Matrine attenuates bleomycin-induced pulmonary injury partially via modulating mononuclear phagocyte phenotype switching in mice

AIM: To investigate the influence of matrine (MA) on the phenotype switching of mouse monocytes and alveolar macrophages induced by bleomycin (BLM).METHODS: All mice were randomly divided into normal saline (NS) group, BLM group, BLM+NS group and BLM+MA group. The mice were administered with BLM at 2.5 mg/kg via oropharyngeal instillation. The mice in BLM+MA group were treated with MA (15 mg·kg^-1·d^-1) by oral gavage following BLM administration. The mice were sacrificed on days 3, 7, 14, and 21. The lungs were removed for pathological analysis. The circulating monocyte subsets and polarization state of bronchoalveolar lavage fluid (BALF)-derived alveolar macrophages were analyzed by flow cytometry.RESULTS: The results of HE and Masson trichrome staining in BLM and BLM+NS groups exhibited classical pathological stages of lung fibrosis, including acute inflammation phase and later fibrosis phase. Compared with BLM+NS group, MA treatment alleviated the inflammatory response and the degree of fibrosis induced by BLM (P<0.05). There was a rapid change of circulating Ly6C^hi monocytes and its magnitude was positively associated with the pulmonary inflammatory response. An expansion of M2-like alveolar macrophages was positively correlated with the magnitude of lung fibrosis. Moreover, MA treatment partially normalized the phenotype switching of monocytes and alveolar macrophages.CONCLUSION: Matrine treatment attenuates BLM-induced pulmonary injury partially via modulating the phenotype switching of monocytes and alveolar mocrophages.     

Effects of Danshensu on TGF-β1/Smads signaling pathways in rats with pulmonary fibrosis

AIM: To study the preventive and curative roles of Danshensu (DA) in bleomycin (BLM)-induced pulmonary fibrosis in rats. METHODS: Pulmonary fibrosis was induced in SD rats by intratracheal instillation of BLM. The rats were intraperitoneally injected with dexamethasone (1 mg·kg-1·d-1, DXM group), DA (15 mg·kg-1·d-1, DA group), or physiological saline (2 mL·d-1, BLM group). Normal controls (NC group) received physiological saline both intratracheally and intraperitoneally. At the 28th day after modeling, the histological changes of the lungs were evaluated by hematoxylin-eosin (HE) and Masson’s trichrome staining. The protein levels of α-smooth muscle actin (α-SMA) in the lung tissues were detected by the method of immunohistochemistry. The mRNA expression of transforming growth factor beta 1 (TGF-β1), Smad3 and Smad7 was assessed by real-time fluorescence quantitative PCR. RESULTS: Compared with BLM group, the degree of inflammation and fibrosis of the lung in DA group was obviously reduced, and so was the expression of α-SMA in the lung tissues. The mRNA expression of TGF-β1 and Smad3 in the lung tissues of the rats decreased and the mRNA expression of Smad7 increased. CONCLUSION: DA alleviates BLM-induced pulmonary fibrosis in rats in the early stage by inhibiting the expression of TGF-β1/Smad3 and stimulating the expression of Smad7 in the lung tissues.     

Effects of lutein on viability of breast cancer cells

AIM:To investigate the effect of lutein on the viability of breast cancer cells and its possible mechanism. METHODS:The human breast cancer T47D cells were divided into control group and lutein (6.25, 12.5, 25, 50 mg/L) treatment groups. The effect of lutein on the viability of T47D cells was measured by MTT assay. The mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione peroxidase 1 (GPx1) and superoxide dismutase 2(SOD2) was detected by RT-qPCR. Fluorescent probes DCFH-DA was used to determine the production of reactive oxygen species (ROS). The protein expression of Nrf2 and p65 was determined by Western blot. RESULTS:The MTT results showed that lutein inhibited T47D breast cancer cell viability in a dose-and time-dependent manner. The RT-qPCR results showed that the mRNA levels of Nrf2, GPx1 and SOD2 were higher in lutein treatment groups than those in the control group (P<0.05), and with the increased concentrations and extension of intervention time of lutein, the relative mRNA levels were all increased. The ROS levels were significantly decreased in the lutein-treated groups (P<0.05). The results of Western blot demonstrated that the protein expression of Nrf2 was significantly increased (P<0.05), and p65 protein was decreased (P<0.05) in a dose-dependent manner with lutein treatment for 48 h. CONCLUSION:Lutein significantly inhibits the viability of breast cancer cells, and the inhibition roles may be related to up-regulation of the expression of Nrf2, antioxidant enzymes GPx1 and SOD2 mRNA expression and down-regulation of oxidative stress, thus blocking the NF-κB signaling pathway.     

