Kaempferol Protects Cell Damage in In Vitro Ischemia Reperfusion Model in Rat Neuronal PC12 Cells

Ischemic cerebral stroke is a severe neurodegenerative disease with high mortality. Ischemia and reperfusion injury plays a fundamental role in ischemic cerebral stroke. To date, the strategy for ischemic cerebral stroke treatment is limited. In the present study, we aimed to investigate the effect of kaempferol (KFL), a natural flavonol, on cell injury induced by oxygen and glucose deprivation (OGD) and reoxygenation (OGD-reoxygenation) in PC12 cells. We found that KFL inhibited OGD-induced decrease of cell viability and the increase of lactate dehydrogenase (LDH) release. OGD-induced activation of mitochondrial dysfunction, mitochondrial apoptotic pathway, and apoptosis was inhibited by KFL. KFL also reduced OGD-induced oxidative stress in PC12 cells. P66shc expression and acetylation were increased by OGD and KFL inhibited these changes. Upregulation of P66shc suppressed KFL-induced decrease of apoptosis, the decrease of LDH release, and the increase of cell viability. Furthermore, KFL inhibited OGD-induced decrease of sirtuin 1 (SIRT1) expression and downregulation of SIRT1 blocked KFL-induced decrease of apoptosis, the decrease of LDH release, and the increase of cell viability. In summary, we identified that KFL exhibited a beneficial effect against OGD-induced cytotoxicity in an ischemia/reperfusion injury cell model. The findings suggest that KFL may be a promising choice for the intervention of ischemic stroke and highlighted the SIRT1/P66shc signaling.

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LncRNA FOXD2-AS1 Protects Against Cerebral Ischemia-Reperfusion Injury Through miR-3144-5p/ KCTD15 Axis

10.21203/rs.3.rs-877342/v1 ◽

2021 ◽

Author(s):

Qi Gao ◽

Yanfeng Wang

Keyword(s):

Cerebral Ischemia ◽

Cell Viability ◽

Reperfusion Injury ◽

Real Time ◽

Pc12 Cells ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Luciferase Reporter ◽

Cerebral Ischemia Reperfusion ◽

Cerebral Ischemia Reperfusion Injury

Abstract Background: Long non-coding RNAs (lnc-RNAs) and microRNAs (miRNAs) play key roles in the development of stroke. LncRNA FOXD2-AS1 has been reported to be important in many cancers. However, the role of lncRNA FOXD2-AS1 in stroke is limited known.Methods: Real-time polymerase chain reaction (real-time PCR) assays were used to measure expressions of lncRNA FOXD2-AS1, miR-3144-5p and KCTD15. Western blot assays were employed to examine Bax and Bcl2 protein expression. The cell viability was measured by cck8 assay. The cell apoptosis was detected by TUNEL staining assay The interaction between FOXD2-AS1, miR-3144-5p and KCTD was confirmed by site-directed mutagenesis and luciferase assays. Results: The expression of lncRNA FOXD2-AS1 was down-regulated in blood sample from stroke patients and MAO mice tissues. In addition, lncRNA FOXD2-AS1 was decreased on OGD/R treated cells. LncRNA FOXD2-AS1 overexpression promoted cell viability and reduced apoptosis in OGD/R-Induced PC12 Cells. LncRNA FOXD2-AS1 could interact with miR-3144-5p. Meanwhile, inhibition of miR-3144-5p alleviated OGD/ R-induced neuronal injury in PC12 Cells. Dual luciferase reporter assay verified that KCTD15 was a target of miR-3144-5p. And level of KCTD15 was positive with LncRNA FOXD2-AS1.Conclusion: Taken together, lncRNA FOXD2-AS1 protected against c cerebral ischemia-reperfusion injury by acting as a sponge of miR-3144-5p to modulate KCTD15 level.

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Anthocyanin attenuates oxygen-glucose deprivation/reperfusion-induced apoptosis of PC12 cells via inactivation of ASK1/JNK/p38 signaling pathway

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v18i10.6 ◽

2021 ◽

Vol 18 (10) ◽

pp. 2037-2043

Author(s):

Hong Zhu ◽

Dan Ren ◽

Lan Xiao ◽

Ting Zhang ◽

Ruomeng Li ◽

...

