Objective Glucocorticoid is the main cause of non-traumatic avascular necrosis of femoral head. To explore the changes of reactive oxygen species (ROS) in the bone microvascular endothel ial cells treated with glucocorticoid so as to investigate the pathogenesis of steroid-induced avascular necrosis of femoral head. Methods The cancellous bone of femoral head was harvested from voluntary donators undergoing total hip arthroplasty, and then the bone microvascular endothel ial cells were isolated by enzyme digestion. The cells at passage 3 were cocultured with different concentrations of hydrocortisone (0, 0.03, 0.10, 0.30, and 1.00 mg/mL) for 24 hours. MTT assay was used for the inhibitory rate of cell prol iferation, flow cytometry for apoptosis rate, and fluorescence probe for the production of ROS and xanthine oxidase (XOD). Results At 2-3 days primary culture, the cells were spindle and arranged l ike cobbles and they reached confluence after 1 week. The inhibitory rates of cell prol iferation in 0.03, 0.10, 0.30, and 1.00 mg/mL groups were 20.22% ± 2.97%, 22.94% ± 4.52%, 43.98% ± 3.35%, and 78.29% ± 3.85%, respectively; and 2 high-concentration groups (0.30 and 1.00 mg/mL groups) were significantly higher (P lt; 0.05) than 2 low-concentration groups (0.03 and 0.10 mg/mL groups). The apoptosis rates in 0, 0.03, 0.10, 0.30, and 1.00 mg/mL groups were 0.10% ± 0.01%, 0.23% ± 0.02%, 1.83% ± 0.04%, 6.34% ± 0.11%, and 15.33% ± 0.53%, respectively; 2 high-concentration groups (0.30 and 1.00 mg/mL groups) were significantly higher (P lt; 0.05) than 0 mg/mL group. In 0, 0.30, and 1.00 mg/ mL groups, the ROS levels were 57.35 ± 7.11, 120.47 ± 15.68, and 166.15 ± 11.57, respectively, and the XOD levels were 0.017 9 ± 0.000 9, 0.028 3 ± 0.001 7, and 0.067 7 ± 0.004 1, respectively; there were significant differences in the levels of ROS and XOD among 3 groups (P lt; 0.05). Conclusion Increasing of ROS production in bone microvascular endothel ial cells can be induced by high concentration glucocorticoid, and it can result in cell injury
ObjectiveTo investigate the effects of interferon gene stimulating protein (STING) inhibitor (C176) on human retinal microvascular endothelial cells (hRMEC) under oxidative stress. MethodsAn animal experimental study. In vivo experiment: 48 healthy male C57BL/6J mice were randomly divided into wild type mice group (WT group) and diabetes (DM) group, with 24 mice in each group. DM mice were induced by streptozotocin to establish DM model. After successful modeling, DM group was divided into DM+dimethyl sulfoxide (DMSO) group and DM+C176 group, with 12 mice in each group. The mice in the DM+DMSO group were intraperitoneally injected with DMSO at the dose of 50 mg/kg. Mice in DM+C176 group were intraperitoneally injected with STING inhibitor C176 750 nmol at the dose of 50 mg/kg. Four weeks after modeling, immunohistochemical staining, Western blot and real-time fluorescence quantitative polymerase chain reaction were used to detect the expression of STING in the retina of WT and DM mice. The leukocyte adhesion test was used to detect the number of leukocytes adhering to hRMEC in mice with WT, DM+DMSO and DM+C176 groups. In vitro experiment: hRMEC was randomly divided into conventional culture cell group (N group), dimethyl sulfoxide (DMSO) group (with DMSO intervention) and C176 group (with C176 intervention). The cells were induced by 150 μg/ml glycation end products for each group. In vitro leukocyte adhesion test combined with 4', 6-diamino-2-phenylindole staining was used to detect the number of leukocytes adhering to hRMEC. The adherent leukocytes were quantitatively analyzed by flow cytometry; H2DCFDA/reactive oxygen species (ROS) fluorescence probe was used to detect ROS expression in cells; Seahorse XFe96 cell energy metabolism analyzer was used to measure the level of intracellular glycolysis. t-test was used to compare the two groups; single factor analysis of variance was used to compare the three groups. ResultsIn vivo experiment: compared with WT group, the expression level of STING (t=73.248) and the relative expression amount of mRNA (t=67.385) in the retina of DM group mice increased significantly (P＜0.05). Compared with WT group, the number of leukocytes adhering to the retinal vessels of mice in DM+DMSO group was significantly increased, while that in DM+C176 group was significantly decreased (F=84.352, P＜0.01). In vitro: compared with N group and DMSO group, the number of leukocyte adhesion on hRMEC in C176 group decreased significantly (F=35.251, P＜0.01). Compared with N group, the number of leukocytes adhering to hRMEC in DMSO group and C176 group decreased significantly (F=26.374, P＜0.01). The ROS level in hRMEC in C176 group was significantly lower than that in N group and C176 group (F=41.362, P＜0.01). Compared with N group and DMSO group, the glycolysis level of hRMEC in C176 group was significantly reduced, with a statistically significant difference (F=68.741, P＜0.01). ConclusionInhibiting the expression of STING in retinal vascular endothelial cells can improve the progress of DM by inhibiting leukocyte adhesion, ROS production and glycolysis level.
