Enhancement of porcine in vitro embryonic development through luteolin‑mediated activation of the Nrf2/Keap1 signaling pathway

Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Luteolin(Lut)has been documented for its protective effects against oxidative stress in various studies.However,its specific role in embryonic development remains unexplored.This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.Results After undergoing parthenogenetic activation(PA)or in vitro fertilization,embryos supplemented with 0.5μmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates,with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control.Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control.Moreover,Lut supplementation significantly augmented mitochondrial content and membrane potential.Intriguingly,activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut,leading to the upregulation of antioxidant-related gene transcription levels.To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development,we cultured PA embryos in a medium supplemented with brusatol,with or without the inclusion of Lut.The positive effects of Lut on developmental competence were negated by brusatol treatment.Conclusions Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence,and offers insight into the mechanisms regulating early embryonic development.

基于Nrf2-Keap1通路探讨小槐花不同提取物对CCl_(4)所致肝损伤的保护机制

目的探讨小槐花不同提取物对CCl_(4)所致肝损伤的保护机制。方法采用0.5%CCl_(4)花生油溶液构建小鼠肝损伤模型。苏木精-伊红(hematoxylin-eosin staining,HE)染色观察肝脏病变;试剂盒检测丙氨酸转氨酶(alanine amin-otransferase,ALT)、天冬氨酸转氨酶(aspartate aminotransferase,AST)、丙二醛(malondialdehyde,MDA)、还原型谷胱甘肽(glutathione,GSH)、超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、活性氧(reactive oxygen species,ROS)水平;Western blot检测肝脏组织中核因子促红细胞生成素2相关因子2(nuclear factor erythropoietin-2-related factor 2,Nrf2)、Kelch样环氧氯丙烷相关蛋白1(Kelch like epichlorohydrin related protein-1,Keap1)、血红素加氧酶1(heme oxyge-nase 1,HO-1)蛋白的相对表达量。结果与模型组相比,小槐花水提物与醇提物中、高剂量组能显著降低肝重指数以及血清中ALT、AST水平(P<0.05,P<0.01)。小槐花水提物与醇提物中、高剂量组小鼠肝组织匀浆MDA、TNF-α、IL-6、ROS含量显著降低(P<0.05,P<0.01),SOD、CAT酶活性显著升高(P<0.05,P<0.01),GSH含量显著升高(P<0.05,P<0.01)。其中小槐花水提物作用效果优于醇提物。小槐花水提物高剂量组显著上调Nrf2、HO-1,下调Keap1蛋白的表达(P<0.05)。结论小槐花不同提取物对CCl_(4)肝损伤小鼠具有保护作用,水提物保肝效果优于醇提物,作用机制与激活Nrf2-Keap1通路,减轻氧化应激反应有关。

干预自噬调控p62-Keap1/Nrf2-GPX4通路对结直肠癌细胞铁死亡及奥沙利铂耐药的影响

目的探讨干预自噬对结直肠癌细胞铁死亡和奥沙利铂(OXA)耐药的影响及分子机制。方法培养人结直肠癌HCT-8、COLO205、HCT-116、SW620和SW480细胞系,筛选LC3表达适中的HCT-116细胞,检测其与OXA耐药HCT-116/OXA细胞株LC3、p62、Keap1、Nrf2、GPX4蛋白及Fe^(2+)、GSH、MDA的表达差异,并应用自噬和铁死亡干预剂并联合OXA干预HCT-116/OXA细胞株,检测LC3、p62、Keap1、Nrf2、GPX4蛋白及Fe^(2+)、GSH、MDA的表达,CCK-8实验检测各组细胞的增殖活性及对OXA的敏感性,平板克隆、Transwell实验检测各组细胞的生长情况和侵袭能力。结果5组细胞中HCT-116细胞的LC3、p62和GPX4均呈中等表达量;与HCT-116细胞相比,HCT-116/OXA细胞对OXA敏感性较低,且p62、Nrf2、GPX4蛋白呈高表达,LC3和Keap1呈低表达,Fe^(2+)、GSH、MDA表达水平均增高(P<0.05);自噬激活剂Rapa组及Rapa+Fer-1组较Fer-1组和对照组的LC3、Keap1蛋白及Fe^(2+)、MDA水平均升高,而p62、Nrf2、GPX4蛋白及GSH水平呈低表达,且Rapa+Fer-1组GPX4蛋白、GSH的水平低于Rapa组(P<0.05)。在自噬抑制剂组中,CQ组及CQ+Erastin组与对照组和Erastin组相比其LC3、p62、Nrf2、GPX4蛋白及GSH水平均呈高表达,Keap1蛋白、Fe^(2+)、MDA均呈低表达,而Erastin组GPX4蛋白、GSH表达低于其余三组,Fe^(2+)、MDA含量高于其余三组(P<0.05)。自噬激活剂联合应用OXA,Rapa干预组与其余三组相比,其对OXA的化学敏感性高、迁移细胞数量少、细胞增殖活性低;Rapa+Fer-1组对OXA的敏感性低于Rapa组,但高于Fer-1组和对照组(P<0.05)。而Fer-1组与对照组相比,差异无统计学意义(P>0.05)。自噬抑制剂联合应用OXA,Erastin、CQ+Erastin和CQ三组与对照组相比其细胞活性、迁移能力和克隆形成能力均降低,其中Erastin组最低,而CQ+Erastin组高于Erastin组、低于CQ组(P<0.05)。结论在结直肠癌中,自噬参与了铁死亡的调节,干预自噬可通过p62-Keap1/Nrf2-GPX4通路调控结直肠癌细胞发生铁死亡,从而逆转OXA耐药。

