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TrkB pathway
Journal Pre-proofs Research paper KLF2 protects BV2 microglial cells against oxygen and glucose deprivation injury by modulating BDNF/TrkB pathway Jingbin Zhou, Muchun Wang, Dongfeng Deng PII: DOI: Reference:
S0378-1119(19)30936-9 https://doi.org/10.1016/j.gene.2019.144277 GENE 144277
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Gene Gene
Received Date: Revised Date: Accepted Date:
8 August 2019 21 November 2019 21 November 2019
Please cite this article as: J. Zhou, M. Wang, D. Deng, KLF2 protects BV2 microglial cells against oxygen and glucose deprivation injury by modulating BDNF/TrkB pathway, Gene Gene (2019), doi: https://doi.org/10.1016/ j.gene.2019.144277
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KLF2 protects BV2 microglial cells against oxygen and glucose deprivation injury by modulating BDNF/TrkB pathway
Jingbin Zhou*, Muchun Wang, Dongfeng Deng
Neurosurgery Department, Affiliated Zhongshan Hospital Dalian University, Dalian, 116001, Liaoling Province, China
*Corresponding
author: Jingbin Zhou
Neurosurgery Department, Affiliated Zhongshan Hospital Dalian University, No.6, Jiefang street, zhongshan district, Dalian, 116001, Liaoling Province, China Tel: +86-411-62893170 Email: [email protected]
Abstract Cerebral ischemia injury is common in cerebral ischemic disease, and treatment options remain limited. Krueppel-like factor 2 (KLF2) is reported to negatively regulate inflammation in several ischemic diseases. Our study aimed to investigate the effects and underlying mechanism of KLF2 in BV2 microglial cells exposed to oxygen and glucose deprivation (OGD). We first found decreased KLF2 and toll-like receptor 2 (TLR2)/TLR4 in these cells. OGD also led to decrease in cell viability and increase in LDH release, apoptosis, the Bax/Bcl-2 ratio, and caspase3/9 expression, as well as production of inflammatory cytokines (e.g., TNFα ， IL-1β and IL-6), reactive oxygen species (ROS), and TLR2/TLR4. To examine KLF2’s effects on these OGD effects, we infected BV2 microglial cells with an ad-KLF2 or negative control vector, and we found that KLF2 reversed all of the effects of OGD exposure. Furthermore, KLF2 significantly increased levels of BDNF and TrkB in these cells, but these effects were blocked by K252a, a BDNF/TrkB inhibitor. K252a also decreased cell viability and increased apoptosis, inflammatory factors, ROS production, and TLR2/TLR4 expression in OGD-exposed BV2 cells that were treated with KLF2, were implying that K252a could reverse the effects of KLF2 on these cells. Taken together, our study results indicate that KLF2 may protect BV2 microglial cells against OGD injury by activating the BDNF/TrkB pathway. Key words Cerebral ischemia, Kruppel-like factor 2, oxygen and glucose deprivation, BDNF/TrkB
Introduction Ischemic stroke is a leading cause of death worldwide. It is caused by interruption of the cerebral blood supply (Feigin et al., 2003). Many well-known and complex pathological mechanisms are involved in the injury of the central nervous system after cerebral ischemia (Lambertsen et al., 2012). Neuronal and glial cell death leads to extensive local inflammation of the brain parenchyma and microvasculature. Microglia is key cells that help detect abnormal alterations in response to internal and external insults during cerebral ischemia injury (Kettenmann et al., 2011; Ginhoux et al., 2013). In fact, microglia can produce many kinds of inflammatory mediators in response to injury or infection, and this inflammation correlates with poor clinical outcomes in central nervous system diseases (Iadecola and Anrather, 2011). However, research indicates that specific microglial actions can be neuro-protective (Prinz and Priller, 2014). Thus, the underlying mechanism of microglia in cerebral ischemia injury remains unclear and disease-dependent. The activation and injury of microglia also have been found in other ischemic and hypoxic conditions (Kaur et al., 2013). In this study, we have established a model of cerebral ischemia injury in vitro by exposing microglial BV2 cells to oxygen and glucose deprivation (OGD). Kruppel-like factors are a zinc finger family of transcription factors that have important roles in modulating cell biological processes (Patel et al., 2013; Fan et al., 2017). Among the 17 highly conserved members (KLF1-KLF17), KLF2 has been most widely studied for its role in the survival and differentiation of lymphocyte biology (Clipson et al., 2015). However, its role isn’t limited to immune cell function and regulation. Studies have indicated that KLF2 also highly expressed in endothelial and hematopoietic cells (McConnell and Yang, 2010) and that it plays important roles in regulating inflammation in these cells (Das et al., 2006). In aneurysmal models of blood flow
dynamics, KLF2 participates in vascular wall reconstruction by negatively regulating inflammation (Wu et al., 2017). Other studies have confirmed the important effects of KLF2 in several biological processes, including systemic vascular permeability (Lin et al., 2010), atherosclerosis (Atkins et al., 2008), and even cerebrovascular function (Shi et al., 2013). However, its role in cell injury under cerebral ischemia injury remains unclear. Brain-derived neurotrophic factor (BDNF) is an important member of the neurotrophin family of proteins. It protects against ischemia-induced brain damage and cognitive impairment via its high-affinity receptor, tropomyosin-related kinase receptor type B (TrkB) (Binder and Scharfman, 2004; Fanaei et al., 2014; Sun et al., 2014). BDNF was reported to promote angiogenesis and neural regeneration by inhibiting oxidative damage, reducing apoptosis, and improving functional recovery in ischemic stroke (Schabitz et al., 2004). Importantly, BDNF protects the neonatal brain from cerebral ischemic injury in vivo (Nakamura et al., 2019). As a BDNF receptor, TrkB has been implicated in regulating central nervous system axon growth by binding to BDNF. The BDNF/TrkB pathway also activates various other intracellular signaling pathways (Bothwell, 2016). Thus, BDNF may provide a promising clinical target for treating cerebral ischemia. Indeed, a recent study has reported that BDNF/TrkB signaling reduces inflammation in a spinal cord injury model (Liang et al., 2019). We investigated whether the effects of KLF2 on cerebral ischemia injury were related to the BDNF/TrkB signaling pathway. Using a cellular model of OGD-injured BV2 cells, we investigated the effects and underlying mechanisms of KLF2 on microglia during cerebral ischemia. This study provides a novel theoretical basis for targeting the prevention and treatment of ischemic stroke. Materials and methods
Cell culture and treatment BV2 microglial cells were purchased from the China Center for Type Culture Collection (Wuhan, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) under 5% CO2 in 37 °C. BV2 microglial cells were infected for 24 hours with the KLF2 virus vector (Ad-KLF2; MOI=10) or a negative control (Ad-GFP; MOI=10), then the cells were plated and cultivated for 12 h in a normoxic (20% O2, 5% CO2) or hypoxic (1% O2, 5% CO2, and 92% N2) chamber at 37 °C. In addition, small interfering RNA (siRNA) oligonucleotides targeting KLF2 (si-KLF2) and the negative control siRNA (si-KLF2) were purchased from Sangon Biotech (Shanghai, China). BV2 microglial cells were seeded into 6-well plates and grown to 70~80% confluence. Then si-KLF2 and si-Ctrl (10 nM) were transfected into BV2 cells using Lipofectamine 2000 reagent according to the manufacture’s protocols, then the cells were plated and cultivated for 12 h in a normoxic (20% O2, 5% CO2) or hypoxic (1% O2, 5% CO2, and 92% N2) chamber at 37 °C. MTT assay Cell viability was assessed via MTT assay according to the manufacture’s protocol. BV2 cells were cultured in a six-well plate and infected with Ad-KLF2 (MOI=10) or transfected with si-KLF2 for 24 hours. Cells then were subjected to normoxia or OGD. Briefly, grouped BV2 cells were seeded in a 96-well plate and cultured for 24 h. Then, MTT (50 µl) was added to each well and incubated for 4 h at 37 °C. Dimethyl sulfoxide (100 µl) was used to dissolve the insoluble formazan crystals. Cell viability was recorded as the optical density at 570 nm according to a Synergy plate reader (Bio-TEK instruments, Winooski, VT, USA). Lactate dehydrogenase assay