ObjectiveTo observe the effect of interleukin-8 (IL-8) on the adhesion and migration of retinal vascular endothelial cells (RCEC). MethodsA cell experiment. Human RCEC (hRCEC) was divided into normal control group (N group), advanced glycation end product (AGE) treatment group (AGE group), and AGE-induced combined IL-8 antagonist SB225002 treatment group (AGE+SB group). The effect of AGE on IL-8 expression in hRCEC was observed by Western blot. The effect of SB225002 on hRCEC migration was observed by cell scratch assay. The effects of SB225002 on leukocyte adhesion and reactive oxygen species (ROS) on hRCEC were detected by flow cytometry. Student-t test was performed between the two groups. One-way analysis of variance was performed among the three groups. ResultsCompared with group N, the expression level of IL-8 in cells of AGE group was significantly increased, with statistical significance (t=25.661, P＜0.001). Compared with N group and AGE+SB group, cell mobility in AGE group was significantly increased (F=29.776), leukocyte adhesion number was significantly increased (F=38.159, 38.556), ROS expression level was significantly increased (F=22.336), and the differences were statistically significant (P＜0.05). ConclusionIL-8 antagonist SB225002 may down-regulate hRCEC adhesion and migration by inhibiting ROS expression.
Lung cancer is the leading cause of cancer-related deaths worldwide. Despite the development and use of several targeting drugs for lung cancer therapy, the five-year survival rate has remained as low as 15% for the past three decades. Cisplatin-based chemotherapy is considered the first-line therapeutic strategy for lung cancer. However, developments of chemoresistance is a major obstacle for the successful treatment. Therefore, the development of novel therapy against cisplatin-resistance lung cancer is imperative. Photodynamic therapy (PDT), which is a non-invasive combinatorial therapeutic modality using light, photosensitizer (PS) and oxygen, may provide an unprecedented tool to develop more effective treatments. To provide experimental basis for its application in cisplatin-resistance lung cancer, we will discuss the biological effects of MPPa-photodynamic therapy in human cisplatin-resistance lung cancer cells in this article. Human cisplatin-resistance lung cancer cells A549/DDP were co-cultured with MPPa (0, 1, 2, 4, 8, 16 μmol/L) and exposed to light (0, 0.6, 1.2, 2.4, 3.6, 4.8 J/cm2), and cell viability was determined with CCK-8 assay. Flow cytometry was used to detect apoptosis, DCFH-DA staining was employed to observe reactive oxygen species (ROS), and Western blot was used to detect the expressions of B-cell lymphoma-2 (Bcl-2) protein and Bcl-2 associated X protein (Bax). The proliferation of A549/DDP cells was suppressed by PDT. The apop-totic rate in the PDT group was significantly higher than that in the control, MPPa or light group (P < 0.05). The level of ROS was increased. The expression of Bax was increased, and that of Bcl-2 was decreased. MPPa-photodynamic therapy can significantly suppress cell viability, and induce apoptosis in human cisplatin-resistance lung cancer cells.
