Activation of macrophage peroxisome proliferator-activated receptor α inhibits macrophage inflammation-induced cardiac fibroblast activation and migration

AIM: To investigate the effect of macrophage peroxisome proliferator-activated receptor α (PPARα) activation on macrophage inflammation-induced activation and migration of cardiac fibroblasts. METHODS: Mouse bone marrow-derived macrophages were treated with vehicle, PPARα agonist WY14643 (10 μmol/L), angiotensin Ⅱ (Ang II; 1 μmol/L) or Ang II+WY14643 for 24 h, and the supernatants were collected as conditioned medium (CM) to stimulate cardiac fibroblasts for additional 24 h. The mRNA levels of PPARα, interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in the macrophages as well as fibrotic markers collagen type Ⅰ alpha 2 chain (Col1a2), collagen alpha 1 chain (Col3a1) and actin alpha 2 (Acta2) in the cardiac fibroblasts were detected by RT-qPCR. The protein levels of IL-6 and IL-1β in the macrophages as well as collagen I, collagen III and α-smooth muscle actin (α-SMA; encoded by Acta2 gene) in the cardiac fibroblasts were determined by Western blot. Wound-healing assay was applied to eva-luate the migration ability of cardiac fibroblasts. RESULTS: Ang II significantly increased the mRNA levels of pro-inflammatory factors, such as IL-6, IL-1α and TNF-α, but decreased the mRNA level of PPARα in the macrophages. Administration of PPARα agonist WY14643 dramatically decreased Ang II-induced mRNA levels of IL-6, IL-1β and TNF-α in the macrophages, and significantly decreased Ang II-induced protein expression of IL-6 and pro-IL-1β in the macrophages. The CM from Ang II-treated macrophages significantly up-regulated the mRNA levels of Col1a2, Col3a1 and Acta2 in the cardiac fibroblasts, which were inhibited by the CM from WY14643-treated macrophages. The same results were observed in the protein levels of collagen I, collagen III and α-SMA in the cardiac fibroblasts. Moreover, the CM from Ang II-treated macrophages significantly promoted cardiac fibroblast migration, whereas the CM from WY14643-treated macrophages markedly inhibited macrophage inflammation-induced cardiac fibroblast migration. CONCLUSION: WY14643-activated PPARα inhibits activation and migration of cardiac fibroblasts by attenuating Ang II-induced macrophage inflammatory response.     

Hydrogen sulfide inhibits inflammatory responses induced by acute myocardial ischemia in isolated rat hearts

AIM：To investigate whether hydrogen sulfide (H2S) protects the hearts against inflammatory responses induced by acute myocardial ischemia in isolated rat hearts. METHODS：Rat acute myocardial ischemia injury was induced by ligation of the left anterior descending coronary artery for 4 h, and the normal perfusate was replaced with NaHS (5 μmol/L, 10 μmol/L and 20 μmol/L) perfusate accordingly in NaHS groups 2 h after ischemia. The changes of cardiac function in the myocardial ischemic injury rats were observed. The mRNA expression of TNF-α, IL-1β, IL-6, IL-10 and ICAM-1 was detected by real-time PCR. The protein level of nuclear factor-κB (NF-κB) in the myocardial tissues was detected by Western blotting. RESULTS：The cardiac function in ischemia group was lower than that in sham group (P<0.01). Compared with ischemia group, perfusion of NaHS resulted in the improvement of the cardiac function (P<0.05 or P<0.01). Compared with sham group, the mRNA expression of TNF-α, IL-1β, IL-6 and ICAM-1 in the cardiac tissues was significantly increased, and the mRNA expression of IL-10 in the cardiac tissues was significantly decreased in ischemia group (P<0.01). Compared with ischemia group, the perfusion of NaHS significantly decreased the mRNA expression of TNF-α, IL-1β, IL-6 and ICAM-1 (P<0.05 or P<0.01). The perfusion of NaHS at concentrations of 10 μmol/L and 20 μmol/L significantly increased the mRNA expression of IL-10 (P<0.01). The protein level of NF-κB in ischemia group was markedly higher than that in sham group (P<0.01). Compared with ischemia group, the perfusion of NaHS at concentrations of 10 μmol/L and 20 μmol/L significantly decreased the expression of NF-κB (P<0.05 or P<0.01). CONCLUSION：H2S protects the hearts against acute ischemia injury through inhibition of NF-κB activation and subsequent down-regulation of NF-κB-dependent inflammatory gene expression.     