Levels of lactate dehydrogenase (LDH) released into the culture medium were used to assess cell membrane integrity. BV2 cells were cultured in a six-well plate, infected with Ad-KLF2 (MOI=10) or transfected with si-KLF2 for 24 hours, and then subjected to normoxia or OGD. LDH release was measured using an LDH Assay Kit (Biotime, Xiamen, China) according to the manufacturer’s instructions. Results were recorded at 490 nm spectrophotometrically. Western blot assay Total protein was extracted from the grouped BV2 cells. The protein concentration was measured using a Pierce BCA Protein Assay Kit (Biotime, Xiamen, China). Proteins were electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). After blocking with 5% nonfat milk, the membrane was incubated overnight at 4 °C with each of the following antibodies: KLF2 (1:1000), TLR2 (1:500), TLR4 (1:500), Bcl-2 (1:1000), Bax (1:500), BDNF (1:2000), TrkB (1:500), caspase 3 (1:800) and caspase 9 (1:500) all from Abcam (Cambridge, UK). The membrane then was washed and incubated in the appropriate alkaline-phosphatase-conjugated secondary antibody for 2 h at room temperature. The blotted protein bands were visualized using enhanced chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Finally, the optical densities of proteins were obtained using Image J software. Flow cytometry assay BV2 cell apoptosis was measured via flow cytometry. BV2 cells were cultured in a six-well plate, infected with Ad-KLF2 (MOI=10) or transfected with si-KLF2 for 24 hours, and then subjected to normoxia or OGD. Following the experiments, the BV2 cells were washed with
phosphate-buffered saline and resuspended in 500 μl of buffer solution. After staining with FITC-Annexin V and propidium iodide (PI) for 15 min in the dark at room temperature, cells were analyzed within 1 h using a FACScan® flow cytometer equipped with Cell Quest software (BD Biosciences, San Jose, CA, USA). BV2 cells were loaded with 5-(and-6)-chloromethyl-2-,7-dichlorofluorescin diacetate (DCHF-DA) for intracellular ROS measurement via flow cytometry. Similarly, OGD-injured cells were infected with Ad-KLF2 or Ad-GFP, washed with phosphate-buffered saline, incubated with DCHF-DA in the dark for 30 min at 37 °C, and then resuspended in plain DMEM. The relative fluorescence levels were quantified using a flow cytometer with excitation at 480 nm and emission at 530 nm. ELISA BV2 cells were seeded in a 24-well plate and exposed to the previously described treatments. Culture supernatants then were collected, and levels of inflammatory cytokines TNFα, IL-1β, and IL-6 were measured using ELISA assay kits according to the manufacturer’s instructions. Statistical analysis All data are presented as the mean ± SD of three independent experiments. Statistical analyses were performed using Graphpad 6.0 using a one-way analysis ANOVA for multiple groups or two-tailed t test for two groups. P<0.05 was considered statistically significant.
Results KLF2 and TLR2/4 were abnormally expressed in oxygen/glucose-deprived BV2 microglial cells
In this study, cell proliferation decreased more in the OGD-injured BV2 microglial cells than in the control group cells (Fig. 1A). LDH release was higher in OGD-injured BV2 cells than in the controls (Fig. 1B). The results indicate that OGD could induce BV2 microglial cell injury. OGD treatment induces overexpression of the TLR pathway, as previously reported (Fig. 1C). Interestingly, we found that KLF2 decreased in BV2 microglial cells after OGD exposure (Fig. 1D), suggesting that KLF2 may play important roles in the microglia during cerebral ischemia. KLF2 inhibited apoptosis in BV2 microglial cells exposed to oxygen-glucose deprivation To investigate the effects of KLF2 on OGD-exposed BV2 microglial cells, cells were infected with Ad-KLF2 or si-KLF2. The protein expression of KLF2 was substantially more elevated in the Ad-KLF2 group than in the control group, and that was decreased in the si-KLF group compared with the control group (Fig. 2A). The MTT assay showed that OGD exposure led to decreased cell proliferation (Fig. 2B), whereas KLF2 overexpression increased it and KLF2 knockout decreased it. LDH release also decreased more in the OGD-exposed Ad-KLF2 group than in the OGD treatment group (Fig. 2C). In addition, si-KLF2 transfection increased the LDH release compared with the OGD group. Moreover, the apoptosis rate and Bax/Bcl-2 ratio increased after OGD but decreased after KLF2 infection, while those were all increased after KLF2 knockout (Fig. 2D-2E). KLF2 infection decreased the OGD-induced increase in caspase 3/9 expression, while si-KLF2 increased the caspase 3/9 expression compared with the OGD group (Fig. 2F). These results indicate that KLF2 may alleviate apoptosis in OGD-exposed BV2 microglial cells. KLF2 inhibited inflammatory factor secretion and ROS production in BV2 microglial cells exposed to oxygen-glucose deprivation We confirmed the effects of KLF2 on the inflammatory reaction of BV2 microglial cells under
OGD conditions. The ELISA results showed that OGD upregulated levels of inflammatory cytokines, which were reduced after Ad-KLF2 infection (Fig. 3A). ROS production also increased after OGD, but Ad-KLF2 infection also reversed that effect (Fig. 3B). Moreover, protein expression of the TLR pathway was detected after OGD or ad-KLF2 treatment (Fig. 3C). The protein expression of TLR2 and TLR4 increased by OGD treatment were decreased after ad-KLF2 infection. These results indicate that KLF2 may alleviate the inflammatory reaction of OGD-exposed BV2 microglial cells. KLF2 protected BV2 microglial cells against oxygen-glucose deprivation injury by modulating the BDNF/TrkB pathway To investigate the underlying molecular mechanism in KLF2’s protection against OGD-induced injury in BV2 microglial cells, we detected the expression of BDNF/TrkB using a western blot assay. The results showed that the protein expression of BDNF and TrkB decreased more in the OGD-injured cells than in the control group cells. However, ad-KLF2 infection upregulated this protein expression in OGD-exposed BV2 cells (Fig. 4A). Thus, the inhibitor of the BDNF/TrkB pathway (K252a) was used in OGD-injured BV2 cells after that were infected with ad-KLF2 (Fig. 4B). K252a treatment reversed KLF2’s effects on cell proliferation and LDH release (Fig. 4C-4D). Similar, the KLF2’s effects on cell apoptosis was also reversed by K252a (Fig.5A). The Bax/Bcl-2 ratio and caspase3/9 protein expression also increased after K252a treatment, implying that inhibition of the BDNF/TrkB pathway reversed the effects of KLF2 on apoptosis in OGD-exposed BV2 microglial cells (Fig. 5B-D). Moreover, K252a increased inflammatory cytokines, ROS production, and the protein expression of TLR2 and TLR4 (Fig. 5E-5F), suggesting that inhibition of the BDNF/TrkB pathway reversed the effects of KLF2 on the inflammatory
reaction in these cells. These results indicate that KLF2 may protect BV2 microglial cells against OGD injury by activating the BDNF/TrkB pathway.
