Objective To study the effect and mechanism of recombinant human brain natriuretic peptide (rh-BNP) in alleviating myocardial ischemia-reperfusion (I/R) injury by regulating mitogen activated protein kinase (MAPK) pathway. Methods A total of 128 adult male Sprague-Dawley (SD) rats with specific pathogen free were selected. The SD rats were divided into groups according to random number table, including, sham operation (Sham) group, I/R group, I/R+rh-BNP group, negative control adenovirus (Ad-NC)+Sham group, Ad-NC+I/R group, Ad-NC+I/R+rh-BNP group, p38 mitogen-activated protein kinase adenovirus (Ad-p38MAPK)+I/R group and Ad-p38MAPK+I/R+rh-BNP group, with 16 SD rats in each group. Myocardial I/R injury model was established by ligation of left anterior descending coronary artery. Before modeling, rh-BNP was injected intraperitoneally or adenovirus was injected into myocardium; 180 minutes after reperfusion, the contents of lactate dehydrogenase (LDH), creatine kinase isoenzyme (CK-MB) in serum, myocardial infarction size, the contents of reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α) and the expression of phosphorylated p38MAPK (p-p38MAPK), phosphorylated JNK (p-JNK) and phosphorylated extracellular regulated protein kinases 1/2 (p-ERK1/2) were detected. Results The contents of LDH, CK-MB, myocardial infarction size, the contents of TNF-α, ROS and the expression of p-p38MAPK and p-JNK in I/R group were higher than those in Sham group, p-ERK1/2 expression level was lower than that in Sham group (P<0.05). The contents of LDH, CK-MB, myocardial infarction size, the contents of TNF-α, ROS and the expression of p-p38MAPK in I/R+rh-BNP group were lower than those in I/R group (P<0.05), the expression of p-JNK and p-ERK1/2 had no significant difference compared with I/R group (P>0.05). The contents of LDH, CK-MB, myocardial infarction size, the contents of TNF-α, ROS and the expression of p-p38MAPK in Ad-p38mapk+I/R+rh-BNP group were higher than those in Ad-NC+I/R-rh-BNP group (P<0.05). Conclusion rh-BNP can alleviate myocardial I/R injury, which is related to inhibiting p38MAPK pathway, reducing inflammation response and oxidative stress response.
ObjectiveTo investigate the effects of MK-801 on antioxidant system activity in the central nervous system of rats with obstructive jaundice. MethodsTwenty rats were divided into four groups: sham operation group, control group, MK-801 low dose group, and MK-801 high dose group. The control group, MK-801 low dose group, and MK-801 high dose group were the obstructive jaundice model groups (OJ groups). From the first day after operation, MK-801 low dose group were processed intraperitoneal injection of MK-801 0.025 mg/(kg·d) and MK-801 high dose group were processed intraperitoneal injection of MK-801 0.25 mg/(kg·d). Meanwhile, sham operation group and control group were injected the same volume of normal saline everyday for 10 days. Three days after operation, rats' tail vein blood were collected for examining the direct bilirubin DBIL) and total bile acids (TBA) in order to determine whether the model were successfully established. And malondialdehyde (MDA), catalase (CAT), total superoxide dismutase (T-SOD), and total antioxidant capacity (T-AOC) were determined on the 10th day to evaluate the oxdative status of the rats. Results①Obstructive jaundice model was established successfully.②The content of MDA in control group, MK-801 low dose group and MK-801 high dose group were significantly increased than the sham operation group, and there was statistical difference (P < 0.05). The content of MDA decreased in MK-801groups compared with the control group (P < 0.05).③Compared with the sham operation group, the activity of CAT in control group decreased significantly (P < 0.05). The activity of CAT in the MK-801 groups increased compared with the control group with significant difference (P < 0.05). There was no statistical difference on the activity of CAT between MK-801 low dose group and high dose group (P > 0.05).④Compared with sham operation group, the activity of T-SOD was decreased significantly in control group with statistical significance (P < 0.05). The activity of T-SOD were increased in the MK-801 groups compared with control group with significant difference (P < 0.05), but the activity of T-SOD was decreased significantly in the high dose group than the low dose group (P < 0.05).⑤In the Oj groups, the T-AOC were significantly increased compared with the sham operation group, and there was statistical significance (P < 0.05). The T-AOC in MK-801 groups were increased compared with the control group with statistical significance (P < 0.05), but there was no statistical difference between the MK-801 groups. Conciusions Oxidative stress exists when obstructive jaundice occurs, and obstructive jaundice can aggravate the oxidative stress damage in the rats' central nervous system and cause increasing expression of enzymes such as CAT which enhance antioxidant capacity of the whole body. MK-801 can decrease lipid peroxidation, and increase activity of CAT and SOD as well as T-AOC in CNS of jaundice rats. But High dose of MK-801 has no better effect than low dose of MK-801. On the contrary, activity of T-SOD decrease in the high dose group than in the low dose group. Further research is needed on the specific mechanism.