Effects of tetrahydroxystilbene-2-O-β-D-glucoside on apoptosis and expression of bcl-2/bax/caspase-3 in HUVECs treated with homocysteine

AIM：To explore the effects of tetrahydroxystilbene-2-O-β-D-glucoside (TSG) from Polygonum multiflorum on the apoptosis and the mRNA expression of bcl-2, bax and caspase-3 in human umbilical vein endothelial cells (HUVECs) treated with homocysteine (Hcy). METHODS：Cultured HUVECs were treated with Hcy (3 mmol/L) to establish a Hcy-damaged model. HUVECs in TSG treated groups were pre-incubated with TSG at concentrations of 1 μmol/L and 10 μmol/L for 2 h before treated with Hcy. Cell nuclear damage was detected by Hoechst 33342 staining. Cell apoptosis was determined by flow cytometry. The mRNA expression of bcl-2, bax and caspase-3 was measured by real-time fluorescence quantitative RT-PCR. RESULTS： After treatment with Hcy at concentration of 3 mmol/L, the nuclear damage and apoptotic rate of HUVECs were higher than that in normal group. The expression of bcl-2 was lower, and the expression of Bax and caspase-3 was higher than that in normal group. On the other hand, pre-incubation with TSG at concentrations of 1 μmol/L and 10 μmol/L decreased the nuclear damage and cell apoptosis, increased the expression of bcl-2, and decreased the expression of bax and caspase-3 as compared with the cells only treated with Hcy. CONCLUSION：TSG reduces the apoptosis of HUVECs induced by Hcy, and the mechanism might be associated with regulating the expression of bcl-2, bax and caspase-3.     

Minocycline inhibits BV-2 cell activation by regulating P2X7 receptor

AIM：To explore the role of P2X7 receptor in inhibition of lipopolysaccharide (LPS)-stimulated BV-2 cell activation by minocycline. METHODS：BV-2 cells were divided into 5 groups: control group, LPS group, LPS+0.1 μmol/L Mino group, LPS+1 μmol/L Mino group and LPS+10 μmol/L Mino group. The expression of P2X7 receptor was determined by real-time PCR and Western blotting. The levels of TNF-α and IL-1β in the microglia culture supernatants were measured by ELISA. The morphological changes of the cells were also observed. RESULTS：After exposed to LPS, the expression of P2X7 receptor increased in BV-2 cells at mRNA and protein levels. The concentrations of TNF-α and IL-1β in the microglia culture supernatants also increased. Meanwhile, 0.1~10 μmol/L minocycline inhibited those changes in a dose-dependent manner. CONCLUSION：Minocycline inhibits the activation of microglia. The mechanism may be related to the P2X7 receptor.     

Effects of tumor necrosis factor alpha on proliferation and apoptosis of HSCs

AIM:To explore the role of tumor necrosis factor alpha(TNF-α) in the pathogenesis of liver fibrosis.METHODS:The proliferation and apoptosis of hepatic stellate cells (HSCs) in vitro were detected with flow cytometry, electron microscopy and TUNEL.RESULTS:The flow cytometry analysis showed that the cell proliferation index (PI) in the TNF-α(0.5 μg/L, 2.0 μg/L, 8.0 μg/L) groups was evidently lower than that in the control group (P＜0.05). In the cell cycle distribution, the portion of G₀/G₁ phase in the TNF-α groups was significantly higher than that in the control group(P＜0.05), but the portion of S phase in the TNF-α groups was evidently lower than that in the control group(P＜0.05). These indicated that TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. The apoptotic rate in the TNF-α groups was evidently higher than that in the control group(P＜0.05). The gene expression of bcl-2 and bax was also detected with flow cytometry. The expression of bcl-2 in the TNF-α groups was evidently lower than that in the control group(P＜0.05), but the expression of bax in the TNF-α groups was significantly higher than that in the control group(P＜0.05). TUNEL analysis showed the apoptotic rate of HSCs in the TNF-α(2.0 μg/L) group was 18.7%±2.5% compared with 5.3%±1.2% in the control group(P＜0.05).CONCLUSIONS:TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. TNF-α down-regulated bcl-2 gene expression and up-regulated bax gene expression whereupon the apoptosis of HSCs was induced.     

