Role of Nrf2-ARE signaling pathway in protective effect of hypoxia or pinacidil postconditioning against hypoxia-reoxygenation injury in adult rat cardiomyocytes

AIM：To investigate whether nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is involved in the protective effect of hypoxia or pinacidil postconditioning against hypoxia-reoxygenation injury in adult rat cardiomyocytes. METHODS：Cardiomyocytes were isolated from the left ventricle of male adult Sprague-Dawley rats (250~300 g) by Langendorff perfusion and collagenase II digestion. The cells were randomly divided into six groups (n=8 in each group). The cells in N group were cultured for 22 h without any treatment. The cells in the other five groups were cultured for 20 h and then underwent 45 min of hypoxia followed by 60 min of reoxygenation (M group), three 5-min cycles of reoxygenation/hypoxia plus 60 min of reoxygenation (IPO group), or treatment with pinacidil (25, 50 and 100 μmol/L) for 5 min plus 60 min of reoxygenation (P25, P50 and P100 groups, respectively). The expression of Nrf2, NAD(P)H:quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1) and superoxide dismutase 1 (SOD1) at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. RESULTS：The mRNA and protein levels of Nrf2, HO-1, NQO1 and SOD1 in M, IPO, P25, P50 and P100 groups were significantly decreased compared with N group (P＜0.05), and those in IPO, P25, P50 and P100 groups were obviously higher than those in M group (P＜0.05). The mRNA levels of Nrf2, HO-1, NQO1 and SOD1 in IPO and P50 groups were obviously higher than those in P25 and P100 groups (P＜0.05). The mRNA expression of SOD1 in P25 group was obviously higher than that in P100 group (P＜0.05). The protein levels of Nrf2, NQO1 and SOD1 in IPO and P50 groups were obviously higher than those in P25 and P100 groups (P＜0.05). The protein expression of HO-1 in P50 group was obviously higher than that in IPO, P25 and P100 groups (P＜0.05). The protein levels of HO-1 and NQO1 in P25 group were obviously higher than those in P100 group (P<0.05). CONCLUSION：Hypoxia or pinacidil postconditioning may attenuate hypoxia-reoxygenation injury in adult rat cardiomyocytes via activation of Nrf2-ARE signaling pathway.     

Effect of cigarette smoking condensate on HDAC2 and inflammatory mediators in mouse myoblast C2C12 cells

AIM: To investigate the effect of cigarette smoking condensate on histone deacetylase 2 (HDAC2) and inflammatory mediators in mouse myoblast C2C12 cells. METHODS: C2C12 cells were treated with cigarette smoke extract (CSE). HDAC2 siRNA was transfected into the cells using Lipofectamine^TM 2000. The levels of interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) in the culture supernatants were measured by ELISA, and the expression of HDAC2 at mRNA and protein levels was determined by real-time PCR and Western blotting. RESULTS: The expression of HDAC2 at mRNA and protein levels in CSE group was lower than that in control group (P<0.05). The supernatant levels of IL-8 and TNF-α in CSE group were significantly higher than those in control group (P<0.05). When the cells were transfected with HDAC2 siRNA followed by CSE stimulation, the expression of HDAC2 at mRNA and protein levels was decreased, and the supernatant levels of IL-8 and TNF-α were significantly increased as compared with CSE group and control group (P<0.05).CONCLUSION: Under the oxidative stress condition, C2C12 cells generate high levels of IL-8 and TNF-α by down-regulating the expression of HDAC2.     

Role of PI3K/Nrf2 signaling pathway in endotoxin-induced acute kidney injury in rabbits