Febrile seizures (FS) are one of the most common neurological disorders in pediatrics, commonly seen in children from three months to five years of age. Most children with FS have a good prognosis, but some febrile convulsions progress to refractory epilepsy (RE). Epilepsy is a common chronic neurological disorder , and refractory epilepsy accounts for approximately one-third of epilepsies. The etiology of refractory epilepsy is currently complex and diverse, and its mechanisms are not fully understood. There are many pathophysiological changes that occur after febrile convulsions, such as inflammatory responses, changes in the blood-brain barrier, and oxidative stress, which can subsequently potentially lead to refractory epilepsy, and inflammation is always in tandem with all physiological changes as the main response. This article focuses on the pathogenesis of refractory epilepsy resulting from post-febrile convulsions.
The aim of this research is to investigate the influence of microencapsulation on the expression of the oxidative stress genes and exogenous regulation of HepG2 cells. We compared the expression of hemeoxygenase-1 (HO-1) and glutathione S-transferases-A1 (GST-A1) in HepG2 cells under different culture conditions through real-time PCR. The effects of exogenous antioxidants on cell viability and albumin levels were also evaluated through MTT assay and ELISA assay. The results showed that after culturing for 6 and 16 days, the expression levels of HO-1 in encapsulated cells were approximately 4.9 and 3.1 times higher than that of monolayer cells at the same culture period; As for the expression levels of GST-A1, they were elevated to 11.2 and 33 times of monolayer cells (P<0.05). Accordingly, we found that NAC at 5-10 mmol/L significantly increased the viability by 40%-70% and the biosynthetic function by 20%-30% in microencapsulated HepG2 cells (P<0.05). GSH increased the viability of the encapsulated cells by 20%-55% and the biosynthetic function by 15% (P<0.05). In conclusion, oxidative stress exists in the microcapsules and affects genes expression. Exogenous antioxidants can prevent the inhibition effects of oxidative stress on cellular growth.
ObjectiveTo explore the protective effects of sodium valproic acid (VPA) on oxidative stress injury of osteoblasts induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and its mechanism. Methods Osteoblasts were isolated from the skulls of 10 newborn Sprague Dawley rats and cultured by tissue block method, and the 1st generation cells were identified by alkaline phosphatase (ALP) and alizarin red staining. The 3rd generation osteoblasts were cultured with 2-18 μmol/L CCCP for 2-18 minutes, and cell counting kit 8 (CCK-8) was used to detect the cell survival rate. An appropriate inhibitory concentration and culture time were selected for the preparation of osteoblasts oxidative stress injury model based on half maximal concentration principle. The cells were cultured with 0.2- 2.0 mmol/mL VPA for 12-72 hours, and CCK-8 was used to detect cell activity, and appropriate concentration was selected for further treatment. The 3rd generation cells were randomly divided into 4 groups, including blank control group (normal cultured cells), CCCP group (the cells were cultured according to the selected appropriate CCCP concentration and culture time), VPA+CCCP group (the cells were pretreated according to the appropriate VAP concentration and culture time, and then cultured with CCCP), VPA+CCCP+ML385 group (the cells were pretreated with 10 μmol/L Nrf inhibitor ML385 for 2 hours before VPA treatment, and other treatments were the same as VPA+CCCP group). After the above treatment was complete, the cells of 4 groups were taken to detect oxidative stress indicators [reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA)], cell apoptosis rate, ALP/alizarin red staining, and the relative expressions of osteogenic related proteins [bone morphogenetic protein 2 (BMP-2), RUNX2], anti-apoptotic family protein (Bcl2), apoptotic core protein (Cleaved-Caspase-3, Bax), channel protein (Nrf2) by Western blot. Results The