V-ATPase Deficiency Aggravates Hypoxia-induced Spermatogenesis Reduction by Promoting Spermatocyte Apoptosis via the JNK/c-Jun Pathway in Mice

Abstract Spermatocyte apoptosis is the primary cause of poor outcome after hypoxia-triggered spermatogenesis reduction (HSR). The vacuolar H+-ATPase (V-ATPase) has been found to be involved in the regulation of hypoxia-induced GC-2 cells apoptosis. However, the mechanism of V-ATPase regulating spermatocyte apoptosis after HSR hasnot been well elucidated. In this study, HSRmodel was established by hypoxia exposure in vivo in V-ATPase-knockout (V-ATPase-/-) and wild-type (WT) mice to investigate theeffectof V-ATPase deficiency on spermatocyte apoptosis. GC-2, amouse pachytene spermatocyte-derived cell line, was introduced in vitro experiments. The sperm count and spermatogenic apoptosis were recorded after 60 d of hypoxia exposure in HSR model. The apoptosis of GC-2 cells was detected by flow cytometry and TUNEL staining. The expression of JNK/c-Jun was evaluated by RNA-seq or western blot. The expression of DR5 and caspase-8 was evaluated by RT-qPCR and western blot. The expression of V-ATPase was determined by western blot in the presence and absence ofLenti-transcription factor EB (TFEB).C-Jun interference was used for evaluating the role of JNK in regulating the apoptosis of GC-2 cells byTUNEL and flow cytometry. The in vivo results suggested that hypoxia induced spermatogenesis reduction and downregulation of V-ATPase. Moreover, V-ATPase deficiency resulted in moresevere spermatogenesis reduction after hypoxia exposure. The spermatogenesis reduction was associated with exacerbation of spermatocyte apoptosis. Hypoxia down-regulated the transcription of V-ATPase through inhibiting TFEB and its nuclear translocation. The mRNA and protein expressions of V-ATPaseincreased after TFEB overexpression in GC-2 cells. Moreover, V-ATPase deficiency enhanced JNK/c-Jun activation and related DR-apoptotic pathwayin GC-2 cells.However,inhibition of c-Jun attenuated V-ATPase deficiency-induced GC-2 cells apoptosis in vitro and HSR in vivo. In conclusion, JNK/c-Jun was involved in the enhancement of V-ATPase-mediated HSR in V-ATPase -/- mice. V-ATPase deficiency aggravates spermatocyte apoptosis, which may account forthe poor spermatogenesis outcomes of V-ATPase-/- mice. The discoveredfunction of V-ATPase modulating spermatocyte apoptosis indicates its potential therapeutic effect against HSR.

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The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520619666191022160024 ◽

2020 ◽

Vol 19 (17) ◽

pp. 2108-2119

Author(s):

Yang Jin ◽

Li Lv ◽

Shu-Xiang Ning ◽

Ji-Hong Wang ◽

Rong Xiao

Keyword(s):

Wound Healing ◽

Western Blot ◽

Morphological Changes ◽

In Vivo Studies ◽

Migration And Invasion ◽

Tunel Staining ◽

Dna Ladder ◽

Western Blot Assay

Background: Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. Methods: MTT assay was used to detect the inhibitory rate of viability. Giemsa’s staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. Results: In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. Conclusion: hese results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

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RIP3 impedes transcription factor EB to suppress autophagic degradation in septic acute kidney injury

Cell Death and Disease ◽

10.1038/s41419-021-03865-8 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Ruizhao Li ◽

Xingchen Zhao ◽

Shu Zhang ◽

Wei Dong ◽

Li Zhang ◽

...

Keyword(s):

