Objective To investigate the expression of transcriptional factors zinc finger Krüppel like transcription factor 2 ( Klf2) and NF-E2 related factor 2 ( Nrf2) /BTB CNC homology 1 ( Bach1) in rat bronchial epithelial cells stimulated by cigarette smoke extract ( CSE) , and explore the regulatingmechanism of γ-glutamylcysteine synthetase ( γ-GCS) expression in the oxidative condition. Methods Rat bronchial epithelial cells were harvested using enzyme digestion method, and intervened by 10% CSE for 6 hours. Then γ-GCS activity was detected by two enzymes method, and the nuclear transfer of Nrf2 /Bach1 in cells was detected by immunohistochemistry. Western blot and reverse transcription-polymerase chain reaction ( RT-PCR) techniques were used for detecting the protein and mRNA expressions of Klf2, Nrf2, Bach1, and γ-GCS in the cells. Results The γ-GCS activity was elevated in the CSE group. Immunohistochemical results showed that Nrf2 translocated from cytoplasm to nucleus in response to stimulation by CSE. On the contrary, Bach1 translocated from nucleus to cytoplasm in the same condition. Western blot results showed that protein levels of Klf2, Nrf2, Bach1, and γ-GCS were higher in the CSE group than those in the control group ( Plt;0.05) . RT-PCR results were the same as the Western blot results ( Plt;0.05) . Linear correlation analysis showed that γ-GCS expression was positively correlated with Klf2, Nrf2, and Bach1 ( Plt;0. 05) . Conclusion CSE might regulate the expression of γ-GCS through the transcription factors of Klf2, Nrf2, and Bach1.
Objective To observe the effect of melatonin (MT) on retinal apoptosis in rats with ischemia-reperfusion injury (RIRI). Methods A total of 54 male healthy Sprague-Dawley adult rats were randomly divided into the normal control (CON) group (6 rats), RIRI group (24 rats) and MT group (24 rats). The rats of RIRI and MT group were induced using suture-occluded methods to establish RIRI model. The rats of MT group were injected with MT in the left carotid artery 30 minutes after RIRI, and RIRI group was injected with the same amount of saline. On 6, 24 hours and 3, 7 days after RIRI, the morphological changes of retina were evaluated by hematoxylin and eosin (HE) staining; the effects of MT on retinal cell apoptosis and Nrf2, HO-1 proteins were examined by immunohistochemistry staining. The correlation between active Caspase-3 and Nrf2 protein, active Caspase-3 and HO-1 protein in MT group were analyzed by linear regression analysis. Results HE staining results showed that the morphology of retinal cells was regular and retinal cells were well arranged in the CON and MT group. In the RIRI group, both the thickness of inner retinal layer and the number of retinal ganglion cells (RGC) were decreased. On 6, 24 hours and 3, 7 days after RIRI, the thickness of inner retinal layer (F=16.710, 62.303, 68.389, 57.132; P＜0.01) and RGC number (F=24.250, 11.624, 14.155, 32.442; P＜0.05) in MT group were more than those in RIRI group. Immunohistochemistry staining results showed that less active Caspase-3+ cells were observed in MT group as compared with those in RIRI group at each time points (F=49.118, 134.173, 76.225, 18.385; P＜0.01). There were more Nrf2+ (F=11.041, 31.480, 59.246, 6.740; P＜0.05) and HO-1+ cells (F=128.993, 21.606, 51.349, 8.244; P＜0.05) in MT group as compared with those in RIRI group at each time points. Linear regression analysis results showed that the difference of active Caspase-3+ cells were all linearly correlated with the Nrf2+ cells and HO-1+cells in the MT group (r2=0.810, 0.730; P＜0.01). Conclusion MT could reduce retinal cell apoptosis in RIRI rats, and its mechanism may be associated with increased Nrf2 and HO-1 expression, reduced active Caspase-3 expression.