Effects of microRNA-141 regulating Nrf2/ARE signaling pathways by targeting Keap1 on viability of T47D breast cancer cells

AIM:To investigate the effects of microRNA-141 (miRNA-141) regulating Nrf2/ARE signaling pathways by targeting Keap1 on the viability of T47D breast cancer cells. METHODS:The breast cancer T47D cells were transfected with miRNA-141 mimic and the negative control sequence (negative control, NC), as miRNA-141 group and NC group, respectively, and the cell without transfection was used as control group. Real-time PCR was used to detect the expression level of miRNA-141. The cell viability was measured by MTT assay. Fluorescent probe 2',7'-dihydrodichlorofluorescein diacetate ester (DCFH-DA) was used to detect cell reactive oxygen species (ROS) level. The protein expression levels of Keap1, nuclear factor E2-related factor 2 (Nrf2), superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPx1) were determined by Western blot. Dual luciferase assay was used to analyze relationship between miRNA-141 and Keap1. RESULTS:After the cells were transfected with miRNA-141 mimic, the expression of miRNA-141 was obviously higher in miRNA-141 group than that in other groups (P<0.05). The cell viability, ROS level and Keap1 protein expression were significantly decreased, while the Nrf2 protein in the nucleus and antioxidants SOD2 and GPx1 expression were up-regulated in miRNA-141 group. Moreover, the luciferase reporter assay demonstrated that Keap1 was the target gene of miRNA-141. CONCLUSION:miRNA-141 may negatively regulates Keap1 and activates Nrf2/ARE signaling pathways, which inhibits the viability of breast cancer cells via inducing the expression of antioxidant enzymes to reduce the oxidative stress levels of the cells.     

Gefitinib prevents bleomycin-induced lung fibrosis in mice

AIM: To evaluate the effect of epidermal growth factor receptor- tyrosine kinase inhibitor (EGFR-TKI) on lung fibrosis induced by bleomycin in mice. METHODS: Forty BALB/c female mice were randomly divided into 4 groups: the mice in control group were given vehicle orally with administering of saline intratracheally; the mice in Ge200 group were given gefitinib (a tyrosine kinase inhibitor) at dose of 200 mg/kg orally with administering of saline intratracheally; the mice in BLM group were given vehicle orally with administering of bleomycin intratracheally; the mice in BLM+Ge20 group was given gefitinib at dose of 20 mg/kg orally with administering of bleomycin intratracheally. All animals were sacrificed by abdominal aortic bleeding 14 days after treatments. The left lung was stained with hematoxylin, eosin and Masson's trichrome respectively for the pathological examination. Total EGFR and phosphorylated EGFR were detected by immunohistochemistry. The tissues of right lung were sampled and the contents of hydroxyproline (HYP) were measured to quantitate the lung fibrosis. RESULTS: Gefitinib prevented lung fibrosis induced by bleomycin with significantly reducing lung collagen accumulation and the level of HYP in BLM+Ge20 group (P<0.05). The phosphorylation of EGFR in lung mesenchymal cells and epithelial cells was inhibited by treating gefitinib after intratracheal administration of bleomycin (P<0.05). Furthermore, in those mice that did not receive bleomycin treatment (Ge200 group), gefitinib neither induced the lung fibrosis nor increased the expression of p-EGFR. CONCLUSION: These findings suggest that, in the preclinical setting, gefitinib has a protective effect on lung fibrosis induced by bleomycin. Gefitinib at high dose (200 mg/kg) dose not induces lung fibrosis.     