Keyword(s):

Cell Viability ◽

Signaling Pathway ◽

Pc12 Cells ◽

Cell Apoptosis ◽

Cell Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Oxygen Glucose Deprivation ◽

Induced Apoptosis

Purpose: To investigate whether the cytoprotective effect of anthocyanin (Anc) on oxygen-glucose deprivation/reperfusion (OGD/R)-induced cell injury is related to apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK)/p38 signaling pathway. Methods: PC12 cells were pre-treated with various concentrations of Anc (10, 50, and 100 μg/mL) in OGD/R-induced cell injury model. The 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide (MTT) assay was used to assess cell viability. Cell apoptosis was measured by lactic acid dehydrogenase (LDH) release assay and flow cytometry. Western blot was employed to determine the protein expressions of BCL-2, BAX, caspase-3, p-ASK1 (Thr845), p-JNK, and p-p38. Results: The results indicate that Anc increased the viability of PC12 cells after OGD/R exposure (p < 0.05), and also efficiently rescued OGD/R-induced apoptosis (p < 0.05). Mechanistic studies showed that these protective roles of Anc are related to the inhibition of ASK1/JNK/p38 signaling pathway. Conclusion: The results indicate Anc protects against OGD/R-induced cell injury by enhancing cell viability and inhibiting cell apoptosis. The underlying mechanism of action is partly via inactivation of ASK1/JNK/p38 signaling pathway. Thus, Anc has promise as a potential natural agent to prevent and treat cerebral ischemia-reperfusion injury.

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Human Mesenchymal Stem Cell-derived Exosomes Reduce Ischemia/Reperfusion Injury by the Inhibitions of Apoptosis and Autophagy

Current Pharmaceutical Design ◽

10.2174/1381612825666190119130441 ◽

2019 ◽

Vol 24 (44) ◽

pp. 5334-5341 ◽

Cited By ~ 13

Author(s):

Xiaofei Jiang ◽

Kar-Sheng Lew ◽

Qiying Chen ◽

Arthur M. Richards ◽

Peipei Wang

Keyword(s):

Stem Cell ◽

Mesenchymal Stem Cell ◽

Cell Viability ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Isolated Heart ◽

Ischemia Reperfusion ◽

Human Mesenchymal Stem Cell ◽

Ldh Release ◽

Induced Apoptosis

Background: Human mesenchymal stem cell-derived exosomes (hMSC-Exo) have been shown to reduce ischemia/reperfusion injury (I/R) in multiple models. I/R-induced apoptosis or autophagy play important roles in cell death. However, little or no reports demonstrate any roles of hMSC-Exo in this regards. Objective: To test the hypothesis that the inhibition of I/R-induced apoptosis and autophagy play a pivotal role in the cardioprotection of hMSC-Exo. Methods: Myoblast H9c2 cells and isolated rat hearts underwent hypoxia/re-oxygenate (H/R) or ischemia/ reperfusion (I/R) respectively. H9c2 were treated with 1.0 μg/ml Exo, in comparison with 3-MA or rapamycin (Rapa), a known anti- or pro-autophagic agent respectively. Hearts were treated with 0.5, 1.0 and 2.0 μg/ml Exo for 20 min in the beginning of reperfusion. Cell viability, WST assay, LDH release, Annexin-V staining apoptosis assay and GFP-LC3 labeled autophagosomes formation, cardiac function and Western blot were measured. Results: Exo significantly reduced H/R injury as indicated by increased cell viability and reduced LDH and apoptosis. 3-MA, while Rapa, showed increased or decreased protective effects. Rapa-induced injury was partially blocked by Exo. Exo decreased LC3-II/I ratio and increased p62, inhibited autophagosome formation, an indication of autophagy inhibition. In isolated heart, Exo increased cardiac functional recovery and reduced LDH release in I/R. Bcl-2 was significantly upregulated by Exo but not 3-MA. Exo downregulated Traf6 and upregulated mTORC1/p-4eBP1. Conclusion: Exo reduce I/R-induced apoptosis and autophagy. Up-regulation of Bcl-2 is the cross-talk between these two processes. The down-regulation of Traf6 and activation of mTORC1 are additional mechanisms in the inhibition of apoptosis and autophagy.