ObjectiveTo observe the expression of probucol on high glucose-induced specificity protein 1(SP1), kelchlike ECH associated protein1 (Keap1), NF-E2-related factor 2 (Nrf2) and glutamate-cysteine ligase catalytic (GCLC) in the cultured human müller cells and preliminary study the antioxidation of the probucol on müller cells.MethodsPrimary cultured human müller cells were randomly divided into four groups: normoglycaemia group (5.5 mmol/L glucose), normoglycaemia with probucol group (5.5 mmol/L glucose+100 μmol/L probucol), hyperglycemia group (25.0 mmol/L glucose), hyperglycemia with probucol group (25.0 mmol/L glucose + 100 μmol/L probucol). Immunofluorescence staining was used to assess distribution of SP1, Keap1, Nrf2, GCLC in human Müller cells. SP1, Keap1, Nrf2 and GCLC messenger RNA (mRNA) expression was evaluated by quantitative real-time RT-PCR (qRT-PCR). Independent sample t test was used to compare the data between the two groups.ResultsAll müller cells expressed glutamine synthetase (＞95%), which confirmed the cultured cells in vitro were the purification of generations of müller cells. The expressions of SP1, Keap1, Nrf2, and GCLC protein were positive in human müller cells. qRT-PCR indicated that SP1 (t=28.30, P＜0.000), Keap1 (t=5.369, P=0.006), and Nrf2 (t=10.59, P=0.001) mRNA in the hyperglycemia group increased obviously compared with the normoglycaemia group; GCLC (t=4.633, P=0.010) mRNA in the hyperglycemia group decreased significantly compared with the normoglycaemia group. However, SP1 (t=12.60, P=0.000) and Keap1 (t=4.076, P=0.015) in the hyperglycemia with probucol group decreased significantly compared with the hyperglycemia group; Nrf2 (t=12.90, P=0.000) and GCLC (t=15.96, P＜0.000) mRNA in the hyperglycemia with probucol group increased obviously compared with with the hyperglycemia group.ConclusionProbucol plays an antioxidant role by inhibiting the expression of SP1, Keap1 and up-regulating the expression of Nrf2, GCLC in müller cells induced by high glucose.
Objective To investigate the cellular viability and mitochondrial reactive oxygen species (ROS) production of the Müller cells under high glucose condition, and explore the protection role of the 5,6-dihydrocyclopenta-1, 2-dithiole-3-thione (CPDT) on Müller cells. Methods Müller cells from Sprague Dawley rats were divided into 5 groups randomly, including 25 mmol/L normal glucose group (group A) and 65 mmol/L high glucose group (group B). High glucose group with 45, 60, 70 μmol/L CPDT and cultured them 72 hour was set as group C, D and E. Water soluble tetrazolium salt (WST)-8 was used to measure the cellular viability. Flow cytometry was used to measure the active oxygen and apoptosis index. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), Bcl-2 and Bax protein were measured by Western blot. Results Compared with group A, the WST-8 showed that the viability of Müller cells apparently decreased in group B (t=39.59,P＜0.05). Compared with the group B, the viability of Müller cells had changes in group C (t=0.97,P＞0.05), but recovered in group D and E (t=?4.17, ?7.52;P＜0.05). Compared with group A, the FCM showed that the mitochondrial ROS levels was higher in group B (t=?30.99,P＜0.05). Compared with group B, the mitochondrial ROS levels were decreased in group D (t=27.68,P＜0.05). Compared with group A, Bax, Nrf2 and HO-1 increased (t=–11.03, –63.17, –11.44;P＜0.05), while the bcl-2 decreased in group B (t=7.861,P＜0.05). Compared with the group B, Nrf2, HO-1 and Bax decreased (t=15.11, 26.59, 6.27;P＜0.05), while the bcl-2 increased in group D (t=?6.53,P＜0.05). Conclusions Under the high glucose, CPDT may reduce the mitochondrial ROS levels and the expression of Nrf2, HO-1 and Bax protein of Müller cells. It may inhibit apoptosis through activating the Nrf2/HO-1 pathway and balancing of level of Bcl-2 protein and mitochondrial ROS.
ObjectiveTo investigate the protective effect and the regulation mechanism of oxaloacetate (OAA) on myocardial ischemia reperfusion injury in rats. MethodsSixty rats, weight ranged from 200 to 250 grams, were randomly divided into 6 groups:a negative control group, a sham operation control group, a model control group, an OAA pretreatment myocardial ischemia-reperfusion model group (three subgroups:15 mg/kg, 60 mg/kg, 240 mg/kg). We established the model of myocardial ischemia reperfusion of rats and recorded the internal pressure of left ventricle (LVSP), the maximal rate of left ventricular pressure change (±dp/dtmax) and left ventricular end diastolic pressure (LVEDP). We restored reperfusion 180 minutes after ligating the left anterior descending coronary artery 30 minutes and determinated cardiac troponin Ⅰ (cTn-I), lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px). We took out heart tissues, stained it and calculated the infarcted size. We used the Western blot to detect the expression of NF-E2 related factor 2 (Nrf2), Kelch-like ECH-associated protein-1 (Keap1) and heme oxygenase-1 (HO-1). ResultsCompared with the sham operation group, heart function indexes in the negative control group had no significant difference (P>0.05). But in the model control group there was a decrease (P<0.05) And the serum levels of LDH, cTn-I, and myocardial infarcted size were significantly increased (P<0.01). Compared with the model control group, heart function indexes in the OAA pretreatment groups improved, the serum LDH, cTn-I activity, and infarct size decreased (P<0.05), SOD and GSH-Px activity increased (P<0.05). And these results were statistically different (P<0.01) in the high dose OAA pretreatment groups. Compared with the model control group, the expression of Keap1 in the OAA pretreatment group was down-regulated (P<0.001) while total Nrf2, nucleus Nrf2 and its downstream HO-1 was up-regulated (P<0.001), which suggested that OAA enhanced antioxidant capacity by (at least in part) Keap1-Nrf2 pathway, resulting in reducing myocardial damage and protecting myocardium after acute myocardial ischemia reperfusion injury. ConclusionOxaloacetate can provide protective effects on myocardial ischemia reperfusion injury through down-regulating the expression of Keap1 and up-regulating the expression of Nrf2 and its downstream peroxiredoxins to improve antioxidant capacity.
