Protective role of 14-3-3γ in burn and LPS-induced rat myocardial injury

AIM: To study the protective role of 14-3-3γ in burn or LPS-induced myocardial injury. METHODS: The rat model of burn or LPS-induced injury was established. The heart functions and 14-3-3γ protein expression were detected 3 h, 6 h, 12 h and 24 h after treatment. Primary neonatal rat cardiomyocytes were used in vitro. pFLAG-14-3-3γ plasmid was constructed and transfected into the cardiomyocytes 24 h before LPS-induced injury. The injury in the cardiomyocytes was evaluated by measuring the cell viability and the level of lactate dehydrogenase(LDH). Apoptosis of cardiomyocytes was detected by flow cytometry. Opening of mitochondrial permeability transition pore (mPTP) was also determined by Ca²⁺-induced swelling of isolated myocardial mitochondria. RESULTS: The expression of 14-3-3γ was elevated following the burn or LPS-induced myocardial injury in vivo. In vitro, transfection with pFLAG-14-3-3γ plasmid in to the cardiomyocytes significantly protected against LPS-induced injury. Compared with the cardiomyocytes without transfection with pFLAG-14-3-3γ plasmid, higher cell viability rate and lower LDH release, cell apoptosis and mPTP opening were observed in the cardiomyocytes transfected with pFLAG-14-3-3γ plasmid. CONCLUSION: The 14-3-3γ protein protects the heart against burn or LPS-induced injury by inhibiting the mPTP opening.     

Role of retinoic acid X receptor in regulation of autophagy in rat alveolar type Ⅱ epithelial cells induced by hypoxia/reoxygenation

AIM To investigate the regulatory effect of retinoic acid X receptor （RXR） on autophagy induced by hypoxia/reoxygenation （H/R） in rat alveolar type Ⅱ epithelial cells （AEC Ⅱ） and its molecular mechanism. METHODS AEC Ⅱ were cultured in normoxia. The cells growing to logarithmic growth phase were randomly divided into 5 groups： （1） control （Con） group： cells were cultured for 30 h under normal operation; （2） H/R group： cells were cultured in hypoxia condition for 6 h and then in reoxygenation condition for 24 h; （3） DMSO group： cells were pretreated 1.5 h with medium containing less than 0.1% DMSO before modeling, and the rest were treated the same as the H/R group; （4） 9-cis-retinoic acid （9-RA） group： cells were pretreated for 1 h with 9-RA （100 nmol/L） before hypoxia; （5） HX531 group： cells were treated with 9-RA （100 nmol/L） for 0.5 h, then treatment with HX531 （2.5 μmol/L） for 1 h. CCK-8 assay was used to detect the cell viability. Immunofluorescence staining was used to observe the expression of RXRα. Transmission electron microscope was used to observe the changes of intracellular ultrastructure, and the mRNA expression of adenosine AMP-activated protein kinase （AMPK）, beclin 1, LC3, mammalian target of rapamycin （mTOR） and P62 was detected by RT-PCR. Western blot was used to detected the protein levels of p-AMPK, beclin 1, LC3-Ⅱ, p-mTOR and P62. RESULTS Compared with Con group, the cell viability in H/R, DMSO, 9-RA and HX531 groups were significantly decreased. The mRNA expression of AMPK, beclin 1 and LC3 was significantly increased, and the protein levels of p-AMPK, beclin 1 and LC3-Ⅱ were also increased. The mRNA expression of mTOR and P62 was decreased, and the protein levels of p-mTOR and P62 were also decreased （P<0.05）. The cell injury in 9-RA group was alleviated and autophagy level was significantly lower than that in H/R, DMSO and HX531 groups （P<0.05）, and no significant difference among H/R, DMSO and HX531 groups was observed （P>0.05）. CONCLUSION H/R induces autophagy of AEC Ⅱ. Activating RXR reduce the damage of AEC Ⅱ cells induced by H/R, and its mechanism may be related to the inhibition of autophagy.     