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Inhibition of LncRNA MALAT1 Attenuates Cerebral Ischemic Reperfusion Injury via Regulating AQP4 Expression

European Neurology ◽

10.1159/000511238 ◽

2020 ◽

pp. 1-10

Author(s):

Jing Jin ◽

Hongwei Wang ◽

Xiaoxiao Zheng ◽

Shangzhi Xie ◽

Li Zheng ◽

...

Keyword(s):

Cell Viability ◽

Reperfusion Injury ◽

Ischemia Reperfusion ◽

Cell Counting ◽

Ischemic Reperfusion Injury ◽

Aqp4 Expression ◽

Ischemic Reperfusion ◽

Ldh Release ◽

Cerebral Ischemic ◽

Cerebral Ischemic Reperfusion

Stroke is one of the leading causes of mortality and disability worldwide. Long noncoding RNAs (lncRNAs) including MALAT1 have been shown to have critical roles in cerebral ischemia reperfusion injury (CIRI). However, the underlying mechanism of MALAT1 in CIRI has not been elucidated. The present study aimed to investigate the function and potential regulatory mechanism of MALAT1 in cerebral ischemic reperfusion injury. We established the middle cerebral artery occlusion (MCAO) model and oxygen-glucose deprivation/reoxygenation (OGD/RX) model in vivo and in vitro, and then Cell Counting Kit-8 (CCK-8), RT-qPCR, flow cytometry analysis, lactate dehydrogenase (LDH) analysis, and 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to examine cell viability, MALAT1, aquaporin-4 (AQP4) expression, LDH release, and infarct volume, respectively. The level of AQP4 was remarkably upregulated in CIRI 24 h/48 h or OGD/RX 24 h/48 h compared with the sham group. Knockdown of AQP4 could alleviate OGD/RX-induced injury through enhancing cell viability and reducing LDH release and the rate of apoptotic cells. Furthermore, we found that MALAT1 was also increased in OGD/RX 24 h/48 h and silencing of MALAT1 could decrease AQP4. Inhibition of MALAT1 could also protect OGD/RX-induced injury, while the protective effect of MALAT1 siRNA on cerebral ischemic reperfusion was disappeared after transfection with AQP4 plasmid, indicating that MALAT1 may play a protective role in brain stroke through regulating AQP4. Taken together, our study provides evidence that MALAT1 is involved in ischemic stroke by inhibiting AQP4. Therefore, MALAT1 may serve as a potential target for therapeutic intervention in ischemic brain injury.

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ATP2B1-AS1 Promotes Cerebral Ischemia/Reperfusion Injury Through Regulating the miR-330-5p/TLR4-MyD88-NF-κB Signaling Pathway

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.720468 ◽

2021 ◽

Vol 9 ◽

Author(s):

Lei Wang ◽

Ying Tan ◽

Ziyu Zhu ◽

Jun Chen ◽

Qiang Sun ◽

...

Keyword(s):

Cerebral Ischemia ◽

Cell Viability ◽

Pc12 Cells ◽

Ischemia Reperfusion Injury ◽

Cell Model ◽

Infarct Volume ◽

Ischemia Reperfusion ◽

Morris Water Maze Test ◽

Cerebral Ischemia Reperfusion

We aim to explore the expression and function of long non-coding RNA (lncRNA) ATP2B1-AS1 in a cerebral ischemia/reperfusion (I/R) injury. In this study, we established a middle cerebral artery occlusion/reperfusion (MCAO/IR) rat model and an OGD/R PC12 cell model to evaluate the expression and role of ATP2B1-AS1 in the cerebral I/R injury. We found that the expression of ATP2B1-AS1 was upregulated in both in vitro and in vivo cerebral I/R injury models. Knockdown of ATP2B1-AS1 increased the cell viability, inhibited apoptosis, and decreased the expressions of inflammation cytokines. The target of ATP2B1-AS1 was predicted and validated to be miR-330-5p. MiR-330-5p abrogated the regulatory effect of ATP2B1-AS1 on cell viability, apoptosis, and cytokines of OGD/R PC12 cells. Furthermore, the results showed that miR-330-5p targeted TLR4, which was also upregulated in the infarcted area of MCAO/IR rats and OGD/R PC12 cells. Overexpression of ATP2B1-AS1 increased the expressions of TLR4, MyD88, and NF-κB p65 of OGD/R PC12 cells, while the effect of ATP2B1-AS1 was abrogated by miR-330-5p. In addition, knockdown of ATP2B1-AS1 decreased the latency time, increased the time of passing the platform position, reduced the cerebral infarct volume, decreased neurological deficit scores, and reduced the number of damaged neurons of MCAO/IR rats that were subjected to the Morris water maze test. Taken together, our study indicates that ATP2B1-AS1 may be an attractive therapeutic target for the treatment of cerebral ischemic injuries.