Reduction of the oxidative damage to H_(2)O_(2)-induced HepG2 cells via the Nrf2 signalling pathway by plant flavonoids Quercetin and Hyperoside

Hyperoside and quercetin are similar in molecular structures.In this study,the antioxidant regulatory targets of hyperoside and quercetin are mainly in the nuclear factor(erythroid-2-derived)-related factor 2(Nrf2)pathway predicted by network pharmacology.And the antioxidant effect and mechanism of hyperoside and quercetin were measured and compared in H_(2)O_(2)-induced Hep G2 cells and Caenorhabditis elegans.The findings indicated that quercetin was more effective than hyperoside in reducing oxidative damage,which was proved by improved cell viability,decreased reactive oxygen species(ROS)production,decreased cellular apoptosis,and alleviated mitochondrial damage.In addition,quercetin was more efficient than hyperoside in enhancing the expression of Nrf2-associated m RNAs,increasing the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and catalase(CAT),and reducing the cellular malondialdehyde(MDA)content.Quercetin was superior to hyperoside in prolonging the lifespan of worms,decreasing the accumulation of lipofuscin,inhibiting ROS production,and increasing the proportion of skn-1 in the nucleus.With the Nrf2 inhibitor ML385,we verified that quercetin and hyperoside primarily protected the cells against oxidative damage via the Nrf2 signalling pathway.Furthermore,molecular docking and dynamics simulations demonstrated that the quercetin-Kelch-like ECH-associated protein 1(Keap1)complex was more stable than the hyperoside-Keap1 complex.The stable structure of the complex might hinder the binding of Nrf2 and Keap1 to release Nrf2 and facilitate its entry into the nucleus to play an antioxidant role.Overall,quercetin had a better antioxidant than hyperoside.

Hesperidin ameliorates H_(2)O_(2)-induced bovine mammary epithelial cell oxidative stress via the Nrf2 signaling pathway

Background Hesperidin is a citrus flavonoid with anti-inflammatory and antioxidant potential. However, its protective effects on bovine mammary epithelial cells(b MECs) exposed to oxidative stress have not been elucidated.Results In this study, we investigated the effects of hesperidin on H_(2)O_(2)-induced oxidative stress in b MECs and the underlying molecular mechanism. We found that hesperidin attenuated H_(2)O_(2)-induced cell damage by reducing reactive oxygen species(ROS) and malondialdehyde(MDA) levels, increasing catalase(CAT) activity, and improving cell proliferation and mitochondrial membrane potential. Moreover, hesperidin activated the Keap1/Nrf2/ARE signaling pathway by inducing the nuclear translocation of Nrf2 and the expression of its downstream genes NQO1 and HO-1, which are antioxidant enzymes involved in ROS scavenging and cellular redox balance. The protective effects of hesperidin were blocked by the Nrf2 inhibitor ML385, indicating that they were Nrf2 dependent.Conclusions Our results suggest that hesperidin could protect b MECs from oxidative stress injury by activating the Nrf2 signaling pathway, suggesting that hesperidin as a natural antioxidant has positive potential as a feed additive or plant drug to promote the health benefits of bovine mammary.

X-Paste improves wound healing in diabetes via NF-E2-related factor/HO-1 signaling pathway

BACKGROUND Diabetic foot ulcers(DFU),as severe complications of diabetes mellitus(DM),significantly compromise patient health and carry risks of amputation and mortality.AIM To offer new insights into the occurrence and development of DFU,focusing on the therapeutic mechanisms of X-Paste(XP)of wound healing in diabetic mice.METHODS Employing traditional Chinese medicine ointment preparation methods,XP combines various medicinal ingredients.High-performance liquid chromatography(HPLC)identified XP’s main components.Using streptozotocin(STZ)-induced diabetic,we aimed to investigate whether XP participated in the process of diabetic wound healing.RNA-sequencing analyzed gene expression differences between XP-treated and control groups.Molecular docking clarified XP’s treatment mechanisms for diabetic wound healing.Human umbilical vein endothelial cells(HUVECs)were used to investigate the effects of Andrographolide(Andro)on cell viability,reactive oxygen species generation,apoptosis,proliferation,and metastasis in vitro following exposure to high glucose(HG),while NF-E2-related factor-2(Nrf2)knockdown elucidated Andro’s molecular mechanisms.RESULTS XP notably enhanced wound healing in mice,expediting the healing process.RNA-sequencing revealed Nrf2 upregulation in DM tissues following XP treatment.HPLC identified 21 primary XP components,with Andro exhibiting strong Nrf2 binding.Andro mitigated HG-induced HUVECs proliferation,metastasis,angiogenic injury,and inflammation inhibition.Andro alleviates HG-induced HUVECs damage through Nrf2/HO-1 pathway activation,with Nrf2 knockdown reducing Andro’s proliferative and endothelial protective effects.CONCLUSION XP significantly promotes wound healing in STZ-induced diabetic models.As XP’s key component,Andro activates the Nrf2/HO-1 signaling pathway,enhancing cell proliferation,tubule formation,and inflammation reduction.

