OBJECTIVE: To investigate the effects and underlying mechanisms of VX765 on osteoarthritis (OA) and chondrocytes inflammation in rats. METHODS: Chondrocytes were isolated from the knee joints of 4-week-old Sprague Dawley (SD) rats. The third-generation cells were subjected to cell counting kit 8 (CCK-8) analysis to assess the impact of various concentrations (0, 1, 5, 10, 20, 50, 100 μmol/L) of VX765 on rat chondrocyte activity. An in vitro lipopolysaccharide (LPS) induced cell inflammation model was employed, dividing cells into control group, LPS group, VX765 concentration 1 group and VX765 concentration 2 group without obvious cytotoxicity. Western blot, real-time fluorescence quantitative PCR, and ELISA were conducted to measure the expression levels of inflammatory factors-transforming growth factor β ₁ (TGF-β ₁), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Additionally, Western blot and immunofluorescence staining were employed to assess the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Thirty-two SD rats were randomly assigned to sham surgery group (group A), OA group (group B), OA+VX765 (50 mg/kg) group (group C), and OA+VX765 (100 mg/kg) group (group D), with 8 rats in each group. Group A underwent a sham operation with a medial incision, while groups B to D underwent additional transverse incisions to the medial collateral ligament and anterior cruciate ligament, with removal of the medial meniscus. One week post-surgery, groups C and D were orally administered 50 mg/kg and 100 mg/kg VX765, respectively, while groups A and B received an equivalent volume of saline. Histopathological examination using HE and safranin-fast green staining was performed, and Mankin scoring was utilized for evaluation. Immunohistochemical staining technique was employed to analyze the expressions of matrix metalloproteinase 13 (MMP-13) and collagen type Ⅱ. RESULTS: The CCK-8 assay indicated a significant decrease in cell viability at VX765 concentrations exceeding 10 μmol/L ( P<0.05), so 4 μmol/L and 8 μmol/L VX765 without obvious cytotoxicity were selected for subsequent experiments. Following LPS induction, the expressions of TGF-β ₁, IL-6, and TNF-α in cells significantly increased when compared with the control group ( P<0.05). However, intervention with 4 μmol/L and 8 μmol/L VX765 led to a significant decrease in expression compared to the LPS group ( P<0.05). Western blot and immunofluorescence staining demonstrated a significant upregulation of Nrf2 pathway-related molecules Nrf2 and HO-1 protein expressions by VX765 ( P<0.05), indicating Nrf2 pathway activation. Histopathological examination of rat knee joint tissues and immunohistochemical staining revealed that, compared to group B, treatment with VX765 in groups C and D improved joint structural damage in rat OA, alleviated inflammatory reactions, downregulated MMP-13 expression, and increased collagen type Ⅱ expression. CONCLUSION VX765 can improve rat OA and reduce chondrocyte inflammation, possibly through the activation of the Nrf2 pathway.
Rats ; Animals ; Chondrocytes/metabolism* ; Matrix Metalloproteinase 13/metabolism* ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha/metabolism* ; Collagen Type II/metabolism* ; Interleukin-6 ; Lipopolysaccharides/pharmacology* ; NF-E2-Related Factor 2/pharmacology* ; Inflammation/drug therapy* ; Osteoarthritis/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Dipeptides ; para-Aminobenzoates

Diabetes has long been considered a risk factor in implant therapy and impaired wound healing in soft and hard oral tissues. Magnesium has been proved to promote bone healing under normal conditions. Here, we elucidate the mechanism by which Mg²⁺ promotes angiogenesis and osseointegration in diabetic status. We generated a diabetic mice model and demonstrated the alveolar bone healing was compromised, with significantly decreased angiogenesis. We then developed Mg-coating implants with hydrothermal synthesis. These implants successfully improved the vascularization and osseointegration in diabetic status. Mechanically, Mg²⁺ promoted the degradation of Kelch-like ECH-associated protein 1 (Keap1) and the nucleation of nuclear factor erythroid 2-related factor 2 (Nrf2) by up-regulating the expression of sestrin 2 (SESN2) in endothelial cells, thus reducing the elevated levels of oxidative stress in mitochondria and relieving endothelial cell dysfunction under hyperglycemia. Altogether, our data suggested that Mg²⁺ promoted angiogenesis and osseointegration in diabetic mice by regulating endothelial mitochondrial metabolism.
