Upregulation of P2Y12 Inhibits Chondrocyte Apoptosis in Lumbar Osteoarthritis Through PI3K/AKT Signaling Pathway

Abstract Lumbar facet osteoarthritis (FJOA) is one of the major causes of severe low back pain and disability worldwide. However, the underlying mechanism of cartilage degeneration in FJOA remains unclear. The purpose of this study was to investigate the expression of P2Y12 in FJOA and its possible role. Changes of chondrocytes in rat facet joints with degenerative changes were observed by HE and ferro red solid green staining. The expression changes of P2Y12, MMP13 and COL2 in FJOA were observed by immunohistochemistry. In vitro, human SW1353 chondrosarcoma cells were stimulated with IL-β to establish chondrocyte apoptosis model. Western blot analysis showed that P2Y12 and cleaved caspase-3 were significantly expressed in SW1353 cells. Co-localization of P2Y12-cleaved Caspase-3 in apoptosis model was detected by double-standard immunofluorescence staining. We demonstrated that P2Y12 may have an anti-apoptotic effect in FJOA chondrocytes apoptosis by inhibiting the expression of P2Y12 by siRNA. In addition, flow cytometry showed that P2Y12 gene knockout enhanced apoptosis induced by P2Y12. Our data suggest that P2Y12 has a chondroprotective effect on FJOA.

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miR-146a-5p Promotes Chondrocyte Apoptosis and Inhibits Autophagy of Osteoarthritis by Targeting NUMB

Cartilage ◽

10.1177/19476035211023550 ◽

2021 ◽

pp. 194760352110235

Author(s):

Hongjun Zhang ◽

Wendi Zheng ◽

Du Li ◽

Jia Zheng

Keyword(s):

Caspase 3 ◽

Cartilage Tissue ◽

Luciferase Reporter ◽

Chondrocyte Apoptosis ◽

Knee Cartilage ◽

Before And After ◽

Related Proteins ◽

Human And Mouse

Objective miR-146a-5p was found to be significantly upregulated in cartilage tissue of patients with osteoarthritis (OA). NUMB was shown to be involved in the autophagy regulation process of cells. We aimed to learn whether NUMB was involved in the apoptosis or autophagy process of chondrocytes in OA and related with miR-146a-5p. Methods QRT-PCR was used to detect miR-146a-5p level in 22 OA cartilage tissues and 22 controls. The targets of miR-146a-5p were analyzed using software and the luciferase reporter experiment. The apoptosis and autophagy, and related proteins were detected in chondrocytes treated with miR-146a-5p mimic/inhibitor or pcDNA3.1-NUMB/si-NUMB and IL-1β, respectively. In vivo experiment, intra-articular injection of miR-146a-5p antagomir/NC was administered at the knee of OA male mice before and after model construction. Chondrocyte apoptosis and the expression of apoptosis and autophagy-related proteins were also detected. Results miR-146a-5p was highly expressed in knee cartilage tissue of patients with OA, while NUMB was lowly expressed and negatively regulated by miR-146a-5p. Upregulation of miR-146a-5p can promote cell apoptosis and reduce autophagy of human and mouse chondrocytes by modulating the levels of cleaved caspase-3, cleaved PARP, Bax, Beclin 1, ATG5, p62, LC3-I, and LC3-II. Increasing the low level of NUMB reversed the effects of miR-146a-5p on chondrocyte apoptosis and autophagy. Intra-articular injection of miR-146a-5p antagomir can also reverse the effects of miR-146a-5p on the apoptosis and autophagy of knee joint chondrocytes in OA mice. Conclusion Downregulation of miR-146a-5p suppresses the apoptosis and promotes autophagy of chondrocytes by targeting NUMB in vivo and in vitro.