PPARγ agonist pioglitazone attenuates cortical neuron lesion and gliosis in rat brain of post-traumatic injury

AIM: To investigate the neuroprotective effect of pioglitazone (Pio), a potent agonist of peroxisome proliferator-activated receptor gamma (PPARγ), on the traumatic brain injury (TBI) in rats. METHODS: SD rats were randomly divided into 4 groups: sham group, vehicle+TBI group, Pio+TBI group and Pio+T0070907+TBI group. TBI was induced by the method of controlled cortical impact (CCI) injury. Neutral red staining technique was used to determine the cortical lesion volume. NeuN, GFAP and OX-42 were measured by immunohistochemical technique to evaluate the morphology of neurons, activation and infiltration of astrocytes and microglia at the edge of cortical lesion. RESULTS: CCI injury in rat elicited activation and proliferation of the astrocytes and microglia. The glial scar wall formation at the edge of cortical lesion, which was accompanied by the loss of neurons, was observed. Pio significantly reduced the cortical lesion volume, the activation and infiltration of the astrocytes and microglia, and the loss of pyramidal neurons at the edge of cortical lesion. T0070907, an antagonist of PPARγ, reversed the effects of Pio. CONCLUSION: Pioglitazone exerts a neuroprotective efficacy, attenuates the loss of neurons and cortical lesion volume following CCI injury by inhibiting the activation and infiltration of astrocytes and microglia, especially glial scar formation.     

Protective effect and functional mechanism of curcumin on TNF-α induced neuronal damage in rat hippocampus

AIM: To observe the protective effect of curcumin on TNF-α induced neuronal damage in rat hippocampus and to explore the functional mechanism of curcumin. METHODS: The excitatory postsynaptic potential (EPSP) was recorded in CA1 pyramidal layer of rat hippocampal slices with in vitro brain slices recording techniques. High frequency stimulation was given on Schaffer branches to induce long-term potentiation (LTP). After treated with drugs, the initial slope of EPSP in each group was measured and calculated. RESULTS: Compared to control group, TNF-α and N-methyl-D-aspartate(NMDA) obviously inhibited the LTP in hippocampal slices of rat brain (P<0.05). Curcumin partly recovered the LTP, which was inhibited by TNF-α or NMDA, to near the control level (P>0.05). No effect of TNF-α, NMDA or curcumin on basal synaptic transmission in hippocampal slices was observed. CONCLUSION: Curcumin has protective effect on hippocampal neurons of rats. Curcumin can partly prevent the over-activation of NMDA receptor on neuronic membrane induced by TNF-α and maintain the long-term potentiation in neurons.     

Effects of curcumin on actinomycin D/TNF-α-induced injury in PC12 cells and rat hippocampal neurons

AIM: To study the possible mechanism of curcumin on actinomycin D (ActD)/tumor necrosis factor α (TNF-α)-induced injury in PC12 cells and rat hippocampal neurons. METHODS: PC12 cells were divided into control group, TNF-α group, ActD group, curcumin group, ActD/TNF-α group and curcumin+ActD/TNF-α group. The cells were cultured for 24 h. Inverted fluorescence microscopy was used to observe the morphological changes of the cells in each group. Annexin V/PI double staining was applied to analyze the apoptosis of PC12 cells. The level of intracellular Ca²⁺ was detected by Fluo-3 AM staining. Rat hippocampal slices were prepared and divided into the same groups as the PC12 cells. Extracellular microelectrode recording technique was used to observe and calculate the changes of long-term potentiation (LTP) in different groups. RESULTS: Apoptosis of PC12 cells was induced by ActD/TNF-α. Curcumin protected the PC12 cells from ActD/TNF-α-induced apoptosis (P<0.05). ActD/TNF-α increased the intracellular Ca²⁺ concentration. Curcumin significantly reduced ActD/TNF-α-induced apoptosis by decreasing the intracellular Ca²⁺ concentration (P<0.05), inversed the effect of ActD/TNF-α on LTP in hippocampal slices, and improved the synaptic plasticity (P<0.05). CONCLUSION: Curcumin protects ActD/TNF-α-induced neuronal damage by depressing the intracellular Ca²⁺ concentration and maintaining the homostasis of intracellular calcium.     

Effects of ginkgolide B on ［Ca2+］i and mitochondrial function of cultured rat retinal neurons in vitro

AIM: To observe the effect of ginkgolide B (GB) on the intracellular calcium ion concentration (［Ca2+］i) and mitochondrial function of cultured rat retinal neurons in vitro.METHODS: in vitro primary culture of rat retinal neurons was used in the experiment. The apoptosis model of glutamate-induced retinal neurons was established and co-cultured with ginkgolide B. The ［Ca2+］i and mitochondrial membrane potential of the retinal neurons were detected by laser scanning confocal microscope.RESULTS: Glutamate decreased the survival rate of retinal neurons, increased the apoptosis and the ［Ca2+］i, lowered the mitochondrial membrane potential. The ［Ca2+］i was clearly diminished and the mitochondrial membrane potential was significantly increased with the GB intervention, and the apoptosis decreased significantly.CONCLUSION: GB protects retinal neurons from glutamate induced neurotoxicity. The effect of GB on retinal neurons might be due to its ability to decrease the ［Ca2+］i and increase mitochondrial membrane potential.     

