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ObjectiveTo observe the effect of Banxia Xiexintang （BXT） on the proliferation of human gastric cancer HGC-27， MKN-45， and AGS cells and its mechanism. MethodCell counting kit-8 （CCK-8） was used to detect the effects of different concentrations of BXT-containing serum （5%， 10%， and 20%） on the proliferation of HGC-27， MKN-45， and AGS cells. A mitochondrial membrane potential probe （TMRE） was used to detect the expression of mitochondrial membrane potential in cells. A kit was used to detect iron ion （Fe2+） content， lipid peroxide （LPO）， and superoxide dismutase （SOD） activity. Western blot was used to detect the protein expression levels of glycogen synthase3β （GSK3β）， phosphorylated GSK3β （p-GSK3β）， nuclear factor E2 related factor 2 （Nrf2）， and glutathione peroxidase 4 （GPX4）. The real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of member 11 of the cystine/glutamic acid reverse transporter solute vector family 7 （SLC7A11）， member 2 of the heavy chain solute vector family 3 （SLC3A2）， transferrin receptor 3 （TFRC）， and tumor protein （TP）53. ResultCCK-8 results showed that BXT and capecitabine could significantly reduce the survival rate of three kinds of gastric cancer cells after treatment with drug-containing serum for 24 h （P<0.01）. After 48 h of intervention with drug-containing serum， the survival rate of three kinds of gastric cancer cells was significantly decreased in both the capecitabine group and the BXT group compared with the blank group. The BXT group was dose-dependent， with 20% BXT having the most significant effect （P<0.01）. In terms of biochemical indicators of ferroptosis， compared with the blank group， BXT and capecitabine significantly decreased the expression of mitochondrial membrane potential （P<0.01） and SOD activity （P<0.01） and significantly increased the contents of LPO and Fe2+ （P<0.01）， so as to improve the sensitivity of gastric cancer cells to ferroptosis. In terms of the Nrf2/GPX4 pathway， compared with the blank group， the BXT group could reduce the protein expressions of p-GSK3β， Nrf2， and GPX4 （P<0.01） in gastric cancer cells and increase mRNA expressions of SLC7A11 and SLC3A2 （P<0.05）. It could also increase the protein expression of GSK3β （P<0.01） and mRNA expression of TP53 and TFRC （P<0.05， P<0.01） in gastric cancer cells. Inhibition of the Nrf2/GPX4 pathway induces ferroptosis in gastric cancer cells. Compared with the capecitabine group， the 20% BXT group showed a more obvious effect. ConclusionBanxia Xiexintang can induce ferroptosis in gastric cancer cells HGC-27， MKN-45， and AGS by inhibiting the Nrf2/GPX4 pathway.
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Acute pancreatitis （AP） is a common clinical acute abdominal disease， which is characterized by acute onset， rapid development， severe disease， many complications， and high mortality rate. It can progress to severe AP （SAP） if not treated promptly in the early stage. The pathogenesis of AP is complex and involves multiple cellular and molecular levels. It is now clear that oxidative stress and reactive oxygen species （ROS） production are involved in the physiopathological process of AP， which is associated with a low quantity and activity of antioxidant enzymes in pancreatic cells. Nuclear factor E2-related factor 2 （Nrf2） serves as the ''golden key'' to maintain redox homeostasis in tissue cells and constitutes an important signaling pathway for antioxidant response and inflammation in vivo by collaborating with downstream antioxidant enzymes such as heme oxygenase-1 （HO-1）. Traditional Chinese medicine has unique efficacy in treating diseases due to its multi-component， multi-target， multi-drug delivery， and multi-formulation characteristics. Based on the concept of synergy between traditional Chinese and Western medicine， traditional Chinese medicine is becoming a new craze in the treatment of AP. The level of oxidative stress and Nrf2/HO-1 signaling pathway in AP pancreatic tissue are in a dynamic change process， and the intervention of traditional Chinese medicine can clean ROS production， affect the inflammatory pathway， and reduce oxidative stress damage， so as to protect against pancreatic injury. This suggests that this pathway plays an important role in AP. This article reviews the recent literature on the regulation of the Nrf2/HO-1 signaling pathway by traditional Chinese medicine for AP and summarizes that the monomers of traditional Chinese medicine targeting this pathway are mainly heat-clearing and detoxifying， blood-activating and blood-stasis-removing， and Qi benefiting and middle warming， and the compounds of traditional Chinese medicine include Yinchenhao Decoction and QingYi Ⅱ， so as to provide a new direction for the prevention and treatment of AP and further drug development.