Exploring the mechanism of electroacupuncture at different acupoints on acute colitis rats based on JAK2/STAT3/SOCS1 signaling pathway

Objective:To investigate the mechanism of JAK2/STAT3/SOCS1 signaling pathway in electroacupuncture of different acupoints on acute colitis rats.Methods:36 SPF SD rats were randomly divided into 6 groups,with 6 rats in each group.The rat model of acute colitis was prepared by enema with glacial acetic acid solution.After the model was established,electroacupuncture was given to each acupoint group,with density wave,frequency 2Hz-50 Hz,intensity 2 mA,muscle tremor as the degree 20 min/time,1 time/day,for 3 consecutive days.Observe the general condition of rats;the pathological changes of colonic mucosa in rats were observed by HE method.The contents of serum interleukin-4(IL-4)and interleukin-8(IL-8)were detected by ELISA.Western blot and RT-PCR were used to detect the expression of JAK2,STAT3,SOCS1 protein and mRNA in rat colon tissue.Results:In contrast to the normal group,the overall condition of the model group was worse,the colonic mucosa was severely damaged,even necrotic,and the ulcer surface was obvious.The content of IL-4 in serum was obviously reduced,and the content of IL-8 was obviously go up(P<0.01).The protein content of JAK2,STAT3 and the expression of JAK2,STAT3 mRNA in colon tissue of rats were obviously go up,while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously reduced(P<0.01).In contrast to the model group,the general condition of rats in each acupoint group was significantly improved,the damage and necrosis of colonic mucosa and ulcer surface were obviously alleviated,the content of IL-4 in serum was obviously go up,and the content of IL-8 was significantly decreased(P<0.01).The protein content of JAK2,STAT3 and the expression of JAK2,STAT3 mRNA in colon tissue of rats were obviously reduced,while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously go up(P<0.05,P<0.01).Comparison of different acupoint groups,the colonic mucosal injury in the Zusanli group was significantly reduced,the content of serum IL-4 was significantly increased,and the content of IL-8 was significantly decreased(P<0.05,P<0.01).The protein content and mRNA expression of JAK2 and STAT3 in colon tissue were significantly down-regulated,while the protein content and mRNA expression of SOCS1 were significantly go up(P<0.05,P<0.01).Conclusion:Electroacupuncture at each acupoint can improve the damage of colonic mucosa and reduce the inflammatory response.The therapeutic effect of Zusanli(ST36)is better than that of Tianshu(ST25),Dachangshu(BL25)and Shangjuxu(ST37).The mechanism may be related to the regulation of JAK2/STAT3/SOCS1 signaling pathway related proteins and inflammatory cytokines IL-4 and IL-8.

丹参多酚酸盐调控Nrf2/HO-1信号通路对膜性肾病大鼠氧化应激的影响

[目的]基于血清核转录因子2(Nrf2)/血红蛋白氧合酶-1(HO-1)信号通路探讨丹参多酚酸盐对膜性肾病(MN)大鼠氧化应激的影响。[方法] 80只SD雄性大鼠随机选取20只为正常对照组,其余60只大鼠均采用尾静脉注射阳离子化牛血清白蛋白(C-BSA)构建MN模型,MN模型大鼠复制成功后随机分为模型组,盐酸贝那普利组(10 mg/kg),丹参多酚酸盐分为低、中、高剂量组(16.7、33.3、66.7 mg/kg)。药物灌胃连续4周,1次/d,正常组和模型组予以相等体积的生理盐水灌胃。治疗后检测大鼠24 h尿蛋白定量(24 h UTP)。给药结束后,经大鼠腹主动脉取血检测血尿素氮(BUN),血清肌酐(Scr),三酰甘油(TG),总胆固醇(TC),总蛋白(TP),白蛋白(ALB)水平;过碘酸-六胺银(PASM)染色观察大鼠肾组织病理形态;免疫荧光检测肾组织免疫球蛋白G(IgG)、补体C3沉积情况;采用酶联免疫吸附测定法(ELISA)检测大鼠血清中超氧化物歧化酶(SOD)、丙二醛(MDA)表达情况;蛋白免疫印迹法(Western Blot)和实时荧光定量聚合酶链式反应(Real-time PCR)观察肾组织Nrf2、HO-1蛋白及Nrf2、HO-1 mRNA表达。[结果]与正常组相比,模型组大鼠肾小球出现体积增大、基底膜增厚、“钉突”形成,沿毛细血管襻有补体C3、IgG弥漫性沉积,24 h UTP、血清TG、TC水平显著升高(P<0.01),TP、ALB水平显著降低(P<0.01),BUN、SCr差异无统计学意义;血清中SOD表达显著降低(P<0.01),MDA表达显著升高(P<0.01);肾组织Nrf2、HO-1 mRNA及蛋白表达显著降低(P<0.01)。与模型组相比,各治疗组大鼠24 h UTP、血清TG、TC水平显著降低(P<0.05或P<0.01),TP、ALB水平显著升高(P<0.01),但丹参多酚酸盐低剂量组改善不明显;各治疗组肾脏病理损害明显改善;SOD表达水平显著升高(P<0.01),MDA表达水平明显降低(P<0.01);Nrf2、HO-1mRNA及蛋白表达显著升高(P<0.05或P<0.01)。[结论]丹参多酚酸盐可能通过调控Nrf2/HO-1信号通路,缓解肾组织氧化应激,进而保护肾脏及延缓疾病进展。