Change of endogenous hydrogen sulfide/cystathionine-γ-lyase system in acute lung injury induced by LPS in rats

AIM: To observe the changes of endogenous hydrogen sulfide/cystathionine-γ-lyase (H2S/CSE) system while acute lung injury induced by LPS in rats. METHODS: Eighty rats were randomly divided into six groups (n=8): Ⅰ, control group;Ⅱ, LPS 1 h group; Ⅲ, LPS 3 h group; Ⅳ, LPS 6 h group; Ⅴ, LPS 9 h group; Ⅵ, LPS 12 h group. The ALI model of rats was prepared with LPS. The rats were respectively killed at 1, 3, 6, 9 or 12 h after administration of LPS. The morphological changes of lung tissues were observed by light and electron microscope. The lung coefficient and the wet-to-dry weight ratio were measured. The contents of IL-1β and IL-10 in serum, the H2S level in plasma and the CSE activity in lung tissue were respectively detected. RESULTS: ⑴ In LPS 1 h group, the morphology, the lung coefficient, the wet-to-dry weight ratio, the H2S level and the CSE activity showed no changes compared with the control group. The contents of IL-1β and IL-10 were increased compared with the control group (IL-1β, P<0.05;IL-10, P<0.01). ⑵ In LPS 3 h, 6 h, 9 h and 12 h groups, compared with the control group, the lung tissues were significantly damaged, the lung coefficient and the wet-to-dry weight ratio were significantly increased respectively (LPS 3 h, P<0.05; LPS 6 h, 9 h, 12 h, P<0.01). The contents of IL-1β and IL-10 in serum were markedly increased (P<0.01). The H2S level in plasma and the CSE activity in lung tissue were significantly decreased (P<0.01).CONCLUSION: The changes of inflammatory cytokines may be the pathological foundation of the ALI induced by LPS and the endogenous hydrogen sulfide/cystathionine-γ-lyase system is possibly involved in the formation of the ALI.     

Proapoptotic mechanism and changes of endogenous TGF-β1 in NB4 cells induced by exogenous TGF-β1

AIM: To study the effects of transforming growth factor-β1 (TGF-β1) on cell apoptosis, cell cycle, production of endogenous TGF-β1, expressions of P27Kip1, cyclin E and bcl-2 mRNA levels in NB4 cells. METHODS: Apoptotic morphological changes were observed by Wright-Giemsa staining. Cell cycle and apoptosis were detected with flow cytometry. Semiquantitative RT-PCR was used to examine the mRNA levels of endogenous TGF-β1, P27Kip1, cyclin E and bcl-2. RESULTS: TGF-β1 significantly restrained the growth and promoted the apoptosis of NB4 cells. The blockage of NB4 cells treated by TGF-β1 at concentration of 5 μg/L was in G1 phase. Endogenous TGF-β1 mRNA expression in NB4 cells was up-regulated when the concentration of exogenous TGF-β1 was <5 μg/L. Meanwhile, the expression of endogenous TGF-β1 mRNA was down-regulated when the concentration of exogenous TGF-β1 was 10 μg/L. After treated with TGF-β1 at concentration of 5 μg/L, P27Kip1 mRNA expression in NB4 cells was up-regulated, cyclin E and bcl-2 were reduced. CONCLUSION: TGF-β1 is able to induce apoptosis and cell cycle distribution abnormally in NB4 cells by (1) Up-regulation of endogenous TGF-β1, so that NB4 cells was induced into apoptosis through consequently high expression of P27Kip1. (2) TGF-β1 may lead to cell cycle arrest by inhibiting the expression of cyclin E directly, or by inhibiting the activity of cyclin E through the increased expression of P27Kip1. (3) Down-regulation of bcl-2 induces apoptosis of NB4 cells.     