AIM: To evaluate the role of phosphatidylinositol 3-kinase/nuclear factor E2-related factor 2 (PI3K/Nrf2) signaling pathway in endotoxin-induced acute kidney injury in rabbits. METHODS: Healthy male New Zealand white rabbits were randomly divided into 5 groups: control group (group C), LPS group (group L), wortmannin+LPS group (group WL), wortmannin group (group W) and dimthyl sulfoxide (DMSO) group (group D). Wortmannin at dose of 0.6 mg/kg was injected via the auricular vein in groups W and WL, DMSO at concentration of 0.08 mL/kg was injected in group D, while normal saline (0.08 mL/kg) was injected in groups C and L. LPS at dose of 5 mg/kg was injected via the auricular vein in groups L and WL 30 min later, and equal volume of normal saline was injected in group C, D and W for control. The rabbits were sacrificed 6 h after LPS or normal saline administration. The kidneys were removed for microscopic examination and the determination of histological scores of kidney (HSK). The concentrations of blood urea nitrogen (BUN) and creatinine (Cr), urinary α1-microglobulin (α1-MG), MDA content, SOD activity, the mRNA expression of Nrf2 and HO-1, and the protein levels of total Akt, p-Akt, total Nrf2, p-Nrf2, nuclear Nrf2 and HO-1 in the renal tissues were also detected. RESULTS: Compared with groups C, D and W, the concentrations of BUN and Cr, urinary α1-MG concentration, MDA content and HSK were significantly increased, while SOD activity was significantly decreased (P<0.05). The mRNA expression of Nrf2 and HO-1, and the protein levels of p-Akt, total Nrf2, p-Nrf2, nuclear Nrf2 and HO-1 in the renal tissues were significantly increased in groups L and WL. No significant change among groups C, D and W was observed. Compared with group L, the concentrations of BUN and Cr, urinary α1-MG concentration, MDA content and HSK were significantly increased, while SOD activity, the mRNA expression of Nrf2 and HO-1, and the protein levels of p-Akt, total Nrf2, p-Nrf2, nuclear Nrf2 and HO-1 in the renal tissues were significantly decreased in group WL. CONCLUSION: Activation of PI3K/Nrf2 signaling pathway may be one of the regulatory mechanisms of the body adapting to the endotoxin-induced acute kidney injury in rabbits.     

Effects of ginsenoside Rg1 of Panax notoginseng on brain tissue injury and Nrf2/HO-1 signal pathway after cerebral ischemia/reperfusion in mice

AIM:To observe the effects of ginsenoside Rg1 of Panax notoginseng on brain tissue injury after mouse cerebral ischemia/reperfusion(I/R), and to explore the mechanisms involving nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signal pathway. METHODS:C57BL/6 mice were randomly divided into sham group, cerebral I/R group, ginsenoside Rg1+cerebral I/R group and edaravone+cerebral I/R group. Ginsenoside Rg1 was successively administered for 3 d. One hour after final administration, bilateral common carotid arteries were ligated to induce brain tissue injury for 20 min, and then reperfusion for 24 h. Edaravone, a drug for anti-oxidative stress injury in the treatment of ischemic cerebro-vascular disease, was used as a positive control. The brain tissues were obtained to determine the neural cellular pathology in hippocampal CA1 region. The mRNA expression of Nrf2 and HO-1 was detected by RT-PCR. The protein levels of Nrf2 in the nucleus and cytoplasm and HO-1 in the whole cells in the brain tissues were measured by Western blotting. RESULTS:After ischemia/reperfusion for 24 h, the pathological injury in the neural cells was obvious, and the cell survival rate decreased. Ginsenoside Rg1 and edaravone attenuated the neural cell injury, and significantly increased the cell survival rate. After ischemia/reperfusion for 24 h, the mRNA expression of Nrf2 and HO-1 significantly increased in the brain tissues. The protein levels of Nrf2 in the nucleus and cytoplasm in the brain tissues were increased, the nuclear translocaition rate and the protein expression of HO-1 also increased. Ginsenoside Rg1 and edaravone both decreased the protein levels of Nrf2 in the cytoplasm of the brain tissues, increased that in the nucleus, and also increased Nrf2 nuclear translocation rate and the protein expression of HO-1. The effect of edaravone was higher than that of ginsenoside Rg1, but they had no significant effect on the mRNA expression of Nrf2 in the brain tissues. CONCLUSION: Ginsenoside Rg1 has the effect of anti-brain tissue injury on cerebral ischemia/reperfusion. The mechanisms may be related to activating the Nrf2/HO-1 signal pathway, promoting Nrf2 synthesis and nuclear translocation, thus promoting the expression of downstream antioxidant protein HO-1.     