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Poly-L-Arginine Induces Apoptosis of NCI-H292 Cells via ERK1/2 Signaling Pathway

Journal of Immunology Research ◽

10.1155/2018/3651743 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Ya-Ni Wang ◽

Ling-Ling Zhang ◽

Xiao-Yun Fan ◽

Sha-Sha Wu ◽

Sheng-Quan Zhang

Keyword(s):

Epithelial Cells ◽

Caspase 3 ◽

Underlying Mechanism ◽

Mitochondrial Pathway ◽

Airway Epithelial Cells ◽

Dependent Manner ◽

Airway Epithelial ◽

Cationic Protein ◽

Concentration Dependent Manner

Cationic protein is a cytotoxic protein secreted by eosinophils and takes part in the damage of airway epithelium in asthma. Poly-L-arginine (PLA), a synthetic cationic protein, is widely used to mimic the biological function of the natural cationic protein in vitro. Previous studies demonstrated the damage of the airway epithelial cells by cationic protein, but the molecular mechanism is unclear. The purpose of this study aimed at exploring whether PLA could induce apoptosis of human airway epithelial cells (NCI-H292) and the underlying mechanism. Methods. The morphology of apoptotic cells was observed by transmission electron microscopy. The rate of apoptosis was analyzed by flow cytometry (FCM). The expressions of the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Bcl-2/Bax, and cleaved caspase-3 were assessed by western blot. Results. PLA can induce apoptosis in NCI-H292 cells in a concentration-dependent manner. Moreover, the phosphorylation of the ERK1/2 and the unbalance of Bcl2/Bax, as well as the activation of caspase-3, were involved in the PLA-induced apoptosis. Conclusions. PLA can induce the apoptosis in NCI-H292 cells, and this process at least involved the ERK1/2 and mitochondrial pathway. The results could have some indications in revealing the apoptotic damage of the airway epithelial cells. Besides, inhibition of cationic protein-induced apoptotic death in airway epithelial cells could be considered as a potential target of anti-injury or antiremodeling in asthmatics.

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Melatonin Relieves Busulfan-Induced Spermatogonial Stem Cell Apoptosis of Mouse Testis by Inhibiting Endoplasmic Reticulum Stress

Cellular Physiology and Biochemistry ◽

10.1159/000486165 ◽

2017 ◽

Vol 44 (6) ◽

pp. 2407-2421 ◽

Cited By ~ 20

Author(s):

Yanhua Cui ◽

Lipeng Ren ◽

Bo Li ◽

Jia Fang ◽

Yuanxin Zhai ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Caspase 3 ◽

Survival Rates ◽

Underlying Mechanism ◽

Pcr Analysis ◽

Caspase 12 ◽

Apoptosis Proteins

Background/Aims: Busulfan is commonly used for cancer chemotherapy. Although it has the advantage of increasing the survival rate of patients, it can cause male infertility via damaging the testes and reducing sperm counts. Therefore, the underlying mechanism should be explored, and new agents should be developed to protect the male reproductive system from busulfan-induced damage. Endoplasmic reticulum stress (ERS) is considered a key contributor to numerous pathologies. Despite several studies linking ERS to toxicants, studies have yet to determine whether ERS is a contributing factor to busulfan-induced testicular damage. Melatonin is a well-known broad-spectrum antioxidant, anti-inflammatory and antitumour agent, but the effects of melatonin on busulfan-induced ERS in mouse testes damage are less documented. Methods: The effects of melatonin were measured by immunofluorescence staining, Western blot, qRT-PCR analysis and flow cytometry assay. The underlying mechanism was investigated by measuring ERS. Results: We found that ERS was strongly activated in mouse testes (in vivo) and the C18-4 cell line (in vitro) after busulfan administration. ERS-related apoptosis proteins such as caspase-12, CHOP and caspase-3 were activated, and the expression of apoptotic proteins such as P53 and PUMA were upregulated. Furthermore, we investigated whether melatonin reduced the extent of damage to mouse testes and improved the survival rates of busulfan-treated mice. When exploring the underlying mechanisms, we found melatonin could counteract ERS by decreasing the expression levels of the ERS markers GRP78, ATF6, pIRE1 and XBP1 in mouse testes and mouse SSCs (C18-4 cells). Moreover, it blocked the activation of ERS-related apoptosis proteins caspase-12, CHOP and caspase-3 and suppressed P53 and PUMA expression stimulated by busulfan both in vivo and in vitro. Conclusion: Our results demonstrate that ERS is an important mediator for busulfan-induced apoptosis. The attenuation of ERS by melatonin can prevent busulfan-treated SSCs apoptosis and protect busulfan-treated testes from damage. Thus, this study suggests that melatonin may alleviate the side effects of busulfan for male patients during clinical treatment.