Acute Kidney Injury ◽

Transcription Factor ◽

Nuclear Translocation ◽

Kidney Injury ◽

Septic Acute Kidney Injury ◽

Transcription Factor Eb ◽

Autophagic Degradation ◽

Lps Treatment

AbstractAutophagy is an important renal-protective mechanism in septic acute kidney injury (AKI). Receptor interacting protein kinase 3 (RIP3) has been implicated in the renal tubular injury and renal dysfunction during septic AKI. Here we investigated the role and mechanism of RIP3 on autophagy in septic AKI. We showed an activation of RIP3, accompanied by an accumulation of the autophagosome marker LC3II and the autophagic substrate p62, in the kidneys of lipopolysaccharide (LPS)-induced septic AKI mice and LPS-treated cultured renal proximal tubular epithelial cells (PTECs). The lysosome inhibitor did not further increase the levels of LCII or p62 in LPS-treated PTECs. Moreover, inhibition of RIP3 attenuated the aberrant accumulation of LC3II and p62 under LPS treatment in vivo and in vitro. By utilizing mCherry-GFP-LC3 autophagy reporter mice in vivo and PTECs overexpression mRFP-GFP-LC3 in vitro, we observed that inhibition of RIP3 restored the formation of autolysosomes and eliminated the accumulated autophagosomes under LPS treatment. These results indicated that RIP3 impaired autophagic degradation, contributing to the accumulation of autophagosomes. Mechanistically, the nuclear translocation of transcription factor EB (TFEB), a master regulator of the lysosome and autophagy pathway, was inhibited in LPS-induced mice and LPS-treated PTECs. Inhibition of RIP3 restored the nuclear translocation of TFEB in vivo and in vitro. Co-immunoprecipitation further showed an interaction of RIP3 and TFEB in LPS-treated PTECs. Also, the expression of LAMP1 and cathepsin B, two potential target genes of TFEB involved in lysosome function, were decreased under LPS treatment in vivo and in vitro, and this decrease was rescued by inhibiting RIP3. Finally, overexpression of TFEB restored the autophagic degradation in LPS-treated PTECs. Together, the present study has identified a pivotal role of RIP3 in suppressing autophagic degradation through impeding the TFEB-lysosome pathway in septic AKI, providing potential therapeutic targets for the prevention and treatment of septic AKI.

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Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development

PLoS ONE ◽

10.1371/journal.pone.0246197 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0246197

Author(s):

Jorge Marquez ◽

Jianping Dong ◽

Chun Dong ◽

Changsheng Tian ◽

Ginette Serrero

Keyword(s):

Flow Cytometry ◽

Western Blot ◽

Cell Lines ◽

Cancer Cell ◽

Cancer Cell Lines ◽

Negative Regulator ◽

Target Validation ◽

Antibody Drug

Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for hematopoietic and lymphoid tumors. However, there is need to identify new targets for ADCs, particularly for solid tumors and cancers with unmet needs. From a hybridoma library developed against cancer cells, we selected the mouse monoclonal antibody 33B7, which was able to bind to, and internalize, cancer cell lines. This antibody was used for identification of the target by immunoprecipitation and mass spectrometric analysis, followed by target validation. After target validation, 33B7 binding and target positivity were tested by flow cytometry and western blot analysis in several cancer cell lines. The ability of 33B7 conjugated to saporin to inhibit in vitro proliferation of PTFRN positive cell lines was investigated, as well as the 33B7 ADC in vivo effect on tumor growth in athymic mice. All flow cytometry and in vitro internalization assays were analyzed for statistical significance using a Welsh’s T-test. Animal studies were analyzed using Two-Way Analysis of Variance (ANOVA) utilizing post-hoc Bonferroni analysis, and/or Mixed Effects analysis. The 33B7 cell surface target was identified as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was confirmed by showing that PTGFRN-expressing cells bound and internalized 33B7, compared to PTGFRN negative cells. Cells able to bind 33B7 were PTGFRN-positive by Western blot analysis. In vitro treatment PTGFRN-positive cancer cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers.

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A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization

Antibodies ◽

10.3390/antib7040037 ◽

2018 ◽

Vol 7 (4) ◽

pp. 37 ◽

Cited By ~ 3

Author(s):

Jennifer Linden ◽

Kiel Telesford ◽

Samantha Shetty ◽

Paige Winokour ◽

Sylvia Haigh ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Monoclonal Antibodies ◽

Western Blot ◽

Clostridium Perfringens ◽

Specificity And Sensitivity ◽

Epsilon Toxin ◽

Blocking Antibodies

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

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Aurora-a confers radioresistance in human hepatocellular carcinoma by activating NF-κB signaling pathway

BMC Cancer ◽

10.1186/s12885-019-6312-y ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 3

Author(s):

Ze-Tian Shen ◽

Ying Chen ◽

Gui-Chun Huang ◽

Xi-Xu Zhu ◽

Rui Wang ◽

...