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ObjectiveTo observe the effect of earthworm protein on the expression of phosphatidylinositol 3-kinase/protein kinase B/nuclear factor E2-related factor 2 （PI3K/Akt/Nrf2） pathway in the aorta of spontaneously hypertensive rats （SHR） and explore mechanism of earthworm protein in treating hypertensive vascular endothelial dysfunction （VED）. MethodTen 10-week-old Wistar Kyoto （WKY） rats and fifty SHR rats were selected for a week of adaptive feeding. WKY rats were selected as the normal group， and fifty SHR rats were randomized according to body weight into model， valsartan （8×10-3 g·kg-1·d-1）， and high-， medium-， and low-dose （0.2， 0.1， 0.05 g·kg-1·d-1， respectively） earthworm protein groups. The normal and model groups were administrated with equal volume of double distilled water by gavage. During the drug intervention period， the general situations of rats in each group were observed and their blood pressure was monitored at specific time points every other week before and after administration. After 8 weeks of drug intervention， enzyme-linked immunosorbent assay was employed to measure the levels of angiotensin-Ⅱ （Ang-Ⅱ） and endothelin-1 （ET-1） in the serum of rats in each group. The corresponding kits were used to determine the levels of nitric oxide （NO）， malondialdehyde （MDA）， glutathione peroxidase （GPX）， superoxide dismutase （SOD）， and ferrous ion （Fe2+）. Hematoxylin-eosin （HE） staining was employed to observe the changes in the intima of the aorta. Fluorescence quantitative polymerase chain reaction （Real-time PCR） was employed to measure the mRNA levels of PI3K， Akt， Nrf2， heme oxygenase-1 （HO-1）， and glutathione peroxidase 4 （GPX4） in the aortic tissue. Western blotting was used to determine the protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 in the thoracic aorta. ResultCompared with the normal group， the model group had decreased body mass， increased irritability， severe endothelial damage， elevated blood pressure and serum levels of Ang-Ⅱ， ET1， MDA， and Fe2+ （P<0.01）， lowered NO level （P<0.01）， and down-regulated mRNA and protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 in the aortic tissue （P<0.01）. Compared with the model group， drug intervention caused no significant change in the body mass， calmed the rats， alleviated the endothelial damage， lowered blood pressure and serum levels of Ang-Ⅱ， ET1， MDA， and Fe2+ （P<0.01）， elevated the NO level （P<0.05）， and up-regulated the mRNA and protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 （P<0.05）. ConclusionThe earthworm protein can exert antihypertensive effects by ameliorating VED in SHR. Specifically， it may regulate the PI3K/Akt/Nrf2 signaling pathway to inhibit oxidative stress and ferroptosis.
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Alzheimer's disease （AD） is a neurodegenerative disease characterized by a progressive decline in memory and cognitive function. β-amyloid protein （Aβ） aggregation and excessive phosphorylation of Tau protein in the brain can increase oxidative stress levels， leading to energy metabolism imbalance， extensive apoptosis of nerve cells， and damage to synaptic function. The nuclear factor E2 related factor 2 （Nrf2） encoded by the Nfe212 gene is known as the "main regulatory factor" of antioxidant response. On the one hand， It can activate antioxidant response elements， such as heme oxygenase-1 （HO-1） and NAD（P）H quinone oxidoreductase1 （NQO1）， increase the expression of antioxidant enzymes glutathione S-transfer （GST） and superoxide dismutase 1 （SOD1）， and reduce the release of reactive oxygen species. On the other hand， it can inhibit immune inflammation， cell apoptosis， and activation of autophagy pathways and delay the progression of AD. Therefore， this article summarized， analyzed， and reviewed the relevant research on the regulation of the Nrf2 signaling pathway by traditional Chinese medicine in the prevention and treatment of AD in the past five years， including its structural characteristics， pathway conduction， mechanism of action in AD， and drug regulation. The results showed that among all reports， research on traditional Chinese medicine compounds occupied a high proportion and mostly focused on flavonoids， with the Nrf2/HO-1 and PI3K/Nrf2 signaling pathways being the most extensively studied. The mechanisms of action were mainly to inhibit oxidative stress， neuroinflammation， and cell apoptosis and improve synaptic function. This indicates that traditional Chinese medicine can regulate multiple Nrf2 signaling pathways and play a role in preventing and treating Alzheimer's disease from multiple mechanisms.
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ObjectiveTo investigate the effect and mechanism of Shenqi Tangluo pill （SQTLP） on oxidative stress injury of skeletal muscle of type 2 diabetes mellitus （T2DM） mice based on nuclear factor erythroid 2-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1）/NAD（P）H quinone oxidoreductase 1 （NQO1） pathway. MethodA total of 60 7-week-old male db/db mice ［specific pathogen-free （SPF） grade］ were selected and fed for one week for adaption. They were divided into the model control group， SQTLP low-， medium- and high-dose （19， 38， and 76 g·kg-1） groups and metformin group （0.26 g·kg-1） by gavage. Each group consisted of 12 mice. Twelve male db/m mice of the same age were selected as the blank group. The intervention was implemented continuously for 8 weeks. Fasting blood glucose （FBG） was detected. Fasting serum insulin （FINS） levels were detected by enzyme-linked immunosorbent assay （ELISA）， and the homeostasis model assessment-insulin resistance （HOMA-IR） index and the homeostasis model assessment-insulin sensitivity index （HOMA-ISI） were calculated. Oral glucose tolerance test （OGTT） and insulin tolerance test （ITT） were conducted. The activities of superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） and the contents of malondialdehyde （MDA） and reduced nicotinamide adenine dinucleotide phosphate （NADPH） in skeletal muscle tissues were detected by biochemical kits. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in skeletal muscle tissues. The levels of reactive oxygen species （ROS） and 4-hydroxynonenal （4-HNE） in skeletal muscle tissue were detected by immunofluorescence （IF）. The expression levels of Nrf2， HO-1， NQO1 and glutamate-cysteine ligase catalytic subunit （GCLC） proteins in skeletal muscle tissues were detected by Western blot. ResultCompared with those in the blank group， FBG， FINS and HOMA-IR in the model group were significantly increased （P<0.05）， while HOMA-ISI was decreased （P<0.05）. The results of OGTT and ITT showed that blood glucose was significantly increased at all time points （P<0.05）， and glucose tolerance and insulin tolerance were significantly impaired. SOD and GSH-Px activities in skeletal muscle tissues were significantly decreased （P<0.05）， and MDA and NADPH contents were significantly increased （P<0.05）. In skeletal muscle tissues， the arrangement of muscle fibers was loose， the nucleus was disordered， and inflammatory cells were infiltrated. The expression levels of ROS and 4-HNE in skeletal muscle tissues were significantly increased （P<0.05）. The protein expression levels of Nrf2， HO-1， NQO1 and GCLC in skeletal muscle tissues were significantly decreased （P<0.05）. Compared with those in the model group， FBG， FINS and HOMA-IR in the metformin group were significantly decreased （P<0.05）， while HOMA-ISI was increased （P<0.05）. The results of OGTT and ITT showed that blood glucose in the metformin group was significantly decreased at all time points （P<0.05）. The activities of SOD and GSH-Px in skeletal muscle tissues were significantly increased （P<0.05）， while the contents of MDA and NADPH were significantly decreased （P<0.05）. No obvious abnormality was found in the skeletal muscle tissue of the metformin group. The expressions of ROS and 4-HNE in skeletal muscle tissues were decreased （P<0.05）. The protein expression levels of Nrf2， HO-1， NQO1 and GCLC in skeletal muscle tissues were significantly increased （P<0.05）. Compared with those in the model group， FBG， FINS and HOMA-IR in the SQTLP medium- and high-dose groups were significantly decreased （P<0.05）， while HOMA-ISI was increased （P<0.05）. The results of OGTT and ITT showed that the glucose tolerance and insulin tolerance of mice were improved in each dose group of SQTLP. The GSH-Px activity in the SQTLP low-dose group was significantly increased （P<0.05）， and the NADPH content was decreased （P<0.05）. The activities of SOD and GSH-Px in the SQTLP medium- and high-dose groups were significantly increased （P<0.05）， while the contents of MDA and NADPH were significantly decreased （P<0.05）. The skeletal muscle tissue injury of mice in each dose group of SQTLP was ameliorated to different degrees. In the SQTLP medium- and high-dose groups， the expressions of ROS and 4-HNE were decreased （P<0.05）， and the protein expression levels of Nrf2， HO-1， NQO1 and GCLC were significantly increased （P<0.05）. Compared with those in the SQTLP low-dose group， FBG and HOMA-IR in the SQTLP high-dose group were significantly decreased （P<0.05）， while HOMA-ISI was increased （P<0.05）. The results of OGTT and ITT showed that the SQTLP high-dose group significantly improved the glucose tolerance and insulin tolerance of mice. The activities of SOD and GSH-Px in skeletal muscle tissues were significantly increased （P<0.05）， while the contents of MDA and NADPH were significantly decreased （P<0.05）. No obvious abnormality was found in the skeletal muscle tissue， the expressions of ROS and 4-HNE were decreased （P<0.05）， and the protein expression levels of Nrf2， HO-1， NQO1 and GCLC were significantly increased （P<0.05） in the skeletal muscle tissue of the SQTLP high-dose group. ConclusionSQTLP can significantly improve IR in T2DM mice， and the mechanism is related to SQTLP activating the Nrf2/HO-1/NQO1 signaling pathway， promoting the expression of antioxidant enzymes， and thus improving the oxidative stress injury in the skeletal muscle.
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ObjectiveTo investigate the effects of Xinjia Congrong Tusizi decoction （XJCTD） on ovarian functions in the rat model of premature ovarian insufficiency （POI） and decipher the mechanism of regulating the tumor suppressor protein （p53）/nuclear factor E2-related factor 2 （Nrf2） pathway to attenuate granulosa cell ferroptosis. MethodForty-eight SPF-grade female SD rats were randomized into control， model， low-， medium-， and high-dose （1.1， 2.2， 4.4 g·kg-1） XJCTD， and Western medicine （coenzyme Q10， 0.002 7 g·kg-1） groups， with eight rats in each group. The rat model of POI was established by gavage of triptolide （TP）， and after successful modeling， each group was administrated with the corresponding drugs by gavage for 14 d. The body weight and ovarian weight of each rat were weighed and the ovarian index was calculated. The morphology of the ovarian tissue was observed by hematoxylin-eosin staining， and the proportions of growing follicles and atretic follicles were calculated. The serum levels of anti-Müllerian hormone （AMM）， estradiol （E2）， and follicle-stimulating hormone （FSH） were measured by enzyme-linked immunosorbent assay （ELISA）. The DCFH-DA fluorescent probe was used to measure the reactive oxygen species （ROS） content in granulosa cells. The content of cellular Ferrous ion （Fe2+）， lipid peroxide （LPO）， malondialdehyde （MDA）， glutathione （GSH）， and superoxide dismutase （SOD） was detected by colorimetry. The expression of the tumor suppressor protein p53，Nrf2， solute carrier family 7 member 11 （SLC7A11）， and glutathione peroxidase 4 （GPX4） was determined by immunohistochemistry and Western blot. ResultCompared with the control group， the model group showed decreased ovarian weight， body weight， and ovarian index （P<0.01）， reduced ovarian tissue volume and proportion of growing follicles （P<0.01）， increased proportion of atretic follicles （P<0.01）， lowered AMH and E2 levels and elevated FSH level in the serum （P<0.01）， and elevated levels of Fe2+， ROS， LPO， and MDA （P<0.01） and lowered levels of GSH and SOD in granulosa cells （P<0.01）. Moreover， the modeling up-regulated the expression of p53 （P<0.01） and down-regulated the expression of Nrf2， SLC7A11， and GPX4 （P<0.05， P<0.01） in the ovarian tissue. Compared with the model group， XJCTD increased the body weight， ovarian weight， and ovarian index （P<0.01）， alleviated the pathological changes in the ovarian tissue， increased the proportion of growing follicles （P<0.01）， decreased the proportion of atretic follicles （P<0.01）， and reduced the content of ROS in granulosa cells （P<0.05， P<0.01）. In addition， medium- and high-dose XJCTD lowered the FSH level （P<0.01） and raised E2 and AMH levels （P<0.01） in the serum， reduced the Fe2+ content （P<0.05， P<0.01）， and increased the SOD content （P<0.01） in granulosa cells. High-dose XJCTD reduced the LPO and MDA content （P<0.01） and increased the SOD content （P<0.01） in the granulosa cells， down-regulated the expression of p53 （P<0.05）， and up-regulated the expression of Nrf2， SLC7A11， and GPX4 in the ovarian tissue （P<0.05， P<0.01）. ConclusionXJCTD may protect the ovarian function in the rat model of POI by regulating the p53/Nrf2 signaling pathway to attenuate the ferroptosis of ovarian granulosa cells.