Discussion In this study, we found reduced cell proliferation and increased LDH release and TLR pathway expression in OGD-exposed BV2 microglial cells. And KLF2 levels were lower in OGD-injured BV2 cells than in the control group cells. Infection with ad-KLF2 led to increased cell viability and reduced apoptosis, inflammatory factor levels, ROS production, and TLR pathway expression in OGD-exposed BV2 cells. Moreover, OGD led to a significantly decreased BDNF and TrkB expression. Ad-KLF2 infection significantly increased the expression of BDNF and TrkB, but these effects were blocked significantly by the BDNF/TrkB inhibitor, K252a. Importantly, K252a treatment decreased cell proliferation and increased apoptosis, inflammatory factor levels, ROS production, and TLR pathway expression in KLF2-infected BV2 cells with OGD injury, implying that K252a could reverse the effects of KLF2 in these cells. Taken together, our study indicates that KLF2 may protect BV2 microglial cells against OGD injury by activating the BDNF/TrkB signaling pathway. Microglia is macrophage-like cells and the main immune cells of the brain. They play a critical role in the brain’s inflammatory reactions to stroke, brain trauma, neurodegeneration, and other nervous system diseases (Kreutzberg, 1996; A et al., 2019). Microglia determine the fate of neurons by modulating cytokines and molecules to alter micro-environmental homeostasis (Kohman and Rhodes, 2013). They act as critical mediators of neuropathology and neuroprotection in many physiological conditions (Puyal et al., 2013; Walker et al., 2014). Microglia also contribute to
neuronal damage by modulating excessive inflammatory cytokines or cytotoxic factors, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and ROS (Lucas et al., 2006; Trettel et al., 2019). These factors participate in the neuronal dysfunction of several brain diseases, such as ischemic stroke (Yang et al., 2010). Thus, promoting the overactive inflammatory response of microglia may provide a therapeutic strategy to alleviate the progression of ischemic stroke. The cell model of OGD-induced microglia injury has been established to investigate the underlying mechanism of cerebral ischemic injury (Xiang et al., 2014). Our results show that cell viability decreased markedly after 3 h of OGD. Kruppel-like factors are implicated in many biological processes including inflammation. KLF2 is widely known to act as a molecular switch that regulates endothelial function (Atkins and Jain, 2007). It is also highly expressed and plays important regulatory roles in pro-inflammatory activation in endothelial and hematopoietic cells (McConnell and Yang, 2010). It induces protective factors’ expression, including vasodilator and anti-oxidant genes, and it inhibits expression of inflammatory genes, including TNFα (Chu et al., 2018; Shi et al., 2018). Importantly, KLF2 has been implicated in the positive effects of simvastatin on liver ischemia-reperfusion injury in rats (Liu et al., 2017), including suppressing inflammatory cytokines, hepatic oxidative stress, and apoptosis. Moreover, targeting the downstream effector of KLF2 improves critical limb ischemia in adults (Caradu et al., 2018). Thus, KLF2 may play an important role in diseases related to ischemic injury. In this study, we found decreased KLF2 levels in OGD-exposed BV2 microglial cells. Overexpression of KLF2 inhibited the apoptosis and inflammatory reaction of BV2 microglial cells. Furthermore, we found the reduced expression of BDNF/TrkB in OGD-exposed BV2 microglial cells. BDNF is a member of the neurotrophin family, which exerts its protective effects
by binding to the endogenous TrkB receptor (Yoshii and Constantine-Paton, 2010; Harward et al., 2016). BDNF has neuroprotective roles and improves functional recovery after ischemic stroke (Wei et al., 2014). In a hypoxia-ischemia model, BDNF has been reported to inhibit apoptosis (Zhang et al., 2012). Reduced expression of BDNF occurs after middle cerebral artery occlusion in mice (Yang et al., 2018) and OGD/reoxygenation in cells (Ye et al., 2017). Continuous treatment with BDNF could protect the brain against neurological damage (Galvin and Oorschot, 2003). The BDNF/TrkB pathway also reportedly participates in the protective effects of mGluR5 in BV2 cells exposed to OGD/R (Ye et al., 2017). BDNF binding to TrkB can activate various downstream intracellular signaling pathways in contact with the biological functions of cells. Additionally, BDNF gene is subject to an extensive autoregulatory-positive feedback loop, in which TrkB signaling induces the expression of all major BDNF transcripts. The TLR pathway has been shown to increase in BV2 microglial cells after hypoxia exposure. TLR4 activation is a key part of microglia functions after hypoxia exposure (Yao et al., 2013). Suppression of TLR4 thus may reveal new opportunities to develop effective therapeutics for inflammatory diseases. Isoflurane protects against OGD injury in microglia by regulating the TLR4 pathway (Xiang et al., 2014). Our results also indicated that KLF2 inhibited increases in TLR2 and TLR4 in BV2 microglial cells. Therefore, microglial TLR4 may be a key factor in KLF2’s protection against hypoxic brain injuries. BDNF level increased by presynaptic stimulation may arise from the presynaptic region (due to the electrical stimulus) or the postsynaptic region (due to local ACh signaling) (Hurtado et al., 2017). BDNF has a complex gene structure (Aid et al., 2007). The rodent BDNF is consisted of one protein-coding 3’ exon and one of the eight noncoding 5’ exons. Besides, two exons (Vh and VIIIh) in humans have been described except that. All the exons have their
promoter, allowing complex spatiotemporal regulation of BDNF expression in different brain regions throughout development or in response to various stimuli (Baj et al., 2013). AP-1 family transcription factors have reported to upregulate exon I, III, and VI transcripts, which further affect BDNF promoter I directly and affect promoters III and VI indirectly (Tuvikene et al., 2016). Interestingly, KLF2 attenuates inflammation through the activation of AP-1 (Shi et al., 2018). In the current study, KLF2 overexpression increased protein expression of BDNF and TrkB in OGD-injured BV2 microglial cells. The BDNF/TrkB pathway inhibitor K252a reversed all of KLF2’s effects on OGD-injured BV2 microglial cells. Thus, we conclude that KLF2 may protect BV2 microglial cells by activating the BDNF/TrkB pathway. However, whether KLF2 activates the BDNF/TrkB pathway by regulating the BDNF promoter or by activating the AP-1 transcription factor requires further investigation. In conclusion, this study identified abnormal expression of KLF2 and TLR pathway protein in OGD-exposed microglia. KLF2 alleviated OGD-induced injury to BV2 cells by promoting cell viability and by reducing apoptosis, inflammatory factors, and ROS production. Moreover, we found that KLF2 conferred this protection by activating the BDNF/TrkB pathway. Therefore, our study provides evidence for a new therapeutic approach for treating patients injured by cerebral ischemia.