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Role of CYP27A1 in progesterone metabolism in vitro and in vivo

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00298.2009 ◽

2009 ◽

Vol 297 (4) ◽

pp. E949-E955 ◽

Cited By ~ 3

Author(s):

Geneviève Escher ◽

Isabelle Vögeli ◽

Robert Escher ◽

Robert C. Tuckey ◽

Sandra Erickson ◽

...

Keyword(s):

Gene Knockout ◽

Human Kidney ◽

Underlying Mechanism ◽

Expression Cloning ◽

Dependent Manner ◽

Expression Library ◽

Concentration Dependent Manner ◽

Adrenodoxin Reductase

In the kidney, progesterone is inactivated to 20α-dihydro-progesterone (20α-DH-progesterone) to protect the mineralocorticoid receptor from progesterone excess. In an attempt to clone the enzyme with 20α-hydroxysteroid activity using expression cloning in CHOP cells and a human kidney expression library, serendipitously cDNA encoding CYP27A1 was isolated. Overexpression of CYP27A1 in CHOP cells decreased progesterone conversion to 20α-DH-progesterone in a dose-dependent manner, an effect enhanced by cotransfection with adrenodoxin and adrenodoxin reductase. Incubation of CHOP cells with 27-hydroxycholesterol, a product of CYP27A1, increased the ratio of progesterone to 20α-DH-progesterone in a concentration-dependent manner, indicating that the effect of CYP27A1 overexpression was mediated by 27-hydroxycholesterol. To analyze whether these observations are relevant in vivo, progesterone and 20α-DH-progesterone were measured by gas chromatography-mass spectometry in 24-h urine of CYP27A1 gene knockout (ko) mice and their control wild-type and heterozygote littermates. In CYP27A1 ko mice, urinary progesterone concentrations were decreased, 20α-DH-progesterone increased, and the progesterone-to-20α-DH-progesterone ratio decreased threefold ( P < 0.001). Thus CYP27A1 modulates progesterone concentrations. The underlying mechanism is inhibition of 20α-hydroxysteroid dehydrogenase by 27-hydroxycholesterol.

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HIF-1α Protects Granulosa Cells From Hypoxia-Induced Apoptosis During Follicular Development by Inducing Autophagy

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.631016 ◽

2021 ◽

Vol 9 ◽

Author(s):

Zonghao Tang ◽

Renfeng Xu ◽

Zhenghong Zhang ◽

Congjian Shi ◽

Yan Zhang ◽

...

Keyword(s):