osteoblasts were successfully extracted. According to the results of CCK-8 assay, the oxidative stress injury model was established by 10 μmol/L CCCP cultured for 10 minutes and 0.8 mmol/mL VPA cultured for 24 hours was selected for subsequent experiments. Compared with blank control group, the activity and mineralization capacity of osteoblasts in CCCP group decreased, the contents of ROS and MDA increased, the activity of SOD decreased, and the apoptosis rate increased. Meanwhile, the relative expressions of BMP-2, RUNX2, and Bcl2 decreased, and the relative expressions of Cleaved-Caspase-3, Nrf2, and Bax increased. The differences were significant (P<0.05). After further VPA treatment, the oxidative stress damage of osteoblasts in VPA+CCCP group was relieved, and the above indexes showed a recovery trend (P<0.05). In VPA+CCCP+ML385 group, the above indexes showed an opposite trend (P<0.05), and the protective effects of VPA were reversed. Conclusion VPA can inhibit the CCCP-induced oxidative stress injury of osteoblasts and promote osteogenesis via Keap1/Nrf2/Are pathway.
ObjectiveTo investigate the protective effect of butylphenyphthalein (NBP) on RPE apoptosis induced by H2O2.MethodsThe human RPE cell line (human ARPE-19 cell line) were used as the experimental cells and were divided as control group, model group, NBP group. Complete medium was used in control group. The model group was stimulated with 200 μmol/L H2O2 for 2 h, and the cells were cultured in complete medium. The NBP group was cultured with 200 μmol/L H2O2 and 1 μmol/L NBP for 2 h. After changing the medium, complete medium was combined with 1 μmol/L NBP to continue the culture of the cells. Cell viability were detected by MTT assay while the morphology of RPE were observed by HE staining. Moreover, Hoechst 33258 was used to detect RPE cell apoptosis. Mitochondrial membrane potential (JC-1) staining were performed to monitor changes in cell membrane potential and the characteristic change of apoptosis in RPE cells. Furthermore, 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) staining were used to analyze the effect of NBP treatment on the expression of ROS. The effect of NBP on the expression of Heme oxygenase-1(HO-1) was analyzed by cellular immunofluorescence and western blotting.ResultsThe results of MTT assay showed that the cells were cultured for 24 and 48 hours, cell viability of control group (t=17.710, 13.760; P＜0.000 1, ＜0.000 1) and treatment group (t=4.857, 9.225; P=0.000 7, ＜0.000 1) were stronger than that of model group, and the difference was statistically significant. HE staining and Hoechst33258 staining showed that compared with the control group, the number of cells in the model group was significantly less, and the cell morphology was incomplete. Compared with the model group, the number of cells in the treatment group was significantly increased, and the cell morphology was better. The results of JC-1 assay showed that the number of apoptotic cells in the model group was significantly higher than that in the control group, and the number of apoptotic cells in the treatment group was significantly lower than that in the model group. DCFH-DA staining showed that the ROS accumulation in the model group was more than that in the control group, and the ROS accumulation in the treatment group was less than that in the model group. Immunostaining observation showed that the HO-1 fluorescence intensity of the cells in the treatment group was significantly higher than that of the control group, and the difference was statistically significant (t=10.270, P=0.000 5). Western blot analysis showed that NBP up-regulated the expression level of HO-1 in a time-dependent manner. The relative expression of HO-1 at 4, 8, and 12 h of NBP showed a clear increase trend compared with 0 h, and the difference was statistically significant (F=164.91, P＜0.05).ConclusionsOxidative stress injury can down-regulate the viability of RPE cells and induce apoptosis. NBP can increase the antioxidant capacity of RPE cells, reduce cell damage and inhibit cell apoptosis by up-regulating HO-1 expression.