Effect of hydrogen molecule on apoptosis-related proteins and Nrf2 signaling pathway in glomerular mesangial cells cultured with high glucose

AIM: To investigate the role of hydrogen molecule on apoptosis-related proteins in glomerular mesangial cells cultured with high glucose and to explore its possible mechanism. METHODS: Mouse glomerular mesangial cells cultured in vitro were divided into 4 groups:normal control group (C group, 5.5 mmol/L glucose), mannitol group (G group, 5.5 mmol/L glucose+19.5 mmol/L mannitol), high glucose group (H group, 25 mmol/L glucose), high glucose+hydrogen-rich water group (HH group, 25 mmol/L glucose+hydrogen-rich water), and cultured for 48 h. The protein levels of Bax, Bcl-2, cleaved caspase-3, nuclear factor E2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO-1) were determined by Western blot, and the mRNA expression of HO-1 and NQO-1 was determined by RT-PCR. The level of intracellular reactive oxygen species (ROS) was detected by dihydroethidium method, and the activity of superoxide dismutase (SOD) was measured by WST-8 assay. RESULTS: Compared with C group, the protein levels of Bax and cleaved caspase-3 were up-regulated, and Bcl-2 was down-regulated in H group (P <0.05). No significantly difference of the protein levels mentioned above between C and HH group was observed. Compared with H group, the protein levels of Bax and cleaved caspase-3 were down-regulated, and Bcl-2 was up-regulated in HH group (P <0.05). The level of intracellular ROS was higher and the activity of SOD was lower in H group than those in C group (P<0.05). However, there was no difference of the SOD activity between C group and HH group. The level of intracellular ROS decreased and the activity of SOD increased in HH group as compared with H group (P<0.05). Compared with C group, clearly reduced protein expression of Nrf2, HO-1 and NQO-1, and decreased mRNA expression of HO-1 and NQO-1 in H group were observed (P<0.05). Compared with H group, the protein levels of Nrf2, HO-1 and NQO-1 as well as the mRNA levels of HO-1 and NQO-1 were obviously increased in HH group (P<0.05).CONCLUSION: Hydrogen molecule inhibits the expression of pro-apoptotic proteins and induces the expression of anti-apoptotic proteins in glomerular mesangial cells cultured with high glucose. The mechanism may be related to activation of Nrf2 signaling pathway.     

Docosahexaenoic acid protects human retinal pigment epithelial cells against oxidative stress-induced apoptosis

AIM: To observe the effect of docosahexaenoic acid(DHA) on H₂O₂-induced apoptosis in human retinal pigment epithelium cells and its molecular mechanism. METHODS: Human retinal pigment epithelium cell line ARPE-19 was cultured in vitro, and 12.5 mmol/L H₂O₂ was used to mimic the oxidative stress condition. The cells were treated with 30~100μmol/L DHA for 4~24 h. The expression of heme oxygenase-1(HO-1) at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The enzymic activity of HO-1 was measured by colorimetry. Production of reactive oxygen species(ROS) was determined by fluorescent probe. Activation of NF-E2-related factor 2(Nrf2) was examined by immunofluorescence method. Apoptosis of ARPE-19 cells was analyzed by flow cytometry. RESULTS: The mRNA and protein expression and the enzymic activity of HO-1 were significantly increased in the ARPE-19 cells after DHA treatment. Meanwhile, nuclear translocation of Nrf2 was also observed. Apoptosis appeared and ROS was produced upon H₂O₂ incubation. In contrast, DHA at 100μmol/L significantly abrogated H₂O₂-induced apoptosis and ROS production. Furthermore, silencing of HO-1 by specific siRNA, or treatment with ZnPP, an inhibitor of HO-1, partly counteracted the protective effect against H₂O₂-induced apoptosis and ROS production. CONCLUSION: DHA protects retinal pigment epithelial cells against oxidative stress via induction of heme oxygenase-1 expression after Nrf2 activation.     

Effects of Nrf2 overexpression on proliferation,activation and collagen type I synthesis in cultured hepatic stellate cells stimulated by ethanol