Release of tumor necrosis factor-α in podocytes and its mechanism by mesangial cell cultured medium with IgA1 from IgA nephropathy

AIM: To explore the effect and mechanism on tumor necrosis factor-α production in podocytes by medium from growth arrested mesangial cells incubated with aIgA1 isolated from IgAN patients and normal control. METHODS: Jacalin affinity chromatography and Sephacryl S-200 molecular sieve chromatography were used to isolate IgA1. Monomeric IgA1 (mIgA1) was transformed to aggregated IgA1 (aIgA1) by heating. IgA-mesangial cell medium was prepared by collecting medium in which growth arrested mesangial cell were incubated with different aIgA1, then the medium with RPMI-1640 containing 0.5% FBS was exposed to podocytes. Real time PCR was used to detect the mRNA expression of Ang and ACE, and TNF-α was measured by ELISA assay. RESULTS: TNF-α level of podocytes by medium from aIgA1 from IgAN cultured with mesangial cells was higher than that in control (6.47±0.45) ng/L vs (1.33±0.65) ng/L, P<0.05. Ang and ACE mRNA in podocytes exposed to the special medium were higher than those in podocytes exposed to control and medium from mesangial cells incubated with aIgA1 from healthy control (P<0.05). Enalaprilat decreased the TNF-α to (7.52±1.12) ng/L (P<0.05), and valsartan decreased TNF-α in the podocytes to (6.64±0.68) ng/L (P<0.05), while enalaprilat and valsartan restored the level of TNF-α in podocytes to normal level (2.72±0.55) ng/L, P>0.05. CONCLUSION: Our findings implicate that medium from mesangial cells cultured with IgA1 from IgA nephropathy stimulates the release of TNF-α in podocytes by the activation of renin-angiotensin system, which might accelerate the progression of IgAN.     

Effect of S-nitrosylation induced by IL-1β and IFN-γ on DNA binding activity of cAMP response element binding protein in fibroblast-like synoviocytes

AIM: To investigate the effect of S-nitrosylation induced by recombinant interleukin-1β (rIL-1β) and recombinant interferon-γ (rIFN- γ) on DNA binding activity of cAMP response element binding protein (CREB) in fibroblast-like synoviocytes (FLSs). METHODS: (1)FLSs were incubated with rIL-1β and rIFN-γ in the presence or absence of inducible nitric oxide synthase inhibitor aminoguanidine (AG) for 12 h. The supernatant of the cell culture was collected to determine the contents of nitric oxide (NO). The total proteins prepared from each group ［only the total proteins prepared from rIL-1β+rIFN-γ group was reacted with dithiothreitol (DTT) for 15 min in vitro］ were subjected to the biotin switch assay, and the level of S-nitrosylation was determined by Western blotting. (2)FLSs were incubated with rIL-1β, rIL-1β+ rIFN-γ and AG respectively for 12 h. The nuclear extracts from each group were prepared. The nuclear extracts from each group were subjected to electrophoresis mobility shift assay to analyze the DNA binding activity of CREB (only the nuclear extracts from rIL-1β +rIFN-γ group was reacted with DTT for 15 min in vitro before assaying). RESULTS: rIL-1β plus rIFN-γ increased the production of NO and the level of S-nitrosylation, which was inhibited by AG. Administration of DTT in the total proteins reversed the induction of S-nitrosylation by rIL-1β and rIFN-γ. Co-incubation with rIL-1β and rIFN-γ inhibited the CREB activity induced by rIL-1β alone, which was reversed by AG. Administration of DTT in the nuclear extracts reversed the effect of co-incubation of the cytokines. CONCLUSION: Co-incubation with rIL-1β and rIFN-γ may increase the level of S-nitrosylation through inducing the production of endogenous NO, leading to reversible thiols modification of CREB and inhibit the DNA binding activity of CREB in FLSs.     

Adiponectin inhibits decrease in autophagy of rat H9c2 cardiomyocytes induced by β1-adrenergic receptor autoantibodies