Effect of leonurine on expression of miR-1 in rats with myocardial fibrosis induced by isoproterenol

AIM: To investigate effect of leonurine on the expression of microRNA-1 (miR-1) in rats with myocardial fibrosis induced by isoproterenol (ISO). METHODS: SD rats (n=10) were used as normal control group, and 80 rats were given ISO by intraperitoneal injection daily for 2 weeks to establish the model of myocardial fibrosis. The model rats were randomly divided into 5 groups:model group, low-dose (7.5 mg·kg^-1·d^-1) leonurine group, middle-dose (15 mg·kg^-1·d^-1) leonurine group, high-dose (30 mg·kg^-1·d^-1) leonurine group and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (0.3 mg·kg^-1·d^-1) group. After the treatment for 2 weeks, the ultrastructure of left ventricular myocardial tissues was observed under electron microscope. Masson staining was used to detect collagen fibrosis, and the expression of collagen I and collagen Ⅲ was determined by the method of immunohistochemistry. The contents of endothelin-1 (ET-1) and angiotensin Ⅱ (Ang Ⅱ) were measured by ELISA. The expression of miR-1 and ET-1 mRNA was detected by real-time PCR, and the protein expression of p38 MAPK, β-myosin heavy chain (MHC) and α-MHC was determined by Western blot. RESULTS: Compared with model group, the ultrastructure of left ventricular myocardial tissues in high-dose leonurine group was attenuated, and the expression of miR-1 and the protein expression of α-MHC in left ventricular myocardial tissues of high-dose leonurine group were increased (P<0.05). Collagen volume fraction, collagen I, collagen Ⅲ, the ratio of collagen Ⅰ/collagen Ⅲ, the contents of ET-1 and Ang Ⅱ, the mRNA expression of ET-1, and the protein expression of p38 MAPK and β-MHC in high-dose leonurine group were lower than those in model group (P<0.05). CONCLUSION: Leonurine attenuates myocardial fibrosis in the rats induced by ISO, and it is potentially associated with affecting the expression of miR-1, and inhibiting ET-1/p38 MAPK signaling pathway.     

Hypoxia-inducible factor-1α attenuates myocardial inflammatory injury induced by ischemia and reperfusion in rats

AIM:To investigate the role of hypoxia-inducible factor-1α (HIF-1α) stable expression in myocardial inflammatory injury induced by ischemia and reperfusion (I/R) in rats. METHODS:Male Wistar rats were randomly divided into 4 groups:sham operation (sham) group, I/R group, HIF-1α stabilizer dimethyloxalyl glycine (DMOG)+I/R group and HIF-1α inhibitor YC-1+I/R group. The protein expression of myocardial Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was determined by Western blot. The mRNA levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, TLR4 and NF-κB were detected by real-time PCR. The myeloperoxidase (MPO) activity in the myocardial tissues was measured. HE staining was used to observe the infiltration of inflammatory cells. RESULTS:HIF-1α decreased the infiltration of inflammatory cells, the MPO activity, and the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the myocardial tissues. HIF-1α also reduced the expression of TLR4 and NF-κB at mRNA and protein levels (P<0.05). CONCLUSION:The stable expression of HIF-1α has an anti-inflammatory effect on the myocardial tissues after I/R injury in rats. The mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway.     

Effects of interferon-γ, tumor necrosis factor-α and interleukin-1β on apoptosis of human airway smooth muscle cellsin vitro