Mice ; Animals ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Magnesium/metabolism* ; Osseointegration ; Diabetes Mellitus, Experimental/metabolism* ; Endothelial Cells/metabolism* ; NF-E2-Related Factor 2/metabolism*

BACKGROUND: Cancer stem-like cells (CSCs) are a small subset of cells in tumors that exhibit self-renewal and differentiation properties. CSCs play a vital role in tumor formation, progression, relapse, and therapeutic resistance. B7-H3, an immunoregulatory protein, has many protumor functions. However, little is known about the mechanism underlying the role of B7-H3 in regulating gastric cancer (GC) stemness. Our study aimed to explore the impacts of B7-H3 on GC stemness and its underlying mechanism. METHODS: GC stemness influenced by B7-H3 was detected both in vitro and in vivo . The expression of stemness-related markers was examined by reverse transcription quantitative polymerase chain reaction, Western blotting, and flow cytometry. Sphere formation assay was used to detect the sphere-forming ability. The underlying regulatory mechanism of B7-H3 on the stemness of GC was investigated by mass spectrometry and subsequent validation experiments. The signaling pathway (Protein kinase B [Akt]/Nuclear factor erythroid 2-related factor 2 [Nrf2] pathway) of B7-H3 on the regulation of glutathione (GSH) metabolism was examined by Western blotting assay. Multi-color immunohistochemistry (mIHC) was used to detect the expression of B7-H3, cluster of differentiation 44 (CD44), and Nrf2 on human GC tissues. Student's t -test was used to compare the difference between two groups. Pearson correlation analysis was used to analyze the relationship between two molecules. The Kaplan-Meier method was used for survival analysis. RESULTS: B7-H3 knockdown suppressed the stemness of GC cells both in vitro and in vivo . Mass spectrometric analysis showed the downregulation of GSH metabolism in short hairpin B7-H3 GC cells, which was further confirmed by the experimental results. Meanwhile, stemness characteristics in B7-H3 overexpressing cells were suppressed after the inhibition of GSH metabolism. Furthermore, Western blotting suggested that B7-H3-induced activation of GSH metabolism occurred through the AKT/Nrf2 pathway, and inhibition of AKT signaling pathway could suppress not only GSH metabolism but also GC stemness. mIHC showed that B7-H3 was highly expressed in GC tissues and was positively correlated with the expression of CD44 and Nrf2. Importantly, GC patients with high expression of B7-H3, CD44, and Nrf2 had worse prognosis ( P = 0.02). CONCLUSIONS B7-H3 has a regulatory effect on GC stemness and the regulatory effect is achieved through the AKT/Nrf2/GSH pathway. Inhibiting B7-H3 expression may be a new therapeutic strategy against GC.