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Figure legends Fig.1 KLF2 and TLR2/4 were abnormally expressed in OGD induced BV2 microglial cells. BV2 cells were exposed to OGD for different times. A. Decreased cell proliferation was detected by MTT assay in OGD treatment BV2 cells compared to control group. B. Increased LDH release was detected by LDH assay in OGD treatment BV2 cells compared to control group. C. Elevated protein expression of TLR2 and TLR4 were determined by western blot assay in OGD treatment BV2 cells compared to control group. D. Reduced protein expression of KLF2 was determined by western blot assay in OGD treatment BV2 cells compared to control group. Data were expressed as the mean ± SD, n=3. These results was performed using the analysis of variance (ANOVA) test.*P<0.05 vs. Ctrl group.
Fig.2 KLF2 inhibited apoptosis in BV2 microglial cells exposed to OGD. BV2 cells were infected with Ad-KLF2 or si-KLF2 before OGD treatment. A. Protein expression of KLF2 was determined by western blot assay. B. Cell proliferation was detected by MTT assay. C. LDH release was detected by LDH assay. D. Cell apoptosis were detected by flow cytometry. E. Protein expression of Bcl-2 and Bax were detected by western blot assay. F. Protein expression of caspase 3 and caspase 9 were determined by western blot assay. Data were expressed as the mean ± SD, n=3. These results were performed using the analysis of variance (ANOVA) test. *P<0.05 vs. Ctrl group; #P<0.05
vs. OGD+ad-GFP group, $P<0.05 vs. OGD+si-Ctrl group.
Fig.3 KLF2 inhibited inflammatory factor secretion and ROS production in BV2 microglial cells exposed to OGD. BV2 cells were infected with Ad-KLF2 or Ad-GFP before OGD treatment.
A. The levels of inflammatory factors (TNF-α, IL-1β, IL-6) were detected by ELISA. B. ROS production was detected by flow cytometry. C. Protein expression of TLR2 and TLR4 were determined by western blot assay. Data were expressed as the mean ± SD, n=3. These results were performed using the analysis of variance (ANOVA) test. *P<0.05 vs. Ctrl group; #P<0.05 vs. OGD+ad-GFP group.
Fig.4 KLF2 protected BV2 microglial cells on cell viability against OGD injury by modulating the BDNF/TrkB pathway. A. Protein expression of BDNF and TrkB were determined by western blot assay in BV2 cells after ad-KLF2 infection. The inhibitor of the BDNF/TrkB pathway (K252a) was used in OGD-injured BV2 cells that were infected with ad-KLF2. Data were expressed as the mean ± SD, n=3. The results were performed using the analysis of variance (ANOVA) test. B. Protein expression of BDNF and TrkB were determined by western blot assay. C. Cell proliferation was detected by MTT assay. D. LDH release was detected by LDH assay. Data were expressed as the mean ± SD, n=3. These results were performed using two-tailed t test analysis. *P<0.05 vs. Ctrl group; #P<0.05 vs. OGD+ad-GFP group. &P<0.05 vs. OGD+ad-KLF2 group.
Fig. 5 KLF2 protected BV2 microglial cells on cell apoptosis and inflammatory response against OGD injury by modulating the BDNF/TrkB pathway. The inhibitor of the BDNF/TrkB pathway (K252a) was used in OGD-injured BV2 cells that were infected with ad-KLF2. A. Cell apoptosis were detected by flow cytometry. B. Protein expression of Bcl-2 and Bax were detected by western blot assay. C. Protein expression of caspase 3 and caspase 9 were determined by western blot assay. H. The levels of inflammatory factors (TNF-α, IL-1β, IL-6) were detected by ELISA. I.
ROS production was detected by flow cytometry. J. Protein expression of TLR2 and TLR4 were determined by western blot assay. Data were expressed as the mean ± SD, n=3. These results were performed using two-tailed t test analysis. &P<0.05 vs. OGD+ad-KLF2 group.
Abbreviation list: KLF2: Krueppel-like factor 2, OGD: Oxygen and glucose deprivation, TLR2: Toll-like receptor 2, ROS: Reactive oxygen species, TNFα: Tumor necrosis factor α, IL-1β: Interleukin 1β, BDNF: Brain-derived neurotrophic factor, TrkB: Tropomyosin-related kinase receptor type B, DMEM: Dulbecco’s Modified Eagle’s Medium, FBS: Fetal bovine serum, LDH: Lactate dehydrogenase, SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Authors' contributions We wish to confirm the authors' contributions and final approval of this manuscript. Jingbin Zhou, Muchun Wang and Dongfeng Deng conceived and designed the study. Jingbin Zhou and Dongfeng Deng performed the experiments. Muchun Wang analyzed the data. Daigang Lu, Jingbin Zhou wrote and reviewed the manuscript. Jingbin Zhou and Dongfeng Deng have overall responsibility for this manuscript.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Highlights 1. KLF2 and TLR2/4 were abnormally expressed in OGD induced BV2 cells. 2. KLF2 inhibits apoptosis in BV2 cells exposed to OGD. 3. KLF2 reduces levels of inflammatory factors and ROS in BV2 cells exposed to OGD. 4. KLF2 protects BV2 cells against OGD injury by modulating the BDNF/TrkB pathway.