表没食子儿茶素没食子酸酯靶向调控Nrf2⁃Keap1信号通路与脑梗死神经保护作用的关系

目的分析表没食子儿茶素没食子酸酯(EGCG)对高血糖诱导的出血性转化(HT)的预防作用,并进一步探索了其潜在机制是否与涉及核因子E2相关因子2/Kelch样环氧氯丙烷相关蛋白1(Nrf2/Keap1)通路。方法将雄性SD大鼠随机分为假手术组(Sham,n=20)、模型组(n=27)、高血糖模型组(HG,n=43)、EGCG组(n=43)。模型组大鼠仅建立电凝脑缺血模型,HG组和EGCG组采用急性高血糖联合电凝脑缺血模型建立HT模型。此外,EGCG组在脑缺血前灌胃EGCG 5 d,剂量为50 mg/(kg·d)。通过网络药理学用于预测EGCG在HT发生中的潜在靶点和通路,进一步研究证实了相关靶点。结果与模型组相比,HG组大鼠死亡率显著升高[21.2%(6/27)vs.51.2%(22/43),P<0.05]。EGCG组大鼠死亡率较HG组显著降低[30.20%(13/43)vs.51.2%(22/43),P<0.05]。其次,EGCG组mNSS、Longa评分和梗死体积显著低于HG组(P<0.05)。HG组的HT发生率高于模型组(59.3%vs.90.7%)。EGCG显著降低高血糖诱导的HT发病率至69.8%。与HG组相比,EGCG使血红蛋白含量分别从(53.42±5.11)mg/dL降低到(37.04±2.39)mg/dL(P<0.05)。网络药理学显示Nrf2-Keap1介导的神经炎症可能与高血糖诱导的HT相关。HG组Nrf2和Keap1的表达显著降低和TLR4的表达、NF-κB的磷酸化显著增加,但EGCG逆转了这一过程。结论EGCG预处理防止了HT的发生,这可能与激活Nrf2-Keap1信号通路介导的神经保护作用有关。

亚慢性铝暴露致大鼠认知障碍的Sirt1-Keap1/Nrf2信号通路机制

目的:观察亚慢性铝暴露对大鼠海马沉默信息调节因子1(silent information regulator 1,Sirt1)、Kelch样环氧氯丙烷相关蛋白(Kelch-like ECH-associated protein-1,Keap1)、核因子E2相关因子2(nuclear factor E2-related factor2,Nrf2)和微小RNA-128-3p(miR-128-3p)表达水平的影响,探讨miR-128-3p和Sirt1-Keap1/Nrf2信号通路在铝致大鼠认知障碍中的机制。方法:选取32只6周龄SPF级健康雄性SD大鼠,体重(190±20)g,按体质量随机分为4组:对照组、低剂量(10μmol/kg)组、中剂量(20μmol/kg)组和高剂量(40μmol/kg)组,每组8只。腹腔注射麦芽酚铝建立大鼠染毒模型。染毒结束后,Morris水迷宫实验来检验大鼠的学习记忆能力,Western blot检测大鼠海马组织中Sirt1、Keap1和Nrf2蛋白的表达,RT-qPCR检测海马组织miR-128-3p的表达,冰冻切片荧光染色检测大脑皮层活性氧(reactive oxygen species,ROS)水平。结果:(1)在定位巡航实验中,第3、4和5天铝暴露组大鼠的逃避潜伏期均显著高于对照组(P<0.05)。第6天,高剂量组与对照组和低剂量组相比穿越平台和平台象限的次数均减少(P<0.01)。(2)各组大鼠海马组织中Sirt1和Nrf2的相对表达水平随着麦芽酚铝暴露剂量的增加逐渐降低;Keap1的相对表达水平随着麦芽酚铝暴露剂量的增加逐渐升高,且高剂量组中miR-128-3p相对表达水平显著高于对照组(P<0.05)。(3)随着染毒剂量的增加,大鼠海马中谷胱甘肽过氧化物酶的含量逐渐减少,ROS水平逐渐升高。结论:亚慢性铝暴露可激活大鼠海马中miR-128-3p的表达,抑制Sirt1-Keap1/Nrf2通路,使Sirt1-Keap1/Nrf2通路不能被激活发挥抗氧化能力,大鼠的抗氧化系统失衡,导致大鼠神经细胞氧化损伤,表现为大鼠的认知功能降低。