Humans ; Cell Line, Tumor ; Neoplasm Recurrence, Local ; NF-E2-Related Factor 2/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction ; Stomach Neoplasms

To investigate the protective effect and the potential mechanism of leonurine(Leo) against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells), an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins. HK-2 cells were cultured in vitro, and the effects of Leo on the viability of HK-2 cells at 10, 20, 40, 60, 80 and 100 μmol·L~(-1) were examined by CCK-8 assay to determine the safe dose range of Leo administration. A ferroptosis cell model was induced by erastin, a common ferroptosis inducer, and the appropriate concentrations were screened. CCK-8 assay was used to detect the effects of Leo(20, 40, 80 μmol·L~(-1)) and positive drug ferrostatin-1(Fer-1, 1, 2 μmol·L~(-1)) on the viability of ferroptosis model cells, and the changes of cell morphology were observed by phase contrast microscopy. Then, the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2) activation, and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis. Flow cytometry was performed to detect reactive oxygen species(ROS), and the level of glutathione(GSH) was measured using a GSH assay kit. The expressions of glutathione peroxidase 4(GPX4), p62, and heme oxygenase 1(HO-1) in each group were quantified by Western blot. RESULTS:: showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100 μmol·L~(-1). The viability of HK-2 cells decreased as the concentration of erastin increased, and 5 μmol·L~(-1) erastin significantly induced ferroptosis in the cells. Compared with the model group, Leo dose-dependently increased cell via-bility and improved cell morphology, and 80 μmol·L~(-1) Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus. Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin, inhibited the release of intracellular ROS, elevated GSH and GPX4, promoted the nuclear translocation of Nrf2, and significantly upregulated the expression of p62 and HO-1 proteins. In conclusion, Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells, which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.
Humans ; Ferroptosis ; Reactive Oxygen Species/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Sincalide/pharmacology* ; Signal Transduction ; Epithelial Cells/metabolism* ; Glutathione

To investigate the antidepressant mechanism of Shenling Kaixin Granules(SLKX) in treating chronic unpredictable mild stress(CUMS) model rats. Ninety male SD rats were randomly divided into control group, model group, Shugan Jieyu Capsules(110 mg·kg~(-1)) group and SLKX low-(90 mg·kg~(-1)), medium-(180 mg·kg~(-1)), and high-dose(360 mg·kg~(-1)) groups. Depression rat model was replicated by CUMS method. After treatment, the behavioral changes of rats were evaluated by sugar preference, open field, elevated cross maze and forced swimming experiments. The contents of interleukin 1 beta(IL-1β), tumor necrosis factor α(TNF-α), brain-derived neurotrophic factor(BDNF) and 5-hydroxytryptamine(5-HT) in serum were determined by enzyme linked immunosorbent assay(ELISA), and the activities of superoxide dismutase(SOD) and catalase(CAT) in hippocampal CA1 region were also detected. Pathological changes in hippocampal CA1 region were detected by hematoxylin-eosin(HE) staining, and Western blot was used to determine the expression of nerve growth factor(NGF), BDNF, phospho-tyrosine kinase receptor(p-TrkB)/TrkB, phospho-cAMP-response element binding protein(p-CREB)/CREB, 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 hippocampal CA1 region. RESULTS:: showed that compared with the control group, the model group had decreased sugar preference, reduced number of entries and time spent in the center of open field and shortened total distance of movement, reduced number of entries and proportion of time spent in open arm, and increased number and time of immobility in forced swimming experiment. Additionally, the serum contents of IL-1β and TNF-α and the expression of caspase-3 were higher, while the contents of BDNF and 5-HT, the activities of SOD and CAT in hippocampal CA1 region, the expressions of NGF, BDNF, p-TrkB/TrkB, p-CREB/CREB, HO-1 and Bcl-2/Bax, and the Nrf2 nuclear translocation were lower in model group than in control group. Compared with the conditions in model group, the sugar preference, the number of entries and time spent in the center of open, total distance of movement, and the number of entries and proportion of time spent in open arm in treatment groups were increased while the number and time of immobility in forced swimming experiment were decreased; the serum contents of IL-1β and TNF-α and the expression of caspase-3 were down regulated, while the contents of BDNF and 5-HT, the activities of SOD and CAT in hippocampal CA1 region, the expressions of NGF, BDNF, p-TrkB/TrkB, p-CREB/CREB, HO-1, Bcl-2/Bax, and Nrf2 nuclear translocation were enhanced. In conclusion, SLKX might regulate the Nrf2 nucleus translocation by activating BDNF/TrkB/CREB pathway, lower oxidative stress damage in hippocampus, inhibit caspase-3 activity, and reduce apoptosis of hippocampal nerve cells, thereby playing an antidepressant role.