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ObjectiveTo explore the mechanism of Buzhong Yiqitang-containing serum in alleviating the cisplatin resistance in human non-small cell lung cancer （A549/DDP） cells via regulating the nuclear factor E2-related factor 2 （Nrf2）/reactive oxygen species （ROS） signaling pathway. MethodThe serum containing Buzhong Yiqitang was prepared and A549/DDP cells were cultured and randomly grouped： blank （10% blank serum）， cisplatin （10% blank serum+20 mg·L-1 cisplatin）， Buzhong Yiqitang （10% Buzhong Yiqitang-containing serum+20 mg·L-1 cisplatin）， ML385 （10% blank serum+5 μmol·L-1 ML385+20 mg·L-1 cisplatin）， Buzhong Yiqitang+ML385 （10% Buzhong Yiqitang-containing serum+5 μmol·L-1 ML385+20 mg·L-1 cisplatin）， tertiary butylhydroquinone （TBHQ） （10% blank serum+5 μmol·L-1 TBHQ+20 mg·L-1 cisplatin）， and Buzhong Yiqitang+TBHQ （10% Buzhong Yiqitang-containing serum+5 μmol·L-1 TBHQ+20 mg·L-1 cisplatin）. The median inhibitory concentration （IC50） of cisplatin in each group was determined by the cell counting kit-8 （CCK-8） method and the resistance index （RI） was calculated. The apoptosis rate was detected by flow cytometry. The ROS content of each group was determined with the DCFH-DA fluorescence probe. Western blot was employed to determine the protein levels of Nrf2， cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3）， cytochrome C （Cyt C）， and B-cell lymphoma-2 （Bcl-2）. ResultCompared with those in the cisplatin group， the IC50 and RI of A549/DDP cells to cisplatin in Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 groups decreased （P˂0.05）. Compared with the blank group， the cisplatin， Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 groups showed increased apoptosis rate of A549/DDP cells （P˂0.05）. Compared with the blank group， cisplatin promoted the expression of Nrf2 （P˂0.05）. Compared with the cisplatin group， Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 inhibited the expression of Nrf2 （P<0.05）， elevated the ROS level （P˂0.05）， up-regulated the protein levels of cleaved Caspase-3 and Cyt C， and down-regulated the protein level of Bcl-2 （P<0.05）， which were the most significant in the Buzhong Yiqitang+ML385 group. Compared with the cisplatin group， the TBHQ group showed increased IC50 and RI of cisplatin （P<0.05）， decreased apoptosis rate of A549/DDP cells （P<0.05）， up-regulated protein levels of Nrf2 and Bcl-2 （P<0.05）， lowered level of ROS （P˂0.05）， and down-regulated protein levels of cleaved Caspase-3 and Cyt C （P<0.05）. Compared with the TBHQ group， Buzhong Yiqitang+TBHQ decreased the IC50 and RI of cisplatin in A549/DDP cells （P<0.05）， increased the apoptosis rate （P<0.05）， down-regulated the protein levels of Nrf2 and Bcl-2 （P<0.05）， increased ROS （P˂0.05）， and up-regulated the protein levels of cleaved Caspase-3 and Cyt C （P<0.05）. ConclusionBuzhong Yiqitang induced apoptosis by inhibiting Nrf2/ROS pathway to alleviate cisplatin resistance in A549/DDP cells.
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Objective:To investigate whether silence information regulator 1 (SIRT1) could regulate nuclear factor E2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway and its role in acute lung injury (ALI) in sepsis rats.Methods:Twenty-four male Sprague-Dawley (SD) rats were randomly divided into sham operation group (Sham group), cecal ligation and puncture (CLP) induced sepsis group (CLP group), sepsis+SIRT1 specific agonist group (CLP+SRT1720 group,10 mg/kg SRT1720 was intraperitoneally injected 2 hours before CLP), sepsis+SIRT1 specific inhibitor group (CLP+EX527 group, 10 mg/kg EX527 was intraperitoneally injected 2 hours before CLP), with 6 rats in each group. The rats were killed 24 hours after modeling and their lung tissues were taken for pathological score (Smith score), superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1β), and SIRT1, Nrf2 and HO-1 mRNA and protein expression were detected.Results:The lung tissue of the CLP group mice was severely damaged, the alveolar interval was widened and a large number of inflammatory cells infiltrated, and there was visible pulmonary capillary hyperemia. The Smith score, the levels of TNF-α, IL-6, IL-1β, MDA and 8-OHdG were significantly increased, the levels of SOD, GSH, SIRT1, Nrf2 and HO-1 were significantly decreased in CLP group. After using SIRT1 specific agonist, the lung injury in CLP+SRT1720 group was significantly alleviated compared with that in CLP group, Smith score and lung tissue TNF-α, IL-6, and IL-1β levels were significantly decreased [Smith score: 2.83±0.75 vs. 5.67±0.52, TNF-α (ng/L): 36.78±5.36 vs. 66.99±5.44, IL-6 (ng/L): 23.97±3.76 vs. 45.70±4.16, IL-1β (ng/L): 16.76±1.39 vs. 39.64±2.59, all P < 0.05], SOD activity and GSH content increased [SOD (kU/g): 115.88±3.31 vs. 101.65±1.09, GSH (μmol/g): 8.42±0.81 vs. 5.74±0.46, both P < 0.05], MDA and 8-OHdG contents decreased [MDA (μmol/g): 5.24±0.33 vs. 9.86±0.66, 8-OHdG (ng/L): 405.76±8.54 vs. 