ObjectiveTo evaluate the effects of N-acetylcysteine (NAC) on lung tissue of Wistar rats, which were tracheally instilled fine particulate matter (PM2.5).MethodsForty-eight male Wistar rats were randomly divided into six groups: two control groups [they were blank group (C1), fake treatment group (C2) separately], four treatment groups [they were PM2.5 group (P), low-dose NAC group (L), medium-dose NAC group (M), high-dose NAC group (H) separately]. C1 received no treatments at all. C2 was instilled with sterile water (1 ml/kg) tracheally once a week for four times. P was instilled equivoluminal PM2.5 suspension (7.5 mg/kg) tracheally once a week for four times. The NAC groups received gavage (10 ml/kg) of different dosage of NAC (125, 250, 500 mg/kg) for six days. At the seventh day, the NAC groups were instilled PM2.5 suspension (7.5 mg/kg) tracheally. The procedures were repeated for three times in the NAC groups. Twenty-four hours later after four weeks or after the last instilling, all rats were sacrificed. Lung tissue was stained by hematoxylin-eosin (HE) staining, and histopathological changes of lung tissue were observed by optical microscope. The levels of C-reactive protein (CRP) as well as tumor necrosis factor-α (TNF-α) of serum, TNF-α of bronchoalveolar lavage fluid (BALF), TNF-α as well as interleukin-1β (IL-1β) of homogenates of lung tissue were detected by enzyme-linked immunosorbent assay. The activity of lactate dehydrogenase (LDH) as well as the levels of malondialhyde (MDA) of serum and BALF were detected by standard colorimetric method.ResultsHE staining showed that the normal structure of lung were destroyed in the groups dealed with PM2.5 and NAC could alleviate these changes. Higher dosage of NAC seemed to provide more powerful protections. Structure of the lung in C1 as well as C2 were nearly normal. The levels of CRP as well as TNF-α of serum, TNF-α of BALF, TNF-α as well as IL-1β of homogenates of lung tissue in the groups of P, L, M, H were higher than that in the groups of C1, C2 (all P<0.05). The levels of CRP as well as TNF-α of serum, TNF-α of BALF, TNF-α as well as IL-1β of homogenates of lung tissue in the groups of L, M, H which groups received NAC treatments were lower than that in P group. More, the groups seemed to have lower levels of CRP, TNF-α, IL-1β when higher dosage of NAC were given. The activity of LDH as well as the levels of MDA of serum, and BALF in the groups of P, L, M, H were higher than that in the groups of C1, C2 (all P<0.05). The activity of LDH as well as the levels of MDA of serum and BALF in the groups of L, M, H which groups received NAC treatments were lower than that in P group (all P<0.05). ConlusionTo some extent, NAC demonstrate antagonistic effects on oxidative stress and inflammatory injury on rats’ lung brought by PM2.5.
