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

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.

scholarly journals 1,8-Cineol Attenuated AΒ25-35-Induced PC12 Cell Injury through Reducing Caspase 3 Expression and NO Production

2019 ◽  
Vol 1 (1) ◽  
pp. 12-17
Author(s):  
Ze-qin Zhang ◽  
Hai-jian Li ◽  
Wan-zhong Li ◽  
Lin Wang ◽  
Zhen-zhen Li ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Viability ◽  
Pc12 Cells ◽  
Pc12 Cell ◽  
Caspase 3 ◽  
Cell Injury ◽  
Control Group ◽  
No Production ◽  
Detection Assay

Objective To investigate the effect of 1,8-cineol on caspase 3 expression and NO production induced by Aβ25-35 in PC12 cells. Methods PC12 cells were cultured in vitro, and cell injury was induced by Aβ25-35 with a concentration of 20 μM. 1,8-cineol (1, 3, 10 μM) was pretreated before Aβ25-35 treatment. PC12 cell viability was evaluated by MTT detection assay. Caspase 3 protein expression was detected by Western blotting. The level of NO production in PC12 cells was measured using ELISA detection assay kit. Results In cultured PC12 cells in vitro, MTT results showed that 20 μM of Aβ25-35 reduced cell viability significantly compared with control group. The cell viability was increased by pretreatment with 1,8-cineol with concentrations of 3 and 10 μM compared with Aβ25-35 only group. Western blotting results showed compared with control group, caspase 3 expression was increased significantly in 20 μM Aβ25-35 group. Compared with Aβ25-35 group, 1,8-cineol of 3 and 10 μM group reduced caspase 3 protein expression significantly. The level of NO production in PC12 cells was increased significantly, which was decreased by pretreatment with 3 and 10 μM of 1,8-cineol. Conclusions: Our results revealed a protective effect of 1,8-cineol on Aβ25-35 induced PC12 cell injury through inhibition of caspase 3 expression and NO production.

scholarly journals MiR-144-3p Aggravated Cartilage Injury in Rheumatoid Arthritis by Regulating BMP2/PI3K/Akt Axis

2021 ◽  
Author(s):  
Mei-Li Mo ◽  
Jin-Mei Jiang ◽  
Xiao-Ping Long ◽  
Li-Hu Xie
Keyword(s):  
Extracellular Matrix ◽  
Cell Viability ◽  
Cell Injury ◽  
Tissue Injury ◽  
Cartilage Injury ◽  
Luciferase Assay ◽  
Tunel Staining ◽  
Western Blots

Abstract Objectives Present study aimed to illustrate the role of miR-144-3p in RA. Methods N1511 chondrocytes were stimulated by IL-1β to mimic RA injury model in vitro. Rats were subjected to injection of type II collagen to establish an in vivo RA model and the arthritis index score was calculated. Cell viability was determined by CCK-8. The expression of cartilage extracellular matrix proteins (Collagen II and Aggrecan) and matrix metalloproteinases protein (MMP-13) were determined by qRT-PCR and western blots. Cell apoptosis was measured by Flow cytometry. ELISA was applied to test the secretion of pro-inflammatory cytokines (IL-1β and TNF-α). Tissue injury and apoptosis were detected by HE staining and TUNEL staining. Interaction of miR-144-3p and BMP2 was verified by dual luciferase assay. Results MiR-144-3p was dramatically increased in IL-1β induced N1511 cells. MiR-144-3p depletion elevated cell viability, suppressed apoptosis, pro-inflammatory cytokine releasing, and extracellular matrix loss in IL-1β induced N1511 cells. Moreover, miR-144-3p targeted BMP2 to modulate its expression negatively. Activation of PI3K/Akt signaling compromised inhibition of BMP2 induced aggravated N1511 cell injury with IL-1β stimulation. Inhibition of miR-144-3p alleviated cartilage injury and inflammatory in RA rats. Conclusion Collectively, miR-144-3p could aggravate chondrocytes injury inflammatory response in RA via BMP2/PI3K/Akt axis.

scholarly journals PRC1 promotes GLI1-dependent osteopontin expression in association with the Wnt/β-catenin signaling pathway and aggravates liver fibrosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Shenzong Rao ◽  
Jie Xiang ◽  
Jingsong Huang ◽  
Shangang Zhang ◽  
Min Zhang ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Western Blot ◽  
Cell Viability ◽  
Signaling Pathway ◽  
Cell Model ◽  
Stellate Cell ◽  
Underlying Mechanism ◽  
Histopathological Analysis ◽  
Osteopontin Expression