Granulosa Cells ◽

Mammalian Cells ◽

Caspase 3 ◽

Ovarian Follicle ◽

Hypoxia Inducible Factor ◽

Follicular Development ◽

B Cell Lymphoma ◽

Underlying Mechanism

Owing to the avascular structure of the ovarian follicle, proliferation of granulosa cells (GCs) and development of follicles occur under hypoxia, which is obviously different from the cell survival requirements of most mammalian cells. We hypothesized that autophagy may exert an inhibitory effect on GC apoptosis. To decipher the underlying mechanism, we constructed a rat follicular development model using pregnant mare serum gonadotropin and a cell culture experiment in hypoxic conditions (3% O2). The present results showed that the autophagy level was obviously increased and was accompanied by the concomitant elevation of hypoxia inducible factor (HIF)-1α and BNIP3 (Bcl-2/adenovirus E1B 19kDa-interacting protein 3) in GCs during follicular development. The levels of Bax (Bcl2-associated X) and Bcl-2 (B-cell lymphoma-2) were increased, while the activation of caspase-3 exhibited no obvious changes during follicular development. However, inhibition of HIF-1α attenuated the increase in Bcl-2 and promoted the increase in Bax and cleaved caspase-3. Furthermore, we observed the downregulation of BNIP3 and the decrease in autophagy after treatment with a specific HIF-1α activity inhibitor (echinomycin), indicating that HIF-1α/BNIP3 was involved in autophagy regulation in GCs in vivo. In an in vitro study, we also found that hypoxia did not obviously promote GC apoptosis, while it significantly enhanced the activation of HIF-1α/BNIP3 and the induction of autophagy. Expectedly, this effect could be reversed by 3-methyladenine (3-MA) treatment. Taken together, these findings demonstrated that hypoxia drives the activation of HIF-1α/BNIP3 signaling, which induces an increase in autophagy, protecting GC from apoptosis during follicular development.

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Polysaccharide from angelica sinensis attenuates SNP-Induced Apoptosis in osteoarthritis Chondrocytes by Inducing Autophagy via ERK1/2 Pathway

10.21203/rs.3.rs-29285/v1 ◽

2020 ◽

Author(s):

Chao Xu ◽

Su Ni ◽

Chao Zhuang ◽

Chenkai Li ◽

Gongyin Zhao ◽

...

Keyword(s):

Western Blot ◽

Specific Inhibitor ◽

Underlying Mechanism ◽

Protective Role ◽

Vital Role ◽

Chondrocyte Apoptosis ◽

Angelica Sinensis ◽

Apoptotic Effect ◽

Osteoarthritis Chondrocytes ◽

Induced Apoptosis

Abstract Objective Chondrocyte apoptosis plays a vital role in osteoarthritis (OA) progression. Angelica sinensis polysaccharide (ASP), a traditional Chinese medicine, possesses anti-inflammatory and anti-apoptotic properties in chondrocytes. This study aimed to determine the protective role of ASP on sodium nitroprusside (SNP)-induced chondrocyte apoptosis, and explore the underlying mechanism. Method Human primary chondrocytes isolated from articular cartilage of OA patients were treated with SNP alone or in combination with different dose of ASP. Cell viability and apoptosis were assessed, and apoptosis-related proteins including Bcl-2 and Bax were detected. Autophagy levels were evaluated by light chain 3 (LC3)-II immunofluorescence staining, mRFP-GFP-LC3 fluorescence localisation and western blot (LC3II, p62, Beclin-1, Atg5). Meanwhile,activation of ERK 1/2 pathway was determined by western blot. The autophagy inhibitors 3-Methyladenine (3-MA), chloroquine (CQ) and a specific inhibitor of ERK1/2, SCH772984, were used respectively to confirm the autophagic effect of ASP. Results The results showed that SNP-induced chondrocyte apoptosis was significantly rescued by ASP, whereas ASP alone promoted chondrocytes proliferation. The anti-apoptotic effect of ASP was related to the enhanced autophagy and depended on the activation of ERK1/2 pathway.Conclusion ASP markedly rescued SNP-induced chondrocyte apoptosis by activating ERK1/2-dependent autophagy in chondrocytes, and it made ASP a potential therapeutic supplementation for OA treatment.