AIM：To investigate the effects of nuclear factor E2-related factor 2 (Nrf2) overexpression on the proliferation, cell cycle distribution, collagen type I (Col I) synthesis and alpha-smooth muscle actin (α-SMA) expression in cultured hepatic stellate cell line HSC-T6 stimulated by ethanol. METHODS：Cultured HSC-T6 cells were transfected with pEGFP-Nrf2 or pEGFP-N1 (empty vector) plasmid by liposome transient transfection. The cells were divided into control group, ethanol group, ethanol+pEGFP-Nrf2 group and ethanol+pEGFP-N1 group. The mRNA expression of Nrf2, α-SMA and Col I was determined by RT-PCR, and their protein expression was detected by Western blotting. Cell proliferation was assessed by MTT assay, and cell cycle was detected by flow cytometry. RESULTS：The pEGFP-Nrf2 plasmid was successfully transfected into HSC-T6 cells, and the mRNA and protein expression of Nrf2 was higher than other three groups 48 h after transfection (P<0.05). Compared with control group, the cell proliferation and the mRNA and protein expression of α-SMA and Col I in ethanol group and ethanol+pEGFP-N1 group were significantly increased (P<0.05), and the numbers of HSC-T6 cells were decreased in G1 phase and increased in S phase (P<0.05), without significant differences between the two groups (P>0.05). Meanwhile, the cells in ethanol+pEGFP-Nrf2 group showed significantly decreased proliferation level, down-regulated mRNA and protein expression of α-SMA and Col I, higher numbers in G1 phase and lower numbers in S phase compared with ethanol group and ethanol+pEGFP-N1 group (P<0.05). CONCLUSION：Nrf2 overexpression could significantly down-regulate the expression of α-SMA and Col I and cause G1/S phase arrest in HSC-T6 cells cultured with ethanol, thus inhibiting the proliferation and activation of the cells.     

Effect of combination of Penthorum chinense Pursh and Puerariae flos on L-02 liver cell injury induced by alcohol

AIM: To explore the effect of Penthorum chinense Pursh and Puerariae flos-containing serum on L-02 liver cell injury induced by alcohol and its possible mechanism. METHODS: After preparing drug-containing serum, the L-02 cells cultured in vitro were divided into 6 groups:blank control group, model group, 1:1 group, 2:1 group and 1:2 group of combination of Penthorum chinense Pursh and Puerariae flos, and tiopronin group. The viability of the L-02 cells was measured by MTT assay. The activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and superoxide dismutase (SOD), and the content of malondialdehyde (MDA) were detected by enzyme label methods. The expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) at mRNA and protein levels was determined by real-time PCR and Western blot, respectively. RESULTS: Compared with control group, the levels of ALT, AST and MDA were increased significantly, and SOD was decreased in model group (P<0.01). Compared with model group, these indexes in all treatment groups were opposite (P<0.01). Compared with control group, the expression of TNF-α and IL-6 at mRNA and protein levels was significantly increased, the mRNA and protein expression of Nrf2 and HO-1 was decreased (P<0.01). Compared with model group, these indexes in combination groups were opposite (P<0.01). CONCLUSION: The therapeutic effects of Pentehorum chinensa Pursh and Puerariae flos-containing serum may affect the expression levels of TNF-α, IL-6, Nrf2 and HO-1, and reduce the inflammatory reaction and oxidative stress in alcohol-induced L-02 liver cells, which plays a role in attenuating alcoholic liver injury.     

Mollugin inhibits viability and collagen synthesis of rat CFSC-2G cells

AIM: To investigate the effects of mollugin on the viability and collagen synthesis of rat hepatic stellate cell line CFSC-2G. METHODS: The activation of CFSC-2G cells was induced with low concentration (10 μmol/L) of hydrogen peroxide (H₂O₂) for 30 min in the experiment. The viability of the CFSC-2G cells after exposed to mollugin at different concentrations (0, 20, 40, 60 and 120 μmol/L) was detected by MTT assay. The mRNA and protein expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), nuclear factor-κB (NF-κB) p65, Bcl-2, Bcl-xL, Bax, and hepatic stellate cell activation markers α-smooth muscle actin (α-SMA) and collagen type I (Col Ⅰ) were detected by real-time PCR and Western blot. The phosphorylation level of p38 mitogen-activated protein kinase (p38 MAPK) was determined by Western blot. RESULTS: Mollugin significantly inhibited the viability and collagen synthesis of activated CSFC-2G cells induced by H₂O₂. The expression of Nrf2, HO-1 and Bax at mRNA and protein levels, and the phosphorylation level of p38 MAPK were promoted, while the levels of NF-κB p65, Bcl-2, Bcl-xL, α-SMA and ColⅠwere inhibited by mollugin (P<0.05). CONCLUSION: Mollugin may inhibit H₂O₂-induced viability and collagen synthesis of the CSFC-2G cells by activating Nrf2 and HO-1, and blocking the NF-κB p65 and Bcl-2 expression.     