Abstract Background PRC1 (Protein regulator of cytokinesis 1) regulates microtubules organization and functions as a novel regulator in Wnt/β-catenin signaling pathway. Wnt/β-catenin is involved in development of liver fibrosis (LF). We aim to investigate effect and mechanism of PRC1 on liver fibrosis. Methods Carbon tetrachloride (CCl4)-induced mice LF model was established and in vitro cell model for LF was induced by mice primary hepatic stellate cell (HSC) under glucose treatment. The expression of PRC1 in mice and cell LF models was examined by qRT-PCR (quantitative real-time polymerase chain reaction), western blot and immunohistochemistry. MTT assay was used to detect cell viability, and western blot to determine the underlying mechanism. The effect of PRC1 on liver pathology was examined via measurement of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and hydroxyproline, as well as histopathological analysis. Results PRC1 was up-regulated in CCl4-induced mice LF model and activated HSC. Knockdown of PRC1 inhibited cell viability and promoted cell apoptosis of activated HSC. PRC1 expression was regulated by Wnt3a signaling, and PRC1 could regulate downstream β-catenin activation. Moreover, PRC1 could activate glioma-associated oncogene homolog 1 (GLI1)-dependent osteopontin expression to participate in LF. Adenovirus-mediated knockdown of PRC1 in liver attenuated LF and reduced collagen deposition. Conclusions PRC1 aggravated LF through regulating Wnt/β-catenin mediated GLI1-dependent osteopontin expression, providing a new potential therapeutic target for LF treatment.

Stachydrine inhibits PC12 cell apoptosis induced by Aβ25-35 in an in vitro cell model of neurocognitive disorders

2020 ◽  
Vol 17 ◽  
Author(s):  
Huan Fu ◽  
Mei Liu ◽  
Jinxiu Yan ◽  
Na Zhao ◽  
Liangchao Qu
Keyword(s):  
Cell Cycle ◽  
Pc12 Cells ◽  
Pc12 Cell ◽  
Cell Apoptosis ◽  
Target Genes ◽  
Cell Model ◽  
Neurocognitive Disorders ◽  
Water Soluble ◽  
Cell Cycle Distribution

Background: Abnormal deposition of amyloid beta (Aβ) is considered the primary cause of neurocognitive disorders (NCDs). Inhibiting cytotoxicity is an important aspect of the treatment of NCDs. Stachydrine (STA) has been widely used for gynecological and cardiovascular disorders. However, whether STA has protective functions in PC12 cells treated with Aβ25–35 remains unclear. Introduction: Traditional Chinese Medicine, stachydrine (STA) is a water-soluble alkaloid of Leonurus heterophyllus, which can inhibit cell apoptosis, suppress tumor growth, maintain homeostasis of myocardial cells, and alleviate endothelial dysfunction. This study will investigate the effect of STA on inhibiting PC12 cell apoptosis induced by Aβ25-35 in an in vitro cell model of neurocognitive disorders. Methods: The differentially expressed genes (DEGs) in cells treated with STA were analyzed according to the Gene Expression Omnibus (GSE) 85871 data, and the STITCH database was used to identify the target genes of STA. PC12 cells were treated with Aβ25–35 and/or STA, an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed and lactate dehydrogenase (LDH) activity was determined. The cell cycle distribution was detected by flow cytometry, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) or Western blotting were used to detect the expression of genes or proteins. Results and Discussion: GSE85871 data showed 37 upregulated and 48 downregulated genes among the DEGs affected by STA. The results from the STITCH database showed that RPS8 and EED were target genes of STA. GSE1297 analysis showed the 13 most significantly upregulated genes. STA might affect the occurrence of NCDs through the interaction of TP53 with EED and RPS8. Finally, Aβ25-35 promoted apoptosis and LDH release of PC-12 cells, arrested the cell cycle in the G2/M phase, and inhibited the expression of the RPS8, EED, Bcl-2 and P53 genes. STA could reverse the effect of Aβ25-35. Conclusion: STA may play an important role in inhibiting apoptosis induced by Aβ25-35 by targeting the RPS8 and EED genes in the NCDs model in vitro.

scholarly journals MiR-513a-5p Aggravates Atherosclerosis Phenotype by Downregulating TFPI2 in Vitro

2021 ◽  
Author(s):  
Chun Wang ◽  
Feng Yao
Keyword(s):  
Cell Viability ◽  
Cell Model ◽  
Expression Level ◽  
Luciferase Assay ◽  
Wound Healing Assay ◽  
Bioinformatical Analysis ◽  
And Migration ◽  
Migration Capacity ◽  
Low Expression