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Chondrocyte apoptosis induced in vitro is caspase-3-mediated, but is not demonstrated in cartilage from patients with rheumatoid arthritis or osteoarthritis

Journal of Allergy and Clinical Immunology ◽

10.1016/s0091-6749(03)81170-3 ◽

2003 ◽

Vol 111 (2) ◽

pp. S321

Author(s):

J.F. Van Offel ◽

I.C. Koster ◽

G.J. Van Elsen ◽

A.J. Schuerwegh ◽

C.H. Bridts ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Caspase 3 ◽

Chondrocyte Apoptosis

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MBNL1 Suppressed Cancer Metastatic of Skin Squamous Cell Carcinoma Via by TIAL1/MYOD1/Caspase-9/3 Signaling Pathways

Technology in Cancer Research & Treatment ◽

10.1177/1533033820960755 ◽

2021 ◽

Vol 20 ◽

pp. 153303382096075

Author(s):

Jiaorong Chen ◽

Jiaqi Wang ◽

Jingyi Qian ◽

Mengying Bao ◽

Xin Zhang ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Signaling Pathways ◽

Squamous Cell ◽

Caspase 3 ◽

Underlying Mechanism ◽

Down Regulation ◽

Skin Squamous Cell Carcinoma ◽

Cell Metastasis

Objective: The incidence of skin squamous cell carcinoma (SSCC) has recently been increasing, with diverse clinical manifestations.SSCC could metastasize to lymph nodes or other organs, posing a great threat to life. The present study was designed to investigate the function and underlying mechanism of muscleblind-like protein 1 (MBNL1) in skin squamous cell carcinoma. Methods: SCL-1 cell was used for vitro model and transfected with MBNL1 or siMBNL1 plasmids. MTT Assays, LDH activity ELISA, and Transwell chamber migration experiment were used to confirm the effects of MBNL1 on cell growth of SCL-1 cell. Western blot analysis was used to analyze the mechanism of MBNL1 in SCL-1 cell. Results: Down-regulation of MBNL1 promoted cell metastasis of SSCC, while up-regulation of MBNL1 reduced cell metastasis of SSCC in vitro. Down-regulation of MBNL1 suppressed the protein expression of T cell intracellular antigen (TIAL1), myogenic determinant 1 (MyoD1) and Caspase-3 in vitro. Consistent with these observations, inhibition of TIAL1 or MYOD1 expression attenuated the effects of MBNL1 in SSCC. Conclusion: The present study revealed that MBNL1 suppressed thecancer metastatic capacity of SSCC via by TIAL1/MYOD1/Caspase-3 signaling pathways.

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Role of midkine in cadmium-induced liver, heart and kidney damage

Human & Experimental Toxicology ◽

10.1177/0960327110372402 ◽

2010 ◽

Vol 30 (5) ◽

pp. 391-397 ◽

Cited By ~ 9

Author(s):

Nuray Yazihan ◽

Mehtap Kacar Kocak ◽

Ethem Akcil ◽

Onur Erdem ◽

Ahmet Sayal

Keyword(s):

Messenger Rna ◽

Caspase 3 ◽

Biological Effects ◽

Toxic Substance ◽

Underlying Mechanism ◽

Enzyme Linked Immunosorbent Assay ◽

Activity Levels ◽

Cd Toxicity ◽

Tnf Α

Accumulation of the widespread environmental toxin cadmium (Cd) in tissues results in toxicity. Cd, which can induce a broad spectrum of biological effects, is a toxic substance and is associated with inflammation and apoptosis. Midkine (MK) has fibrinolytic, antiapoptotic, transforming, angiogenetic and chemotactic activities. After Cd toxicity, we found increased MK expression in liver cells in an in vitro cell culture model. The aim of this study was to determine the possibility of relationship between tissue MK expression levels, tumor necrosis factor α(TNF-α) levels and apoptosis in a chronic Cd toxicity model in rats. Male Wistar rats were exposed to Cd at the dose of 15 parts per million (ppm) for 8 weeks. MK levels were measured in kidney, heart and liver tissue by enzyme-linked-immunosorbent assay (ELISA). MK messenger RNA (mRNA) expression was evaluated by RT-PCR. Tissue apoptosis level was evaluated with tissue caspase-3 activity levels. Accumulation of Cd in liver is higher than the kidney and heart. Cd-treated rats had significantly higher tissue TNF-α and caspase-3 levels when compared with the control rats (p < 0.001). MK mRNA and protein levels were also significantly upregulated in the Cd-treated group (p < 0.05, p < 0.001, respectively). When compared with apoptosis in tissues, it was more prominent in the liver than kidney and heart. MK level is found increased 3, 1.7 and 1.3× folds in liver, kidney and heart, respectively. Our results showed that chronic Cd administration induces inflammation and apoptosis in rat liver, kidney and heart. MK involved in damage mechanisms of Cd-induced tissues. Further studies will show the underlying mechanism of increased MK expression in Cd toxicity.