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.     

Inhibition of plasminogen activator inhibitor-1 by small interfering RNA attenuates pulmonary fibrosis in rats

AIM: To examine the effects of silencing of plasminogen activator inhibitor-1 (PAI-1) expression by small interfering RNA (siRNA) on bleomycin (BLM)-induced rat pulmonary fibrosis. METHODS: Total 72 Wistar rats were divided into 4 groups: control, BLM, BLM+non-specific siRNA (BLM+N), and BLM+ PAI-1 siRNA (BLM+P). Pulmonary fibrosis was induced by intratracheal injection of BLM (5 mg/kg), whereas equal volume of normal saline was used in control group. After the administration of BLM or normal saline, the rats were treated with tracheal injection of PAI-1-siRNA (7.5 nmol/0.2 mL per rat) in BLM+P group, non-specific siRNA (7.5 nmol/0.2 mL per rat) in BLM+N group, and 0.2 mL normal saline in BLM group and control group, twice a week, 8 times in 28 d. On day 7, 14, and 28, the rats (n=6 at each time point) were sacrificed. The bronchoalveolar lavage fluid (BALF) from the left lung was harvested to examine the activity of PAI-1. The mRNA expression of collagen type Ⅲ, α-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the middle lobe of the right lung was detected by RT-PCR. RESULTS: PAI-1 activity and the expression of collagen type Ⅲ, α-SMA and TIMP-1 were increased in BLM group on day 7, 14 and 28. Intratracheal injection of PAI-1 siRNA twice a week continuously reduced PAI-1 activity in the BALF (P<0.05),and decreased the expression of collagen type Ⅲ, α-SMA and TIMP-1 in the fibrotic lung tissues on day 7, 14 and 28. Statistical differences in the expression of collagen type Ⅲ, α-SMA and TIMP-1 between BLM+P group and BLM group at the same time point were observed. CONCLUSION: Intratracheal injection of PAI-1 siRNA twice a week continuously inhibits the expression of PAI-1. PAI-1 siRNA ameliorates BLM-induced pulmonary fibrosis by down-regulation of TIMP-1 expression.     

Expression of PPARγ, Nrf2 and γ-GCS-h in inflammatory cells in bronchoalveolar lavage fluid of guinea pigs with bronchial asthma

AIM: To investigate the expression of PPARγ and Nrf2/γ-GCS-h in inflammatory cells in bronchoalveolar lavage fluid(BALF) of guinea pig with bronchial asthma of acute episode, and to explore the roles of PPARγ on Nrf2/γ-GCS-h expression. METHODS: Forty adult male guinea pigs were randomly divided into 4 groups (10 guinea pigs in each group): control group (group A), asthmatic group (group B), dexamethasone treatment group (group C) and rogridone treatment group (group D). The asthmatic model was established by an ovalbumin challenge method. BALF was collected, and the total cell count and the proportion of the inflammatory cells were measured. After centrifugation, the concentrations of ROS and MDA in the clear supernatant were detected. The methods of in situ hybridization and immunohistochemistry were used for detecting the expression of PPARγ and Nrf2/γ-GCS-h at mRNA and protein levels. RESULTS: The proportion of eosinophils (EOS) in BALF in group B was significantly higher than that in groups A, C and D (P<0.01). The concentrations of ROS and MDA in BALF of group B was the highest. The difference of ROS and MDA was statistically significant (all P<0.05) as compared to the control. The results of immunohistochemistry and in situ hybridization indicated that the A value was the lowest in group B as compared to that in groups A, C and D (all P<0.01). In group B, the positive correlations were observed between PPARγ and Nrf2/γ-GCS-h, between γ-GCS-h and Nrf2. A negative correlation between the proportion of EOS in BALF and the expression of PPARγ and Nrf2/γ-GCS-h was also observed (all P<0.05). CONCLUSION: In acute asthmatic models induced by ovalbumin, the expression of PPARγ and Nrf2/γ-GCS-h is decreased, and PPARγ may up-regulate the expression of Nrf2/γ-GCS-h to inhibit the inflammatory and oxidative reactions, indicating a new way for prevention and treatment of bronchial asthma.     