Objective To explore the regulation of peroxisome proliferator-activated receptor γ coactivator 1α( PGC-1α) and NF-E2-related factor 2( Nrf2) on expression of γ-glutamylcysteine synthetase ( γ-GCS) , and their roles in chronic obstructive pulmonary disease( COPD) . Methods Twenty-four SD rats were randomly divided into a COPD group and a normal control group. COPD model was established by intratracheal instillation of lipopolysaccharide ( LPS) and daily exposure to cigarette smog in the COPD group. The lung function was measured and the pathological changes were observed. The protein and mRNA expressions of PGC-1α, Nrf2, and γ-GCS in lung tissue were measured by immunohistochemistry, Western blot, in site hybridization ( ISH) , and reverse transcription-polymerase chain reaction ( RT-PCR ) ,respectively. Results In the COPD group, the pulmonary function ( FEV0. 3, FEV0. 3 /FVC, PEF) damage and lung pathological changes were conformed as morphological characteristics of COPD. The mRNA of PGC-1α and Nrf2 expressed in lung tissues of two group rats in the region consistent with γ-GCS mRNA. The protein and mRNA expressions of PGC-1αand γ-GCS were markedly increased in the COPD group( all P lt;0. 05) ,and the protein expression of Nrf2 was obviously up-regulated ( P lt; 0. 01) , while Nrf2 mRNA had no significant difference between the two groups( P gt;0. 05 ) . Linear correlation analysis showed that the level ofPGC-1αprotein was positively correlated with the levels of Nrf2 protein and mRNA ( r = 0. 775, 0. 515, all P lt; 0. 01) , and the levels of PGC-1αand Nrf2 protein were positively correlated with the levels of γ-GCS protein ( r = 0. 531, 0. 575, all P lt; 0. 01) and mRNA ( r = 0. 616, 0. 634, all P lt; 0. 01) . Conclusions PGC-1α, which may serve as a co-activator of Nrf2, can up-regulate the expression of γ-GCS gene cooperatively with Nrf2 through a common pathway, which might involve in the oxidative and antioxidative mechanism in the pathogenesis of COPD.
ObjectiveTo observe the effect of phase Ⅱenzyme inducer 5, 6-dihydrocyclopenta 1, 2-dithiole-3-thione (CPDT) on nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signal pathway and oxidative stress in the retina of type 2 diabetic rats. MethodsThirty-five male Wistar rats were randomly divided into two group, normal group and model group. Model group were further randomly divided into two group, diabetic group and CPDT intervention group. There were 8 rats in the normal group and 27 rats in the model group. Diabetic group and CPDT intervention group were given high fat and high sugar diet for 2 months. After 12 hours of fasting, type 2 diabetic rat model was induced by intraperitoneal injection of low dose of streptozotocin. CPDT was added into the high fat and high sugar diets at 1 week after the diabetic model was established in the CPDT intervention group. Eight weeks after CPDT treatment, blood glucose, serum malondialdehyde (MDA), blood lipid, Nrf2 and hemeoxygenase-1 (HO-1) expression were evaluated. ResultsType 2 diabetic model was successfully established in 25 rats, the success rate was 92.6%.The level of blood lipid of diabetic group was higher than those of the normal group (FTC=65.866, FTG=25.441, FLDL-C=38.889; P=0.000). Blood glucose was significant different between all groups (χ2=25.812, P=0.000), and was significantly higher in diabetic group than that in normal group and CPDT intervention group. The serum MDA content was significant different between all groups (F=59.545, P=0.000), and was significantly higher in diabetic group than that in normal group (t=10.523, P=0.000) and CPDT intervention group (t=7.766, P=0.000). The mRNA level of retinal Nrf2 and HO-1 was significant different between all groups (FNrf2=19.503, PNrf2=0.000;FHO-1=9.737, PHO-1=0.001), and was higher in CPDT intervention group than the diabetic group (tNrf2=3.399, PNrf2=0.002;tHO-1=2.167, PHO-1=0.039). The protein level of retinal Nrf2 and HO-1 was significant different between all groups (FNrf2=112.823, FHO-1=119.361; P=0.000), and was higher in CPDT intervention group than the diabetic group (tNrf2=6.203, tHO-1=6.388; P=0.000). Immuno-staining showed that Nrf2 and HO-1 were mainly expressed in retinal ganglion cell layer, inner plexiform layer and inner nuclear layer, and were significant different between all groups (FNrf2=16.206, FHO-1=46.790; P=0.000). They also were higher in CPDT intervention group than the diabetic group (tNrf2=3.172, PNrf2=0.003;tHO-1=6.321, PHO-1=0.000), was higher in diabetic group than that in normal group (tNrf2=2.679, PNrf2=0.011;tHO-1=3.482, PHO-1=0.001). ConclusionCPDT may activate Nrf2/ARE pathway, induce Nrf2 and HO-1 expression, decrease serum MDA and blood glucose, and thus reduce oxidative stress injury in the retina of type 2 diabetic rats.