Water Extract of Rice False Smut Balls Activates Nrf2/HO-1 and Apoptosis Pathways,Causing Liver Injury

Ustiloxins are vital cyclopeptide mycotoxins originally isolated from rice false smut balls that form in rice spikelets infected by the fungal pathogen Ustilaginoidea virens.The toxicity of the water extract of rice false smut balls(RBWE) remains to be investigated.Studies have shown that RBWE may be toxic to animals,but toxicological evidence is still lacking.In this study,we found that the IC50 values of RBWE to BNL CL.2 cells at 24 and 48 h were 40.02 and 30.11 μg/m L,respectively,with positive correlations with dose toxicity and time toxicity.After treatment with RBWE,the number of BNL CL.2 cells decreased significantly,and the morphology of BNL CL.2 cells showed atrophy and wall detachment.RBWE induced DNA presynthesis phase arrest of BNL CL.2 cells,increased the proportion of apoptotic cells and inhibited cell proliferation.RBWE up-regulated reactive oxygen species(ROS) levels and lowered mitochondrial membrane potentials.Additionally,Western blot and q RT-PCR results suggested that RBWE exerted the above effects by promoting the Nrf2/HO-1 and caspase-induced apoptosis pathways in vitro and in vivo.The contents of alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase,and total bile acids in the serum of mice from Institute of Cancer were significantly up-regulated by RBWE.At the same time,RBWE can lead to increases in ROS and malondialdehyde contents,decreases in contents of oxidized glutathione,glutathione and reduced glutathione,as well as decrease in catalase and superoxide dismutase activities in mouse liver tissues,demonstrating that oxidative stress occurred in mice.Moreover,liver damage was further detected by haematoxylin-eosin staining and electron microscopy to verify the damage to the mice caused by RBWE.In general,RBWE may cause hepatotoxicity in vivo and in vitro via the apoptosis pathway,which provides a reference for hepatotoxicity and its mechanism of action.

Scutellarin alleviates complete freund’s adjuvant-induced rheumatoid arthritis in mice by regulating the Keap1/Nrf2/HO-1 pathway

Scutellarin(SCU)is a herbal flavonoid glucuronide with multiple pharmacological activities,including antioxidant,anti-inflammation,vascular relaxation,anti-platelet,and myocardial protection.However,the effect of SCU on complete Freund’s adjuvant(CFA)-induced rheumatoid arthritis(RA)had not been studied.In this study,we investigated the beneficial effects of SCU in the CFA-induced RA mice model and the anti-arthritic activity was evaluated by paw edema.Enzyme-linked immunosorbent assay(ELISA)was carried out to evaluate the plasma levels of immunoglobulin(Ig)G,IgE,tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6,receptor activator of nuclear factor-κB ligand(RANKL),and osteoprotegerin(OPG).Histological slides were prepared from the harvested paws of mice to determine the pathological changes in the joints.The proportions of T helper type 1(Th1)and T helper type 2(Th2)cells of CD4+T lymphocyte subsets were analyzed by flow cytometry.The expression of Kelch-like ECHassociated protein 1(Keap1),nuclear factor erythroid 2-related factor 2(Nrf2),and heme oxygenase-1(HO-1)was analyzed using real-time quantitative PCR(RT-qPCR)and western blotting assays.The present study demonstrated that SCU prevented CFA-induced RA,and inhibited the expression of inflammation factors,IgG,IgE,TNF-α,IL-1β,and IL-6.While SCU also reduced the RANKL level,it increased OPG expression in RA mice.The Th1/Th2 ratio was significantly lower in mice treated with SCU.Additionally,HO-1 expression was reduced while the expression of Keap1 and Nrf2 was elevated following SCU treatment.Results provide preliminary evidence to employ SCU in arthritis treatment which might be related to the regulation of Th1/Th2 balance and the Keap1/Nrf2/HO-1 pathway.