AIM:To clarify if interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)can induce apoptosis of human airway smooth muscle cells (ASMCs) in vitro.METHODS:Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4-6 cell was used in the experiment. IFN-γ,TNF-α and IL-1β, were used separately or together in the treatment of human ASMCs. The effects of IFN-γ,TNF-α and IL-1β on the growth of the cells was detected by MTT method at the hour 0,24,48 and 72. Light microscopy and electron microscopy were used to examine the morphological change. DNA fragmentation was analyzed by agarose gel electrophoresis. SP immunohistological staing method was performed to detect the change of expressions of p 53, bcl- 2 and bax gene. The apoptosis cell percentage were detected by in situ end labeling technique (TUNEL)of fragmental DNA. RESULTS:(1)IFN-γ or IFN-γ together with TNF-α and IL-1β decreased the number of viable cells in a time dependent manner. (2) Light and electron microscopic examination showed cell shrinkage, membrane blebbing, nuclear contraction, chromatin condensation and nuclear fragmentation in human ASMCs. (3) Agarose gel electrophoresis showed a characteristic"ladder"of DNA bands representing integer multiples of the internucleosomal fragments (about 180-200 bp) in cytokine cotreated human ASMCs. (4)The expression of p 53 and bax gene in cytokine cotreated group was significantly higher than in control group, but the expression of bcl-2 gene was lower than in control group. (5)Stimultaneous treatment with IFN-γ(4×10⁵ U/L),TNF-α(4×10⁵ U/L)and /or IL-1β (10×10⁴ U/L) induced apoptosis of human ASMCs. Apoptotic index of human ASMCs in cytokine co-treated group was significantly higher than in control group (P＜0.01).CONCLUSION:Stimultaneous treatment with IFN-γ,TNF-α and /or IL-1β induced apoptosis of human ASMCs. These immune cytokines may play an important role in airway remodeling of asthma and of chronic obstructive pulmonary disease.     

Protective role of carnosine on cardiac dysfunction in type 1 diabetes mellitus rat model

AIM:To explore the effects of carnosine (CAR) on cardiac dysfunction in type 1 diabetic mellitus rats and the underlying mechanism. METHODS:The SD rats were randomly divided into 4 groups:control (C) group, control+carnosine (C+CAR) group, diabetes mellitus (DM) group and diabetes mellitus+carnosine (DM+CAR) group (n=10). The rats were sacrificed after 12 weeks. The cardiac function was assessed by ventricular cannulation. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were assessed by ELISA. The mRNA levels of tumor necrosis factor α(TNF-α), interleukin-1β(IL-1β) and IL-6 were measured by real-time PCR. The distribution of connexin 43 (Cx43) was examined by immunofluorescence. The protein levels of Cx43 and protein kinase C (PKC) were determined by Western blot. RESULTS:Compared with the C group, the left ventricular end diastolic pressure (LVEDP) was increased whereas the left ventricular pressure maximum rise/fall velocity (±dp/dt_max) was decreased in the DM group (P<0.01). The activity of SOD decreased while the MDA increased in the left ventricular tissues (P<0.01). The mRNA levels of TNF-α, IL-1β and IL-6 were increased (P<0.01). The Cx43 distribution was irregular. The protein levels of phosphorylated Cx43 and PKCε were elevated (P<0.01). Compared with the DM group, the cardiac function of LVEDP and ±dp/dt_max in DM+CAR group was ameliorated (P<0.01), with increased SOD activity and decreased MDA content (P<0.05). The mRNA levels of TNF-α, IL-1β and IL-6 were reduced (P<0.01). The Cx43 distribution was improved and the protein levels of phosphorylated Cx43 and PKCε were decreased (P<0.01). CONCLUSION:CAR treatment can improve the cardiac function by its anti-oxidative and anti-inflammation effects and suppression of Cx43 abnormalities through PKCε in DM rats.     

Experimental study on anti-endotoxin activity of a tetrahydropyrimidine derivative,ZL-5015

AIM: To investigate the protective effect of 1, 3-dicyclopentyl-1, 2, 3, 6-tetrahydropyrimidine-4, 5-dicarboxylic acid diethyl ester (ZL-5015) on lethal endotoxin-challenged mice and to explore the underlying mechanism. METHODS: Mouse model of lethal endotoxin challenge and endotoxemia were established by intraperitoneal administration of lipopolysaccharide (LPS) at a dose of 70 mg/kg to the C57BL/6J mice. Mouse peritoneal macrophages stimulated with LPS (10 mg/L) were used as an in vitro inflammatory model. The levels of interleukin-1β (IL-1β), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was used to evaluate the mRNA expression of the cytokines. RESULTS: Prophylactic treatment of the mice with ZL-5015 (100 and 200 mg/kg, ig) slightly increased the survival rate, extended the survival time, decreased the serum levels of IL-1β and TNF-α, and increased the serum level of IL-10 in the early stage of endotoxemia as compared with model group. The results of in vitro study demonstrated that treatment of the endotoxin-stimulated mouse peritoneal macrophages with ZL-5015 (10, 20 and 40 μmol/L) inhibited the expression of IL-1β and TNF-α at both mRNA and protein levels but promoted the expression of IL-10 at both mRNA and protein levels. CONCLUSION: The tetrahydropyrimidine derivative ZL-5015 shows a moderate anti-endotoxin effect by increasing the survival rate and extending the survival time of the mice challenged by endotoxin, which may result from inhibition of the expression of pro-inflammatory cytokines such as IL-1β and TNF-α, and promotion of the expression of anti-inflammatory cytokine IL-10.     