Erianin induces apoptosis of human lung cancer A549 cells via ROS/p38 MAPK pathway

AIM:To investigate the effect of erianin on the viability and apoptosis of human lung cancer A549 cells and its possible mechanism. METHODS:A549 cells and BEAS-2B cells were cultured in vitro and treated with erianin at 10, 20, 40, 80 and 160 nmol/L for 48 h. The cell viability was measured by CCK-8 assay. The apoptosis and reactive oxygen species (ROS) were analyzed by flow cytometry. The activity of superoxide dismutase (SOD) was detected by WST-8 method, and the content of malondialdehyde (MDA) was detected by barbituric acid method. The protein levels of nuclear factor E2-related factor 2 (Nrf2), NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), p38 MAPK, p-p38 MAPK, caspase-3 and cleaved caspase-3 were determined by Western blot.RESULTS:Erianin remarkably reduced the viability of A549 cells in a dose-dependent manner (P<0.05) with IC₅₀ at 52.64 nmol/L. Erianin also induced apoptosis (P<0.05), increased ROS level and MDA content (P<0.05), diminished SOD activity (P<0.05), and down-regulated the protein levels of Nrf2, NQO1 and HO-1 (P<0.05), in a dose-dependent manner. Meanwhile, erianin up-regulated the levels of p-p38 MAPK and cleaved caspase-3 (P<0.05), and these effects were inhibited by N-acetyl-L-cysteine and SB203580 (P<0.05).CONCLUSION:Erianin may induce apoptosis of human lung cancer A549 cells most likely via inhibiting SOD activity and down-regulating the protein levels of Nrf2, NQO1 and HO-1, thus resulting in an increase in ROS and activation of p38 MAPK.     

Etanercept attenuates bleomycin-induced lung fibrosis in mice

AIM: To evaluate the effect of tumor necrosis factor α (TNF-α) antagonist etanercept on bleomycin-induced lung fibrosis in mice.METHODS: Forty-five Kunming female mice were randomly divided into 3 groups: the mice in control group were intraperitoneally injected with vehicle and intratracheally administered with saline aerosol, the mice in bleomycin group were intraperitoneally injected with vehicle and intratracheally administered with bleomycin (3 mg/kg) aerosol, and the mice in bleomycin+etanercept group were intraperitoneally injected with etanercept (4 mg/kg) every 3 d and intratracheally administered with bleomycin aerosol. All animals were sacrificed 28 d after treatments. The left lung was fixed in 10% neutral formalin and then stained with hematoxylin-eosin or Masson’s trichrome for the pathological examination. The tissues of right lung were sampled for measuring the content of hydroxyproline (HYP) by the method of alkaline hydrolysis. The serum concentrations of TNF-α and TGF-β were detected by ELISA. Total tissue protein was extracted for examination of ERK1/2, JNK and p38 by Western blotting.RESULTS: Etanercept prevented the collagen accumulation under the airway epithelium and decreased the scores of lung inflammation and fibrosis induced by bleomycin with significantly reduced the levels of tissue HYP, serum TNF-α and serum TGF-β. The protein phosphorylations of ERK/JNK/p38 in the lung tissues were remarkably decreased compared with BLM group.CONCLUSION: Etanercept decreases the phosphorylations of ERK1/2/JNK/p38 via inhibiting the expression of TNF-α and TGF-β. Etanercept might be useful in the treatment of pulmonary fibrosis.     