647.12±10.64, both P < 0.05], the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 were increased [SIRT1 mRNA (2 -ΔΔCT): 1.49±0.15 vs. 0.64±0.03, Nrf2 mRNA (2 -ΔΔCT): 1.19±0.08 vs. 0.84±0.02, HO-1 mRNA (2 -ΔΔCT): 1.80±0.41 vs. 0.64±0.11, SIRT1 protein (SIRT1/β-actin): 1.03±0.06 vs. 0.52±0.05, Nrf2 protein (Nrf2/β-actin): 1.14±0.10 vs. 0.63±0.05, HO-1 protein (HO-1/β-actin): 1.01±0.11 vs. 0.73±0.03, all P < 0.05]. The lung injury in CLP+EX527 group was more severe than that in CLP group, Smith score and lung tissue TNF-α, IL-6, IL-1β levels were significantly increased [Smith score: 8.00±0.89 vs. 5.67±0.52, TNF-α (ng/L): 87.15±4.23 vs. 66.99±5.44, IL-6 (ng/L): 66.79±2.93 vs. 45.70±4.16, IL-1β (ng/L): 58.99±2.12 vs. 39.64±2.59, all P < 0.05], SOD activity and GSH content decreased [SOD (kU/g): 72.84±3.85 vs. 101.65±1.09, GSH (μmol/g): 3.30±0.67 vs. 5.74±0.46, both P < 0.05], the contents of MDA and 8-OHdG were increased [MDA (μmol/g): 14.14±0.70 vs. 9.86±0.66, 8-OHdG (ng/L): 927.66±11.47 vs. 647.12±10.64, both P < 0.05], the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 were decreased [SIRT1 mRNA (2 -ΔΔCT): 0.40±0.07 vs. 0.64±0.03, Nrf2 mRNA (2 -ΔΔCT): 0.48±0.07 vs. 0.84±0.02, HO-1 mRNA (2 -ΔΔCT): 0.27±0.14 vs. 0.64±0.11, SIRT1 protein (SIRT1/β-actin): 0.20±0.05 vs. 0.52±0.05, Nrf2 protein (Nrf2/β-actin): 0.45±0.01 vs. 0.63±0.05, HO-1 protein (HO-1/β-actin): 0.36±0.08 vs. 0.73±0.03, all P < 0.05]. Conclusions:In the rat model of ALI induced by sepsis, SIRT1 can regulate the activation of Nrf2/HO-1 signaling pathway, upregulate the expression of downstream antioxidant enzymes, reduce oxidative stress injury, and then alleviate the ALI induced by sepsis in rats.
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ObjectiveTo explore the mechanism of plumbagin as a novel ferroptosis inducer in bladder cancer inhibition. MethodBladder cancer T24 cells were used in this study. The effect of different concentrations of plumbagin （0.1， 1， 2， 3， 6， 12， 24， 48 μmol·L-1） on the viability of T24 cells was detected by cell counting kit-8 （CCK-8）. The effect of different concentrations of plumbagin （1.5， 3， 6 μmol·L-1） on the apoptosis of T24 cells was detected by annexin V-fluorescein isothiocyanate （Annexin V FITC）/PI apoptosis kit. Different inhibitors （ferroptosis inhibitor Fer-1， apoptosis inhibitor VAD， and necroptosis inhibitor Nec-1） were used in combination with plumbagin （6 μmol·L-1）. Reactive oxygen species （ROS） fluorescent probe （DCFH-DA）， malonaldehyde （MDA）， and glutathione （GSH） kits were used to detect the effects of different concentrations of plumbagin （1.5， 3， 6 μmol·L-1） on the level of ROS and the content of MDA and GSH in T24 cells， respectively. The effect of different concentrations of plumbagin （1.5， 3， 6 μmol·L-1） on peroxide levels in T24 cells was detected by C11-BODIPY fluorescent probe. Western blot was used to detect the effect of different concentrations of plumbagin （1.5， 3， 6 μmol·L-1） on the protein expression of solute carrier family 7 member 11 （SLC7A11）， glutathione peroxidase 4 （GPX4）， nuclear factor E2-related factor-2 （Nrf-2）， and Kelch-like ECH-associated protein 1 （Keap1）. ResultCompared with the blank group， plumbagin could inhibit the activity of T24 cells （P<0.05） with IC50 of 3.52 μmol·L-1. At the concentrations of 1.5， 3， 6 μmol·L-1， plumbagin significantly promoted the apoptosis of T24 cells （P<0.05） as compared with the blank group. Compared with the plumbagin group at 6 μmol·L-1， the ferroptosis inhibitor and apoptosis inhibitor groups could reverse the inhibitory effect of 6 μmol·L-1 plumbagin on the proliferation of T24 cells （P<0.05）. Compared with the blank group， the plumbagin groups at 1.5， 3， 6 μmol·L-1 showed increased content of ROS， MDA， and lipid peroxides in T24 cells， decreased GSH level， and reduced SLC7A11， GPX4， and Nrf-2/Keap1 （P<0.05）. Conclusionplumbagin can induce ferroptosis， and its mechanism is related to the Nrf-2/Keap1 signaling pathway.
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Renal ischemia-reperfusion injury (RIRI) is the main cause of acute kidney injury (AKI), which commonly occurs in surgery, severe trauma, shock and drug-induced kidney injury. At present, effective treatment for RIRI is still lacking. Oxidative stress is the major pathological injury mechanism of RIRI. Nuclear factor E2-related factor 2 (Nrf2) is the key transcription factor of anti-oxidative stress response, which may activate various cytoprotective genes related to redox and detoxification. Recent studies have shown that Nrf2 may play a protective role in the protection and treatment of RIRI by regulating oxidative stress, inflammation, cell apoptosis and autophagy, etc. Consequently, the structure and biological function of Nrf2, related signaling pathways, its role in the incidence and development of RIRI and potential mechanism were reviewed in this article, aiming to provide novel ideas for the prevention and treatment of RIRI.