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Typhae pollen polysaccharides protect hypoxia-induced PC12 cell injury via regulation of miR-34a/SIRT1

International Journal of Immunopathology and Pharmacology ◽

10.1177/2058738420910005 ◽

2020 ◽

Vol 34 ◽

pp. 205873842091000

Author(s):

Shichun Wang ◽

Qianqian Tang ◽

Fuchao Ge ◽

Qing Guo

Keyword(s):

Western Blot ◽

Cell Viability ◽

Pc12 Cells ◽

Pc12 Cell ◽

Cell Injury ◽

Cell Model ◽

Luciferase Assay ◽

Signal Pathways ◽

Qrt Pcr

This current research was performed to investigate the role of typhae pollen polysaccharides (TPP) in hypoxia-treated PC12 cell which was an in vitro cell model of cerebral ischemia. Hypoxia-treated cells were treated with TPP for 12 h. Cell viability and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-2 5-diphenyl-2H-tetrazolium bromide (MTT) assay and flow cytometry, respectively. Cell apoptotic proteins and PI3K/AKT and Ras/Raf/MEK/ERK signal pathway–associated proteins were also examined by western blot. Furthermore, abnormal expression of miR-34a and silent information regulator 1 (SIRT1) was achieved by transfection. Besides, the expression of miR-34a and SIRT1 was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of SIRT1 was detected by qRT-PCR and western blot. The relationship between miR-34a and SIRT1 was verified by luciferase assay. We found that TPP enhanced cell viability and inhibited apoptosis in hypoxia-treated PC12 cells. Moreover, TPP increased the accumulated levels of Bcl-2 while decreased expression of Bax, cleaved Caspase-3, and cleaved PARP. TPP downregulated miR-34a expression while induced by hypoxia. Further results showed that miR-34a overexpression reversed the results led by TPP in cell viability, apoptosis, and its related proteins. In addition, SIRT1 was upregulated by TPP and was verified to be a target of miR-34a. Silence of SIRT1 led to the opposite results led by TPP. In the end, TPP activated PI3K/AKT and Ras/Raf/MEK/ERK signal pathways. In conclusion, TPP plays important roles in regulating cell viability and apoptosis in hypoxia-treated PC12 cells via modulating miR-34a/SIRT1, as well as activating PI3K/AKT and Ras/Raf/MEK/ERK signal pathways.

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LncRNA LSINCT5/miR-222 regulates myocardial ischemia‑reperfusion injury through PI3K/AKT pathway

Journal of Thrombosis and Thrombolysis ◽

10.1007/s11239-021-02506-3 ◽

2021 ◽

Author(s):

Xueying Tong ◽

Jiajuan Chen ◽

Wei Liu ◽

Hui Liang ◽

Hezhong Zhu

Keyword(s):

Myocardial Ischemia ◽

Cell Viability ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Akt Signaling ◽