Effects of icariin preconditioning on inflammatory responses in myocar-dial ischemia-reperfusion injury

AIM:To observe the effects of icariin on myocardial ischemia-reperfusion injury. METHODS:The left anterior descending coronary artery was ligated for 30 min and then loosened for 2 h to establish the rat model of myocardial ischemia-reperfusion injury. Forty-eight healthy adult male SD rats weighing 250~300 g were randomly divided into sham group, model group, low-, middle-and high-dose icariin groups, and aspirin group. The morphological changes of the myocardium were observed by HE staining. The protein expression of NF-κB p65 in the myocardial nucleus was determined by the method of immunohistochemistry. The content of tumor necrosis factor α (TNF-α) in the myocardial tissues was detected by Western blotting. The level of interleukin 1β (IL-1β) in the serum was measured by ELISA. The activity of myeloperoxidase (MPO) in the myocardial tissues was assayed by colorimetry. RESULTS:Compared with sham group, TNF-α content, IL-1β concentration, NF-κB expression and MPO activity in all other groups increased. Compared with model group, TNF-α content, IL-1β concentration, NF-κB expression and MPO activity in low-, middle- and high-dose icariin groups and aspirin group all decreased. No significant difference of the above parameters between high-dose icariin group and aspirin group was observed. CONCLUSION: Icariin preconditioning reduces inflammatory responses in the process of myocardial ischemia-reperfusion injury in a dose-dependent manner.     

Long-term insulin treatment improves postischemic cardiac structure and function via increasing serum BNP levels

AIM：To investigate the influence of long-term insulin treatment on postischemic cardiac structural and functional changes, and to further explore the underlying mechanisms. METHODS：Adult male SD rats were randomly divided into 4 groups (8~10 rats per group): sham group, myocardial infarction (MI) + saline (1 mL·kg-1·d-1, hypodermic injection for 4 weeks) group, MI + insulin (2 U·kg-1·d-1, hypodermic injection for 4 weeks) group and MI + insulin (2 U·kg-1·d-1, hypodermic injection for 4 weeks) + wortmannin ［a phosphatidylinositol 3-kinase (PI3K) inhibitor; 15 μg·kg-1·d-1, intraperitoneal injection 15 min before each insulin treatment］ group. The rats in the latter 3 groups were subject to ligation of the left anterior descending coronary artery, while those in sham group underwent the same surgical procedures without tying the sutures. The cardiac structural and functional changes were observed by echocardiogram, heart catheterization and microscopy with HE and Masson trichrome staining. Blood glucose was determined by Roche blood glucose meter, and the serum levels of insulin and brain natriuretic peptide (BNP) were detected by ELISA. The protein expression and phosphorylation of PI3K, Akt, glycogen synthase kinase 3β (GSK3β) and p38 mitogen-activated protein kinase (p38 MAPK) in myocardial tissues were detected by Western blotting. The mRNA expression of BNP, β-myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP) in myocardial tissues was determined by real-time fluorescence quantitative PCR. RESULTS：At the end of the 4th week, MI rats receiving long-term insulin treatment showed decreased ratio of heart length/heart weight, smaller systolic left ventricle cavity, thicker systolic interventricular septum, and increased cardiac ejection fraction, left ventricular development pressure and instantaneous first derivate of left ventricle pressure (P<0.05 vs MI + saline group). Moreover, insulin treatment significantly increased the phosphorylation of PI3K and Akt and the serum level of BNP, and inhibited the phosphorylation of p38 MAPK (P<0.05 vs MI + saline group), but did not change the mRNA expression of BNP in myocardial tissues. The effects of insulin on BNP were not blocked by wortmannin (P>0.05 vs MI + insulin group). CONCLUSION：Insulin improves postischemic cardiac structure and function by increasing serum BNP levels possibly independent of PI3K-Akt signaling pathway.     