Diabetic retinopathy (DR) constitutes a major retinal vascular disorder leading to blindness in adults. Current therapeutic approaches for DR exhibit certain degrees of efficacy but are constrained by a spectrum of limitations. Hence, there is a pressing need to deeply investigate the underlying pathogenesis of DR and explore novel therapeutic targets. Ferroptosis, a distinctive form of programmed cell death, has emerged as a pertinent phenomenon in recent years. Notably, ferroptosis has been implicated in the progression of DR through mechanisms involving the induction of retinal oxidative stress, provocation of anomalous retinal vascular alterations, exacerbation of retinal neural damage, and elicitation of immune dysregulation. Thus, elucidating the mechanistic role of ferroptosis in DR holds the potential to establish a robust foundational rationale. This could potentially facilitate the clinical translation of ferroptosis inhibitors as promising agents for the prevention and treatment of DR, thereby forging novel avenues in the landscape of DR management.
Epilepsy is a heterogeneous disease with a very complex etiological mechanism, characterized by recurrent and unpredictable abnormal neuronal discharge. Epilepsy patients mainly rely on oral antiseizure medication (ASMs) the for treatment and control of disease progression. However, about 30% patients are resistance to ASMs, leading to the inability to alleviate and cure seizures, which gradually evolve into refractory epilepsy. The most common type of intractable epilepsy is temporal lobe epilepsy. Therefore, in-depth exploration of the causes and molecular mechanisms of seizures is the key to find new methods for treating refractory epilepsy. Mitochondria are important organelles within cells, providing abundant energy to neurons and continuously driving their activity. Neurons rely on mitochondria for complex neurotransmitter transmission, synaptic plasticity processes, and the establishment of membrane excitability. The process by which the autophagy system degrades and metabolizes damaged mitochondria through lysosomes is called mitophagy. Mitophagy is a specific autophagic pathway that maintains cellular structure and function. Mitochondrial dysfunction can produce harmful reactive oxygen species, damage cell proteins and DNA, or trigger programmed cell death. Mitophagy helps maintain mitochondrial quality control and quantity regulation in various cell types, and is closely related to the occurrence and development of epilepsy. The imbalance of mitophagy regulation is one of the causes of abnormal neuronal discharge and epileptic seizures. Understanding its related mechanisms is crucial for the treatment and control of the progression of epilepsy in patients.
ObjectiveTo investigate the effects of leptin on the oxidative damage in human retinal pigment epithelial (RPE) cells. MethodsHuman RPE cells (ARPE-19) were cultured in vitro, and randomly divided into control group and insulin resistance group. RPE cells were treated with 0, 10, 100 ng/mL leptin for 24, 48, 72 hours respectively. Then the levels of reactive oxygen species (ROS) expression in RPE cells were detected by 2', 7'-dichlorofluorescin-diacetate (DCFH-DA), and the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression in RPE cells were observed by immunocytochemistry (ICC), and the levels of human 8-oxoguanine DNA glycosylase l (hOGG1) expression in lysate were measured by Western blot. ResultsAfter 24, 48, 72 hours, the level of ROS (Control group:F=37.136, 37.178, 49.634; P < 0.05. Insulin resistance group:F=9.822, 28.881, 71.150;P < 0.05), 8-OHdG (Control group:F=88.643, 390.920, 1039.276;P < 0.05.Insulin resistance group:F=273.311, 299.155, 82.237;P < 0.05) and hOGGl (Control group:F=470.062, 1073.113, 295.456;P < 0.05. Insulin resistance group:F=240.032, 592.389, 527.760;P < 0.05) expression increased significantly with the increase of leptin concentration in control group and insulin resistance group. Under the same leptin concentration, the level of 8-OHdG has a trend that it was higher in the insulin resistance group than the control group. After 24 hours, the difference of hOGGl expression between control group and insulin resistance group was not significant (F=23.392, P > 0.05). After 72 hours, the level of hOGGl expression was significantly higher in the insulin resistance group than the control group (F=129.394, P < 0.05). The level of hOGGl expression was significantly higher at 48 hours than that at 24 hours and 72 hours (P < 0.05). ConclusionLeptin could induce the oxidative damage of RPE cells in normal and insulin resistance status. With the increase of leptin concentration and time extended, the degree of oxidative damage and its repair were both increased. The degree of oxidative repair increased with the increase of leptin concentration, but decreased with time extended.