Objective Bone marrow mesenchymal stem cells (BMSCs) transplantation can potentially regenerate the degenerated intervertebral disc, with the underlying regenerating mechanism remaining largely unknown. To investigate the potential of human BMSCs protecting nucleus pulposus cells (NPCs) from oxidative stress-induced apoptosis in a coculturesystem, and to illustrate the possible mechanisms of BMSCs transplantation for intervertebral disc regeneration. Methods BMSCs collected by density gradient centrifugation in Percoll solution were cultured and sub-cultured till passage 3, and the surface molecules of CD34, CD45, and CD13 were identified. NPCs were isolated by collagenase digestion and the chondrocyte l ike phenotype was confirmed by morphologic observation after HE staining, inverted phase contrast microscope, proteoglycan, and collagen type II expression after toluidine blue and immunocytochemistry staining. The 3rd passage BMSCs and the 1st passage NPCs were divided into four groups: group A, NPCs (1 × 106 cells) were cultured alone without apoptosis inducing (negative control); group B, NPCs (1 × 106 cells) were co-cultured with BMSCs (1 × 106 cells) with apoptosis inducing; group C, NPCs (1 × 106 cells) were co-cultured with BMSCs (3 × 105 cells) with apoptosis inducing; group D, NPCs (1 × 106 cells) were cultured alone with apoptosis inducing (positive control). After 3 or 7 days of culture or co-culture, the NPCs in groups B, C, and D were exposed to 0.1 mmol hydrogen peroxide for 20 minutes to induce apoptosis. With DAPI staining cellular nucleus, Annexin-V/propidium iodide staining cellular membrane for flow cytometry analysis, the apoptosis of NPCs in each group was studied both qual itatively and quantitatively. Besides, the changes in Bax/Bcl-2 gene transcription and Caspase-3 protein content, were analyzed with semi-quantitative RT-PCR and Western blot. Results BMSCs were successfully isolated and CD34-, CD45-, and CD13+ were demonstrated; after isolated from degenerated intervertebral discs and sub-cultured, the spindle-shaped 1st passage NPCs maintained chondrocyte phenotype with the constructive expressions of proteoglycan and collagen type II in cytoplasm. DAPI staining showed the nucleus shrinkage of apoptosis NPCs. Co-cultured with BMSCs for 3 days and 7 days, the apoptosis rates of NPCs in groups B (29.26% ± 8.90% and 18.03% ± 2.25%) and C (37.10% ± 3.28% and 13.93% ± 1.25%) were lower than that in group D (54.90% ± 5.97% and 26.97% ± 3.10%), but higher than that of groupA (15.67% ± 1.74% and 8.87% ± 0.15%); all showing significant differences (P lt; 0.05). Besides, semi-quantitative RT-PCR showed Bcl-2 gene transcription up-regulated (P lt; 0.05) and no significant change of Bax (P gt; 0.05); Western blot result showed that the Caspase-3 protein expression of groups B and C was lower than that of group D, and was higher than that of group A; all showing significant differences (P lt; 0.05). Conclusion In a co-culture system without direct cellular interactions, the oxidative stress-induced apoptosis of human NPCs was amel iorated by BMSCs. The enhanced anti-apoptosis abil ity of NPCs preconditioned by co-culturing with BMSCs might come from the decreased Bax/Bcl-2 gene transcription ratio.
Objective To observe the effects of cigarette smoke extract ( CSE) on the proliferation and secretion of hydrogen peroxide ( H2O2 ) in human lung fibroblasts ( HLFs) induced by transforming growth factor-β1 ( TGF-β1 ) . Methods Cultured HLFs were divided into a normal group and a model group induced by TGF-β1 ( 5 ng/mL) , then intervened with CSE at different concentrations ( 0% , 2. 5% , 5% ,10% , respectively) . Brdu ELISA assay was used to detect cell proliferation. H2O2 release from cultured cells was assayed using a fluorimetric method. Cellular localization of H2O2 and expression of α-SMA were performed using a fluorescent-labeling strategy. Results TGF-β1 stimulated group showed positive expression of α-SMA, implying TGF-β1 had induced fibroblasts to differentiate into myofibroblasts. In TGF-β1 stimulated group, 2. 5% and 5% CSE promoted cell proliferation ( P lt; 0. 01 or 0. 05) , while 10% CSE inhibited cell proliferation ( P lt; 0. 01) . In the normal group, both low and high concentration of CSE inhibited cell proliferation ( P lt; 0. 01 or P lt; 0. 05) , and the inhibition effect was dose-dependent. HLF induced by TGF-β1 generated low constitutive levels of extracellular H2O2 that was markedly enhanced by CSE treatment ( P lt; 0. 01) . Pretreatment with DPI, an inhibitor of NADPH oxidase, abolished secretion of H2O2 . Cellular localization of H2O2 by a fluorescent-labeling strategy demonstrated that extracellular secretion of H2O2 is specific to the myofibroblast. Conclusions Low concentration of CSE can promote myofibroblast proliferation, and markedly increase extracellular secretion of H2O2 . CSE possibly take part in the development and progress of idiopathic pulmonary fibrosis by increasing oxidative stress.