Astragaloside IV attenuates apoptosis of H9c2 cardiomyocytes induced by angiotensin II

AIM: To investigate the protective effect of astragaloside IV (ASIV) on angiotensin II (Ang II)- induced apoptosis of H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were treated with different concentrations of Ang II and ASIV. The effects of Ang II and ASIV on the viability of H9c2 cells was measured by CCK-8 assay. The optimum concentration of Ang II was 1 μmol/L and the concentrations of ASIV were 25, 50 and 100 μmol/L. The H9c2 cells was divided into 6 groups:control group, ASIV group, Ang II group, Ang II+ASIV (25 μmol/L) group, Ang II+ASIV 50 (μmol/L) group and Ang II+ASIV (100 μmol/L) group. The morphological changes of the H9c2 cells were observed under inverted phase-contrast microscope. Apoptosis was detected by TUNEL assay. The generation of reactive oxygen species (ROS) was detected by DCFH-DA staining. The protein expression of Bax, Bcl-2, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was determined by Western blot. H9c2 cardiomyocytes were transfected with negative control shRNA (NC) or Nrf2-shRNA (shRNA), and the cells were divided into 8 groups:NC+control group, NC+AngⅡgroup, NC+ASIV group, NC+AngⅡ+ASIV group, shRNA+control group, shRNA+AngⅡgroup, shRNA+ASIV group and shRNA+AngⅡ+ASIV group. ROS level was detected by ROS detection kit. The protein expression of Nrf2 and HO-1 was determined by Western blot. RESULTS: Ang II decreased the viability of H9c2 cells in a concentration-dependent manner (P<0.05). ASIV reversed the effect of Ang II on the viability of H9c2 cells in a concentration-dependent manner (P<0.05). Compared with control group, the apoptotic rate, the level of ROS and the protein expression of Bax in Ang II group were increased significantly, while the protein expression of Bcl-2, Nrf2 and HO-1 was decreased significantly (P<0.05). Compared with Ang II group, ASIV reversed the increase in apoptotic rate of H9c2 cells induced by Ang II in a concentration-dependent manner, reduced ROS level, down-regulated the protein expression of Bax and up-regulated the protein expression of Bcl-2, Nrf2 and HO-1 (P<0.05). After shRNA transfection, the effects of ASIV decreasing ROS production induced by Ang II and up-regulating the expression of Nrf2 and HO-1 were eliminated. CONCLUSION: ASIV protects H9c2 cardiomyocytes from apoptosis induced by Ang II, which may be related to reducing ROS generation and mediating the Nrf2/HO-1 signaling pathway.     

Role of TRIM8 in mouse cardiomyocyte apoptosis induced by high glucose and high free fatty acid

AIM: To investigate the effects of tripartite motif-containing protein 8 (TRIM8) on the apoptosis of mouse cardiomyocytes (MCMs) induced by high glucose and high free fatty acid (HGHF) and the underlying mechanism. METHODS: The MCMs were divided into normal glucose (NG) group (glucose at 5.5 mmol/L), high glucose (HG) group (glucose at 33 mmol/L), high free fatty acid (HF) group (sodium palmitate at 300 μmol/L) and HGHF group (glucose at 33 mmol/L and sodium palmitate at 300 μmol/L). The expression of TRIM8 in the MCMs was knocked down by siRNA, and the MCMs was further divided into control group, scrambled siRNA (Scra-siRNA)/PBS group, TRIM8-siRNA/PBS group, Scra-siRNA/HGHF group and TRIM8-siRNA/HGHF group. To further confirm the specific mechanism of TRIM8 in the MCM injury induced by HGHF, the MCMs were subgrouped into HGHF/DMSO group, HGHF+TRIM8-siRNA+DMSO (HGHF+Ts/DMSO) group, HGHF/ML385 group and HGHF+Ts/ML385 group. Accordingly, apoptosis was analyzed by flow cytometry, and the levels of reactive oxygen species (ROS) were measured by flow cytometry and DHE staining. The expression of TRIM8, nuclear factor E2-related factor 2 (Nrf2), glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO-1) at mRNA and protein levels was determined by qPCR and Western blot. RESULTS: HGHF increased the expression of TRIM8, and suppressed the expression of Nrf2, GCLC, HO-1 and NQO-1 in the MCMs (P < 0.05). Compared with Scra-siRNA/HGHF group, the intracellular ROS content and apoptotic rate were decreased in TRIM8-siRNA/HGHF group (P < 0.05). Correspondingly, the expression of the antioxidant molecule Nrf2 and its downstream genes GCLC, HO-1 and NQO-1 was increased (P < 0.05). In contrast, the addition of Nrf2 inhibitor ML385 partially reversed the inhibitory effect of TRIM8 expression knock-down on HGHF-induced apoptosis of MCMs. CONCLUSION: TRIM8 exacerbates the HGHF-induced cardiomyocyte apoptosis by modulating Nrf2 antioxidative pathway.     