Effect of Ginkgo biloba extract on the expression of connective tissue growth factor in the initiating stage of fibrosis in lungs of rats

AIM: To study the effect of Ginkgo biloba extract (GbE) on the expression of connective tissue growth factor (CTGF) in the initiating stage of pulmonary fibrosis of rats after administration of bleomycin (BLM).METHODS: The expression of CTGF in lungs was detected by Western blotting. The content of hydroxyproline was assayed by the method of chloramines T. The content of malondialdehyde (MDA) in plasma was investigated by colorimetry.RESULTS: On day 14 after administration of BLM, the contents of CTGF in lungs and MDA in plasma in BLM+NS group were higher than those in NS group, respectively (P<0.05; P<0.01). On day 30 after BLM, the contents of hydroxyproline in lungs and MDA in plasma in BLM+NS group were higher than those in NS group, respectively (both P<0.01). Treatment with GbE ameliorated the above changes induced by BLM. CONCLUSION: GbE ameliorates the up-regulation of CTGF in the initial stage of fibrosis in lungs of rats after administration of BLM. GbE prevents the hyperoxidative injury in lungs of rats after BLM, which might be one of mechanisms underling the effect of GbE on CTGF.     

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.     

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     

Lipid peroxidation precedes TNF and PDGF release in alveolar macrophage of the experimental pulmonary fibrosis rats

AIM:To evaluate the role of alveolar macrophage(AM)lipid peroxidation in bleomycin(BLM)-induced TNF-and PDGF release from AM in rats.METHODS:Male SD rats were intratracheal y instilled with saline or BLM(5 mg/kg body weight), and AM were isolated and analyzed for malondialdehyde(MDA)and glutathione peroxidase(GSH-Px)following 1, 3, 7, 14 or 28 day(s).Simultaneously, TNF-and platelet-derived growth factor(PDGF)were also determined in AM-conditoned media by ELISA and bioassay respectively.RESULTS:(1)MDA in AM was markedly increased on day1, peaked on day 3 and then decreased to near control level on day 7;on the other hand, GSH-Px activity in AM were decreased to non-detectable levels during day 1-7 after BLM instillat ion, returned to the control level by day 14.(2)TNF-and PDGF were increased significantly from the third day on and then exhibited a consectutive changes.(3)AM lipid peroxidation preceded TNF-and PDGF release.CONCLUSION:Prior oxidative changes leading to AM lipid peroxidation, depletion of antioxidative pool might be essential for the subsequent mediators release in BLM-induced pulmonary fibrosis.     

Effect of arsenic trioxide on bleomycin-induced pulmonary fibrosis in rats

AIM: To observe the effect of arsenic trioxide (ATO) on bleomycin-induced pulmonary fibrosis in rats. METHODS: Pulmonary fibrosis was induced in Sprague-Dawley (SD) rats by intratracheal instillation of bleomycin (BLM). The rats in ATO treatment group, steroid treatment group and model group were intraperitoneally injected with ATO, dexamethasone or normal saline (NS), respectively, while the control rats received NS both intratracheally and intraperitoneally. The effects of ATO were evaluated by analyzing the median survival time, hydroxyproline level in the lung, semi-quantitative grading of alveolitis and pulmonary fibrosis, and quantitative analysis of the collagen in lung tissue (Masson’s trichrome staining). Apoptosis index (AI) of the lung was detected by using the terminal transferase dUTP-digoxygenin nick end-labeling (TUNEL) method. The results of immunohistochemical staining for some cytokines were quantitatively analyzed. RESULTS: ATO (1) prolonged the median survival time of rats with BLM-induced pulmonary fibrosis at some extent; (2) attenuated the alveolitis and pulmonary fibrosis, reduced hydroxyproline level and collagen deposition in the lung tissue; (3) increased the AI of lung tissue at a certain phase; and decreased the levels of transforming growth factor-β1 (TGF-β1) and tissue inhibitor of metalloproteinase-1 (TIMP-1), increased the content of interferon-γ (IFN-γ), but did not influence the concentration of matrix metalloproteinase-9 (MMP-9) significantly. CONCLUSION: ATO might attenuate BLM-induced pulmonary fibrosis in rats via increasing the AI in the lung tissue.     