Rats ; Male ; Animals ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Nerve Growth Factor/metabolism* ; Brain-Derived Neurotrophic Factor/metabolism* ; Signal Transduction ; Tumor Necrosis Factor-alpha/metabolism* ; Serotonin/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Rats, Sprague-Dawley ; Antidepressive Agents/pharmacology* ; Hippocampus/metabolism* ; Superoxide Dismutase/metabolism* ; Sugars/pharmacology* ; Depression/genetics* ; Stress, Psychological/metabolism*

This study aims to explore the neuroprotective mechanism of ginsenoside Re(GS-Re) on drosophila model of Parkinson's disease(PD) induced by rotenone(Rot). To be specific, Rot was used to induce PD in drosophilas. Then the drosophilas were grouped and respectively treated(GS-Re: 0.1, 0.4, 1.6 mmol·L~(-1); L-dopa: 80 μmol·L~(-1)). Life span and crawling ability of drosophilas were determined. The brain antioxidant activity [content of catalase(CAT), malondialdehyde(MDA), reactive oxygen species(ROS), superoxide dismutase(SOD)], dopamine(DA) content, and mitochondrial function [content of adenosine triphosphate(ATP), NADH:ubiquinone oxidoreductase subunit B8(NDUFB8) Ⅰ activity, succinate dehydrogenase complex, subunit B(SDHB) Ⅱ activity] were detected by enzyme-linked immunosorbent assay(ELISA). The number of DA neurons in the brains of drosophilas was measured with the immunofluorescence method. The levels of NDUFB8 Ⅰ, SDHB Ⅱ, cytochrome C(Cyt C), nuclear factor-E2-related factor 2(Nrf2), heme oxygenase-1(HO-1), B-cell lymphoma/leukemia 2(Bcl-2)/Bcl-2-assaciated X protein(Bax), and cleaved caspase-3/caspase-3 in the brain were detected by Western blot. The results showed that model group [475 μmol·L~(-1) Rot(IC_(50))] demonstrated significantly low survival rate, obvious dyskinesia, small number of neurons and low DA content in the brain, high ROS level and MDA content, low content of SOD and CAT, significantly low ATP content, NDUFB8 Ⅰ activity, and SDHB Ⅱ activity, significantly low expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax, large amount of Cyt C released from mitochondria to cytoplasm, low nuclear transfer of Nrf2, and significantly high expression of cleaved caspase-3/caspase-3 compared with the control group. GS-Re(0.1, 0.4, and 1.6 mmol·L~(-1)) significantly improved the survival rate of PD drosophilas, alleviated the dyskinesia, increased DA content, reduced the loss of DA neurons, ROS level, and MDA content in brain, improved content of SOD and CAT and antioxidant activity in brain, maintained mitochondrial homeostasis(significantly increased ATP content and activity of NDUFB8 Ⅰ and SDHB Ⅱ, significantly up-regulated expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax), significantly reduced the expression of Cyt C, increased the nuclear transfer of Nrf2, and down-regulated the expression of cleaved caspase-3/caspase-3. In conclusion, GS-Re can significantly relieve the Rot-induced cerebral neurotoxicity in drosophilas. The mechanism may be that GS-Re activates Keap1-Nrf2-ARE signaling pathway by maintaining mitochondrial homeostasis, improves antioxidant capacity of brain neurons, then inhibits mitochondria-mediated caspase-3 signaling pathway, and the apoptosis of neuronal cells, thereby exerting the neuroprotective effect.