S0378-1119(19)30936-9 https://doi.org/10.1016/j.gene.2019.144277 GENE 144277
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Received Date: Revised Date: Accepted Date:
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Please cite this article as: J. Zhou, M. Wang, D. Deng, KLF2 protects BV2 microglial cells against oxygen and glucose deprivation injury by modulating BDNF/TrkB pathway, Gene Gene (2019), doi: https://doi.org/10.1016/ j.gene.2019.144277
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KLF2 protects BV2 microglial cells against oxygen and glucose deprivation injury by modulating BDNF/TrkB pathway
Jingbin Zhou*, Muchun Wang, Dongfeng Deng
Neurosurgery Department, Affiliated Zhongshan Hospital Dalian University, Dalian, 116001, Liaoling Province, China
*Corresponding
author: Jingbin Zhou
Neurosurgery Department, Affiliated Zhongshan Hospital Dalian University, No.6, Jiefang street, zhongshan district, Dalian, 116001, Liaoling Province, China Tel: +86-411-62893170 Email: [email protected]
Abstract Cerebral ischemia injury is common in cerebral ischemic disease, and treatment options remain limited. Krueppel-like factor 2 (KLF2) is reported to negatively regulate inflammation in several ischemic diseases. Our study aimed to investigate the effects and underlying mechanism of KLF2 in BV2 microglial cells exposed to oxygen and glucose deprivation (OGD). We first found decreased KLF2 and toll-like receptor 2 (TLR2)/TLR4 in these cells. OGD also led to decrease in cell viability and increase in LDH release, apoptosis, the Bax/Bcl-2 ratio, and caspase3/9 expression, as well as production of inflammatory cytokines (e.g., TNFα ， IL-1β and IL-6), reactive oxygen species (ROS), and TLR2/TLR4. To examine KLF2’s effects on these OGD effects, we infected BV2 microglial cells with an ad-KLF2 or negative control vector, and we found that KLF2 reversed all of the effects of OGD exposure. Furthermore, KLF2 significantly increased levels of BDNF and TrkB in these cells, but these effects were blocked by K252a, a BDNF/TrkB inhibitor. K252a also decreased cell viability and increased apoptosis, inflammatory factors, ROS production, and TLR2/TLR4 expression in OGD-exposed BV2 cells that were treated with KLF2, were implying that K252a could reverse the effects of KLF2 on these cells. Taken together, our study results indicate that KLF2 may protect BV2 microglial cells against OGD injury by activating the BDNF/TrkB pathway. Key words Cerebral ischemia, Kruppel-like factor 2, oxygen and glucose deprivation, BDNF/TrkB
Introduction Ischemic stroke is a leading cause of death worldwide. It is caused by interruption of the cerebral blood supply (Feigin et al., 2003). Many well-known and complex pathological mechanisms are involved in the injury of the central nervous system after cerebral ischemia (Lambertsen et al., 2012). Neuronal and glial cell death leads to extensive local inflammation of the brain parenchyma and microvasculature. Microglia is key cells that help detect abnormal alterations in response to internal and external insults during cerebral ischemia injury (Kettenmann et al., 2011; Ginhoux et al., 2013). In fact, microglia can produce many kinds of inflammatory mediators in response to injury or infection, and this inflammation correlates with poor clinical outcomes in central nervous system diseases (Iadecola and Anrather, 2011). However, research indicates that specific microglial actions can be neuro-protective (Prinz and Priller, 2014). Thus, the underlying mechanism of microglia in cerebral ischemia injury remains unclear and disease-dependent. The activation and injury of microglia also have been found in other ischemic and hypoxic conditions (Kaur et al., 2013). In this study, we have established a model of cerebral ischemia injury in vitro by exposing microglial BV2 cells to oxygen and glucose deprivation (OGD). Kruppel-like factors are a zinc finger family of transcription factors that have important roles in modulating cell biological processes (Patel et al., 2013; Fan et al., 2017). Among the 17 highly conserved members (KLF1-KLF17), KLF2 has been most widely studied for its role in the survival and differentiation of lymphocyte biology (Clipson et al., 2015). However, its role isn’t limited to immune cell function and regulation. Studies have indicated that KLF2 also highly expressed in endothelial and hematopoietic cells (McConnell and Yang, 2010) and that it plays important roles in regulating inflammation in these cells (Das et al., 2006). In aneurysmal models of blood flow
dynamics, KLF2 participates in vascular wall reconstruction by negatively regulating inflammation (Wu et al., 2017). Other studies have confirmed the important effects of KLF2 in several biological processes, including systemic vascular permeability (Lin et al., 2010), atherosclerosis (Atkins et al., 2008), and even cerebrovascular function (Shi et al., 2013). However, its role in cell injury under cerebral ischemia injury remains unclear. Brain-derived neurotrophic factor (BDNF) is an important member of the neurotrophin family of proteins. It protects against ischemia-induced brain damage and cognitive impairment via its high-affinity receptor, tropomyosin-related kinase receptor type B (TrkB) (Binder and Scharfman, 2004; Fanaei et al., 2014; Sun et al., 2014). BDNF was reported to promote angiogenesis and neural regeneration by inhibiting oxidative damage, reducing apoptosis, and improving functional recovery in ischemic stroke (Schabitz et al., 2004). Importantly, BDNF protects the neonatal brain from cerebral ischemic injury in vivo (Nakamura et al., 2019). As a BDNF receptor, TrkB has been implicated in regulating central nervous system axon growth by binding to BDNF. The BDNF/TrkB pathway also activates various other intracellular signaling pathways (Bothwell, 2016). Thus, BDNF may provide a promising clinical target for treating cerebral ischemia. Indeed, a recent study has reported that BDNF/TrkB signaling reduces inflammation in a spinal cord injury model (Liang et al., 2019). We investigated whether the effects of KLF2 on cerebral ischemia injury were related to the BDNF/TrkB signaling pathway. Using a cellular model of OGD-injured BV2 cells, we investigated the effects and underlying mechanisms of KLF2 on microglia during cerebral ischemia. This study provides a novel theoretical basis for targeting the prevention and treatment of ischemic stroke. Materials and methods
Cell culture and treatment BV2 microglial cells were purchased from the China Center for Type Culture Collection (Wuhan, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) under 5% CO2 in 37 °C. BV2 microglial cells were infected for 24 hours with the KLF2 virus vector (Ad-KLF2; MOI=10) or a negative control (Ad-GFP; MOI=10), then the cells were plated and cultivated for 12 h in a normoxic (20% O2, 5% CO2) or hypoxic (1% O2, 5% CO2, and 92% N2) chamber at 37 °C. In addition, small interfering RNA (siRNA) oligonucleotides targeting KLF2 (si-KLF2) and the negative control siRNA (si-KLF2) were purchased from Sangon Biotech (Shanghai, China). BV2 microglial cells were seeded into 6-well plates and grown to 70~80% confluence. Then si-KLF2 and si-Ctrl (10 nM) were transfected into BV2 cells using Lipofectamine 2000 reagent according to the manufacture’s protocols, then the cells were plated and cultivated for 12 h in a normoxic (20% O2, 5% CO2) or hypoxic (1% O2, 5% CO2, and 92% N2) chamber at 37 °C. MTT assay Cell viability was assessed via MTT assay according to the manufacture’s protocol. BV2 cells were cultured in a six-well plate and infected with Ad-KLF2 (MOI=10) or transfected with si-KLF2 for 24 hours. Cells then were subjected to normoxia or OGD. Briefly, grouped BV2 cells were seeded in a 96-well plate and cultured for 24 h. Then, MTT (50 µl) was added to each well and incubated for 4 h at 37 °C. Dimethyl sulfoxide (100 µl) was used to dissolve the insoluble formazan crystals. Cell viability was recorded as the optical density at 570 nm according to a Synergy plate reader (Bio-TEK instruments, Winooski, VT, USA). Lactate dehydrogenase assay