Akt Pathway ◽

Akt Signaling Pathway ◽

Myocardial Ischemia Reperfusion Injury ◽

Myocardial Ischemia Reperfusion

AbstractCardiovascular diseases rank the top cause of morbidity and mortality worldwide and are usually associated with blood reperfusion after myocardial ischemia/reperfusion injury (MIRI), which often causes severe pathological damages and cardiomyocyte apoptosis. LSINCT5 expression in the plasma of MI patients (n = 53), healthy controls (n = 42) and hypoxia-reoxygenation (HR)-treated cardiomyocyte AC16 cells was examined using qRT-PCR. The effects of LSINCT5 on cell viability and apoptosis were detected by MTT and flow cytometry, respectively. The expression of apoptosis-related proteins Bcl2, Bax and caspase 3 were tested by Western blot. The interaction between LSINCT5 and miR-222 was predicted by bioinformatic analysis. Moreover, changes in viability and apoptosis of AC16 cells co-transfected with siLSINCT5 and miR-222 inhibitor after HR treatment were examined. At last, the expression of proteins in PI3K/AKT pathway, namely PTEN, PI3K and AKT, was examined to analyze the possible pathway participating in LSINCT5-mediated MI/RI. Our study showed that LSINCT5 expression was upregulated in the plasma of MI patients and HR-treated AC16 cells. LSINCT5 overexpression significantly decreased cell viability and apoptosis. Luciferase reporter gene assay and RNA pulldown assay showed that LSINCT5 was a molecular sponge of miR-222. MiR-222 silencing in AC16 cells simulated the phenotypes of MIRI patients and HR-treated cells, indicating that LSINCT5 functions via miR-222 to regulate proliferation and apoptosis of HR-treated AC16 cells. We also showed that proteins of PI3K/AKT signaling pathway were affected in HR-treated AC16 cells, and LSINTC5 knockdown rescued these effects. LncRNA LSINCT5 was upregulated during MI pathogenesis, and LSINCT5 regulated MIRI possibly via a potential LSINCT5/miR-222 axis and PI3K/AKT signaling pathway. Our findings may provide novel evidence for MIRI prevention.

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Expression of SSAT, a novel biomarker of tubular cell damage, increases in kidney ischemia-reperfusion injury

AJP Renal Physiology ◽

10.1152/ajprenal.00318.2002 ◽

2003 ◽

Vol 284 (5) ◽

pp. F1046-F1055 ◽

Cited By ~ 56

Author(s):

Kamyar Zahedi ◽

Zhaohui Wang ◽

Sharon Barone ◽

Anne E. Prada ◽

Caitlin N. Kelly ◽

...

Keyword(s):

Renal Failure ◽

Acute Renal Failure ◽

Reperfusion Injury ◽

Cell Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Atp Depletion ◽

Tubular Cell ◽

Mrna Levels ◽

Tubular Injury

Ischemia-reperfusion injury (IRI) is the major cause of acute renal failure in native and allograft kidneys. Identifying the molecules and pathways involved in the pathophysiology of renal IRI will yield valuable new diagnostic and therapeutic information. To identify differentially regulated genes in renal IRI, RNA from rat kidneys subjected to an established renal IRI protocol (bilateral occlusion of renal pedicles for 30 min followed by reperfusion) and time-matched kidneys from sham-operated animals was subjected to suppression subtractive hybridization. The level of spermidine/spermine N 1-acetyltransferase (SSAT) mRNA, an essential enzyme for the catabolism of polyamines, increased in renal IRI. SSAT expression was found throughout normal kidney tubules, as detected by nephron segment RT-PCR. Northern blots demonstrated that the mRNA levels of SSAT are increased by greater than threefold in the renal cortex and by fivefold in the renal medulla at 12 h and returned to baseline at 48 h after ischemia. The increase in SSAT mRNA was paralleled by an increase in SSAT protein levels as determined by Western blot analysis. The concentration of putrescine in the kidney increased by ∼4- and ∼7.5-fold at 12 and 24 h of reperfusion, respectively, consistent with increased functional activity of SSAT. To assess the specificity of SSAT for tubular injury, a model of acute renal failure from Na+depletion (without tubular injury) was studied; SSAT mRNA levels remained unchanged in rats subjected to Na+ depletion. To distinguish SSAT increases from the effects of tubular injury vs. uremic toxins, SSAT was increased in cis-platinum-treated animals before the onset of renal failure. The expression of SSAT mRNA and protein increased by ∼3.5- and >10-fold, respectively, in renal tubule epithelial cells subjected to ATP depletion and metabolic poisoning (an in vitro model of kidney IRI). Our results suggest that SSAT is likely a new marker of tubular cell injury that distinguishes acute prerenal from intrarenal failure.

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