Protective effect of catalpol on diabetic rat aorta and its antioxidant mechanisms

AIM：To investigate the protective effect of catalpol on Goto-kakizaki (GK) rat aorta and to explore its antioxidant mechanisms. METHODS：Six-month-old GK rats (n=45) were randomly divided into diabetic model group, metformin (100 mg·kg-1·d-1) group, and high-dose (100 mg·kg-1·d-1), medium-dose (50 mg·kg-1·d-1) and low-dose (10 mg·kg-1·d-1) catalpol groups. The healthy male Wistar rats (n=10) were used as control group. The rats in control and model groups were given a same volume of saline. All reagents were administered by oral gavage for 12 weeks. Blood glucose and lipids were detected by an automatic biochemical analyzer. Serum reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) levels were detected by commercial kits. The expression of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the thoracic aorta was determined by Western blotting. The pathological changes of the thoracic aorta were observed by HE staining. The ultrastructural changes of the thoracic aorta were observed under electron microscope. RESULTS：After catalpol treatment, the levels of blood glucose and blood lipids were decreased significantly, and serum levels of ROS and MDA were significantly decreased, but the activity of SOD and T-AOC were significantly enhanced. The protein expression of Nrf2 and HO-1 in the thoracic aorta were significantly increased, the thoracic aortic lesions indicated by HE staining significantly reduced, and the thoracic aortic damage under ultrastructural observation was attenuated slightly. CONCLUSION：Catalpol effectively protects GK rat thoracic aorta, which may be associated with decreasing blood lipids, reducing oxidative stress and activating Nrf2/ARE/HO-1 signaling pathways     

Effects of FHL1 on proliferation and migration of rat pulmonary arterial smooth muscle cells stimulated by cigarette smoke

AIM:To explore the role of four-and-a-half LIM domain 1 (FHL1) in the proliferation and migration of rat distal pulmonary arterial smooth muscle cells (PASMCs) stimulated by cigarette smoke extract (CSE). METHODS:Rat distal PASMCs were isolated and primarily cultured. The cells were divided into 6 groups: blank control group, negative transfection group, FHL1 siRNA transfection group, CSE group, CSE + negative transfection group and CSE + FHL1 siRNA transfection group. The mRNA and protein expression of FHL1 was detected by real-time PCR and Western blotting, respectively. Cell proliferation and migration were determined by CCK-8 and Transwell assays, respectively. RESULTS:CSE promoted the proliferation and migration of PASMCs, and increased the expression of FHL1 protein (P<0.01), but did not change the expression of FHL1 mRNA (P>0.05). FHL1 siRNA transfection attenuated the proliferation and migration of PASMCs induced by CSE (P<0.01). CONCLUSION: FHL1 protein is involved in CSE-induced proliferation and migration of rat PASMCs.     

Effect of CHOP expression knock-down on apoptosis of renal tubular epithelial cells

AIM:To study the effect of C/EBP homologous protein (CHOP) on the apoptosis of renal tubular epithelial HK2 cells. METHODS:The serum mRNA levels of CHOP in the patients with acute kidney injury and healthy controls were detected by qPCR. In vitro, renal tubular epithelial HK2 cells were divided into control group, negative group (transfected with negative control siRNA), si-CHOP group (transfected with CHOP siRNA), and induced by transforming growth factor-β1 (TGF-β1). The viability of the cells was measured by MTT assay, and the apoptotic rate was analyzed by flow cytometry. The protein levels of nuclear antigen Ki-67, proliferating cell nuclear antigen (PCNA), caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with the healthy controls, the serum mRNA levels of CHOP in the patients with acute kidney injury were increased significantly (P<0.05). Transfection with CHOP siRNA significantly decreased the expression of CHOP in the renal tubular epithelial HK2 cells (P<0.05). Knock-down of CHOP expression by siRNA significantly increased the viability of renal tubular epithelial HK2 cells (P<0.05), decreased the apoptotic rate (P<0.05), increased the expression of Ki-67 and PCNA (P<0.05), and down-regulated the protein level of cleaved caspase-3 (P<0.05). CONCLUSION:The serum mRNA levels of CHOP were increased in the patients with acute kidney injury. Knock-down of CHOP expression inhibits the apoptosis of renal tubular epithelial cells by regulating the expression of proliferation-and apoptosis-related proteins.     