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ObjectiveTo explore the effect and underlying mechanism of alcohol extract of Phyllanthi Fructus on silicosis mice induced by silicon dioxide （SiO2）. MethodThirty-six male Kunming mice of SPF grade were randomly divided into a blank group，a model group，high-， medium， and low-dose Phyllanthi Fructus groups （800， 400， 200 mg·kg-1），and a tetrandrine group （0.039 mg·kg-1），with six mice in each group. The silicosis model was induced by static SiO2 exposure in mice except for those in the blank group. After 28 days of administration by gavage，the lung tissues were collected and the organ coefficient was calculated. Hematoxylin-eosin（HE）staining and Masson staining were used to detect the morphology of lung tissues. The content of hydroxyproline （HYP），superoxide dismutase （SOD），malondialdehyde （MDA）， and catalase （CAT） in serum was detected by enzyme-linked immunosorbent assay （ELISA）. Western blot and Real-time polymerase chain reaction（Real-time PCR） were used to detect the protein and mRNA expression of nuclear factor E2-related factor 2 （Nrf2），heme oxygenase-1 （HO-1），NAD（P）H：quinone oxidoreductase 1 （NQO1），and Kelch-like ECH-associated protein 1 （Keap1）， respectively. ResultCompared with the blank group，the model group showed seriously damaged morphological structure of lung tissues with inflammatory cell infiltration and fibrous tissue proliferation， reduced serum content of SOD and CAT（P<0.01），increased content of HYP and MDA（P<0.01）， down-regulated protein and mRNA expression of Nrf2，HO-1， and NQO1（P<0.01），and up-regulated protein and mRNA expression of Keap1 （P<0.05，P<0.01）. Compared with the model group，the high- and medium-dose Phyllanthi Fructus groups showed significantly restored morphological structure of lung tissues with reduced collagen deposition， increased serum content of SOD and CAT（P<0.05，P<0.01），decreased content of HYP and MDA（P<0.01）， up-regulated protein and mRNA expression of Nrf2，HO-1， and NQO1 （P<0.05，P<0.01），and down-regulated protein and mRNA expression of Keap1（P<0.05，P<0.01）. ConclusionThe alcohol extract of Phyllanthi Fructus can inhibit pulmonary fibrosis in silicosis mice，and the underlying mechanism may be related to the regulation of the Nrf2/antioxidant response element （ARE） signaling pathway.
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The pathological manifestations of neurodegenerative diseases， such as Alzheimer's disease， Parkinson's disease， and multiple sclerosis， are abnormal protein aggregation and accumulation， microglia activation， and mitochondrial dysfunction， which eventually lead to the gradual loss of neuronal structure or function and deteriorate over time. These pathological processes are related to the production of reactive oxygen species （ROS）， which can cause oxidative stress and damage proteins， lipids， and DNA， leading to cell and tissue injuries. The Kelch-like ECH-associated protein 1 （Keap1）/nuclear factor E2-related factor 2 （Nrf2）/antioxidant response element （ARE） signaling pathway is the main mechanism to maintain the redox balance of the body and defend against oxidative stress injury. Nrf2 activates the expression of a series of antioxidant genes related to ARE through the dissociation of Keap1 and nuclear transfer in the cytoplasm to protect the body from oxidative damage. Therefore， the discovery and study of the Keap1/Nrf2/ARE signaling pathway activator is of great significance for the prevention and treatment of neurodegenerative diseases. Because of the remarkable biological activity and slight side effects， natural products are a treasure trove for new drug research and development. Studies have shown that a variety of natural products can activate the Keap1/Nrf2/ARE signaling pathway and play a neuroprotective role. According to the structural characteristics， natural products can be divided into flavonoids， terpenoids， volatile oils， polyphenols， and phenylpropanoids. This study summarized the underlying mechanism of the Keap1/Nrf2/ARE signaling pathway in regulating diseases and reviewed the research progress on natural products based on this signaling pathway in neuroprotection to provide references for the development of clinical drugs for the prevention and treatment of neurodegenerative diseases.
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ObjectiveTo verify the anti-oxidative stress effect of Huangqintang based on the nuclear factor E2-related factor 2 （Nrf2） signaling pathway by using Caco-2 cells as a carrier and RNA interference （RNAi） technology with in vitro experiments. MethodThe Caco-2 cells in the logarithmic growth phase were transfected with siRNA to construct siRNA Caco-2 cells. After normal Caco-2 cells and siRNA Caco-2 cells were incubated with Huangqintang of different doses， RNA and protein were extracted. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the mRNA and protein expression of heme oxygenase-1 （HO-1）， NAD（P）H quinone oxidoreductase 1 （NQO1）， glutathione S-transferase （GST）， Kelch-like ECH-associated protein 1 （Keap1）， and Nrf2. Meanwhile， the activities of superoxide dismutase （SOD） and GSH-Px， as well as the expression levels of malondialdehyde （MDA） and reactive oxygen species （ROS）， were detected by the colorimetric method and the probe method. ResultCompared with the results in the normal group， only the 400 mg·L-1 Huangqintang group and the sulforaphane （SFN） group could reduce the content of ROS and MDA in Caco-2 cells （P<0.01）， while the activities of SOD and GSH-Px in the cells of the Huangqintang groups and the SFN group showed an upward trend. Furthermore， there were significant differences in the 400 mg·L-1 Huangqintang group/the SFN group and the normal group （P<0.01）. Meanwhile， the protein and mRNA expression levels of HO-1， GST， Keap1， NQO1， and Nrf2 showed an upward trend in all groups （P<0.05， P<0.01）. After transfection， compared with the normal group， the model group showed increased content of MDA and ROS， blunted activities of GSH-Px and SOD， and reduced protein and mRNA expression of HO-1， GST， Keap1， and NQO1 （P<0.05， P<0.01）. After drug incubation， compared with the model group， the SFN group showed potentiated SOD activity， and the SFN group and the Huangqintang groups showed enhanced GSH-Px activity （P<0.01）. Moreover， the activities of SOD and GSH-Px in the 400 and 200 mg·L-1 Huangqintang groups and the SFN group showed an upward trend （P<0.01）， and the content of MDA in the 400 mg·L-1 Huangqintang group and the SFN group showed a downward trend. ROS decreased in all groups with drug intervention （P<0.01）， and the protein and mRNA expression of HO-1， GST， Keap1， NQO1， and Nrf2 increased to varying degrees （P<0.05， P<0.01）. ConclusionHuangqintang can play an anti-oxidative stress role by regulating the Nrf2 pathway.