Levels of lactate dehydrogenase (LDH) released into the culture medium were used to assess cell membrane integrity. BV2 cells were cultured in a six-well plate, infected with Ad-KLF2 (MOI=10) or transfected with si-KLF2 for 24 hours, and then subjected to normoxia or OGD. LDH release was measured using an LDH Assay Kit (Biotime, Xiamen, China) according to the manufacturer’s instructions. Results were recorded at 490 nm spectrophotometrically. Western blot assay Total protein was extracted from the grouped BV2 cells. The protein concentration was measured using a Pierce BCA Protein Assay Kit (Biotime, Xiamen, China). Proteins were electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). After blocking with 5% nonfat milk, the membrane was incubated overnight at 4 °C with each of the following antibodies: KLF2 (1:1000), TLR2 (1:500), TLR4 (1:500), Bcl-2 (1:1000), Bax (1:500), BDNF (1:2000), TrkB (1:500), caspase 3 (1:800) and caspase 9 (1:500) all from Abcam (Cambridge, UK). The membrane then was washed and incubated in the appropriate alkaline-phosphatase-conjugated secondary antibody for 2 h at room temperature. The blotted protein bands were visualized using enhanced chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Finally, the optical densities of proteins were obtained using Image J software. Flow cytometry assay BV2 cell apoptosis was measured via flow cytometry. BV2 cells were cultured in a six-well plate, infected with Ad-KLF2 (MOI=10) or transfected with si-KLF2 for 24 hours, and then subjected to normoxia or OGD. Following the experiments, the BV2 cells were washed with
phosphate-buffered saline and resuspended in 500 μl of buffer solution. After staining with FITC-Annexin V and propidium iodide (PI) for 15 min in the dark at room temperature, cells were analyzed within 1 h using a FACScan® flow cytometer equipped with Cell Quest software (BD Biosciences, San Jose, CA, USA). BV2 cells were loaded with 5-(and-6)-chloromethyl-2-,7-dichlorofluorescin diacetate (DCHF-DA) for intracellular ROS measurement via flow cytometry. Similarly, OGD-injured cells were infected with Ad-KLF2 or Ad-GFP, washed with phosphate-buffered saline, incubated with DCHF-DA in the dark for 30 min at 37 °C, and then resuspended in plain DMEM. The relative fluorescence levels were quantified using a flow cytometer with excitation at 480 nm and emission at 530 nm. ELISA BV2 cells were seeded in a 24-well plate and exposed to the previously described treatments. Culture supernatants then were collected, and levels of inflammatory cytokines TNFα, IL-1β, and IL-6 were measured using ELISA assay kits according to the manufacturer’s instructions. Statistical analysis All data are presented as the mean ± SD of three independent experiments. Statistical analyses were performed using Graphpad 6.0 using a one-way analysis ANOVA for multiple groups or two-tailed t test for two groups. P<0.05 was considered statistically significant.
Results KLF2 and TLR2/4 were abnormally expressed in oxygen/glucose-deprived BV2 microglial cells
In this study, cell proliferation decreased more in the OGD-injured BV2 microglial cells than in the control group cells (Fig. 1A). LDH release was higher in OGD-injured BV2 cells than in the controls (Fig. 1B). The results indicate that OGD could induce BV2 microglial cell injury. OGD treatment induces overexpression of the TLR pathway, as previously reported (Fig. 1C). Interestingly, we found that KLF2 decreased in BV2 microglial cells after OGD exposure (Fig. 1D), suggesting that KLF2 may play important roles in the microglia during cerebral ischemia. KLF2 inhibited apoptosis in BV2 microglial cells exposed to oxygen-glucose deprivation To investigate the effects of KLF2 on OGD-exposed BV2 microglial cells, cells were infected with Ad-KLF2 or si-KLF2. The protein expression of KLF2 was substantially more elevated in the Ad-KLF2 group than in the control group, and that was decreased in the si-KLF group compared with the control group (Fig. 2A). The MTT assay showed that OGD exposure led to decreased cell proliferation (Fig. 2B), whereas KLF2 overexpression increased it and KLF2 knockout decreased it. LDH release also decreased more in the OGD-exposed Ad-KLF2 group than in the OGD treatment group (Fig. 2C). In addition, si-KLF2 transfection increased the LDH release compared with the OGD group. Moreover, the apoptosis rate and Bax/Bcl-2 ratio increased after OGD but decreased after KLF2 infection, while those were all increased after KLF2 knockout (Fig. 2D-2E). KLF2 infection decreased the OGD-induced increase in caspase 3/9 expression, while si-KLF2 increased the caspase 3/9 expression compared with the OGD group (Fig. 2F). These results indicate that KLF2 may alleviate apoptosis in OGD-exposed BV2 microglial cells. KLF2 inhibited inflammatory factor secretion and ROS production in BV2 microglial cells exposed to oxygen-glucose deprivation We confirmed the effects of KLF2 on the inflammatory reaction of BV2 microglial cells under
OGD conditions. The ELISA results showed that OGD upregulated levels of inflammatory cytokines, which were reduced after Ad-KLF2 infection (Fig. 3A). ROS production also increased after OGD, but Ad-KLF2 infection also reversed that effect (Fig. 3B). Moreover, protein expression of the TLR pathway was detected after OGD or ad-KLF2 treatment (Fig. 3C). The protein expression of TLR2 and TLR4 increased by OGD treatment were decreased after ad-KLF2 infection. These results indicate that KLF2 may alleviate the inflammatory reaction of OGD-exposed BV2 microglial cells. KLF2 protected BV2 microglial cells against oxygen-glucose deprivation injury by modulating the BDNF/TrkB pathway To investigate the underlying molecular mechanism in KLF2’s protection against OGD-induced injury in BV2 microglial cells, we detected the expression of BDNF/TrkB using a western blot assay. The results showed that the protein expression of BDNF and TrkB decreased more in the OGD-injured cells than in the control group cells. However, ad-KLF2 infection upregulated this protein expression in OGD-exposed BV2 cells (Fig. 4A). Thus, the inhibitor of the BDNF/TrkB pathway (K252a) was used in OGD-injured BV2 cells after that were infected with ad-KLF2 (Fig. 4B). K252a treatment reversed KLF2’s effects on cell proliferation and LDH release (Fig. 4C-4D). Similar, the KLF2’s effects on cell apoptosis was also reversed by K252a (Fig.5A). The Bax/Bcl-2 ratio and caspase3/9 protein expression also increased after K252a treatment, implying that inhibition of the BDNF/TrkB pathway reversed the effects of KLF2 on apoptosis in OGD-exposed BV2 microglial cells (Fig. 5B-D). Moreover, K252a increased inflammatory cytokines, ROS production, and the protein expression of TLR2 and TLR4 (Fig. 5E-5F), suggesting that inhibition of the BDNF/TrkB pathway reversed the effects of KLF2 on the inflammatory
reaction in these cells. These results indicate that KLF2 may protect BV2 microglial cells against OGD injury by activating the BDNF/TrkB pathway.