Nrf2-Keap1-ARE信号通路在复发性流产绒毛组织中的表达及其与氧化应激和炎症反应关系研究

目的探讨核因子E2相关因子2(Nrf2)-Kelch样环氧氯丙烷相关蛋白1(Keap1)-抗氧化反应元件(ARE)信号通路在复发性流产绒毛组织中的表达水平及其与氧化应激和炎症反应的关系。方法纳入2019年3月至2021年9月广东医科大学附属医院收治的59例复发性流产孕妇作为病例组,于同期随机选取65例行人工流产孕妇作为对照组。采用化学比色法检测绒毛组织丙二醛(MDA)含量及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPX)活性,酶联免疫吸附试验法检测血清白介素(IL)-6和IL-10水平,免疫组织化学法检测绒毛组织Nrf2、Keap1、ARE阳性表达情况,实时荧光定量聚合酶链反应(RT-qPCR)法检测绒毛组织Nrf2、Keap1、ARE mRNA相对表达量。结果病例组绒毛组织中MDA含量高于对照组,SOD、CAT、GPX活性低于对照组(P<0.05)。病例组血清IL-6、IL-10水平低于对照组(P<0.05)。病例组绒毛组织中Nrf2、ARE阳性表达率低于对照组,Keap1阳性表达率高于对照组(P<0.05)。病例组绒毛组织中Nrf2、ARE mRNA相对表达量低于对照组,Keap1 mRNA相对表达量高于对照组(P<0.05)。经Pearson积矩相关分析,病例组Nrf2、ARE mRNA相对表达量与MDA均呈负相关(P<0.05),而与SOD、CAT、GPX均呈正相关(P<0.05);Keap1 mRNA相对表达量与MDA呈正相关(P<0.05),而与SOD、CAT、GPX均呈负相关(P<0.05)。病例组Nrf2、ARE mRNA相对表达量与IL-6、IL-10呈正相关(P<0.05),Keap1 mRNA相对表达量与IL-6、IL-10呈负相关(P<0.05)。结论复发性流产孕妇绒毛组织中Nrf2、ARE表达下调,Keap1表达上调,且与氧化应激和炎症反应密切相关;激活Nrf2-Keap1-ARE信号通路有望通过调节氧化应激和炎症反应防治复发性流产。

Effects of Cigu Xiaozhi Formula on miR-378a-3p Expression and Hh Signaling Pathway in TGF-β1 Induced LX2 Cells

[Objectives]To observe the effects of Cigu Xiaozhi Formula on miR-378a-3p expression and Hh signaling pathway in TGF-β1 induced and activated LX2 cells.[Methods]Cells were divided into control group,induction group,drug-containing serum group,miR-378a-3p inhibitor group,and miR inhibitor NC group.CCK-8 method was used to detect the cell viability of each group,and flow cytometry was used to detect the apoptosis rate of each group.RT-qPCR was used to detect the expression of miR-378a-3p in each group s cells,and RT-qPCR and Western blot were used to detect mRNA and protein expression of Shh,Gli1,Gli2,Col-I,andα-SMA in each group s cells.[Results]Compared with the control group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,andα-SMA mRNA and protein in induction group increased(P<0.01),while the expression of miR-378a-3p decreased(P<0.01).Compared with the induction group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA andα-SMA and Gli2 protein decreased in drug-containing serum group(P<0.05),while cell apoptosis rate and miR-378a-3p expression increased(P<0.01).In miR-378a-3p inhibitor group,cell viability and the expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA and Gli1,Gli2,α-SMA protein increased(P<0.05,P<0.01),while the apoptosis rate and miR-378a-3p expression decreased(P<0.05,P<0.01).[Conclusions]Cigu Xiaozhi Formula containing serum can upregulate miR-378a-3p expression and downregulate the expression of Gli2 andα-SMA in TGF-β1 induced LX2 cells,thereby inhibiting the activation of LX2 cells and exerting the effects of anti liver fibrosis.

活化蛋白C通过调控Nrf-2/HO-1信号通道改善大鼠皮瓣缺血再灌注损伤的作用研究

目的:研究活化蛋白C(Activated protein C,APC)对大鼠皮瓣缺血再灌注损伤的作用及可能机制。方法:将80只SD雄性大鼠随机分为四组:对照组、药物对照组、模型组和治疗组,每组20只。观察术后72 h内模型大鼠皮瓣形态变化,HE染色观察大鼠皮瓣组织病理学变化,TUNEL法染色观察皮瓣组织细胞凋亡,ELISA法检测血清中TNF-α、IL-6水平,用黄嘌呤氧化酶法和硫代巴比妥酸TBA比色法分别测定皮瓣组织中超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量,Westernblot法检测皮瓣组织中Nrf-2、HO-1、γ-GCS蛋白表达水平。结果:与模型组比较,治疗组皮瓣红肿、坏死程度、病理损伤程度减弱;TUNEL法染色观察,与模型组比较,治疗组皮瓣组织细胞凋亡率减少(P<0.05);ELISA法检测发现,与模型组比较,治疗组血清中TNF-α、IL-6降低(P<0.05);与模型组比较,治疗组SOD活性升高(P<0.05),MDA含量降低(P<0.05);Westernblot法检测发现,与模型组比较,治疗组Nrf-2、HO-1、γ-GCS蛋白相对表达水平上升(P<0.05)。结论:APC能改善大鼠皮瓣缺血再灌注损伤,其机制可能与激活Nrf-2/HO-1信号通路,抑制氧化应激反应和减少细胞凋亡、炎症反应有关。