Animals ; Reactive Oxygen Species/metabolism* ; Antioxidants/pharmacology* ; Oxidative Stress ; NF-E2-Related Factor 2/metabolism* ; Caspase 3/metabolism* ; Parkinson Disease/genetics* ; bcl-2-Associated X Protein/metabolism* ; Neuroprotective Agents/pharmacology* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Drosophila/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Superoxide Dismutase/metabolism* ; Adenosine Triphosphate/pharmacology*

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia seen in clinical settings, which has been associated with substantial rates of mortality and morbidity. However, clinically available drugs have limited efficacy and adverse effects. We aimed to investigate the mechanisms of action of andrographolide (Andr) with respect to AF. We used network pharmacology approaches to investigate the possible therapeutic effect of Andr. To define the role of Andr in AF, HL-1 cells were pro-treated with Andr for 1 h before rapid electronic stimulation (RES) and rabbits were pro-treated for 1 d before rapid atrial pacing (RAP). Apoptosis, myofibril degradation, oxidative stress, and inflammation were determined. RNA sequencing (RNA-seq) was performed to investigate the relevant mechanism. Andr treatment attenuated RAP-induced atrial electrophysiological changes, inflammation, oxidative damage, and apoptosis both in vivo and in vitro. RNA-seq indicated that oxidative phosphorylation played an important role. Transmission electron microscopy and adenosine triphosphate (ATP) content assay respectively validated the morphological and functional changes in mitochondria. The translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus and the molecular docking suggested that Andr might exert a therapeutic effect by influencing the Keap1-Nrf2 complex. In conclusions, this study revealed that Andr is a potential preventive therapeutic drug toward AF via activating the translocation of Nrf2 to the nucleus and the upregulation of heme oxygenase-1 (HO-1) to promote mitochondrial bioenergetics.
Animals ; Rabbits ; Atrial Fibrillation/metabolism* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Signal Transduction ; NF-E2-Related Factor 2/pharmacology* ; Molecular Docking Simulation ; Oxidative Stress ; Energy Metabolism ; Mitochondria/metabolism* ; Inflammation/metabolism* ; Heme Oxygenase-1

OBJECTIVE: To investigate the role and mechanism of silent information regulator 1 (SIRT1) in regulating nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in oxidative stress and inflammatory response to sepsis-induced liver injury. METHODS: A total of 24 male Sprague-Dawley (SD) rats were randomly divided into sham operation (Sham) group, cecal ligation and puncture (CLP) group, SIRT1 agonist SRT1720 pretreatment (CLP+SRT1720) group and SIRT1 inhibitor EX527 pretreatment (CLP+EX527) group, with 6 rats in each group. Two hours before operation, SRT1720 (10 mg/kg) or EX527 (10 mg/kg) were intraperitoneally injected into the CLP+SRT1720 group and CLP+EX527 group, respectively. Blood was collected from the abdominal aorta at 24 hours after modeling and the rats were sacrificed for liver tissue. The serum levels of interleukins (IL-6, IL-1β) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by microplate method. Hematoxylin-eosin (HE) staining was used to observe the pathological injury of rats in each group. The levels of malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), glutathione (GSH) and superoxide dismutase (SOD) in liver tissue were detected by corresponding kits. The mRNA and protein expressions of SIRT1, Nrf2 and HO-1 in liver tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. RESULTS: Compared with the Sham group, the serum levels of IL-6, IL-1β, TNF-α, ALT and AST in the CLP group were significantly increased; histopathological results showed that liver cords were disordered, hepatocytes were swollen and necrotic, and a large number of inflammatory cells infiltrated; the contents of MDA and 8-OHdG in liver tissue increased, while the contents of GSH and SOD decreased; and the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 in liver tissues were significantly decreased. These results suggest that sepsis rats have liver dysfunction, and the levels of SIRT1, Nrf2, HO-1 and antioxidant protein in liver tissues were decreased, while the levels of oxidative stress and inflammation were increased. Compared with the CLP group, the levels of inflammatory factors and oxidative stress were significantly decreased in the CLP+SRT1720 group, the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 were significantly increased [IL-6 (ng/L): 34.59±4.21 vs. 61.84±3.78, IL-1β (ng/L): 41.37±2.70 vs. 72.06±3.14, TNF-α (ng/L): 76.43±5.23 vs. 130.85±5.30, ALT (U/L): 30.71±3.63 vs. 64.23±4.59, AST (U/L): 94.57±6.08 vs. 145.15±6.86, MDA (μmol/g): 6.11±0.28 vs. 9.23±0.29, 8-OHdG (ng/L): 117.43±10.38 vs. 242.37±11.71, GSH (μmol/g): 11.93±0.88 vs. 7.66±0.47, SOD (kU/g): 121.58±5.05 vs. 83.57±4.84, SIRT1 mRNA (2^-ΔΔCt): 1.20±0.13 vs. 0.46±0.02, Nrf2 mRNA (2^-ΔΔCt): 1.21±0.12 vs. 0.58±0.03, HO-1 mRNA (2^-ΔΔCt): 1.71±0.06 vs. 0.48±0.07, SIRT1 protein (SIRT1/β-actin): 0.89±0.04 vs. 0.58±0.03, Nrf2 protein (Nrf2/β-actin): 0.87±0.08 vs. 0.51±0.09, HO-1 protein (HO-1/β-actin): 0.93±0.14 vs. 0.54±0.12, all P < 0.05], these results indicated that SIRT1 agonist SRT1720 pretreatment could improve liver injury in sepsis rats. However, pretreatment with SIRT1 inhibitor EX527 showed the opposite effect [IL-6 (ng/L): 81.05±6.47 vs. 61.84±3.78, IL-1β (ng/L): 93.89±5.83 vs. 72.06±3.14, TNF-α (ng/L): 177.67±5.12 vs. 130.85±5.30, ALT (U/L): 89.33±9.52 vs. 64.23±4.59, AST (U/L): 179.59±6.44 vs. 145.15±6.86, MDA (μmol/g): 11.39±0.51 vs. 9.23±0.29, 8-OHdG (ng/L): 328.83±11.26 vs. 242.37±11.71, GSH (μmol/g): 5.07±0.34 vs. 7.66±0.47, SOD (kU/g): 59.37±4.28 vs. 83.57±4.84, SIRT1 mRNA (2^-ΔΔCt): 0.34±0.03 vs. 0.46±0.02, Nrf2 mRNA (2^-ΔΔCt): 0.46±0.04 vs. 0.58±0.03, HO-1 mRNA (2^-ΔΔCt): 0.21±0.03 vs. 0.48±0.07, SIRT1 protein (SIRT1/β-actin): 0.47±0.04 vs. 0.58±0.03, Nrf2 protein (Nrf2/β-actin): 0.32±0.07 vs. 0.51±0.09, HO-1 protein (HO-1/β-actin): 0.19±0.09 vs. 0.54±0.12, all P < 0.05]. CONCLUSIONS SIRT1 can inhibit the release of proinflammatory factors and alleviate the oxidative damage of hepatocytes by activating Nrf2/HO-1 signaling pathway, thus playing a protective role against CLP-induced liver injury.
