an important role in Ca2+ influx. (2) The pathophysiological function of MCFs is influenced by regulation of Ca2+ influx mediated by TRPM7L in the condition of acidosis.
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To investigate the effect of sea anemone toxin anthopleurin-Q (AP-Q) on potassium currents in isolated rats and guinea pig ventricular myocytes.Methods The ventricular cells of guinea pigs and SD rats were obtained by enzymatic dissociation method.Whole cell patch clamp technique was used to record potassium currents (Ito,IK,and IK1).Results AP-Q 3-100 nmol/L increased Ito in a concentration-dependent manner,with an EC50 value of 12.7 nmol/L.At a potential of +50mV,AP-Q 10nmol/L increased Ito from (13.3±3.4) pA pF-1 to (19.46±4.3) pA pF-1.AP-Q 0.1-100 nmol/L increased IK and IK tail in a concentration-dependent manner with EC50 values of 4.7 nmol/L and 5.0 nmol/L,respectively.AP-Q 1 pmol/L-100 nmol/L increased IK1 in dose-dependent manner,with an EC50 of 0.2 nmol/L.Conclusions The effect of AP-Q on Ito,IK and IK1 may partly explain its mechanism in shortening APD and increasing RP.(J Geriatr Cardiol 2008;5:243-247)
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Objective To investigate the changes of TRPM 7-like current in mouse cardiac fibroblast(CFs) after myocardial infarction and the effect of myocardial ischemia on the TRPM 7 expression and current. Methods (1) The model of myocardial infarction was made and CFs were isolated;(2) CFs were cultured and infected by TRPM 7 siRNA;(3) The effects of myocardial ischemia on TRPM 7 current were recorded by whole cell patch clamp technique;(4) The influences of myocardial isehemia on Ca2+ influx in CFs were recorded by Ca2+ fluorescence imaging. (5) The effects of ischemia on total collagen content of CFs were studied. Results (1) Ca2+ inward current of CFs was increased after myocardial infarction [(16.2±1.7) vs. (7.4±0.7) pA/pF, P<0.053];(2) TRPM 7 current was reduced by 90% after siRNA infection;(3) The total collagen content of CFs after ischemia was approximately 2.3-fold higher than basic value. Conclusions Ca2+ influx in CFs plays an important role in the pathophysiological process of myocardial fibrosis.
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0.05),while those of the cells treated with 6?10-5,6?10-6,6?10-7 mol/L of exemestane were significantly different from that of controls(P
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Objective To investigate the pathological contribution of serological markers of collagen metabolism including matrix-metalloproteinases(MMPs)and its tissue inhibitors(TIMPs),the markers for type I collagen degradation(ICTP)and synthesis(PICP)to cardiac remodeling of ischemic cardiomyopathy(ICM)as well as the pharmacological regulation effect.Methods Plasma levels of MMP-9,TIMP-1,ICTP and PICP were determined by ELISA and RIA in 86 consecutive ICM patients and 25 age-matched control subjects.Patients were divided into two subgroups according to ACEI-taking or not.Echocardiography were performed in all cases.Results The plasma concentration of MMP-9、TIMP-1 and MMP-9/TIMP-1 was significantly increased in ICM group(all P