Keyword(s):

Flow Cytometry ◽

Western Blot ◽

Luciferase Reporter ◽

Aurora A ◽

Western Blot Assay ◽

Induced Apoptosis ◽

Mtt Assays ◽

Hcc Cells

Abstract Background Radiotherapy failure is a significant clinical challenge due to the development of resistance in the course of treatment. Therefore, it is necessary to further study the radiation resistance mechanism of HCC. In our early study, we have showed that the expression of Aurora-A mRNA was upregulated in HCC tissue samples or cells, and Aurora-A promoted the malignant phenotype of HCC cells. However, the effect of Aurora-A on the development of HCC radioresistance is not well known. Methods In this study, colony formation assay, MTT assays, flow cytometry assays, RT-PCR assays, Western blot, and tumor xenografts experiments were used to identify Aurora-A promotes the radioresistance of HCC cells by decreasing IR-induced apoptosis in vitro and in vivo. Dual-luciferase reporter assay, MTT assays, flow cytometry assays, and Western blot assay were performed to show the interactions of Aurora-A and NF-κB. Results We established radioresistance HCC cell lines (HepG2-R) and found that Aurora-A was significantly upregulated in those radioresistant HCC cells in comparison with their parental HCC cells. Knockdown of Aurora-A increased radiosensitivity of radioresistant HCC cells both in vivo and in vitro by enhancing irradiation-induced apoptosis, while upregulation of Aurora-A decreased radiosensitivity by reducing irradiation-induced apoptosis of parental cells. In addition, we have showed that Aurora-A could promote the expression of nuclear IkappaB-alpha (IκBα) protein while enhancing the activity of NF-kappaB (κB), thereby promoted expression of NF-κB pathway downstream effectors, including proteins (Mcl-1, Bcl-2, PARP, and caspase-3), all of which are associated with apoptosis. Conclusions Aurora-A reduces radiotherapy-induced apoptosis by activating NF-κB signaling, thereby contributing to HCC radioresistance. Our results provided the first evidence that Aurora-A was essential for radioresistance in HCC and targeting this molecular would be a potential strategy for radiosensitization in HCC.

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Altered Iron Metabolism Is a New Targetable Hallmark for Multiple Myeloma

Blood ◽

10.1182/blood-2019-123473 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3059-3059

Author(s):