Effect of shikonin on high glucose-induced apoptosis and oxidative stress in vascular endothelial cells

AIM:To investigate the effect of shikonin on the apoptosis and oxidative stress induced by high concentration of glucose in vascular endothelial cells. METHODS:Rat thoracic aortic endothelial cells were randomly divided into 5 groups:normal control group (with glucose at concentration of 5.5 mmol/L in cell culture medium), high glucose group (with glucose at concentration of 33 mmol/L in cell culture medium), high glucose+low shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 0.1 μmol/L in cell culture medium), high glucose+medium shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 1 μmol/L in cell culture medium), and high glucose+high shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 10 μmol/L in cell culture medium). After treatments, the cell viability was measured by CCK-8 assay and cell apoptotic rate was analyzed by flow cytometry. In addition, the status of oxidative stress was evaluated by determining the levels of malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The activation of Nrf2/HO-1 signaling pathway was determined by Western blot. RESULTS:Compared with high glucose group, shikonin reversed high glucose-induced decrease in cell viability and increase in apoptosis in a concentration-dependent manner. High concentration of glucose induced high levels of MDA and ROS, while decreased the levels of SOD and GSH-Px. However, after treatment with shikonin, the contents of MDA and ROS were decreased, while the activities of SOD and GSH-Px were increased as compared with high glucose group. Furthermore, the high concentration of glucose up-regulated the protein levels of cleaved caspase-3, HO-1 and Nrf2 (nuclear). Compared with high glucose group, the protein levels of cleaved caspase-3, HO-1 and Nrf2 (nuclear) were partly decreased after treatment with shikonin. CONCLUSION:Shikonin alleviates high glucose-induced vascular endothelial cell apoptosis. Its mechanism may be related to activation of Nrf2/HO-1 signaling pathway and down-regulation of oxidative stress in vascular endothelial cells.     

Protective effect of procyanidin single active ingredient B2 on human endothelial progenitor cells under high glucose stimulation

AIM: To study the protective effect of procyanidin single active ingredient B2(PC-B2) on human endothelial progenitor cells(EPCs) stimulated with high glucose. METHODS: The human EPCs were isolated from peripheral blood of healthy people and identified. The EPCs were divided into control group(PBS treatment), hypertonic control group(25 mmol/L mannitol treatment), high glucose(30 mmol/L) group, and different concentrations(2, 10 and 50 mg/L) of PC-B2+30 mmol/L glucose groups. The viability of EPCs was detected by CCK-8 assay. The levels of LDH, MDA, SOD and GSH in the EPCs were detected. The changes of NO, ET-1, ICAM-1 and VCAM-1 in the EPCs cultured medium were measured by ELISA. The cell apoptotic rate and reactive oxygen species(ROS) in the EPCs were analyzed by flow cytometry. The expression of VEGF and VEGFR-2 in the EPCs were determined by Western blot. RESULTS: Compared with control group, the viability of human EPCs was decreased significantly in 30 mmol/L glucose group(P<0.05). The LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were induced significantly(P<0.05), but SOD and GSH activity and NO production were decreased significantly(P<0.05). The ROS and cell apoptotic rate were increased significantly(P<0.05). The expression of VEGF and VEGFR-2 in the EPCs were decreased(P<0.05). When human EPCs were treated with different concentrations of PC-B2 and 30 mmol/L glucose, the viability was obviously rebounded(P<0.05), the LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were decreased gradually(P<0.05), the SOD, GSH activity and NO production were increased significantly(P<0.05), the ROS and cell apoptotic rate were decreased significantly(P<0.05), and the expression of VEGF and VEGFR-2 in the EPCs was increased gradually(P<0.05).CONCLUSION: PC-B2 enhances the viability of human EPCs under high glucose condition, reduces high glucose-induced oxidative damage, restores the EPCs normal function, and reduces the releases of inflammatory cytokines and apoptosis, thus playing a protective effect on human EPCs through inducing the expression of VEGF and VEGFR-2.     