目的 观察黄芪三七合剂（Aamp;R）对肾缺血再灌注损伤（IRI）大鼠血液活性氧（ROS）变化的影响，探讨其抗IRI损伤的机制。 方法 雄性Sprague-Dawley（SD）大鼠30只，随机分为正常组（n=5）、假手术组（SG）（n=5）和IRI 24 h组（n=10），Aamp;R组（n=10）。造模：采用微血管夹夹闭双侧肾蒂，22 min后松开动脉夹，用5/0尼龙缝合线缝合腹部。再灌注24 h后将小鼠行麻醉处死。Aamp;R组给予Aamp;R（3 mL/d），假手术组及IRI 24 h组给予同等体积的生理盐水。采用全自动生化分析仪检测各组大鼠的肾功能，苏木精-伊红染色了解肾脏病理损害，流式细胞仪检测红细胞ROS。 结果 IRI 24 h组和Aamp;R组肾小管出现不同程度的管腔扩张、变性与坏死，间质炎性细胞浸润、充血水肿等变化。IRI后24 h时，IRI 24 h组、Aamp;R组血清尿素氮（BUN）和肌酐（Cr）均高于假手术组、正常组，差异有统计学意义（P＜0.05）；Aamp;R组ROS荧光强度阳性率显著低于IRI 24 h组，差异有统计学意义（P＜0.05）。Aamp;R组肾小管损伤评分明显低IRI 24 h组（P＜0.05）。相关性分析发现，红细胞ROS荧光强度阳性率与肾小管损伤评分、肌酐、尿素氮水平成正相关（r=0.917，P＜0.01；r=0.897，P＜0.01；r=0.896，P＜0.01）。 结论 Aamp;R对肾脏缺血再灌注损伤具有明显的保护作用，其机制可能为抑制血液中ROS的活性，从而抑制氧化应激对肾脏的损伤。
Objective To observe and preliminarily explore the effect of mogroside on oxidative stress of retinal pigment epitheliaum (RPE) cells induced by hydrogen peroxide (H2O2) and its possible mechanism. MethodsA experimental study. The RPE cells were divided into control group, H2O2 group, silent information regulator of transcription 1 (SIRT1) inhibitor EX527 group (EX527 group), mogroside group, mogroside+EX527 group. Methyl thiazolete trazolium method was used to detect cell survival rate. Flow cytometry was used to detect cell apoptosis rate. 2',7'-dichlorodihydrofluorescein diacetate fluorescent probe method, xanthine method and enzyme-linked immunosorbent assay method were used to detect the level of reactive oxygen species (ROS), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in cells respectively. Real-time quantitative polymerase chain reaction and Western blot were used to detect relative expressions of SIRT1, nuclear factor erythroid-2-related actor 2 (Nrf2), heme oxygenase-1 (HO-1) mRNA and protein in cells. One-way ANOVA was used for comparison among groups. The pairwise comparison between groups was tested by the least significant difference t test. Results Compared with the control group, the H2O2 group cell survival rate decreased, the apoptosis rate increased, the ROS level in the cells increased, the SOD activity decreased, the MDA content increased, and the relative expression of SIRT1, Nrf2, HO-1 mRNA and protein decreased (P＜0.05). Compared with H2O2 group, the cell survival rate decreased, apoptosis rate increased, the cell ROS level increased, SOD activity decreased, MDA content increased, SIRT1, Nrf2, HO-1 mRNA and protein expression decreased in EX527 group (P＜0.05); the cell survival rate increased, apoptosis rate decreased, ROS level decreased, SOD activity increased, MDA content decreased, and the relative expression of SIRT1, Nrf2, HO-1 mRNA and protein increased in mogroside group (P＜0.05). Compared with the mogrosides group, the cell survival rate decreased, the apoptosis rate increased, the level of ROS increased, SOD activity decreased, MDA content increased, SIRT1, Nrf2, HO-1 mRNA and protein decreased in mogrosides+EX527 group (P＜0.05). ConclusionsMogrosides can alleviate the oxidative stress response of visual RPE cells induced by H2O2, promote cell proliferation, and reduce cell apoptosis. Mogrosides may exert antioxidant effects by activating the SIRT1/Nrf2 signaling pathway.