Can Li ◽

Xuelian Tan ◽

Qierra Brockman ◽

Yogesh Jethava ◽

Marta Chesi ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Western Blot ◽

Plasma Cells ◽

Macrophage Polarization ◽

Mouse Bone Marrow ◽

Iron Dextran

Conventional therapies to multiple myeloma (MM) are not aimed at specific molecular targets leading ultimately to treatment resistance. Recent reports have shown that iron is instrumental in cancer development and progression and that high intracellular iron levels are associated with poor prognosis. We have demonstrated that MM cells exhibit dysregulated iron homeostasis and that limitation of cytosolic iron inhibits MM cell growth both in vitro and in vivo. The potential therapeutic role of iron should be further investigated to better understand how targeting high-iron MM cells could prevent or delay MM development and recurrence. Our study will provide crucial insights into the iron biology of MM pathogenesis and may lead to novel MM therapy. In this study, two mouse models, young Vk*MYC and old KaLwRij mice, were injected with iron dextran (1.25 mg/kg, IP, once a week). Tumor burden was monitored by serial Serum Protein Electrophoresis (SPEP) tests, flow cytometry, and immunohistochemistry. In vitro co-culturing of ARP1 MM cells with macrophages was employed to determine iron transfer. To determine iron's roles in MM evolution, we injected iron dextran into Vk*MYC mice at 8-week age. Vk*MYC mice develop MGUS around 40-50 weeks with plasma cell (PC) bone marrow infiltration and kidney damage etc. Iron-dextran was used because it is primarily taken up by macrophages. After 14-16 weeks of iron injection, M spike was detected in the injected Vk*MYC mice. The percentage of bone marrow plasma cells (CD138+) were significantly increased to 9% in the Vk*MYC mice injected with iron compared to control mice injected with vehicle by flow cytometry and immunohistochemistry. The acceleration of disease progression via iron injection was also tested in KaLwRij mice, which also spontaneously develops MGUS in old age. M protein was detected in 12 of 15 mice (80%) injected with iron dextran for 10 weeks and 1 of 5 KaLwRij (20%) control mice at 18-months of age. CD138+ B220- plasma cells were determined by flow cytometry. A significant increase of CD138+B220- plasma cells in iron treated mice (4% versus 2%) was observed compared to vehicle control mice. Deparaffined sections of bone marrow from the above mice were stained with Prussian blue and confirmed positive staining of macrophages from iron administrated mice. These results indicate that iron accelerates MGUS development in vivo. We next evaluated whether MM cells accumulate iron from the microenvironment. ARP1 MM cells were co-cultured with primary macrophages derived from mouse bone marrow to mimic disease environment in vitro. Under these conditions, MM cells induced macrophage polarization from M0 to M1 and M2. Furthermore, increased macrophage polarization was confirmed in vivo from the KaLwRij mice injected with 5TGM1 MM cells. To confirm that MM cells uptake iron from macrophages, increased intracellular ferritin levels were observed by western blot in ARP1 MM cells following co-culture with iron-loaded macrophages. We observed that this increase in intracellular ferritin was mediated via the transferrin receptor. This iron mobilization was prevented by iron chelation. Additionally, we confirmed that ferritin levels were higher in CD138+ primary human MM cells compared to CD138- non-MM cells by western blot. Our data indicate that MM cells promote macrophage polarization resulting in the transferring of iron into MM cells. The blockade of iron trafficking between MM cells and macrophages might hold a promise for the prevention and therapy in MM. Disclosures Bergsagel: Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Zhan:BIPHARM LLC: Consultancy, Other: % Allocation of Profit.

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Regulated Phosphatidylserine Exposure on Platelets Mediates Fibrin Formation in Hemostasis and Thrombosis.

Blood ◽

10.1182/blood.v106.11.1645.1645 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1645-1645

Author(s):

Jialan Shi ◽

Steven W. Pipe ◽

Jan T. Rasmussen ◽

Christian W. Heegaard ◽

Gary E. Gilbert

Keyword(s):

Flow Cytometry ◽

Platelet Rich Plasma ◽

Factor Xa ◽

Annexin V ◽

Vascular Wall ◽

Fibrin Deposition ◽

Exposure In Vivo

Abstract Thrombin-stimulated platelets support activity of phosphatidylserine (PS)-dependent blood coagulation reactions. However, only 2–6% of stimulated platelets expose sufficient PS to bind annexin V, leading to the supposition that procoagulant reactions are localized to the annexin V positive platelets and to microparticles shed by platelets. We hypothesized that thrombin-stimulated platelets expose sufficient PS to support the prothrombinase and factor Xase complexes but insufficient to meet the threshold for annexin V binding. We evaluated lactadherin, a PS-binding milk protein, as a reagent to detect platelet PS exposure. Thrombin or TRAP-treated platelets bound lactadherin with 3200 ± 700 sites/plt, but did not bind annexin V, as detected by flow cytometry. To confirm that lactadherin binding truly reported PS exposure, we performed activation experiments upon platelets loaded with 1-Palmitoyl-2- [6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-Glycero-3-Phospho-L-Serine (NBD-PS). Stimulation of platelets with 10 μM TRAP led to 8 ± 2 % PS exposure vs > 90% PS exposure for 10 μM A23187. PS exposure was maximal within 1 min of exposure to TRAP and was reversible with >70% of exposed PS re-internalized within 30 min. In situ platelet fibrin deposition was monitored utilizing a novel flow cytometry assay. Platelet rich plasma was recalcified and supplemented with 50 pM factor Xa, 100 μM GPRP. Platelet-bound fibrin, lactadherin and annexin V binding sites were measured at time intervals. Fibrin accumulated on lactadherin + platelets at a rate greater than or equal to the rate on platelet microparticles or annexin V + platelets. Lactadherin inhibited > 98% of prothrombinase and factor Xase activity on platelets while annexin V inhibition reached a plateau of ~ 80%. The localization and importance of regulated PS exposure in vivo was evaluated in mouse models. Anesthetized mice were injected intravenously with 1 μg of lactadherin and 1 μg annexin V. Three minutes after FeCl3 injury of exposed mesentery, mice were perfused with fixative. Immunohistochemistry showed veins edged with fibrin and decorated with platelets. Lactadherin co-localized with CD41+ platelets along the vascular wall but was less intense on platelets lodged within fibrin aggregates that extended into the vessel lumen and did not stain platelets in sequestered blood. Annexin V stained only scattered platelets and endothelial cells and did not correlate well to sites of fibrin deposition. To evaluate the hemostatic importance of platelet PS, 10 μg of lactadherin was injected intravenously prior to tail snip; bleeding volumes increased from 33 ± 29 μl to 128 ± 39 μl, n experimental = 8. Rose bengal/laser-induced carotid thrombosis delayed from 34 ± 9 min to 80 ± 20 min, n = 10 after 8–21 μg lactadherin. Four treated animals did not develop a thrombosis. In summary, use of lactadherin as a PS probe has revealed that the capacity for regulated PS exposure, at levels below the annexin V binding threshold, is a general platelet property and that low level PS exposure is sufficient to support thrombin and fibrin generation in vitro and in vivo. These results have implications about the mechanism through which PS exposure is regulated as well as for the potential value of PS exposure as a diagnostic or therapeutic target.