AIMTo investigate whether adiponectin inhibits the decrease in autophagy of rat H9c2 cardiomy?ocytes induced by β₁-adrenergic receptor (β₁-AR) autoantibodies (β₁-AA), and to explore its mechanism. METH?ODS: SD rats were actively immunized with β₁-AR extracellular second loop (β₁-AR-ECII) antigen peptide. Affinity chromatography was used to purify β₁-AA in serum of the SD rats. The viability of H9c2 cells was measured by CCK-8 assay. The mRNA levels of LC3B and beclin-1 in the H9c2 cells were detected by real-time PCR. The protein levels of LC3-II, P62, AMP-activated protein kinase (AMPK) and phosphorylated AMPK (p-AMPK) were determined by Western blot. RESULTSPretreatment with adiponectin at 10 μg/L for 1 h reversed the decreased viability of H9c2 cells induced by β₁-AA. Compared with control group, β₁-AA decreased the mRNA expression of LC3B and beclin-1, decreased the protein level of LC3-II, and increased the expression of P62 protein in the H9c2 cells, suggesting that β₁-AA decreased the autophagic flux in cardiomyocytes. Adiponectin obviously reversed β₁-AA-induced decline in autophagic flux, and up-regulated the phosphorylation level of AMPK decreased by β₁-AA. Treatment with AMPK inhibitor Compound C for 30 min, we observed that the mRNA expression of LC3B and beclin-1 and the protein level of LC3-II in the H9c2 cells decreased by β₁-AA were not effectively reversed by adiponectin, but the increase in P62 protein expression was still effectively reversed, indicating that adiponectin increased autophagosome production dependent on the AMPK pathway, but increased autophagosome clearance independent on the AMPK pathway. CONCLUSION Adiponectin inhibits the decreased autophagy of H9c2 cardiomyocytes induced by β₁-AA.     

Role of TGF-β1/Smads and ERK expression in vascular remodeling induced by high-salt diet in Wistar rats

AIM: To investigate the role of transforming growth factor β₁ (TGF-β₁)/Smads and extracellular signal-regulated kinase(ERK) expression in vascular remodeling induced by high-salt diet in Wistar rats. METHODS: Wistar rats were randomly divided into 3 groups: normal control group (n=13), high salt (8%) model group and high salt+telmisartan group (n=13). Tail-cuff arterial pressure was determined every 2 weeks. After 24 weeks, the rats in high salt model group were divided into model animals with hypertension group (MH, n=12) and model animals without hypertension group (MN, n=12). The remodeling of aorta and mesenteric artery was observed by HE and Masson staining. In addition, the techniques of immunohistochemistry and real-time PCR were applied to detect the expression of proliferating cell nuclear antigen (PCNA), TGF-β₁, p-Smad2/3, p-ERK1/2 and Smad7 at both protein and mRNA levels. RESULTS: Compared with normal control group, blood pressure in MH group was much higher, and media thickness (MT) and collagen volume fraction (CVF) of arteries in MH and MN groups were higher.The mRNA expression of TGF-β₁, Smad2 and Smad7 in the aorta was significantly increased, and the protein levels of PCNA, p-ERK1/2, TGF-β₁ and p-Smad2/3 in the aorta and mesenteric artery media were elevated, but Smad7 decreased. After telmisartan treatment, MT and CVF were much lower,and the protein levels of PCNA, TGF-β₁, p-Smad2/3 and p-ERK1/2 were significantly reduced, whereas Smad7 was increased. CONCLUSION: The abnormal expression of TGF-β₁/Smads and ERK may be involved in the mechanism of remodeling of aorta and mesenteric artery induced by high-salt diet. Telmisartan prevents the vascular remodeling via regulating TGF-β₁/Smads and ERK signal pathways mediated by angiotensinⅡ type 1 (AT₁) receptor, at least in part.     

Effects of IOX1 on proliferation,apoptosis and extracellular matrix-related protein expression in LX2 cells induced by TGF-β

AIM To investigate the effects of histone demethylase inhibitor IOX1 (5-carboxy-8-hydroxyquinoline) on the proliferation, apoptosis and extracellular matrix (ECM)-related protein expression in transforming growth factor-β (TGF-β)-induced human hepatic stellate LX2 cells. METHODS The proliferation and apoptosis of the LX2 cells were determined by real-time cell analysis and flow cytometry, respectively. The level of histone H3 lysine 9 dimethylation (H3K9me2) and the protein expression of ECM-related molecules [α-smooth muscle actin (α-SMA), collagen type I (Col I), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1)] in the LX2 cells were detected by Western blot. RESULTS Treatment with IOX1 at 50~300 μmol/L significantly inhibited LX2 cell proliferation, and 300 μmol/L IOX1 significantly promoted the apoptosis of the LX2 cells. In addition, different concentrations of IOX1 increased the levels of H3K9me2 and MMP-1, and down-regulated the expression of α-SMA, Col I and TIMP-1 in TGF-β-induced LX2 cells (P<0.05). CONCLUSION Treatment with IOX1 inhibits the proliferation of LX2 cells induced by TGF-β, promotes the cell apoptosis, and regulates the synthesis and metabolism of ECM by elevating H3K9me2 level, thus attenuating hepatic fibrosis.