Discussion In this study, we found reduced cell proliferation and increased LDH release and TLR pathway expression in OGD-exposed BV2 microglial cells. And KLF2 levels were lower in OGD-injured BV2 cells than in the control group cells. Infection with ad-KLF2 led to increased cell viability and reduced apoptosis, inflammatory factor levels, ROS production, and TLR pathway expression in OGD-exposed BV2 cells. Moreover, OGD led to a significantly decreased BDNF and TrkB expression. Ad-KLF2 infection significantly increased the expression of BDNF and TrkB, but these effects were blocked significantly by the BDNF/TrkB inhibitor, K252a. Importantly, K252a treatment decreased cell proliferation and increased apoptosis, inflammatory factor levels, ROS production, and TLR pathway expression in KLF2-infected BV2 cells with OGD injury, implying that K252a could reverse the effects of KLF2 in these cells. Taken together, our study indicates that KLF2 may protect BV2 microglial cells against OGD injury by activating the BDNF/TrkB signaling pathway. Microglia is macrophage-like cells and the main immune cells of the brain. They play a critical role in the brain’s inflammatory reactions to stroke, brain trauma, neurodegeneration, and other nervous system diseases (Kreutzberg, 1996; A et al., 2019). Microglia determine the fate of neurons by modulating cytokines and molecules to alter micro-environmental homeostasis (Kohman and Rhodes, 2013). They act as critical mediators of neuropathology and neuroprotection in many physiological conditions (Puyal et al., 2013; Walker et al., 2014). Microglia also contribute to
neuronal damage by modulating excessive inflammatory cytokines or cytotoxic factors, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and ROS (Lucas et al., 2006; Trettel et al., 2019). These factors participate in the neuronal dysfunction of several brain diseases, such as ischemic stroke (Yang et al., 2010). Thus, promoting the overactive inflammatory response of microglia may provide a therapeutic strategy to alleviate the progression of ischemic stroke. The cell model of OGD-induced microglia injury has been established to investigate the underlying mechanism of cerebral ischemic injury (Xiang et al., 2014). Our results show that cell viability decreased markedly after 3 h of OGD. Kruppel-like factors are implicated in many biological processes including inflammation. KLF2 is widely known to act as a molecular switch that regulates endothelial function (Atkins and Jain, 2007). It is also highly expressed and plays important regulatory roles in pro-inflammatory activation in endothelial and hematopoietic cells (McConnell and Yang, 2010). It induces protective factors’ expression, including vasodilator and anti-oxidant genes, and it inhibits expression of inflammatory genes, including TNFα (Chu et al., 2018; Shi et al., 2018). Importantly, KLF2 has been implicated in the positive effects of simvastatin on liver ischemia-reperfusion injury in rats (Liu et al., 2017), including suppressing inflammatory cytokines, hepatic oxidative stress, and apoptosis. Moreover, targeting the downstream effector of KLF2 improves critical limb ischemia in adults (Caradu et al., 2018). Thus, KLF2 may play an important role in diseases related to ischemic injury. In this study, we found decreased KLF2 levels in OGD-exposed BV2 microglial cells. Overexpression of KLF2 inhibited the apoptosis and inflammatory reaction of BV2 microglial cells. Furthermore, we found the reduced expression of BDNF/TrkB in OGD-exposed BV2 microglial cells. BDNF is a member of the neurotrophin family, which exerts its protective effects
by binding to the endogenous TrkB receptor (Yoshii and Constantine-Paton, 2010; Harward et al., 2016). BDNF has neuroprotective roles and improves functional recovery after ischemic stroke (Wei et al., 2014). In a hypoxia-ischemia model, BDNF has been reported to inhibit apoptosis (Zhang et al., 2012). Reduced expression of BDNF occurs after middle cerebral artery occlusion in mice (Yang et al., 2018) and OGD/reoxygenation in cells (Ye et al., 2017). Continuous treatment with BDNF could protect the brain against neurological damage (Galvin and Oorschot, 2003). The BDNF/TrkB pathway also reportedly participates in the protective effects of mGluR5 in BV2 cells exposed to OGD/R (Ye et al., 2017). BDNF binding to TrkB can activate various downstream intracellular signaling pathways in contact with the biological functions of cells. Additionally, BDNF gene is subject to an extensive autoregulatory-positive feedback loop, in which TrkB signaling induces the expression of all major BDNF transcripts. The TLR pathway has been shown to increase in BV2 microglial cells after hypoxia exposure. TLR4 activation is a key part of microglia functions after hypoxia exposure (Yao et al., 2013). Suppression of TLR4 thus may reveal new opportunities to develop effective therapeutics for inflammatory diseases. Isoflurane protects against OGD injury in microglia by regulating the TLR4 pathway (Xiang et al., 2014). Our results also indicated that KLF2 inhibited increases in TLR2 and TLR4 in BV2 microglial cells. Therefore, microglial TLR4 may be a key factor in KLF2’s protection against hypoxic brain injuries. BDNF level increased by presynaptic stimulation may arise from the presynaptic region (due to the electrical stimulus) or the postsynaptic region (due to local ACh signaling) (Hurtado et al., 2017). BDNF has a complex gene structure (Aid et al., 2007). The rodent BDNF is consisted of one protein-coding 3’ exon and one of the eight noncoding 5’ exons. Besides, two exons (Vh and VIIIh) in humans have been described except that. All the exons have their
promoter, allowing complex spatiotemporal regulation of BDNF expression in different brain regions throughout development or in response to various stimuli (Baj et al., 2013). AP-1 family transcription factors have reported to upregulate exon I, III, and VI transcripts, which further affect BDNF promoter I directly and affect promoters III and VI indirectly (Tuvikene et al., 2016). Interestingly, KLF2 attenuates inflammation through the activation of AP-1 (Shi et al., 2018). In the current study, KLF2 overexpression increased protein expression of BDNF and TrkB in OGD-injured BV2 microglial cells. The BDNF/TrkB pathway inhibitor K252a reversed all of KLF2’s effects on OGD-injured BV2 microglial cells. Thus, we conclude that KLF2 may protect BV2 microglial cells by activating the BDNF/TrkB pathway. However, whether KLF2 activates the BDNF/TrkB pathway by regulating the BDNF promoter or by activating the AP-1 transcription factor requires further investigation. In conclusion, this study identified abnormal expression of KLF2 and TLR pathway protein in OGD-exposed microglia. KLF2 alleviated OGD-induced injury to BV2 cells by promoting cell viability and by reducing apoptosis, inflammatory factors, and ROS production. Moreover, we found that KLF2 conferred this protection by activating the BDNF/TrkB pathway. Therefore, our study provides evidence for a new therapeutic approach for treating patients injured by cerebral ischemia.