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.
ObjectiveTo investigate the inhibitory effect of lentivirus-mediated polypyrimidine bundle binding protein-associated splicing factor (PSF) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR).MethodsOne hundred and twelve 5-day-old C57BL/6J mice were randomly divided into normal control group, simple OIR model group, OIR model + lentivirus empty vector treatment group (Vec group) and OIR model + PSF lentivirus treatment group (PSF group), with 16, 32, 32 and 32 mice, respectively. When the mice were 7 days old, the mice in the normal control group were fed in a routine environment, and the mice in the OIR model group, Vec group and PSF group were established OIR model. The mice in the Vec group and PSF group were given an intravitreal injection of 1 μl of lentiviral vector and PSF lentivirus (titer 1×1011 TU/ml) at the age of 12 days. No injection was performed in the normal control group and simple OIR group. RNV was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR was applied to detect the mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Western blot analysis was applied to detect the protein expression of Nrf2, HO-1 and PSF. Results Of the normal control group, simple OIR model group, Vec group and PSF group, the number of pre-retinal neovascular cell nuclei were 0.00, 14.36±5.50, 15.67±4.96, 8.13±2.09, the non-perfusion area were 0.00%, (35.71±2.81)%, (36.57±4.53)%, (15.33±4.75)%, respectively. The differences of the number of pre-retinal neovascular cell nuclei and non-perfusion area among 4 groups were significant (F=24.87, 165.70; P＜0.05). Compared with the normal control group, there were more pre-retinal neovascular cell nucleis and larger non-perfusion area in the simple OIR model group and Vec group (P＜0.05). Compared with the simple OIR model group and Vec group, there were lower pre-retinal neovascular cell nucleis and smaller non-perfusion area in the PSF group (P＜0.05). Real-time quantitative PCR and Western blot showed that the mRNA expression of Nrf2, HO-1 (F=53.66, 83.54) and protein expression of Nrf2, HO-1 and PSF (F=58.38, 52.69, 24.79) among 4 groups were significant (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the simple OIR model group and Vec group decreased significantly than those in the normal control group (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the PSF group were increased significantly than those in the simple OIR model group and Vec group (P＜0.05). model group and Vec group (P＜0.05).ConclusionIntravitreal injection of lentivirus-mediated PSF inhibits RNV in mice model of OIR possibly through up-regulating the expression of Nrf2 and HO-1.
ObjectiveTo summarize the research progress of risk factors related to early recurrence and late recurrence of hepatocellular carcinoma (HCC) after radical resection.MethodsReviewed and summarized recent literatures on factors related to early and late recurrence of HCC after radical resection.ResultsRadical resection was the most effective treatment for HCC, but the postoperative recurrence rate was high, which seriously affected the treatment effect. Current research divided the recurrence after radical resection of HCC into early recurrence (≤2 years) and late recurrence (>2 years). Early recurrence was considered to be mainly caused by intrahepatic metastasis (IM), which was related to the tumor itself, while late recurrence was mainly caused by multicentric occurrence (MO) and was related to background liver factors. Factors of the tumor itself, including tumor diameter and number, invasion of tumor large vessels and microvessels, anatomical and non-anatomical resection, tumor margin, residual liver ischemia (RLI), intermittent total entry hepatic blood flow interruption method (IPM), the expression level of circulating microRNA in serum and long-chain non-coding RNA, circulating tumor cells, and circulating tumor DNA were related to early recurrence; background liver factors, including liver cirrhosis, high viral load, and liver inflammatory activity, were associated with late recurrence.ConclusionsBoth the tumor factors associated with early recurrence and the background liver factors associated with late recurrence can affect the recurrence after radical resection of HCC.