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Anti-Apoptotic Effect of Angelica Polysaccharide (APS) on Cryopreservation of Platelets

Blood ◽

10.1182/blood-2021-151963 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3250-3250

Author(s):

Mo Yang ◽

Weiqing Su ◽

Liuming Yang ◽

Huimin Kong ◽

Huiling Wei ◽

...

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Annexin V ◽

Control Group ◽

Apoptotic Effect ◽

Vitro Effect

Abstract Background: Angelica Polysaccharide (APS) is from the root of Radix Angelicae Sinensis (Danggui). Danggui has been used for centuries to treat blood-deficiency related diseases. The hematopoietic effect of Danggui may be related to its constituent, polysaccharide. The effects of angelica polysaccharide on cryopreservation of platelets and megakaryocytes have not been well studied. This study focused on anti-apoptotic effect of APS and TPO on cryopreservation of platelets and megakaryocytes and provided new methods for prolonging the preservation time of platelets in vitro. Methods: The expression of platelet membrane glycoprotein CD41 and CD61, as well as the platelet apoptotic rate, Caspase 3 expression and mitochondrial membrane potential (MMP) were detected by flow cytometry; the anti-apoptotic mechanism of APS by PI3K /AKT signaling pathway was analyzed by Western blot assay. CFU assays were used to determine the effects of APS on megakaryocytic progenitor cells. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were conducted in megakaryocytic cell line M-07e. The effects of APS on cells treated with Ly294002, PI3K inhibitor and the effect of APS on the p-AKT were also studied. Results: The platelets were divided into 4 group: control group (4 ℃ stored platelets), APS group (APS-treated platelets stored at 4 ℃), LY294002 group (LY294002-treated platelets stored at 4 ℃) and LY294002+APS group (LY294002+APS treated platelets stored at 4 ℃). The apoptotic rate of platelets in LY294002 group was obviously increased. Compared with control group, the expression of CD41 and CD61 gradually decreased along with the enhancement of LY294002 concentrations (r=-0.953). The apoptotic rate of platelets in LY294002 group was enhanced significantly (P<0.05). While in LY294002+APS group, the apoptotic rate of platelets was significantly reduced (P<0.05) as compare with LY294002 group, which suggest that APS has an anti-apoptotic effect on the cryopreserved platelets. APS decreased the expression of Caspase-3 and inhibited the reduction of mitochondrial membrane potential induced by LY294002. Moreover, APS increased the activation of PI3K /AKT pathway in Platelets . We further analyzed the in vitro effect of APS on CFU-MK formation. APS (50 ug/ml) enhanced TPO (50 ng/ml) -induced CFU-MK formation (p=0.06, n=4). APS also significantly enhanced PDGF, bFGF and VEGF-induced CFU-MK formation (n=4). Moreover, the anti-apoptotic effect of APS in M-07e cells was also demonstrated by Annexin-V, Caspase-3, and JC-1 assays. Adding LY294002 alone increased the percentage of cells undergoing apoptosis. However, additional of APS to LY294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of APS significantly increased the p-AKT. Conclusion: APS, like TPO, has an anti-apoptotic effect on the cryopreserved platelets and megakaryocytes through activating PI3K/AKT, decreasing the expression of Caspase-3 and inhibiting the reduction of MMP. Disclosures No relevant conflicts of interest to declare.

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