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CD137 Signaling Promotes Endothelial Apoptosis by Inhibiting Nrf2 Pathway, and Upregulating NF-κB Pathway

Mediators of Inflammation ◽

10.1155/2020/4321912 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15

Author(s):

Tianxin Geng ◽

Yang Yan ◽

Yue Zhang ◽

Liangjie Xu ◽

Guangyao Zang ◽

...

Keyword(s):

Endothelial Cells ◽

Recombinant Protein ◽

Western Blot ◽

Nuclear Translocation ◽

Umbilical Vein ◽

In Vivo Studies ◽

Control Group ◽

Nrf2 Pathway

Background. Endothelial dysfunction and apoptosis resulting from oxidative stress can lead to the development of atherosclerosis. Our group has previously showed that CD137 signaling contributes to the progression of atherosclerosis and the vulnerability of plaques. The aim of this study is to investigate the effects of CD137 signaling in atherosclerosis on endothelial cells (ECs) apoptosis and to explore the underlying mechanisms. Methods. Serum samples were collected from 11 patients with acute myocardial infarction and 4 controls. Peritoneal injection of agonist-CD137 recombinant protein in ApoE−/− mice was used to determine whether CD137 signaling can promote apoptosis in vivo, and human umbilical vein endothelial cells treated with agonist-CD137 recombinant protein, M5580 (a Nrf2 pathway agonist) and CAPE (a NF-κB pathway inhibitor) were used to explore the effect of Nrf2 and NF-κB pathway in CD137 signaling-induced ECs apoptosis in vitro. Results. ELISA showed that Bcl-2 in the serum of AMI patients was lower than that of the control group, while TNF-α and sCD137 were higher than that of the control group. Confocal microscopy and Western blot analysis showed that the nuclear translocation of Nrf2 in the agonist-CD137 group was significantly inhibited, and the expression of its downstream antioxidant enzymes was also decreased when compared with control. Immunofluorescence and Western blot results showed that the nuclear translocation of NF-κB in the agonist-CD137 group was enhanced, and ELISA results showed that the secretion of proinflammatory cytokines in the agonist-CD137 group was increased. Immunofluorescence results revealed that ROS production in the agonist-CD137 group was higher than that in control, M5580 (a Nrf2 pathway agonist) and CAPE (a NF-κB pathway inhibitor) groups. In vitro studies using HUVECs and in vivo studies using high-fat-fed ApoE−/− mice showed that the number of apoptotic endothelial cells was the highest in the agonist-CD137 group. By contrast, both M5580 and CAPE treatments were able to reduce CD137 induced ECs apoptosis. Conclusions. Our results showed that CD137 signaling promotes ECs apoptosis through prooxidative and proinflammatory mechanisms, mediated by Nrf2 and NF-κB pathways, respectively.

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KLF2 Protects against Osteoarthritis by Repressing Oxidative Response through Activation of Nrf2/ARE Signaling In Vitro and In Vivo

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/8564681 ◽

2019 ◽

Vol 2019 ◽

pp. 1-18 ◽

Cited By ~ 1

Author(s):

Xiang Gao ◽

Shuangpeng Jiang ◽

Zhangzhen Du ◽

Angtin Ke ◽

Qingwei Liang ◽

...