Animals ; Male ; Rats ; Actins/metabolism* ; Chemical and Drug Induced Liver Injury, Chronic ; Heme Oxygenase-1/metabolism* ; Interleukin-6 ; NF-E2-Related Factor 2/metabolism* ; Rats, Sprague-Dawley ; RNA, Messenger ; Sepsis/metabolism* ; Signal Transduction ; Sirtuin 1/metabolism* ; Superoxide Dismutase/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

Lignans derived from Eucommia ulmoides Oliver (Eucommia lignans) inhibit the progression of inflammatory diseases, while their effect on the progression of diabetic nephropathy (DN) remained unclear. This work was designed to assess the function of Eucommia lignans in DN. The major constituents of Eucommia lignans were analyzed by UPLC-Q-TOF-MS/MS. The binding between Eucommia lignans and aldose reductase (AR) was predicted by molecular docking. Eucommia lignans (200, 100, and 50 mg·kg^-1) were used in model animals to evaluate their renal function changes. Rat glomerular mesangial cells (HBZY-1) were transfected with sh-AR, sh-AMPK, and oe-AR in the presence of high glucose (HG) or HG combined with Eucommia lignans to evaluate whether Eucommia lignans affected HG-induced cell injury and mitochondrial dysfunction through the AR/Nrf2/HO-1/AMPK axis. Eucommia lignans significantly attenuated the progression of DN in vivo. Eucommia lignans notably reversed HG-induced upregulation of inflammatory cytokines and mitochondrial injury, while downregulating the levels of Cyto c, caspase 9, AR, and NOX4 in HBZY-1 cells. In contrast, HG-induced downregulation of Nrf2, HO-1 and p-AMPKα levels were abolished by Eucommia lignans. Meanwhile, knockdown of AR exerted similar therapeutic effect of Eucommia lignans on DN progression, and AR overexpression reversed the effect of Eucommia lignans. Eucommia lignans alleviated renal injury through the AR/Nrf2/HO-1/AMPK axis. Thus, these findings might provide evidence for the use of Eucommia lignans in treating DN.
Animals ; Rats ; AMP-Activated Protein Kinases/genetics* ; Diabetes Mellitus ; Diabetic Nephropathies/prevention & control* ; Eucommiaceae/metabolism* ; Lignans/therapeutic use* ; Molecular Docking Simulation ; NF-E2-Related Factor 2/metabolism* ; Tandem Mass Spectrometry

OBJECTIVE: To explore the mechanism underlying the inhibitory effect of quercetin against testicular oxidative damage induced by a mixture of 3 commonly used phthalates (MPEs) in rats. METHODS: Forty male Sprague-Dawley rats were randomly divided into control group, MPEs exposure group, and MPEs with low-, median- and high-dose quercetin treatment groups. For MPEs exposure, the rats were subjected to intragastric administration of MPEs at the daily dose of 900 mg/kg for 30 consecutive days; Quercetin treatments were administered in the same manner at the daily dose of 10, 30, and 90 mg/kg. After the treatments, serum levels of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH), and testicular malondialdeyhde (MDA), catalase (CAT) and superoxide dismutase (SOD) were detected, and testicular pathologies of the rats were observed with HE staining. The expressions of nuclear factor-E2-related factor 2 (Nrf2), Kelch-like ECH2 associated protein 1 (Keap1) and heme oxygenase 1 (HO-1) in the testis were detected using immunofluorescence assay and Western blotting. RESULTS: Compared with the control group, the rats with MPEs exposure showed significant reductions of the anogenital distance, weight of the testis and epididymis, and the coefficients of the testis and epididymis with lowered serum testosterone, LH and FSH levels (P < 0.05). Testicular histological examination revealed atrophy of the seminiferous tubules, spermatogenic arrest, and hyperplasia of the Leydig cells in MPEs-exposed rats. MPEs exposure also caused significant increments of testicular Nrf2, MDA, SOD, CAT and HO-1 expressions and lowered testicular Keap1 expression (P < 0.05). Treatment with quercetin at the median and high doses significantly ameliorated the pathological changes induced by MPEs exposure (P < 0.05). CONCLUSION Quercetin treatment inhibits MPEs-induced oxidative testicular damage in rats possibly by direct scavenging of free radicals to lower testicular oxidative stress and restore the regulation of the Nrf2 signaling pathway.
Rats ; Male ; Animals ; Testis ; Quercetin/pharmacology* ; Rats, Sprague-Dawley ; NF-E2-Related Factor 2/metabolism* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Oxidative Stress ; Testosterone/pharmacology* ; Superoxide Dismutase/metabolism* ; Follicle Stimulating Hormone ; Luteinizing Hormone