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Ulcerative colitis （UC） is a commonly seen digestive system disease with unclear pathogenesis. The condition is complex and variable， often chronic， and has a long treatment period with no specific cure. Currently， the treatment of UC often involves the use of corticosteroids， aminosalicylates， and biologics in western medicine， which provide fast-acting and definite efficacy in the short term. However， with prolonged medication， some patients may develop drug resistance and worsening of the disease， leading to the occurrence of colon cancer. Research has found that oxidative stress is one of the important pathogenic factors in UC and influences its onset and development. Oxidative stress is a state of imbalance between oxidative products and the antioxidant system in the body， characterized by overexpression of oxidative products such as malondialdehyde （MDA）， reactive oxygen species （ROS）， nitric oxide （NO）， or deficiency of antioxidant enzymes such as superoxide dismutase （SOD）， catalase （CAT）， and glutathione （GSH）. It is worth noting that traditional Chinese medicine （TCM）， as a unique characteristic medicine of China， has achieved significant efficacy in the treatment of UC. Studies have shown that TCM effectively inhibits the occurrence of UC by suppressing the accumulation of metabolites and antagonizes the development of UC by enhancing the antioxidant system. Therefore， using TCM to regulate the oxidative balance as a diagnostic and therapeutic approach may be a new method and direction for the treatment of UC in the future. Based on the above research， this article summarized the mechanisms of key pathogenic proteins in oxidative stress and the occurrence and development of UC， and compiled the effective ingredients of Chinese medicine， single drugs， prescriptions， and acupuncture and moxibustion in regulating upstream and downstream target proteins of oxidative stress. These interventions can reduce pathological damage to the intestinal mucosa， lower the colon injury index， enrich the intestinal microbiota， increase colon length， and improve clinical symptoms of UC. The article is expected to expand the application of TCM in the treatment of UC and provide a reliable scientific theoretical basis.
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ObjectiveTo investigate the effect and mechanism of Shenling Baizhusan on the treatment of oligoasthenospermia with hyperuricemia （HUA）. MethodThirty-two male Kunming （KM） mice were randomly divided into blank group （n=6）， model group （n=6）， high-dose Shenling Baizhusan group （n=7）， low-dose Shenling Baizhusan group （n=7）， and febuxostat group （n=6）. Except for the blank group， all other groups received intraperitoneal injection of potassium oxazinate suspension （600 mg·kg-1） for 7 days. After modeling， the high-dose Shenling Baizhusan group and the low-dose Shenling Baizhusan group were orally administered with 20.14 g·kg-1 and 10.07 g·kg-1 of Shenling Baizhusan， respectively. The Febuxostat group was orally administered with 0.25 g·kg-1 of Febuxostat， while the blank group and model group were orally administered with the same volume of physiological saline. Oral administration was performed once a day for 14 consecutive days， after which samples were collected. Biochemical methods were used to measure serum uric acid （UA）， superoxide dismutase （SOD） and malondialdehyde （MDA） in testicular tissue. Hematoxylin-eosin （HE） staining was used to observe the histopathological changes in testicular tissue and evaluate the spermatogenesis function. Automated sperm analyzer was used to measure sperm density and motility. Single-cell gel electrophoresis （SCGE） was used to assess sperm DNA integrity. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） was used to detect testicular cell apoptosis rate. Western blot analysis was performed to measure the protein expression levels of Kelch-like ECH-associated protein 1 （Keap1）， nuclear factor E2-related factor 2 （Nrf2）， heme oxygenase-1 （HO-1）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and Caspase-3 in testicular tissue. Real-time polymerase chain reaction （PCR） was conducted to evaluate the mRNA expression levels of Keap1， Nrf2， and HO-1 in testicular tissue. ResultCompared with the blank group， the model group showed elevated serum UA level （P<0.01）， decreased testicular spermatogenesis function， sperm density， and motility （P<0.01）， and increased sperm trailing rate and testicular cell apoptosis rate （P<0.01）. Compared with the model group， the high-dose Shenling Baizhusan group showed significant improvements in the above-mentioned indicators （P<0.05， P<0.01）. Additionally， the expression levels of Keap1， Bax， and Caspase-3 in testicular tissue were reduced， while the expression levels of Nrf2， HO-1， and Bcl-2 increased （P<0.05， P<0.01）. The mRNA level of Keap1 decreased （P<0.05， P<0.01）， while the mRNA levels of Nrf2 and HO-1 increased （P<0.05， P<0.01）. ConclusionShenling Baizhusan can significantly improve HUA oligoasthenospermia， and its mechanism may be related to the Nrf2/antioxidant response element （ARE） signaling pathway.