Keyword(s):

Nuclear Translocation ◽

Inflammatory Diseases ◽

Cartilage Degradation ◽

Cartilage Destruction ◽

Chondrocyte Apoptosis ◽

Tunel Staining ◽

Heme Oxygenase 1 ◽

Related Factor

Osteoarthritis (OA) is a multifactorial and inflammatory disease characterized by cartilage destruction that can cause disability among aging patients. There is currently no effective treatment that can arrest or reverse OA progression. Kruppel-like factor 2 (KLF2), a member of the zinc finger family, has emerged as a transcription factor involved in a wide variety of inflammatory diseases. Here, we identified that KLF2 expression is downregulated in IL-1β-treated human chondrocytes and OA cartilage. Genetic and pharmacological overexpression of KLF2 suppressed IL-1β-induced apoptosis and matrix degradation through the suppression of reactive oxygen species (ROS) production. In addition, KLF2 overexpression resulted in increased expression of heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) through the enhanced nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Further, Nrf2 inhibition abrogated the chondroprotective effects of KLF2. Safranin O/fast green and TUNEL staining demonstrated that adenovirus-mediated overexpression of KLF2 in joint cartilage protects rats against experimental OA by inhibiting cartilage degradation and chondrocyte apoptosis. Immunohistochemical staining revealed that KLF2 overexpression significantly decreases MMP13 expression caused by OA progression in vivo. This in vitro and in vivo study is the first to investigate the antioxidative effect and mechanisms of KLF2 in OA pathogenesis. Our results collectively provide new insights into OA pathogenesis regulated by KLF2 and a rationale for the development of effective OA intervention strategies.

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Resveratrol Inhibits Ischemia-Induced Myocardial Senescence Signals and NLRP3 Inflammasome Activation

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/2647807 ◽

2020 ◽

Vol 2020 ◽

pp. 1-20

Author(s):

Hong Feng ◽

Shan-qi Mou ◽

Wen-jing Li ◽

Nan Zhang ◽

Zi-ying Zhou ◽

...

Keyword(s):

Nuclear Translocation ◽

Sirtuin 1 ◽

Neonatal Rat ◽

Tunel Staining ◽

Mouse Heart ◽

Inflammasome Activation ◽

Coronary Artery Ligation ◽

Rat Cardiomyocytes

Aims. The aim of this study was to investigate whether resveratrol (RSV) could ameliorate ischemia- and hypoxia-associated cardiomyocyte apoptosis and injury via inhibiting senescence signaling and inflammasome activation. Materials and Methods. Mice were treated with RSV by gastric tube (320 mg/kg/day) or vehicle one week before left coronary artery ligation or sham surgery until the end of the experiments. After pressure–volume loop analysis, mouse hearts were harvested for histopathological (including PSR, TTC, TUNEL staining, immunohistochemistry, and immunofluorescence) and molecular analysis by western blotting and RT-PCR. In addition, neonatal rat cardiomyocytes (NRCMs), cardiac fibroblasts (CFs), and macrophages were isolated for in vitro experiments. Key Findings. RSV treatment decreased mortality and improved cardiac hemodynamics. RSV inhibited the expression of senescence markers (p53, p16, and p19), inflammasome markers (NLRP3 and Cas1 p20), and nuclear translocation of NF-κB, hence alleviating infarction area, fibrosis, and cell apoptosis. RSV also inhibited expression of interleukin- (IL-) 1β, IL-6, tumor necrosis factor-α, and IL-18 in vivo. In in vitro experiment, RSV prevented hypoxia-induced NRCM senescence and apoptosis. After inhibition of sirtuin 1 (Sirt1) by EX27, RSV failed to inhibit p53 acetylation and expression. Moreover, RSV could inhibit expression of NLRP3 and caspase 1 p20 in NRCMs, CFs, and macrophages, respectively, in in vitro experiments. Significance. Our findings revealed that RSV protected against ischemia-induced mouse heart injury in vivo and hypoxia-induced NRCM injury in vitro via regulating Sirt1/p53-mediated cell senescence and inhibiting NLRP3-mediated inflammasome activation.

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