Effects of renal denervation on inflammatory factors in a rabbit model of early atherosclerosis

AIM: To evaluate the effects of renal denervation (RDN) on the expression of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-6 (IL-6) in a rabbit model of early atherosclerosis. METHODS: New Zealand male rabbits were divided into control group, RDN+ high-fat diet (HFD) group (RDN group), sham+HFD group (sham group) and HFD group. The rabbits in later 3 groups were fed with 2% cholesterol for 8 weeks to establish an early atherosclerosis model. The blood samples were collected to test the levels of lipids, norepinephrine (NE), TNF-α, IL-1α and IL-6. The protein expression of angiotensin Ⅱ (Ang Ⅱ) was detected by the method of immunohistochemistry. The levels of nuclear factor-κB (NF-κB) and Ang II 1 type receptor (AT1R) were evaluated by Western blot. The mRNA expression of TNF-α, IL-1α and IL-6 was determined by real-time PCR. RESULTS: After 1 d of RDN procedure, the NE level was lower in RDN group than that in sham group (P<0.01). After 8 weeks, the NE level was lower in RDN group than that in sham group and HFD group (P<0.05), and triglyceride (TG) was lower in RDN group than that in HFD group (P<0.05). The protein expression of Ang II was decreased in RDN group compared with sham group and HFD group (P<0.01). The protein expression of NF-κB was lower in RDN group than that in sham group (P<0.05). The plasma levels of TNF-α and IL-1α were reduced in RDN group compared with sham group and HFD group (P<0.05). The mRNA expression of TNF-α, IL-1α and IL-6 was reduced in RDN group compared with sham group (P<0.05). CONCLUSION: RDN inhibits sympathetic activity, decreases the plasma level of TG, and alleviates inflammatory reactions in the rabbits with atherosclerosis.     

Endogenous hydrogen sulfide results in increase of reactive oxygen species induced by angiotensin II in neurons

AIM：To determine the effect of endogenous hydrogen sulfide (H2S) on the production of reactive oxygen species (ROS) in medullary neurons induced by angiotensin II (Ang II). METHODS：Primary cultured rat medullary neurons were used in the study. Identification of medullary neurons and the co-expression of cystathionine β-synthetase (CBS) were detected by double-labeling immunofluorescence. Medullary neurons were treated with Ang II in the presence or absence of sodium butyrate (NaBu, a CBS agonist; 100 μmol/L, 250 μmol/L and 500 μmol/L). ROS production was measured by dihydroethidium staining. The activity of total superoxide dismutase (SOD) was detected by ELISA. The mRNA expression of CBS was determined by real-time PCR. RESULTS：The medullary neurons in the cultured cells were over 90%. Ang II (1 μmol/L) significantly increased ROS level in the medullary neurons. Ang II inhibited the activity of total SOD in the medullary neurons. CBS was expressed in the medullary neurons. Ang II decreased the mRNA expression of CBS. NaBu (250 μmol/L and 500 μmol/L) inhibited ROS production induced by Ang II with a dose-dependent manner, while NaBu alone had no influence on the ROS level in the medullary neurons. CONCLUSION：Ang II increases the level of ROS in medullary neurons partly by inhibiting the activity of total SOD and the mRNA expression of CBS. Endogenous H2S inhibits the ROS level increased by Ang II in the medullary neurons.     

Protective effects and mechanisms of losartan on apoptosis of H9c2 cells induced by β-adrenergic stimulation in vitro

AIM: To investigate the protective effect of losartan (Los) on apoptosis of H9c2 cells induced by isoprenaline (ISO), and to discover its related mechanism. METHODS: H9c2 cells cultured on plastic plates were divided into control, ISO, ISO+Los, ISO+Los+LY294002 and DMSO groups. Cell apoptosis was evaluated by flow cytometery and agarose gel electrophoresis. The mRNA levels of bax, bcl-2 and caspase-9 were detected by RT-PCR and the expressions of phosphorylated and total Akt (p-Akt and t-Akt) were assessed by Western blotting. The cAMP was measured by radioimmunoassay. RESULTS: ISO at concentration of 10 μmol/L induced apoptosis of H9c2 with an increase in bax/bcl-2, caspase-9 and cAMP. Addition of 10 μmol/L losartan inhibited apoptosis obviously with a decrease in bax/bcl-2, caspase-9 and cAMP. A significant increase in p-Akt was observed, and its protein level was elevated. LY294002 at concentration of 1 μmol/L abolished the protective effects of losartan on ISO-induced apoptosis in H9c2 cells. CONCLUSION: ISO might induce H9c2 cell apoptosis through stimulation of β-adrenergic receptor (β-AR). Los inhibits downstream signaling of β-AR, and promotes the activation of Akt. Subsequently it might attenuate the apoptosis induced by β-adrenergic stimulation of ISO.     

