ObjectiveTo investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD+) levels.MethodsThe bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H2O2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H2O2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H2O2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD+ and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment.ResultsThe rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased (P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H2O2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD+ and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant (P<0.05).ConclusionNMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD+ levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.
【摘要】 目的 观察长期大量酒精摄入对大鼠心肌结构及心肌组织中丙二醛(MDA)、超氧化物歧化酶(SOD)和金属硫蛋白(MT)含量的影响,探讨氧化应激在酒精性心肌病大鼠中的作用。 方法 雄性健康SD大鼠45只,随机分为2组,即对照组20只和模型组25只。模型组酒精浓度从5%、10%、20%和30%依次各自由饮1周,然后递增至36%后以该浓度维持饲喂。对照组每日饮用与模型组酒精同等热量的葡萄糖水。6个月后,观察大鼠心肌组织的形态学改变及超微结构的变化,测定心肌组织中MDA、SOD及MT的含量。结果 模型组大鼠心肌细胞排列紊乱、间质充血、炎细胞浸润、线粒体肿胀、空泡形成、肌丝溶解、核膜不规则和核仁裂解。心肌组织中MDA含量明显升高(Plt;0.01),SOD活力含量明显降低(Plt;0.01),MT含量明显降低(Plt;0.01)。 结论 长期摄入大量酒精可使氧自由基代谢失衡,导致心肌损伤。氧化应激在酒精性心肌病发病机制中发挥着重要的作用。【Abstract】 Objective To observe the effect of longterm and large quantities of alcohol intake on myocardial structure of rats and the content of malondialdehyde (MDA), superoxide dismutase (SOD) and metallothionein (MT) in myocardium tissue. To study the effect of oxidative stress on the rats with alcoholic cardiomyopathy. Methods Fortyfive male and healthy SD rats were randomly divided into the control group (20 rats) and model group (25 rats).The alcoholic concentrate in model group was increased from 5%,10%,20% to 30% every week, and maintain free drinking mass concentration of 36% alcohol. The control group drink the same calories of glucose water. Six months later, the myocardial tissues were observed both in light microscope and electron microscope .The level of MDA、SOD and MT were tested in myocardium tissue. Results In the model rats, the cells of myocardial disarray, interstitial congestion, inflammatory cell infiltration, mitochondrial swelling, vacuole formation, melt filaments, irregular nuclear membrane and nucleolus cracking. The content of MDA incresed(Plt;0.01)and the activities of SOD decreased(Plt;001),levels of MT decreased (Plt;0.01) in the cardiac muscular tissues in the model group compared with the control group. Conclusion Longterm intake of large amounts of alcohol can break the balance of oxygen free radicals, which leading to the damage of myocardial. Oxidative stress plays an important role in the etiopathogenesis of alcoholic cardiomyopathy.
ObjectiveTo investigate the expression changes and the repair effect of mitogen and stress- activated protein kinase 1 (MSK1) on spinal cord injury (SCI) in rats.MethodsOne hundred and twenty male Sprague Dawley (SD) rats (weighing 220-250 g) were used for the study, 70 of them were randomly divided into sham-operation group and SCI group (n=35), the rats in SCI group were given SCI according to Allen’s method, and the sham-operation group only opened the lamina without injuring the spinal cord; spinal cord tissue was collected at 8 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, and 7 days after invasive treatment, each group of 5 rats was used to detect the expression of MSK1 and proliferating cell nuclear antigen (PCNA) by Western blot assay. Another 20 SD rats were grouped by the same method as above (n=10). In these rats, a negative control lentiviral LV3NC dilution was injected at a depth of approximately 0.8 mm at the spinal cord T10 level. The results of transfection at 1, 3, 5, 7, and 14 days after injection were observed under an inverted fluorescence microscope to determine the optimal transfection time of the virus. The other 30 SD rats were randomly divided into group A with only SCI, group B with a negative control lentiviral LV3NC injected after SCI, and group C with MSK1 small interfering RNA (siRNA) lentivirus injected after SCI, with 10 rats each group. The Basso, Beatlie, Bresnahan (BBB) score of hind limbs was measured at 1, 3, 5, 7, and 14 days after treatment; spinal cord tissue collected at the optimal time point for lentivirus transfection was detected the expression changes of MSK1 and PCNA by Western blot and the localization by immunofluorescence staining of MSK1 and PCNA proteins.ResultsWestern blot assay showed that there was no significant changes in the expression of MSK1 and PCNA at each time points in the sham-operation group. In the SCI group, the expression of MSK1 protein was gradually decreased from 8 hours after injury to the lowest level at 3 days after injury, and then gradually increased; the expression change of PCNA protein was opposite to MSK1. The expression of MSK1 in SCI group was significantly lower than that in the sham-operation group at 1, 2, 3, and 5 days after injury (P<0.05), and the expression of PCNA protein of SCI group was significantly higher than that of the sham-operation group at 8 hours and 1, 2, 3, 5, and 7 days after injury (P<0.05). The fluorescence expression of both the SCI group and the sham-operation group has be found and peaked at 7 days. There was a positive correlation between fluorescence intensity and time in 7 days after transfection. With the prolongation of postoperative time, the BBB scores of groups A, B, and C showed a gradually increasing trend. The BBB score of group C was significantly lower than those of groups A and B at 5, 7, and 14 days after treatment (P<0.05). After transfection for 7 days, Western blot results showed that the relative expression of MSK1 protein in group C was significantly lower than that in groups A and B (P<0.05); and the relative expression of PCNA protein was significantly higher than that in groups A and B (P<0.05). Immunofluorescence staining showed that MSK1 was expressed in the nuclei of the spinal cord and colocalized with green fluorescent protein, neuronal nuclei, and glial fibrillary acidic protein (GFAP). The relative expression area of MSK1 positive cells in group C was significantly higher than that in group B (P<0.05), and the relative expression areas of PCNA and GFAP positive cells were significantly lower than those in group B (P<0.05).ConclusionLentivirus-mediated MSK1 siRNA can effectively silence the expression of MSK1 in rat spinal cord tissue. MSK1 may play a critical role in the repair of SCI in rats by regulating the proliferation of glial cells.