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Ulcerative colitis （UC） is a common chronic digestive system disease in clinic. The disease is repeated and difficult to treat， and the pathogenesis is complex， which is related to oxidative stress response. Nuclear factor E2-related factor 2 （Nrf2） is an important factor in antioxidant reaction， which regulates the expression of downstream heme oxygen-1 （HO-1）， and plays a role in maintaining redox homeostasis. In the course of UC， the biological activity and content of Nrf2 and HO-1 are decreased， the antioxidant and anti-inflammatory abilities of tissues are weakened， the intestinal epithelial cells are damaged， and the intestinal mucosal barrier is destroyed. At present， western medicine mainly focuses on controlling inflammation and alleviating symptoms in clinical treatment. Although it has a certain effect， there are many problems such as easy recurrence after drug withdrawal and long-term side effects. Studies have shown that Chinese medicine has rich and flexible therapeutic methods and has broad application prospects in the prevention and treatment of UC. In recent years， with Nrf2/HO-1 pathway as the entry point， a large number of basic and clinical experiments on this signal in UC have been carried out in the field of Chinese medicine， and the results show that the intervention of Nrf2/HO-1 pathway is an important potential target for the treatment of UC by Chinese medicines. Based on the etiology and pathogenesis of deficiency-excess in complexity， Chinese medicine regulates Nrf2/HO-1 pathway by clearing heat and detoxifying dampness， activating blood circulation to remove stasis and relieve pain， invigorating Qi， tonifying middle， and warming interior， and treating both symptoms and root causes， to improve the tissue's ability to resist oxidative stress， maintain the balance between pro-inflammatory factors and anti-inflammatory factors， relieve inflammatory response， and play a therapeutic role in UC. This paper summarized and analyzed the effect of Chinese medicine targeting the Nrf2/HO-1 signaling pathway on interfering with UC and its mechanism. The purpose of this study is to provide references for researchers to have a more comprehensive understanding of the mechanism of Chinese medicines in the Nrf2/HO-1 signaling pathway in UC and promote the rational application of Chinese medicines in the prevention and treatment of UC in the future.
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Engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties. In this study, we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32 (WB800-KR32) using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli (ETEC) K88 in weaned piglets. Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet. The feed of the control group (CON) was infused with normal sterilized saline; meanwhile, the ETEC, ETEC+WB800, and ETEC+WB800-KR32 groups were orally administered normal sterilized saline, 5×1010 CFU (CFU: colony forming units) WB800, and 5×1010 CFU WB800-KR32, respectively, on Days 1‒14 and all infused with ETEC K88 1×1010 CFU on Days 15‒17. The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance, improved the mucosal activity of antioxidant enzyme (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) and decreased the content of malondialdehyde (MDA). More importantly, WB800-KR32 downregulated genes involved in antioxidant defense (GPx and SOD1). Interestingly, WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum. WB800-KR32 markedly changed the richness estimators (Ace and Chao) of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces. The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway, providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.
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Animals , Swine , Enterotoxigenic Escherichia coli , Kelch-Like ECH-Associated Protein 1 , Bacillus subtilis , NF-E2-Related Factor 2 , Antioxidants , Oxidative StressABSTRACT
Myocardial infarction （MI） is a common cardiovascular disease in clinical practice and one of the main causes of cardiovascular mortality. Its pathogenesis is complex and associated with oxidative stress reactions. Nuclear factor E2-related factor 2 （Nrf2） is a key factor in regulating oxidative stress reactions. It can regulate the expression of heme oxygenase-1 （HO-1）， playing a role in maintaining the oxidative-reductive homeostasis in the body. During the course of MI， the biological activity and levels of Nrf2 and HO-1 decrease， leading to weakened tissue antioxidant and anti-inflammatory capabilities， endothelial damage in myocardial blood vessels， release of vascular cell adhesion factors， and impaired endothelial function. In recent years， many basic research studies have explored the role and mechanisms of traditional Chinese medicine （TCM） in treating MI by modulating the Nrf2/HO-1 signaling pathway. The results have indicated that the Nrf2/HO-1 signaling pathway is an important potential target for TCM in the treatment of MI. This article reviewed the mechanism of the Nrf2/HO-1 signaling pathway in MI and the research progress of TCM in targeting and regulating this pathway， aiming to provide a theoretical basis for the prevention and treatment of MI and further drug development.
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Cell-free hemoglobin refers to the rupture of erythrocyte membrane due to instability of erythrocyte membrane caused by various causes, and hemoglobin is released free in the plasma.High concentration of plasma cell-free hemoglobin can cause intracellulariron ion deposition, oxidative damage, damage of lung, kidney and other related tissue, and seriously affect the treatment and prognosis of patients with sepsis.Nuclear factor E2-related factor 2(NRF2) as an antioxidant cytokine at the same time, plays important roles in the process of adjusting the resistance to oxidative stress, but the mechanism is not thorough.This review summarized the sepsis cell-free hemoglobin production, metabolism, and the activation of NRF2 and antioxidant mechanisms, to provide reference for further research.
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Since the production and use of paraquat was banned in China in 2016, the use of diquat (DQ) has been increasing and the clinical cases of DQ poisoning have also shown an increasing trend every year. The treatment of DQ poisoning is a worldwide medical problem, and there is no specific antidote. Studies have found that oxidative stress, lipid peroxidation, neurotoxicity, reproductive and developmental toxicity play an important role in DQ poisoning. Nuclear factor E2-related factor 2 (Nrf2) can inhibit oxidative stress, lipid peroxidation and inflammation by regulating the protein expression of upstream and downstream signaling molecules. Therefore, the role of Nrf2 signaling pathway in the poisoning and treatment of DQ has become a hot spot of attention for emergency critical care researchers in recent years. This paper reviews the relationship between Nrf2 signal pathway and DQ poisoning, in order to provide a theoretical basis for improving the treatment strategy for DQ poisoning.