Effect of rhynchophylline on TGF-β1/Smad pathway for processing ventricular remodeling in spontaneously hypertensive rats

AIM: To investigate the effect of rhynchophylline (Rhy) on blood pressure, cardiac hypertrophy and myocardial fibrosis in spontaneously hypertensive rats (SHR). METHODS: Spontaneously hypertensive rats were randomly divided into model group, high dose (10 mg·kg^-1·d^-1) and low dose (2.5 mg·kg^-1·d^-1) group of rhynchophylline, captopril group (17.5 mg·kg^-1·d^-1). Wistar-Kyoto rats were used as normal control. Respectively, systolic blood pressure was measured by tail cuff every 2 weeks. After 10 weeks, heart weight index and left ventricular weight index were calculated. The myocardial hydroxyproline and plasma angiotensin Ⅱ were detected. Moreover, basic myocardial histopathological changes and myocardial collagen fibres were observed by HE staining and Masson staining, respectively. The protein expression of TGF-β₁ and Smad3 in the myocardium was measured by the methods of immunohistochemistry and Western blot. RESULTS: Compared with SHR model group, Rhy significantly reduced blood pressure (P<0.05), the levels of HYP in the myocardium (P<0.05) and the levels of AngⅡ in the plasma (P<0.01). The pathological damages of the myocardial tissues and collagen deposition were attenuated. The protein expression of TGF-β₁ and Smad3 was significantly reduced by the treatment with Rhy (P<0.01). CONCLUSION: Rhynchophylline reduces blood pressure and adjusts to improve ventricular remodeling of SHR. The mechanism may be involved in the TGF-β₁/Smad pathway and reducing AngⅡ content.     

Dexmedetomidine attenuates acute alcoholic hepatic injury in mice

AIM: To investigate the effects of dexmedetomidine (DEX) on acute alcoholic hepatic injury in mice and to explore the possible mechanisms. METHODS: Kunming mice (n=50) were randomly divided into 5 groups (n=10): normal saline control (NS) group, acute alcoholic hepatic injury model (E) group, low-dose (10 μg/kg) DEX (E+L) group, medium-dose (50 μg/kg) DEX (E+M) group and high-dose (100 μg/kg) DEX (E+H) group. The animals were sacrificed at 6 h after gavage of alcohol or normal saline. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) were measured. The livers were removed for evaluation of histological characteristics and determining the content of tumor necrosis factor-α (TNF-α) amd interleukin-1β (IL-1β) in the liver tissues by ELISA. The expression levels of cytochrome P450 2E1 (CYP2E1) and nuclear factor-κB (NF-κB) in the liver tissues were evaluated by Western blot. RESULTS: Compared with NS group, the levels of ALT, AST and TG were obviously increased in E group, which were obviously decreased in E+M and E+H groups. Compared with NS group, the levels of TNF-α, IL-1β and MDA were obviously increase in E group, which were obviously decreased in E+M and E+H groups. Compared with NS group, the activity of SOD and the content of GSH were obviously decreased in E group, which were obviously increased in E+M and E+H groups. Compared with NS group, the expression of CYP2E1 and NF-κB was obviously increase in E group, which was obviously decreased in E+M and E+H groups. Compared with NS group, ethanol induced marked liver histological injury, which was less pronounced in E+M and E+H groups. CONCLUSION: DEX has a protective effect on mouse liver with acute alcoholic injury by the involvement in the processes of antioxidation and antiinflammation, and its mechanism may be associated with the inhibition of CYP2E1 and NF-κB expression.     