槲皮素通过影响Nrf2-Keap1信号通路减缓镉中毒大鼠的生殖机能损伤

本试验旨在探索联合使用槲皮素(QE)可以减轻镉(Cd)致雄性大鼠生殖系统损伤及在此过程Nrf2-Keap1信号通路发挥的作用。试验选取32只3月龄、体重相近的SPF级Wistar雄性大鼠,随机分为4组,每组8只,分别为对照组[0.5mL·(kg·d)^(-1)生理盐水]、Cd组[5mg·(kg·d)^(-1) CdCl_2]、QE组[15mg·(kg·d)^(-1) QE]、Cd+QE组[5mg·(kg·d)^(-1) CdCl_2+15mg·(kg·d)^(-1) QE],连续灌胃1个月。试验结束后,计算各组大鼠睾丸及附睾脏器系数,检测精子质量及睾丸组织抗氧化水平等变化,利用RT-qPCR及Western blot法检测Nrf2及其相关基因mRNA和蛋白的表达。结果表明:与对照组相比,Cd组大鼠生殖机能受损严重。与Cd组相比,Cd+QE组大鼠睾丸附睾脏器系数(P<0.01)和睾酮分泌量(P<0.05)增加明显;精子质量提高;睾丸组织中MDA、H_2O_2含量极显著降低(P<0.01),GSH含量(P<0.05)和CAT、T-SOD、GSH-Px酶活性(P<0.01)明显提高;Nrf2及其相关基因NQO1、γ-GCS、HO-1、GSH-Px的mRNA和蛋白的表达呈现明显上升趋势(P<0.01或P<0.05),而Keap1基因和蛋白质的表达显著降低(P<0.05)。综上,QE可能通过激活Nrf2-Keap1信号通路有效减轻Cd引起的大鼠生殖系统损伤。

生姜提取物对苯并芘染毒小鼠Ⅱ相酶的诱导作用及对Nrf2-Keap1通路的影响

研究生姜提取物对苯并芘(benzo[α]pyrene,BαP)染毒小鼠抗氧化酶及Ⅱ相酶的影响及调控机制。用100、200、400 mg/(kg·d)的生姜提取物(分别标记为LG、MG和HG)对昆明小鼠连续灌胃给药14 d,然后腹腔注射50 mg/(kg·d) BαP,测定小鼠血清及肝肾中谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、过氧化氢酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase,SOD)以及醌氧化还原酶(quinone reductase,QR)、血氧合酶1(hemooxygenase 1,HO-1)和谷胱甘肽-S-转移酶(glutathione-S-transferase,GST)的活力及相应蛋白和mRNA的表达水平,分析了核转录因子E2相关因子2(nuclear factor (erythroid-derived 2)-like 2,Nrf2)和Kelch样环氧氯丙烷相关蛋白1(Kelch like ECH associated protein 1,Keap1)的表达情况。结果表明:与BαP损伤组相比,BαP+MG和BαP+HG能显著诱导小鼠血清中CAT和GSH-Px活力及肝肾中CAT、SOD和GSH-Px的活力(P<0.05);BαP+MG和BαP+HG处理导致动物肝脏中QR、HO-1和GST的活力分别提高了21.7%和29.05%、23.99%和56.39%、32.40%和15.08%。与BαP处理组相比,BαP+MG极显著提高了Nrf2的蛋白表达水平(21.9%),抑制了Keap1蛋白表达(34.7%)(P<0.01)。高效液相色谱-轨道离子阱质谱联用方法从生姜提取物中共鉴定出19种成分。因此,生姜提取物能显著诱导BαP染毒小鼠中抗氧化酶和Ⅱ相酶,并且该过程与Nrf2-Keap1信号通路相关。

An exploration on the protective mechanism of Xuduan Zhongzi prescription against epididymis oxidative damage in oligoasthenospermia model rats based on Nrf2-NQO1/γ-GCS signaling pathway

Objective:To investigate the protective mechanism of Xuduan Zhongzi prescription against epididymal oxidative damage in oligoasthenospermia model rats.Methods:Forty SD rats were randomly divided into blank group,model group,Xuduan Zhongzi prescription group(10g/kg)and L-carnitine group(0.1g/kg).Except blank group,all induced oligoasmospermia.The blank group and model group were given normal saline intragastric administration,the Xuduan Zhongzi prescription group was given Xuduan Zhongzi prescription solution intragastric administration,and the L-carnitine group was given L-carnitine intragastric administration.HE staining was used to observe the epididymis structure after 8 weeks.The concentration and activity rate of epididymis sperm were measured by sperm quality.MRNA and protein expression levels of Nrf2,NQO1 andγ-GCs in epididymis were detected by RT-qPCR and immunohistochemistry.Results:①HE staining:in the blank group,the epididymis tubes were arranged tightly and regularly,the tissue structure was complete,the epithelial cells were arranged orderly,and the lumen sperm were numerous and evenly distributed.The epididymis of model group showed structural atrophy,loose arrangement,enlarged mesenchyme,increased cell debris and significantly reduced sperm cells.Compared with the model group,the lumen lesions of epididymis in Xuduan Zhongzi prescription group and L-carnitine group were significantly improved,and the amount of normal sperm in lumen was increased and the distribution was uniform.②Results of sperm quality comparison among each group:sperm density and sperm motility rate:compared with blank group,sperm density and sperm motility rate in other groups were significantly decreased(P<0.05),and sperm density and sperm motility rate in model group were significantly decreased(P<0.05);Compared with model group,the sperm density and motility rate in Xuduan Zhongzi prescription group and L-carnitine group were significantly increased(P<0.05).③RT-qPCR and immunohistochemistry:Compared with the blank group,the mRNA and protein levels of Nrf2,NQO1 andγ-GCs in epididymal rats in model group were significantly decreased(P<0.05),while the mRNA and protein levels of Nrf2,NQO1 andγ-GCs were significantly increased in L-carnitine group and Continua seed formula group(P<0.05).Conclusion:Xuduan Zhongzi prescription can reduce oxidative stress damage and improve sperm quality of oligoasthenospermia.The mechanism may related to promoting the activation of Nrf2-NQO1/γ-GCS pathway in epididymis of oligoasthenospermia rats,and up-regulate the expressions of Nrf2,NQO1 andγ-GCS proteins.