ObjectiveTo investigate the influence of Ataxia-telangiectasia mutated (ATM) activation on cellular oxidative stress induced by high glucose in bovine retinal capillary endothelial cells(BRECs). Methods The BRECs were treated by different culture medium with various glucose concentrations (5 mmol/L glucose, 30 mmol/L glucose, 30 mmol/L glucose+10 μmol/L KU55933) as normal glucose group, high glucose group and treatment group respectively.After the cells incubated for 48 hours, the protein expression of ATM, P-ATM, Mitogen-Activated Protein Kinase P38(P38), P-P38, Extracellular signal-regulated kinases(ERKs), P-ERKs was detected by Western blot; cellular ROS level was detected by Reactive Oxygen Species Assay Kit; propidium iodide/Hoechst staining was used for analysis of apoptosis; the expression of vascular endothelial growth factor (VEGF) in the supernatant was determined by Enzyme-Linked Immunosorbent Assay (ELISA); the paracellular permeability between endothelium cells was detected by FITC-dextran. ResultsCompared with the protein level of P-ATM, P-P38 and P-ERKs in high glucose group increased. Especially, P-P38, P-ERKs expressed much more than in high glucose group. The secretion of VEGF in high glucose group was higher than that in the normal glucose group but less than that in treatment group. The same tendency existed in ROS assay, apoptosis assay and paracellular permeability measuring. ConclusionsHigh glucose induced altered activation of ATM which might play a protective role in cellular oxidative stress. Deficiency of ATM might lead to ROS explosion, cell apoptosis and dysfunction of endothelial barrier. The mechanism might be associated with P38, ERKs and VEGF.
Objective To explore effect of enhanced recovery after surgery (ERAS) on maintaining homeostasis of patient body and role of ERAS in alleviating stress response of physiological and psychological of patient and promoting recovery of patient from operative trauma as soon as possible. Method The related literatures published at home and abroad about the ERAS and its influence on the perioperative stress degree of patient were reviewed and analyzed. Results The ERAS was a new perioperative management mode established under the guidance of evidence-based medicine, whose core was to reduce the perioperative physiological and psychological stress level of the patient through a series of optimized measures, and to promote the postoperative rehabilitation. At the same time, the ERAS had been more and more widely accepted by the surgeons and patients because of its unique advantages, especially in shortening the hospital stay and reducing the operating costs. Conclusions Although concept of ERAS is not yet accepted by most clinicians, ERAS does provide a more optimal perioperative management strategy for patient, could obviously reduce perioperative stress level, improve patient outcome, accelerate postoperative recovery of patient, and provide benefit for patient underwent surgery.
Objective To study the changes of receptor activator of nuclear factor-κB ligand (RANKL, an osteoclastogenesis-promoting factor) and osteoprotegerin (OPG, the decoy receptor for RANKL), oxidative stress and bone turnover markers in obstructive sleep apnea-hypopnea syndrome (OSAHS), in order to understand the potential mechanisms underlying bone loss in OSAHS patients. Methods Ninety-eight male patients with OSAHS, confirmed by polysomnography (PSG) study, were enrolled. The patients were divided into mild-moderate groups and severe groups. Forty-two male subjects who were confirmed as not having OSAHS served as the controls. The subjects’ bone mineral density (BMD) and T-score were assessed in lumbar spine and femoral neck using dual-energy X-ray absorptiometry. Blood samples were collected from all subjects for measurement of RANKL, OPG, the bone formation marker bone-specific alkaline phosphatase (BAP), the bone resorption marker tartrate-resistant acid phosphatase-5b (TRAP-5b), total antioxidant capacity (TAOC). Twenty-eight severe OSAHS patients accepted continuous positive airway pressure (CPAP) treatment voluntarily. After 6 months, PSG was conducted, and serum RANKL, OPG, TAOC, TRAP-5b, BAP was measured after six months treatment. Results The BMD, T-score of the femoral neck and the lumbar spine were significantly lower in OSAHS patients as compared to the control group. The level of BAP was significantly decreased in the OSAHS group as compared to the control group, and there was no significant difference in TRAP-5b level between two groups. As compared with the control group, levels of OPG, TAOC and the OPG/RANKL ratio decreased significantly. None of these parameters (BMD, T-score, RANKL, OPG, TRAP-5b, BAP) showed significant difference between patients with mild-moderate and severe OSAHS group. Correlation analysis showed that the apnea hypopnea index and oxygen desaturation index were correlated with TAOC. BAP level was positively correlated with TAOC and lowest pulse oxygen saturation. The serum level of TAOC was lower in the OSAHS group after CPAP therapy, but the levels of RANKL, OPG, TRAP-5b, BAP were not different. As compared with the OSAHS group before CPAP therapy, the BMD of the femoral neck and the lumbar spine were not significant difference. Conclusions In patients with OSAHS, the oxidative stress response is enhanced, and imbalance of OPG/RANKL is shifted, which participates in the occurrence of osteoporosis. The oxidative stress injury of severe OSAHS patients was relieved after non-invasive ventilation treatment, but the effect of oxidative stress response on bone metabolism still needs further evaluation.