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Figure legends Fig.1 KLF2 and TLR2/4 were abnormally expressed in OGD induced BV2 microglial cells. BV2 cells were exposed to OGD for different times. A. Decreased cell proliferation was detected by MTT assay in OGD treatment BV2 cells compared to control group. B. Increased LDH release was detected by LDH assay in OGD treatment BV2 cells compared to control group. C. Elevated protein expression of TLR2 and TLR4 were determined by western blot assay in OGD treatment BV2 cells compared to control group. D. Reduced protein expression of KLF2 was determined by western blot assay in OGD treatment BV2 cells compared to control group. Data were expressed as the mean ± SD, n=3. These results was performed using the analysis of variance (ANOVA) test.*P<0.05 vs. Ctrl group.
Fig.2 KLF2 inhibited apoptosis in BV2 microglial cells exposed to OGD. BV2 cells were infected with Ad-KLF2 or si-KLF2 before OGD treatment. A. Protein expression of KLF2 was determined by western blot assay. B. Cell proliferation was detected by MTT assay. C. LDH release was detected by LDH assay. D. Cell apoptosis were detected by flow cytometry. E. Protein expression of Bcl-2 and Bax were detected by western blot assay. F. Protein expression of caspase 3 and caspase 9 were determined by western blot assay. Data were expressed as the mean ± SD, n=3. These results were performed using the analysis of variance (ANOVA) test. *P<0.05 vs. Ctrl group; #P<0.05
vs. OGD+ad-GFP group, $P<0.05 vs. OGD+si-Ctrl group.
Fig.3 KLF2 inhibited inflammatory factor secretion and ROS production in BV2 microglial cells exposed to OGD. BV2 cells were infected with Ad-KLF2 or Ad-GFP before OGD treatment.
A. The levels of inflammatory factors (TNF-α, IL-1β, IL-6) were detected by ELISA. B. ROS production was detected by flow cytometry. C. Protein expression of TLR2 and TLR4 were determined by western blot assay. Data were expressed as the mean ± SD, n=3. These results were performed using the analysis of variance (ANOVA) test. *P<0.05 vs. Ctrl group; #P<0.05 vs. OGD+ad-GFP group.
Fig.4 KLF2 protected BV2 microglial cells on cell viability against OGD injury by modulating the BDNF/TrkB pathway. A. Protein expression of BDNF and TrkB were determined by western blot assay in BV2 cells after ad-KLF2 infection. The inhibitor of the BDNF/TrkB pathway (K252a) was used in OGD-injured BV2 cells that were infected with ad-KLF2. Data were expressed as the mean ± SD, n=3. The results were performed using the analysis of variance (ANOVA) test. B. Protein expression of BDNF and TrkB were determined by western blot assay. C. Cell proliferation was detected by MTT assay. D. LDH release was detected by LDH assay. Data were expressed as the mean ± SD, n=3. These results were performed using two-tailed t test analysis. *P<0.05 vs. Ctrl group; #P<0.05 vs. OGD+ad-GFP group. &P<0.05 vs. OGD+ad-KLF2 group.
Fig. 5 KLF2 protected BV2 microglial cells on cell apoptosis and inflammatory response against OGD injury by modulating the BDNF/TrkB pathway. The inhibitor of the BDNF/TrkB pathway (K252a) was used in OGD-injured BV2 cells that were infected with ad-KLF2. A. Cell apoptosis were detected by flow cytometry. B. Protein expression of Bcl-2 and Bax were detected by western blot assay. C. Protein expression of caspase 3 and caspase 9 were determined by western blot assay. H. The levels of inflammatory factors (TNF-α, IL-1β, IL-6) were detected by ELISA. I.
ROS production was detected by flow cytometry. J. Protein expression of TLR2 and TLR4 were determined by western blot assay. Data were expressed as the mean ± SD, n=3. These results were performed using two-tailed t test analysis. &P<0.05 vs. OGD+ad-KLF2 group.
Abbreviation list: KLF2: Krueppel-like factor 2, OGD: Oxygen and glucose deprivation, TLR2: Toll-like receptor 2, ROS: Reactive oxygen species, TNFα: Tumor necrosis factor α, IL-1β: Interleukin 1β, BDNF: Brain-derived neurotrophic factor, TrkB: Tropomyosin-related kinase receptor type B, DMEM: Dulbecco’s Modified Eagle’s Medium, FBS: Fetal bovine serum, LDH: Lactate dehydrogenase, SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Authors' contributions We wish to confirm the authors' contributions and final approval of this manuscript. Jingbin Zhou, Muchun Wang and Dongfeng Deng conceived and designed the study. Jingbin Zhou and Dongfeng Deng performed the experiments. Muchun Wang analyzed the data. Daigang Lu, Jingbin Zhou wrote and reviewed the manuscript. Jingbin Zhou and Dongfeng Deng have overall responsibility for this manuscript.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Highlights 1. KLF2 and TLR2/4 were abnormally expressed in OGD induced BV2 cells. 2. KLF2 inhibits apoptosis in BV2 cells exposed to OGD. 3. KLF2 reduces levels of inflammatory factors and ROS in BV2 cells exposed to OGD. 4. KLF2 protects BV2 cells against OGD injury by modulating the BDNF/TrkB pathway.