Naringenin attenuates myocardial injury by regulating AMPK/Nrf2/HO-1 signaling pathways in diabetic mice

AIM: To investigate the effects of naringenin (NAR) on the myocardium as well as its effects on adenosine monophosphate-activated protein kinase (AMPK)/nuclear factor (NF)-E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling pathways in diabetic mice. METHODS: C57BL/6 mice (n=50) were randomly divided into normal group (N group) and model group. The mouse model of type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin (STZ), then the mice were divided into diabetes group (D group), diabetes+low dose of NAR intervention group (D+L-NAR group), diabetes+middle dose of NAR intervention group (D+M-NAR group) and diabetes+high dose of NAR intervention group (D+H-NAR group). The mice in intervention groups were received NAR at low, middle and high doses respectively by gavage, and the mice in N group and D group were received equal volume of normal saline. After 6 weeks, the mice were sacrificed to observe the effects of NAR at different doses on the body weight and blood glucose. The histopathological changes of the cardiac tissues were observed with HE staining. The myocardial collagen volume fraction (CVF) was calculated by Masson staining. Immumohistochemical staining was used to test the protein levels of interleukin-6 (IL-6) and IL-10, and the TUNEL was used to observe the apoptosis of myocardial tissues. The production of reactive oxygen species (ROS) in the myocardial cells was analyzed by fluorescence probe of DHE, and superoxide dismutase (SOD) activity and malondiodehyde (MDA) content in the myocardial cells were measured by SOD and MDA kits. Western blot was applied to determine the protein levels of p-AMPKα, AMPKα, Nrf2, HO-1, NAD(P)H:quinone oxidoreductase 1 (NQO1) and cleaved caspase-3 in the myocardial tissues. RESULTS: Compared with N group, the blood glucose of the mice in D groups was increased and the body weight was decreased significantly. Compared with D group, the blood glucose of the mice in NAR intervention groups was decreased and the body weight was increased. Compared with N group, the CVF, apoptotic rate and the protein levels of IL-6, cleaved caspase-3 were increased, while the protein levels of IL-10, p-AMPKα, Nrf2, HO-1, NQO1 and SOD activity were decreased, the ROS production rate and MDA content was increased significantly in D group (P < 0.05). Compared with D group, the CVF, apoptotic rate and the protein levels of IL-6, cleaved caspase-3 were relatively decreased, conversely the protein levels of IL-10, p-AMPKα, Nrf2, HO-1, NQO1 were increased in NAR intervention groups(P < 0.05). No significantly difference of the ROS production rate, SOD activity and MDA content between D group and D+L-NAR group was observed. However, the ROS production rate and MDA content was decreased,SOD activity were increased in D+M-NAR group and D+H-NAR group as compared with D group. CONCLUSIONS: NAR attenuates myocardial injury in diabetic mice, and its mechanism may be related to regulation of AMPK/Nrf2/HO-1 signaling pathway, enhancement of the antioxidant reaction, reduction of myocardial fibrosis, apoptosis and inflammation.     

Effect of cigarette smoke extract on adhesion and migration of human airway epithelial cells

AIM: To study the effect of cigarette smoke extract (CSE) on adhesion and migration of human airway epithelial cells and it's mechanism. METHODS: After 24 h culture, the airway epithelial cells were treated with different concentrations of CSE. The rate of cell attachment and the velocity of cell migration were measured. The expression of FIP200 mRNA and protein were analyzed by RT-PCR and Western blotting. RESULTS: CSE inhibited the rate of cell attachment and the velocity of cell migration. Meanwhile CSE increased the expression level of FIP200 mRNA and FIP200 protein. The effects of CSE became more evident with increased concentration of CSE. Expression of FIP200 mRNA and FIP200 protein were positively correlated to the decreased rate of cell attachment and the velocity of cell migration. CONCLUSION: CSE inhibits the rate of cell attachment, the velocity of cell migration and increases the expression of FIP200.