Mechanism of hesperidin improving myocardial ischemia/reperfusion injury in type 2 diabetic rats through SIRT1/Nrf2/HO-1 signaling pathway

Objective:To observe the protective effect of hesperidin on myocardial ischemia/reperfusion injury in type 2 diabetes mellitus and its effect on SIRT1/Nrf2/HO-1 signaling pathway.Methods:50 Sprague-Dawley(SD)rats were randomly assigned to the normal control group(NC),model group,ischemia-reperfusion group(IR),hesperidin group,SIRT1 inhibitor group and hesperidin plus SIRT1 inhibitor group.In addition to NC,the rats in the remaining groups were replicated by intraperitoneal of high-fat diet combined with injection of streptozotocin for type 2 diabetic rats.After then,the myocardial ischemia/reperfusion injury(MIRI)rat model was established by LAd for 30 minutes with 2 hours reperfusion.He staining was used to observe the pathological changes of myocardial tissue,and the levels of serum LDH,CK-MB and SOD,GSH and MDA in myocardial tissue were detected by kit methods,and the expression abundance of related proteins in 4-HNE and SIRT1/Nrf2/HO-1 signal pathway were detected by immunohistochemistry and Western blot;Results:Hesperidin could significantly inhibit cardiomyocyte necrosis and inflammatory cell infiltration,reduce LDH activity,CK-MB and MDA level,and increase SOD activity,GSH and 4-HNE level,the differences were statistically significant when compared with IR group(P<0.01).In addition,compared with the ischemia-reperfusion group,the expressions of SIRT1,Nrf2 and HO-1 proteins in hesperidin group were significantly up-regulated,the differences were statistically significant(P<0.01);Conclusion:Hesperidin inhibits oxidative stress by activating SIRT1/Nrf2/HO-1 signaling pathway,and play a protective effect of myocardial ischemia reperfusion injury in diabetic rats.

针刺疗法对腹泻型肠易激综合征大鼠Nrf2-Keap1-ARE信号通路的影响

目的:观察针刺通过Nrf2-Keap1-ARE信号通路对IBS-D大鼠的作用机制。方法:50只Wistar大鼠随机分为5组,每组10只。大鼠实施强行应激法(CAS)刺激获得IBS-D模型;大鼠CAS刺激前1 h灌胃番木液(6 g/kg),其后实施CAS刺激获得药物致泻+CAS刺激模型;大鼠CAS刺激后,取ST25(天枢)、ST36(足三里)和LR3(太冲)针刺,获得CAS+针刺组模型;体外构建siRNA,进行鼠尾静脉注射后进行CAS刺激和针刺治疗得到CAS刺激+针刺+Nrf2 siRNA(Nrf2^(sh))模型;正常组正常饲养。检测各组5-HT、NPY、MDA、GSH/GSSG、SOD和ROS水平以及Nrf2信号通路中基因表达水平和蛋白表达水平。结果:IBS模型组、药物致泻模型+CAS刺激组和CAS+针刺+Nrf2^(sh)组的ROS和MDA水平高于CAS+针刺组(P<0.01)和正常组(P<0.01),GSH水平均低于CAS+针刺组(P<0.01)和正常对照组(P<0.01),而GSSG水平呈相反趋势(P<0.01);正常组和CAS+针刺组的SOD水平均与其他组有差异(P<0.01),5-HT和NPY的水平以及JNK的基因和蛋白质表达均低于其他组(P<0.01),Nrf2、Keap-1和HO-1的基因和蛋白质表达均与其他组有差异(P<0.01)。结论:Nrf2在IBS-D治疗中具有重要作用,针刺可上调Nrf2的表达,从而抑制肠道氧化性损伤和脂质过氧化,并能在针刺后上调HO-1的表达,为针刺疗法治疗IBS-D的分子机制做出了一定的理论基础。