Background: Atherosclerosis (AS) induced cardiology disease is largely associated with morbidity and mortality. The dysfunction of vascular smooth muscle cells (VSMCs) is considered to contribute to the etiology of AS. However, the mechanism underlying VSMCs dysfunction remains largely unclear. Our study aimed to explore novel molecules mediating VSMCs function. Methods: Bioinformatical analysis was applied to identify the key miRNAs that was aberrantly expressed in AS mouse and potentially targeted TFPI2. The AS-like cell model was generated by treating VSMCs with ox-LDL. The expression level of miR-513a-5p and TFPI2 in VSMCs and the serum of AS patients was evaluated by RT-qPCR, and the expression level of TFPI2 and PCNA was measured by western blot. The cell viability and migration capacity of VSMCs were determined by CCK-8 and wound healing assay, respectively. The target relationship between miR-513a-5p and TFPI2 was validated by dual-luciferase assay. Results: MiR-513a-5p was highly expressed while TFPI2 presented a low expression in AS patient serum. Treatment with 100 μg/mL ox-LDL overtly facilitated the cell viability and migration of VSMCs, also promoted miR-513a-5p expression while limit the expression of TFPI2. Moreover, silencing miR-513a-5p inhibited the cell viability, migration and the expression of proliferative marker in ox-LDL treated VSMCs, while inhibition of TFPI2 enhanced that. It was further found that miR-513a-5p could target TFPI2 and silencing miR-513a-5p compromised the aggresive effect of TFPI2 inhibition on the viability and migration ox-LDL treated VSMCs. Conclusion: miR-513a-5p could contribute to the dysfunction of VSMCs in AS through targeting and inhibiting TFPI2.

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

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Ya-ping Zhou ◽  
Guo-chun Li
Keyword(s):  
Cell Viability ◽  
Reperfusion Injury ◽  
Pc12 Cells ◽  
Cell Injury ◽  
Cell Model ◽  
Ischemia Reperfusion ◽  
Sirtuin 1 ◽  
Cerebral Stroke ◽  
Stroke Treatment ◽  
Ldh Release

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.

MiR-582-5p attenuates neonatal hypoxic-ischemic encephalopathy by targeting high mobility group box 1 (HMGB1) through inhibiting neuroinflammation and oxidative stress

2021 ◽  
Vol 18 ◽  
Author(s):  
Guang Yang ◽  
Zhimin Xue ◽  
Yuan Zhao
Keyword(s):  
Oxidative Stress ◽  
Cell Viability ◽  
Pc12 Cells ◽  
Pc12 Cell ◽  
Cell Injury ◽  
High Mobility ◽  
High Mobility Group ◽  
Hypoxic Ischemic Encephalopathy ◽  
And Oxidative Stress ◽  
Ischemic Encephalopathy

Background: MiR-582-5p has been demonstrated to protect against ischemic stroke. However, its implication in the progression of neonatal hypoxic-ischemic encephalopathy (HIE) has not been explored. Methods: In this study, we used an in vitro model of oxygen-glucose deprivation (OGD) to investigate the protective effect of miR-582-5p on PC12 cells. OGD-induced inhibition of cell viability and promotion of cell death was assessed by CCK-8 assay and flow cytometry. Real-time PCR and enzyme-linked immunosorbent assay (ELISA) were utilized to examine the levels of inflammatory cytokines. The effects of miR-582-5p on OGD-induced oxidative injury were assessed by the determination of oxidative stress indicators. Furthermore, dual-luciferase reporter assay and gain-offunction assay were used to determine the mechanism of miR-582-5p in OGD-induced cell injury. Results : The expression of miR-582-5p was reduced upon OGD treatment in PC12 cells. Overexpression of miR-582-5p inhibited OGD-induced PC12 cell injury by regulating cell viability, apoptosis, inflammatory responses, and oxidative stress. MiR-582-5p targeted and negatively regulated high mobility group box 1 (HMGB1). MiR-582-5p presented protective effects on OGD-induced PC12 cell injury by targeting HMGB1. Conclusion: Our results indicated that miR-582-5p ameliorates neuronal injury by inhibiting apoptosis, inflammation, and oxidative stress through targeting HMGB1.