ObjectiveTo investigate the protective effect and mechanism of arbutin on LPS induced Acute lung injury in mice. Methods SPF BCLB/C mice were randomly divided into control group, model group,arbutin group, and arbutin+PI3K inhibitor group.arbutin group and arbutin+PI3K group were intervened with corresponding drugs respectively; Constructing an ALI model by intranasal instillation of LPS into mice; After modeling for 6 hours, the mice were killed. After staining the lung tissue slices, observe the pathological changes of the lung tissue and evaluate the lung injury score, and calculate the wet to dry weight ratio (W/D); ELISA method was used to determine the levels of TNF-a and IL-6 in serum and bronchoalveolar lavage fluid (BALF); Measure the ROS content, MDA level,and MPO activity in the lungs; Western blot method was used to detect the expression of PI3K/Akt/mTOR pathway related proteins and autophagy related proteins Beclin-1 and LC3II/I. ResultsCompared with the control group, the pathological changes in the lung tissue of model group mice worsened, and the W/D and lung injury scores increased, The levels of IL-6 and TNF-a in BALF and serum was increased, The ROS content, MDA expression and MPO activity in the lungs was increased,the expression of Beclin-1 and LC3II/I in the lungs was increased. The expression of PI3K/Akt/mTOR pathway related proteins in the lungs decreased (P<0.05). Compared with the model group, the pathological changes in the lung tissue of arbutin group mice were alleviated, with a decrease in W/D and lung injury score, The levels of IL-6 and TNF-a in BALF and serum decrease,ROS content, MDA expression and MPO activity in lung were decreased.The expression of PI3K/Akt/mTOR pathway related proteins in the lung was increased, The expression of Beclin-1 and LC3II/I decreased. However, the appeal performance was partially blocked in the arbutin+PI3K group after the administration of LY294002.ConclusionsArbutin regulates autophagy through PI3K/Akt/mTOR pathway to inhibit inflammatory response and oxidative stress in LPS-induced ALI mice, and plays a protective role in LPS-induced ALI.
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 To explore the variation about the application of fast-track surgery and laparoscopy in treatment for colorectal cancer in recent years． To investigate the probability of combining protocols of the two for treatment for colorectal cancer. Methods The clinical and basic literatures of related researches about colorectal treatment of laparoscopy and fast-track surgery were collected and reviewed. Results Compared with the traditional treatment modalities, both of fast-track surgery and laparoscopy used for the treatment of colorectal cancer have better clinical effects. Conclusions Fast-track surgery and laparoscopic techniques used for the treatment of colorectal cancer are feasible, but the combination of the two should be confirmed by further randomized controlled trials.
ObjectiveTo investigate the association between the stress-induced hyperglycemia ratio (SHR) and all-cause, cardiovascular, and diabetes-related mortality in patients with advanced cardiovascular-kidney-metabolic (CKM) syndrome, and to evaluate the value of SHR as an independent prognostic marker. MethodsThis retrospective cohort study used data from the 1999–2018 U.S. National Health and Nutrition Examination Survey (NHANES). A total of 2 135 patients with advanced CKM (stages 3 and 4) were included. Kaplan-Meier analysis and multivariable Cox regression models were applied to assess the relationship between SHR and mortality outcomes. Restricted cubic spline (RCS) analysis was employed to explore potential non-linear associations. Subgroup analyses were conducted to identify possible effect modifiers. ResultsOver a mean follow-up of 248 months, 674 all-cause, 198 cardiovascular, and 31 diabetes-related deaths occurred. Elevated SHR was significantly associated with diabetes-related mortality (HR=3.48, P<0.001) in a dose-response manner. SHR exhibited a U-shaped relationship with both all-cause and cardiovascular mortality (non-linearity P<0.001), indicating increased risk at both low and high SHR levels. Subgroup analyses revealed that sex, BMI, and hyperlipidemia significantly modified the association between SHR and diabetes-related death. ConclusionSHR is an independent predictor of mortality risk in patients with advanced CKM syndrome, particularly for diabetes-related death. These findings support the integration of SHR into risk stratification of high-risk CKM populations and provide a basis for metabolic stress-targeted interventions.