Activation of Nrf2 by 18α-GA affects proliferation of adult neural stem cells

AIM: To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) activation by 18α-glycyrrhetinic acid (18α-GA) on the proliferation and self-renewal of adult neural stem cells (aNSCs), and to explore an effective way of maintaining the viability of aNSCs. METHODS: NSCs were dissociated from subventricular zone of the mice at postnatal days 0, 60, and 300. The expression levels of Nrf2 in the NSCs at various ages were compared. After treatment with 18α-GA, the expression of Nrf2 was examined by real-time PCR and Western blot. shRNA lentiviral vector (LV) carrying green fluorescent protein (GFP) gene was constructed to knock down Nrf2 expression. The knockdown efficiency in the aNSCs was detected by real-time PCR and Western blot. Subsequently, the aNSCs were divided into DMSO group, 18α-GA group, LV-GFP group and LV-Nrf2-shRNA group. BrdU incorporation assay, Tuj1 staining, CCK-8 assay, Hoechst 33342/PI staining and detection of reactive oxygen species (ROS) were performed to analyze the proliferation, differentiation, viability, apoptosis and oxidative stress levels of the NSCs. RESULTS: The mRNA expression level of Nrf2 in adult and aged NSCs was significantly lower than that in newborn NSCs (P<0.01), while the ROS level of aNSCs was significantly higher (P<0.05). After treatment with 18α-GA, the expression level of Nrf2 in the aNSCs was significantly up-regulated as compared with DMSO group (P<0.01). Increased number of BrdU⁺ and Tuj1⁺ cells was observed in 18α-GA group, indicating that 18α-GA-treated cells had higher viability (P<0.05). Meanwhile, there were fewer apoptotic cells and lower ROS level in 18α-GA group than those in DMSO group (P<0.05). After knockdown of Nrf2 in aNSCs and then treated with 18α-GA, there were less BrdU⁺ and Tuj1⁺ cells, as well as the aNSCs with lower viability in LV-Nrf2-shRNA group (P<0.05). Moreover, the ROS level was increased in LV-Nrf2-shRNA group as compared with LV-GFP group (P<0.05). CONCLUSION: Activation of Nrf2 by 18α-GA elevates the antioxidant capacity of aNSCs, thus ameliorating the cell proliferation and differentiation potentials.     

Effects of UTI on oxidative stress damage in rabbit brain tissues after cardiopulmonary resuscitation

AIM: To investigate the effects of ulinastatin (UTI) on oxidative stress damage in rabbit brain tissues after cardiopulmonary resuscitation (CPR). METHODS: The model of cardiac arrest (CA) in adult male New Zealand rabbits was established and the levels of malondialdehyde (MDA) and glutathione (GSH) in plasma 24 h after restoration of spontaneous circulation (ROSC) were detected. In another experiment, 48 CA New Zealand rabbits were used. After ROSC, the animals were randomly divided into model group and UTI treatment group. The effects of UTI on MDA, GSH and the apoptosis of neurons in the cortex and hippocampus were observed. The effect of UTI on the expression of nuclear factor E2-related factor 2 (Nrf2) was also analyzed. RESULTS: The concentration of MDA in plasma increased rapidly after CA and peaked at 2 h after ROSC, whereas the content of GSH dropped to a lower level. UTI restrained the rise of MDA in the cortex and hippocampus and increased the level of GSH. UTI also accelerated the activation of Nrf2. In the cortex and hippocampus CA1~CA3 areas, the number of apoptotic neurons in model group was more than that in UTI group. CONCLUSION: UTI evidently increases the expression of Nrf2 in rabbit brain tissues, elevates the level of GSH and decreases the concentration of MDA to protect cortical and hippocampal neurons.