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

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.

scholarly journals IL-6/STAT3 Induced Neuron Apoptosis in Hypoxia by Downregulating ATF6 Expression

2021 ◽  
Vol 12 ◽  
Author(s):  
Simin Zhou ◽  
Zhifeng Zhong ◽  
Pei Huang ◽  
Bin Xiang ◽  
Xiaoxu Li ◽  
...  
Keyword(s):  
Western Blot ◽  
Er Stress ◽  
Pc12 Cells ◽  
Hypobaric Hypoxia ◽  
Cell Model ◽  
Enzyme Linked Immunosorbent Assay ◽  
Qrt Pcr ◽  
Stat3 Phosphorylation ◽  
Induced Neuron ◽  
Neuron Apoptosis

Background: Neuron apoptosis, regulated by endoplasmic reticulum (ER) stress in the hippocampus, is an essential factor influencing the cognitive impairment induced by hypobaric hypoxia. Hypoxia mainly changes the activating transcription factor (ATF6) pathway of ER stress. However, the role of ATF6 in neuron survival, apoptosis, and upstream regulation is still controversial.Methods: We established a hypobaric hypoxia-induced C57BL/6 murine model and cell lines exposed to 1% hypoxia, including PC12 and HT22. First, we tested the expressions of interleukin 6 (IL-6), IL-1β, and IL-10 in C57BL/6 mice’s hippocampus under hypoxia using enzyme-linked immunosorbent assay (ELISA). We determined the signal transducer and activator of transcription 3 (STAT3) phosphorylation at tyrosine (Tyr)705 by western blot and the expression of ATF6, 78-kDa glucose-regulated protein (GRP78), and C/-EBP homologous protein (CHOP) related to ER stress by immunofluorescence (IF), western blot, and qRT-PCR; they were then verified on the cell model. Additionally, IL-6 (40 ng/mL) and STAT3 siRNA were used to treat the PC12 cells for 48 and 4 h to activate or silence STAT3, respectively. Subsequently, the cells of siRNA group were exposed to 1% hypoxia for 48 h. Furthermore, the ATF6 and CHOP expressions were detected with western blot and qRT-PCR. Finally, we examined the binding of STAT3 to the ATF6 promoter by chromatin immunoprecipitation (ChIP)-seq.Results: The results showed that IL-6 increased, IL-10 decreased in the hypoxia group, and IL-1β showed no difference between the hypoxia and the normoxia groups. Neuron apoptosis was significantly elevated by exposure to hypoxia for 48h in PC12 cells. The hypobaric hypoxia-induced ER stress proteins, ATF6, GRP78, and CHOP, and the p-STAT3 (Tyr705) expressions increased both in in vivo and in vitro. Besides, STAT3 silencing significantly promoted the ATF6 expression and inhibited CHOP, while STAT3 activation downregulated the expression of ATF6 and upregulated CHOP in PC12 cells. The ChIP-seq assay demonstrated that p-STAT3 (Tyr705) protein could bind to the ATF6 promoter region in HT22 cells.Conclusion: Phosphorylation of STAT3 at the Tyr705 site contributes to hypoxia-induced neuron apoptosis by downregulating ATF6, which might explain the inflammatory reaction and apoptosis of the hippocampal neurons induced by ER stress.

Geniposide alleviates lipopolysaccharide (LPS)-induced inflammation by downregulation of miR-27a in rat pancreatic acinar cell AR42J

2019 ◽  
Vol 400 (8) ◽  
pp. 1059-1068 ◽  
Author(s):  
Xiaofen Zhang ◽  
Taishan Gao ◽  
Yanhua Wang
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Cell Apoptosis ◽  
Cell Injury ◽  
Control Cell ◽  
Cell Counting ◽  
Pancreatic Acinar Cells ◽  
Protective Effects ◽  
Qrt Pcr ◽  
Ar42j Cells

Abstract Pancreatitis is a disease caused by inflammation of pancreatic acinar cells. Geniposide (GEN) possesses anti-inflammation activities. Hence, we investigated the effects of GEN on lipopolysaccharide (LPS)-stimulated AR42J cells. AR42J cells were stimulated by LPS and then treated with GEN and/or transfected with miR-27a mimic or negative control. Cell viability and cell apoptosis were detected using the Cell Counting Kit-8 and flow cytometry, respectively. All related proteins were measured by Western blot. The expression of miR-27a was detected by quantitative real time-polymerase chain reaction (qRT-PCR). Moreover, the expression of inflammatory cytokines interleukin-6 (IL-6) and monocyte chemoattractant protein (MCP)-1 was analyzed by qRT-PCR and Western blot. LPS significantly decreased cell viability, and enhanced cell apoptosis and IL-6, MCP-1 expression. Then GEN administration alleviated inflammatory injury by increasing cell viability, while reducing apoptosis, and IL-6 and MCP-1 expression. GEN downregulated miR-27a expression which was induced by LPS. Transfection with miR-27a mimic partially eliminated the protective effects of GEN. The phosphorylation of JNK and c-Jun was downregulated by GEN while upregulated by miR-27a overexpression. GEN alleviates LPS-induced AR42J cell injury as evidenced by promoting cell growth, and upregulation of IL-6 and MCP-1. This process might be modulated by down-regulating miR-27a and inactivation of JNK pathway.

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