Role of TGF-β1/Smad signaling in angiotensin Ⅱ mediated down-regulation of connexin 43

AIM: To analyze the alterations of angiotensin Ⅱ (Ang Ⅱ), connexin 43 (Cx43), angiotenisin Ⅱ receptor type 1 (AT1) and signaling molecules in the TGF-β1/Smad pathway in different regions of the left ventricular heart tissue for exploring whether Ang Ⅱ regulates Cx43 expression via the TGF-β1/Smad signaling pathway in myocardial infarction (MI) rats. METHODS: MI was induced in 20 male Sprague-Dawley rats by the left anterior descending coronary artery ligation. The rats were then randomized into 2 groups. In the losartan group, 20 mg·kg^-1·d^-1 of losartan were administered for 2 weeks. Heart functions were assessed after surgery and 2 weeks later again following the above treatments. All the rats were sacrificed and relevant molecules, including Ang Ⅱ, AT1, and Cx43 were determined thereafter in diffe-rent areas of the left ventricle. TGF-β1 and its downstream signaling molecules, including Smad 2, Smad 3 and Smad 7, were also detected. RESULTS: In losartan group, both left ventricular internal dimension diastole (LVIDd) and left ventricular internal dimension systole (LVIDs) were smaller, with diminished interventricular septal thickness (IVSd) and left ventricular posterior wall depth (LVPWd) and distinct improvement of left ventricular ejection fraction (LVEF) (P<0.05). Losartan therapy exhibited a reduction of Ang Ⅱ in the infarct zone and the border zone in the cardiac tissues. AT1 was obviously attenuated in the infarct zone with an enhanced expression of Cx43, which was also elevated in the border zone and none infarct zone. TGF-β1, Smad 2 and Smad 3 were decreased in different zones of the left ventricle, while Smad 7, in contrary to the above factors, presented a converse alteration.CONCLUSION: The activation of Ang Ⅱ provokes downregulation of Cx43 through TGF-β1/Smad signaling pathway in MI rats.     

Inhibitory effects of right cervical sympathetic trunk transection on inflammatory response after acute myocardial infarction in rats and its influence on HMGB1/TLR4/NF-κB signaling pathway

AIM: To observe the effects of transection of right cervical sympathetic trunk (TCST) on inflammatory response and expression of high mobility group box 1 (HMGB1) and TLR4/NF-κB signaling pathway in the rats after acute myocardial infarction (AMI). METHODS: AMI model was established by ligation of left anterior descending coronary artery in SD rats, then the model rats were randomly divided into MI group and MI+TCST group. MI+TCST model was performed by transection of right cervical sympathetic trunk after left anterior descending coronary artery ligation. The rats in MI group and MI+TCST group were divided into 1, 3, 7, 14 and 28 d subgroups, and another sham operation group threading without ligation, with 8 rats in above each group. After modeling for 4 weeks, the cardiac function was measured by echocardiography. All rats were killed to harvest the hearts for mesuring cardiac hypertrophy index. The myocardial tissue close to infarction was observed with HE staining. The relative mRNA expression levels of HMGB1, tumor necrosis factor α(TNF-α) and interleukin (IL)-6 at different time points were detected by real-time PCR. The protein expression of HMGB1 and TLR4 at different time points after AMI was determined by Western blot. The effect of transection of right cervical sympathetic trunk on the expressions of HMGB1 and TLR4/NF-κB signaling pathway was also analyzed.RESULTS: Compared with the MI group, left ventricular ejection fraction (LVEF) and left ventricular shorterning fraction (LVFS) were significantly higher (P<0.05), left ventricular end-diastole dimension (LVEDd), left ventricular end-systole dimension (LVESd) and cardiac hypertrophy index were significantly lower (P<0.05), and the mRNA levels of HMGB1, TNF-α and IL-6 decreased significantly in MI+TCST group (P<0.05). Western blot results revealed that the protein expression level of HMGB1 increased in the infarct border zone at 3 d, and reached its peak at 7 d, then gradually decreased, and at 28 d after MI in MI group was still significantly higher than that in sham group (P<0.05). The protein expression of TLR4 was consistent with that of HMGB1. Transection of right cervical sympathetic trunk reduced protein expression of HMGB1 and TLR4/NF-κB signaling pathway-related proteins (P<0.05).CONCLUSION: Transection of right cervical sympathetic trunk improves ventricular remodeling and maintaining cardiac function. The mechanism may be related to inhibiting HMGB1/TLR4/NF-κB signaling pathway to reduce inflammatory response.